發(fā)布時間：2018-03-27 05:22

  本文選題：骨髓間充質干細胞　切入點：軟骨細胞　出處：《大連醫(yī)科大學》2012年碩士論文

【摘要】：目的：在微團培養(yǎng)條件下探討不同離心力對骨髓間充質干細胞向軟骨細胞分化的影響與作用。 方法：取6周齡的SD大鼠，在無菌條件下分離出股骨和脛骨并抽取骨髓，使用密度梯度離心法提取原代BMSCs，并經過反復換液和傳代得到純化的BMSCs，使用流式細胞儀測出CD29、CD90和CD45的含量，鑒定細胞的純度。取第6代BMSCs采用微團方式進行立體培養(yǎng)，在培養(yǎng)過程中更換以TGF-β1,ITS為主要成分的誘導培養(yǎng)基并根據離心力強度將細胞分為4組：無應力對照組、100g應力組、200g應力組和300g應力組。誘導培養(yǎng)28天后，對細胞團塊進行鑒定和比較。鑒定指標主要為大體觀察細胞團塊的形態(tài)、光澤度等；臺盼藍染色測定細胞活度；甲苯胺藍染色測定蛋白聚糖的合成和II型膠原免疫組化檢測II型膠原含量。 結果： 1、BMSCs原代細胞早期接種時，細胞呈類圓形，48小時后少許細胞貼壁，并逐漸延伸成長梭性。約7日后即可見明顯的集落形成，與周圍的集落漸漸的融合，2周后，細胞基本融合達90%,排列緊密而有序，至第5代時細胞形態(tài)更加規(guī)矩，單個細胞已成一致的狹長梭形，排列緊密有序，集落中心呈旋渦狀生長。 2、BMSCs流式細胞儀檢測結果： CD29表達率為97.36％，CD90表達率為99.61％，均為強陽性反應；CD45表達率為0.44％為陰性反應。說明此BMSCs細胞樣本純度很高，符合本實驗要求。 3、BMSCs經28天誘導后，于離心管底部肉眼可見的銀白色小質塊，,球形,略有光澤,用鑷子夾之韌性較差，各分組間細胞團塊的大小，色澤無明顯差異。 4、BMSCs經28天誘導后臺盼藍染色結果：無應力，100g，200g三組之間細胞活力無明顯差異，而300g應力組的細胞活力則較前三組明顯降低，，具有統(tǒng)計學意義（p0.05）。 5、BMSCs經28天誘導后甲苯胺藍染色：200g應力組胞外基質有較明顯的紫紅色異染，100g組和300g組異染性較差，無應力對照組最差。 6、BMSCs經28天誘導后Ⅱ型膠原免疫組化結果：200g應力組呈強陽性反應，可見大量棕黃色的軟骨細胞特異性基質，100g及300g組可見少量淡棕黃色的軟骨細胞特異性基質，但較200g組明顯減少，無應力組含量最少。 結論：100g應力強度過小，不足以使BMSCs充分的轉化。300g的應力強度過大，超出了BMSCs所能承受的最大范圍，反而對細胞造成了傷害，200g的離心力較適宜BMSCs向軟骨細胞定向分化。
[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

本文編號：1670176