摘要
目的
研究COX-2 shRNA对大鼠肝星状细胞肾上腺素受体表达的影响。
方法
1应用已构建的带有荧光的三条不同COX-2 shRNA片段的质粒,通过脂质体Liplofectamine~(TM) 2000转染大鼠HSC,荧光显微镜下评估细胞的质粒转染效率。
2分别在转染HSC后24h、48h、72h提取RNA,用RT-PCR法检测肝星状细胞中COX-2、β_1-AR、β_2-AR、β_3-AR的mRNA表达,同时筛选出三条COX-2 shRNA序列中沉默效果最好的序列。
3用沉默效果最好的COX-2 shRNA片段转染肝星状细胞,免疫细胞化学法检测肝星状细胞中COX-2、β_1-AR、β_2-AR、β_3-AR的蛋白表达。
4计量资料数据均以均数±标准差((x|-)±S)表示,多组间比较采用单因素方差分析(one-way ANOVA),组内比较采用LSD-t检验,用SPSS17.0软件进行统计分析,P﹤0.05作为差异显著性标准。
结果
1 COX-2 shRNA三条序列转染HSC后的24h、48h、72h三个时间段,在荧光显微镜下观察到HSC内均有荧光表达,而空载组和空白对照组无荧光表达。
2 RT-PCR法证实HSC可表达COX-2、β_1-AR、β_2-AR、β_3-AR mRNA,三条片段的COX-2 shRNA在转染HSC后均可不同程度的减少COX-2、β_1-AR、β_2-AR、β_3-AR mRNA的表达,其中以COX-2 shRNA序列1组转染HSC后的72h作用最明显。
3免疫细胞化学法证实HSC可以表达COX-2、β_1-AR、β_2-AR、β_3-AR蛋白,且COX-2、β_1-AR、β_2-AR、β_3-AR在HSC中的分布基本一致,主要分布于细胞膜,细胞浆也有少量表达,在COX-2 shRNA序列1组转染HSC后的72h,COX-2、β_1-AR、β_2-AR、β_3-AR蛋白表达减弱最明显。
结论
1 HSC上可表达COX-2、β_1-AR、β_2-AR、β_3-AR。
2 COX-2 shRNA能够沉默大鼠HSC中COX-2、β_1-AR、β_2-AR、β_3-AR的mRNA及其蛋白的表达,抑制COX-2可减少HSC上肾上腺素受体的表达。
Objective
Research the expression of adrenaline receptors in HSC by the way of COX-2 shRNA in rat .
Methods
1 Application has been built with three different fluorescent fragments of COX-2 shRNA plasmid, liposome LiplofectamineTM 2000 Transfection of Rat HSC, cell fluorescence microscopy to assess the efficiency of plasmid transfection.
2 HSC were transfected after 24h, 48h, 72h extraction of RNA, RT-PCR assay using hepatic stellate cells COX-2,β_1-AR,β_2-AR,β_3-AR mRNA expression, while selected the best sequence of silence between the three of COX-2 shRNA sequence.
3 Silence the best results with the COX-2 shRNA fragments transfected hepatic stellate cells, detected by immunocytochemistry of hepatic stellate cells COX-2,β_1-AR,β_2-AR,β_3-AR protein.
4 Measurement information and data are mean±standard deviation ((x|-)±S), said many groups were compared by single factor analysis of variance (one-way ANOVA), group compared with LSD-t test, using statistical analysis software SPSS17.0, P <0.05 as significant difference criteria.
Results
1 COX-2 shRNA transfected HSC three series after 24h, 48h, 72h three time periods, observed under a fluorescence microscope fluorescence were expressed in HSC, while no-load group and control group had no fluorescent expression.
2 RT-PCR technique showed that HSC can express COX-2,β_1-AR,β_2-AR,β_3-AR mRNA, the three segments of the COX-2 shRNA transfected HSC at different levels can be reduced after the COX-2,β_1-AR,β_2-AR,β_3-AR mRNA expression, in which COX-2 shRNA sequences 1 group 72h after transfection of HSC the most significant effect.
3 Immunocytochemistry confirmed that HSC can express COX-2,,β_1-AR,β_2-AR,β_3-AR protein, and COX-2,β_1-AR,β_2-AR,β_3-AR in the distribution of HSC is consistent Mainly distributed in the cell membrane, a small amount of cytoplasm is also expressed in the COX-2 shRNA sequences of Group 1 after transfection of HSC 72h, COX-2,β_1-AR,β_2-AR,β_3-AR protein expression decreased the most obvious.
Conclusions
1 HSC can be expressed COX-2,β_1-AR,β_2-AR,β_3-AR.
2 COX-2 shRNA to silence rat HSC in the COX-2,β_1-AR,β_2-AR,β_3-AR in mRNA and protein expression, inhibition of COX-2 adrenergic receptors can reduce the expression on the HSC.
引文
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