本文選題：5HT1B　切入點：NF-κB通路　出處：《昆明醫(yī)科大學》2017年碩士論文

【摘要】：[目的]探討重組質粒對血管平滑肌細胞增殖的影響和血管平滑肌細胞NF-κB通路活化的影響因素。[方法]制作重組的5-HT1B質粒,構建具有不同5-HT1B受控表達水平的血管平滑肌細胞模型。根據(jù)各自不同的干擾水平將細胞模型分成:完全空白對照組、高劑量脂質體組(20ul)、低劑量脂質體組(5ul)和5HT1B質粒組,待培養(yǎng)條件穩(wěn)定后利用蛋白免疫印跡法測定各組VSMC的p42/44MAPK、PKC、IKK-α蛋白表達量,采用Tanon GIS圖像分析軟件分析各蛋白的表達水平。[結果]血管平滑肌細胞培養(yǎng)及轉染鑒定:用熒光顯微鏡(×400)下觀察5HT1B重組質粒轉染VSMC48h后,發(fā)現(xiàn)綠色熒光位于胞漿中,說明重組質粒已成功轉染進入人VSMC;高劑量脂質體處理的VSMC細胞內IKK-α蛋白的表達被抑制(p0.001),低劑量處理的VSMC細胞與未經(jīng)任何處理的VSMC細胞(完全培養(yǎng)基組)的IKK-α蛋白水平無統(tǒng)計學差異(p0.05);未經(jīng)任何處理的VSMC細胞內的PKC蛋白的表達量與高劑量處理的VSMC細胞內PKC表達無統(tǒng)計學差異(p0.05),但低劑量脂質體處理組的PKC表達顯著增加(p0.01); 5-HT1B質粒組、高劑量脂質體處理組以及低劑量脂質體處理組的p42/44MAPK蛋白的表達量均明顯低于完全空白對照組(完全培養(yǎng)基組)(p0.001),且5-HT1B質粒組的p42/44MAPK蛋白減少程度高于與高劑量脂質體組和脂質體組。[結論]發(fā)現(xiàn)血管平滑肌細胞MAPK通路蛋白轉染質粒組有明顯低于各組的趨勢,5HTIB受體可能影響血管平滑肌細胞MAPK通路活化;未發(fā)現(xiàn)血管平滑肌細胞PKC及IKK-α蛋白的實驗組有低于各組的趨勢,5HTIB受體可能不參與血管平滑肌細胞NF-κB通路活化;發(fā)現(xiàn)高劑量脂質體組的IKKα和MAPK蛋白水平均被顯著抑制,推測脂質體濃度可能影響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- 偽 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- 偽 protein was inhibited in VSMC cells treated with high dose liposome, and IKK- 偽 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- 偽 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 偽 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.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R135

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