人脐带血间充质干细胞向成肌细胞分化的体外研究
发布时间：2019-07-04 19:44
【摘要】： 研究背景和目的人脐带血间充质干细胞(human umbilical cord bloodmesenchymal stem cells,hUCB-MSCs)具有其他组织来源的间充质干细胞的生物学特性。目前的研究表明hUCB-MSCs含量较少,培养体系众多,但培养效率较低。对UCB-MSCs体外向成肌细胞分化的研究不够深入。因此,我们选择hUCB-MSCs作为研究的种子细胞,优化体外培养条件,探讨其体外诱导成肌细胞的能力,为肌源性疾病的治疗提供理想的种子细胞。 实验方法①hUCB-MNCs培养体系的建立:应用淋巴细胞分离液法、羟乙基淀粉沉降法和羟乙基淀粉+淋巴细胞分离液两步法三不同方法分别从脐带血中获得单个核细胞(mononuclear cells,MNCs),应用L-DMEM、DMEM/F12和MesencultTM不同培养基,在不同胎牛血清(fetal bovine serum,FBS)浓度和细胞接种密度下,培养UCB-MNCs。比较所获得的MNCs数量、原代培养时间和不同培养条件下hUCB-MSCs的生长情况。流式细胞仪对P3细胞进行细胞表面标记鉴定,并诱导成骨,观察其生物学特征。②基于上一部分的UCB-MSCs培养体系,建立以5-氮杂胞苷(5-azacytidine,5-Aza)为诱导剂的诱导成肌分化体系:MTT法选择5-Aza适宜的诱导浓度,对P3 hUCB-MNCs进行体外向成肌细胞诱导分化,于诱导后第7d和第14d进行MyoD1、myogenin和myosin heavy chain免疫荧光染色和Real-time PCR检测。 实验结果建立了hUCB-MSCs稳定有效的培养体系:应用羟乙基淀粉+淋巴细胞分离液两步法采集单个核细胞的数量较多(P＜0.01);10%FBS DMEM/F12和MesencultTM培养基,T25培养瓶中细胞接种密度为5×10~7时培养效率高于各对照组(P＜0.01);贴壁细胞表达CD29、CD105,不表达CD34、CD45、CD90、CD133;经成骨诱导培养21d后,细胞Yon Kossa染色可见矿化结节。P3 UCB-MNCs经5μmmol/L5-Aza诱导7d后,免疫荧光检测仅表达MyoD1;当诱导后14d时,可见MyoD1和myogenin阳性表达。始终无myosin heavy chain的表达。Real-time PCR检测显示诱导后14d较7d,MyoD1和myogenin的相对浓度增高,而无myosin heavy chain的表达。另外,在未诱导条件下UCB-MNCs同样能表达MyoD1。 实验结论脐带血中存在一定数量的间充质干细胞,通过改善hUCB-MSCs分离方法,优化培养条件,提高了培养效率。UCB-MSCs不表达造血干细胞和内皮细胞表面标记,表达与骨髓间充质干细胞表面标记,并能够诱导成骨,符合间充质干细胞的特点。在成肌细胞分化中,hUCB-MSCs经5μmmol/L5-Aza诱导后,表达成肌细胞分化过程中的调节因子,能够向成肌细胞分化,而且在未诱导条件下能够保持成肌细胞分化的潜能。
[Abstract]:The research background and the human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) have the biological characteristics of other tissue-derived mesenchymal stem cells. The present study shows that the content of hUCB-MSCs is less, the culture system is numerous, but the culture efficiency is low. The research on the differentiation of UCB-MSCs in vitro to myoblasts is not in-depth. Therefore, we selected hUCB-MSCs as the seed cells of the study, to optimize the in vitro culture conditions, to explore its ability to induce myoblasts in vitro, and to provide the ideal seed cells for the treatment of myogenic diseases. The establishment of hUCB-MNCs culture system was carried out by using a two-step method of lymphocyte separation, hydroxyethyl starch sedimentation and hydroxyethyl starch + lymphocyte separation. The UCB-M was cultured under different fetal bovine serum (FBS) concentrations and cell seeding density. NCs. Comparison of the number of MNCs, primary culture time and the generation of hUCB-MSCs under different culture conditions In that long case, the cell surface mark of the P3 cell was identified by flow cytometry, and the bone formation was induced and the organism was observe. Based on the UCB-MSCs culture system, the induction of 5-azacytidine (5-Aza) as an inducer was established. The appropriate induction concentration of 5-Aza was selected by the MTT method, and the differentiation of P3 hUCB-MNCs in vitro to myoblasts was carried out. oD1, mygenin, and myosin heavy chain immunofluorescence staining and Real-time PC R. The results of the experiment established a stable and effective culture system of hUCB-MSCs: the number of mononuclear cells was much higher than that of the two-step method with hydroxyethyl starch + lymphocyte separation (P <0.01);10% FBS DMEM/ F12 and Mesenu In the ltTM culture medium, the culture efficiency of the cells in the T25 culture flask was 5-10-7, and the culture efficiency was higher than that of the control group (P <0.01). The expression of CD29 and CD105 in the adherent cells did not express CD34, CD45, CD90 and CD133. After 21 days of bone-induced culture, the cell Yon Kossa was stained. Color-visible mineralized nodules. After 7 days of induction of P3 UCB-MNCs via 5. m mmol/ L 5-Aza, only MyoD1 was expressed by immunofluorescence test; when 14 d after induction, MyoD1 and myoge were visible. nin positive expression. No myosin heavy c at all times The expression of hain was detected by Real-time PCR and the relative concentrations of MyoD1 and mygenin increased compared to 7 d after induction, without myosin heavy c In addition, UCB-MNCs can also be used in uninduced conditions The expression of MyoD1. The experiment concluded that there was a certain number of mesenchymal stem cells in the cord blood, and by improving the method of separation of hUCB-MSCs, the tissue culture was optimized. the UCB-MSCs do not express the surface marks of the hematopoietic stem cells and the endothelial cells, and the expression is marked with the surface of the bone marrow mesenchymal stem cells and can induce the osteogenesis, In the differentiation of myoblasts, hUCB-MSCs were induced by 5. mu. mmol/ L 5-Aza, and the regulation factors in the differentiation of myoblasts can be expressed.