脐血间充质干细胞的生物学特性及其多向诱导分化潜能的实验研究
发布时间：2019-04-07 19:47
【摘要】： 目的 探讨影响人脐血间充质干细胞(UCB-MSCs)成功分离培养的相关因素及其生物学特性,并对其向成骨、成脂肪细胞诱导分化的能力进行研究。 方法 (1)分别从不同胎龄(≥40周、37周和≤32周)、脐血中单个核细胞(MNCs)的数量(≥2.5×10~9/L、＜2.5×10~9/L)、不同细胞接种密度(1×10~4 cells/ml、1×10~6 cells/ml、1×10~8 cells/ml)、不同胎牛血清(FBS)浓度(5%、10%、15%、20%)以及培养瓶是否被FBS包被等方面对UCB-MSCs的分离培养成功率进行分析比较;通过倒置相差显微镜及透射电镜观察细胞的形态、生长增殖情况及其超微结构;并描绘细胞生长曲线;计算并分析比较不同胎龄组UCB-MSCs的集落形成能力和细胞倍增时间差异;用流式细胞仪对细胞表面标志物进行检测。 (2)分别用成骨及成脂诱导液对UCB-MSCs进行诱导,通过茜素红染色、碱性磷酸酶染色及活性测定等检测UCB-MSCs向成骨细胞诱导分化的能力;通过油红O染色检测其向脂肪细胞诱导分化的能力。 结果 (1)UCB-MSCs的分离培养成功率为58.3%,且不同胎龄组培养成功率随胎龄的增高而降低,差异有统计学意义(x~2=9.87,P=0.007);脐血中MNCs含量在2.5×10~9/L以上者培养成功率高达76.9%,与MNCs含量低于2.5×10~9/L组(36.4%)比较,差异有统计学意义(x~2=8.07,P=0.005);相同容量的脐血中MNCs与胎龄的相关性分析显示,两者呈负相关(n=20,r=-0.95,P＜0.01);在接种密度低于1×10~8 cells/ml组中,原代及传代培养的MSCs生长及扩增情况均不如1×10~8 cells/ml组;FBS浓度为5%组与10%、15%、20%组相比,MSCs贴壁速度略慢,但MSCs的纯度较高,破骨样细胞含量明显要少,后三组之间未见明显差别,且5%FBS浓度组细胞传代速度与其他三组相比无明显差别;用FBS对培养瓶进行包被后,UCB-MSCs不仅在原代和传代后的纯度增加了,而且其扩增能力也明显快于未包被组。倒置相差显微镜下观察UCB-MSCs贴壁生长,呈成纤维细胞样外观,细胞呈螺旋状排列;透射电镜观察UCB-MSCs细胞核大,胞核比例大,细胞器少,为低分化细胞;原代及传代培养的UCB-MSCs生长曲线均呈S型,第3、5代细胞增殖能力最强;3个不同胎龄组的UCB-MSCs集落形成能力存在统计学差异(F=8.53,P＜0.05),低胎龄组集落形成能力大于高胎龄组;不同胎龄的UCB-MSCs细胞群体倍增时间比较差异无统计学意义(F=2.68,P＞0.05);流式细胞仪检测结果显示,UCB-MSCs均稳定的表达与MSCs相关的表面抗原标志物CD29、CD44和CD90等,不表达造血标志CD34和CD45。 (2) UCB-MSCs成骨诱导后ALP活性逐渐增强,3周时诱导细胞ALP染色强阳性、茜素红染色可检测到大量钙化基质的形成;成脂诱导3周时油红O染色可检测到胞质中脂滴的形成。 结论 (1)不同胎龄的脐血中均含有MSCs,其分离培养成功率受多种因素的影响;通过选择较低胎龄的胎儿、采集足够量的脐血、以较高的细胞密度接种、培养基中添加较低浓度的FBS、并将培养瓶预先用FBS进行包被、且在细胞培养的适当时机进行换液和传代等,能在体外建立稳定的UCB-MSCs培养体系。 (2) UCB-MSCs的形态特征、生长增殖特点及细胞表面标志物等生物学特性与骨髓MSCs相似,具有强大的生长增殖与自我更新能力。 (3) UCB-MSCs在体外诱导条件下可以向成骨细胞及脂肪细胞等间质组织细胞定向分化,为其在临床上的应用奠定了理论基础。
[Abstract]:Purpose The related factors and their biological characteristics of the successful isolation and culture of human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) were discussed. line-and-study Methods (1) The number of mononuclear cells (MNCs) in the umbilicus was 10-4 cells/ ml,1-10-6 cells/ ml,1-10,10-4 cells/ ml,1-10-6 cells/ ml,10-9/ L,10-9/ L,10-9/ L,10-9/ L,10-9/ L, respectively. The success rate of the separation and culture of UCB-MSCs was analyzed by means of inverted phase contrast microscope and transmission electron microscope. The cell growth curve, colony-forming ability and cell doubling time difference of UCB-MSCs in different gestational age groups were calculated and analyzed, and flow cytometry was used. The cell surface markers were tested. (2) UCB-MSCs were induced by bone and fat-forming induction solution, and the ability of UCB-MSCs to induce differentiation into osteoblasts was detected by the staining of fluorescein red, alkaline phosphatase staining and activity determination. detection Results (1) The success rate of the separation and culture of UCB-MSCs was 58.3%, and the success rate of the different gestational age groups decreased with the increase of the gestational age, the difference was statistically significant (x ~ 2 = 9.87, P = 0.007), and the content of MNCs in the umbilical blood was 2. The results showed that the culture success rate was 76.9%, the content of MNCs was lower than that of 2.5-10-9/ L (36.4%), the difference was statistically significant (x ~ 2 = 8.07, P = 0.005), and the correlation between MNCs and gestational age in the umbilical blood of the same volume showed negative correlation (n = 20, r =-0.95, P <0.01). In the group of 10 ~ 8 cells/ ml, the growth and amplification of MSCs in primary and subcultured cells were less than