rBMSC和rLFs做为韧带组织工程种子细胞的特性比较  

Comparison of rat BMSCs and LFs as cell source for ligaments tissue engineering

作　　者：张承昊[1] 金瑛[1] 范青洪[1] 孙鹏鹏[1] 吴术红[1] 刘毅[1] 

机构地区：[1]遵义医学院附属医院骨一科,贵州遵义563099

出　　处：《遵义医学院学报》2015年第1期54-59,共6页Journal of Zunyi Medical University

基　　金：贵州省科学技术基金资助项目(NO:黔科合SY字[2010]2091)

摘　　要：目的比较大鼠骨髓间充质干细胞(r BMSCs)和韧带成纤维细胞(r LFs)做为韧带组织工程种子细胞来源的价值。方法采用密度梯度离心结合贴壁培养法获得r BMSCs,通过组织消化法获取培养r LFs。分别取第3代r BMSCs和r LFs,根据是否添加TGF-β1和b FGF-1生长因子诱导分为诱导组和非诱导组(共4组)。用MTT法检测细胞增殖情况,天狼猩红和固绿检测细胞总胶原蛋白量和总非胶原蛋白量,ELISA法检测细胞内外Ⅰ、Ⅲ型胶原蛋白浓度,实时荧光定量PCR检测细胞韧带特异性基因Tnc、Fn1、Mmp2的mRNA表达水平。结果 r BMSCs原代时的活性和增殖能力高于r LFs,第3代r BMSCs未诱导组的细胞活性明显高于r LFs未诱导组(P<0.05),且r BMSCs诱导组也明显高于r LFs诱导组(P<0.05),2种细胞诱导后的细胞活性均比诱导前提高。细胞总胶原分泌量诱导前后r LFs均高于同条件下r BMSCs。诱导后细胞内Ⅰ、Ⅲ型胶原蛋白浓度r BMSCs增长速度比r LFs快(P<0.05),但其Ⅰ型胶原蛋白增长速度明显高于Ⅲ型胶原蛋白(P<0.05),而r LFs两者成比例增长。诱导前后韧带特异性基因mRNA表达量以r BMSCs增长速度最快(P<0.05),但诱导后的表达量仍未超过诱导后r LFs的表达。结论 r BMSCs具有较高的细胞活性和增殖能力,对外界诱导反应明显高于r LFs,但其韧带特异性基因的表达诱导前后均较r LFs低。Objective To compare cellular activities, proliferation and extracellular matrix synthesis between rat bone mesenehymal stem cells （rBMSCs） and fibroblasts （rLFs） as cell source for ligament tissue engineering. Methods rBMSCs were isolated and separated by explants method, rLFs were isolated and separated by digestion method. Primary cell activities and proliferation were observed by inverted microscope. The third generation cells with and without the application of TGF- β1and bFGF- 1 were divided into the induced and the non -induced groups. After 12 -day culture, cells activities and proliferation were observed by MTr assay. Intracellular and extracellular collagen type Ⅰand typeⅢ concentration were determined by ELISA. Quantification of collagen and total extracellular matrix proteins were analyzed by selective binding of Sirius Red and Fast Green FCF. The mRNA expressions of Fibronectin, Tenasein - C and MPP - 2 were analyzed by real - time RT - PCR. Results rBMSCs had much higher activities and proliferation ability than rLFs at prime generation. The third generation cell activity in non- induced rBMSCs group and induced rBMSCs group was higher than that in non -induced rLFs group and induced rLFs group, respectively, in which the cell activity in the induced group was higher than that in the non -induced group. The total collagen in the induced and non -induced rLFs group was more than that in the induced and non - induced rBMSCs group. The density of collagen type Ⅰ and type Ⅲin rBMSCs group induced by growth factor was increased faster than that in induced rLFs group, where the density of colla- gen type Ⅰ in rBMSCs group was increased higher than that of collagen type Ⅲ and the density of collagen type Ⅰ and type Ⅲin rLFs group was increased proportionally. Ligament- related gene mRNA expressions in rBM- SCs group were increased faster than that in rLFs group, while the level of gene mRNA expressions in rBMSCs group was lower than that in rLFs group. Conclusion As the cell source

关 键 词：BMSC LF 生长因子 韧带 组织工程 

分 类 号：R686.5[医药卫生—骨科学]