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
本文编号：2510175
[Abstract]:The research background and the human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) have the biological characteristics of other tissue-derived mesenchymal stem cells. The present study shows that the content of hUCB-MSCs is less, the culture system is numerous, but the culture efficiency is low. The research on the differentiation of UCB-MSCs in vitro to myoblasts is not in-depth. Therefore, we selected hUCB-MSCs as the seed cells of the study, to optimize the in vitro culture conditions, to explore its ability to induce myoblasts in vitro, and to provide the ideal seed cells for the treatment of myogenic diseases. The establishment of hUCB-MNCs culture system was carried out by using a two-step method of lymphocyte separation, hydroxyethyl starch sedimentation and hydroxyethyl starch + lymphocyte separation. The UCB-M was cultured under different fetal bovine serum (FBS) concentrations and cell seeding density. NCs. Comparison of the number of MNCs, primary culture time and the generation of hUCB-MSCs under different culture conditions In that long case, the cell surface mark of the P3 cell was identified by flow cytometry, and the bone formation was induced and the organism was observe. Based on the UCB-MSCs culture system, the induction of 5-azacytidine (5-Aza) as an inducer was established. The appropriate induction concentration of 5-Aza was selected by the MTT method, and the differentiation of P3 hUCB-MNCs in vitro to myoblasts was carried out. oD1, mygenin, and myosin heavy chain immunofluorescence staining and Real-time PC R. The results of the experiment established a stable and effective culture system of hUCB-MSCs: the number of mononuclear cells was much higher than that of the two-step method with hydroxyethyl starch + lymphocyte separation (P <0.01);10% FBS DMEM/ F12 and Mesenu In the ltTM culture medium, the culture efficiency of the cells in the T25 culture flask was 5-10-7, and the culture efficiency was higher than that of the control group (P <0.01). The expression of CD29 and CD105 in the adherent cells did not express CD34, CD45, CD90 and CD133. After 21 days of bone-induced culture, the cell Yon Kossa was stained. Color-visible mineralized nodules. After 7 days of induction of P3 UCB-MNCs via 5. m mmol/ L 5-Aza, only MyoD1 was expressed by immunofluorescence test; when 14 d after induction, MyoD1 and myoge were visible. nin positive expression. No myosin heavy c at all times The expression of hain was detected by Real-time PCR and the relative concentrations of MyoD1 and mygenin increased compared to 7 d after induction, without myosin heavy c In addition, UCB-MNCs can also be used in uninduced conditions The expression of MyoD1. The experiment concluded that there was a certain number of mesenchymal stem cells in the cord blood, and by improving the method of separation of hUCB-MSCs, the tissue culture was optimized. the UCB-MSCs do not express the surface marks of the hematopoietic stem cells and the endothelial cells, and the expression is marked with the surface of the bone marrow mesenchymal stem cells and can induce the osteogenesis, In the differentiation of myoblasts, hUCB-MSCs were induced by 5. mu. mmol/ L 5-Aza, and the regulation factors in the differentiation of myoblasts can be expressed.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
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