中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2013年
4期
273-276
,共4页
徐岩%沈学飞%宋年华%吴道诩
徐巖%瀋學飛%宋年華%吳道詡
서암%침학비%송년화%오도후
细胞增殖%细胞周期%黏着斑蛋白酪氨酸激酶类%细胞周期蛋白质依赖激酶类%Cyr61蛋白%肾小管上皮细胞
細胞增殖%細胞週期%黏著斑蛋白酪氨痠激酶類%細胞週期蛋白質依賴激酶類%Cyr61蛋白%腎小管上皮細胞
세포증식%세포주기%점착반단백락안산격매류%세포주기단백질의뢰격매류%Cyr61단백%신소관상피세포
Cell proliferation%Cell cycle%Focal adhesion protein-tyrosine kinases%Cyclin-dependent kinases%Cysteine rich protein 61%Human renal tubular epithelial cells
目的 观察富含半胱氨酸蛋白61(Cyr61)蛋白对人肾小管上皮细胞(HK-2)增殖与细胞周期的影响.方法 细胞被分为空白对照、阴性对照和Cyr61转染组.克隆Cyr61cDNA,构建pEGFP-N2-Cyr61重组质粒,利用脂质体分别将重组质粒与pEGFP-N2空质粒瞬时转染HK-2.四唑盐比色实验(MTT)检测细胞增殖；流式细胞分析术检测转染效率和细胞凋亡率；Western印迹法检测Cyr61蛋白、磷酸化黏着斑激酶(p-FAK)、细胞周期蛋白依赖性激酶2 (CDK2)的表达.结果 pEGFP-N2-Cyr61重组质粒可高效转染HK-2缅胞.与空白对照、阴性对照组相比,Cyr61转染组细胞和培养液中的Cyr61蛋白表达量显著提高,细胞增殖活力明显增强,细胞中G1时相的比例显著减少,S时相显著增加,细胞凋亡率下降(均P＜0.01).Cyr61转染组细胞中p-FAK、CDK2表达量亦显著提高(P＜0.01).结论 高表达的Cyr61蛋白可通过FAK途径促进CDK2表达,从而促进HK-2细胞进入S期,促进细胞增殖,抑制细胞凋亡.
目的 觀察富含半胱氨痠蛋白61(Cyr61)蛋白對人腎小管上皮細胞(HK-2)增殖與細胞週期的影響.方法 細胞被分為空白對照、陰性對照和Cyr61轉染組.剋隆Cyr61cDNA,構建pEGFP-N2-Cyr61重組質粒,利用脂質體分彆將重組質粒與pEGFP-N2空質粒瞬時轉染HK-2.四唑鹽比色實驗(MTT)檢測細胞增殖；流式細胞分析術檢測轉染效率和細胞凋亡率；Western印跡法檢測Cyr61蛋白、燐痠化黏著斑激酶(p-FAK)、細胞週期蛋白依賴性激酶2 (CDK2)的錶達.結果 pEGFP-N2-Cyr61重組質粒可高效轉染HK-2緬胞.與空白對照、陰性對照組相比,Cyr61轉染組細胞和培養液中的Cyr61蛋白錶達量顯著提高,細胞增殖活力明顯增彊,細胞中G1時相的比例顯著減少,S時相顯著增加,細胞凋亡率下降(均P＜0.01).Cyr61轉染組細胞中p-FAK、CDK2錶達量亦顯著提高(P＜0.01).結論 高錶達的Cyr61蛋白可通過FAK途徑促進CDK2錶達,從而促進HK-2細胞進入S期,促進細胞增殖,抑製細胞凋亡.
목적 관찰부함반광안산단백61(Cyr61)단백대인신소관상피세포(HK-2)증식여세포주기적영향.방법 세포피분위공백대조、음성대조화Cyr61전염조.극륭Cyr61cDNA,구건pEGFP-N2-Cyr61중조질립,이용지질체분별장중조질립여pEGFP-N2공질립순시전염HK-2.사서염비색실험(MTT)검측세포증식；류식세포분석술검측전염효솔화세포조망솔；Western인적법검측Cyr61단백、린산화점착반격매(p-FAK)、세포주기단백의뢰성격매2 (CDK2)적표체.결과 pEGFP-N2-Cyr61중조질립가고효전염HK-2면포.여공백대조、음성대조조상비,Cyr61전염조세포화배양액중적Cyr61단백표체량현저제고,세포증식활력명현증강,세포중G1시상적비례현저감소,S시상현저증가,세포조망솔하강(균P＜0.01).Cyr61전염조세포중p-FAK、CDK2표체량역현저제고(P＜0.01).결론 고표체적Cyr61단백가통과FAK도경촉진CDK2표체,종이촉진HK-2세포진입S기,촉진세포증식,억제세포조망.
Objective To investigate the effect of cysteine-rich protein 61 (Cyr61) on proliferation and cell cycle in human renal tubular epithelial cells (HK-2).Methods Cyr61 cDNA was cloned into pEGFP-N2,then HK-2 cells were transfected with the recombinant plasmid pEGFP-N2-Cyr61 by Lipofectamine.The cell proliferation was measured by MTT.The expression level of Cyr61,p-FAK and cyclin dependent cyclin-dependent kinase 2 (CDK2) protein were detected by Western blotting.The cell cycle and cell apoptosis were analyzed by flow cytometry.Results The recombinant plasmid pEGFP-N,-Cyr61 could be transfected into HK-2 efficiently.After transfection,the proliferative activity was significantly increased,the proportion of HK-2 cells in G1 phase decreased and in S-phase increased significantly,the level of cell apoptosis decreased markedly (all P ＜ 0.01).The expressions of Cyr61,p-FAK and CDK2 in Cyr61-transfected group were all amplified significantly (all P ＜ 0.01).Conclusions Cyr61 protein over-expressed in HK-2 cells can increase CDK2 expression throngh FAK pathway,resulting in the promotion of HK-2 cells entering into S phase,cell proliferation and the reduction of cell apoptosis.
