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目的研究重组hTGF-β1腺病毒(AdhTGF-β1)转染人脂肪干细胞(ADSCs)对其向软骨分化的作用。方法重组AdhTGF-β1转染人ADSCs,对照组转染AdLacZ,腺病毒的量以200pfu/细胞计算,体外细胞团聚集连续诱导培养21d,酶联免疫吸附定量检测(ELISA)hTGF-β1蛋白的表达,然后分别从大体观察、组织学和II型胶原蛋白免疫组化的检测对形成组织进行评价。结果 hTGF-β1蛋白量在14d时达最高峰,随后逐渐降低。连续细胞团聚集诱导培养21d,细胞团收缩成近似小球形的组织块,外观成乳白色。HE染色可见细胞团外周为由数层扁平状成纤维样细胞组成的纤维软骨膜,下部区域有巢状软骨样细胞组成,有些区域可见软骨样细胞包埋在软骨陷窝内。Safranin’O染色显示,形成的软骨组织区域有被染成桔红色蛋白多糖类基质分泌。而对照组苏木素-伊红染色观察见无软骨样组织形成或有向软骨分化现象。Ⅱ型胶原免疫组化染色检测显示实验组细胞团出现较明显的阳性染色区域,可见棕黄色的颗粒分布于胞浆内。对照组Ⅱ型胶原免疫组化染色检测显示无明显的阳性染色区。结论重组hTGF-β1腺病毒转染人ADSCs诱导人ADSCs向软骨细胞表型分化形成软骨样组织,为hTGF-β1基因转染的人ADSCs在软骨组织工程应用中奠定了基础。
Objective To study the effect of AdhTGF-β1 transfected human adipose-derived stem cells (ADSCs) on the differentiation of cartilage. Methods Recombinant AdhTGF-β1 was transfected into human ADSCs. The control group was transfected into AdLacZ. The amount of adenovirus was 200pfu / cell. The cells were aggregated and cultured continuously for 21 days. The expression of hTGF-β1 protein was detected by enzyme-linked immunosorbent assay (ELISA) , And then the formation of tissue was evaluated from gross observation, histology and type II collagen immunohistochemistry, respectively. Results The amount of hTGF-β1 protein peaked at 14 days and then gradually decreased. Continuous cell mass aggregation induced culture 21d, the cell mass contraction into an approximately spherical tissue, the appearance of milky white. HE staining showed that the periphery of the cell mass was composed of several layers of flat fibroblast-like fibroblasts, and the lower region was composed of nevival cartilage-like cells. In some regions, cartilage-like cells were embedded in the cartilage lacunae. Safranin’O staining revealed that the area of cartilage formed was excreted as a mahogany polysaccharide matrix. The control group hematoxylin-eosin staining showed no cartilage-like tissue formation or directed chondrogenic differentiation. Type Ⅱ collagen immunohistochemical staining showed that the experimental group of cell clusters appear more obvious positive staining area, visible brown particles distributed in the cytoplasm. The control group type Ⅱ collagen immunohistochemical staining showed no obvious positive staining area. Conclusions Recombinant hTGF-β1 adenovirus transfected human ADSCs induces phenotypic differentiation of human ADSCs into chondrocytes to form cartilage-like tissue. The human ADSCs transfected with hTGF-β1 gene have laid the foundation for the engineering application of cartilage.