TRPC6蛋白表达在子痫前期发病机制中的相关性研究
发布时间：2018-10-12 08:03
【摘要】：第一部分:化学缺氧对滋养细胞中TRPC6表达的影响目的:研究应用化学试剂二氯化钴(CoCl2)来构建化学缺氧模型后,绒毛外滋养细胞(本研究中应用的是JEG3)中瞬时感受器阳离子通道蛋白6(transient receptor potential channel 6,TRPC6)表达的改变,以及细胞内外钙离子平衡的变化,探讨细胞缺氧后TRPC6的表达及其在子痫前期发病机制中的相关作用。方法:1.绒毛外滋养细胞选取JEG3细胞株2.采用化学物质二氯化钴(CoCl2)建立JEG3细胞化学缺氧模型,将细胞分为3组进行处理,以缺氧时间不同来分组,分为对照组,缺氧24h和缺氧48h组。3.采用实时的荧光定量pcr来检测各组TRPC6的mRNA含量,westen-blot的方法检测各组TRPC6蛋白的表达情况,采用fluo-3来检测细胞内钙离子的浓度变化。结果:1.在缺氧存在的条件下,TRPC6 mRNA表达增加,并且在缺氧24和48小时均增加明显。此时,与对照相比,暴露于缺氧环境中的JEG3细胞,它的TRPC6mRNA的表达量增加大约9倍。RT-PCR的实验揭示了同一批细胞中缺氧组和对照组之间TRPC6 mRNA,存在显著差异(P0.005)。2.在缺氧24h和48h时,使用Western Blot方法,检测JEG3细胞中的TRPC6表达,我们发现在缺氧24h和48h时,TRPC6的蛋白表达水平均升高,且与对照组相比有显著差异(P0.005)。并且TRPC6蛋白表达水平在缺氧后随时间增加保持在稳定水平。3.在本研究中,我们使用了荧光探针Fluo-3/AM来标记细胞内的钙离子,用Pro-plus图像的分析软件来分析荧光图像,用平均光密度(IOD SUM/areaSUM)来表示每组中钙离子的浓度。结果表明,正常条件下,图片均显示相似的低荧光强度。而缺氧细胞的Fluo-3/AM的荧光强度增加,表明TRPC6高表达可能导致细胞内游离钙的变化。结论:应用二氯化钴进行化学缺氧培养时,JEG3细胞的TRPC6表达增加,同时细胞内的钙离子浓度也相应的增加。我们用JEG3细胞系建立了缺氧诱导的TRPC6高表达的细胞模型,发现缺氧可以诱导TRPC6高表达,而缺氧本身就是子痫前期发病的重要原因,因此我们可以认为该细胞模型与子痫前期患者体内滋养细胞有相似之处,应用它我们可以进一步解释TRPC6高表达的作用。第二部分过表达TRPC6蛋白对JEG3生物学行为的影响及其在子痫前期发病机制中的研究目的:探究TRPC6蛋白对这滋养细胞(JEG3)的增殖、凋亡的影响,进而再探讨TRPC6对子痫前期的滋养细胞模型的生物学行为的影响及其在子痫前期中发病的机制的作用。方法:1.利用慢病毒包装含有rs3824934序列的过表达TRPC6蛋白干预正常培养条件下绒毛外滋养细胞JEG3。2.荧光显微镜下观察慢病毒转染情况,检测转染效率达80%以上,遂对携带有TRPC6蛋白的绒毛外滋养细胞JEG3继续培养。3.采用荧光定量的pcr检测各组细胞中的TRPC6的表达情况,再次验证过表达实验是否成功。对确定了TRPC6蛋白过表达的细胞应用荧光定量的pcr检测凋亡因子caspase3、12表达的情况,再利用western blot的方法检测来各组细胞不同蛋白的表达情况。应用CCK8的检测方法来检测滋养细胞的增殖改变情况。结果:1.将携带有TRPC6过表达的蛋白转染后分别将空白组与阴性对照结果及阴性对照与TRPC6过表达组结果进行统计学分析,发现前一组之间无明显差异,(t=2.061,p(29)0.05),而阴性对照组与TRPC6过表达组之间差异显著(t=9.390,p(27)0.05),提示慢病毒转染成功。2.过表达TRPC6后,与阴性对照组相比,凋亡因子caspase3及caspase12 mRNA表达也相应的增加,前者增加约32倍,后者增加约8倍。RT-PCR实验揭示了同一批细胞中的TRPC6过表达组和阴性对照组之间caspase3(t=14.97,p(27)0.05)及caspase12(t=6.827,p(27)0.05)的表达有显著差异。3.在获得TRPC6过表达稳转株后,使用Western Blot方法分别检测两组细胞中的caspase3及caspase12蛋白表达情况,发现在TRPC6过表达组caspase3(t=13.08,p(27)0.05)及caspase12(t=15.45,p(27)0.05)蛋白表达水平均升高,且与阴性对照组相比有显著差异。4.CCK8细胞增殖实验结果显示:与阴性对照组相比,TRPC6过表达组滋养细胞OD值均显著降低,差异具有统计学意义(t=5.378,P0.05)。并且我们还发现滋养细胞增殖能力与培养时间长短有关,培养24h时,滋养细胞OD值最高,其增殖能力最强,随后随着培养时间延长,滋养细胞OD值逐渐降低,其增殖能力逐步降低,差异具有统计学意义(one-way ANOVA,F=40.76,p(27)0.05)。结论:在本阶段中我们将携带有过表达TRPC6蛋白的慢病毒转染至滋养细胞内,获取过表达该蛋白的细胞稳转株。进而对稳转株进行分析检测,我们发现过表达TRPC6蛋白后,细胞中凋亡因子表达明显升高,细胞凋亡因子的表达升高,相应的即提示细胞凋亡数量的增加,而绒毛外滋养细胞的增殖的能力则明显下降,因而我们可以推测TRPC6可能是通过影响滋养细胞的增殖和凋亡能力,进而导致胎盘浅着床,影响胎盘的血管的重塑,导致患者高血压的发生,从而参与子痫前期的发生。
[Abstract]:In the first part, the effect of chemical hypoxia on the expression of TRPC6 in trophoblast cells was studied: the effect of chemical hypoxia on the expression of TRPC6 in trophoblast cells was studied. The expression of TRPC6 and the relationship between the expression of TRPC6 and the pathogenesis of preeclampsia were discussed. Method: 1. JEG3 cell line was selected from the trophoblast cells of villi. The chemical hypoxia model of JEG3 cells was established by cobalt chloride (CoCl2), the cells were divided into 3 groups for treatment, and the groups were divided into two groups: control group, hypoxia 24h and hypoxia 48h group. The mRNA content of TRPC6 in each group was detected by real-time fluorescence quantitative polymerase chain reaction (PCR). The expression of TRPC6 protein in each group was detected by the method of senen-blot. Fluo-3 was used to detect the concentration of intracellular calcium ions. Result: 1. In the presence of hypoxia, the expression of TRPC6 mRNA was increased, and the expression of TRPC6 mRNA increased significantly in 24 and 48 hours. At this time, compared to the control, the expression of TRPC6mRNA was increased by about 9 times in the JEG3 cells exposed to the hypoxic environment. RT-PCR revealed that there was significant difference in TRPC6 mRNA between hypoxia group and control group (P0.05). After hypoxia 24h and 48h, the expression of TRPC6 in JEG3 