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目的重组hTGF-β1腺病毒(adeno-hTGF-β1)转染的BMSCs在体内成软骨能力的初步研究,探讨其作为组织工程化软骨的种子细胞的可行性。方法重组adeno-hTGF-β1转染猪BMSCs,酶联免疫吸附定量检测(ELISA法)重组腺病毒转染hTGF-β1蛋白的表达。然后消化收集重组腺病毒转染后的BMSCs,均匀接种于圆盘状PGA支架上,对照组转染adeno-LacZ,然后植入裸鼠背部皮下,在植入后第3周取材,分别行大体观察、组织学、II型胶原免疫组化和蛋白聚糖含量检测对形成组织进行评价。结果 ELISA结果显示adeno-hTGF-β1转染的BMSCs,其hTGF-β1表达量是转染adeno-lacZ 的BMSCs 2.65倍( P<0.05)。植入裸鼠体内后3周取材,实验组HE染色观察可见有软骨形成,但较不均匀,并被纤维组织分割,形成的软骨组织区域可见软骨细胞包埋在软骨陷窝内;对照组可见仅有少量软骨形成,被大量的纤维组织和未降解的PGA支架包裹,实验组和对照组形成软骨占总组织百分比,分别为(41.83±4.64)%和(17.50±2.85)%,P<0.05。Safranin’O染色显示,实验组形成的软骨组织区域都有被染成桔红色蛋白多糖类基质分泌,着色比对照组更深。实验组形成的软骨组织区域有大量棕黄色的II型胶原颗粒,而对照组仅有少量的棕黄色的II型胶原颗粒,实验组形成的软骨组织中的蛋白聚糖含量多于正常猪耳软骨。结论重组hTGF-β1腺病毒转染BMSCs作为种子细胞,在裸鼠体内能促使软骨组织形成,从而为hTGF-β1基因转染的BMSCs在软骨组织工程应用中奠定了基础。
OBJECTIVE: To study the ability of BMSCs transfected with hTGF-β1 (adeno-hTGF-β1) into chondrocytes in vivo to explore their feasibility as seed cells for tissue-engineered cartilage. Methods Recombinant adeno-hTGF-β1 was transfected into porcine BMSCs and the expression of hTGF-β1 protein was detected by enzyme-linked immunosorbent assay (ELISA). Then, the BMSCs transfected with the recombinant adenovirus were digested and collected, and then uniformly inoculated on a disc-shaped PGA scaffold. The control group was transplanted with adeno-LacZ, then transplanted into the back of the nude mice subcutaneously. The rats were harvested at the third week after implantation. Observations, histology, type II collagen immunohistochemistry, and proteoglycan levels were used to assess the formation of tissue. Results The results of ELISA showed that the expression of hTGF-β1 in adeno-hTGF-β1 transfected BMSCs was 2.65 times that of BMSCs transfected with adeno-lacZ (P <0.05). Three weeks after implantation in nude mice, the experimental group showed cartilage formation with HE staining, but it was less uniform and was separated by fibrous tissue. The cartilage tissue formed in the experimental group showed chondrocytes embedded in the cartilage lacunae. In the control group, Only a small amount of cartilage was formed, which was surrounded by a large amount of fibrous tissue and undegraded PGA scaffold. The percentages of cartilage in the experimental group and the control group were (41.83 ± 4.64)% and (17.50 ± 2.85)%, respectively, P <0.05 . Safranin’O staining showed that the cartilage tissue regions formed by the experimental group were all secreted by the tangerine proteoglycan matrix and the coloration was deeper than that of the control group. The experimental group formed a large number of cartilaginous tissue area of collagen type II collagen particles, while the control group only a small amount of brown yellow type II collagen particles, experimental group formed cartilage tissue proteoglycan content than normal pig ear cartilage . Conclusion Recombinant hTGF-β1 adenovirus transfected BMSCs as seed cells can promote the formation of cartilage in nude mice, which laid the foundation for the engineering application of hTGF-β1 transfected BMSCs in cartilage tissue engineering.