不同年龄层SD大鼠骨髓间充质干细胞生物学特性的比较

发布时间：2018-03-06 19:29

本文选题：骨髓间充质干细胞　切入点：年龄相关　出处：《中南大学》2014年硕士论文　论文类型：学位论文

【摘要】：由于骨髓间充质干细胞(Mesenchymal Stromal Cells, MSCs)具有多向分化潜能,而且易于获得,所以近年来随着基因工程兴起,不少学者把骨髓间充质干细胞作为基因治疗靶细胞,治疗某些难治疾病,比如骨缺损、椎间盘退行性疾病、心力衰竭等等,而这些疾病的患病人群多为老年患者,可能跟衰老机体中的骨髓间充质干细胞的生长和分化发生一定改变有关,因此,探讨间充质干细胞的生物学特性,以及其功能随着年龄增长是否发生改变尤为重要。本次实验通过对不同年龄层SD大鼠的骨髓间充质干细胞的生物学特性进行比较分析,探讨年龄对间充质干细胞的影响。目的：研究不同年龄SD大鼠的骨髓间充质干细胞的生物学特性,并探讨不同年龄SD大鼠骨髓间充质干细胞生物学特性的影响。方法：分别取幼年(21天)、中年(3个月)、老年(14个月)的SD大鼠,取四肢骨髓,采用冲骨髓细胞贴壁培养法,用含10%体积分数胎牛血清(FCS)的低糖DMEM (L-DMEM)培养基,贴壁、传代、培养、纯化出MSC,使其在体外扩增生长,在倒置显微镜下观察生长特性,瑞氏染色观察细胞形态。流式细胞术检测细胞表面抗原。分别取每个年龄阶段细胞的第3代、第6代、第9代、第12代和第15代做MTT,绘制生长曲线,比较细胞的增殖能力。取每个年龄层的第3代的MSCs做成骨诱导分化和成脂肪诱导分化,观察年龄差异对其成成骨细胞的能力和成脂肪细胞的能力的影响。通过茜素红半定量法比较钙盐沉积情况。通过油红O定量法比较3个年龄层SD鼠MSCs的成脂能力。通过检测ALP的值,来比较年龄对MSCs成骨能力的影响。结果： (1)经流式细胞术鉴定出细胞表面抗原95.27±0.52CD29, CD44为阳性,4.06±0.29CD34, CD45为阴性,证明通过冲骨髓细胞贴壁培养法培养出的细胞为骨髓间充质干细胞。 (2)倒置显微镜下观察细胞形态,幼年组,中年组,老年组的SD大鼠的MSC的原代都是呈集落样生长,并且其中有许多杂细胞,经过一次胰酶消化传代后,细胞均呈梭形,不再有集落产生,第3,4代,细胞形态趋于稳定,杂细胞逐渐减少,各组间没有差异。 (3)MTT结果显示,第3、6代的3个年龄组比较,增殖能力无显著差异,但在第9代、第12代和第15代,幼年组的增殖能力显著高于中年和老年组。 (4)成脂肪细胞诱导和成骨细胞诱导的结果显示,幼年SD大鼠的间充质干细胞诱导出的钙结节明显,且数目显著多于中年组和老年组,而中年组多于老年组。诱导出的成脂肪细胞,脂泡在数量上幼年组显著多于中年组和老年组,并且更加饱满圆润。 (5)ALP检测结果得出幼年组MSCs的成骨能力优于中年组,中年组要优于老年组。结论：幼年鼠,中年鼠和老年鼠的MSC的生物学特性都具有较强的增殖能力,及成骨、成脂诱导分化能力,但是幼年组的MSC的增殖能力、成脂和成骨诱导分化能力要明显优于中年组和老年组。图9幅,参考文献106篇
[Abstract]:Because bone marrow mesenchymal Stromal cells (MSCs) have multidirectional differentiation potential and are easy to obtain, in recent years, with the rise of genetic engineering, many scholars have used bone marrow mesenchymal stem cells as target cells for gene therapy to treat some refractory diseases. For example, bone defects, intervertebral disc degenerative diseases, heart failure and so on, and most of the patients with these diseases are elderly patients, which may be related to the growth and differentiation of bone marrow mesenchymal stem cells in the aging organism, so, It is particularly important to investigate the biological characteristics of mesenchymal stem cells and whether their functions change with age. The biological characteristics of bone marrow mesenchymal stem cells in SD rats of different age groups were compared and analyzed. Objective: to investigate the effect of age on mesenchymal stem cells (MSCs). To study the biological characteristics of bone marrow mesenchymal stem cells (BMSCs) in SD rats of different ages and to explore the effects of different ages on the biological characteristics of BMSCs. Sprague-Dawley rats with 21 days of infancy, middle age (3 months, 14 months) were taken from bone marrow of limbs. Bone marrow cells were flushed and cultured on low sugar DMEM L-DMEM medium containing 10% volume fraction fetal bovine serum. MSCI was purified to grow in vitro, growth characteristics were observed under inverted microscope, cell morphology was observed by Rayleigh staining, and cell surface antigen was detected by flow cytometry. MTTs of the 12th and 15th generations were used to draw the growth curve to compare the proliferation ability of the cells. The third generation of MSCs from each age group was used to induce bone differentiation and adipogenic differentiation. To observe the effect of age difference on the ability of osteoblasts and adipoblasts. To compare the calcium deposition by alizarin red semi-quantitative method, and to compare the fat-forming ability of MSCs of three age groups SD rats by oil red O method. By detecting the value of ALP, To compare the effect of age on MSCs osteogenesis. Results:. Cell surface antigen 95.27 卤0.52CD29, CD44 positive 4.06 卤0.29CD34, CD45 negative, and bone marrow mesenchymal stem cells (BMSCs) identified by flow cytometry were proved to be bone marrow mesenchymal stem cells. (2) the morphology of the cells was observed under inverted microscope. The primary MSC of SD rats in juvenile group, middle age group and aged group all had colony like growth, and there were many mixed cells in them. After one passage of trypsin digestion, the cells were all fusiform. There was no colony formation, but in the 3rd and 4th generation, the cell morphology tended to be stable, and the hybrids decreased gradually, but there was no difference among the three groups. The results of MTT showed that there was no significant difference in proliferative ability among the three age groups in the 3rd and 6th generation, but in the 9th, 12th and 15th generation, the proliferative ability of the young group was significantly higher than that of the middle age group and the old age group. 4) the results of adipoblast induction and osteoblast induction showed that the number of calcium nodules induced by mesenchymal stem cells in young SD rats was significantly higher than that in middle-aged and elderly groups. But the number of adipoblasts induced in middle age group was more than that in old age group, and the number of lipid bubble in juvenile group was significantly more than that in middle age group and old age group, and it was more full and round. The results showed that the osteogenic ability of MSCs in young group was superior to that in middle age group, and that in middle age group was superior to that in old group. The biological characteristics of MSC in juvenile, middle-aged and aged mice have strong proliferative ability, osteogenesis and adipogenic differentiation ability, but the proliferation ability of MSC in juvenile group. The ability of adipogenic and osteogenic induction was better than that of middle age group and old age group.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R329

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