Egr-1诱骗寡核苷酸下调MMP-9抑制原代培养大鼠血管平滑肌细胞的迁移

发布时间：2018-07-08 21:00

本文选题：早期生长反应因子-1 + 基质金属蛋白酶-9　；参考：《中国医科大学》2010年硕士论文

【摘要】： 目的通过转染针对早期生长反应因子-1(early growth response factor-1,Egr-1)的诱骗寡核苷酸(Egr-1 decoy ODNs)到大鼠动脉血管平滑肌细胞中,检测各组早期生长反应因子-1、基质金属蛋白酶-9(Matrix metalloproteinases-9,MMP-9)表达变化及测量各组平滑肌细胞(vascular smooth muscle cell, VSMC)的迁移,探讨针对早期生长反应因子-1的诱骗寡核苷酸对体外培养的大鼠平滑肌细胞迁移的影响和作用机制。方法通过组织块贴壁法进行原代大鼠动脉血管平滑肌细胞培养、传代培养和鉴定,设计合成Egr-1诱骗、诱骗对照寡核苷酸。转染人工合成的针对Egr-1的诱骗寡核苷酸(Egr-1 decoy ODN),将培养的平滑肌细胞分为对照组(正常培养细胞)、诱骗组(转染Egr-1的诱骗寡核苷酸)、诱骗对照组(转染Egr-1诱骗杂码寡核苷酸)。免疫组织化学染色法检测各组不同时间Egr-1及基质金属蛋白酶-9(MMP-9)蛋白的表达,进行图像分析。对各组采用划痕法测定平滑肌细胞的迁移距离。所有数据均采用SPSS13.0统计软件进行统计分析,计量资料以均值±标准差(x±S)表示,各组间比较应用单因素方差分析,P0.05有统计学意义。结果成功进行原代VSMC培养,经细胞鉴定培养细胞为高纯度的血管平滑肌细胞。转染Egr-1诱骗寡核苷酸后,用免疫组织化学法分别观察各组不同时间点的Egr-1和MMP-9蛋白的表达情况。实验发现：Egr-1诱骗寡核苷酸转染后诱骗组的迁移距离明显低于诱骗对照组和对照组(P0.05),诱骗对照组和对照组没有明显的差异。同时发现Egr-1诱骗寡核苷酸转染后Egr-1及MMP-9蛋白在诱骗组的表达较低,与诱骗对照组和对照组比较具有统计学差异。结论本实验通过体外转染Egr-1的诱骗寡核苷酸在一定程度上能够通过抑制Egr-1蛋白的表达,抑制其下游MMP-9基因的表达,减少血清诱导的平滑肌细胞的迁移,从而达到抑制细胞迁移的目的。
[Abstract]:Objective to transfect Egr-1 decoy ODNs targeting early growth response factor-1 (Egr-1) into rat vascular smooth muscle cells. The expression of early growth response factor-1, matrix metalloproteinases-9 (MMP-9) and the migration of smooth muscle cells (vascular smooth muscle cell, VSMC) in each group were measured. To investigate the effect and mechanism of decoy oligonucleotides targeting early growth response factor-1 on the migration of rat smooth muscle cells in vitro. Methods the primary vascular smooth muscle cells of rat artery were cultured, cultured and identified by tissue mass adherence. Egr-1 decoy was designed and synthesized, and the control oligonucleotides were decoded. The cultured smooth muscle cells were divided into control group (normal cultured cells), decoy group (Egr-1 decoy oligonucleotide) and control group (transfected Egr-1 decoy oligonucleotide). The expression of Egr-1 and matrix metalloproteinase-9 (MMP-9) were detected by immunohistochemical staining. The migration distance of smooth muscle cells was measured by scratch method. All the data were analyzed by SPSS 13.0 statistical software. The measurement data were expressed as mean 卤standard deviation (x 卤S). Results the primary VSMC was successfully cultured and the cultured cells were identified as high purity vascular smooth muscle cells. The expression of Egr-1 and MMP-9 were observed by immunohistochemical method after transfection of Egr-1 decoded oligonucleotides. It was found that the migration distance of the decoy group was significantly lower than that of the decoy control group and the control group after transfection (P0.05), but there was no significant difference between the decoy control group and the control group. It was also found that the expression of Egr-1 and MMP-9 protein was lower in the decoy group after transfection with Egr-1 decoy oligodeoxynucleotides, which was significantly different from that in the decoy control group and the control group. Conclusion Egr-1 induced oligonucleotides can inhibit the expression of MMP-9 gene in the downstream of Egr-1 and reduce the migration of smooth muscle cells induced by serum to a certain extent by inhibiting the expression of Egr-1 protein and the expression of MMP-9 gene in the downstream of Egr-1. In order to achieve the purpose of inhibiting cell migration.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

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