血管平滑肌细胞5-羟色胺受体1B基因差异表达对NF-κB通路活化的影响

发布时间：2018-03-31 18:07

本文选题：5HT1B　切入点：NF-κB通路　出处：《昆明医科大学》2017年硕士论文

【摘要】：[目的]探讨重组质粒对血管平滑肌细胞增殖的影响和血管平滑肌细胞NF-κB通路活化的影响因素。[方法]制作重组的5-HT1B质粒,构建具有不同5-HT1B受控表达水平的血管平滑肌细胞模型。根据各自不同的干扰水平将细胞模型分成:完全空白对照组、高剂量脂质体组(20ul)、低剂量脂质体组(5ul)和5HT1B质粒组,待培养条件稳定后利用蛋白免疫印迹法测定各组VSMC的p42/44MAPK、PKC、IKK-α蛋白表达量,采用Tanon GIS图像分析软件分析各蛋白的表达水平。[结果]血管平滑肌细胞培养及转染鉴定:用荧光显微镜(×400)下观察5HT1B重组质粒转染VSMC48h后,发现绿色荧光位于胞浆中,说明重组质粒已成功转染进入人VSMC;高剂量脂质体处理的VSMC细胞内IKK-α蛋白的表达被抑制(p0.001),低剂量处理的VSMC细胞与未经任何处理的VSMC细胞(完全培养基组)的IKK-α蛋白水平无统计学差异(p0.05);未经任何处理的VSMC细胞内的PKC蛋白的表达量与高剂量处理的VSMC细胞内PKC表达无统计学差异(p0.05),但低剂量脂质体处理组的PKC表达显著增加(p0.01); 5-HT1B质粒组、高剂量脂质体处理组以及低剂量脂质体处理组的p42/44MAPK蛋白的表达量均明显低于完全空白对照组(完全培养基组)(p0.001),且5-HT1B质粒组的p42/44MAPK蛋白减少程度高于与高剂量脂质体组和脂质体组。[结论]发现血管平滑肌细胞MAPK通路蛋白转染质粒组有明显低于各组的趋势,5HTIB受体可能影响血管平滑肌细胞MAPK通路活化;未发现血管平滑肌细胞PKC及IKK-α蛋白的实验组有低于各组的趋势,5HTIB受体可能不参与血管平滑肌细胞NF-κB通路活化;发现高剂量脂质体组的IKKα和MAPK蛋白水平均被显著抑制,推测脂质体浓度可能影响NF-κB通路和MAPK通路的活化。
[Abstract]:[objective] to investigate the effects of recombinant plasmids on the proliferation of vascular smooth muscle cells and the factors affecting the activation of NF- 魏 B. [methods] Recombinant 5-HT1B plasmids were prepared. Vascular smooth muscle cell models with different levels of controlled expression of 5-HT1B were constructed. According to different interference levels, the cell models were divided into three groups: blank control group, high dose liposome group, low dose liposome group and 5HT1B plasmid group. After stable culture conditions, the expression of p42 / 44 MAPK- 伪 protein in VSMC was determined by Western blot. Tanon GIS image analysis software was used to analyze the expression level of each protein. [results] VSMCs were cultured and identified. After transfection of VSMC48h with 5HT1B recombinant plasmid under fluorescence microscope (脳 400), it was found that the green fluorescence was located in the cytoplasm. These results indicate that the recombinant plasmid has been successfully transfected into human VSMC, the expression of IKK- 伪 protein was inhibited in VSMC cells treated with high dose liposome, and IKK- 伪 protein in VSMC cells treated with low dose and in VSMC cells without any treatment (complete medium group) was inhibited. The expression of PKC protein in VSMC cells without any treatment was not significantly different from that in VSMC cells treated with high dose, but the expression of PKC in low dose liposome treatment group was significantly higher than that in 5-HT1B plasmid group. The expression of p42/44MAPK protein in the high dose liposome treatment group and the low dose liposome treatment group was significantly lower than that in the complete blank control group (complete culture medium group), and the decrease of p42/44MAPK protein in the 5-HT1B plasmid group was higher than that in the high dose liposome group. [conclusion] it was found that the MAPK pathway activation of vascular smooth muscle cells in plasmid group was significantly lower than that in plasmid group (P < 0.05). PKC and IKK- 伪 protein in vascular smooth muscle cells in experimental group were lower than those in each group. 5 HTIB receptor may not participate in the activation of NF- 魏 B pathway in vascular smooth muscle cells, and the levels of IKK 伪 and MAPK protein in high dose liposome group were significantly inhibited. It is speculated that the concentration of liposome may affect the activation of NF- 魏 B pathway and MAPK pathway.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R135

【参考文献】