DeltaFosB ka tsela e sa tobang e laola mosebetsi oa Cck promoter (2010)

Brain Res. 2010 May 6; 1329: 10-20.

E phatlalalitsoe marang-rang 2010 March 11. Doi:  10.1016 / j.brainres.2010.02.081

PMCID: PMC2876727

John F. Enwright, III,1 Megan Wald,1 Madison Paddock,1 Elisabeth Hoffman,1 Rachel Arey,2 Scott Edward,2 Sade Spencer,2 Eric J. Nestler,3 'me Colleen A. McClung2, *

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Moetso oa khatiso oa ho qetela oa mohatisi o fumaneha ho Resin ea Boko

Sheba lihlooho tse ling ho PMC hore Qotsa sehlooho se hatisitsoeng.

Eya ho:

inahaneloang

Tse ling tsa liphetoho tsa bohlokoa tsa biochemical, sebopeho, le boits'oaro bo susumetsoang ke ho pepesehela lithethefatsi tsa tlhekefetso ho bonahala li tsamaisoa ke ntho e tsitsitseng ea mongolo oa ΔFosB. Mosebetsi oa nakong e fetileng o bonts'itse hore osFosB overexpression ea litoeba bakeng sa libeke tsa 2 e lebisa ho keketseho ea polelo ea mefuta e mengata ho striatum, boholo ba eona hamorao e theotsoeng kamora ho latela libeke tsa 8 tsa polelo ea ΔFosB. Ho khahlisang, palo e kholo ea mefuta ena e ile ea ngolisoa ka mokhoa o fetisang taba ea sengoloa sa CREB. Ho ne ho sa hlaka ho tsoa thutong ena, hore na -FosB ea nakoana e laola mefuta ena ka CREB. Mona re fumana hore libeke tsa 2 tsa ΔFosB overexpression e eketsa polelo ea CREB ho striatum, phello e phatlohang ke libeke tsa 8. Ho kenella pele ho nako ho amana le ho eketseha ha CREB ho tlatselletsa ho ba bang ba buoang ka mofuta oa mofuta. Ho makatsang ke hore, mofuta o le mong o neng o belaelloa oa CREB o thehiloe litlalehong tse fetileng, cholecystokinin (Cck), e ne e sa laoloe ke CREB ka striatum. Ho etsa lipatlisiso tse ling tsa tsamaiso ea Cck Kamora ho ΔFosB ho tebisa maikutlo, re netefalitse hore nako e khuts'oanyane ea ΔFosB e tebile ka bobeli Cck ts'ebetso ea papatso le polelo ea mofuta. E eketsa ts'ebetso e tlamang sebakeng se tlamang sa CREB se tlamang Cck morekisi. Leha ho le joalo, leha sebaka sa CRE se hlokahala bakeng sa polelo e tloaelehileng ea basal ea Cck, ha e hlokahale bakeng sa tlhahiso ea ΔFosB ea Cck. Ha li kopantsoe hammoho, liphetho tsena li fana ka maikutlo a hore le ha ts'ebetso ea nakoana ea ΔFosB e eketsa polelo le tšebetso ea CREB ho baphatlalatsi ba bang ba gene, ha se ona feela mokhoa oo mefuta ea ona e ngolisitsoeng tlasa maemo ana.

Keywords: li-bokellase

Eya ho:

SELELEKELA

Ho pepesehela lithethefatsi tsa tlhekefetso ho baka liphetoho tse biochemical le sebopeho libakeng tse 'maloa tsa boko. Liphetoho tsena ho nahanoa hore li kenyelletsa liphetoho lipakeng tsa mofuta oa polelo ea gene tsamaisong ea mesolimbic. Sistimi ena e entsoe ka li-neuron tsa dopaminergic tse projeke e tsoang sebakeng sa "ventral tegmental" (VTA) bohareng ba "mele" ho fihlela bohareng ba "" spiny neurons "bohareng ba methapo ea kutlo (NAc) (ventral striatum) hammoho le likarolo tse ling tsa boko. Liphetoho tšebetsong ea lintlha tse peli tse hatisitsoeng, protheine ea karabo ea cAMP (CREB) le ΔFosB (protheine ea lelapa la Fos), e reretsoe ho kopanya liphetoho tse ngata tsa biochemical, sebopeho, le boitšoaro tse bonts'ang khafetsa ho lithethefatsi tsa tlhekefetso ( e hlahlojoe ke Nestler, 2005).

CREB ke karolo e fumanehang hangata ea mongolo eo e leng setho sa lelapa la CREB / ATF. Li-Homodimers tsa CREB li hokahana mefuteng ea tatellano ea CRE (tumellano: TGACGTCA) e fumanoang ho khothalletso ea mefuta e mengata. Phosphorylation ea CREB (haholo serine 133, leha libaka tse ling li fumanoe) li ka susumetsoa ke li-cascade tse 'maloa tse saenneng ho kenyelletsa le tse theohelang li-receptor tsa dopamine (Cole et al., 1995). Kamora phosphorylation ho serine 133, CREB e hira protheine e kopanyang CREB (protein) e kopanyang kapa liproteine ​​tse amanang le eona tse lebisang polelong ea liphatsa tsa lefutso (e hlahlojoe ke Johannessen et al., 2004). Ho pepeseha ho sa feleng lithethefatsi tsa opiate kapa psychostimulant tsa tlhekefetso ho etsa hore ho be le keketseho ea nako le nako mesebetsing ea CREB ka har'a nucleus (Barrot et al., 2002; Shaw-Lutchman et al., 2002, 2003), mohopolo o ikamahanyang le litheko tsa ho theola lithethefatsi ka mokhoa o thabisang (Nestler, 2005).

Lliphetoho tsa ka mehla tsa lithethefatsi tsa tlhekefetso ho nahanoa hore li ka arolelanoa ka karolo ke ΔFosB, mofuta o tsitsitseng oa mofuta oa FosB (Nestler et al., 1999; McClung et al., 2004; Nestler, 2008). Litho tsa lelapa la Fos li ikhula le litho tsa lelapa la Jun ho theha mochine oa protheine oa 1 (AP-1). Tlhatlhobo ea AP-1 e kopanya likarolo tsa karolo ea karabo ea AP-1 (tumellano: TGACTCA) ho laola sengoloa (se hlahlojoe ke Chinenov le Kerppola, 2001). Le ha ho pepesetsoa hoa lithethefatsi tsa tlhekefetso ho lebisa ho ho kenngeng ha nako e khuts'oane (li-hrs) tsa litho tsa lelapa la Fos (hammoho le ts'ebetso e eketsehang ea CREB), ho pepesetsoa lithethefatsi khafetsa ho lebisa ho bokelleng hoa ΔFosB e tsitsitseng haholo (Hope et al., 1994; Perrotti et al., 2008), polelo ea eona e tsoelang pele libeke.

Litoeba tsa bi-transgenic ka mokhoa o sa hlokeng ho fetelletsa kapa ΔFosB kapa CREB ka hara striatum e kholo li bonts'a mefuta e mengata ea biochemical le boits'oaro bo bonoang liphoofolong tse pepesetsoang hangata ho li-psychostimulants kapa lithethefatsi tse ling tsa tlhekefetso. Aliphetoho tsa polelo ea gene liphoofolong tsena tse fetotsoeng li bonts'a mefuta e mengata e ntlafalitsoeng ke libeke tsa nakoana tsa (2 libeke) oxpxpression ea ΔFosB, liphetoho tse amanang le phokotso e tšoanang ea moputso oa lithethefatsi. Liphetoho tse hlahisoang ke mofuta oa gene le karabelo ea boits'oaro bo nang le osFosB e ne e ts'oana hantle le tse bonoang ho liphoofolo CREB tse fetang tekano bakeng sa libeke tsa 2 kapa 8 (McClung le Nestler, 2003). Ka lehlakoreng le leng le hlollang, overexpression ea nako e telele (libeke tsa 8) tsa ΔFosB e fokotse polelo ea bongata ba mefuta ena e ts'oanang mme e lebisa keketsehong ea moputso oa lithethefatsi (Kelz et al., 1999; McClung le Nestler, 2003).

