發(fā)布時間：2018-04-17 08:38

  本文選題：肝臟庫普弗細(xì)胞 + 轉(zhuǎn)染效率��； 參考：《南方醫(yī)科大學(xué)學(xué)報》2015年12期

【摘要】：目的對比不同的轉(zhuǎn)染試劑轉(zhuǎn)染RIP140-siRNA至肝臟庫普弗細(xì)胞的轉(zhuǎn)染效率及它們對肝臟庫普弗細(xì)胞的細(xì)胞毒性,從而尋找出最佳的肝臟庫普弗細(xì)胞試劑轉(zhuǎn)染方法和條件。方法以肝臟庫普弗細(xì)胞為研究對象,以綠色熒光蛋白(GFP)標(biāo)記的RIP140-siRNA為報告基因(reporter gene),采用lipofectamine 2000,羅氏試劑(X-treme GENE siRNA Transfection Reagent)及嘌呤霉素篩選的慢病毒(1.0×108TU/m L)作為轉(zhuǎn)染試劑,熒光倒置顯微鏡下觀察細(xì)胞轉(zhuǎn)染效果,激光掃描共聚焦顯微鏡分析不同試劑轉(zhuǎn)染后細(xì)胞RIP140的表達,流式細(xì)胞術(shù)檢測各組細(xì)胞凋亡,CCK-8檢測各組細(xì)胞增殖抑制情況。收集細(xì)胞并進行裂解,提取細(xì)胞RNA與蛋白質(zhì),運用RT-RCR和Western blot實驗法檢測轉(zhuǎn)染RIP140-siRNA后的基因及蛋白質(zhì)表達情況。結(jié)果對于肝臟庫普弗細(xì)胞而言,在轉(zhuǎn)染效率方面:嘌呤霉素篩選的慢病毒轉(zhuǎn)染效率最高,可達90%以上;羅氏試劑其次;lipofectamine2000效果最差。在試劑的細(xì)胞毒性方面:流式細(xì)胞術(shù)及CCK-8檢測結(jié)果顯示羅氏試劑的細(xì)胞毒性最小,細(xì)胞可見其原有形態(tài);慢病毒其次;lipofectamine 2000細(xì)胞毒性最大,可見多數(shù)細(xì)胞失去原有形態(tài),并存在細(xì)胞裂解狀態(tài)。RT-RCR和Western blot實驗顯示慢病毒轉(zhuǎn)染RIP140-siRNA的肝臟庫普氏細(xì)胞組無論在基因方面還是在蛋白質(zhì)水平均明顯低表達于lipofectamine 2000和羅氏試劑所轉(zhuǎn)染的肝臟庫普弗細(xì)胞組(P0.05)。結(jié)論對于原代細(xì)胞肝臟庫普弗細(xì)胞,在試劑轉(zhuǎn)染方面,慢病毒轉(zhuǎn)染方法可以達到理想的轉(zhuǎn)染效率和較小的細(xì)胞毒性,且條件可控性與穩(wěn)定性方面更加優(yōu)越。
[Abstract]:Objective to compare the transfection efficiency and cytotoxicity of different transfection reagents (RIP140-siRNA) into liver Kupffer cells, and to find out the best transfection methods and conditions of liver Kupffer cells.Methods Hepatic Kupffer cells were studied. RIP140-siRNA labeled with green fluorescent protein (GFP) was used as reporter gene. Lipofectamine 2000, X-treme GENE siRNA Transfection Reagentand lentivirus selected by purine mycin were used as transfection reagents.The effect of cell transfection was observed under fluorescence inverted microscope. The expression of RIP140 was analyzed by laser scanning confocal microscopy (LSCM) and apoptosis was detected by flow cytometry (FCM) and the inhibition of cell proliferation was detected by flow cytometry (FCM) and CCK-8 respectively.The RNA and protein were extracted from the cells, and the expression of genes and proteins after transfection of RIP140-siRNA was detected by RT-RCR and Western blot.Results for liver Kupffer cells, the transfection efficiency of lentivirus screened by purine mycin was the highest (over 90%), and that of lipofectamine 2000 was the worst.The results of flow cytometry and CCK-8 analysis showed that the cytotoxicity of Roche reagent was the least and the original morphology of the cells was observed, while the cytotoxicity of lentivirus was the highest in lipofectamine 2000, and most of the cells lost their original morphology.The cell cleavage state. RT-RCR and Western blot experiments showed that the expression of Lentivirus transfected RIP140-siRNA in Kupffer cell group was significantly lower than that in lipofectamine 2000 and Roche reagent transfected Kupffer cells group in both gene and protein level.Conclusion in the aspect of reagent transfection, lentivirus transfection can achieve ideal transfection efficiency and less cytotoxicity, and the conditional controllability and stability are better for the primary cell liver Kupffer cells.
【作者單位】： 重慶醫(yī)科大學(xué)第二臨床學(xué)院;重慶醫(yī)科大學(xué)附屬第二醫(yī)院肝膽外科;
【基金】：國家自然科學(xué)基金(81470899,81170442)~~
【分類號】：R575

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