TRPC6蛋白表達在子癇前期發(fā)病機制中的相關性研究
發(fā)布時間：2018-10-12 08:03
【摘要】：第一部分:化學缺氧對滋養(yǎng)細胞中TRPC6表達的影響目的:研究應用化學試劑二氯化鈷(CoCl2)來構(gòu)建化學缺氧模型后,絨毛外滋養(yǎng)細胞(本研究中應用的是JEG3)中瞬時感受器陽離子通道蛋白6(transient receptor potential channel 6,TRPC6)表達的改變,以及細胞內(nèi)外鈣離子平衡的變化,探討細胞缺氧后TRPC6的表達及其在子癇前期發(fā)病機制中的相關作用。方法:1.絨毛外滋養(yǎng)細胞選取JEG3細胞株2.采用化學物質(zhì)二氯化鈷(CoCl2)建立JEG3細胞化學缺氧模型,將細胞分為3組進行處理,以缺氧時間不同來分組,分為對照組,缺氧24h和缺氧48h組。3.采用實時的熒光定量pcr來檢測各組TRPC6的mRNA含量,westen-blot的方法檢測各組TRPC6蛋白的表達情況,采用fluo-3來檢測細胞內(nèi)鈣離子的濃度變化。結(jié)果:1.在缺氧存在的條件下,TRPC6 mRNA表達增加,并且在缺氧24和48小時均增加明顯。此時,與對照相比,暴露于缺氧環(huán)境中的JEG3細胞,它的TRPC6mRNA的表達量增加大約9倍。RT-PCR的實驗揭示了同一批細胞中缺氧組和對照組之間TRPC6 mRNA,存在顯著差異(P0.005)。2.在缺氧24h和48h時,使用Western Blot方法,檢測JEG3細胞中的TRPC6表達,我們發(fā)現(xiàn)在缺氧24h和48h時,TRPC6的蛋白表達水平均升高,且與對照組相比有顯著差異(P0.005)。并且TRPC6蛋白表達水平在缺氧后隨時間增加保持在穩(wěn)定水平。3.在本研究中,我們使用了熒光探針Fluo-3/AM來標記細胞內(nèi)的鈣離子,用Pro-plus圖像的分析軟件來分析熒光圖像,用平均光密度(IOD SUM/areaSUM)來表示每組中鈣離子的濃度。結(jié)果表明,正常條件下,圖片均顯示相似的低熒光強度。而缺氧細胞的Fluo-3/AM的熒光強度增加,表明TRPC6高表達可能導致細胞內(nèi)游離鈣的變化。結(jié)論:應用二氯化鈷進行化學缺氧培養(yǎng)時,JEG3細胞的TRPC6表達增加,同時細胞內(nèi)的鈣離子濃度也相應的增加。我們用JEG3細胞系建立了缺氧誘導的TRPC6高表達的細胞模型,發(fā)現(xiàn)缺氧可以誘導TRPC6高表達,而缺氧本身就是子癇前期發(fā)病的重要原因,因此我們可以認為該細胞模型與子癇前期患者體內(nèi)滋養(yǎng)細胞有相似之處,應用它我們可以進一步解釋TRPC6高表達的作用。第二部分過表達TRPC6蛋白對JEG3生物學行為的影響及其在子癇前期發(fā)病機制中的研究目的:探究TRPC6蛋白對這滋養(yǎng)細胞(JEG3)的增殖、凋亡的影響,進而再探討TRPC6對子癇前期的滋養(yǎng)細胞模型的生物學行為的影響及其在子癇前期中發(fā)病的機制的作用。方法:1.利用慢病毒包裝含有rs3824934序列的過表達TRPC6蛋白干預正常培養(yǎng)條件下絨毛外滋養(yǎng)細胞JEG3。2.熒光顯微鏡下觀察慢病毒轉(zhuǎn)染情況,檢測轉(zhuǎn)染效率達80%以上,遂對攜帶有TRPC6蛋白的絨毛外滋養(yǎng)細胞JEG3繼續(xù)培養(yǎng)。3.采用熒光定量的pcr檢測各組細胞中的TRPC6的表達情況,再次驗證過表達實驗是否成功。對確定了TRPC6蛋白過表達的細胞應用熒光定量的pcr檢測凋亡因子caspase3、12表達的情況,再利用western blot的方法檢測來各組細胞不同蛋白的表達情況。應用CCK8的檢測方法來檢測滋養(yǎng)細胞的增殖改變情況。結(jié)果:1.將攜帶有TRPC6過表達的蛋白轉(zhuǎn)染后分別將空白組與陰性對照結(jié)果及陰性對照與TRPC6過表達組結(jié)果進行統(tǒng)計學分析,發(fā)現(xiàn)前一組之間無明顯差異,(t=2.061,p(29)0.05),而陰性對照組與TRPC6過表達組之間差異顯著(t=9.390,p(27)0.05),提示慢病毒轉(zhuǎn)染成功。2.過表達TRPC6后,與陰性對照組相比,凋亡因子caspase3及caspase12 mRNA表達也相應的增加,前者增加約32倍,后者增加約8倍。RT-PCR實驗揭示了同一批細胞中的TRPC6過表達組和陰性對照組之間caspase3(t=14.97,p(27)0.05)及caspase12(t=6.827,p(27)0.05)的表達有顯著差異。3.在獲得TRPC6過表達穩(wěn)轉(zhuǎn)株后,使用Western Blot方法分別檢測兩組細胞中的caspase3及caspase12蛋白表達情況,發(fā)現(xiàn)在TRPC6過表達組caspase3(t=13.08,p(27)0.05)及caspase12(t=15.45,p(27)0.05)蛋白表達水平均升高,且與陰性對照組相比有顯著差異。4.CCK8細胞增殖實驗結(jié)果顯示:與陰性對照組相比,TRPC6過表達組滋養(yǎng)細胞OD值均顯著降低,差異具有統(tǒng)計學意義(t=5.378,P0.05)。并且我們還發(fā)現(xiàn)滋養(yǎng)細胞增殖能力與培養(yǎng)時間長短有關,培養(yǎng)24h時,滋養(yǎng)細胞OD值最高,其增殖能力最強,隨后隨著培養(yǎng)時間延長,滋養(yǎng)細胞OD值逐漸降低,其增殖能力逐步降低,差異具有統(tǒng)計學意義(one-way ANOVA,F=40.76,p(27)0.05)。結(jié)論:在本階段中我們將攜帶有過表達TRPC6蛋白的慢病毒轉(zhuǎn)染至滋養(yǎng)細胞內(nèi),獲取過表達該蛋白的細胞穩(wěn)轉(zhuǎn)株。進而對穩(wěn)轉(zhuǎn)株進行分析檢測,我們發(fā)現(xiàn)過表達TRPC6蛋白后,細胞中凋亡因子表達明顯升高,細胞凋亡因子的表達升高,相應的即提示細胞凋亡數(shù)量的增加,而絨毛外滋養(yǎng)細胞的增殖的能力則明顯下降,因而我們可以推測TRPC6可能是通過影響滋養(yǎng)細胞的增殖和凋亡能力,進而導致胎盤淺著床,影響胎盤的血管的重塑,導致患者高血壓的發(fā)生,從而參與子癇前期的發(fā)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.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R714.244
本文編號：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.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R714.244
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