Mofuta o mong o khethiloeng lithutong tsena ke cholecystokinin (Cck), neuropeptide e hlahisitsoeng libakeng tse 'maloa tsa boko, ho kenyelletsa le striatum (Hoktima et al., 1980). CCK e khona ho fetisa phetiso ea dopaminergic (Vaccarino, 1992), ea amehang moputsong oa lithethefatsi le ntlafatso (Josselyn et al., 1996; Josselyn et al., 1997; Hamilton, et al., 2000; Beinfeld et al., 2002; Rotzinger et al., 2002), hape e kenelletsoa ho striatum ke cocaine e sa foleng (Ding le Mocchetti, 1992). Ntle le moo Cck Phatlalatso e bontšitsoe hore e arabela ka bobeli ho CREB le AP-1 complexes (Haun le Dixon, 1990; Deavall, et al., 2000; Hansen, 2001). Kaha boholo ba liphatsa tsa lefutso (ho kenyeletsa Cck) e bonts'itseng polelo e eketsehang ka mor'a CREB le polelo ea nakoana ea ΔFosB li ne li tsejoa kapa li belaelloa ka mofuta oa sepheo sa CREB (McClung le Nestler, 2003), re ne re batla ho tseba hore na polelo ea nakoana ea ΔFosB e tlisa taolo ea mefuta ena (e shebane haholo le eona Cck) ka taelo ea CREB kapa ka mekhoa e rarahaneng ea tsamaiso ea liphatsa tsa lefutso.

Eya ho:

LIPOTSO

ΔFosB overexpression butle-butle e eketsa maemo a CREB

Re sebelisitse blotting ea Bophirimela ho fuputsa hore na ΔFosB everexpression e eketsa maemo a protheine ea CREB maemong a litoeba. Bakeng sa thuto ena, re sebelisitse litoeba tsa bi-transgenic (mola 11A) tse tsamaisang transmit ea NSE-tTA le transetic ea TetOp-ΔFosB. Ha ho na le doxycycline, litoeba tsena li bonts'a keketseho e matla ea polelo ea ΔFosB methapong e ntle ea dynorphin in the striatum (Kelz et al., 1999). Mokhoa ona oa ho fetelletsa litaba oa ΔFosB o ngotsoe haholo ebile o lumella ho natsoa ka mokhoa o ikhethileng tikolohong, o tsamaisanang le kenyelletso ea ΔFosB e bonoang liphoofolong tse pepesitsoeng cocaine e sa foleng (Hope et al., 1994; Kelz et al., 1999). Re fumane hore maemong a 11A mice overexpressing ΔFosB, maemo a liprotheine tsa CREB a ntlafalitsoe haholo kamora libeke tsa 2 tsa polelo mme maemo ana a ile a khutlela ho basement kamora libeke tsa 8 tsa polelo (Setšoantšo sa 1A, n = 8). Ho tseba hore na liphetoho tse maemong a CREB ka khatello ea maikutlo ea nako e khuts'oane ea ΔFosB li ka phetoa ts'ebetsong e ngoe, re ile ra hatella ΔFosB hanyane ka hanyane ho lisele tsa PC12 mme ra fumana hore maemo a CREB a eketsehile haholo (p <0.05) ha a bapisoa le litsamaiso (Setšoantšo sa 1B, n = 4). Liphetho tsena li bonts'a hore polelo ea nakoana ea ΔFosB e eketsa maemo a protheine ea CREB.

Setšoantšo sa 1

Setšoantšo sa 1

Maemo a CREB a eketseha ha vereFosB overexpression. Tlhahlobo ea blot ea bophirima ea lysates ho tsoa (A) mouse striata ho tloha 11A litoeba tsa dox bakeng sa 2 kapa 8 libeke kapa (B) Lisele tsa PC12 li tebisa khatello ea ΔFosB. Lintlha mo A li bonts'oa joalo ka phetoho ea liperesente ho off dox ha e bapisoa ...

Ka bobeli ΔFosB le CREB li tlama ho bahlahisi ba mofuta o itseng oa sepheo ho striatum kamora nako e khuts'oanyane ea ΔFosB

Re sebelisitse ChIP (chromatin immunoprecipitation) lits'oants'o tsa lithaelese tsa striatal ho bona hore na ΔFosB kapa CREB binding e hlahile ho tse itseng tse reretsoeng ho holisa liphatsa tsa lefutso mme haeba ho tlama hona ho ne ho eketseha ho latela nako e khuts'oanyane ea ΔFosB overexpression. Re ile ra sekaseka tlama ho BDNF 'me Cck barekisi, hobane liphatsa tsa lefutso ka bobeli li khothalelitsoe haholo kamora nako e khutšoanyane ea ΔFosB overexpression, CREB overexpression, kapa kalafo e sa foleng ea koae (McClung le Nestler, 2003). Re boetse re ile a sekaseka tlamang ho CDK5 morekisi, kaha ke sepheo se tsebahalang sa ΔFosB (Chen le al., 2000; Bibb et al., 2001; Kumar et al., 2005). Qetellong, re lekanya ho tlama prodynorphin papatso, kaha lithuto tse fetileng li fumane hore e ka tlangoa ke ΔFosB le CREB tlasa maemo a fapaneng (Andersson et al., 2001; Zachariou et al., 2006).

Tlhahlobo ea ChIP e entsoe ka striata ho tloha 11A litoeba tse fetisang ΔFosB bakeng sa libeke tsa 2 li sebelisa li-antibodies tse hlokomelang CREB kapa ΔFosB. Maemong a tloaelehileng, re fumane hore ΔFosB e tlamahanya le CDK5 'me prodynorphin baphatlalatsi, ha ho se na lintho tse tlamang BDNF or Cck baphatlalatsi (Lethathamo 1). Ntle le moo, ΔFosB oxpxpression e eketse tlamo ea ΔFosB ka CDK5 papatso, empa eseng ho prodynorphin morekisi. Ka mor'a moo re ile ra lekanya CREB e tlamang ho baphatlalatsi bana mme ra fumana hore CREB e tlameha ho CDK5, BDNF 'me prodynorphin bahlahisi, empa eseng ho Cck papatso, maemong a tloaelehileng, le hore ΔFosB overexpression ea libeke tsa 2 e eketsa CREB e tlamang ho BDNF 'me prodynorphin batsebi, empa eseng CDK5 morekisi. Ha li kopantsoe hammoho, liphetho tsena li bonts'a hore ΔFosB le CREB ka bobeli ba tsona li ka tlatsana le ho tse ling tsa lipapatso tsa mofuta o kang prodynorphin 'me CDK5leha ho le joalo, ho tlama CREB ho ikhethile ho barekisi ba bang ba kang BDNF. Ntle le moo, ΔFosB overexpression e lebisa keketseho ho ΔFosB e tlamang ho baphatlalatsi ba bang, joalo ka ha ho ka lebelloa, empa hape le ho CREB e tlama ho barekisi ba ikhethang, e lumellanang le tlhahiso ea ΔFosB-Mediated CREB e hlokometsoeng nakong ena.

Lethathamo 1

Lethathamo 1

Ho tlangoa ha liprotheine tsa kemiso e tlamang ho batho ba bang ba ntlafatsang ho litheko tse fetisang forFosB bakeng sa libeke tse peli.

