大鼠骨髓间充质干细胞的改良法分离纯化及鉴定

发布时间：2018-04-20 05:44

  本文选题：骨髓间充质干细胞 + 细胞培养　； 参考：《泸州医学院》2011年硕士论文

【摘要】：目的：探讨改良法分离纯化SD大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)的可行性,并评价改良法的分离纯化效果,为下一步实验研究提供高纯度的BMSCs。 方法：(1)无菌条件下分离SD大鼠股骨、胫骨,清除附于骨上的肌肉和筋膜,磷酸盐缓冲液(Phosphate buffered saline, PBS)洗净后从股骨干和胫骨干中间剪断,用5ml注射器抽取5ml L-DMEM培养基反复冲洗骨髓腔,冲洗液经200目不锈钢标准筛过滤后以2000rpm速度离心5 min,弃上清及脂肪层后加入完全培养液反复吹打制成单细胞悬液,以106个/ml密度接种于4个25cm2细胞培养瓶中,随机分为A、B两组细胞,每组2瓶,置于37℃、5%C02细胞培养箱内培养；(2)48h后首次换液,以后每3天换液一次；(3)细胞生长至80%～90%融合时,以0.25%胰蛋白酶1ml消化2～3min后加入5ml含12%胎牛血清(fetal bull serum,FBS)的L-DMEM完全培养液,吹打制成单细胞悬液后以1：2比例传代扩增；(4)第1代细胞融合达80%～90%时,A组细胞PBS洗涤、0.25%胰蛋白酶1ml消化后制成单细胞悬液,缓慢加入盛有同样体积Percoll分离液(密度为1.073)的离心管,以2000rpm速度离心20 min。用吸管小心吸取中间白色絮状层加入另一离心管,再加入完全培养液,充分吹打洗涤细胞后离心,弃上清液后加入完全培养液重悬细胞,调整细胞密度为105/ml后接种5ml入25cm2细胞培养瓶,继续置细胞培养箱内培养；B组细胞PBS洗涤、0.25%胰蛋白酶1ml消化后制成单细胞悬液,调整细胞密度为105/ml后直接接种5ml于25cm2细胞培养瓶(未经Percoll分离液分离),置培养箱内培养；(5)每日在倒置相差显微镜下观察两组各代细胞形态变化及生长增殖状况并拍照记录,采用MTT法绘制两组第4、第9代BMSCs的生长曲线；(6)A、B组第3代BMSCs用免疫组织化学染色鉴定细胞表型；(7)流式细胞仪检测A、B两组第3代细胞表达CD34、CD44、CD45、CD90阳性细胞的百分比；(8)A、B组第3代大鼠BMSCs进行细胞周期检测；(9)对A、B组所得的BMSCs进行成骨诱导分化培养及鉴定。 结果：(1)A、B两组第4、第9代BMSCs生长曲线显示两种分离方法分离纯化的BMSCs的增殖能力无明显差异(P0.05)(2)免疫细胞化学染色结果显示：A组第3代BMSCs的CD29、CD44、CD90、CD 106检测均呈阳性表达,细胞膜和胞浆呈棕褐色,而CD34、CD45呈阴性表达,胞膜及胞浆未着色；B组第3代BMSCs的CD29、CD44、CD90、CD 106检测呈阳性表达,大部分细胞CD34、CD45阴性表达,少部分阳性表达,提示A组BMSCs的纯度高于B组；(3)用流式细胞仪分别检测A、B两组第3代BMSCs,结果：A组细胞均一的阳性表达CD44、CD90,而几乎不表达CD34和CD45,A组细胞表达CD44、CD90阳性率明显高于B组(P0.05),而表达CD34、CD45阳性率明显低于B组(P0.05),提示A组BMSCs的纯度高于B组；(4)对A、B两组第3代细胞进行细胞周期检测,结果显示大部分细胞处于静止期,只有少数的细胞处于活跃的增殖期,均符合干细胞特征；(5)A、B两组第3代BMSCs经成骨诱导培养液培养3周后经茜素红染色均可见大量深红色钙结节,两种方法分离纯化的大鼠间充质干细胞均具有良好的成骨分化潜能,无明显差异。 结论：(1)本研究采用改良法分离纯化SD大鼠骨髓间充质干细胞,成功在单只大鼠分离获得大量高纯度的BMSCs,解决了大鼠BMSCs研究的同源性、细胞相容性、排斥性问题,同时节约大量骨髓与干细胞资源；(2)改良法具有操作简便、快速、不影响细胞增殖能力的优点；(3)改良法分离纯化获得的BMSCs形态和生物学稳定性好、具有强大的增殖能力和良好的成骨分化潜能；(4)改良法分离纯化SD大鼠骨髓间充质干细胞效果优于传统的全骨髓贴壁筛选法。
[Abstract]:Objective: To explore the feasibility of separating and purifying SD rat bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) by the improved method, and to evaluate the purification effect of the modified method and provide a high purity BMSCs. for the next experimental study.
Methods: (1) the femur and tibia of SD rats were separated from the femur and tibia under aseptic conditions, and the muscles and fasciae attached to the bone were removed. The phosphate buffer solution (Phosphate buffered saline, PBS) was washed from the middle of the femoral shaft and tibial shaft, and the bone marrow cavity was washed by the 5ml syringe 5ml L-DMEM medium. The rinse solution was filtered through the 200 mesh stainless steel standard sieve to 2. 000rpm speed centrifugation 5 min, discard the supernatant and fat layer after adding complete culture fluid and repeatedly blow into a single cell suspension, 106 /ml density inoculated in 4 25cm2 cell culture bottles, randomly divided into A, B two groups, each group of 2 bottles, at 37, 5%C02 cell culture box culture; (2) 48h after the first liquid, and then every 3 days to change liquid once; (3) fine. (3) fine (3) fine When the cell growth was 80% ~ 90% fusion, 0.25% trypsin 1ml was digested for 2 ~ 3min and added to the L-DMEM complete culture of 5ml containing 12% fetal bovine serum (fetal bull serum, FBS). After being blown into a single cell suspension, it was amplified in a 1:2 proportion. (4) when the first generation of cell fusion reached 80% to 90%, the A group cells PBS washing and 0.25% trypsin digested after digestion. The single cell suspension was slowly added to the centrifuge tube with the same volume Percoll separation liquid (density 1.073). At the 2000rpm speed centrifuge, the white floc layer was carefully absorbed into the other centrifuge tube with a sucker, and the complete culture fluid was added to the centrifuge. The centrifuge was fully blown after the washing cells were blown, and the supernatant was