本文選題：血清反應(yīng)因子 + 血清反應(yīng)因子N端片段��； 參考：《臨床與實(shí)驗(yàn)病理學(xué)雜志》2017年10期

【摘要】：目的探討血清反應(yīng)因子全長(SRF-Full)及N端片段(SRF-N,1-254aa)對TGF-β1誘導(dǎo)c-Kit+心臟干細(xì)胞(cardiac stem cell,CSC)分化的影響。方法從c DNA文庫擴(kuò)增大鼠SRF-Full及SRF-N表達(dá)序列并克隆入酶切線性化慢病毒載體GV358(Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin),轉(zhuǎn)化感受態(tài)細(xì)胞篩選出陽性克隆并測序鑒定。將SRF-Full及SRF-N過表達(dá)質(zhì)粒與病毒包裝輔助質(zhì)粒共轉(zhuǎn)染工具細(xì)胞HEK293T,包裝慢病毒。磁激活細(xì)胞分選法分離乳SD大鼠c-Kit+CSC,將SRF-Full及SRF-N過表達(dá)慢病毒感染CSC并經(jīng)TGF-β1誘導(dǎo),定量PCR分析下游分化基因mRNA水平的變化。結(jié)果成功擴(kuò)增出SRF-Full及SRF-N編碼序列并正確連接入線性化載體,獲得的陽性轉(zhuǎn)化子經(jīng)測序無誤。將轉(zhuǎn)化子質(zhì)粒與病毒包裝輔助質(zhì)粒共轉(zhuǎn)染HEK293T后,獲得滴度為2×108TU/m L的慢病毒顆粒,Western blot檢測到預(yù)期Flag融合蛋白。重組慢病毒感染CSC后細(xì)胞核中見e GFP信號。TGF-β1誘導(dǎo)CSC出現(xiàn)心肌細(xì)胞分化基因(Nkx2.5、Gata4、c Tn I)及平滑肌細(xì)胞分化基因(SM22α)的表達(dá),內(nèi)皮細(xì)胞分化(v WF)無影響,SRF-Full促進(jìn)CSC的心肌細(xì)胞分化,而SRF-N則抑制這種分化。結(jié)論成功構(gòu)建SRFFull及SRF-N過表達(dá)重組慢病毒質(zhì)粒。SRF-Full促進(jìn)而SRF-N減弱TGF-β1誘導(dǎo)CSC的心肌細(xì)胞分化。
[Abstract]:Objective to investigate the effects of SRF-Full and N-terminal SRF-NU (1-254 aa) on the differentiation of c-Kit cardiac stem cells induced by TGF- 尾 _ 1. Methods Rat SRF-Full and SRF-N expression sequences were amplified from the cDNA library and cloned into the vector GV358 Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycinin. The positive clones were screened and sequenced. SRF-Full and SRF-N overexpression plasmids were co-transfected with viral packaging assistant plasmid HEK293T to package lentivirus. The c-Kit CSCs of SD rats were isolated by magnetic activated cell sorting method. SRF-Full and SRF-N overexpression lentiviruses were infected with CSC and induced by TGF- 尾 1. The mRNA levels of downstream differentiation genes were analyzed by quantitative PCR. Results the SRF-Full and SRF-N coding sequences were successfully amplified and correctly ligated into the linearized vector. The obtained positive transformants were sequenced correctly. After the transformant plasmid was co-transfected into HEK293T with virus packaging assistant plasmid, the expected Flag fusion protein was detected by Western blot with a titer of 2 脳 108TU / mL lentivirus particles. E GFP signal. TGF- 尾 1 induced the expression of differentiation gene Nkx2. 5 and smooth muscle cell differentiation gene SM22 偽 in the nucleus of recombinant lentivirus infected with CSC, but endothelial cell differentiation v WF did not affect SRF-Full in promoting the differentiation of CSC cardiomyocytes. SRF-N inhibits this differentiation. Conclusion SRFFull and SRF-N overexpression recombinant lentivirus plasmids. SRF-Full promoted SRF-Full and SRF-N attenuated TGF- 尾 1 induced myocardial differentiation of CSC.
【作者單位】： 廣東醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院病理學(xué)系;廣東醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院病理生理學(xué)教研室;海南醫(yī)學(xué)院第一附屬醫(yī)院心血管研究所;
【基金】：國家自然科學(xué)基金(81460042) 廣東省教育廳科技創(chuàng)新項(xiàng)目基金(KJCX20130088) 廣東省“揚(yáng)帆計(jì)劃”高層次人才項(xiàng)目(4YF16007G) 2015年國家留學(xué)人員科技活動擇優(yōu)資助項(xiàng)目
【分類號】：R54

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