Mosebetsi oa nakong e fetileng o bontšitse hore liphetoho liphatlalatsong tsa gene tse hlahisitsoeng ke nako e telele ea ΔFosB overexpression li amahanngoa le liphetoho tsa chromatin, haholo, acetylation ea histone H3, ho li-promoters tsa gene tse ikhethang.Kumar et al., 2005). Ho fumana hore na hona ho ka baka liphetoho phetohong ea sebopeho sa mofuta oa mofuta oa nakoana oa ΔFosB overexpression, ra lekanya ho tlangoa ha histone H3 (phetoho ea histone e amanang le chromatin e sebetsang), kapa methylated histone H3 (Lys9, phetoho ea histone e amanang le ho fetisoa. chromatin e sa sebetseng). Re fumane hore overexpression ea ΔFosB bakeng sa libeke tsa 2 ha ea ka ea tlisa liphetoho tse tlisoang ke H3 ea acetylated ho mofuta ofe kapa ofe oa bahlahisi ba ithutetseng (Lethathamo 1). Re fumane phokotseho e kholo ea ho tlama ha methylated histone H3 ho prodynorphin morekisi, empa ha ho na tlamo ho Cck papatso le ha ho na phetoho ea ho tlama ho CDK5 'me BDNF bahlahisi, ba fana ka maikutlo a hore ona ha se mochini o akaretsang oo ΔFosB, ka nako e khuts'oane, e laolang polelo ea mofuta. Re ne re maketse re bile re thahasella taba ea hore ha rea ​​fumana liphapang lipapisong tsa rona tsa ChIP tse ka bakang keketseho ea Cck polelo ea mRNA ka har'a litoeba tsa 11A kamora libeke tsa 2 tsa polelo ea ΔFosB mme ea etsa qeto ea ho fuputsa mochine ona ho ea pele.

Nakoana ea nakoanaFFB ea eketseha Cck polelo e methating e mengata

Boitšoaro ba Microarray ba bi-transgenic 11A litoeba tse feteletseng tsa ΔFosB ho striatum li fumane hore Cck maemo a polelo a eketseha ka mor'a libeke tsa 2 tsa ho qeta nako ea ho qeta nako o tebile mme butle-butle o fokotseha ka linako tse telele tsa polelo ea ΔFosB (Setšoantšo sa 2A, n = 3). Re netefatsa phello ena bakeng sa Cck o sebelisa PCR ea nako ea nnete ho sehlopha se arohaneng sa liphoofolo ka libeke tsa 2 le 8 mme o fumane liphetho ka linako tse peli hore li ts'oane le tlhahlobo ea microarray (data e sa bontsitsoeng).

Setšoantšo sa 2

Setšoantšo sa 2

Ho qeta nako e khutšoanyane ho qojoa ha ΔFosB hoa eketseha Cck polelo ea mofuta. (A) Cck polelo ea mRNA ka striatum e latelang ΔFosB overexpression ho litoeba tsa 11A bakeng sa 1, 2, 4, kapa libeke tsa 8. Tlhahlobo ea Microarray e entsoe ka lisampuli tsa striatal le Cck maemo ...

Ho etsa lipatlisiso tse ling ka bokhoni ba ΔFosB ba ho laola Cck polelo, re sebelisitse lisele tsa PC12 fetoleloa nako le nako ka a Cck-lucitterase motlalehi oa plasmid le mohlomong eitherFosB expression plasmid kapa palo e lekanang ea pCDNA. Ka bobeli matsatsi a (n = 5-9) le matsatsi a mararo (n = 11-13) ea ΔFosB overexpression e bakile keketseho e kholo ho CckPolelo Ho feta moo, matsatsi a mararo a oFosB overexpression a hlahisitse haholo Cck- ho kenella ka matla ha ho bapisoa le matsatsi a mabeli a ho feta tekong (Setšoantšo sa 2B). Ho tebella hoa ofFosB ha hoa ka ha fana ka tlhahiso ea tlhahiso e sa thekeseleng e sa hlahiseng (ya data e sa bontsitsoeng). Tlas'a maemo a kalafo ha ho na phokotso ea Cck- Ketsahalo e bonoang e bonahala. Liphetho tsena li bonts'a hore polelo ea nakoana ea ΔFosB e eketsa ts'ebetso ea Cck papatso, mme e siea e sa arabe mochine oo khatello ea khatello ea nako e telele ea osFosB e hatellang Cck polelo.

OFosB oxpxpression e eketsa tlama setsing se tlamang sa CREB ho Cck moetlisisi

Litlhahlobo tsa rona li fumane hore Cck polelo e eketseha kamora polelo ea nakoana ea ΔFosB, leha ho le joalo, CHIP ea rona e fumane hore CREB kapa ΔFosB ha e tlangoe ho Cck morekisi. Ho fumana hore na ho na le liphetoho tse teng tsamaisong ea protheine ho Cck morekisi ea latelang ΔFosB overexpression, re sebelisitse lits'ebetso tsa motlakase oa motlakase oa ho falla (EMSA) ka projeke e nang le sebaka sa mofuta oa CRE-putative se teng kahare ho Cck morekisi. Re sebelisa litlatsetso tsa nyutlelie tse tsoang holofe ea 11A litoeba tse fetisang vereFosB bakeng sa libeke tsa 2, re fumane keketseho ea ho tlama setsi sa tlatsetso sa CRE sebakeng sa Cck morekisi (Setšoantšo sa 3 A, C, n = 4). Ho khahlisang, 11A litoeba tse feteletseng tsa ΔFosB bakeng sa libeke tsa 8, tse bonts'ang ho fokotseha ha Cck polelo, hape e bonts'itse tlamo e eketsehileng setsing sena (Setšoantšo sa 3B, C, n = 4). Ha re etsa papiso, re ile ra hlahloba tlamo ea sebaka sa tumellano ea CRE maemong a makatsang a ΔFosB bakeng sa libeke tsa 2 mme ra fumana CER e matla e kopantseng litlatsetso tse ntle, empa ha ho na keketseho e tlamang ea ho qalella ha ΔFosB (Setšoantšo sa 3F, n = 4). Ka hona, leha ho na le keketseho ea ΔFosB e bakiloeng ke maemo ohle a CREB a bonoang nakong ena, liphetho tsena li tsamaellana le litekanyetso tsa rona tsa ChIP tse fumanang hore ho eketseha ha CREB ho batsetedi ho latela ΔFosB overexpression ho ikhethile ho mefuta e itseng feela mme ha se ketsahalo ea lefats'e. . Ho fumana hore na CREB e tlameletsoe ho Cck motšehetsi oa tlhahlobo ea rona ea EMSA, re sebelisitse lits'ebetso tsa boemo bo holimo le anti-anti-CREB e khethehileng. Ka tumellano le litlatsetso tsa rona tsa ChIP, ha rea ​​fumana tlamo ea CREB ho Cck probe e nang le sebaka sa "CRE" se tlatsetsang lits'ebetsong tsa EMSA, athe CREB e ile ea tiisa le ho tiisa sebaka sa tumellano ea CRE (Setšoantšo sa 3D, E, n = 4). Ketsahalo e tlamang ea DNA ho Cck morekisi o ne a boetse a sa angoe ke anti anti to ΔFosB (data e sa bonts'ang), tlasa maemo a bonts'itsoeng lithutong tse 'maloa tse fetileng ho thibela ΔFosB e tlamang libaka tsa AP-1 tsa netefal (Hope et al., 1994a, 1994b; Chen le al., 1995, Hiroi et al., 1998). Re boetse ra etsa liteko tsa ChIP litšoantšong tsa liphoofolo tsa 11A kamora libeke tsa 8 tsa polelo ea ΔFosB mme ha re sa fumana tlamo ea CREB kapa ΔFosB Cck morekisi (ya data a sa bontšoa). Kahoo, polelo ea "ΔFosB e lebisa hoketseho ea liprotheine tse tlamang ho Cck papatso ka mor'a libeke tsa 2 le 8 tsa ho buoa; leha ho le joalo, hore na mabaka ana ke afe ho ntse ho sa tsejoe.