abandoned after the supernatant was discarded, and the suspension cells were added and adjusted. After the cell density was 105/ml, 5ml was inoculated into 25cm2 cell culture bottle and continued to be cultured in cell culture box; B group cells were washed by PBS, 0.25% trypsin 1ml was digested to make single cell suspension, and the cell density was 105/ml, and 5ml was directly inoculated with 5ml in 25cm2 cell culture bottle (not separated by Percoll separation solution) and incubated in culture box; (5) daily in the culture box. In the inverted phase contrast microscope, the morphological changes and growth and proliferation of two groups of cells were observed and recorded. The growth curves of two groups of fourth, ninth generation BMSCs were drawn by MTT; (6) A, B group third BMSCs was used to identify the cell phenotype by immunohistochemical staining; (7) flow cytometry was used to detect A, B two and third generation cells expressed CD34, CD44, CD45, CD90 Yang The percentage of sex cells; (8) A, B group third generation rats BMSCs cell cycle detection; (9) A, B group BMSCs induced osteogenic differentiation, culture and identification.
Results: (1) A, B two groups fourth, ninth generation BMSCs growth curve showed that two separate methods of separation and purification of BMSCs proliferation capacity of no significant difference (P0.05) (2) immunocytochemical staining results showed that the third generation of BMSCs CD29, CD44, CD90, CD 106 of the third generation of A group were positive expression, cell membrane and cytoplasm were brown, CD34, negative expression, The cell membrane and cytoplasm were not stained, and the CD29, CD44, CD90, CD 106 of the third generation of B group showed positive expression. Most of the cells were expressed in CD34, CD45 negative and few positive expressions, suggesting that the purity of BMSCs in the A group was higher than that in the B group; (3) the A was detected by flow cytometry and two groups and third generations respectively. No expression of CD34 and CD45, A group cells expressed CD44, CD90 positive rate was significantly higher than that of B group (P0.05), but the expression of CD34, CD45 positive rate was significantly lower than B group (P0.05), suggesting that the purity of the A group was higher than that of the group. (4) the cell cycle detection of the two groups third generation cells showed that most of the cells were in the stationary phase, only a few cells were active. The proliferation period was in line with the characteristics of stem cells. (5) A, B two group third generation of third generation of third generations after culture medium culture 3 weeks after the culture of alizarin red stained with a large number of dark red calcium nodules, two methods of purified rat mesenchymal stem cells have good osteogenic differentiation potential, no significant difference.
Conclusions: (1) the modified method was used to separate and purify SD rat bone marrow mesenchymal stem cells, and successfully isolated a large number of high purity BMSCs in a single rat, and solved the homology, cytocompatibility and rejection of the rat BMSCs study, and saved a large number of bone marrow and stem cell resources. (2) the improved method was easy to operate, fast and no shadow. The advantages of the proliferation ability of ringing cells; (3) the BMSCs morphology and biological stability obtained by the modified method were good, with strong proliferation ability and good osteogenic differentiation potential, and (4) the effect of the modified method for the separation and purification of bone marrow mesenchymal stem cells in SD rats was better than that of the traditional bone marrow adherence screening method.

【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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