尿毒症血清激活NLRP3炎症体信号通路介导平滑肌细胞增殖的机制研究

发布时间：2019-06-24 20:03

【摘要】：目的:体外培养人主动脉平滑肌细胞,明确尿毒症血清可激活NLRP3炎症体信号通路介导VSMCs增殖,并探索NLRP3抑制剂对VSMCs增殖的抑制作用。方法:收集尿毒症患者及健康人血清,以10%的血清浓度作用于VSMCs,将实验分为4组:尿毒症血清组(U)、健康血清组(N)、尿毒症血清+格列苯脲组(U+Gli)、健康血清+格列本脲组(N+Gli)。1.VSMCs培养与处理:人主动脉平滑肌细胞(HASMCs)购于美国ATCC公司,用10%尿毒症血清刺激,以健康血清作对照;进一步尝试格列本脲对VSMCs增殖的抑制作用。2.VSMCs NLRP3 m RNA表达水平检测:RT-PCR方法检测NLRP3 m RNA表达量。3.VSMCs NLRP3、Caspase-1活性检测:Western Blots法检测NLRP3、Caspase-1 p20蛋白表达量。4.VSMCs增殖活性检测:使用ELISA检测试剂盒测定溴脱氧尿苷(Brdu)摄取量,通过分析Brdu摄取量来判断VSMCs的增殖活性。结果:与健康血清组比较,尿毒症血清组VSMCs NLRP3 m RNA表达量增多,NLRP3与Caspase-1 p20蛋白表达量也升高,细胞培养上清液中Brdu含量增多,两组对照分析,差别有统计学意义(P0.05)。而格列本脲处理前后,尿毒症血清组和健康血清组相比较,NLRP3 m RNA、NLRP3蛋白、Caspase-1 p20蛋白表达量均减少,Brdu摄取量也随之减少,差别具有统计学意义(P0.05)。结论:尿毒症血清通过激活NLRP3炎症复合体信号通路,可以促进人主动脉平滑肌细胞增殖,而格列苯脲可通过抑制NLRP3炎症体活化进而抑制血管平滑肌细胞的增殖。
[Abstract]:Aim: to culture human aortic smooth muscle cells in vitro, to confirm that uremic serum can activate NLRP3 inflammatory body signal pathway mediated VSMCs proliferation, and to explore the inhibitory effect of NLRP3 inhibitor on VSMCs proliferation. Methods: the serum of uremic patients and healthy people was collected and treated with 10% serum concentration of VSMCs,. The experiment was divided into four groups: uremic serum group (U), healthy serum group (N), uremic serum glibenclamide group (U Gli), healthy serum glibenclamide group (N Gli). 1.VSMCs culture and treatment: human aortic smooth muscle cell (HASMCs) was purchased from ATCC Company of the United States and stimulated with 10% uremic serum. Healthy serum was used as control. The inhibitory effect of glibenclamide on the proliferation of VSMCs was further tried. 2. The expression of NLRP3 m RNA was detected by RT-PCR method. 3. The expression of NLRP3,Caspase-1 p20 protein was detected by: Western Blots assay. 4. The (Brdu) uptake of bromodeoxyuridine was measured by ELISA detection kit, and the proliferation activity of VSMCs was judged by analyzing Brdu uptake. Results: compared with the healthy serum group, the expression of VSMCs NLRP3 m RNA, the expression of NLRP3 and Caspase-1 p20 protein and the content of Brdu in the culture medium of uremic serum group were also increased, and the difference between the two groups was statistically significant (P 0.05). Before and after glibenclamide treatment, compared with the healthy serum group, the expression of NLRP3 m RNA,NLRP3 protein and Caspase-1 p20 protein decreased, and the Brdu uptake also decreased, the difference was statistically significant (P 0.05). Conclusion: uremic serum can promote the proliferation of human aortic smooth muscle cells by activating NLRP3 inflammatory complex signal pathway, while glibenclamide can inhibit the proliferation of vascular smooth muscle cells by inhibiting the activation of NLRP3 inflammation.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692.5

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