Setšoantšo sa 3

Setšoantšo sa 3

Protein e tlamang ho Cck morekisi. (A, B) Electrophoretic motsamao oa phetoho o sebetsa o sebelisa Cck Sebaka sa marang-rang sa CRE se nang le lithaelese tse tsoang ho liphoofolo ho feta animalsFosB bakeng sa libeke tsa 2 (A) kapa 8 libeke (B). Ho (A), tlholisano le tlholisano ea bao e seng morui ...

Sebaka sa putlama CRE ho Cck morekisi ha a ikarabelle bakeng sa tšebetso e eketsehileng ea papatso

Kaha re fumane keketseho ea liprotheine tse tlamang re sebelisa sekhechana sa Cck papatso, e nang le sebaka sa "CRE" se tlatsetsang, kamora ΔFosB oxpxpression, re ne re batla ho tseba hore na sebaka sena sa marang-rang sea hlokahala bakeng sa ho eketseha. Cck polelo e latelang ΔFosB oxpxpression. Ho hlahloba monyetla ona, re fetotse lisele tsa PC12 ka Cck-luciferase plasmid e nang le sebaka sa eona sa CRE-se tšoanang kapa se nang le phetoho sebakeng se ka felisang tšebelisano efe kapa efe le CREB. Ho khahlisang, phetoho ea sebaka sa marang-rang sa CRE e theohile haholo Cck Ts'ebetso ea papatso ke 32% (Setšoantšo sa 4A, n = 9), empa ha e a ama Cck Ho kenyelletsoa ha morekisi ke ΔFosB overexpression (Setšoantšo sa 4B, n = 11-13). Sena se fana ka maikutlo a hore, leha ho le joalo Cck Phatlalatso e hloka sebaka se ts'oanang le CRE-like site bakeng sa ts'ebetso e felletseng ea basal, ts'ebetso ea papatso e eketsehang e hlahisoang ke ΔFosB overexpression ha e hloke tatellano ea CRE.

Setšoantšo sa 4

Setšoantšo sa 4

The CckSebaka se kang CRE ha se hlokahale Cck tlhahiso ka byFosB. (A) CckTs'ebetso ea -luciferase e lekantsoe matsatsi a 2 kamora phetiso ka mokhoa o tloaelehileng Cck-luciferase kapa eo ho eona sebaka sa marang-rang se kang CRE se fetotsoeng. * p <0.05 (B) Cck-motshelo ...

cFos e tlamang ho Cck moetlisisi

Lithuto tse fetileng li fumane seo cFos mRNA e eketseha ka polelo ea nakoana ea ΔFosB, leha ho le joalo, ka mor'a polelo ea nako e telele ea ΔFosB, bokhoni ba kalafo ea koae bo kenyelletsa cFos ho striatum bo fokotsehile (McClung le Nestler, 2003; Renthal et al., 2008). Ka hona, ho ka etsahala hore li-cFos li kenye letsoho keketseho ea Cck polelo e latelang poleloana ea poleloana ea ΔFosB haufinyane. Re sebelitse lits'oants'o tsa ChIP ka antibody e khethehileng ea li-cFos le ho lekanya li-cFos tse tlamelletsoeng ho Cck papatso le ntle le polelo ea nakoana ea andFosB. Ha re ntse re fumane hore li-cFos li tlamehile ho Cck morekisi, ho tlama hona ha hoa ka ha eketsa haholo ho latela ΔFosB overexpression (Setšoantšo sa 5, n = 5). Sena se fana ka maikutlo a hore kaha li-cFos li tlama Cck papatso, e ka kenya letsoho taolong ea kakaretso ea Cck polelo, empa ha e a ameha morerong oa Cck papatso ke ΔFosB.

Setšoantšo sa 5

Setšoantšo sa 5

cFos e tlamang ho Cck morekisi. Lits'oants'o tsa thibelo ea tšireletso ea Chromatin li entsoe ka antibody e khethehileng bakeng sa li-cFos li sebelisa lithane tsa striatal ho tsoa ho litoeba tsa ΔFosB overexpressing e kanna ea ba ka Dox, kapa kamora libeke tsa 2 tsa ho tlosoa hoa Dox. Tlhahlobo ea PCR ea nako ea nnete e ne e le ...

Eya ho:

TŠOHLOA

Boithuto bona bo tiisa le ho hola molemong oa liphumano tse fetileng bo bonts'ang hore ΔFosB e laola mofuta oa mofuta oa mofuta ho striatum mme re fumana hore e etsa joalo ka mekhoa e mengata kamora ho bua ha nakoana. Re bonts'a hore, kamora libeke tsa 2 tsa overexpression, ΔFosB e tlama ka kotloloho ho bahlahisi ka mefuta e meng e lebisang phetohong ea polelo (ke hore. CDK5). Ho feta moo, e eketsa maemo a liprotheine tsa CREB, e le phello e bonoang liseleng tse nang le cultured le li-striatum, e lebisang ho eketseheng ha CREB ho babapatsi ba bang ba mofuta. dynorphin 'me BDNF). Phuputsong e fetileng, re fumane hore nako e khuts'oanyane ea ΔFosB ho striatum e lebisa ho liphetoho tse ngata tse tšoanang tsa liphatsa tsa lefutso tse fumanoang ha CREB e tebile, mme e lebisa likarabo tse ts'oanang tsa boits'oaro mehatong ea khetho ea cocaine (McClung le Nestler, 2003). Kahoo, sephetho sa hona joale sa hore ΔFosB e lebisa ho kenngoeng ha CREB, hammoho le ho tlama CREB ho bahlahisi ba bang ba mofuta oa gene, ho thusa ho hlalosa hore na ke hobaneng ha liphetoho tse ngata tsa polelo ea mofuta oa tsona li arotsoe ke lintlha tse peli tse ngotsoeng.

Ho kenella ha CREB ke ΔFosB hoa khahla, kaha ho bontšitsoe hore lithethefatsi tsa tlhekefetso li baka liphetoho maemong a CineB a serine 133-phosphorylatedMattson et al., 2005) le ho eketsa sengoloa sa CRE-Mediated (Barrot et al., 2002; Shaw-Lutchman et al., 2002, 2003), ntle le ho fetola maemo ohle a CREB. Ho ka etsahala hore liphetoho tse maemong a CREB li ka ba tsa nakoana, ka hona, li hlotsoe habonolo lithutong tse ling. Ka matsohong a rona, re bone keketseho e khothalletsoang ke koae ka CREB mRNA ka ho khetheha liteko, leha ho le joalo, phello ena e fapana haholo (ho sa bonoe e sa phatlalatsoang). Kaha litoeba ka bobeli tsa 11A transgenic le lisele tsa PC-12 li bonts'a ts'ebetso ea CREB kamora nako e khuts'oanyane ea ΔFosB, sena se fana ka maikutlo a hore CREB e hlile e ts'oaroa ke ΔFosB (kapa sepheo sa ΔFosB), e fana ka mokhoa o mong hape oa ho hlalosa liphetoho tse susumetsoang ke lithethefatsi ho gene polelo.

Ho makatsang ke hore re fumane hore CREB kapa ΔFosB ha e tlamele ho Cck papatso leha e le Cck Polelo e ntlafalitsoe ka mokhoa o hlakileng ka mor'a polelo ea nakoana ea ΔFosB. Cck ke neuropeptide e ngata e hlahisitsoeng ho VTA le NAc (Hoktima et al., 1980) hape o ameha karabong ea boitšoaro ho lithethefatsi tsa tlhekefetso (Josselyn et al., 1996; Josselyn et al., 1997; Hamilton, et al., 2000; Beinfeld et al., 2002; Rotzinger et al., 2002). Litloaelong tsa seleng, Cck morekisi o bontšitse haholo 'me o bontšitsoe ho arabela ho litho tsa lelapa tsa CREB le AP-1 (e hlahlojoe ke Hansen, 2001). Haun le Dixon (1990) e bonts'itse hore li-AP-1 complexes li ka tlama ho Cck Sebaka sa marang-rang se bitsoang CRE a vitro, 'me e ile ea bonts'oa hamorao, ho sebelisoa lisele tsa SK-N-MC neuroblastoma, hore phetoho ea sebaka sa marang-rang sa CRE e ile ea fokotsa karabelo ea morekisi ho cFos / cJun (Rourke et al., 1999). Ka 'nete, re boetse re eketseha Cck ts'ebetso ea papatso (Setšoantšo sa 2) le ho tlama ntho ea CRE-kapa e haufi le eonaSetšoantšo sa 3) holim'a overexpression ea ΔFosB, setho se seng sa lelapa sa AP-1, empa ha re fumane tlamo e tobileng ea ΔFosB Cck moetlisisi Ka vivo or a vitro, leha e tebeloa le ho feta.