that of 1-10 ~ 8 cells/ ml, and the concentration of the FBS was 5% and 10%,15% and 20%, and the adherent rate of MSCs was slightly slower, but the purity of MSCs was higher. The content of osteoclast-like cells was significantly lower, and there was no significant difference between the three groups, and the cell passage speed of 5% FBS group was not significantly different from the other three groups; after the culture flask was coated with FBS, the UCB-MSCs were not only after primary and passage The purity of UCB-MSCs was increased, and the amplification ability of UCB-MSCs was significantly faster than that of uncoated group. UCB-MSCs were observed under the inverted phase-contrast microscope. The appearance of the fibroblast-like appearance and the cell-like appearance were observed by transmission electron microscope. The nucleus of UCB-MSCs was observed by transmission electron microscope. The growth curve of UCB-MSCs was the strongest in the third and fifth generation, and the colony-forming ability of the three different gestational age groups was higher than that of the high-gestational age group. The UCB-MSCs of different gestational age The difference of population doubling time was not significant (F = 2.68, P> 0.05). The results of flow cytometry showed that the expression of UCB-MSCs was stable to the surface antigen markers CD29 and CD44 associated with MSCs. And CD90 and the like, and the hematopoietic marker CD34 and the CD45 are not expressed. (2) the ALP activity of the UCB-MSCs is gradually enhanced after the osteogenesis induction, and the ALP staining of the induction cells is strongly positive at 3 weeks, and a large amount of the calcified matrix can be detected by the fluorescein red staining. to form Conclusion (1) The successful rate of separation and culture of MSCs in different gestational age is affected by many factors. And the culture flask is pre-coated with FBS and cultured in cell culture. A stable UCB-MSCs culture system can be established in vitro. (2) The morphological characteristics, growth and proliferation characteristics of UCB-MSCs and the growth and proliferation characteristics of UCB-MSCs can be established in vitro. The biological characteristics of the cell surface markers are similar to that of the bone marrow MSCs, and have strong growth and proliferation and self-renewal capacity. (3) UCB-MSCs are induced in vitro
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
本文编号：2454372
[Abstract]:Purpose The related factors and their biological characteristics of the successful isolation and culture of human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) were discussed. line-and-study Methods (1) The number of mononuclear cells (MNCs) in the umbilicus was 10-4 cells/ ml,1-10-6 cells/ ml,1-10,10-4 cells/ ml,1-10-6 cells/ ml,10-9/ L,10-9/ L,10-9/ L,10-9/ L,10-9/ L, respectively. The success rate of the separation and culture of UCB-MSCs was analyzed by means of inverted phase contrast microscope and transmission electron microscope. The cell growth curve, colony-forming ability and cell doubling time difference of UCB-MSCs in different gestational age groups were calculated and analyzed, and flow cytometry was used. The cell surface markers were tested. (2) UCB-MSCs were induced by bone and fat-forming induction solution, and the ability of UCB-MSCs to induce differentiation into osteoblasts was detected by the staining of fluorescein red, alkaline