转化生长因子β1基因修饰兔B型滑膜细胞复合Pluronic-F127体内构建组织工程软骨

背景:兔B型滑膜细胞在转化生长因子的作用下,具有向软骨细胞分化的潜力,形成的细胞在体外可保持软骨细胞的表型,但转染后的细胞能否与支架材料一起形成软骨组织尚没有深入的研究.目的:以脂质体法转染兔B型滑膜细胞,将转染后细胞复合Pluronic-F127在裸鼠体内构建组织工程软骨,观察转染后细胞体内向软骨组织方向分化的可行性.设计、时间及地点:细胞学试验,随机分组动物实验,于2007-04/2008-05在中山大学附属第二医院医学研究中心完成.材料:3月龄健康新西兰大白兔,雌雄不限,4周龄体质量为20 g左右的BALB/c裸鼠共12只,雌雄不限.方法:取新西兰大白兔膝关节腔内面的滑膜组织,以酶消化法进行分离培养、纯化、传代,以脂质体法进行转染,以G418筛选阳克隆,同时检测转化生长因子β1、Ⅱ型胶原的表达;4℃将Pluronic-F127溶解,配成质量分数为30%液体,然后与转染后稳定表达的细胞进行混合,同时取软骨细胞混合Pluronic-F127、空载体转染细胞混合Pluronic-F127作为对照组,各组细胞浓度均为5×1010L-1的复合物,迅速以0.2mL注射器行裸鼠背部皮下注射,分别在术后4,6,8周处死实验裸鼠,取出裸鼠背部组织进行观察.主要观察指标:细胞生长曲线、转染后细胞表型变化观察、裸鼠皮下组织块组织学观察.结果:生长曲线显示B型滑膜细胞在脂质体法转染后生长活性降低,至转染后6,7d,细胞基本恢复正常的增殖能力,转染后第4天,滑膜细胞转化生长因子β1表达阳性,转染后第7天,滑膜细胞胞浆内抗Ⅱ型胶原染色为阳性,PluronicF-127与B型滑膜细胞混合后在裸鼠皮下4周形成不成熟的软骨样组织,8周形成成熟的软骨样组织,抗Ⅱ型胶原染色为阳性.结论:转染转化生长因子β1基因的B型滑膜细胞在体外可以表达软骨细胞的表型,形成软骨样细胞,而在裸鼠体内复合Pluronic F-127可保持软骨细胞表型,形成软骨样组织.

Type B synoviocytes induced by transforming growth factor beta 1 combined with Pluronic-F127 to construct tissue engineering cartilage in vivo

BACKGROUND:The type B synoviocytes induced by transforming growth factor β1(TGF-β1) have the potential to differentiate into chondrocyte,which can remain the phenotype in vitro. However,whether the transfected cell combined with scaffold can form cartilage tissues need further research. OBJECTIVE:Rabbit type B synoviocytes was transfected by liposome method in vitro,combined the cells with Pluronic-F127,and then implanted into nude mouse to construct tissue engineering cartilage. Additionally,to explore the feasibility of synoviocytes differentiate into chondrocytes.DESIGN,TIME AND SETTING:The randomized animal experiment of cytology observation. The experiment was performed at the Medical Research Center,the Second Affiliated Hospital of Sun Yat-sen University between April 2007 and May 2008.MATERIALS:Healthy New Zealand white rabbits,aged 3 months,and 12 BALB/c nude mice,aged 4 weeks,weighted 20 g.METHODS:The synovial membrane tissues were taken out from the rabbit knee,isolated by enzyme digestion,and then transfected. The positive cloning was screened by G418,and the expression of TGF-β1 and collagen Ⅱ was detected. The Pluronic-F127 was dissolved at 4℃ and prepared fluid with concentration of 30%. The fluid was mixed with cells. At the same time,there were 2 groups as the control group:chondrocytes with Pluronic-F127,and synoviocytes transfeced by pcDNA3.1(+)and Pluronic-F127. The cell density was 5x1010/L. Each compound (0.2 mL) was injected into the subcutaneous of the nude mouse back. Rats were sacrificed at weeks 4,6 and 8 to harvest samples.MAIN OUTCOME MEASURES:Cell growth curve;phenotype change of the cells after transfection;histological observation of the tissue in the subcutaneous of the nude mouse back. RESULTS:Cell growth curve demonstrated that the living activity of the transfected cells was temporary decreased,which returned into normal level at days 6,7 after transfection. At day 4 after transfection,TGF β1 were positive expressed,and at day 7,the collagen Ⅱ staining were positive. The compounds in the subcutaneous of the nude mouse back formed immature chondroid tissues at week 4,which turned to mature chondroid tissue at week 8,and the collagen Ⅱ staining were positive.CONCLUSION:The transfected synoviocytes can express the phenotype of chondrocytes in vitro,and form chondrocyte-likecells. The transfected synoviocytes with Pluronic-F127 can form chondroid tissues in nude mice.