Mosebetsi o mongata o bonts'a karolo ea CREB taolong ea Cck ts'ebetso ea papatso. The Cck Sebaka sa C--C se bolokiloe hohleHansen, 2001) le, litsong tse ling tsa setso sa lisele, mefuta ea CREB le AP-1 li tlama sebakeng sena sa marang-rang mme li bohlokoa bakeng sa Cck ts'ebetso ea papatso (Haun le Dixon, 1990; Deavall, et al., 2000; Hansen, 2001). Ntle le moo, bahlahisi ba 'maloa ba tsebahalang ba Cck polelo (ho kenyelletsa bFGF, PACAP, peptones, le depolarization) li bontšitsoe ho sebetsa ka CREB (Hansen, et al., 1999; Deavall, et al, 2000; Bernard, et al., 2001; Gevrey, et al., 2002; Hansen, et al., 2004). Rona Cck-datha ea moqolotsi oa litaba ea mofuta oa tlhahlobo ea tšebetso e tšehetsa karolo ea bohlokoa molemong oa sebaka sa CRE joalo ka taolo ea Cck ts'ebetso ea papatso, kaha phetoho ea sebaka sena sa marang-rang e fokotsa basal Cck ts'ebetso ea papatso le CckPolelo ea -luciferase e susumetsoang ke VP16-CREB, sebopeho se sebetsang sa CREB, sea lahleha ha sebaka sena sa marang-rang se fetotsoe (tlhokomeliso e sa phatlalatsoang). Kahoo, re ile ra makala ha re fumana hore CREB ha e bonahale e le tlamo Cck khothaletso lithutong tsa striatal ebang ke motheong oa motheo kapa holima nako e khuts'oanyane ea ΔFosB ha maemo a CREB a eketseha. Sena se tiisa hore maemo a CREB ka bobeli ha se eona feela taba ea ho khetha hore na ho tlangoa bokae sebakeng sena, 'me sena se tšehetsoa ke mosebetsi oa ba bang (Cha-Molstad, et al., 2004). Ho tloha ka barekisi ba mefuta e meng e neng e tsejoa esale pele ea CREB, joalo ka BDNF 'me prodynorphin, ha e tlama CREB, re na le tshepo ea ho fumana hore CREB ha e tlangoe ho Cck phaelletsang mesebetsing e nyarosang ea nyutlelie. Ho feta moo, ho kenyelletsoa ha CckTs'ebetso ea -luciferase ea ΔFosB e ne e sa itšetleha ka sebaka sa marang-rang sa CRE, a fana ka maikutlo a hore ΔFosB ha e laole Cck polelo ka ho laola tlamahano e tobileng ea CREB ho Cck morekisi.

Ho sitoa ha rona ho bona CREB e sebelisana le Cck papatso e tšehelitsoe ke Renthal et al. (2009), ea sebelisitseng mokhoa oa ChIP-chip ho sheba liphetoho tsa lefats'e ho phosphorylated CREB (pCREB) le ΔFosB e tlamang ka striatum kamora ho pepesetsoa ha koae. Litekong tsena, li-protein tsa li-protein tsa DNA li ile tsa emisoa ka li-antibodies tsa ΔFosB kapa pCREB mme DNA ea pele, kamora ho ngola, e ile ea koptjoa ho ba morekisi ea holisang. Ha mefuta e mengata ea CREB ea mehleng ea khale e ne e khethollotsoe ka mokhoa ona (mohlala, BDNF, prodynorphin), Cck ha ea ka ea khetholloa. Ntle le moo, ho bonahetse hore tlamo ea CREB sebakeng sa tumellano ea CRE e fapana haholo pakeng tsa mela ea sele e ratoang (Cha-Molstad, et al., 2004). Lithuto tsohle tse fetileng tse fumaneng tšebelisano ea CREB le Cck Phatlalatso e entsoe moetlong oa sele (eseng boko ho tsoa moo sampole ea rona ea EMSA le ChIP) mme ba sebelisa phetoho ea li-gel ho etsa lipatlisiso ka ts'ebelisano ea protheine-DNA. Le ha EMSA e ka lekola monyetla oa ntho e tlamang ho latela tatellano ea DNA, ChIP e fana ka pono e hlakileng ea litšebelisano tsena. Ka vivo. Ntle le moo, boholo ba data bo bonts'a ho kenyelletsa ha Cck ts'ebetso ea papatso kapa CREB e tlamang ho Cck Promoter e fumanoe liseleng tse neng li khothalelitsoe ke lintlha tse kang li-peptones (Bernard et al., 2001), depolarization (Hansen, et al., 2004), kapa baetsi ba mefuta e fapa-fapaneng ba li-cascade tse sa tsebeng letho tse kenyeletsang cAMP le ERK (Hansen et al., 1999). Litekong tsa rona, "ts'usumetso" e le 'ngoe feela e sebelisitsoeng e ne e le ho hlonama ha ressionFosB, ho lekaneng ho e susumetsa Cck polelo. Ha ho kopantsoe hammoho, sena se fana ka maikutlo a hore bokhoni ba CREB ho tlama ho (le ho ka laola) Cck morekisi o ipapisitse le mofuta oa sele le ts'ebetso ea litsela tse itseng tse tšoaeang. Ntle le moo, the Cck CRE-joalo ka morekisi (bonyane liseleng tsa PC12 tse sa buloang le ho striatum ea mouse) ha se sepheo se tobileng sa CREB kapa ΔFosB. Ho khahlisang, molao oa FosB Polelo ea CREB e boetse e ikhethile ho mofuta oa sele le mofuta oa boitšisinyo. Phuputso e entsoeng ke Andersson et al a. fumana hore ente ea CREB antisense oligonucleotides ho toeba striatum ka mokhoa o fokolang o thibetse tlhahiso ea FosB latela tsamaiso ea koaeAndersson et al., 2001). Leha ho le joalo, ba boetse ba fumane hore bokhoni ba L-Dopa ba ho susumetsa FosB expression in 6-OHDA-lesioned striatum ha e ea susumetsoa ke boteng ba li-oligonucleotides tsa CREB.

