肿瘤研究与临床
腫瘤研究與臨床
종류연구여림상
CANCER RESEARCH AND CLINIC
2015年
5期
305-309
,共5页
李鑫%张娜娜%岳冠军%由江峰%王华
李鑫%張娜娜%嶽冠軍%由江峰%王華
리흠%장나나%악관군%유강봉%왕화
Id2基因%shRNA%尤文肉瘤细胞%稳定转染
Id2基因%shRNA%尤文肉瘤細胞%穩定轉染
Id2기인%shRNA%우문육류세포%은정전염
Inhibitor of differentiation 2 gene%shRNA%Ewing sarcoma cell%Stable transfection
目的 应用短发夹RNA(shRNA)技术,构建分化抑制因子(Id2)基因稳定干扰的人尤文肉瘤细胞系,为进一步研究Id2在尤文肉瘤细胞生长、分化中的作用提供有用模型.方法 体外合成3条Id2序列特异性shRNA,构建pGPU6/GFP/Neo-shRNA-Id2真核表达载体,通过脂质体转染尤文肉瘤RD-ES细胞,采用Western blot方法检测Id2蛋白表达水平,采用细胞计数法检测shRNA转染后对细胞生长的影响,采用流式细胞术检测转染前后细胞周期的变化.结果 Western blot结果显示,稳定转染shRNA-Id2-543及shRNA-Id2-593的RD-ES细胞中Id2表达水平降低,与对照组相比分别下降54％及57％;转染组细胞的生长速度明显减慢,与对照组相比,差异有统计学意义(P＜0.05);细胞周期S期细胞比例分别为(36.60±1.53)％及(44.89±2.46)％,高于对照组的(29.73±2.03)％,差异有统计学意义(P＜0.05),G2期比例无明显变化甚至降低.结论 成功建立Id2基因沉默的人尤文肉瘤RD-ES稳定转染细胞系.Id2表达下调可抑制尤文肉瘤细胞增殖,细胞周期阻滞于S期.Id2可能在尤文肉瘤细胞生长增殖中扮演重要角色.
目的 應用短髮夾RNA(shRNA)技術,構建分化抑製因子(Id2)基因穩定榦擾的人尤文肉瘤細胞繫,為進一步研究Id2在尤文肉瘤細胞生長、分化中的作用提供有用模型.方法 體外閤成3條Id2序列特異性shRNA,構建pGPU6/GFP/Neo-shRNA-Id2真覈錶達載體,通過脂質體轉染尤文肉瘤RD-ES細胞,採用Western blot方法檢測Id2蛋白錶達水平,採用細胞計數法檢測shRNA轉染後對細胞生長的影響,採用流式細胞術檢測轉染前後細胞週期的變化.結果 Western blot結果顯示,穩定轉染shRNA-Id2-543及shRNA-Id2-593的RD-ES細胞中Id2錶達水平降低,與對照組相比分彆下降54％及57％;轉染組細胞的生長速度明顯減慢,與對照組相比,差異有統計學意義(P＜0.05);細胞週期S期細胞比例分彆為(36.60±1.53)％及(44.89±2.46)％,高于對照組的(29.73±2.03)％,差異有統計學意義(P＜0.05),G2期比例無明顯變化甚至降低.結論 成功建立Id2基因沉默的人尤文肉瘤RD-ES穩定轉染細胞繫.Id2錶達下調可抑製尤文肉瘤細胞增殖,細胞週期阻滯于S期.Id2可能在尤文肉瘤細胞生長增殖中扮縯重要角色.
목적 응용단발협RNA(shRNA)기술,구건분화억제인자(Id2)기인은정간우적인우문육류세포계,위진일보연구Id2재우문육류세포생장、분화중적작용제공유용모형.방법 체외합성3조Id2서렬특이성shRNA,구건pGPU6/GFP/Neo-shRNA-Id2진핵표체재체,통과지질체전염우문육류RD-ES세포,채용Western blot방법검측Id2단백표체수평,채용세포계수법검측shRNA전염후대세포생장적영향,채용류식세포술검측전염전후세포주기적변화.결과 Western blot결과현시,은정전염shRNA-Id2-543급shRNA-Id2-593적RD-ES세포중Id2표체수평강저,여대조조상비분별하강54％급57％;전염조세포적생장속도명현감만,여대조조상비,차이유통계학의의(P＜0.05);세포주기S기세포비례분별위(36.60±1.53)％급(44.89±2.46)％,고우대조조적(29.73±2.03)％,차이유통계학의의(P＜0.05),G2기비례무명현변화심지강저.결론 성공건립Id2기인침묵적인우문육류RD-ES은정전염세포계.Id2표체하조가억제우문육류세포증식,세포주기조체우S기.Id2가능재우문육류세포생장증식중분연중요각색.
Objective To construct short hairpin RNA (shRNA) expression plasmids against the inhibitor of differentiation 2 (Id2) gene and establish a suitable cell model for the role of Id2 in proliferation and differentiation of human Ewing sarcoma cell.Methods Three shRNA sequences targeting Id2 gene were designed and inserted into the pGPU6/GFP/Neo (-shRNA-Id2) expression vectors.The recombinant pGPU6/GFP/Neo-shRNA-Id2 plasmids were introduced into Ewing sarcoma RD-ES cells by liposome-mediated transfection.The knock-down efficiency of Id2 in infected RD-ES cells was verified by Western blot assay.The cell growth and cell cycle changes were evaluated by cell counting and flow cytometry between transfected cells and control cells.Results The Id2 expression decreased 54 ％ and 57 ％,respectively,in RD-ES cell line which were transfected with the shRNA-Id2-543 and shRNA-Id2-593 plasmids compared with the control group cells by Western blot analysis.The cell growth assay demonstrated that the cell number in transfected cells was significantly decreased during 6-7 d compared with the control group (P ＜ 0.05).The cells at the S-phase of cell cycle were increased [(36.60±1.53) ％ and (44.89E2.46) ％ vs (29.73±2.03) ％,P ＜ 0.05],and no significant changes at the G2 phase or even reduction in the transfected cells.Conclusions Id2 stable knock-down cell lines are successfully established.The reduced expression of Id2 is related with decreased cell growth and cell cycle arrest in the S-phase.Id2 maybe plays an important role in proliferation of Ewing sarcoma cell.