cells was detected by Western blot. We found that the expression level of TRPC6 increased in hypoxia 24h and 48h, and the expression level of TRPC6 was significantly different from that of control group (P0.05). and the expression level of TRPC6 was maintained at a stable level with time after hypoxia. In this study, we used fluorescence probe Fluo-3/ AM to label calcium ions in cells, analyzed fluorescence images with the analysis software of Pro-plus images, and expressed the concentration of calcium ions in each group by mean optical density (IOD SUM/ araeSUM). The results showed that the images showed similar low fluorescence intensity under normal conditions. However, the fluorescence intensity of Fluo-3/ AM in hypoxic cells increased, indicating that the high expression of TRPC6 could lead to changes in intracellular free energy. Conclusion: The expression of TRPC6 in JEG3 cells increased while the concentration of calcium ions in the cells increased. We established a cell model of hypoxia-induced TRPC6 expression by JEG3 cell line, and found that hypoxia can induce high expression of TRPC6, and hypoxia is an important cause of preeclampsia. Therefore, we can conclude that the cell model is similar to trophoblast cells in preeclampsia. Using it, we can further explain the high expression of TRPC6. The second part studies the effect of TRPC6 protein on the biological behavior of JEG3 and its research aim in the pathogenesis of preeclampsia: to explore the effect of TRPC6 protein on the proliferation and apoptosis of this trophoblast cell (JEG3). Furthermore, the effect of TRPC6 on the biological behavior of trophoblast model in preeclampsia was discussed and the mechanism of TRPC6 in preeclampsia was discussed. Method: 1. An over-expression TRPC6 protein containing rs3824934 sequence in slow virus packaging was used to interfere with JEG3. 2 under normal culture conditions. Under the fluorescence microscope, slow virus transfection was observed, the transfection efficiency was more than 80%, and then JEG3, which carries the TRPC6 protein, continued to be cultured. The expression of TRPC6 in each group was detected by fluorescence quantitative polymerase chain reaction (pcr), and whether the expression experiment was successful was verified again. The expression of caspase-3 and 12 was detected by fluorescent quantitative polymerase chain reaction (pcr), and the expression of different proteins in each group was detected by western blot. The proliferation of trophoblast cells was detected by the detection method of CCK8. Result: 1. There was no significant difference between the negative control group and the negative control group (t = 2.061, p (29) 0.05), and the difference between the negative control group and the TRPC6 over-expression group was significant (t = 9.390, P (29) 0.05). p (27) 0. 05) showed that lentiviral transfection was successful. Compared with the negative control group, the expression of caspase3 and caspase12 mRNA increased correspondingly, the former increased by about 32 times and the latter increased by about 8 times compared with the negative control group. RT-PCR revealed significant differences in the expression of caspase3 (t = 14.97, p (27) 0.05) and caspas12 (t = 6.827, p (27) 0. 05) in the same batch of cells. The expression of caspase3 and caspase12 in the two groups were detected by Western blot, and the expression levels of caspase3 (t = 13. 08, p (27) 0. 05) and caspas12 (t = 15. 45, p (27) 0. 