Kaha CREB le ΔFosB li bonahala li laola ka tsela e sa tobang Cck khothaletso, le liphetoho phetohong ea chromatin li tlalehiloe ho araba mefuta e fapaneng e susumetsang e khothalletsang ΔFosB (Tsankova et al., 2004; Kumar et al., 2005; Renthal et al., 2008), re nahanne hore ΔFosB e kanna ea feto-fetoha ka mokhoa o sa sebetseng ka ho ntlafatsa ts'ebetso ea papatso ka ho fetola sebopeho sa chromatin. Leha ho le joalo, ho iceFosB ea litoeba ka ho feteletseng ea libeke tsa 2, ho ne ho sena phetoho ho histone H3 acetylation ho Cck morekisi (Lethathamo 1). Sena se tšehetsoa ke data ea ChIP-chip ea Renthal et al. (2009), ea tlalehileng hore ha ho na phetoho ea h3 ea acetylated Cck papatso ea litoeba e pepesetsoeng cocaine e sa foleng. Ho tloha ka Cck morekisi o ntse a le mafolofolo striatum, ha ho mofuta o hatellang oa methylated H3 e neng e lebelletsoe kapa e bonoe. Ho khahlisang, hape re bone ho sena phetoho ka lebaka la ΔFosB overexpression ho H3 ea acetylated BDNF papatso (e neng e bonts'a ho eketsa CREB binding) kapa ho CDK5 'me prodynorphin Barekisi (ba bonts'itseng ΔFosB e tlamang). Kaha ho na le liphetoho tse ngata tse ngata tse amanang le liphetoho tsa tšebetso ea ntlafatso (e hlahlojoe ke Rando le Chang, 2009), mohlomong ho na le liphetoho tse ling tsa chromatin tse amanang le ho kenngoa hoa mefuta ena. Phetoho efe kapa efe ea chromatin e le 'ngoe, leha e le hangata e hakanyetsang boemo ba ts'ebetso ea papatso, e kanna ea se fetohe nakong ea ts'ebetso ea mofuta o itseng. Mosebetsing oa nako e tlang, ho tla ba monate ho sheba liphetoho tse ling tse ka bang sebopeng sa chromatin ho potoloha Cck papatso e latelang followingFosB overexpression. Ho khahlisang, koae e sa foleng (e ka 'nang ea etsahala dessyecik UctionFosB induction) e bonts'itsoe ho kenya letsoho polelo ea Sirtuin 1 le 2, sehlopha sa li-deacetylase tsa sehlopha sa III tse shebahalang li fetola physiology ea neuronal, ho supa ha ERK, le karabelo ea boitšoaro ho cocaine (Renthal et al., 2009). Ho hlahisoa ha histone deacetylase ho tla ba le bokhoni ba ho laola ka nako e tšoanang polelo ea palo e kholo ea liphatsa tsa lefutso dessyecik liphetoho tsa lefats'e ka sebopeho sa chromatin.

Motho a le mong ea ka nkang karolo Cck molawana o latelang ΔFosB ouffxpression e ne e le cFos ea setho sa lelapa sa AP-1. Pontšo ea cFos e laoloa ke CREB (Sheng et al., 1990; Impey et al., 2004), le cFos overexpression (concordant le overexpression ea molekane oa eona ea tlamang cJun) ea eketseha Cck ts'ebetso ea papatso (Rourke et al., 1999). Ka hona, maemo a eketsehileng a CREB ka lebaka la ho qeta nako ea ΔFosB a qeta nako a ka eketsa maemo a CF mme a baka tlamahanyo e eketsehileng ho Cck Sebaka sa marang-rang se bitsoang CRE. cfos mRNA e kenyellelitsoe likhoto tse latelang libeke tse peli tsa ΔFosB overexpression (McClung le Nestler, 2003) hape ea fokotseha ka litoeba tse pepesitsoeng ke cocaine e sa foleng kapa nako e telele ea ΔFosB overexpression (Renthal et al., 2008). Mona re fumana hore cFos e tlama ka kotloloho ho Cck papatso ea lisele tsa striatal, leha ho le joalo, ΔFosB oxpxpression ha e eke haholo ho tlama. Sena se fana ka maikutlo a hore le ha li-cFos li ka ameha ho laola Cck polelo ka kakaretso, phetoho ea ho tlama li-cFos feela ha se mochini o sebelisang whichFosB Cck polelo. Ho ka etsahala, leha ho le joalo, ΔFosB e ka susumetsa, liphetoho tsa morao-rao tsa cFos (mohlala, phosphorylation) kapa ea susumetsa polelo ea molekane ea tlamang (joalo ka cJun) kapa protheine ea tšebetso. Leha ho le joalo, ho tloha ka sebaka sa marang-rang se tšoanang le sa CRE (se kileng sa bontšoa e le sebaka sa tlamo bakeng sa mechini ea AP-1, bona Haun le Dixon, 1990) ha e hlokehe bakeng sa keketseho Cck Ts'ebetso ea papatso e bonoang ka ΔFosB overexpression (joalo ka ha e lekiloe litekong tsa rona tsa mofuta oa reporter), hoa utloahala hore mabaka a mang a ho fetisa a sebetsa le ona a laoloa ke ΔFosB.

The Cck Sekhechana sa papatso se sebelisitsoeng boitekong ba mofuta oa rona oa lucifase oa tlhahlobo se na le sebaka sa polokelo sa Sp1 se kopantsoeng le lebokose la E (le hlahlojoang ke Hansen, 2002). Haholo-holo, tatellano ea E-lebokose e bontšitsoe ho kopanya lintlha tse ngata tsa sengoloa (se hlahlojoe ke Forrest le McNamara, 2004). Re sebelisa lisele tsa PC12, re bone hore phetoho ea lebokose la E e ea fokotseha Cck Ts'ebetso ea papatso, empa ha e fetole karabelo ea morekisi ho ΔFosB (ya data e sa bonts'ang). Ho khahlisang, a CckMoqolotsi oa litaba oa -cluciferase ea tšetseng liphetoho sebakeng sa CRE le lebokoseng la E ha a na tšebetso e bonoang ebile ha a arabele ΔFosB overexpression (tlhokomeliso e sa phatlalatsoang). Mokena-lipakeng e mong ea ka etsang ketso ea ΔFosB ho Cck papatso ke li-ATF, mefuta e meng ea eona e tsejoang hore e kenyelletsa striatum ka ho pepeseha ho sa feleng ha psychostimulant (Green le al., 2008). Leha ho le joalo, ha re fumane bopaki ba ho kenella ha BFosB ka li-ATF tsena (data e sa bonts'itsoeng), 'me li-ATF li ne li ke ke tsa lebelloa ho tlama sebakeng sa phetoho se tšoanang sa CRE ho Cck morekisi.

Lebaka le leng la thuto ena ke hore re sebelisa sistimi ea bi-transgenic ho overexpress ΔFosB mme ka hona motho o tlameha ho ba le tšireletso ho ho bona ho tšoana pakeng tsa paradigm ena le tsamaiso ea cocaine e sa foleng. Leha ho le joalo, litoeba tsa transgenic tsa 11A li fana ka monyetla o ikhethang oa ho sheba litlamorao tse ikhethang tsa ΔFosB ho striatum, kaha ho ts'etleha hoa kelello ho lekanyelitsoe sebakeng sena sa boko (Chen le al., 1998), ha ts'ebetso ea cocaine e etsa hore ho be le liphetoho maemong a mang a mangata a kelello a ka amang striatum ka mokhoa o sa tobang. Ntle le moo, liphuputso tse 'maloa li tlalehile tšebetso e tšoanang ea boits'oaro le limolek'hule tse tsoang ho linoko tsa 11A ha li bapisoa le liphoofolo tse sa feteleng tšoaetso tse tšoaroang ka koae e sa foleng.Kelz et al., 1999; McClung le Nestler, 2003; Renthal et al., 2009). Ho phaella moo, Bibb le al. (2001) e tlaleha maemo a tšoanang a striatal Cdk5 mRNA le ts'ebetso ea protheine ka har'a khatello ena e le 'ngoe ea 11A ha e bapisoa le kalafo e sebelisitsoeng ka koae, e sa fetiseng kahare, hammoho le liphetoho tse tšoanang ho li-tarig tsa CDK5, p35 le DARPP-32.

Qetellong, re fumana hore poleloana ea ΔFosB ea nakoana e lebisa ho kenngoeng ha liphatsa tsa lefutso ka striatum ka mekhoa e mengata. Tsena li kenyeletsa tlhabollo ea morekisi ka kotlolloho, ho kenya protheine ea CREB le ts'ebetso, phetoho ea chromatin, ho kenyelletsa litsela tse ntseng li ikemiselitse ho etsoa.

