Scleraxis和碱性成纤维细胞生长因子-1联合修饰对单层骨髓间充质干细胞向韧带成纤维细胞特异性分化的影响

Effects of combined Scleraxis and basic fibroblast growth factor - 1 gene modification on the specific differentiation of single layer bone marrow mesenchymal stem cells to fibroblasts

目的探讨共培养的骨髓间充质干细胞（BMSCs）和韧带成纤维细胞（LFs）经Scleraxis和人碱性成纤维细胞生长因子-1（bFGF-1）联合修饰后，细胞活性、增殖能力和韧带特异性基因及蛋白表达和合成情况。方法取3个月龄SD大鼠12只，通过密度梯度离心法结合贴壁法分离培养BMSCs，胶原酶消化法获得LFs。取第3代BMSCs和LFs，实验分为6组，分别为未诱导单纯BMSCs组（A组）、未诱导单纯LFs组（B组）、未诱导单层共培养组（C组）和诱导后单纯BMSCs组（D组）、诱导后单纯EFs组（E组）、诱导后单层共培养组（F组）。倒置相差显微镜及噻唑蓝（MTT）法观测各组细胞生长情况；酶联免疫吸附试验（ELISA）法检测细胞内Ⅰ、Ⅲ型胶原蛋白浓度；实时荧光定量聚合酶链反应（FQ-PCR）检测韧带特异性蛋白（Ⅰ型胶原、Ⅲ型胶原、纤维连接蛋白、细胞黏合素C、基质金属蛋白酶-2）基因表达。结果倒置相差显微镜观察，共培养细胞可形成一单细胞层生长，F组最快融合至90％以上。MTY检测结果显示，培养9d时，F组吸光度（A）值最高，D组次之，B组最低，与其余组比较差异均有统计学意义（F=1．281，1．593，P〈0．05）；A、C、E组间比较差异无统计学意义（F=0．482，0．529，P〉0．05）。ELISA检测结果显示细胞内Ⅰ、Ⅲ型胶原蛋白浓度中，F组浓度显著高于其余各组（F=2．282，P〈0．05）；比较Ⅰ型胶原蛋白浓度，B、C组间及D、E组间比较差异无统计学意义（F=0．482，0．529，P〉0．05）。比较Ⅲ型胶原蛋白浓度，C组显著高于B组，E组高于D组，差异均有统计学意义（F=1．830，1．938，P〈0．05）。A～F组Ⅰ、Ⅲ型胶原蛋白浓度比值分别为1．17、1．19、1．10、1．25、1．17、1．18，D组明显高于其余组。FQ．PCR检测结果显示，Ⅰ、Ⅲ型胶原及纤维连接蛋白基因表达量F组最高，细胞黏合素CD组最高，基质金属蛋白酶2E组最高，与其余各组比较差异均有统计学意义（F=1．689，P〈0．05）。结论单层共培养BMSCs和LFs经Scleraxis和bFGF-1诱导后，细胞活性、增殖能力、韧带特异性基因及蛋白均增高，可作为韧带组织工程种子细胞来源之一。

Objective To investigate the co culture of bone marrow mesenchymal stem ceils （BMSCs） and ligament fibroblasts （LFs） after Scleraxis and basic fibroblast growth factor - 1 （ bFGF - 1 ） co - modification, cell activity and proliferation ability and ligament specific of gene and protein expression and synthesis conditions. Methods 12 SD rats were isolated by density gradient centrifugation combined with the method of density gradient centrifugation, and BMSCs was obtained by the method of density gradient centrifugation and the collagenase digestion method was used to obtain LFs. The third generation of BMSCs and LFs. The experiments were divided into 6 groups, respectively, did not induce BMSCs group （group A）, did not induce LFs alone group （group B）, did not induce monolayer co cuhure group （Group C ） and induced simple BMSCs group （ Group D）, induced simple LFs group （ E group）, after induction of monolayer co culture group （Group F）. Inverted phase contrast microscope and methyl thiazol tetrazolium （MTT） assay were observed cell growth; were detected by enzyme linked immunosorbent assay （ELISA） of type I and m collagen concentration; Real-time fluorescent quantitative polymerase chain reaction （FQ- PCR） detection of ligament specific proteins （type I collagen, type m collagen, fibronectin, cell adhesion factor C, the matrix metal egg white enzyme 2 ） gene expression. Results Under phase contrast microscope, the cells can grow a monolayer formation of co culture, F group is the fastest fusion to more than 90%. MTT assay showed that： After culture 9 d, Group F absorbanee value is the highest, D group and group B were the lowest, with the other groups there were statistically significant differences （F = 0. 482, 0. 529, P 〉 0. 05 ）. ELISA detection showed that cells in type Ⅰ and Ⅲ collagen concentration in, group F concentration was significantly higher than that of the other groups （ F = 2. 282, P 〈 0. 05 ）. In group D and E type I collagen concentration were no statistical significance （ F = 0. 482, 0. 529, P 〉 0. 05） and group E type Ⅲ collagen concentration higher than group D （ F = 1. 830, 1. 938, P 〈 0. 05 ）. A - F group of type Ⅰ and Ⅲ collagen concentration ratio were 1.17, 1.19, 1.10, 1.25, 1.17, 1.18, D group was significantly higher than that of other groups. FQ - PCR detection showed that type Ⅰ and collagen and fibroneetin gene expression was highest in group F, cell adhesion prime C, D group was the highest, matrix metalloproteinase 2E group was the highest, with the rest of the group differences were statistically significant （ F = 1. 689, P 〈 0. 05 ）. Conclusion bFGF - 1 and LFs were induced by Scleraxis and BMSCs in monolayer culture, cell viability, proliferation capacity, ligament specific gene and protein were increased, which can be used as one of the sources of seed cells in ligament tissue engineering.