反转录病毒载体介导转化生长因子β_1基因体外转染兔膝关节软骨细胞的表达

Expression of articular chondrocytes in rabbits transfected by retroviral vector-mediated transforming growth factor beta 1 gene in vitro

背景:将功能基因片段整合到基因载体内,再将基因载体转染入靶细胞中或转染入关节腔内,通过转基因的靶细胞持续大量分泌功能基因产物,在一个较长的时期内保持局部治疗浓度,可修复关节软骨损伤。目的:以重组反转录病毒载体介导转化生长因子β1体外转染兔膝关节软骨细胞,观察其表达情况及其对软骨细胞生物学性状的影响。方法:采用胰酶消化法分离培养兔软骨细胞。载体经PLNCX2HindⅢ/NotⅠ双酶切、去磷酸化后,与载体pDsRed2双酶切得到大的部分多克隆位点和RFP基因连接,构建PLNCX2-RFP。转化生长因子β1基因从PGEMT-TGF中扩增,经双酶切后与PLNCX2-RFP连接,构建PLNCX2-TGFβ1-RFP。包装反转录病毒载体,并检测病毒上清滴度。将培养的兔膝关节软骨细胞分组转染:对照组(不做任何转染)、转染PLNCX2组、转染PLNCX2-TGFβ1-RFP组,持续筛选2周,观察细胞变化。收集稳定转染的细胞上清液,以NO检测试剂盒检测基因转染对软骨细胞的影响,以ELISA法检测细胞培养上清液中人转化生长因子β1表达。结果与结论:重组基因PLNCX2-TGFβ1-RFP经酶切鉴定测序TGFβ1、RFP、序列均正确,表明构建成功预期的真核表达载体PLNCX2-TGFβ1-RFP。转染到包装细胞并筛选培养后,病毒滴度为1×106CFU。稳定转染软骨细胞后可观察到红色荧光,证明转染成功。持续筛选2周,散在贴壁细胞形成阳性克隆,逐渐弥漫融合,并有细胞簇出现,双核多见,细胞增生活跃。转染PLNCX2-TGFβ1-RFP组NO浓度高于对照组、转染PLNCX2组(P<0.05),对照组、转染PLNCX2组间差异无显著性意义。对照组与转染PLNCX2组均无转化生长因子β1表达,转染PLNCX2-TGFβ1-RFP组转化生长因子β1质量浓度为(28.08±3.73)ng/L。提示反转录病毒载体PLNCX2介导的人转化生长因子β1能有效转染到兔膝关节软骨细胞并获得稳定表达,同时转染后的软骨细胞增生活跃。

BACKGROUND：The functional gene fragments integrate into gene vector,which is then transfected into target cells or joint cavity,through the transgenic target cells continue to secrete a large number of functional gene product,local therapeutic concentrations could be maintained within a long period of time,thus repairing articular cartilage injury. OBJECTIVE：To transfect rabbit articular chondrocytes using recombinant retroviral vector-mediated transforming growth factor-β1 （TGFβ1） in vitro,and to observe its expression and its effect on biological characters of chondrocytes. METHODS：Rabbit chondrocytes were isolated by use of trypsin digestion method. Vector was PLNCX2 Hind Ⅲ/Not Ⅰ doubly digested and dephosphorylated,connected with some multiple cloning sites and RFP gene following pDsRed2 double digestion,to build PLNCX2-RFP. TGFβ1 gene was amplified from the PGEMT-TGF and connected with PLNCX2-RFP following double digestion,to build PLNCX2-TGFβ1-RFP. Subsequent to packaging retroviral vector,viral supernatant titer was detected. The cultured and transfected chondrocytes in rabbit knee joint were divided into 3 groups：control group （without any transfection）,transfected PLNCX2 group and transfected PLNCX2-TGFβ1-RFP group,continued screening 2 weeks to observe the cellular changes. Cell supernatant transfected stably were collected for detecting the effect of gene transfection on the chondrocytes with NO detection kit,ELISA assay was applied to determine human TGFβ1 expression in cell culture supernatant. RESULTS AND CONCLUSION：The recombinant gene PLNCX2-TGFβ1-RFP was identified correct sequence by the enzyme digestion sequencing TGFβ1 and RFP,which showed that the eukaryotic expression vector PLNCX2-TGFβ1-RFP had been successfully built as expectation. They were then transfected into packaging cells and cultured,the virus titer was defined as 1×106 CFU. Following stable transfection of cartilage cells,red fluorescence can be observed,proving successful transfection. After continuous screening 2 weeks,the scattered adherent cells formed positive clones,and gradually diffusely integrated,cell clusters appeared with common dual cores,the cells proliferated actively. NO concentration in the transfected PLNCX2-TGFβ1-RFP group was higher than that of transfected PLNCX2 group （P 0.05）,no difference was significant between control group and transfected PLNCX2 group. The control group and the group transfected PLNCX2 showed no TGFβ1 expression,while TGFβ1 concentration was （28.08±3.73） ng/L in the transfected PLNCX2-TGFβ1-RFP group. PLNCX2 retroviral vector-mediated human TGFβ1 can be effectively transfected into rabbit knee joint cartilage cells and obtain stable expression,while the transfected cartilage cells proliferate actively.