【摘要】：目的：研究ClC-3 siRNA沉默C1C-3氯通道基因后對低分化鼻咽癌細(xì)胞周期的影響。 方法：采用siRNA技術(shù)抑制低分化鼻咽癌(CNE-2Z)細(xì)胞ClC-3基因的表達(dá),流式細(xì)胞術(shù)檢測siRNA的轉(zhuǎn)染率,全細(xì)胞膜片鉗技術(shù)記錄容積激活性氯電流,圖像分析軟件Q500MC測量并計(jì)算細(xì)胞容積,免疫熒光檢測C1C-3蛋白和cyclin Dl蛋白的細(xì)胞內(nèi)分布,Western blot檢測ClC-3蛋白和cyclin D1蛋白的表達(dá),流式細(xì)胞術(shù)檢測細(xì)胞周期分布。 結(jié)果：(1)ClC-3 siRNA轉(zhuǎn)染CNE-2Z細(xì)胞的轉(zhuǎn)染率達(dá)到(63.8±3.8)%(n= 3)。Western blot結(jié)果顯示,與空白對照組相比,用100nmol/L的ClC-3 siRNA轉(zhuǎn)染鼻咽癌細(xì)胞后,ClC-3氯通道蛋白的表達(dá)減少(60.9±4.0)%(n=3,P0.05),而無序siRNA組、轉(zhuǎn)染試劑對照組的C1C-3蛋白的表達(dá)無明顯改變(n=3,P0.05)；(2)以47%低滲液作細(xì)胞外灌流時(shí),空白對照組細(xì)胞容積激活性氯電流平均電流密度為(74.2±6.5) pA/pF (+80 mV), ClC-3 siRNA組細(xì)胞容積激活性氯電流平均電流密度為(13.7±4.1) pA/pF,比空白對照組減少(81.5±4.7)%(n=5,P0.05);(3)ClC-3 siRNA組細(xì)胞的調(diào)節(jié)性容積回縮(regulatory volume decrease, RVD)能力顯著減弱,低滲刺激25min時(shí)的RVD為(10.5±4.8)%(n=16),與對照組的(42.6±2.8)%(n=20)相比, RVD減少75.4%(P0.01)；(4)與空白對照組相比,ClC-3 siRNA組G0/G1期細(xì)胞從(56.8±2.8)%增加到(69.9±3.0)%,S期細(xì)胞從(32.1±1.7)%減少到(23.5±1.5)%(n=3,P0.05,而無序siRNA陰性對照組、轉(zhuǎn)染試劑對照組的細(xì)胞周期分布無明顯改變(P0.05),表明沉默C1C-3氯通道基因的表達(dá)可使細(xì)胞周期停滯在G0/G1期；(5)免疫熒光分析和Western blot結(jié)果顯示,與空白對照組細(xì)胞相比,ClC-3 siRNA組細(xì)胞的cyclin Dl蛋白表達(dá)減少了(40.3±2.0)%(n=3,P0.05),而轉(zhuǎn)染試劑對照組和無序siRNA組的cyclin D1蛋白表達(dá)無明顯變化(n=3,P0.05),表明ClC-3 siRNA可抑制cyclin D1蛋白的表達(dá)。 結(jié)論：低分化鼻咽癌CNE-2Z細(xì)胞表達(dá)C1C-3氯通道蛋白。ClC-3氯通道蛋白參與細(xì)胞周期調(diào)控,是調(diào)節(jié)細(xì)胞從G0/G1期進(jìn)入S期的重要因素之一。C1C-3氯通道可能通過影響調(diào)節(jié)性容積回縮和調(diào)控cyclin D1蛋白的表達(dá)而影響細(xì)胞周期進(jìn)程。
[Abstract]:Aim: to study the effect of ClC-3 siRNA silencing C1C-3 chloride channel gene on the cell cycle of poorly differentiated nasopharyngeal carcinoma (NPC). Methods: siRNA technique was used to inhibit the expression of ClC-3 gene in poorly differentiated nasopharyngeal carcinoma (CNE-2Z) cells, the transfection efficiency of siRNA was detected by flow cytometry, and the volume-activated chloride currents were recorded by whole-cell patch clamp technique. The cell volume was measured by image analysis software Q500MC, the intracellular distribution of C1C-3 protein and cyclin Dl protein was detected by immunofluorescence, the expression of ClC-3 protein and cyclin D1 protein was detected by, Western blot, and the cell cycle distribution was detected by flow cytometry. Results: (1) the transfection efficiency of CNE-2Z cells transfected with ClC-3 siRNA was (63.8 鹵3.8)% (n = 3). Western blot). Compared with the control group, the transfection efficiency of 100nmol/L ClC-3 siRNA was (63.8 鹵3.8)%. The expression of ClC-3 chloride channel protein decreased (60.9 鹵4.0)% (P 0.05), but the expression of C1C-3 protein did not change in the control group (P 0.05). (2) the average current density of volumetric activated chlorine current in the blank control group was (74.2 鹵6.5) pA/pF (80 mV),) when 47% hypotonic solution was used as extracellular perfusion. The average current density of volume-activated chlorine current in ClC-3 siRNA group was (13.7 鹵4.1) pA/pF, lower than that in control group (81.5 鹵4.7)% (P 0.05). (3) in ClC-3 siRNA group, the ability of regulating volume retraction (regulatory volume decrease, RVD) was significantly decreased. The RVD of 25min stimulated by hypotonic stimulation was (10.5 鹵4.8)% (n ~ (16), compared with that of control group (42.6 鹵2.8)% (n ~ (20). RVD decreased by 75.4% (P0.01). (4) compared with the control group, G0/G1 phase cells in ClC-3 siRNA group increased from (56.8 鹵2.8)% to (69.9 鹵3.0)%, S phase cells decreased from (32.1 鹵1.7)% to (23.5 鹵1.5)%. However, the cell cycle distribution in the control group was not significantly changed (P0.05), which indicated that the silencing of C1C-3 chloride channel gene could make the cell cycle arrest in the G0/G1 phase. (5) the results of immunofluorescence analysis and Western blot showed that the expression of cyclin Dl protein in ClC-3 siRNA group was decreased by (40.3 鹵2.0)% (P 0.05) compared with the control group. However, the expression of cyclin D1 protein in the control group and disordered siRNA group was not significantly changed (nong3p0.05), indicating that ClC-3 siRNA could inhibit the expression of cyclin D1 protein. Conclusion: C1C-3 chloride channel protein is expressed in CNE-2Z cells of poorly differentiated nasopharyngeal carcinoma. ClC-3 chloride channel protein is involved in cell cycle regulation. C1C-3 chloride channel may affect cell cycle progression by affecting regulatory volume retraction and regulating the expression of cyclin D1 protein.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R739.63

