CAJ | 학술논문

目的观察RNA干扰(RNAi)沉默RhoC基因表达对人脐静脉内皮细胞(HUVEC)生物学行为的影响.方法构建RhoC基因特异性短发夹状RNA(shRNA)真核表达载体,以脂质体介导转染HUVEC,采用逆转录-聚合酶链反应(RT-PCR)和Western blot法检测RNAi后细胞中RhoCmRNA及蛋白表达水平变化；划痕试验检测细胞迁移能力,噻唑蓝(MTT)比色法测定转染前后细胞增殖及黏附能力的变化.胶原基质胶三维培养检测内皮细胞体外血管生成能力.结果转染RhoCshRNA的实验组与空载体对照组比较,RhoC mRNA的表达分别为(10.93±4.72)％和(56.77±13.79)％,蛋白质的表达分别为(19.95±7.31)％和(62.06±22.75)％,细胞愈合百分比分别为(45.08±12.93)％和(73.49±10.83)％,增殖能力[吸光度(4)值]分别为1.002±0.283和1.629±0.192,血管样结构分别为2.85±1.08和4.46±1.38,而黏附能力(A值)分别为0.795±0.097和0.907 ±0.178.实验组较对照组迁移能力、增殖能力和血管样结构生成能力显著降低(P＜0.05),转染后细胞的体外黏附能力无明显变化(P＞0.05).结论抑制RhoC表达能降低HUVEC体外增殖、迁移及血管生成能力.
목적 관찰RNA간우(RNAi)침묵RhoC기인표체대인제정맥내피세포(HUVEC)생물학행위적영향.방법 구건RhoC기인특이성단발협상RNA(shRNA)진핵표체재체,이지질체개도전염HUVEC,채용역전록-취합매련반응(RT-PCR)화Western blot법검측RNAi후세포중RhoCmRNA급단백표체수평변화；화흔시험검측세포천이능력,새서람(MTT)비색법측정전염전후세포증식급점부능력적변화.효원기질효삼유배양검측내피세포체외혈관생성능력.결과 전염RhoCshRNA적실험조여공재체대조조비교,RhoC mRNA적표체분별위(10.93±4.72)％화(56.77±13.79)％,단백질적표체분별위(19.95±7.31)％화(62.06±22.75)％,세포유합백분비분별위(45.08±12.93)％화(73.49±10.83)％,증식능력[흡광도(4)치]분별위1.002±0.283화1.629±0.192,혈관양결구분별위2.85±1.08화4.46±1.38,이점부능력(A치)분별위0.795±0.097화0.907 ±0.178.실험조교대조조천이능력、증식능력화혈관양결구생성능력현저강저(P＜0.05),전염후세포적체외점부능력무명현변화(P＞0.05).결론 억제RhoC표체능강저HUVEC체외증식、천이급혈관생성능력.
Objective To investigate the effects of RhoC gene silencing by RNA interference (RNAi) on behaviors of human umbilical vein endothelial cells (HUVECs) in vitro.Methods The vector containing shRNA targeting RhoC gene was constructed and transfected into HUVECs by Lipofectamine-2000.The expression of Rhoc mRNA and protein in HUVECs was detected respectively by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Wound-healing assay in vitro and three-dimensional cultures were used to evaluate the migration and angiogenesis capacity of HUVECs.The changes in the adhesion and proliferation of the cells were examined by methyl thiazol tetrazolium (MTT)assay.Results In RhoCshRNA-transfected group and control group,the expression of RhoC mRNA and protein was ( 10.93 ± 4.72 ) ％ and ( 19.95 ± 7.31 ) ％,and ( 56.77 ± 13.79 ) ％ and ( 62.06 ± 22.75 ) ％respectively,and the per-cent wound closure was (45.08 ± 12.93 )％ and (73.49 ± 10.83 )％ respectively.The absorbance (A) value of proliferation was 1.002 ± 0.283 and 1.629 ± 0.192.The number of cordlike was 2.85 ± 1.08 and 4.46 ± 1.38,and the adhesion vasue was 0.795 ± 0.097 and 0.907 ±0.178.The expression of RhoC was suppressed by transfecting RhoCshRNA into HUVEs,meanwhile,the migration,proliferation and angiogenesis capacity of HUVECs were decreased. No significant difference was found in the adhesion of the cells between RhoCshRNA-transfected group and control group.Conclusion RNAi-induced RhoC gene silencing may decrease the capacity of HUVE migration,proliferation and angiogenesis in vitro.