BMP-2、TGF-β_3重组腺病毒载体的构建及其在滇南小耳猪BMSCs中的表达  

CONSTRUCTION AND IDENTIFICATION OF ADENOVIRUS VECTOR EXPRESSING BONE MORPHOGENETIC PROTEIN 2 AND TRANSFORMING GROWTH FACTOR β_3 GENES AND THEIR EXPRESSION IN BONE MARROW MESENCHYMAL STEM CELLS OF DIANNAN SMALL-EAR PIGS

作　　者：王鑫[1] 李彦林[1] 金耀峰 陈建明[3] 王慧建[1] 何川[1] 曹树海[1] 赵沣凯 

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]嘉兴市人民医院骨科 [3]湖南省第二人民医院骨科

出　　处：《中国修复重建外科杂志》2014年第7期896-902,共7页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：教育部博士点基金资助项目(20115317110001);云南省联合专项基金资助项目(2010FB);云南省医学学科带头人培养基金(D-201207)~~

摘　　要：目的构建含BMP-2和TGF-β3基因的重组腺病毒载体,双基因共转染滇南小耳猪BMSCs并检测其表达,为组织工程软骨支架提供改良的种子细胞。方法用PCR方法扩增人BMP-2和TGF-β3目的基因,将2个基因亚克隆至穿梭载体pEC3.1(+)中,构建pEC-GIE3.1-BMP-2和pEC-GIE3.1-TGF-β3重组质粒,通过体外同源重组反应将pEC-GIE3.1-BMP-2、pEC-GIE3.1-TGF-β3重组至腺病毒骨架质粒pGSadeno中,构建重组腺病毒表达质粒pGSadenoBMP-2、pGSadeno-TGF-β3,线性化后转染HEK293细胞进行包装,获得重组腺病毒Ad-BMP-2和Ad-TGF-β3。取成年滇南小耳猪的骨髓,采用离心加贴壁法分离BMSCs,分别用基因Ad-BMP-2(A组)、Ad-TGF-β3(B组)和Ad-BMP-2+AdTGF-β3(C组)转染,以未转染细胞作为对照(D组)。采用免疫荧光、Western blot、PCR检测目的基因和蛋白表达,Ⅱ型胶原免疫组织化学染色观察成软骨分化情况。结果 Ad-BMP-2和Ad-TGF-β3经PCR及测序鉴定正确,带BMP-2、TGF-β3基因的载体在HEK293细胞中包装成功,病毒滴度分别为5.6×108、1.6×108 pfu/mL。双基因共转染滇南小耳猪BMSCs 72 h后,PCR示A组在310 bp处可见条带,B组在114 bp处可见条带,C组在310、114 bp处同时可见条带,D组未见目的条带表达;免疫荧光示A、B组细胞质内有分别有红色、绿色荧光表达,C组同时表达红色及绿色荧光,D组未见荧光表达;Western blot示A组在相对分子质量为18×103处有阳性条带,B组在50×103处有阳性条带,C组在18×103、50×103处同时可见条带,D组未见目的条带表达。Ⅱ型胶原免疫组织化学染色示A、B、C组呈阳性,C组染色强于A、B组,D组染色呈阴性。结论成功构建含BMP-2和TGF-β3基因的重组腺病毒载体。Ad-BMP-2和Ad-TGF-β3双基因联合转染滇南小耳猪BMSCs后目的基因和蛋白均可成功表达,并促进BMSCs成软骨分化,可作为组织工程软骨研究的改良种子细胞。Objective To construct and identify the recombinant adenovirus vector expressing bone morphogenetic protein 2（BMP-2） and transforming growth factor β3（TGF-β3） genes, to observe the expressions of BMP-2 and TGF-β3 after transfected into bone marrow mesenchymal stem cells（BMSCs） of the Diannan small-ear pigs. Methods BMP-2 cDNA and TGF-β3 cDNA were amplified by PCR, and were subcloned into the pEC3.1（＋） plasmid to obtain pEC-GIE 3.1-BMP-2 and pEC-GIE3.1-TGF-β3 plasmid respectively. They were subcloned into pGSadeno vector by homologous recombination reaction and HEK293 cells were transfected after linearization to obtain Ad-BMP-2 and Ad-TGF-β3. The BMSCs were isolated from the bone marrow of Diannan small-ear pig and cultured. The 3rd passage BMSCs were transfered with Ad-BMP-2（group A）, AdTGF-β3（group B）, Ad-BMP-2＋ Ad-TGF-β3（group C）, and untransfected cells served as a control（group D）. The expressions of BMP-2 and TGF-β3 genes and proteins were detected by PCR, immunol uorescence, and Western blot. The chondrogenic differentiation of BMSCs was evaluated by immunohistochemical of collagen type II. Results The Ad-BMP-2 and AdTGF-β3 were constructed successfully and coni rmed by PCR and sequencing. The expression clones of Ad-BMP-2 and AdTGF-β3 were packaged into maturated adenovirus successfully, the titer was 5.6 × 10^8 and 1.6 × 10^8 pfu/mL respectively. The PCR results showed a light band at 310 bp in group A and at 114 bp in group B, and both 310 bp and 114 bp bands in group C, but no band in group D. The image of immunol uorescence showed that there were red l uorescence and green l uorescence expressions in the cytoplasm of BMSCs at 72 hours after transfection in groups A and B, respectively; in group C, both red and green l uorescence expressions were detected, and no red or green l uorescence was detected in group D. The results of Western blot showed that there was a light band at 18 × 10^3 in group A and at 50 × 10^3 in group B; both 18 × 10^3 and 50 ×

关 键 词：组织工程软骨 腺病毒 BMSCS 基因转染 BMP-2 TGF-Β3 猪 

分 类 号：R346[医药卫生—基础医学]