phosphatase staining and activity determination. detection Results (1) The success rate of the separation and culture of UCB-MSCs was 58.3%, and the success rate of the different gestational age groups decreased with the increase of the gestational age, the difference was statistically significant (x ~ 2 = 9.87, P = 0.007), and the content of MNCs in the umbilical blood was 2. The results showed that the culture success rate was 76.9%, the content of MNCs was lower than that of 2.5-10-9/ L (36.4%), the difference was statistically significant (x ~ 2 = 8.07, P = 0.005), and the correlation between MNCs and gestational age in the umbilical blood of the same volume showed negative correlation (n = 20, r =-0.95, P <0.01). In the group of 10 ~ 8 cells/ ml, the growth and amplification of MSCs in primary and subcultured cells were less than that of 1-10 ~ 8 cells/ ml, and the concentration of the FBS was 5% and 10%,15% and 20%, and the adherent rate of MSCs was slightly slower, but the purity of MSCs was higher. The content of osteoclast-like cells was significantly lower, and there was no significant difference between the three groups, and the cell passage speed of 5% FBS group was not significantly different from the other three groups; after the culture flask was coated with FBS, the UCB-MSCs were not only after primary and passage The purity of UCB-MSCs was increased, and the amplification ability of UCB-MSCs was significantly faster than that of uncoated group. UCB-MSCs were observed under the inverted phase-contrast microscope. The appearance of the fibroblast-like appearance and the cell-like appearance were observed by transmission electron microscope. The nucleus of UCB-MSCs was observed by transmission electron microscope. The growth curve of UCB-MSCs was the strongest in the third and fifth generation, and the colony-forming ability of the three different gestational age groups was higher than that of the high-gestational age group. The UCB-MSCs of different gestational age The difference of population doubling time was not significant (F = 2.68, P> 0.05). The results of flow cytometry showed that the expression of UCB-MSCs was stable to the surface antigen markers CD29 and CD44 associated with MSCs. And CD90 and the like, and the hematopoietic marker CD34 and the CD45 are not expressed. (2) the ALP activity of the UCB-MSCs is gradually enhanced after the osteogenesis induction, and the ALP staining of the induction cells is strongly positive at 3 weeks, and a large amount of the calcified matrix can be detected by the fluorescein red staining. to form Conclusion (1) The successful rate of separation and culture of MSCs in different gestational age is affected by many factors. And the culture flask is pre-coated with FBS and cultured in cell culture. A stable UCB-MSCs culture system can be established in vitro. (2) The morphological characteristics, growth and proliferation characteristics of UCB-MSCs and the growth and proliferation characteristics of UCB-MSCs can be established in vitro. The biological characteristics of the cell surface markers are similar to that of the bone marrow MSCs, and have strong growth and proliferation and self-renewal capacity. (3) UCB-MSCs are induced in vitro
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
【引证文献】
相关硕士学位论文 前1条
1 叶兴德;CRIF1在骨髓间充质干细胞辐射氧化应激中的作用及其机制初步研究[D];第三军医大学;2013年
，本文编号：2454372
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