sirna抑制stgc3基因表達(dá)對(duì)nih3t3細(xì)胞增殖的影響

《sirna抑制stgc3基因表達(dá)對(duì)nih3t3細(xì)胞增殖的影響》由會(huì)員上傳分享，免費(fèi)在線(xiàn)閱讀，更多相關(guān)內(nèi)容在學(xué)術(shù)論文-天天文庫(kù)。

1、siRNA抑制STGC3基因表達(dá)對(duì)NIH3T3細(xì)胞增殖的影響碩士研究生：廖銀花指導(dǎo)教師：賀修勝教授中文摘要目的：STGC3基因是新近克隆的一個(gè)候選抑瘤基因。本研究以STGC3基因陽(yáng)性表達(dá)的鼠成纖維細(xì)胞系NIH3T3細(xì)胞為模型，采用siRNA技術(shù)，使NIH3T3細(xì)胞的STGC3基因表達(dá)下調(diào)，觀(guān)察STGC3基因表達(dá)下調(diào)對(duì)NIH3T3細(xì)胞增殖的影響。方法：構(gòu)建了針對(duì)STGC3基因mRNA的pSilencer3．1-STGC3一siRNA表達(dá)載體，經(jīng)脂質(zhì)體轉(zhuǎn)染，導(dǎo)入NIH3T3細(xì)胞，G418篩選，建立穩(wěn)定的pSilencer3．1．STGC3一siRNA／NIH3T

2、3細(xì)胞系。RT-PCR、Westernblotting和免疫細(xì)胞化學(xué)方法，檢測(cè)STGC3基因mRNA和蛋白表達(dá)。通過(guò)HE染色、繪制生長(zhǎng)曲線(xiàn)、軟瓊脂集落形成實(shí)驗(yàn)及流式細(xì)胞儀分析，觀(guān)察STGC3基因表達(dá)下調(diào)后，NIH3T3細(xì)胞形態(tài)、增殖速度、克隆形成率和細(xì)胞周期分布變化。結(jié)果：重組質(zhì)粒DNA經(jīng)酶切和測(cè)序鑒定，結(jié)果均證實(shí)pSilencer3．1-STGC3一siRNA表達(dá)載體構(gòu)建成功。將pSilencer3．1-STGC3．siRNA和pSilencer3．1質(zhì)粒分別轉(zhuǎn)染NIH3T3細(xì)胞，G418篩選陽(yáng)性細(xì)胞克隆，經(jīng)RT-PCR、Westemblotting及免疫

3、細(xì)胞化學(xué)方法檢測(cè)結(jié)果表明，成功建立了STGC3基因表達(dá)下調(diào)的NIH3T3細(xì)胞系。普通光學(xué)顯微鏡下觀(guān)察，pSilencer3．1-STGC3一siRNA／NIH3T3細(xì)胞與兩對(duì)照組相比，核染色加深，細(xì)胞變得細(xì)長(zhǎng)；細(xì)胞生長(zhǎng)曲線(xiàn)結(jié)果顯示：pSilencer3．1-STGC3-siRNA／NIH3T3細(xì)胞較pSilencer3．1／NIH3T3細(xì)胞和NIH3T3細(xì)胞生長(zhǎng)速度快(P

4、4+1．O)％、(2．2+0．3)％和(2．1+0．3y％，4pSilencer3．1一STGC3．siRNA／NIH3T3組克隆形成能力較兩對(duì)照組顯著增強(qiáng)(P<0．05)；流式細(xì)胞儀檢測(cè)結(jié)果：pSilencer3．1一STGC3一siRNA／NIH3T3組較對(duì)照組細(xì)胞群體中處于S期和G2的細(xì)胞數(shù)目增加，Go／Gl期細(xì)胞數(shù)目減少。結(jié)論：1．構(gòu)建了pSilencer3．1-STGC3一siRNA真核表達(dá)載體。2．建立了STGC3基因表達(dá)下調(diào)的pSilencer3．1．STGC3．siRNA／NIH3T3細(xì)胞系。3．siRNA抑制STGC3基因表達(dá)可促進(jìn)NIH3

5、T3細(xì)胞增殖。關(guān)鍵詞：小干擾RNA，STGC3基因，NIH3T3細(xì)胞，細(xì)胞增殖5EffectofSTGC3．siRNAontheGrowthinNIH3T3CellLineAbstractObjective：STGC3geneisacandidatetumorsuppressorgeneclonedrecently．NIH3T3celllinewasselectedas．a(chǎn)targetwithpositiveexpressionofSTGC3geneandobservedtheproliferationeffectafterSTGC3geneexpressi

6、oninhibitedbysiRNA．Methods：pSilencer3．1-STGC3-siRNAexpressionvectortargettingSTGC3geneWasconstructedandtransfectedintoNIH3T3celllinebycationicliposome．ThestablepSilencer3．1-STGC3-siRNA／NIH3T3celllineWasobtainedbyG418selection．ThelevelsofSTGC3mRNAandproteinweredetectedbyRT-PCR，weste

7、rnblottingandimmunohistochemistry．Cellmorphology,growthcurves，colonyformationrateandcellcycledistributionweredetectedinNIH3T3celllineafterSTGC3geneexpressiondown—regulated．Results：TheresultsofrestrictionenzymecuttingandDNAsequencingdemonstratethatthepSilencer3．1一STGC3一siRNAexpressi

8、onvectorWassuccessfullycon