CIC3在碘离子致脐静脉内皮细胞增殖和凋亡中的作用

发布时间：2018-01-10 08:16

本文关键词：CIC3在碘离子致脐静脉内皮细胞增殖和凋亡中的作用　出处：《山东大学》2011年硕士论文　论文类型：学位论文

【摘要】：[目的] 碘离子可以致内皮细胞等多种细胞增殖和凋亡,而经典的碘离子通道就是分布在甲状腺上的钠-碘同向转运体(NIS),NIS主要在甲状腺及神经组织等腺外组织表达,而内皮细胞上是否也有NIS表达至今未见报导。但是,内皮细胞上却有氯离子通道-3(CLC3)的表达,并且CLC3在通常情况下对碘离子的选择性大于氯离子。由此,本实验通过体外培养脐静脉内皮细胞,运用RT-PCR、Western Blot、流式细胞术等方法研究CLC3通道在不同碘离子浓度及时相促进脐静脉内皮细胞增殖和凋亡中的作用,探讨碘离子是否经过CLC3进入内皮细胞,丰富碘离子通道的研究。 [方法] 1.细胞株：人脐静脉内皮(HUVEC)细胞株,购于南京凯基生物有限公司 2.实验分组：KI 3.流式细胞术：用不同浓度的碘离子培养液处理脐静脉内皮细胞24小时和48小时后,经离心—PBS冲洗—吹匀—固定—再离心—冲洗—吹打—染色(4℃,20～30分钟)后,再用400目尼龙网将之过滤之后上机检测。用ModFit分析软件分析,细胞相对增殖活力用细胞分裂增殖指数表示。 4.钠碘同向转运体(NIS) mRNA的RT-PCR:将脐静脉内皮细胞培养于含不同浓度的碘离子培养液中24小时和48小时后,提取细胞的总RNA,经反转录—扩增—电泳,检测钠碘同向转运体(NIS) mRNA的表达。 5.钠碘同向转运体(NIS)蛋白Western Blot:用不同浓度的碘离子培养液培养脐静脉内皮细胞24小时和48小时后,提取总蛋白,经电泳—转膜—封闭—孵育一抗—孵育二抗—显色等过程,检测目的蛋白NIS的表达。 6.氯离子通道(CLC3) mRNA的RT-PCR:将脐静脉内皮细胞用含不同浓度的碘离子培养液处理24小时和48小时后,提取其总RNA,再经反转录—扩增—电泳等步骤,检测氯离子通道(CLC3) mRNA的表达。 7.氯离子通道(CLCN3)蛋白的Western Blot:搜集含不同浓度碘离子培养液处理24小时和48小时后的脐静脉内皮细胞,先用蛋白裂解液提取总蛋白,再经电泳—转膜—封闭—孵育—抗—孵育二抗—显色,检测目的蛋白CLCN3的表达。 [结果] 1.流式细胞术：24小时,碘离子浓度为100μg/L～5000μg/L实验组细胞增殖指数明显高于对照组,而细胞凋亡率明显低于对照组；碘离子浓度为7000μg/L的实验组细胞增殖指数与对照组比较没有明显差别,而细胞凋亡率明显高于对照组；碘离子浓度≥9000μg/L的实验组细胞增殖指数明显低于对照组,而细胞凋亡率高于对照组。48小时,碘离子浓度为100μg/L-3000μg/L实验组细胞增殖指数明显高于对照组,同时细胞凋亡率明显低于对照组；碘离子浓度为5000μg/L的实验组细胞增殖指数跟对照组比较没有差别,而细胞凋亡率高于对照组；碘离子浓度≥7000μg/L的实验组细胞增殖指数低于对照组,同时细胞凋亡率明显高于对照组。 2.钠碘同向转运体(NIS) mRNART-PCR:钠碘同向转运体(NIS) mRNA在HUVEC表达缺失。 3.钠碘同向转运体(NIS)蛋白western:钠碘同向转运体(NIS)蛋白在HUVEC表达缺失。 4.氯离子通道(CLC3)mRNA RT-PCR:与对照组相比,24h时1000μg/L-5000μg/L实验组,和48h时100μg/L实验组CLC3的mRNA表达上调,P0.05,差异有统计学意义；7000μg/L实验组(24h)和5000μg/L实验组(48h)CLC3的mRNA表达差异无统计学意义,P0.05；9000μg/L实验组在24h时及7000μg/L-9000μg/L实验组在48h时CLC3的mRNA表达下调,P0.05,差异有统计学意义。 5.氯离子通道(CLCN3)蛋白western blot:与对照组相比,1000～5000μg/L实验组(24h)及1000μg/L实验组(48h)在细胞CLCN3蛋白表达增强,P0.05,差异有统计学意义；而7000μg/L实验组(24h)及5000μg/L实验组(48h)CLCN3蛋白表达与对照组没有差别,无统计学意义,P0.05；9000μg/L实验组(24h)及7000μg/L-9000μg/L(48h)CLCN3蛋白表达减弱,P0.05,差异有统计学意义。 [结论] 1.碘离子对脐静脉内皮细胞增殖活性具有促进和抑制两方面的作用,其作用效果与作用时间和作用浓度密切相关,即适宜浓度的碘离子对脐静脉内皮细胞增殖活性存在时间-剂量-效应关系。 2.人脐静脉内皮细胞上钠碘转运体(NIS)表达缺失。 3.人脐静脉内皮细胞上氯离子通道-3(CLC3)表达阳性,碘离子可以通过CLC3进入到脐静脉内皮细胞内,从而促进内皮细胞的增殖和凋亡。
[Abstract]:[Objective]
Iodine ion can be induced to endothelial cells in a variety of cell proliferation and apoptosis, and the iodine ion channel is the classic distribution in the thyroid sodium iodide symporter (NIS), NIS is mainly expressed in thyroid gland tissue and nerve tissue, and endothelial cells have NIS expression has not been reported. However, endothelial the cell has -3 chloride channel (CLC3) expression and CLC3 normally selectivity for iodine ion chloride ion. Thus, this experiment in vitro cultured human umbilical vein endothelial cells by RT-PCR, Western, Blot, flow cytometry and other methods of CLC3 channel in different iodine ion concentration in promote the proliferation and apoptosis of human umbilical vein endothelial cells in the role of iodine ion through CLC3 into endothelial cells, rich in iodine ion channel research.
[method]
1. cell lines: human umbilical vein endothelial cells (HUVEC) were purchased from Nanjing keygen Co. Ltd.
2. group of experiments: KI
3. flow cytometry: cultured with different concentrations of iodine ion solution treatment of human umbilical vein endothelial cells for 24 hours and 48 hours after the centrifugal PBS - uniform - Fixed - flushing blowing centrifugal - Rinse - percussion - staining (4 degrees, 20~30 minutes), the machine filter with 400 mesh nylon net will be the detection software. Analyzed by ModFit analysis, the relative cell proliferation activity of cell proliferation index.
4. sodium iodide co transporter (NIS) mRNA RT-PCR:, cultured umbilical vein endothelial cells in different concentrations of iodide medium for 24 hours and 48 hours, extracted the total RNA of cells, and detected the expression of sodium iodine co transporter (NIS) mRNA by reverse transcription amplification electrophoresis.
5. sodium iodide symporter (NIS) protein Western Blot: with different concentrations of iodide ions cultured human umbilical vein endothelial cells for 24 hours and 48 hours, the total protein extracted by electrophoresis transfer membrane incubated in anti - closed - were incubated for two anti color process, detection of the expression of the target protein NIS.
