鼠骨髓间充质干细胞同种异体关节内和静脉内的移植研究

Study on Intra-Articular and Intravenous Transplantation of Mouse Bone Marrow Mesenchymal Stem Cells

摘要: 目的：研究鼠骨髓间充质干细胞(Bone marrow mesenchymal stem cells, BMSCs)同种异体膝关节内移植，静脉移植后在膝关节内的存活与分布情况，为BMSCs在软骨损伤治疗中应用提供理论基础。方法：采用全骨髓培养法分离培养大鼠骨髓间充质干细胞，用5-溴-2-脱氧尿苷标记细胞，计算标记阳性率，通过关节腔注射或鼠尾静脉植入大鼠体内，免疫组化观察术后2 w、4 w标记细胞存活、分布，HE染色观察植入后组织的免疫反应。结果：培养48 h后，BMSCs贴壁大部分呈长梭形，个别为多角形，7 d后细胞融合达90%以上。细胞生长曲线显示细胞生长潜伏期较长，接种后第3~6 d进入对数生长期，第7 d后进入平台期，传代后细胞在24 h内贴壁，以均一的长梭形为主，且生长增殖速度比原代快。标记的BMSCs呈绿色，经计数标记率为95%。同种异体BMSCs植入后均表现出较强的迁移能力，进入关节软骨内。随时间延长，关节软骨内的标记细胞逐渐增多，在异体关节软骨中至少可以存活4 w，关节腔注射和鼠尾静脉植入BMSCs均未观察到明显的免疫反应，且两组在植入2 w和4 w后Brdu标记阳性的BMSCs表达差异无统计学意义(P 0.05)。结论：异体BMSCs通过关节内注射和静脉移植对于治疗关节软骨损伤均具有一定的临床应用价值。

Abstract: Purpose: To study the survival and distribution of bone marrow mesenchymal stem cells (BMSCs) in the knee joint after allogeneic intra-articular knee transplantation, and intravenous transplantation, so as to provide theoretical basis for the application of BMSCs in the treatment of cartilage injury. Methods: Rat bone marrow mesenchymal stem cells were isolated and cultured by whole bone marrow culture method. 5-Bromo-2-deoxyuridine was used to label the cells. The positive rate of labeling was calculated. The survival and distribution of labeled cells were observed by immunohistochemistry at 2 and 4 weeks after operation, and the immune response of implanted tissues was observed by HE staining. Results: After 48 hours of culture, most of BMSCs adhered to the wall in a long spindle shape, and a few were polygonal. After 7 days of culture, more than 90% of the cells were fused. The cell growth curve showed that the incubation period of cell growth was longer. The logarithmic growth phase entered 3~6 days after inoculation, and entered the plateau stage after 7 days. After passage, the cells adhered to the wall within 24 hours, and the growth and proliferation rate was faster than that of the primary generation. The labeled BMSCs were green, and the labeling rate was 95%. Allogeneic BMSCs showed strong migration ability and entered into articular cartilage. The number of labeled cells in articular cartilage increased gradually with time, and could survive at least 4 weeks in allogeneic articular cartilage. No obvious immune response was observed in intra-articular injection and tail vein implantation of BMSCs. There was no significant difference in the expression of BrdU positive BMSCs between the two groups at 2 and 4 weeks after implantation (P < 0.05). Conclusion: Allogeneic BMSCs can be used in the treatment of articular cartilage injury by intra-articular injection and intravenous transplantation.