不同血清对间充质干细胞衰老进程的影响
发布时间：2018-10-16 18:24
【摘要】：间充质干细胞（Mesenchymal stem cells，MSCs）在临床治疗方面有广泛的应用价值。然而，MSCs在体外扩增培养过程中由于多次传代培养会进入复制性衰老，进而发生一些生物学性状的改变，，影响临床应用与研究。已有研究证明干细胞微环境与干细胞的衰老有密切联系，但是不同血清微环境对细胞衰老的影响尚不清楚。目前实验室体外培养MSCs常用到胎牛血清（fetal bovine serum，FBS）或小牛血清（calf serum，CS），两种血清对MSCs的衰老进程有何影响，至今未见相关报道。因此，本实验拟在探究胎牛血清和小牛血清对MSCs衰老进程的影响。 首先采用组织块贴壁培养法获取人脐带源MSCs，并对传统的组织块贴壁法进行了改良，将本应丢弃的组织块进行二次贴壁培养，以获得更多的原代MSCs。首次组织块贴壁培养第5-7天可见有细小梭形细胞从组织块边缘爬出，到10天左右，可见细胞克隆形成。将组织块转移到新的培养瓶中进行二次贴壁培养，2天后即有细胞爬出，5天即可形成细胞克隆。对二次贴壁培养得到的MSCs进行流式检测，结果显示，细胞高表达CD90、CD105，不表达CD34、CD45、HLA-DR，说明第二次组织块贴壁培养得到的细胞也是间充质干细胞。同时，我们将得到的细胞与胎盘来源MSCs进行了生物学特性的比较，发现两种细胞没有明显的差异性。 对于分离的脐带源MSCs，从第一代开始，分别采用胎牛血清和小牛血清进行体外长期培养，直至细胞衰老。两组采用相同的接种密度，并在传代培养时计算所得细胞数和群体倍增数，进而累加得到累计群体倍增数。选取处于相同累计群体倍增数的三个阶段，对两组细胞分别进行增殖、分化、凋亡以及衰老相关指标的检测。结果发现，随着传代次数的增加，MSCs的增殖分化能力下降，凋亡率升高，同时，衰老相关的β-半乳糖苷酶活性增强，细胞内ROS水平升高，p53、p21、p16等基因表达量增高。结果还显示胎牛血清组MSCs的增殖和分化能力要优于小牛血清组，而细胞的凋亡比率、细胞内ROS水平以及p53等衰老相关基因的表达水平要低于小牛血清组。说明胎牛血清能够对MSCs的生长提供一个更好的微环境，有助于保持其生物学特性，延缓衰老，而小牛血清则会加速细胞的衰老进程。
[Abstract]:Mesenchymal stem cell (Mesenchymal stem cells,MSCs) has been widely used in clinical treatment. However, in the process of MSCs amplification in vitro, the repeated passage culture will enter into replicative senescence, and then some biological characters will change, which will affect the clinical application and research. It has been proved that stem cell microenvironment is closely related to stem cell senescence, but the effect of different serum microenvironment on cell senescence is not clear. At present, fetal bovine serum (fetal bovine serum,FBS (FBS) or calf serum (calf serum,CS) are commonly used in laboratory culture of MSCs in vitro. There are no reports on the effects of the two serums on the aging process of MSCs. Therefore, the effect of fetal bovine serum and calf serum on the aging process of MSCs was studied. Firstly, the human umbilical cord derived MSCs, was obtained by using tissue mass adherent culture method, and the traditional tissue block adherent method was improved to obtain more primary MSCs. by secondary adherent culture of tissue mass that should have been discarded. On the 5-7 days after the first tissue mass adherent culture, there were small fusiform cells crawling out from the edge of the tissue mass, and about 10 days later, the cell clone formation was observed. The tissue mass was transferred to a new culture bottle for secondary adherent culture. After 2 days, the cells crawled out, and the cell clone was formed in 5 days. The MSCs obtained from the secondary adherent culture was detected by flow cytometry. The results showed that the cells with high expression of CD90,CD105, did not express CD34,CD45,HLA-DR, indicating that the cells obtained from the second tissue mass adhesion culture were also mesenchymal stem cells. At the same time, we compared the biological characteristics of the obtained cells with placental MSCs, and found that there was no significant difference between the two kinds of cells. For isolated umbilical cord MSCs, fetal bovine serum and calf serum were used for long-term culture in vitro from the first generation, respectively, until cell senescence. The two groups used the same inoculation density and calculated the cell number and the population multiplication number during the passage and then accumulated the cumulative population multiplication number. The cell proliferation, differentiation, apoptosis and senescence were detected in three stages of the same cumulative population multiplication. The results showed that with the increase of passage times, the proliferation and differentiation ability of MSCs decreased and the apoptosis rate increased. At the same time, the activity of 尾 -galactosidase increased, the level of ROS increased, and the expression of p53, p21, p16 and other genes increased. The results also showed that the proliferation and differentiation ability of MSCs in fetal bovine serum group was better than that in calf serum group, while the apoptosis ratio, intracellular ROS level and the expression level of senescence related genes such as p53 in fetal bovine serum group were lower than those in calf serum group. The results showed that fetal bovine serum could provide a better microenvironment for the growth of MSCs, which could help to maintain its biological characteristics and delay senescence, while calf serum could accelerate the process of cell senescence.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R329.2
