本文關(guān)鍵詞：CIC3在碘離子致臍靜脈內(nèi)皮細(xì)胞增殖和凋亡中的作用　出處：《山東大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 氯離子通道-3(CLC3) 碘離子 人臍靜脈內(nèi)皮細(xì)胞 增殖 凋亡

【摘要】：[目的] 碘離子可以致內(nèi)皮細(xì)胞等多種細(xì)胞增殖和凋亡,而經(jīng)典的碘離子通道就是分布在甲狀腺上的鈉-碘同向轉(zhuǎn)運(yùn)體(NIS),NIS主要在甲狀腺及神經(jīng)組織等腺外組織表達(dá),而內(nèi)皮細(xì)胞上是否也有NIS表達(dá)至今未見報(bào)導(dǎo)。但是,內(nèi)皮細(xì)胞上卻有氯離子通道-3(CLC3)的表達(dá),并且CLC3在通常情況下對(duì)碘離子的選擇性大于氯離子。由此,本實(shí)驗(yàn)通過(guò)體外培養(yǎng)臍靜脈內(nèi)皮細(xì)胞,運(yùn)用RT-PCR、Western Blot、流式細(xì)胞術(shù)等方法研究CLC3通道在不同碘離子濃度及時(shí)相促進(jìn)臍靜脈內(nèi)皮細(xì)胞增殖和凋亡中的作用,探討碘離子是否經(jīng)過(guò)CLC3進(jìn)入內(nèi)皮細(xì)胞,豐富碘離子通道的研究。 [方法] 1.細(xì)胞株：人臍靜脈內(nèi)皮(HUVEC)細(xì)胞株,購(gòu)于南京凱基生物有限公司 2.實(shí)驗(yàn)分組：KI 3.流式細(xì)胞術(shù)：用不同濃度的碘離子培養(yǎng)液處理臍靜脈內(nèi)皮細(xì)胞24小時(shí)和48小時(shí)后,經(jīng)離心—PBS沖洗—吹勻—固定—再離心—沖洗—吹打—染色(4℃,20～30分鐘)后,再用400目尼龍網(wǎng)將之過(guò)濾之后上機(jī)檢測(cè)。用ModFit分析軟件分析,細(xì)胞相對(duì)增殖活力用細(xì)胞分裂增殖指數(shù)表示。 4.鈉碘同向轉(zhuǎn)運(yùn)體(NIS) mRNA的RT-PCR:將臍靜脈內(nèi)皮細(xì)胞培養(yǎng)于含不同濃度的碘離子培養(yǎng)液中24小時(shí)和48小時(shí)后,提取細(xì)胞的總RNA,經(jīng)反轉(zhuǎn)錄—擴(kuò)增—電泳,檢測(cè)鈉碘同向轉(zhuǎn)運(yùn)體(NIS) mRNA的表達(dá)。 5.鈉碘同向轉(zhuǎn)運(yùn)體(NIS)蛋白Western Blot:用不同濃度的碘離子培養(yǎng)液培養(yǎng)臍靜脈內(nèi)皮細(xì)胞24小時(shí)和48小時(shí)后,提取總蛋白,經(jīng)電泳—轉(zhuǎn)膜—封閉—孵育一抗—孵育二抗—顯色等過(guò)程,檢測(cè)目的蛋白NIS的表達(dá)。 6.氯離子通道(CLC3) mRNA的RT-PCR:將臍靜脈內(nèi)皮細(xì)胞用含不同濃度的碘離子培養(yǎng)液處理24小時(shí)和48小時(shí)后,提取其總RNA,再經(jīng)反轉(zhuǎn)錄—擴(kuò)增—電泳等步驟,檢測(cè)氯離子通道(CLC3) mRNA的表達(dá)。 7.氯離子通道(CLCN3)蛋白的Western Blot:搜集含不同濃度碘離子培養(yǎng)液處理24小時(shí)和48小時(shí)后的臍靜脈內(nèi)皮細(xì)胞,先用蛋白裂解液提取總蛋白,再經(jīng)電泳—轉(zhuǎn)膜—封閉—孵育—抗—孵育二抗—顯色,檢測(cè)目的蛋白CLCN3的表達(dá)。 [結(jié)果] 1.流式細(xì)胞術(shù)：24小時(shí),碘離子濃度為100μg/L～5000μg/L實(shí)驗(yàn)組細(xì)胞增殖指數(shù)明顯高于對(duì)照組,而細(xì)胞凋亡率明顯低于對(duì)照組；碘離子濃度為7000μg/L的實(shí)驗(yàn)組細(xì)胞增殖指數(shù)與對(duì)照組比較沒(méi)有明顯差別,而細(xì)胞凋亡率明顯高于對(duì)照組；碘離子濃度≥9000μg/L的實(shí)驗(yàn)組細(xì)胞增殖指數(shù)明顯低于對(duì)照組,而細(xì)胞凋亡率高于對(duì)照組。