Eya ho:

MELAO EA TŠEBELETSO

Animals

Liphoofolo tsa bi-transgenic 11A tsa banna (NSE-tTA x TetOP-ΔFosB) li sebelisitsoe thutong ena 'me li tsebahala ka Kelz et al., 1999. Ho overexpress ΔFosB, litoeba li tlositsoe ho doxycycline lipakeng tsa libeke tsa 3 le 6, ha litoeba tsa taolo li ne li bolokiloe ho doxycycline. Limpha tsohle li ne li bolokiloe ka sehlopha 'me li bolokoa mochineng oa 12: 12 lebone / lefifi, mabone a 7 hoseng le mabone a 7 pm, ka lib lib phihlello ea lijo le metsi. Liteko tsohle tsa mouse li ne li tsamaisana le liprotheine tse lumelletsoeng ke komiti ea tlhokomelo ea liphoofolo le tšebeliso ea The University of Texas Southwestern Medical Center ho Dallas.

Motlalehi le li-plasmids tsa polelo

Kholling (WT) Cck Moqapi oa papatso o ntlafalitsoe ka ho kenya sekhechana sa 200 bp PCR ka har'a pGL3-luc vector (Promega). Sekhechana sena se fumanoe ho tsoa ho genomic DNA (li-primers: 5 'TATCCTCATTCACTGGGGG 3' holimo, le 5 'TACCTTTGGATGGGAAATCG 3' tlase) 'me qalong e kentsoe ho pGEM-T Easy vector (PromeUMX, #AXN. Sekhechana sa papatso se ne se kenngoa libakeng tsa enzyme tsa Kpn1360 / Xho1 thibelo ea pGL1-luc.

Ho etsa motsoako oa CRE oa phetoho ho Cck promoter, a mutagenic primer lebisa khahlano le sebaka sa pejana se tlalehiloeng sa CRE (sense primer: 5'CGTGTCCTGCTGGACTGAGCTCGCACTGGTAAACA 3 ', antisense primer: 5'CTGTTTACCCAGTGCGCGCTGAGTCCAGCAGGACACG 3') e sebelisitsoe hammoho le Strata . Sena se fetola sebaka sa marang-rang se tlalehiloeng sa CRE (ACTGCGTCAGC) ho ACTGAGCTCC. Li-plasmids tsohle tsa moqolotsi li netefalitsoe ke tatellano ea DNA. Polelo ea ΔFosB plasmid e na le tatellano ea bolelele bo felletseng ba ΔFosB e kentsoeng sebakeng sa "cloning" sa pCDNA 3.1 mme e hlalositsoe pejana (Ulery le Nestler, 2007).

Setso sa lisele le phetisetso ea DNA

Lisele tsa Rat pheochromocytoma (PC12) li ile tsa bolokoa ka har'a li-Eagle F-12 tse fetotsoeng tsa Dulbecco, tsa tlatsetsoa ka 10% serum ea pere, serum ea likhomo tse 5% le 1% penicillin / streptomycin ho 37 ° C le 5% CO2. Lisele li ile tsa fetisoa ka motlakase ka motlakase oa motlakase o sebelisang BTX 360 electroporator (350V, 0 ohms, le 850 μF) ho 800 μL phosphate ea phosphate e nang le letsoai le nang le letsoai la 4 mm le 10 μg ea moqolotsi oa litaba le 5 μg ea polelo e hahiloeng. Empty vector plasmid (pCDNA) e ne e sebelisetsoa ho hlophisa palo e felletseng ea DNA. Kamora phetisetso, lisele li ne li holisoa lijaneng tse koahetsoeng ka collagen tse 35 mm bakeng sa nako e bontšitsoeng.

Luciferase o re

Matsatsi a mabeli kapa a mararo kamora ho fetisoa, lisele li ile tsa hlatsuoa makhetlo a mararo ho letsoai la Dulbecco's phosphate, e lysed (e sebelisa 3 mM glycylglycine, 25 mM MgSO4, 4 mM EGTA, 1% Triton X-100, pH 7.8, 1 mM DTT), e bokelletse, 'me e hlakotsoe ka centrifugation. 30 μL ea lysate e ne e kopantsoe le 140 μL luciferase assay buffer (25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, 1 mM DTT, 1 mM ATP, 1 mM potasium phosphate, 1 mM coenzyme A, pH 7.8). Ts'ebetso ea Luminescence e lekantsoe ho sebelisoa sebali sa FLx-800 microplate fluorescence ka mor'a ho kenngoa ka ente ea 40 μL 1 mM luciferin ka sediba. Ts'ebetso ea Lusifase e ne e etselitsoe maemo a fapakaneng ho latela sepheo sa protheine ea BioRad.

Electrophoretic motsamao battlefield thlahlobo

Lits'oants'o tsa nyutlelie tse tsoang ho likhaba tsa linaha tse peli tsa striatum ho litoeba tsa bi-transgenic 11A (tseo ho tsona li bolokiloeng kapa li koaletsoe doxycycline bakeng sa libeke tsa 2 kapa 8) (McClung le Nestler, 2003) li ne li lokisitsoe ho latela Huang le Walters (1996). 32Mefuta ea li-oligonucleotide e nang le mela e habeli e hlophisitsoeng e sebelisitsoe protocol ea Promega Gel Shift Assay system (#E3300) mme li-probes li ile tsa hloekisoa li sebelisa Roche Quick Spin Columns. Liphetoho tsa tumellano ea CRE le AP-1 tsa probe li ne li tsoa ho Promega (#E328A le E320B, ka ho latellana) le Cck Mekhahlelo ea CRE e ne e le (kutloisiso ea Cck-CRE: CTAGCGAGCTCTGGACTGCGTCAGCACTGGGTGCA; Cis-CRE antisense: CCCAGTGCTGACGCAGTCCAGAGCCCTCTCTTT).

Boitšoaro bo kopanyang le li-electrophoresis li ile tsa etsoa ho sebelisoa liphetoho tsa Ts'ebetso ea Promega Gel Shift Assay system (#E3300). 50,000 CPM ea probe e ngotsoeng e kopantsoe le 10 μg striatal nuclear extens. DNA ea tlholisano ea likhalase kapa li-antibodies li ile tsa eketsoa pele ho hlahisoa lipatlisiso tsa radiolabeled. Bakeng sa liteko tsa supershift, 2 μg ea CREB antibody (Upstate Biotechnology # 06-083) e sebelisitsoe. Reaction were electrophoresed on 4% polyacrylamide gels, omisitsoeng, 'me ba pepesetsoa filimi (sebelisa skrini tse matlafatsang bakeng sa hora ea 1 ho isa matsatsing a 2).

Ho ts'oara

Bakeng sa lisele tsa PC12, lipoleiti tse 35 mm tsa lisele tse fetisitsoeng li ile tsa hlatsuoa ka letsoai le batang la Dulbecco's phosphate-buffered saline le lysates li lokiselitsoe ka har'a leqhoa le batang la RIPA lysis (50 mM Tris pH 7.4, 5 mM NaCl, 5 mM EDTA, 1% deoxycholate, 1 % Triton X-100, 0.1% ya sodium dodecyl sulfate) e nang le protease inhibitors. Kamora sonase ea sonication, clearing, le Bradford, li-lysate li ile tsa senoloa ka botlalo mme 50 μg ea sampole ka 'ngoe ea khethoa ka motlakase ho li-gel tsa 10% SDS polyacrylamide. Liprotheine li ile tsa fetisetsoa lera la PVDF, tsa thibeloa hora e le 'ngoe lebeseng le ommeng le se nang mafura a 1% ho letsoai le omisitsoeng la Tris le nang le 3% Tween-0.1 (lebese la TBS-T), mme ea hlahlojoa bosiu bo le bong ho 20 ° C le li-antibodies tsa mantlha (CREB- Upstate Biotechnology # 4-06, e sebelisitsoeng ho 083: 1; GAPDH- Sigma # G1,000, e sebelisitsoeng ho 8795: 1) e hlapolotsoeng ka lebeseng la TBS-T. Kamora ho hlatsoa ka makhetlo a mangata ho TBS-T, ho ile ha batloa blots bakeng sa hora e le 'ngoe mochesong oa kamore ho sebelisoa li-antibodies tsa alkaline phosphatase-conjugated secondary (Sigma) tse hlapolotsoeng 80,000: 1 lebeseng la TBS-T. Kamora ho hlatsoa makhetlo a mangata ka letsoai le nang le buffer ea Tris, phetoho ea 'mala e ile ea etsoa ho latela litaelo tsa BioRad (# 5,000-170). Lera le omisitsoe ka bosiu bo le bong, li hlahlojoa ho skena sa flatbed le densitometry e etsoang ka ImageJ (bona ka tlase).