本文编号：2275244
[Abstract]:Mesenchymal stem cell (Mesenchymal stem cells,MSCs) has been widely used in clinical treatment. However, in the process of MSCs amplification in vitro, the repeated passage culture will enter into replicative senescence, and then some biological characters will change, which will affect the clinical application and research. It has been proved that stem cell microenvironment is closely related to stem cell senescence, but the effect of different serum microenvironment on cell senescence is not clear. At present, fetal bovine serum (fetal bovine serum,FBS (FBS) or calf serum (calf serum,CS) are commonly used in laboratory culture of MSCs in vitro. There are no reports on the effects of the two serums on the aging process of MSCs. Therefore, the effect of fetal bovine serum and calf serum on the aging process of MSCs was studied. Firstly, the human umbilical cord derived MSCs, was obtained by using tissue mass adherent culture method, and the traditional tissue block adherent method was improved to obtain more primary MSCs. by secondary adherent culture of tissue mass that should have been discarded. On the 5-7 days after the first tissue mass adherent culture, there were small fusiform cells crawling out from the edge of the tissue mass, and about 10 days later, the cell clone formation was observed. The tissue mass was transferred to a new culture bottle for secondary adherent culture. After 2 days, the cells crawled out, and the cell clone was formed in 5 days. The MSCs obtained from the secondary adherent culture was detected by flow cytometry. The results showed that the cells with high expression of CD90,CD105, did not express CD34,CD45,HLA-DR, indicating that the cells obtained from the second tissue mass adhesion culture were also mesenchymal stem cells. At the same time, we compared the biological characteristics of the obtained cells with placental MSCs, and found that there was no significant difference between the two kinds of cells. For isolated umbilical cord MSCs, fetal bovine serum and calf serum were used for long-term culture in vitro from the first generation, respectively, until cell senescence. The two groups used the same inoculation density and calculated the cell number and the population multiplication number during the passage and then accumulated the cumulative population multiplication number. The cell proliferation, differentiation, apoptosis and senescence were detected in three stages of the same cumulative population multiplication. The results showed that with the increase of passage times, the proliferation and differentiation ability of MSCs decreased and the apoptosis rate increased. At the same time, the activity of 尾 -galactosidase increased, the level of ROS increased, and the expression of p53, p21, p16 and other genes increased. The results also showed that the proliferation and differentiation ability of MSCs in fetal bovine serum group was better than that in calf serum group, while the apoptosis ratio, intracellular ROS level and the expression level of senescence related genes such as p53 in fetal bovine serum group were lower than those in calf serum group. The results showed that fetal bovine serum could provide a better microenvironment for the growth of MSCs, which could help to maintain its biological characteristics and delay senescence, while calf serum could accelerate the process of cell senescence.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R329.2
【参考文献】
相关期刊论文 前1条
1 徐燕;李长虹;孟恒星;郝牧;邱录贵;;人脐带间充质干细胞分离培养条件的优化及其生物学特性[J];中国组织工程研究与临床康复;2009年32期
本文编号：2275244
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2275244.html
最近更新
教材专著