48小時(shí),碘離子濃度為100μg/L-3000μg/L實(shí)驗(yàn)組細(xì)胞增殖指數(shù)明顯高于對(duì)照組,同時(shí)細(xì)胞凋亡率明顯低于對(duì)照組；碘離子濃度為5000μg/L的實(shí)驗(yàn)組細(xì)胞增殖指數(shù)跟對(duì)照組比較沒(méi)有差別,而細(xì)胞凋亡率高于對(duì)照組；碘離子濃度≥7000μg/L的實(shí)驗(yàn)組細(xì)胞增殖指數(shù)低于對(duì)照組,同時(shí)細(xì)胞凋亡率明顯高于對(duì)照組。 2.鈉碘同向轉(zhuǎn)運(yùn)體(NIS) mRNART-PCR:鈉碘同向轉(zhuǎn)運(yùn)體(NIS) mRNA在HUVEC表達(dá)缺失。 3.鈉碘同向轉(zhuǎn)運(yùn)體(NIS)蛋白western:鈉碘同向轉(zhuǎn)運(yùn)體(NIS)蛋白在HUVEC表達(dá)缺失。 4.氯離子通道(CLC3)mRNA RT-PCR:與對(duì)照組相比,24h時(shí)1000μg/L-5000μg/L實(shí)驗(yàn)組,和48h時(shí)100μg/L實(shí)驗(yàn)組CLC3的mRNA表達(dá)上調(diào),P0.05,差異有統(tǒng)計(jì)學(xué)意義；7000μg/L實(shí)驗(yàn)組(24h)和5000μg/L實(shí)驗(yàn)組(48h)CLC3的mRNA表達(dá)差異無(wú)統(tǒng)計(jì)學(xué)意義,P0.05；9000μg/L實(shí)驗(yàn)組在24h時(shí)及7000μg/L-9000μg/L實(shí)驗(yàn)組在48h時(shí)CLC3的mRNA表達(dá)下調(diào),P0.05,差異有統(tǒng)計(jì)學(xué)意義。 5.氯離子通道(CLCN3)蛋白western blot:與對(duì)照組相比,1000～5000μg/L實(shí)驗(yàn)組(24h)及1000μg/L實(shí)驗(yàn)組(48h)在細(xì)胞CLCN3蛋白表達(dá)增強(qiáng),P0.05,差異有統(tǒng)計(jì)學(xué)意義；而7000μg/L實(shí)驗(yàn)組(24h)及5000μg/L實(shí)驗(yàn)組(48h)CLCN3蛋白表達(dá)與對(duì)照組沒(méi)有差別,無(wú)統(tǒng)計(jì)學(xué)意義,P0.05；9000μg/L實(shí)驗(yàn)組(24h)及7000μg/L-9000μg/L(48h)CLCN3蛋白表達(dá)減弱,P0.05,差異有統(tǒng)計(jì)學(xué)意義。 [結(jié)論] 1.碘離子對(duì)臍靜脈內(nèi)皮細(xì)胞增殖活性具有促進(jìn)和抑制兩方面的作用,其作用效果與作用時(shí)間和作用濃度密切相關(guān),即適宜濃度的碘離子對(duì)臍靜脈內(nèi)皮細(xì)胞增殖活性存在時(shí)間-劑量-效應(yīng)關(guān)系。 2.人臍靜脈內(nèi)皮細(xì)胞上鈉碘轉(zhuǎn)運(yùn)體(NIS)表達(dá)缺失。 3.人臍靜脈內(nèi)皮細(xì)胞上氯離子通道-3(CLC3)表達(dá)陽(yáng)性,碘離子可以通過(guò)CLC3進(jìn)入到臍靜脈內(nèi)皮細(xì)胞內(nèi),從而促進(jìn)內(nèi)皮細(xì)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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363.1

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