骨髓间充质干细胞移植在小鼠卵巢功能重建中的应用

发布时间：2018-06-03 22:11

本文选题：骨髓间充质干细胞 + 干细胞移植　；参考：《安徽医科大学》2014年硕士论文

【摘要】：目的：建立一个高效的骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs）体外分离和纯化的方法。方法:取4周龄雌性ICR小鼠的胫骨和股骨进行全骨髓细胞培养，，采用培养初期换液和减少细胞传代时胰蛋白酶消化时间（1min）富集BMSCs，通过形态学观察和流式细胞术检测细胞表面特异性干细胞标记物（CD29、CD44、CD45），鉴定其性质和纯度。结果:①形态学观察原代细胞培养24h可见少量散在分布、呈梭形的贴壁细胞；培养3d后，贴壁细胞呈集落样生长，贴壁面积达30%，集落间可见以红细胞为主的圆形未贴壁细胞；培养7d后，贴壁细胞排列紧密，呈梭形或树突状，细胞贴壁面积达80%。第1次传代培养7d后，贴壁细胞几乎铺满培养皿底，排列密集，贴壁面积达90%；第3次传代培养5d后，梭形贴壁细胞铺满皿底，贴壁面积达100%，细胞饱满、大小均一，呈旋涡状排列。②B MSCs细胞表面标志物鉴定第3代BMSCs特异性表面标志物CD29、CD44的阳性率分别为99.24%和97.43%，而CD45呈低表达，仅为0.21%。结论：原代培养的BMSCs经传代后生长更加迅速和稳定；在培养初期通过增加换液次数、减少细胞传代胰蛋白酶消化时间可获取高纯度的BMSCs。目的：探讨骨髓间充质干细胞（BMSCs）移植在卵巢功能重建中的应用价值。方法：采用环磷酰胺（CTX）120mg/kg和白消安（BUS）12mg/kg一次性腹腔注射建立卵巢早衰小鼠模型。按处理方案分为建模移植组、建模对照组和空白对照组，建模移植组于建模14d经尾静脉注射0.1ml绿色荧光蛋白（green fluorescentprotein, GFP）转基因鼠BMSCs细胞悬液（5×106），建模对照组于建模14d尾静脉注射等量生理盐水，以均注射生理盐水的正常健康雌性小鼠作为空白对照组。观察小鼠的一般情况、卵巢内卵泡数量、血清性激素FSH和E2水平，镜下观察建模移植组小鼠卵巢的组织学变化以及是否有绿色荧光分布。结果：①小鼠一般情况建模组小鼠食欲明显减退，嗜睡及活动减少，体重随着时间推移明显下降，建模14d体重约减轻至建模前的一半，显著低于空白对照组；BMSCs移植后，小鼠体重平缓上升，食欲增强，活动增多。②血清FSH和E2水平测定空白对照组注射生理盐水前后无明显变化；建模后14d，FSH水平显著高于空白对照组，E2水平则显著降低；BMSCs移植后，FSH水平呈逐渐下降、E2呈逐渐上升趋势，移植后28d FSH和E2水平与空白对照组间无显著性差异。③卵巢组织学观察建模14d后，总卵泡数、原始卵泡、生长卵泡和成熟卵泡均低于空白对照组，且闭锁卵泡增多。移植后28d，各级卵泡数均显著高于建模对照组，闭锁卵泡减少。④荧光显微镜观察BMSCs移植后28d，荧光显微镜下可见卵泡周围散在分布颗粒状绿色荧光。结论：骨髓间充质干细胞可定位于卵巢组织，对受损卵巢组织有修复作用，并可明显改善卵巢的内分泌功能。
[Abstract]:Aim: to establish an efficient method for isolation and purification of bone marrow mesenchymal stem cells (BMSCs) in vitro. Methods: the whole bone marrow cells were cultured from the tibia and femur of 4-week-old female ICR mice. BMSCs were enriched with trypsin digestion time (1 min). The cell surface specific stem cell marker, CD29, CD44and CD45, was detected by morphological observation and flow cytometry, and its properties and purity were identified. Results morphological observation showed that a small number of adherent cells were scattered in primary culture for 24 hours, and the adherent cells grew like colony after 3 days of culture, and the adhesion area was 30%, and the round unadherent cells mainly red blood cells could be seen in the colonies. After 7 days of culture, the adherent cells were closely arranged, fusiform or dendritic, and the adherent area of the cells was 80%. After the first passage for 7 days, the adherent cells were almost covered with the bottom of the petri dish, arranged densely, and the adherent area reached 90. After the third passage for 5 days, the fusiform adherent cells were covered with the bottom of the dish, and the adherent area was 100. The cells were full and uniform in size. The positive rates of CD29 and CD44 in the third generation of BMSCs were 99.24% and 97.43%, respectively, while the expression of CD45 was low (0.21%). Conclusion: the primary cultured BMSCs can grow faster and more steadily after passage, and high purity BMSCs can be obtained by increasing the number of liquid exchange at the beginning of culture and reducing the digestion time of passage trypsin. Objective: to investigate the value of bone marrow mesenchymal stem cells (BMSCs) transplantation in ovarian functional reconstruction. Methods: the mouse model of premature ovarian failure was established by intraperitoneal injection of CTX 120 mg / kg and BuUS 12 mg / kg. According to the treatment plan, it is divided into three groups: modeling transplantation group, modeling control group and blank control group. The BMSCs cell suspension of 0.1ml green fluorescent protein (GFP) transgenic mice was injected into the tail vein for 14 days in the model transplantation group and the model control group was injected with the same amount of normal saline through the tail vein on the 14th day. The normal female mice injected with normal saline were used as the blank control group. The general situation of the mice, the number of follicles in the ovary, the serum levels of FSH and E2, the histological changes of the ovaries and the distribution of green fluorescence in the transplanted mice were observed under microscope. Results in the control group, the appetite, lethargy and activity decreased significantly, and the body weight decreased significantly with time. The weight of the model group decreased to about half of that of the control group at the 14th day, which was significantly lower than that of the control group after BMSCs transplantation. The level of serum FSH and E2 in the blank control group did not change significantly before and after the injection of normal saline. On the 14th day after modeling, the level of FSH was significantly higher than that of the control group (P < 0.05), but the level of FSH decreased gradually after transplantation of BMSCs, and the level of FSH increased gradually after transplantation. There was no significant difference in the levels of FSH and E2 between the control group and the control group on the 28th day after transplantation, the total number of follicles, primordial follicles, growth follicles and mature follicles were lower than those in the control group, and the number of atretic follicles increased. On the 28th day after transplantation, the number of follicles at all levels was significantly higher than that in the model control group. At 28 days after BMSCs transplantation, the number of follicles at all levels was significantly higher than that in the model control group. Conclusion: bone marrow mesenchymal stem cells can locate in ovarian tissue, repair damaged ovarian tissue and improve endocrine function of ovary.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R711.75

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