人羊膜间充质干细胞分离培养鉴定及抑制异基因淋巴细胞增殖的实验研究

发布时间：2018-06-08 02:06

本文选题：羊膜 + 间充质干细胞　；参考：《苏州大学》2009年硕士论文

【摘要】： 研究目的: 1、体外分离培养、纯化并鉴定人羊膜间充质干细胞(hAMSCs)。 2、研究hAMSCs对异基因混合淋巴细胞反应(MLR)体系中淋巴细胞增殖及相关细胞因子的影响。研究方法: 1、采用酶消化法从羊膜分离出hAMSCs,进行传代培养、逐步纯化,流式细胞仪检测细胞表面标志,并通过条件培养基定向诱导,最后根据形态学、生物学特征、细胞表面标志以及分化潜能鉴定hAMSCs纯度。 2、建立双向MLR体系,加入丝裂霉素处理过的不同比例的hAMSCs,用3H掺入法测定淋巴细胞增殖率并用ELISA法测定混合培养上清液中白细胞介素-2(IL-2)和干扰素-γ(IFN-γ)的含量,观察hAMSCs对淋巴细胞增殖和相关细胞因子的影响。研究结果: 1、从羊膜中分离的细胞接种24小时后便有少量的细胞贴壁,经1周后逐渐形成扁平单层细胞,呈漩涡状生长或成簇生长,随着细胞密度的增加,胞体变得细长,形态类似成纤维细胞。培养的细胞可以稳定生长传代,而且体外培养10代以后,细胞的增殖速度无明显减慢。 2、hAMSCs的原代细胞在接种后2~8天后进入对数生长期,相差显微镜下观察细胞突起向周围伸展,出现两个核细胞分裂相的hAMSCs多见,细胞密度增大,彼此相连;11~14天进入平台期,hAMSCs铺满瓶底,细胞扩增趋缓,原代培养结束。hAMSCs传代后P3代和P15代的生长曲线显示,随着hAMSCs的逐渐纯化,细胞倍增速度基本稳定。传代后6~12小时,相差显微镜下即可观察到细胞贴壁;2~4天后进入到对数生长期,扩增速度明显快于原代培养;6~7天即可铺满培养板底。 3、hAMSCs表达有BMSCs相似的表面标志,流式细胞仪检测显示,hAMSCs表达CD29、CD44和CD105,不表达CD34、CD45、HLA-DR和CD106。 4、hAMSCs向神经细胞诱导24h后,细胞形态明显改变,胞体回缩,胞核部分折光性增强,出现类似于树突及轴突样结构,染色可见兔抗人神经元特异性烯醇化酶(NSE)和兔抗人胶质纤维酸性蛋白(GFAP)阳性。hAMSCs向成骨细胞诱导分化2周后,细胞形态由纺锤形转变为多角形,染色可见I型胶原阳性;3～4周后可见钙结节形成。 5、用3H-TdR掺入法对淋巴细胞增殖进行检测。hAMSCs以不同的hAMSCs /外周血单个核细胞(PBMC)比例加入MLR体系共同培养后,各组cpm值均较培养前明显下降(P均0.05);hAMSCs /PBMC比例为1?5和1?10的两组,其增殖抑制率均明显强于1?500组(P均0.05)。 6、hAMSCs与未经活化的淋巴细胞混合培养( PBMC1+hAMSCs组、PBMC2+hAMSCs组)时,培养上清中的IL-2、IFN-γ水平与单独淋巴细胞培养(PBMC1组或PBMC2组)上清中的水平无明显变化(P均0.05);hAMSCs加入MLR体系(PBMC1+ PBMC2+hAMSCs组)中培养时,细胞因子IL-2和IFN-γ的分泌均明显低于单纯MLR体系(PBMC1+PBMC2组)中的量(P0.05)。研究结论: 1、体外可以分离培养hAMSCs,hAMSCs具有与BMSCs相似的细胞生物学特性以及多向分化潜能。 2、hAMSCs可以抑制MLR体系中淋巴细胞的增殖,呈剂量依赖性;并可下调MLR体系中IL-2和IFN-γ水平,对同种异体免疫反应具有负调节作用,为日后临床联合应用MSCs和造血干细胞移植以预防和治疗移植物抗宿主病(GVHD)提供理论基础。 3、hAMSCs可以作为间充质干细胞(MSCs)基础及临床应用研究的全新来源。
[Abstract]:The purpose of the study is:
1, in vitro isolation and culture, purification and identification of human amniotic mesenchymal stem cells (hAMSCs).
2, we studied the effects of hAMSCs on lymphocyte proliferation and related cytokines in allogeneic mixed lymphocyte reaction (MLR) system.
Research methods:
1, hAMSCs was isolated from amniotic membrane by enzyme digestion. It was cultured and purified gradually. Flow cytometry was used to detect the cell surface markers, and it was directed by the conditioned medium. Finally, the purity of hAMSCs was identified according to morphology, biological characteristics, cell surface markers and differentiation potential.
2, a bi-directional MLR system was established, and hAMSCs treated with mitomycin was added. The proliferation rate of lymphocyte was measured by 3H incorporation and the content of interleukin -2 (IL-2) and interferon gamma (IFN- gamma) in mixed culture supernatant were measured by ELISA method. The effects of hAMSCs on the proliferation of lymphoblastic cells and related cytokines were observed.
The results of the study:
1, the cells separated from the amniotic membrane were inoculated for 24 hours and then a small number of cells were adhered to the wall. After 1 weeks, the flat monolayer cells were gradually formed to grow or grow in a whirlpool. With the increase of cell density, the cells became elongated and similar to fibroblasts. The cultured cells could be passaged with stable growth, and after 10 generations in vitro culture, the cells were cultured in vitro. The speed of cell proliferation did not slow down.