6. RT-PCR: of chloride channel (CLC3) mRNA, the umbilical vein endothelial cells were treated with iodide ion solution containing different concentrations for 24 hours and 48 hours, the total RNA was extracted, and then the expression of chloride channel (CLC3) mRNA was detected by reverse transcription amplification electrophoresis.
7. chloride channel protein (CLCN3) Western Blot: collected with different iodine ion concentration in cultured human umbilical vein endothelial cells was 24 hours and 48 hours after the first extraction of total protein lysates, followed by electrophoresis and transfer membrane incubated with anti - - closed incubation for two anti color detection the expression of CLCN3 protein.
[results]
1. flow cytometry: 24 hours, iodine ion concentration is 100 g/L ~ 5000 g/L experimental group, the cell proliferation index was significantly higher than the control group, while the apoptosis rate was significantly lower than the control group; the iodine ion concentration for cell proliferation index in the experimental group was 7000 g/L compared with the control group have no obvious difference, and the rate of cell apoptosis the experimental group was significantly higher than the control group; the proliferation index of iodine ion concentration of more than 9000 g/L was significantly lower than the control group, while the apoptosis rate was higher than the control group.48 hours, iodine ion concentration of 100 g/L-3000 g/L experimental group cell proliferation index was significantly higher than the control group, while the apoptosis rate was significantly lower than the control group; iodine ion concentration 5000 g/L cell proliferation index in the experimental group compared with the control group have no difference, but the apoptosis rate was higher than the control group; the experimental group cell proliferation index of iodine ion concentration of more than 7000 g/L lower than the control group, at the same time The rate of apoptosis was significantly higher than that of the control group.
The expression of 2. sodium iodide co transporter (NIS) mRNART-PCR: sodium iodide co transporter (NIS) mRNA is absent in HUVEC.
3. the expression of sodium iodide co transporter (NIS) protein western: sodium iodide co transporter (NIS) protein is absent in HUVEC.
4. chloride channel (CLC3 mRNA RT-PCR:) compared with the control group, 24h 1000 g/L-5000 g/L experimental group, and 48h 100 g/L CLC3 in the experimental group the expression of mRNA, P0.05, the difference was statistically significant; 7000 g/L experimental group (24h) and 5000 g/L experimental group (48h) CLC3 the expression of mRNA was not statistically significant, P0.05; 9000 g/L experimental group at 24h and 7000 g/L-9000 g/L in the experimental group 48h CLC3 expression of mRNA and P0.05, the difference was statistically significant.
5. chloride channel protein western blot: (CLCN3) compared with the control group, 1000 ~ 5000 g/L experimental group (24h) and 1000 g/L experimental group (48h) enhanced the expression of CLCN3 protein in P0.05 cells, the difference was statistically significant; and 7000 g/L experimental group (24h) and 5000 g/L (experimental group 48h) the expression of CLCN3 protein and the control group have no difference, no statistical significance, P0.05; 9000 g/L experimental group (24h) and 7000 g/L-9000 g/L (48h) CLCN3 protein expression decreased, P0.05, the difference was statistically significant.
[Conclusion]
1. iodine ions can promote and inhibit the proliferation of umbilical vein endothelial cells in two aspects. The effect is closely related to the time and concentration of action. That is, there is a time dose effect relationship between the appropriate concentration of iodide and the proliferation activity of umbilical vein endothelial cells.
The expression of sodium iodide transporter (NIS) was absent in 2. human umbilical vein endothelial cells.
The expression of -3 (CLC3) was positive on 3. human umbilical vein endothelial cells, and iodide could enter CLC3 through umbilical vein endothelial cells, thereby promoting the proliferation and apoptosis of endothelial cells.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363.1

【参考文献】