沉默ClC-3氯通道基因对低分化鼻咽癌细胞周期的影响
发布时间：2019-02-24 18:20
【摘要】：目的：研究ClC-3 siRNA沉默C1C-3氯通道基因后对低分化鼻咽癌细胞周期的影响。 方法：采用siRNA技术抑制低分化鼻咽癌(CNE-2Z)细胞ClC-3基因的表达,流式细胞术检测siRNA的转染率,全细胞膜片钳技术记录容积激活性氯电流,图像分析软件Q500MC测量并计算细胞容积,免疫荧光检测C1C-3蛋白和cyclin Dl蛋白的细胞内分布,Western blot检测ClC-3蛋白和cyclin D1蛋白的表达,流式细胞术检测细胞周期分布。 结果：(1)ClC-3 siRNA转染CNE-2Z细胞的转染率达到(63.8±3.8)%(n= 3)。Western blot结果显示,与空白对照组相比,用100nmol/L的ClC-3 siRNA转染鼻咽癌细胞后,ClC-3氯通道蛋白的表达减少(60.9±4.0)%(n=3,P0.05),而无序siRNA组、转染试剂对照组的C1C-3蛋白的表达无明显改变(n=3,P0.05)；(2)以47%低渗液作细胞外灌流时,空白对照组细胞容积激活性氯电流平均电流密度为(74.2±6.5) pA/pF (+80 mV), ClC-3 siRNA组细胞容积激活性氯电流平均电流密度为(13.7±4.1) pA/pF,比空白对照组减少(81.5±4.7)%(n=5,P0.05);(3)ClC-3 siRNA组细胞的调节性容积回缩(regulatory volume decrease, RVD)能力显著减弱,低渗刺激25min时的RVD为(10.5±4.8)%(n=16),与对照组的(42.6±2.8)%(n=20)相比, RVD减少75.4%(P0.01)；(4)与空白对照组相比,ClC-3 siRNA组G0/G1期细胞从(56.8±2.8)%增加到(69.9±3.0)%,S期细胞从(32.1±1.7)%减少到(23.5±1.5)%(n=3,P0.05,而无序siRNA阴性对照组、转染试剂对照组的细胞周期分布无明显改变(P0.05),表明沉默C1C-3氯通道基因的表达可使细胞周期停滞在G0/G1期；(5)免疫荧光分析和Western blot结果显示,与空白对照组细胞相比,ClC-3 siRNA组细胞的cyclin Dl蛋白表达减少了(40.3±2.0)%(n=3,P0.05),而转染试剂对照组和无序siRNA组的cyclin D1蛋白表达无明显变化(n=3,P0.05),表明ClC-3 siRNA可抑制cyclin D1蛋白的表达。 结论：低分化鼻咽癌CNE-2Z细胞表达C1C-3氯通道蛋白。ClC-3氯通道蛋白参与细胞周期调控,是调节细胞从G0/G1期进入S期的重要因素之一。C1C-3氯通道可能通过影响调节性容积回缩和调控cyclin D1蛋白的表达而影响细胞周期进程。
[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 卤3.8)% (n = 3). Western blot). Compared with the control group, the transfection efficiency of 100nmol/L ClC-3 siRNA was (63.8 卤3.8)%. The expression of ClC-3 chloride channel protein decreased (60.9 卤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 卤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 卤4.1) pA/pF, lower than that in control group (81.5 卤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 卤4.8)% (n ~ (16), compared with that of control group (42.6 卤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 卤2.8)% to (69.9 卤3.0)%, S phase cells decreased from (32.1 卤1.7)% to (23.5 卤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 卤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.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.63
本文编号：2429806
[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 卤3.8)% (n = 3). Western blot). Compared with the control group, the transfection efficiency of 100nmol/L ClC-3 siRNA was (63.8 卤3.8)%. The expression of ClC-3 chloride channel protein decreased (60.9 卤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 卤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 卤4.1) pA/pF, lower than that in control group (81.5 卤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 卤4.8)% (n ~ (16), compared with that of control group (42.6 卤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 卤2.8)% to (69.9 卤3.0)%, S phase cells decreased from (32.1 卤1.7)% to (23.5 卤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 卤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.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.63
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