混合人脐带血清对骨髓间充质干细胞增殖的影响

发布时间：2018-05-30 23:41

本文选题：间充质干细胞 + 脐带血清　；参考：《中南大学》2012年硕士论文

【摘要】：研究背景及目的：间充质干细胞(Mesenchymal stem cells, MSCs)是一种来源于中胚层,具有自我复制能力和多向分化潜能的成体干细胞。目前已成为组织修复工程和再生医学理想的种子细胞,具有巨大的临床应用前景。目前,国内外间充质干细胞的体外培养方法有多种,但传统的体外培养都需要添加胎牛血清或小牛血清。但胎牛血清增加了病毒或细菌感染风险,异体蛋白输入人体同时也增加了发生过敏反应的危险。因此,寻找“人源化”胎牛血清替代品成为研究热点。人脐带血血清中含有丰富的细胞因子,具有采集方便,来源广泛,对母亲及胎儿无影响,CMV、EB病毒污染率低,低免疫性等优势,因此为临床应用提供了一种更安全高效的培养方法。本研究工作分两部分：第一部分：混合人脐带血清、自体血清与胎牛血清培养的骨髓间充质干细胞的生物学特性的鉴定及各组增殖效能的比较方法：无菌条件下取10例人骨髓标本,采用密度梯度离心法提取单个核细胞,分别用含体积分数为10%的混合人脐带血清、10%的自体血清、10%的胎牛血清的DMEM/F12培养基,行骨髓间充质干细胞培养,观察细胞形态,取第3代细胞,测定细胞表面标志及行成骨、脂肪诱导分化,以鉴定MSCs的生物学特性,并比较在三组培养体系中MSCs细胞的增殖效能。结果：混合人脐带血清组、自体血清组与胎牛血清组培养的MSCs细胞形态上无明显差异；混合人脐带血清组培养的第3代MSCs表达CD73、CD105的阳性率均在95%以上,高于自体血清组、FBS血清组(P0.05),而CD34、CD45的阳性率各组均在2%以下；混合人脐带血清组、自体血清组、FBS组培养的骨髓MSCs诱导为成骨细胞、脂肪细胞阳性率差异无统计学意义(P0.05)。混合人脐带血清组在增殖速度和细胞数量上的优势明显,胎牛血清次之,自体血清组较差。结论：混合人脐带血清组、自体血清组、FBS组培养的MSCs经鉴定均符合MSCs诊断标准；在混合人脐带血清培养体系中,MSCs增殖效能优于FBS、自体血清组。第二部分：不同体积分数混合人脐带血清对间充质干细胞增殖的影响方法：无菌条件下取10例人骨髓标本,采用密度梯度离心法提取单个核细胞,分别用体积分数为5%、7.5%、10%、15%、20%的脐血清+DMEM/F12培养基,行骨髓间充质细胞培养,在原代培养及第三代培养末行细胞计数,取第3代细胞,用MTT法观察不同体积分数的脐血清对MSCs的增殖能力的影响。结果：在7.5%、10%体积分数的脐血清组,扩增的细胞数量以及细胞增殖能力优于5%、15%、20%体积分数的脐血清组,差异有统计学意义(P0.05)。结论：在7.5%、10%体积分数脐血清培养体系中,MSCs增殖能力较佳。
[Abstract]:Background and objective: mesenchymal stem cells, MSCs) is a kind of adult stem cells derived from mesoderm, which has the ability of self-replication and multi-differentiation. At present, it has become an ideal seed cell for tissue repair engineering and regenerative medicine, and has a great prospect of clinical application. At present, there are many methods in vitro culture of mesenchymal stem cells at home and abroad, but the traditional culture in vitro all need to add fetal bovine serum or calf serum. But fetal bovine serum increases the risk of viral or bacterial infection, and the risk of allergic reactions is also increased by the introduction of alloproteins. Therefore, the search for a "humanized" fetal bovine serum substitute has become a research hotspot. Human umbilical cord blood serum is rich in cytokines, which has the advantages of convenient collection, wide source, low contamination rate of CMV EB virus and low immunity to mother and fetus. Therefore, it provides a more safe and efficient culture method for clinical application. This research is divided into two parts: Part one: identification of biological characteristics of bone marrow mesenchymal stem cells cultured with mixed human umbilical cord blood serum autologous serum and fetal bovine serum and comparison of proliferative efficacy among groups Methods: the mononuclear cells were extracted from 10 human bone marrow specimens under aseptic condition by density gradient centrifugation. The DMEM/F12 medium containing 10% autologous serum and 10% fetal bovine serum containing 10% volume fraction of human umbilical cord blood was used to extract mononuclear cells. Bone marrow mesenchymal stem cells (BMSCs) were cultured, cell morphology was observed, cell surface markers were measured, osteogenesis and adipogenic differentiation were performed to identify the biological characteristics of MSCs, and the proliferative efficiency of MSCs cells in the three groups was compared. Results: there was no significant difference in morphology of MSCs cells between autologous serum group and fetal bovine serum group in mixed human umbilical cord blood serum group, and the positive rate of CD73 CD105 expression in the third generation of mixed human umbilical cord blood serum group was over 95%. The positive rate of CD34 + CD45 was less than 2% in all groups, and the positive rate of bone marrow MSCs was induced into osteoblasts in mixed human umbilical cord blood serum group. There was no significant difference in the positive rate of adipocytes between the two groups. The mixed human umbilical cord blood serum group had obvious advantages in proliferation rate and cell number, followed by fetal bovine serum and autologous serum group. Conclusion: the MSCs cultured in the mixed human umbilical cord blood serum group and autologous serum group were all in accordance with the diagnostic criteria of MSCs, and the proliferative efficacy of MSCs in the mixed human umbilical cord blood serum culture system was better than that in the autologous serum group. The second part: the effect of mixed human umbilical cord blood serum with different volume fraction on the proliferation of mesenchymal stem cells Methods: the mononuclear cells were extracted from 10 human bone marrow specimens under aseptic condition by density gradient centrifugation. Bone marrow mesenchymal cells were cultured in DMEM/F12 medium with volume fraction of 5 and 7.5% respectively. Cells were counted at the end of primary and third generation culture. The effect of cord blood serum with different volume fraction on the proliferation of MSCs was observed by MTT method. Results: in 7. 5% 10% volume fraction umbilical cord blood serum group, the number of expanded cells and the cell proliferation ability were better than those of 5 15% and 20% volume fraction umbilical cord blood serum group, the difference was statistically significant (P 0. 05). Conclusion: in the culture system of 7.5% 10% volume fraction of umbilical cord blood, the proliferative ability of MSCs is better.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

【相似文献】