05) were all increased after TRPC6 overexpression. Compared with the negative control group, the OD value of trophoblast cells in TRPC6 was significantly lower than that in the negative control group (t = 5.378, P0.05). We also found that the proliferative ability of trophoblast cells was related to the length of culture time. When cultured for 24h, the OD value of trophoblast cells was the highest, and the proliferative ability of trophoblast cells was the strongest, then the OD value of trophoblast decreased gradually with the increase of culture time, and the proliferative ability of trophoblast decreased gradually. The difference was statistically significant (one-way ANOVA, F = 40. 76, p (27) 0. 05). Conclusion: In this stage, we will be able to transfect lentivirus expressing TRPC6 protein into trophoblastic cells, and obtain the cells stably expressing the protein. Furthermore, we found that after the expression of TRPC6 protein, the expression of apoptosis factor increased and the expression of apoptosis factor increased. So we can speculate that TRPC6 may affect the proliferation and apoptosis of trophoblast cells, which leads to a shallow implantation of the placenta, which affects the remodeling of the blood vessels of the placenta, leading to the occurrence of hypertension in the patient, thus participating in the occurrence of preeclampsia.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.244
本文编号：2265387
[Abstract]:In the first part, the effect of chemical hypoxia on the expression of TRPC6 in trophoblast cells was studied: the effect of chemical hypoxia on the expression of TRPC6 in trophoblast cells was studied. The expression of TRPC6 and the relationship between the expression of TRPC6 and the pathogenesis of preeclampsia were discussed. Method: 1. JEG3 cell line was selected from the trophoblast cells of villi. The chemical hypoxia model of JEG3 cells was established by cobalt chloride (CoCl2), the cells were divided into 3 groups for treatment, and the groups were divided into two groups: control group, hypoxia 24h and hypoxia 48h group. The mRNA content of TRPC6 in each group was detected by real-time fluorescence quantitative polymerase chain reaction (PCR). The expression of TRPC6 protein in each group was detected by the method of senen-blot. Fluo-3 was used to detect the concentration of intracellular calcium ions. Result: 1. In the presence of hypoxia, the expression of TRPC6 mRNA was increased, and the expression of TRPC6 mRNA increased significantly in 24 and 48 hours. At this time, compared to the control, the expression of TRPC6mRNA was increased by about 9 times in the JEG3 cells exposed to the hypoxic environment. RT-PCR revealed that there was significant difference in TRPC6 mRNA between hypoxia group and control group (P0.05). After hypoxia 24h and 48h, the expression of TRPC6 in JEG3 cells was detected by Western blot. We found that the expression level of TRPC6 increased in hypoxia 24h and 48h, and the expression level of TRPC6 was significantly different from that of control group (P0.05). and the expression level of TRPC6 was maintained at a stable level with time after hypoxia. In this study, we used fluorescence probe Fluo-3/ AM to label calcium ions in cells, analyzed fluorescence images with the analysis software of Pro-plus images, and expressed the concentration of calcium ions in each group by mean optical density (IOD SUM/ araeSUM). The results showed that the images showed similar low fluorescence intensity under normal conditions. However, the fluorescence intensity of Fluo-3/ AM in hypoxic cells increased, indicating that the high expression of TRPC6 could lead to changes in intracellular free energy. Conclusion: The expression of TRPC6 in JEG3 cells increased while the concentration of calcium ions in the cells increased. We established a cell model of hypoxia-induced TRPC6 expression by JEG3 cell line, and found that hypoxia can induce high expression of TRPC6, and hypoxia is an important cause of preeclampsia. Therefore, we can conclude that