中华传染病杂志
中華傳染病雜誌
중화전염병잡지
CHINESE JOURNAL OF INFECTIOUS DISEASES
2013年
2期
66-70
,共5页
陈达之%宋梅%王玉群%曹肃婷%林镯%许烂漫%施可庆%王雅琴%陈永平
陳達之%宋梅%王玉群%曹肅婷%林鐲%許爛漫%施可慶%王雅琴%陳永平
진체지%송매%왕옥군%조숙정%림탁%허란만%시가경%왕아금%진영평
微小核糖核酸%星形细胞%肝硬化%转化生长因子β1%肌动蛋白类%基质金属蛋白酶3
微小覈糖覈痠%星形細胞%肝硬化%轉化生長因子β1%肌動蛋白類%基質金屬蛋白酶3
미소핵당핵산%성형세포%간경화%전화생장인자β1%기동단백류%기질금속단백매3
miRNA%Astrocytes%Liver cirrhosis%Transforming growth factor beta 1%Actins%Matrix metalloproteinase 3
目的 观察慢病毒表达载体介导微小核糖核酸-34a(miRNA-34a)体外转染肝星状细胞-T6(HSC-T6)后对肝星状细胞增殖率及活化的影响,探讨miRNA-34a在肝纤维化中可能的作用.方法 体外培养HSC-T6细胞,分别对miRNA-34a重组慢病毒组和空白对照病毒组转染肝星状细胞,细胞计数试剂盒(CCK-8试剂盒)检测细胞增殖抑制率,实时荧光定量PCR(RT-PCR)、Western印迹法检测HSC-T6细胞转化生长因子-β1 (TGF-β1)、a-平滑肌肌动蛋白(α-SMA)和基质金属蛋白酶3 (MMP3) mRNA及蛋白表达.组间差异采用单因素方差分析,方差齐性者两两比较采用LSD法,方差不齐者采用Dunnett T3检验,相关性检验采用Pearson直线相关分析.结果 转染效率为80％；与空白对照病毒组相比,转染后RT-PCR显示,miRNA-34a重组慢病毒组miRNA-34a mRNA的含量是3.6,其表达量明显升高；miRNA-34a重组慢病毒组肝星状细胞增殖率明显降低(24、48及72 h的F值分别为9.048、168.070、812.390,均P＜0.05)；TGF-β1和α-SMA mRNA的表达量均降低,相对于空白对照病毒组的含量分别为0.28和0.13；TGF-β1和α-SMA蛋白表达量均降低(F=64.444、90.577,均P＜0.05)；而MMP3表达量明显升高(F=374.584,P＜0.05).结论miRNA-34a能抑制肝星状细胞增殖和活化,有望在治疗肝纤维化中发挥作用.
目的 觀察慢病毒錶達載體介導微小覈糖覈痠-34a(miRNA-34a)體外轉染肝星狀細胞-T6(HSC-T6)後對肝星狀細胞增殖率及活化的影響,探討miRNA-34a在肝纖維化中可能的作用.方法 體外培養HSC-T6細胞,分彆對miRNA-34a重組慢病毒組和空白對照病毒組轉染肝星狀細胞,細胞計數試劑盒(CCK-8試劑盒)檢測細胞增殖抑製率,實時熒光定量PCR(RT-PCR)、Western印跡法檢測HSC-T6細胞轉化生長因子-β1 (TGF-β1)、a-平滑肌肌動蛋白(α-SMA)和基質金屬蛋白酶3 (MMP3) mRNA及蛋白錶達.組間差異採用單因素方差分析,方差齊性者兩兩比較採用LSD法,方差不齊者採用Dunnett T3檢驗,相關性檢驗採用Pearson直線相關分析.結果 轉染效率為80％；與空白對照病毒組相比,轉染後RT-PCR顯示,miRNA-34a重組慢病毒組miRNA-34a mRNA的含量是3.6,其錶達量明顯升高；miRNA-34a重組慢病毒組肝星狀細胞增殖率明顯降低(24、48及72 h的F值分彆為9.048、168.070、812.390,均P＜0.05)；TGF-β1和α-SMA mRNA的錶達量均降低,相對于空白對照病毒組的含量分彆為0.28和0.13；TGF-β1和α-SMA蛋白錶達量均降低(F=64.444、90.577,均P＜0.05)；而MMP3錶達量明顯升高(F=374.584,P＜0.05).結論miRNA-34a能抑製肝星狀細胞增殖和活化,有望在治療肝纖維化中髮揮作用.
