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转化生长因子β/骨形成蛋白应答元件表达质粒的构建及功能检测
  • ISSN号:1001-1633Call Number: 31-1285
  • 期刊名称:解剖学杂志
  • 时间:2014
  • 页码:625-628
  • 分类:R619.6[医药卫生—临床医学;医药卫生—外科学]
  • 作者机构:[1]三峡大学医学院,宜昌443002, [2]三峡大学肿瘤微环境与靶向治疗湖北省重点实验室,宜昌443002, [3]湖北工业大学校医院,武汉430068
  • 相关基金:国家自然科学基金(81200307/H0317);湖北省自然科学基金(2013cfb026)
  • 相关项目:运用decoy ODN策略同时干预TGF-β和Notch信号通路逆转肝纤维化进程
中文摘要:

目的:研究活化型肝星状细胞(HSC)中miRNA-193(miR-193)下调肝纤维化相关基因的表达。方法:将大鼠miR-193的前体序列(pre-miR-193)克隆到真核表达载体pcDNA3.1(+)中,经酶切和DNA测序鉴定,获得质粒pcDNA3.1-miR-193;通过脂质体转染法将质粒转染到大鼠的活化型肝星状细胞(HSC-T6)中,采用荧光素酶报告基因分析法和免疫印迹检测肝纤维相关基因的表达。结果:构建的真核表达载体pcDNA3.1-miR-193经酶切鉴定及DNA测序显示,目的片段的大小与预期结果一致;荧光素酶报告基因分析法和免疫印迹检测显示,重组质粒转染到活化型HSC-T6中后,肝纤维化相关基因的α-平滑肌肌动蛋白(a-SMA)的表达呈明显下降趋势,而胶原蛋白IαI和Iα2的下调作用不明显。结论:miR-193能抑制活化型肝星状细胞中肝纤维化相关基因α-SMA的表达。

英文摘要:

Objective: To study how miRNA-193 down-regulates the expression of hepatic fibrosis related genes in activative hepatic steLLate ceils (MS(Z). Methods: After searching for the precursor sequence of miR-193 (pre-miR-193) of rats in Genbank from NCBI, we constructed eukaryotic expression plasmid pcDNA3.1-miR-193 through cloning the pre-miR-193 into the eukaryotic expression vector, pcDNA3. 1 (+), and then identified it using the methods of restriction and DNA sequencing assays. The expressions of hepatic fibrosis-ralated genes (smooth muscle actin (a-SMA), collagen IαI/a2) were evaluated in activative HSC line of rats (HSC-T6) through Western bolt assay when the plasmid pcDNA3.1-miR-193 was transfeted into HSC-T6. Results: The eukaryotic expression plasmid pcDNA3.1-miR-193, which was almost consistent with the result of anticipation, was constructed successfully through the methods of restriction and DNA sequencing assays. The expression of gene α-SMA presented dramatic decline through luciferase activity and Western blot assays after the plasmid pcDNA3.1-miR- 193 was transfected into HSC-T6, but the expressions were not obviously changed for genes of collagen [ α1/α2. Conclusion: miRNA-193 can down-regulate the expression of gene α-SMA in aetivative HSC.

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