干细胞治疗宫腔粘连抗纤维化作用机制的探索

发布时间：2018-04-12 09:16

本文选题：脐带间充质干细胞 + 输卵管内膜干细胞　；参考：《内蒙古民族大学》2017年硕士论文

【摘要】：目的评估人脐带间充质干细胞(UCMSC)、输卵管内膜干细胞(FMMSC)和子宫内膜干细胞(En MSC)的抗纤维化作用潜能,初步探索干细胞治疗宫腔粘连抗纤维化的作用机制,寻求一种更好的干细胞用于宫腔粘连抗纤维化的治疗。方法体外分离、培养及鉴定人UCMSC、En MSC和FMMSC,倒置显微镜观察其细胞形态,流式细胞仪进行细胞表型分析和周期测定,平板细胞克隆形成试验检测其增殖能力,CCK-8法判断其生长特性,核型分析检测其遗传稳定性,体外诱导分化实验证明其多向分化潜能,Real Time PCR方法检测UCMSC、En MSC和FMMSC抗纤维化相关蛋白相对表达量。结果1.三种不同组织来源的细胞接种于MSC完全培养基后,约12h即见贴壁细胞,培养至5d时,细胞融合达80%左右,所有细胞传至P5代,倒置显微镜下观察,表现为均一长梭形,并呈漩涡状生长,UC来源的细胞形态更为均一,细长,并且排列紧密,FM和En来源细胞形态立体感更强,二者形态更为相似。2.三种MSC均阳性表达CD90、CD73、CD105、CD29、CD44、CD166和HLA-ABC,阴性表达CD14、CD19、CD45、CD34和HLA-DR。3.细胞周期检测结果显示,FMMSC中处于DNA合成期(G2+S期)的细胞比例高于UCMSC和En MSC。4.平板细胞克隆形成试验结果显示,UCMSC形成的克隆团为136±26.32个,FMMSC形成的克隆团为89±5.34个,En MSC形成的克隆团为91.6±2.7个,克隆形成率分别为136%±26.32%、89%±5.34%、91.6%±2.7%。UCMSC和FMMSC、UCMSC和En MSC克隆团数目及克隆形成率差异有统计学意义(P0.01),FMMSC和En MSC克隆团数目及克隆形成率差异无统计学意义。5.三种细胞增殖曲线均呈S型。UCMSC潜伏时间较短,于24h左右即进入对数生长期。FMMSC于4-6d进入对数生长期后生长快速,于7-8d进入平台期。6.核型结果符合样本来源,此方法所培养的细胞具有遗传稳定性。7.三种干细胞在体外均具有成脂、成骨和成软骨诱导分化的能力。8.抗纤维化相关蛋白m RNA表达结果显示,FMMSC和En MSC分泌的HGF、IL-10和IFN-γ均高于UCMSC(P0.05),而FMMSC和En MSC分泌HGF、IL-10和IFN-γ无差别。三组干细胞分泌b FGF、Adrenomedullin和lefty无统计学差异,但根据数值目测应该是有差异的,目测FMMSC和En MSC分泌的b FGF相对表达量多于UCMSC,En MSC分泌的Adrenomedullin显著多于FMMSC和En MSC,FMMSC分泌的lefty多于En MSC,En MSC分泌的lefty多于UCMSC。结论1.UCMSC的增殖能力高于FMMSC和En MSC,但FMMSC的增殖速度较快;2.FMMSC和En MSC生物学特性高度相似;3.UCMSC、FMMSC和En MSC均分泌抗纤维化相关蛋白;4.FMMSC和En MSC在抗纤维化作用中,HGF、IL-10和IFN-γ发挥的比重可能要多于b FGF、Adrenomedullin和lefty,其可能主要通过HGF、IL-10和IFN-γ参与的通路来发挥抗纤维化作用。5.对于缺失En MSC的患者,FMMSC可实现MSC的自体移植。在自体移植方面,UCMSC和FMMSC比较,对于子宫内膜损伤后抗纤维化的治疗,作为更优的替代干细胞,FMMSC具有更高的临床应用价值。
[Abstract]:Objective to evaluate the antifibrotic potential of UCM SCC (UCM SCC), FMMSCC (endometrial stem cell) and en (endometrial stem cell) of human umbilical cord mesenchymal stem cells (UCM), and to explore the mechanism of anti fibrosis effect of stem cells in the treatment of intrauterine adhesions.To seek a better stem cell for the treatment of intrauterine adhesion and anti-fibrosis.Methods Human UCM SCN MSC and FMMSCS were isolated, cultured and identified in vitro. The cell morphology was observed by inverted microscope, the cell phenotype was analyzed by flow cytometry and cell cycle was determined.Karyotype analysis was used to detect its genetic stability, and its multidirectional differentiation potential was tested by Real Time PCR method. The relative expression of UCM SCN MSC and FMMSC anti-fibrosis related proteins was detected by Real Time PCR method.Result 1.The adherent cells were observed after inoculation of three different tissue sources in MSC culture medium for 12 hours. After 5 days of culture, the cells were fused to 80%, all the cells were transferred to the P5 passage, and observed under inverted microscope, the cells showed uniform spindle shape.The cells derived from UC were more uniform and slender, and the cells derived from FM and en were more stereosensitive, and the morphology of them was more similar