목적 관찰만병독표체재체개도미소핵당핵산-34a(miRNA-34a)체외전염간성상세포-T6(HSC-T6)후대간성상세포증식솔급활화적영향,탐토miRNA-34a재간섬유화중가능적작용.방법 체외배양HSC-T6세포,분별대miRNA-34a중조만병독조화공백대조병독조전염간성상세포,세포계수시제합(CCK-8시제합)검측세포증식억제솔,실시형광정량PCR(RT-PCR)、Western인적법검측HSC-T6세포전화생장인자-β1 (TGF-β1)、a-평활기기동단백(α-SMA)화기질금속단백매3 (MMP3) mRNA급단백표체.조간차이채용단인소방차분석,방차제성자량량비교채용LSD법,방차불제자채용Dunnett T3검험,상관성검험채용Pearson직선상관분석.결과 전염효솔위80％；여공백대조병독조상비,전염후RT-PCR현시,miRNA-34a중조만병독조miRNA-34a mRNA적함량시3.6,기표체량명현승고；miRNA-34a중조만병독조간성상세포증식솔명현강저(24、48급72 h적F치분별위9.048、168.070、812.390,균P＜0.05)；TGF-β1화α-SMA mRNA적표체량균강저,상대우공백대조병독조적함량분별위0.28화0.13；TGF-β1화α-SMA단백표체량균강저(F=64.444、90.577,균P＜0.05)；이MMP3표체량명현승고(F=374.584,P＜0.05).결론miRNA-34a능억제간성상세포증식화활화,유망재치료간섬유화중발휘작용.
Objective To observe the influence of puromycin lentiviral vector (pLV)-miRNA34a transfection on proliferation and activation of rat hepatic stellate cells,and to investigate the mechanism of miRNA-34a on liver fibrosis in rat.Methods Hepatic stellate cells-T6 (HSC-T6) were transfected with pLV-EGFP or pLV-miRNA-34a,respectively.Cell proliferation was measured by C0038 cell counting kit-8 (CCK-8).The level of miRNA 34a,transforming growth factor (TGF)-β1,α-smooth muscle actin (α-SMA) and matrix metalloproteinasex3 (MMP3) expression were detected by real-time fluorescent quantitative-polymerase chain reaction (RT-PCR) and Western-blot.The comparison of means among groups was done by univariate ANOVA.Results The transfection efficiency was 80％.After hepatic stellate cells transfected with pLV-miRNA-34a,the expression of miRNA-34a mRNA was significantly increased compaired with the control group,and the content of miRNA-34a mRNA relative to the control group was 3.6.Cell proliferation of pLV-enhanced green fluorescent protein (EGFP)-miRNA-34a tranfected cells was lower than the control group (the value of F was 9.048,168.070,812.390 at 24,48,72 h respectively,all P＜0.05).After hepatic stellate cells were infected with pLV-EGEP-miRNA 34a,the expressions of TGF-β1 and α-SMA mRNA were lower than the control group,and the content of TGF-β1 and α SMA was 0.28 and 0.13 respectively.The expression of TGF-β1 and α-SMA proteins were significantly decreased (F=64.444 and 90.577,respectively； both P＜0.05),and the expression of MMP3 was significantly increased (F=374.584,P＜0.05).Conclusion miRNA-34a significantly inhibits the proliferation and activation of hepatic stellate cells,providing potential therapeutic implication for hepatic fibrosis.
