中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2012年
5期
841-843,封3
,共4页
王川%洪天姿%张捷%郭雯珲%傅芳萌%卢辉山
王川%洪天姿%張捷%郭雯琿%傅芳萌%盧輝山
왕천%홍천자%장첩%곽문혼%부방맹%로휘산
乳腺肿瘤%基因表达%细胞增殖%血管内皮生长因子
乳腺腫瘤%基因錶達%細胞增殖%血管內皮生長因子
유선종류%기인표체%세포증식%혈관내피생장인자
Breast carcinoma%Gene expression%. Cell proliferation%Vascular endothelial growth factor
目的 观察分化和DNA结合抑制因子1(Id1)基因对人乳腺癌细胞增殖活性以及分泌血管内皮生长因子(VEGF)能力的影响.方法 将正义、反义Id1基因真核表达载体以及空载体分别转染乳腺癌MCF-7细胞株,经潮霉素筛选获得稳定细胞株；逆转录-聚合酶链反应(RT-PCR)和免疫细胞化学染色检测Id1 mRNA及Id1蛋白、VEGF蛋白表达；细胞计数、噻唑蓝(MTT)比色法及流式细胞仪检测细胞增殖活性及细胞周期；酶联免疫吸附试验( ELISA)检测细胞上清液中VEGF浓度.结果 与空载体转染细胞比较,转染正义Id1载体的MCF-7细胞中Id1 mRNA和Id1蛋白、VEGF蛋白表达水平上调,细胞生长加速(P＜0.05)；而反义Id1载体转染组细胞Id1、VEGF表达下降,细胞增殖速度降低(P＜0.05).流式细胞检测结果显示,正义Id1转染组增殖期细胞(S+G2+M)比例为(48.45 ±2.97)％,空载体组为(32.40±0.49)％,反义Id1组为(23.08±1.56)％,各组间差异有统计学意义(P＜0.01)；3组细胞凋亡指数分别为(3.26±1.28)％、(7.42±1.04)％和(11.83 ±1.59)％,组间差异有统计学意义(P＜0.01)；正义Id1转染组细胞上清液中VEGF浓度较空载体组显著增高(P＜0.01),反义Id1组VEGF分泌量则较空载体组显著降低(P＜0.05).结论 Id1基因与乳腺癌MCF-7细胞增殖活性、细胞周期调控及VEGF分泌能力相关.
目的 觀察分化和DNA結閤抑製因子1(Id1)基因對人乳腺癌細胞增殖活性以及分泌血管內皮生長因子(VEGF)能力的影響.方法 將正義、反義Id1基因真覈錶達載體以及空載體分彆轉染乳腺癌MCF-7細胞株,經潮黴素篩選穫得穩定細胞株；逆轉錄-聚閤酶鏈反應(RT-PCR)和免疫細胞化學染色檢測Id1 mRNA及Id1蛋白、VEGF蛋白錶達；細胞計數、噻唑藍(MTT)比色法及流式細胞儀檢測細胞增殖活性及細胞週期；酶聯免疫吸附試驗( ELISA)檢測細胞上清液中VEGF濃度.結果 與空載體轉染細胞比較,轉染正義Id1載體的MCF-7細胞中Id1 mRNA和Id1蛋白、VEGF蛋白錶達水平上調,細胞生長加速(P＜0.05)；而反義Id1載體轉染組細胞Id1、VEGF錶達下降,細胞增殖速度降低(P＜0.05).流式細胞檢測結果顯示,正義Id1轉染組增殖期細胞(S+G2+M)比例為(48.45 ±2.97)％,空載體組為(32.40±0.49)％,反義Id1組為(23.08±1.56)％,各組間差異有統計學意義(P＜0.01)；3組細胞凋亡指數分彆為(3.26±1.28)％、(7.42±1.04)％和(11.83 ±1.59)％,組間差異有統計學意義(P＜0.01)；正義Id1轉染組細胞上清液中VEGF濃度較空載體組顯著增高(P＜0.01),反義Id1組VEGF分泌量則較空載體組顯著降低(P＜0.05).結論 Id1基因與乳腺癌MCF-7細胞增殖活性、細胞週期調控及VEGF分泌能力相關.