【參考文獻(xiàn)】

相關(guān)期刊論文 前8條

1 葉東;張海峰;朱林燕;王立偉;陳麗新;;ClC-3 siRNA抑制鼻咽癌細(xì)胞調(diào)節(jié)性容積回縮[J];南方醫(yī)科大學(xué)學(xué)報(bào);2011年02期

2 田晶,沙建慧,周士勝,陳艷明;容積調(diào)控氯通道的研究進(jìn)展[J];生理科學(xué)進(jìn)展;2004年03期

3 王立偉,陳麗新,Tim JACOB;鼻咽癌細(xì)胞CIC-3在細(xì)胞周期中的表達(dá)(英文)[J];生理學(xué)報(bào);2004年02期

4 毛建文,王立偉,孫雪榮,朱林燕,李攀,鐘平,聶思槐,Tim JACOB,陳麗新;遷移的鼻咽癌細(xì)胞容積激活性氯電流(英文)[J];生理學(xué)報(bào);2004年04期

5 何慶豐,王立偉,毛建文,孫雪榮,李攀,鐘平,聶思槐,Tim JACOB,陳麗新;ATP激活鼻咽癌細(xì)胞氯電流并減小細(xì)胞容積(英文)[J];生理學(xué)報(bào);2004年06期

6 Tim JACOB;Involvement of regulatory volume decrease in the migration of nasopharyn-geal carcinoma cells[J];Cell Research;2005年05期

7 陳麗新,王立偉,朱林燕,聶思槐,張謹(jǐn),鐘平,羅海兵,蔡波,李攀,Tim Jacob;Cl~-在鼻咽癌細(xì)胞調(diào)節(jié)性容積回縮中的作用[J];中國病理生理雜志;2002年05期

8 王立偉,陳麗新,朱林燕,毛建文,孫雪榮,聶思槐,張瑾,鐘平,蔡波,李攀;抑制氯通道阻抑鼻咽癌細(xì)胞周期和細(xì)胞增殖[J];中國病理生理雜志;2004年05期

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