to that of En. 2. The morphology of the cells derived from UC was more uniform and slender, and the morphology of FM and en derived cells was more stereosensitive.All three kinds of MSC were positive for CD90, CD73, CD105, CD29, CD44, CD166 and HLA-ABC, negative for CD14, CD19, CD45, CD34 and HLA-DR.3.The results of cell cycle detection showed that the proportion of FM-MSC in DNA synthesis phase was higher than that in UCMSC and en MSC.4.The results of plate cell clone formation test showed that the colony formation of UCMSC was 136 卤26.32 m MSC, 89 卤5.34 en MSC, and 91.6 卤2.7 clones formed from UCMSC.The clone formation rates were 13.6% 卤26.32 + 89% 卤5.34 and 91.6% 卤91.6% and 91.6% 卤2.7%.UCMSC, respectively. There was no significant difference in the number of clones and the clone formation rate between FMMSC-UCMSC and en MSC. There was no significant difference in colony number and colony formation rate between P0.01FM-FMMSC and en MSC.All the three cell proliferation curves showed S-type. UCMSC had a short latent time, which entered the logarithmic growth phase at about 24 h. FMMSC grew rapidly at 4-6 days after logarithmic growth, and entered the plateau stage at 7-8 days.The results of karyotype were consistent with the source of the sample, and the cells cultured by this method had genetic stability.All of the three stem cells have the ability of adipogenic, osteogenic and chondrogenic differentiation in vitro.The expression of anti-fibrosis related protein m RNA showed that the HGFN- 10 and IFN- 纬 secreted by FMMSC and en MSC were higher than those of UCMSC-P0.05, but the secretion of HGFIL-10 and IFN- 纬 by FMMSC and en MSC had no difference.There was no significant difference in the secretion of bFGFA Adomedullin and lefty among the three groups of stem cells, but there should be differences according to the numerical results.The relative expression of b FGF in FMMSC and en MSC was significantly higher than that in Adrenomedullin secreted by FMMSC and en MSC than in FMMSC and en MSCT FM MSC. The lefty secreted by EMC FMMSC and en MSC was higher than that by en MSCN MSC than by UCM SCC.Conclusion the proliferative ability of 1.UCMSC is higher than that of FMMSC and en MSC, but the proliferation rate of FMMSC is faster. 2.The biological characteristics of FMMSC and en MSC are highly similar. 3. The ratio of IFN- 纬 and IL-10 produced by FMMSC and en MSC in anti-fibrosis.The weight may be more than that of bFGFN Adrenomedullin and lefty, and it may play an anti-fibrosis role mainly through the pathway of HGF IL-10 and IFN- 纬.For patients without en MSC, MSC autografts can be achieved by FMMSC.Compared with FMMSC in autologous transplantation, UCMSC has higher clinical application value as a better substitute for stem cell FM-MSC in the treatment of endometrial injury.
【学位授予单位】：内蒙古民族大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R711.74

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