목적 관찰분화화DNA결합억제인자1(Id1)기인대인유선암세포증식활성이급분비혈관내피생장인자(VEGF)능력적영향.방법 장정의、반의Id1기인진핵표체재체이급공재체분별전염유선암MCF-7세포주,경조매소사선획득은정세포주；역전록-취합매련반응(RT-PCR)화면역세포화학염색검측Id1 mRNA급Id1단백、VEGF단백표체；세포계수、새서람(MTT)비색법급류식세포의검측세포증식활성급세포주기；매련면역흡부시험( ELISA)검측세포상청액중VEGF농도.결과 여공재체전염세포비교,전염정의Id1재체적MCF-7세포중Id1 mRNA화Id1단백、VEGF단백표체수평상조,세포생장가속(P＜0.05)；이반의Id1재체전염조세포Id1、VEGF표체하강,세포증식속도강저(P＜0.05).류식세포검측결과현시,정의Id1전염조증식기세포(S+G2+M)비례위(48.45 ±2.97)％,공재체조위(32.40±0.49)％,반의Id1조위(23.08±1.56)％,각조간차이유통계학의의(P＜0.01)；3조세포조망지수분별위(3.26±1.28)％、(7.42±1.04)％화(11.83 ±1.59)％,조간차이유통계학의의(P＜0.01)；정의Id1전염조세포상청액중VEGF농도교공재체조현저증고(P＜0.01),반의Id1조VEGF분비량칙교공재체조현저강저(P＜0.05).결론 Id1기인여유선암MCF-7세포증식활성、세포주기조공급VEGF분비능력상관.
Objective To study the influences of differentiation and DNA binding inhibition factor gene1 ( Id1 ) gene expression on cell proliferation and vascular endothelial growth factor (VEGF) secretion in MCF-7 breast cancer cell line.Methods Positive or negative Id1 eukaryotic expression vector (Id1-pcDNA3.1 +/Hygro,Id1-pcDNA3.1-/Hygro),as well as empty vector (pcDNA3.1/Hygro),was transfected into the MCF-7 cells,respectively.Hygromycin was used to select stabilized transfected cell lines.Reverse transcription-polymerase chain reaction (RT-PCR) and cell immunocytochemical staining were performed to detect the expression of Id1 mRNA,Id1 and VEGF proteins.Cell proliferation activity was analyzed by using cell counting,methyl thiazol tetrazolium (MTT) assay and flow cytometry (FCM).The concentration of VEGF in the supernatant from cell cultures was tested by using enzyme-linked immunosorbent assays ( ELISA ).Results In MCF-7 cells transfected with positive Id1 eukaryotic expression vector ( Id1 -pcDNA3.1 +/Hygro),the Id1 expression was up-regulated.Significantly higher cell proliferating activity was found in Id1-pcDNA3.1 +/Hygro group than in the empty vector transfected group (P ＜0.05).Negative Id1 eukaryotic vector (Id1-pcDNA3.1-/Hygro) transfection inhibited the Id1 expression and proliferating activity ( P ＜ 0.05 ).FCM revealed that the proportion of proliferating cells ( S + G2 + M )was (48.45 ± 2.97 ) ％ in the positive Id1 transfected group,( 32.40 ± 0.49 ) ％ in the empty vector trans-fected group,and (23.08 ± 1.56)％ in the negative Id1 transfected group.There were significant differences among these three groups ( P ＜0.01 ).The apoptotic index in the positive Id1 transfected group,the empty vector transfected group,and the negative Id1 transfected group was (3.26 ± 1.28)％,(7.42 ±1.04 ) ％ and ( 11.83 ± 1.59 ) ％,respectively ( P ＜ 0.01 ).Compared to that in the empty vector trans-fected group,the concentrations of VEGF were increased in the positive Id1 transfected group (P ＜0.01 ),and decreased in the negative Id1 transfected group (P ＜ 0.05 ).Conclusion We demonstrated that the proliferation and VEGF secretion of MCF-7 cells were closely correlated with the Id1 gene expression.