Bakeng sa lintlha tse qotsitsoeng tsa setereke, litekanyetso tsa Bophirimela li ile tsa etsoa joalo ka ha li phatlalalitsoe pele (Hope et al., 1994). Letlapa le tlositsoe ho litoeba tse kentsoeng, li beoa holim'a leqhoa ebe li kenngoa ka mokokotlong oa bokong ka botenya ba 1 mm. Methapo ea lithapo e ne e nkuoa ebe e hoamisoa ka -80 ° C ho fihlela e sebelisoa. Tissue e ne e hlahisoa leqhoeng ka sekontiri se fetotsoeng sesepa se nang le phosphatase le proteinase inhibitors (Roche, Sigma). Kamora sonication, mehlala e ne e emisitsoe metsing a belang ebe e kenngoa lipakeng tsa 15,000xg bakeng sa metsotso ea 15; supernatant e ile ea bokelloa mme ea sebetsoa kamora moo; liprotheine tsa mahloriso li ile tsa emisoa ho sebelisoa bradford assay (Bio-Rad). Mehlala e ne e tsamaisoa ka lehare la 10% acrylamide / bisacrylamide, e fetisetsoa ho membrane ea PVDF, e koetsoeng lebese la 5% mme e kentsoe li-antibodies tsa mantlha (Anti-CREB, Upstate, Lake Placid, NY). Li-blots li ile tsa bonoa hamorao ka ts'ebeliso ea sistimi ea chemiluminescence (Pierce). Mefuta eohle e ne e etselitsoe GAPDH (Fitzgerald, Concord, MA). Li-curve tse tloaelehileng li ile tsa tsamaisoa ho netefatsa hore re maemong a lekanang a palo eo.

Tlhatlhobo ea densitometry

Bakeng sa li-immunoblots tsa PC12, tlhahlobo ea densitometry e entsoe ho sebelisoa ImageJ ka molumo oa rodbard. Pontšo ea boholo bo ka morao e ne e tlositsoe tekong ka 'ngoe' me boholo ba CREB ho letšoao la GAPDH bo ile ba balloa sampole ka 'ngoe. Bakeng sa li-immunoblots tsa striatal le tlhahlobo ea EMSA, Scion Image 1.62c e sebelisitsoe ka tšusumetso ea morao.

Chromatin immunoprecipitation (ChIP)

Litekanyetso tsa ChIP li entsoe ho latela mekhoa ea Tsankova et al. (2004) 'me Kumar et al. (2005). Ka bokhutšoanyane, mehlala ea linaha tse peli tse tsoang ho litoeba tsa 11A tse bolokiloeng kapa tse tima doxycycline li ne li hokahantsoe le 1% formaldehyde mme crosslinking e ile ea tima ka glycine (mohopolo oa ho qetela oa 0.125 M). Disampole tsena li ne li tsoa lilapong tsohle tsa boko tse nkuoeng boemong ba li-nucleus accumbens le cortex e tlosoa. Chromatin e ne e kutiloe likhechana tse ka bang 0.2 ho isa ho 1 kb ka sonication, e hlakotsoe ka lifaha tsa Protein G (Thermo Scientific # 22852), le lisampole tse kentsoeng tse hoammeng ka -80 ° C. Pakeng tsa pula le metsi ho ne ho sebelisoa lipakeng tsa 60 le 100 μg ea chromatin. 5-10 μg ea antibody e 'ngoe le e' ngoe ea mantlha e sebelisitsoe (CREB: Biotechnology e kaholimo # 06-863, ΔFosB: Santa Cruz Biotechnology # SC-48x, acetylated H3: Upstate Biotechnology # 06-599, methylated H3 (LYS9): Theknoloji ea ho saena sele, cFos: Santa Cruz Biotechnology # SC-7202x). Mefuta ea Antibody-chromatin e ile ea fokotsoa kahare ho protheine G hammoho le lifaha ho latela litaelo tsa proizvodnje (Thermo Scientific # 22852). Kamora ho fapanya marang-rang ka morao ho lisampole tse kenyellelitsoeng le tse pepesehileng, sampole ka 'ngoe e ne e beoa ho PCR (qPCR) e ngata. Ts'ebeliso ea lithibela-mafu tsena kaofela bakeng sa ChIP e netefalitsoe haholo (Tsankova et al., 2004; Kumar et al., 2005; Renthal et al., 2009).

Maemo a liprotheine tse tlamang molemong o mong le o mong oa phahlo ea liphatsa tsa lefutso o ile oa khethoa ka ho lekanya palo ea DNA e amanang le qPCR (Applied Biosystems (ABI) Prism 7700, Foster City, CA). Ho kenngoa kapa ho kenella ka botlalo ho DNA (nonimmunoprecipended) le DNA e hlahisitsoeng e ile ea eketsoa ka makhetlo a mararo ka pela SYBR Green (Applied Biosystems, CA). Lits'ebetso tsa Ct tse tsoang mohlaleng ka mong li fumanoe ka software ea Sequence Detector 1.1. Quanifying e amanang le template ea DNA e entsoe ka mokhoa oa ΔΔCt (Tsankova et al., 2004). Li-Primers tse sebelisitsoeng: BDNF morekisi 4: CTTCTGTGTGCGTGAATTTGCT; Morekisi oa AGTCCACGAGAGGGCTCCA CDK5: GCTGAAGCTGTCAGGAGGTC; GTGCCCCGCTCTTGTTATTA Cck Promoter: CTTGGGCTAGCCTCATTCACTG; Morekisi oa TTAAATAGCCCCCCGGTTGG Prodynorphin: GGCTTCCTTGTGCTTCAGAC; GCGCTGTTTGTCACTTTCAA.

Tlhahlobo ea statistical

Lintlha tsohle li hlahisoa e le mekhoa ± phoso e tloaelehileng ea se boleloang. Phapang ea lipalo-palo e ile ea khethoa ke t-teko e 'meli ea moithuti ea t (t <0.05). Ha ho bapisoa makhetlo a mangata, litekanyetso tsa p li ile tsa fetoloa ho sebelisoa khalemelo ea Bonferroni.

Eya ho:

Liteboho

Re ka rata ho leboha Will Renthal le Arvind Kumar ka lipuisano tse molemo. Re rata hape ho leboha NIDA bakeng sa ho tšehetsa liteko tsena.

Eya ho:

Mongolo o botlaaseng ba leqephe

Boitlhotlhollo ba Mohoeletsi: Ena ke faele ea PDF ea mongolo o ngotsoeng ka letsoho o sa amoheloang o amoheletsoeng bakeng sa ho hatisoa. Joaloka tšebeletso ho bareki ba rona re fana ka phetolelo ena ea pele ea mongolo o ngotsoeng ka letsoho. Mongolo o ngotsoeng ka letsoho o tla ba le ho kopitsa, ho kenya lihlopha le ho hlahloba bopaki bo hlahang pele o hatisoa ka mokhoa oa oona oa ho qetela. Ka kopo hlokomela hore nakong ea mekhoa ea tlhahiso ea lihlahisoa e ka fumanoa e ka amang litaba, le litsebiso tsohle tsa molao tse sebetsang koranteng e amanang le eona.

Mantsoe a karohano:

Karolo: #1 Cellular le Molecular Biology ea Nervous system

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