TGF-β_3基因转染诱导滇南小耳猪BMSCs向软骨分化的初步研究  

CHONDROGENESIS OF BONE MARROW MESENCHYMAL STEM CELLS INDUCED BY TRANSFORMING GROWTH FACTOR β_3 GENE IN DIANNAN SMALL-EAR PIGS

作　　者：王慧建[1] 李彦林[1] 陈建明[1,2] 王鑫[1] 赵沣凯 曹树海[1] 

机构地区：[1]昆明医科大学第一附属医院运动医学科,昆明650032 [2]湖南省永州市中心医院骨科

出　　处：《中国修复重建外科杂志》2014年第2期149-154,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：云南省医学学科带头人培养基金资助项目(D-201207);云南省自然科学基金资助项目(2010FB);高等学校博士学科点专项科研基金联合资助项目(20115317110001)~~

摘　　要：目的观察TGF-β3基因转染滇南小耳猪BMSCs后目的基因的表达和向软骨细胞分化的作用。方法以血清5型重组腺病毒（recombinantadenovirus5，rAd5）构建系统为基因载体，包装为重组腺病毒rAd5．TGF-β3，行双酶切及PCR鉴定。采集2月龄滇南小耳猪（体重12～15kg）骨髓分离培养制备BMSCs，取第2代细胞用于实验。将实验分为3组，实验组和对照组分别用rAd5-TGF-β3人腺病毒阴性空病毒载体转染BMSCs，以未转染的BMSCs为空白对照组。流式细胞仪检测转染效率，免疫荧光染色、Westernblot检测各组TGF-β3蛋白表达情况；培养后各时间点倒置相差显微镜观察细胞形态变化，Westernblot检测各组II型胶原蛋白表达情况。结果rAd5-TGF．瞰重组腺病毒成功构建并转染BMSCs，在细胞内表达并迅速扩增，免疫荧光显微镜下可观察到强绿色荧光。流式细胞仪检测示转染72h时病毒达最佳转染效果（转染效率为84．86％）。免疫荧光染色示，rAd5．TGF-β3转染BMSCs72h，细胞质内有明显TGF-β3蛋白表达；转染后7、21dWesternblot检测示，实验组在相对分子质量为30×10^3处有TGF-β3阳性条带，而对照组和空白对照组均呈阴性。转染后体外培养，倒置相差显微镜下示实验组向软骨细胞分化明显。培养21d时Westernblot检测示，实验组在相对分子质量为130×10^3处有II型胶原阳性条带，对照组和空白对照组均呈阴性。结论重组腺病毒tAd5．TGF-β3可成功转染BMSCs,TGF-β3蛋白可稳定表达，并促使BMSCs向软骨细胞方向分化，为软骨缺损修复的局部基因治疗奠定了实验基础。Objective To observe transforming growth factor β3 （TGF-β3） gene expression and the chondrogenesis of bone marrow mesenchymal stem cells （BMSCs） after TGF-β3 gene is transfected into BMSCs of Diannan small-ear pig. Methods Recombinant adenovirus 5 （rAd5） was extracted as gene vector and packed into recombinant adenovirus rAd5-TGF-β3, double enzyme digestion and PCR identification were performed. BMSCs were isolated and cultured from bone marrow of 2-month-old Diannan small-ear pigs （weighing, 12-15 kg）, and the 2nd generation of BMSCs were harvested for experiments. The experiments were divided into 3 groups. BMSCs were transfected with rAdS-TGF-β3 as experimental group and with empty vector as control group, and non-transfected BMSCs were used as blank control group. The transfection efficiency of exogenous gene was identified by flow cytometry, TGF-β3 protein expression by immunofluorescence and Western blot. The cell morphology of experimental group was observed by inverted phase contrast microscope, and the expression of collagen type II in each group was detected by Western blot. Results The rAd5-TGF-β3 recombinant adenovirus was successfully constructed and transfected into BMSCs. Green fluorescence was observed by immunofluorescence microscope. Flow cytometry test showed the best transfection at 72 hours （transfection efficiency of 84.86%）. Immunofluorescence staining showed that the expression of TGF-β3 protein was obvious at 72 hours; Western blot showed that there was a TGF-β3 positive band with a relative molecular mass of 30 × 10^3, while the control group and blank control group had no positive band. Obvious chondrogenic differentiation was observed in the experimental group after transfection in vitro, while the control group and blank control group had no obvious chondrogenic differentiation. Western blot showed that there was collagen type II positiveband with a relative molecular mass of 130 × 10^3 at 21 days after culture, while the control group and blank control

关 键 词：软骨组织工程TGF—β3 BMSCS 软骨分化 基因转染 滇南小耳猪 

分 类 号：R684[医药卫生—骨科学]