力学刺激对骨髓间充质干细胞向软骨细胞分化的研究

发布时间：2018-03-27 05:22

  本文选题：骨髓间充质干细胞　切入点：软骨细胞　出处：《大连医科大学》2012年硕士论文

【摘要】：目的：在微团培养条件下探讨不同离心力对骨髓间充质干细胞向软骨细胞分化的影响与作用。 方法：取6周龄的SD大鼠，在无菌条件下分离出股骨和胫骨并抽取骨髓，使用密度梯度离心法提取原代BMSCs，并经过反复换液和传代得到纯化的BMSCs，使用流式细胞仪测出CD29、CD90和CD45的含量，鉴定细胞的纯度。取第6代BMSCs采用微团方式进行立体培养，在培养过程中更换以TGF-β1,ITS为主要成分的诱导培养基并根据离心力强度将细胞分为4组：无应力对照组、100g应力组、200g应力组和300g应力组。诱导培养28天后，对细胞团块进行鉴定和比较。鉴定指标主要为大体观察细胞团块的形态、光泽度等；台盼蓝染色测定细胞活度；甲苯胺蓝染色测定蛋白聚糖的合成和II型胶原免疫组化检测II型胶原含量。 结果： 1、BMSCs原代细胞早期接种时，细胞呈类圆形，48小时后少许细胞贴壁，并逐渐延伸成长梭性。约7日后即可见明显的集落形成，与周围的集落渐渐的融合，2周后，细胞基本融合达90%,排列紧密而有序，至第5代时细胞形态更加规矩，单个细胞已成一致的狭长梭形，排列紧密有序，集落中心呈旋涡状生长。 2、BMSCs流式细胞仪检测结果： CD29表达率为97.36％，CD90表达率为99.61％，均为强阳性反应；CD45表达率为0.44％为阴性反应。说明此BMSCs细胞样本纯度很高，符合本实验要求。 3、BMSCs经28天诱导后，于离心管底部肉眼可见的银白色小质块，,球形,略有光泽,用镊子夹之韧性较差，各分组间细胞团块的大小，色泽无明显差异。 4、BMSCs经28天诱导后台盼蓝染色结果：无应力，100g，200g三组之间细胞活力无明显差异，而300g应力组的细胞活力则较前三组明显降低，，具有统计学意义（p0.05）。 5、BMSCs经28天诱导后甲苯胺蓝染色：200g应力组胞外基质有较明显的紫红色异染，100g组和300g组异染性较差，无应力对照组最差。 6、BMSCs经28天诱导后Ⅱ型胶原免疫组化结果：200g应力组呈强阳性反应，可见大量棕黄色的软骨细胞特异性基质，100g及300g组可见少量淡棕黄色的软骨细胞特异性基质，但较200g组明显减少，无应力组含量最少。 结论：100g应力强度过小，不足以使BMSCs充分的转化。300g的应力强度过大，超出了BMSCs所能承受的最大范围，反而对细胞造成了伤害，200g的离心力较适宜BMSCs向软骨细胞定向分化。
[Abstract]:Aim: to investigate the effects of different centrifugal forces on the differentiation of bone marrow mesenchymal stem cells into chondrocytes under micromass culture. Methods: femur and tibia were isolated from 6-week-old SD rats and bone marrow was extracted under aseptic condition. Primary BMSCs were extracted by density gradient centrifugation, and purified BMSCs were obtained by repeated liquid exchange and subculture. The contents of CD29, CD90 and CD45 were measured by flow cytometry, and the purity of the cells was identified. The sixth generation of BMSCs was cultured stereoscopically by micronucleus. In the course of culture, the induction medium with TGF- 尾 _ 1its as the main component was replaced and the cells were divided into four groups according to the intensity of centrifugal force: no stress control group, 100g stress group, 200g stress group and 300g stress group. After 28 days of induction, the cells were cultured for 28 days. The cell mass was identified and compared. The main identification indexes were observed cell mass morphology, luster, trypan blue staining, cell activity, etc. The synthesis of proteoglycan and the content of type II collagen were detected by toluidine blue staining. Results:. 1 when primary BMSCs were inoculated in the early stage, a few cells adhered to the wall 48 hours after inoculation, and gradually extended the growth spindle. After about 7 days, the colony formed obviously and fused with the surrounding colony gradually for 2 weeks. The cells were arranged closely and orderly, and at the fifth generation, the cells were more regular, and the single cells were in the same narrow spindle shape, arranged closely and orderly, and the center of the colony grew in a swirl shape. 2 the results of flow cytometry showed that the expression rate of CD29 was 97.36% and the positive rate of CD45 was 0.44%. The results showed that the purity of the BMSCs cells was high, which was in accordance with the requirements of this experiment. 3After 28 days of induction, the silvery white lumps of BMSCs were found in the bottom of the centrifuge tube. They were spherical and slightly glossy. The toughness of the tweezers clamp was poor. There was no significant difference in the size and color of the cell masses among the groups. 4The results of Pan-blue staining induced by BMSCs for 28 days showed that there was no significant difference in cell viability among the three groups, but the cell viability in the 300g stress group was significantly lower than that in the former three groups (P 0.05). 5After 28 days of induction, the extracellular matrix of 20% 200g stress group of BMSCs stained with toluidine blue had obvious amaranth heterochromaticity of 100g group and 300g group, and the worst of stress free control group. After 28 days induction of BMSCs, the type 鈪

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