CAJ | 학술논문

目的观察针对结缔组织生长因子(CTGF)所构建的短发卡RNA(shRNA)对转化生长因子β1(TGF-β1)诱导大鼠肌源性干细胞(MDSCs)表达CTGF的影响.方法针对CTGF mRNA上的序列,体外合成表达shRNA的DNA质粒载体pGenesil-3-shRNA并行测序鉴定;从新生SD大鼠骨骼肌中分离培养MDSCs并鉴定.实验分成对照组(MDSCs+TGF-β1培养),实验组(MDSCs+ TGF-β1+pGenesil-3-shRNA培养).观察shRNA质粒转染结果.利用Real-Time PCR及Western Blot检测CTGF mRNA和CTGF蛋白表达.结果正确构建了靶向CTG,F基因的质粒载体pCenesil-3-shCTGF;大鼠MDSCs 48 h、72h和96h时实验组的CTGF mRNA及蛋白质水平均低于对照组(P＜0.05).结论针对CTGF构建的pGenesil-3-shCTGF能够转染MDSCs,并在48h、72h和96h时能明显降低TGF-β1刺激MDSCs所产生CTGF的表达.
목적 관찰침대결체조직생장인자(CTGF)소구건적단발잡RNA(shRNA)대전화생장인자β1(TGF-β1)유도대서기원성간세포(MDSCs)표체CTGF적영향.방법 침대CTGF mRNA상적서렬,체외합성표체shRNA적DNA질립재체pGenesil-3-shRNA병행측서감정;종신생SD대서골격기중분리배양MDSCs병감정.실험분성대조조(MDSCs+TGF-β1배양),실험조(MDSCs+ TGF-β1+pGenesil-3-shRNA배양).관찰shRNA질립전염결과.이용Real-Time PCR급Western Blot검측CTGF mRNA화CTGF단백표체.결과 정학구건료파향CTG,F기인적질립재체pCenesil-3-shCTGF;대서MDSCs 48 h、72h화96h시실험조적CTGF mRNA급단백질수평균저우대조조(P＜0.05).결론 침대CTGF구건적pGenesil-3-shCTGF능구전염MDSCs,병재48h、72h화96h시능명현강저TGF-β1자격MDSCs소산생CTGF적표체.
Objective To observe the influence of the short hairpin RNA (shRNA) on the expression of connective tissue growth factor (CTGF) by transforming growth factor β1 (TGF-β1) induced rat muscle-derived stem cells (MDSCs).Methods CTGF-targeted mRNA sequences were designed to synthesize shRNA expressing DNA plasmid pGenesil-3-shRNA in vitro.Then the sequences were analyzed.Rat MDSCs were obtained from newborn rat skeletal muscles with the two-step method of collagenase and trypsin digestion and dissociation.The cells were identified with cellular immunochemical staining.The MDSCs were cultured with TGF-β1 in the control group,and were transfected with pGenesil-3-shRNA plasmid and then cultured with TGF-β1in the experimental group.The levels of CTGF mRNA and protein were measured by Real-Tune PCR and Western Blot,respectively.Results The plasmid vector with target CTGF gene was designed correctly.The CTGF mRNA and protein expression in the experimental group were significantly lower than the control group at 48,72and 96 hours afier transfection and culture ( P ＜ 0.05 ).Conclusion CTGF-targeted pG,enesil-3-shRNA plasmid can transfect MDSCs and reduce TGF-β1-induced expression of CTGF by MDSCs.