摘要
目的 观察特异性miRNA-200b抑制剂对肝星状细胞增殖、活化、胶原合成的影响.方法 设计并合成miR-200b抑制剂,利用脂质体将miRNA-200b抑制剂转入肝星状细胞中,培养48 h后,收集肝星状细胞和上清液,采用qRT-PCR探针的方法检测miR-200b的表达水平、Westernblotting检测细胞α-平滑肌肌动蛋白（α-SMA）表达、噻唑蓝（MTT）法检测细胞增殖活性和放射免疫法检测培养上清液中Ⅲ型前胶原和透明质酸含量.结果 与阴性对照组相比,miRNA-200b抑制剂转染48 h后HSC-T6细胞miR-200b组表达下调82％;α-SMA蛋白表达降低（19±3）％（P＜0.05）;细胞增殖活性降低（33±5）％（P＜0.01）;培养上清中Ⅲ型前胶原和透明质酸含量分别降低（35±4）％和（31±2）％（均P ＜0.01）.结论 miRNA-200b抑制剂下调HSC-T6细胞中miR-200b表达,抑制肝星状细胞增殖与活化,减少细胞外基质的合成和分泌.
Objective To investigate the effect of miRNA-200b-specific inhibitor on hepatic stellate cells（HSCs） activation,proliferation,and extracellular matrix production.Methods The miRNA-200b-specific inhibitors were designed,synthesized,and transfected into HSCs with lipofectamine 2000.The supernatant and HSCs were collected after incubation for 48 h.The expression of miR-200b was detected by quantitative reverse transcription polymerase chain reaction （qRT-PCR）.The expression ofα-smooth muscle actin （oα-SMA） protein in HSCs was detected by Western blotting.The cell proliferation was assayed by methyl thiazolyl tetrazolium （MTT） method.Contents of type Ⅲ procollagen and hyaluronic acid in supernatant were determined by radioimmunoassay.Results Compared to the control group,miRNA-200b expression was decreased in the miRNA-200b inhibitor group by 82% （P 〈 0.01）,α-SMA protein expression was reduced in the miRNA-200b inhibitor group by （19 ± 3） % （P 〈 0.05）,and the activity of HSCs proliferation was reduced by（33 ± 5）% （P 〈0.01）,and the contents of type Ⅲ procollagen and hyaluronic acid in supernatant were reduced in miRNA-200b inhibitor group by （35 ± 4）% and （31 ± 2）%,respectively（P 〈0.01）.Conclusions The miRNA-200b-specific inhibitor could significantly reduce the expression of miRNA-200b,and inhibit HSC proliferation,activation,and extracellular matrix production.
出处
《中国医师杂志》
CAS
2015年第5期682-684,688,共4页
Journal of Chinese Physician
基金
浙江省自然科学基金资助项目（LY14H030010）
温州市科技局资助项目（Y20120032）
