中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2013年
4期
722-724
,共3页
邓钢%郭振涛%田道锋%刘宝辉%朱晓楠%冀保卫%易伟%吴立权%陈谦学
鄧鋼%郭振濤%田道鋒%劉寶輝%硃曉楠%冀保衛%易偉%吳立權%陳謙學
산강%곽진도%전도봉%류보휘%주효남%기보위%역위%오립권%진겸학
胶质瘤%多亮氨酸重复区免疫球蛋白样蛋白1%信号转导和转录激活因子3%脱噬作用
膠質瘤%多亮氨痠重複區免疫毬蛋白樣蛋白1%信號轉導和轉錄激活因子3%脫噬作用
효질류%다량안산중복구면역구단백양단백1%신호전도화전록격활인자3%탈서작용
Glioma%Leucine-rich repeats and immunoglobulin-like domains 1%Signal transducers and activators of transcription 3%Apoptosis
目的 探讨信号转导和转录激活因子3(STAT3)信号通路在多亮氨酸重复区免疫球蛋白样蛋白1(LRIG1)诱导人脑胶质瘤细胞株U251细胞凋亡中的作用及机制.方法 传代培养U251细胞,随机分为对照组、空载体组及实验组,应用脂质体介导的基因转染技术分别将磷酸盐缓冲液(PBS)、PEGFP-N1质粒体、PEGFP-LRIG1质粒体转染入各组细胞,应用细胞计数试剂盒(CCK-8)法检测细胞体外生长活性,膜联蛋白V-异硫氰酸荧光素(Annexin V-FITC)/碘化丙锭(PI)法经流式检测各组细胞凋亡率,逆转录-聚合酶链反应(RT-PCR)法测定各组细胞中STAT3的mRNA表达,Westernblot法测定各组细胞中STAT3、磷酸化STAT3(p-STAT3)、B淋巴细胞/白血病-2(bcl-2)、bax的蛋白表达.结果 成功转染LRIG1,实验组细胞中LRIG1基因表达明显上调(P＜0.05)；实验组细胞增殖显著受抑,48 h抑制率为(45.12±0.68)％,72 h抑制率为(52.24±1.77)％,总凋亡率由(2.54±0.43)％增高至(22.51±2.12)％ (P ＜0.05)；RT-PCR检测显示实验组细胞中STAT3 mRNA表达明显降低；Western blot检测显示实验组细胞中STAT3蛋白表达及磷酸化水平明显降低,bcl-2表达明显降低,bax表达明显升高.结论 LRIG1可促进U251细胞凋亡,其机制可能是通过下调STAT3信号通路,进而抑制STAT3激活的抗凋亡途径.
目的 探討信號轉導和轉錄激活因子3(STAT3)信號通路在多亮氨痠重複區免疫毬蛋白樣蛋白1(LRIG1)誘導人腦膠質瘤細胞株U251細胞凋亡中的作用及機製.方法 傳代培養U251細胞,隨機分為對照組、空載體組及實驗組,應用脂質體介導的基因轉染技術分彆將燐痠鹽緩遲液(PBS)、PEGFP-N1質粒體、PEGFP-LRIG1質粒體轉染入各組細胞,應用細胞計數試劑盒(CCK-8)法檢測細胞體外生長活性,膜聯蛋白V-異硫氰痠熒光素(Annexin V-FITC)/碘化丙錠(PI)法經流式檢測各組細胞凋亡率,逆轉錄-聚閤酶鏈反應(RT-PCR)法測定各組細胞中STAT3的mRNA錶達,Westernblot法測定各組細胞中STAT3、燐痠化STAT3(p-STAT3)、B淋巴細胞/白血病-2(bcl-2)、bax的蛋白錶達.結果 成功轉染LRIG1,實驗組細胞中LRIG1基因錶達明顯上調(P＜0.05)；實驗組細胞增殖顯著受抑,48 h抑製率為(45.12±0.68)％,72 h抑製率為(52.24±1.77)％,總凋亡率由(2.54±0.43)％增高至(22.51±2.12)％ (P ＜0.05)；RT-PCR檢測顯示實驗組細胞中STAT3 mRNA錶達明顯降低；Western blot檢測顯示實驗組細胞中STAT3蛋白錶達及燐痠化水平明顯降低,bcl-2錶達明顯降低,bax錶達明顯升高.結論 LRIG1可促進U251細胞凋亡,其機製可能是通過下調STAT3信號通路,進而抑製STAT3激活的抗凋亡途徑.
목적 탐토신호전도화전록격활인자3(STAT3)신호통로재다량안산중복구면역구단백양단백1(LRIG1)유도인뇌효질류세포주U251세포조망중적작용급궤제.방법 전대배양U251세포,수궤분위대조조、공재체조급실험조,응용지질체개도적기인전염기술분별장린산염완충액(PBS)、PEGFP-N1질립체、PEGFP-LRIG1질립체전염입각조세포,응용세포계수시제합(CCK-8)법검측세포체외생장활성,막련단백V-이류청산형광소(Annexin V-FITC)/전화병정(PI)법경류식검측각조세포조망솔,역전록-취합매련반응(RT-PCR)법측정각조세포중STAT3적mRNA표체,Westernblot법측정각조세포중STAT3、린산화STAT3(p-STAT3)、B림파세포/백혈병-2(bcl-2)、bax적단백표체.결과 성공전염LRIG1,실험조세포중LRIG1기인표체명현상조(P＜0.05)；실험조세포증식현저수억,48 h억제솔위(45.12±0.68)％,72 h억제솔위(52.24±1.77)％,총조망솔유(2.54±0.43)％증고지(22.51±2.12)％ (P ＜0.05)；RT-PCR검측현시실험조세포중STAT3 mRNA표체명현강저；Western blot검측현시실험조세포중STAT3단백표체급린산화수평명현강저,bcl-2표체명현강저,bax표체명현승고.결론 LRIG1가촉진U251세포조망,기궤제가능시통과하조STAT3신호통로,진이억제STAT3격활적항조망도경.
Objective To investigate the effects of signal transducers and activators of transcription 3 (STAT3) signaling pathway on leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1)induced apoptosis of U251 cells and the mechansim.Methods U251 cells were cultured and randomly divided into three groups:control group,N1-U251 and LRIG1-U251,which were respectively transfected with PBS,PEGFP-N1 plasmid and PEGFP-LRIG1 plasmid by liposome transfection.Cell counting kit-8 (CCK-8) assay was used to assess the proliferation of U251 cells,and the apoptosis rate by flow cytometry Annexin V-FITC/PI.The mRNA expression of STAT3 was detected by using reverse transcription-polymerase chain reaction (RT-PCR),and the protein expression of STAT3,p-STAT3,B lymphocytes/leukemia-2 (bcl-2) and bax by using Westem blotting.Results LRIG1 gene was transfected into U251 cells,and the protein expression level of LRIG1 was significantly upregulated (P ＜ 0.05).After transfection with LRIG1,proliferation of U251 cells was obviously inhibited with the inhibition rate at 48 and 72 h being (45.12 ±0.68)％ and (52.24 ± 1.77)％ respectively,and the total apoptosis rate was increased from (2.54 ± 0.43) ％ to (22.51 ± 2.12) ％ (P ＜ 0.05) ; The mRNA expression level of STAT3 was significantly downregulated.The protein expression level of STAT3,phosphorylated STAT3 and Bcl-2 was significantly downregulated,and the protein expression level of Bax was significantly upregulated.Conclusion LRIG1 can downregulate the STAT3 signaling pathway,resulting in block of the antiapoptotic way,to promote apoptosis of U251 cells.