2, the primary cells of hAMSCs entered the logarithmic growth period after 2~8 days after inoculation. Under the phase contrast microscope, the cell protrusions were observed to extend around, and the hAMSCs of two nuclear cell division phases appeared, the cell density increased, and the cell density was connected to each other; the 11~14 days entered the platform stage, the hAMSCs was filled with the bottle bottom, the cell amplification slowed down, and the primary culture ended the.HAMSCs passage after P3 generation. The growth curve of the P15 generation showed that the cell multiplication speed was basically stable with the gradual purification of hAMSCs. After 6~12 hours, the cell wall was observed under the phase contrast microscope; 2~4 days entered the logarithmic growth period, and the amplification rate was faster than that of the original culture; 6~7 days could fill the bottom of the culture plate.
3, hAMSCs showed BMSCs similar surface markers. Flow cytometry showed that hAMSCs expressed CD29, CD44 and CD105, and did not express CD34, CD45, HLA-DR and CD106..
4, after hAMSCs induced 24h to the nerve cells, the cell morphology changed obviously, the cell body retracted and the nucleus was partially refracted, which appeared similar to the dendrite and axon like structure. The staining showed that the Rabbit anti human neuron specific enolase (NSE) and the Rabbit anti human glial fibrillary acidic protein (GFAP) positive.HAMSCs were induced to differentiate into osteoblasts for 2 weeks. The spindle was transformed into polygon, and I collagen was positive in staining. Calcium nodules were formed after 3~4 weeks.
5, the proliferation of lymphocyte proliferation was detected by 3H-TdR incorporation and.HAMSCs was co cultured with different hAMSCs / peripheral blood mononuclear cells (PBMC) in MLR system. The CPM values of each group were significantly lower than those before the culture (P 0.05); hAMSCs /PBMC ratio was 1? 5 and two groups of 1 10. The proliferation inhibition rate was significantly stronger than the 1? 500 group (P 0.05).
6, when hAMSCs was mixed with unactivated lymphocytes (group PBMC1+hAMSCs, group PBMC2+hAMSCs), the level of IL-2 in the culture supernatant and the level of IFN- gamma in the supernatant of individual lymphocyte culture (group PBMC1 or PBMC2 group) had no significant changes (P 0.05); hAMSCs joined the MLR body system (PBMC1+ PBMC2+hAMSCs group). The secretion was significantly lower than that in the simple MLR system (group PBMC1+PBMC2) (P0.05).
The conclusions are as follows:
1, hAMSCs can be isolated and cultured in vitro. HAMSCs has similar biological characteristics and multipotential differentiation with BMSCs.
2, hAMSCs can inhibit the proliferation of lymphocytes in the MLR system and be dose-dependent, and can reduce the level of IL-2 and IFN- gamma in the MLR system, and have a negative regulatory effect on the allogeneic immune response. It provides a theoretical basis for the combined application of MSCs and hematopoietic stem cell transplantation to prevent and treat graft-versus-host disease (GVHD) in the future.
3, hAMSCs can be used as a new source of mesenchymal stem cells (MSCs) for basic and clinical applications.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

【参考文献】