本文選題：臍帶間充質(zhì)干細胞 + 輸卵管內(nèi)膜干細胞��； 參考：《內(nèi)蒙古民族大學(xué)》2017年碩士論文

【摘要】：目的評估人臍帶間充質(zhì)干細胞(UCMSC)、輸卵管內(nèi)膜干細胞(FMMSC)和子宮內(nèi)膜干細胞(En MSC)的抗纖維化作用潛能,初步探索干細胞治療宮腔粘連抗纖維化的作用機制,尋求一種更好的干細胞用于宮腔粘連抗纖維化的治療。方法體外分離、培養(yǎng)及鑒定人UCMSC、En MSC和FMMSC,倒置顯微鏡觀察其細胞形態(tài),流式細胞儀進行細胞表型分析和周期測定,平板細胞克隆形成試驗檢測其增殖能力,CCK-8法判斷其生長特性,核型分析檢測其遺傳穩(wěn)定性,體外誘導(dǎo)分化實驗證明其多向分化潛能,Real Time PCR方法檢測UCMSC、En MSC和FMMSC抗纖維化相關(guān)蛋白相對表達量。結(jié)果1.三種不同組織來源的細胞接種于MSC完全培養(yǎng)基后,約12h即見貼壁細胞,培養(yǎng)至5d時,細胞融合達80%左右,所有細胞傳至P5代,倒置顯微鏡下觀察,表現(xiàn)為均一長梭形,并呈漩渦狀生長,UC來源的細胞形態(tài)更為均一,細長,并且排列緊密,FM和En來源細胞形態(tài)立體感更強,二者形態(tài)更為相似。2.三種MSC均陽性表達CD90、CD73、CD105、CD29、CD44、CD166和HLA-ABC,陰性表達CD14、CD19、CD45、CD34和HLA-DR。3.細胞周期檢測結(jié)果顯示,FMMSC中處于DNA合成期(G2+S期)的細胞比例高于UCMSC和En MSC。4.平板細胞克隆形成試驗結(jié)果顯示,UCMSC形成的克隆團為136±26.32個,FMMSC形成的克隆團為89±5.34個,En MSC形成的克隆團為91.6±2.7個,克隆形成率分別為136%±26.32%、89%±5.34%、91.6%±2.7%。UCMSC和FMMSC、UCMSC和En MSC克隆團數(shù)目及克隆形成率差異有統(tǒng)計學(xué)意義(P0.01),FMMSC和En MSC克隆團數(shù)目及克隆形成率差異無統(tǒng)計學(xué)意義。5.三種細胞增殖曲線均呈S型。UCMSC潛伏時間較短,于24h左右即進入對數(shù)生長期。FMMSC于4-6d進入對數(shù)生長期后生長快速,于7-8d進入平臺期。6.核型結(jié)果符合樣本來源,此方法所培養(yǎng)的細胞具有遺傳穩(wěn)定性。7.三種干細胞在體外均具有成脂、成骨和成軟骨誘導(dǎo)分化的能力。8.抗纖維化相關(guān)蛋白m RNA表達結(jié)果顯示,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無差別。三組干細胞分泌b FGF、Adrenomedullin和lefty無統(tǒng)計學(xué)差異,但根據(jù)數(shù)值目測應(yīng)該是有差異的,目測FMMSC和En MSC分泌的b FGF相對表達量多于UCMSC,En MSC分泌的Adrenomedullin顯著多于FMMSC和En MSC,FMMSC分泌的lefty多于En MSC,En MSC分泌的lefty多于UCMSC。結(jié)論1.UCMSC的增殖能力高于FMMSC和En MSC,但FMMSC的增殖速度較快;2.FMMSC和En MSC生物學(xué)特性高度相似;3.UCMSC、FMMSC和En MSC均分泌抗纖維化相關(guān)蛋白;4.FMMSC和En MSC在抗纖維化作用中,HGF、IL-10和IFN-γ發(fā)揮的比重可能要多于b FGF、Adrenomedullin和lefty,其可能主要通過HGF、IL-10和IFN-γ參與的通路來發(fā)揮抗纖維化作用。5.對于缺失En MSC的患者,FMMSC可實現(xiàn)MSC的自體移植。在自體移植方面,UCMSC和FMMSC比較,對于子宮內(nèi)膜損傷后抗纖維化的治療,作為更優(yōu)的替代干細胞,FMMSC具有更高的臨床應(yīng)用價值。
[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 鹵26.32 m MSC, 89 鹵5.34 en MSC, and 91.6 鹵2.7 clones formed from UCMSC.The clone formation rates were 13.6% 鹵26.32 + 89% 鹵5.34 and 91.6% 鹵91.6% and 91.6% 鹵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- 緯 secreted by FMMSC and en MSC were higher than those of UCMSC-P0.05, but the secretion of HGFIL-10 and IFN- 緯 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- 緯 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- 緯.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.
【學(xué)位授予單位】：內(nèi)蒙古民族大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R711.74

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