中国组织工程研究
中國組織工程研究
중국조직공정연구
Journal of Clinical Rehabilitative Tissue Engineering Research
2015年
14期
2133-2137
,共5页
干细胞%胚胎干细胞%连接蛋白43%基因干扰%鼠胎肝干细胞%细胞增殖%细胞周期
榦細胞%胚胎榦細胞%連接蛋白43%基因榦擾%鼠胎肝榦細胞%細胞增殖%細胞週期
간세포%배태간세포%련접단백43%기인간우%서태간간세포%세포증식%세포주기
Hepatocytes%Connexin 43%RNA,Smal Interfering%Cel Proliferation
背景：胎肝干细胞具有分化成肝细胞、胆管细胞的潜能，参与肝脏的修复与重建，是肝细胞的重要来源，但是人体的胎肝干细胞含量极少，如何获取一定数量和较高纯度的胎肝干细胞是目前研究的热点。目的：构建能有效抑制大鼠胎肝干细胞Cx43基因表达的siRNA载体，探讨抑制 Cx43表达对体外培养的鼠胎肝干细胞增殖及细胞周期的影响。方法：体外培养鼠胎肝干细胞，设计及合成靶向 Cx43的 siRNA 序列(Cx43-siRNA)以及阴性对照序列(NC-siRNA)，采用电转法转染大鼠胎肝干细胞，即为实验组和对照组，未转染的胎肝干细胞为空白组。应用real-time PCR和Western blot法检测转染前后鼠胎肝干细胞Cx43基因和蛋白的表达；细胞生长曲线、CCK-8法观察细胞生长增殖情况；流式细胞术测定细胞周期分布的变化。结果与结论：转染Cx43-siRNA后，实验组与对照组、空白组相比Cx43基因和蛋白水平表达均明显降低，细胞的生长速度明显增快，G 0/G 1期细胞减少，S期细胞数增多，差异有显著性意义(P <0.05)，结果表明通过电转法转染靶向Cx43的siRNA能促进体外培养的鼠胎肝干细胞增殖，对其培养有优化作用。
揹景：胎肝榦細胞具有分化成肝細胞、膽管細胞的潛能，參與肝髒的脩複與重建，是肝細胞的重要來源，但是人體的胎肝榦細胞含量極少，如何穫取一定數量和較高純度的胎肝榦細胞是目前研究的熱點。目的：構建能有效抑製大鼠胎肝榦細胞Cx43基因錶達的siRNA載體，探討抑製 Cx43錶達對體外培養的鼠胎肝榦細胞增殖及細胞週期的影響。方法：體外培養鼠胎肝榦細胞，設計及閤成靶嚮 Cx43的 siRNA 序列(Cx43-siRNA)以及陰性對照序列(NC-siRNA)，採用電轉法轉染大鼠胎肝榦細胞，即為實驗組和對照組，未轉染的胎肝榦細胞為空白組。應用real-time PCR和Western blot法檢測轉染前後鼠胎肝榦細胞Cx43基因和蛋白的錶達；細胞生長麯線、CCK-8法觀察細胞生長增殖情況；流式細胞術測定細胞週期分佈的變化。結果與結論：轉染Cx43-siRNA後，實驗組與對照組、空白組相比Cx43基因和蛋白水平錶達均明顯降低，細胞的生長速度明顯增快，G 0/G 1期細胞減少，S期細胞數增多，差異有顯著性意義(P <0.05)，結果錶明通過電轉法轉染靶嚮Cx43的siRNA能促進體外培養的鼠胎肝榦細胞增殖，對其培養有優化作用。
배경：태간간세포구유분화성간세포、담관세포적잠능，삼여간장적수복여중건，시간세포적중요래원，단시인체적태간간세포함량겁소，여하획취일정수량화교고순도적태간간세포시목전연구적열점。목적：구건능유효억제대서태간간세포Cx43기인표체적siRNA재체，탐토억제 Cx43표체대체외배양적서태간간세포증식급세포주기적영향。방법：체외배양서태간간세포，설계급합성파향 Cx43적 siRNA 서렬(Cx43-siRNA)이급음성대조서렬(NC-siRNA)，채용전전법전염대서태간간세포，즉위실험조화대조조，미전염적태간간세포위공백조。응용real-time PCR화Western blot법검측전염전후서태간간세포Cx43기인화단백적표체；세포생장곡선、CCK-8법관찰세포생장증식정황；류식세포술측정세포주기분포적변화。결과여결론：전염Cx43-siRNA후，실험조여대조조、공백조상비Cx43기인화단백수평표체균명현강저，세포적생장속도명현증쾌，G 0/G 1기세포감소，S기세포수증다，차이유현저성의의(P <0.05)，결과표명통과전전법전염파향Cx43적siRNA능촉진체외배양적서태간간세포증식，대기배양유우화작용。
BACKGROUND:Fetal liver stem cel s have the potential to differentiate into hepatocytes and bile duct cel s, and participate in the repair and reconstruction of the liver, which is an important source of hepatocytes. But there are a little amount of fetal liver stem cel s in human body, and how to obtain a certain number of high-purity fetal liver stem cel s is currently a hot research. OBJECTIVE:To construct a siRNA carrier that can effectively inhibit the expression of connexin 43 (Cx43) in rat fetal liver stem cel s, and to investigate the effect of Cx43 inhibition on the proliferation and cel cycle of fetal liver stem cel s cultured in vitro. METHODS:Fetal liver stem cel s were cultured by the suspension culture in vitro, siRNA sequences targeting Cx43 (Cx43-siRNA) and negative control sequence (NC-siRNA) were designed and synthesized. Then, rat fetal liver stem cel s were transferred electrophoretical y and divided into three groups:blank group, NC-siRNA group, Cx43-siRNA group. Real-time PCR and western blot were used to assess the knockdown efficiency. Cel ular proliferation was determined by cel growth curve and cel counting kit-8 assay. The cel cycle was analyzed by flow cytometry. RESULTS AND CONCLUSION:After transfection, the Cx43 gene and protein expression levels were declined dramatical y in the Cx43-siRNA, NC-siRNA and blank groups, and the cel s grew faster. The number of cel s at G0/G1 phase decrease, but the number of cel s in S phase increased. There were significant differences between the groups (P<0.05). Electrophoretic transfer of Cx43-siRNA can promote the proliferation of cultured fetal liver stem cel s and optimize the cel culture.
