小鼠多能成体祖细胞的培养及冷冻保存

发布时间：2018-04-25 21:14

本文选题：骨髓细胞 + 原代培养　；参考：《华中科技大学》2010年硕士论文

【摘要】：第一部分BALB/c小鼠多能成体祖细胞的分离培养目的建立获得大量BALB/c小鼠多能成体祖细胞(MAPC)的细胞分离和原代培养方法,为下一步获得纯化的MAPC奠定基础。方法从BALB/c小鼠胫骨和股骨中分离得到骨髓细胞悬液,采用全骨髓差异贴壁法,在含EGF、PDGF、LIF和低血清的培养基中培养,2周后以一定比例传代,继续培养,将不同时期细胞进行Giemsa染色,观察细胞形态,通过MTT比色法比较不同周龄小鼠、原代接种密度和传代接种密度对原代MAPC生长的影响。结果取材自3周小鼠所获得原代细胞均一性和增殖情况最好,原代接种密度在(6～12)×10~5/cm~2,传代接种密度在(20-30)×10~3/cm~2最有利于原代MAPC的生长。Giemsa染色可见梭形和三角形细胞。结论在本实验条件下,BALB/c小鼠的原代MAPC能较稳定生长和扩增,给后续MACS分选提供了大量的细胞来源。第二部分BALB/c小鼠多能成体祖细胞的纯化和扩增目的获得纯化的MAPC并探索MAPC体外稳定传代扩增的培养条件,建立MAPC细胞系,为探索MAPC向生殖细胞的诱导分化潜能打下基础。方法取传2代的骨髓细胞,磁珠阴性分选去除CD45+和Ter119+细胞,将所获目的细胞以一定密度接种,观察细胞生长情况,并绘制MAPC生长曲线。台盼蓝检测MACS分选前后细胞活力有无差异,MTT比色法比较不同接种密度对MAPC生长的影响。结果MACS分选前后细胞活性无显著性差异(P㧐0.05),MAPC的最适接种密度为(6-10)×10~3/cm~2,MAPC接种后1-2天处于生长停滞期,第3天进入对数增长期,约持续3天,以后进入平台期。分选后MAPC核大,胞质少,呈梭形或三角形。结论MACS阴性分选能获得相对单一并具有良好增殖活性的MAPC。第三部分BALB/c小鼠多能成体祖细胞的鉴定和冷冻保存目的鉴定本实验培养的MAPC,并对MAPC的冷冻保存方法的效果进行了评价,为诱导分化实验提供足够的细胞来源。方法流式细胞术检测MAPC表面标记CD 13、CD44和CD34的表达情况。采用文献报道的方法对MAPC进行冷冻保存,对温度稍作调整,比较冻存前后细胞活力。结果MAPC表面CD13~+、CD44~-和CD34~-。细胞冻存前后活力无显著性差异(P㧐0.05)。结论全骨髓贴壁结合磁性分选是获得MAPC的有效方法,采用文献报道的冷冻方法能较好的保存MAPC,保持其生长活性。
[Abstract]:The first part: isolation and culture of BALB/c mouse pluripotent adult progenitor cells Objective to establish a method for isolation and primary culture of a large number of BALB/c mouse multipotent adult progenitor cells (MAPCs) so as to lay a foundation for the further purification of MAPC. Methods Bone marrow cell suspensions were isolated from the tibia and femur of BALB/c mice. The bone marrow cells were cultured in a certain proportion for 2 weeks in the culture medium containing EGFN PDGFGF-LIF and low serum. The cells were stained with Giemsa at different stages. The effects of primary inoculation density and passage inoculation density on the growth of primary MAPC were compared by MTT colorimetry. Results the homogeneity and proliferation of primary cells were the best obtained from 3 weeks old mice. The primary inoculation density was 612 脳 10 ~ (5) / cm ~ (-2), and the inoculation density was 20 ~ 30 脳 10~3/cm~2, which was most favorable to the growth of primary MAPC. Giemsa staining showed fusiform cells and triangular cells. Conclusion the primary MAPC of BALB / c mice can grow and amplify stably under the condition of this experiment, which provides a large number of cell sources for subsequent MACS sorting. The second part: purification and amplification of BALB/c mouse pluripotent adult progenitor cells Objective to obtain purified MAPC and explore the culture conditions of stable passage and amplification of MAPC in vitro, and to establish MAPC cell line, which will lay a foundation for exploring the potential of inducing and differentiating MAPC into germ cells. Methods CD45 and Ter119 cells were removed by magnetic bead negative sorting. The target cells were inoculated with a certain density to observe the growth of the cells and draw the MAPC growth curve. Trypan blue was used to detect the difference of cell viability before and after MACS sorting. The effects of different inoculation densities on the growth of MAPC were compared by MTT colorimetry. Results there was no significant difference in cell activity before and after MACS sorting. The optimum inoculation density of MACS was 6-10) 脳 10 ~ (-3) / cm ~ (2) C ~ (2) C ~ (2) C ~ (2) ~ (-1) ~ (-1) ~ (-1) days after inoculation, and 3 days entered logarithmic growth period, which lasted for 3 days, and then entered the plateau phase. After sorting, the nucleus of MAPC is large and the cytoplasm is less, which is fusiform or triangular. Conclusion MACS negative sorting can obtain relatively single MAPCs with good proliferative activity. The third part: identification and cryopreservation of BALB/c mouse pluripotent adult progenitor cells Objective to identify MAPCs cultured in this experiment and evaluate the effect of cryopreservation method of MAPC in order to provide sufficient cell source for inducing differentiation experiment. Methods flow cytometry was used to detect the expression of CD 13 CD 44 and CD34 on MAPC surface. The cryopreservation of MAPC was carried out by using the method reported in the literature, and the temperature was adjusted slightly to compare the cell viability before and after cryopreservation. Results on the surface of MAPC, CD13 ~ + CD44 ~-and CD _ 34 ~ +-were observed. There was no significant difference in cell viability before and after cryopreservation. Conclusion\\\
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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