miR-34a联合棕榈酸促进肝细胞衰老的实验研究

发布时间：2018-05-05 10:10

本文选题：micro-RNA + miR-34a　；参考：《三峡大学》2014年硕士论文

【摘要】：目的：随着生活习惯及饮食结构的变化，近年来脂肪性肝病的发病率越来越高，成为危害公共卫生的重要因素。而饱和游离脂肪酸可以诱导肝细胞脂肪变性，，通过“两次打击”导致脂肪性肝病的发生发展。有研究发现细胞内非编码微小RNAmiR-34a在脂肪性肝病中高表达，因此，本实验拟应用饱和游离脂肪酸及参与脂肪性肝病调控的miR-34a建立肝细胞损伤模型，研究它们对肝细胞生物学活性的影响，为进一步探索脂肪性肝病的发病机制，以及寻找有效防治脂肪性肝病措施提供前期基础。方法：(1)瞬时转染pEGFP-C1和pEGFP-C1/miR-34a质粒到HepG2细胞中，24h后在荧光显微镜下观察GFP绿色荧光的表达；36h后收细胞通过RT-PCR检测miR-34a的表达。(2)不同浓度的棕榈酸处理正常、瞬时转染pEGFP-C1和pEGFP-C1/miR-34a质粒的HepG2细胞，MTT法检测棕榈酸和miR-34a对HepG2细胞增殖的影响。(3)实验分为空白对照、棕榈酸处理、miR-34a转染、miR-34a转染+棕榈酸处理四个组，用DAPI分别给四个组的细胞染色，显微镜下观察细胞核的形态学变化；并用细胞衰老特异性β-半乳糖苷酶试剂盒检测细胞衰老情况。(4)收集四个处理组的细胞，用RT-PCR检测细胞衰老相关基因P21，P53，细胞周期相关基因CDK6，CyclinE2，E2F1以及细胞炎性因子TNF-α，MCP的表达。(5) western blot检测四个处理组细胞周期蛋白CDK6和细胞衰老相关蛋白Sirt1的表达。结果：(1)由于载体pEGFP-C1带有GFP基因，因此表达质粒在荧光显微镜下可以观察到绿色荧光。瞬时转染pEGFP-C1和pEGFP-C1/miR-34a质粒到HepG2细胞中，24h后在荧光显微镜下观察到了绿色荧光的表达；RT-PCR结果显示，36h后miR-34a在其转染的HepG2细胞中高表达，而在空载组中表达为阴性。(2)不同浓度的棕榈酸处理HepG2细胞，MTT法检测棕榈酸可抑制HepG2细胞增殖，并且呈剂量依赖关系；而棕榈酸和miR-34a联合处理细胞可明显抑制HepG2细胞增殖。(3)研究表明，衰老细胞的细胞核体积增大，细胞内β-半乳糖苷酶在酸性条件下被催化生成深蓝色的沉积产物。本实验结果显示棕榈酸和miR-34a联合处理细胞DAPI染色发现细胞核体积增大，应用试剂盒检测也发现，实验组细胞特异性β-半乳糖苷酶呈深蓝色。(4) RT-PCR结果显示，与对照细胞相比较，miR-34a和棕榈酸联合作用于HepG2细胞，使与细胞衰老相关的基因p21，p16，p53，以及炎性细胞因子MCP，TNF-α的表达明显升高，细胞周期相关基因CDK6为低表达。(5) Western blot发现棕榈酸和miR-34a联合作用使CDK6和Sirt1的表达受到抑制。结论：研究结果表明miR-34a和棕榈酸联合作用于HepG2细胞，可以诱导细胞内β-半乳糖苷酶，阻滞细胞周期，促进HepG2细胞的衰老，提示高脂及miR-34a在肝细胞衰老中的重要作用及药物干预的潜在靶点。
[Abstract]:Objective: with the changes of living habits and dietary structure, the incidence of fatty liver disease is increasing in recent years, which is an important factor to endanger public health. Saturated free fatty acids can induce fatty degeneration of hepatocytes and lead to the occurrence and development of fatty liver disease through "two blows". Some studies have found that there is a high expression of non-coding micro in fatty liver disease. Therefore, we intend to use saturated free fatty acids and miR-34a involved in the regulation of fatty liver disease to establish hepatocyte injury model. The study of their effects on the biological activity of hepatocytes provides a basis for further exploring the pathogenesis of fatty liver disease and for finding effective measures to prevent and treat fatty liver disease. Methods pEGFP-C1 and pEGFP-C1/miR-34a plasmids were transfected into HepG2 cells for 24 h. The expression of GFP green fluorescence was observed under fluorescence microscope for 36 h. The expression of miR-34a was detected by RT-PCR. The cells were treated with palmitic acid at different concentrations. The effect of palmitic acid and miR-34a on the proliferation of HepG2 cells was assayed by MTT assay. The HepG2 cells transfected with pEGFP-C1 and pEGFP-C1/miR-34a plasmid were divided into four groups: palmiR-34a transfected with miR-34a and palmiR-34a transfected with palmiR-34a. The cells of the four groups were stained with DAPI. The morphologic changes of the nucleus were observed under microscope and the senescence specific 尾 -galactosidase kit was used to detect the senescence of the cells. The expressions of cyclin CDK6 and senescence related protein Sirt1 were detected by RT-PCR, CDK6, cyclin E2E2F1 and TNF- 伪 MCP, respectively. Results because the vector pEGFP-C1 contained GFP gene, green fluorescence could be observed under fluorescence microscope. After transient transfection of pEGFP-C1 and pEGFP-C1/miR-34a plasmids into HepG2 cells for 24 hours, the expression of green fluorescence was observed under fluorescence microscope. The results of RT-PCR showed that miR-34a was highly expressed in HepG2 cells after 36 hours of transfection. HepG2 cells treated with different concentrations of palmitic acid could inhibit the proliferation of HepG2 cells in a dose-dependent manner. However, palmitic acid combined with miR-34a could significantly inhibit the proliferation of HepG2 cells. The results showed that the nuclear volume of aging cells increased and 尾 -galactosidase was catalysed to produce dark blue deposition products under acidic conditions. The results showed that the cell nuclear volume was increased by DAPI staining with palmitic acid and miR-34a, and the cell specific 尾 -galactosidase of the experimental group was dark blue. RT-PCR showed that the cell specific 尾 -galactosidase in the experimental group was dark blue. Compared with the control cells, the combination of miR-34a and palmitic acid on HepG2 cells increased the expression of p21, p16, p53, and the inflammatory cytokine MCP-TNF- 伪. The expression of CDK6 and Sirt1 was inhibited by palmitic acid and miR-34a combined with Western blot. Conclusion: miR-34a combined with palmitic acid can induce 尾 -galactosidase in HepG2 cells, block cell cycle and promote the senescence of HepG2 cells. It is suggested that hyperlipidemia and miR-34a may play an important role in hepatocyte aging and potential targets for drug intervention.
【学位授予单位】：三峡大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.5

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