the cell model is similar to trophoblast cells in preeclampsia. Using it, we can further explain the high expression of TRPC6. The second part studies the effect of TRPC6 protein on the biological behavior of JEG3 and its research aim in the pathogenesis of preeclampsia: to explore the effect of TRPC6 protein on the proliferation and apoptosis of this trophoblast cell (JEG3). Furthermore, the effect of TRPC6 on the biological behavior of trophoblast model in preeclampsia was discussed and the mechanism of TRPC6 in preeclampsia was discussed. Method: 1. An over-expression TRPC6 protein containing rs3824934 sequence in slow virus packaging was used to interfere with JEG3. 2 under normal culture conditions. Under the fluorescence microscope, slow virus transfection was observed, the transfection efficiency was more than 80%, and then JEG3, which carries the TRPC6 protein, continued to be cultured. The expression of TRPC6 in each group was detected by fluorescence quantitative polymerase chain reaction (pcr), and whether the expression experiment was successful was verified again. The expression of caspase-3 and 12 was detected by fluorescent quantitative polymerase chain reaction (pcr), and the expression of different proteins in each group was detected by western blot. The proliferation of trophoblast cells was detected by the detection method of CCK8. Result: 1. There was no significant difference between the negative control group and the negative control group (t = 2.061, p (29) 0.05), and the difference between the negative control group and the TRPC6 over-expression group was significant (t = 9.390, P (29) 0.05). p (27) 0. 05) showed that lentiviral transfection was successful. Compared with the negative control group, the expression of caspase3 and caspase12 mRNA increased correspondingly, the former increased by about 32 times and the latter increased by about 8 times compared with the negative control group. RT-PCR revealed significant differences in the expression of caspase3 (t = 14.97, p (27) 0.05) and caspas12 (t = 6.827, p (27) 0. 05) in the same batch of cells. The expression of caspase3 and caspase12 in the two groups were detected by Western blot, and the expression levels of caspase3 (t = 13. 08, p (27) 0. 05) and caspas12 (t = 15. 45, p (27) 0. 05) were all increased after TRPC6 overexpression. Compared with the negative control group, the OD value of trophoblast cells in TRPC6 was significantly lower than that in the negative control group (t = 5.378, P0.05). We also found that the proliferative ability of trophoblast cells was related to the length of culture time. When cultured for 24h, the OD value of trophoblast cells was the highest, and the proliferative ability of trophoblast cells was the strongest, then the OD value of trophoblast decreased gradually with the increase of culture time, and the proliferative ability of trophoblast decreased gradually. The difference was statistically significant (one-way ANOVA, F = 40. 76, p (27) 0. 05). Conclusion: In this stage, we will be able to transfect lentivirus expressing TRPC6 protein into trophoblastic cells, and obtain the cells stably expressing the protein. Furthermore, we found that after the expression of TRPC6 protein, the expression of apoptosis factor increased and the expression of apoptosis factor increased. So we can speculate that TRPC6 may affect the proliferation and apoptosis of trophoblast cells, which leads to a shallow implantation of the placenta, which affects the remodeling of the blood vessels of the placenta, leading to the occurrence of hypertension in the patient, thus participating in the occurrence of preeclampsia.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.244
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