本文選題：羊膜 + 間充質(zhì)干細(xì)胞　； 參考：《蘇州大學(xué)》2009年碩士論文

【摘要】： 研究目的: 1、體外分離培養(yǎng)、純化并鑒定人羊膜間充質(zhì)干細(xì)胞(hAMSCs)。 2、研究hAMSCs對(duì)異基因混合淋巴細(xì)胞反應(yīng)(MLR)體系中淋巴細(xì)胞增殖及相關(guān)細(xì)胞因子的影響。 研究方法: 1、采用酶消化法從羊膜分離出hAMSCs,進(jìn)行傳代培養(yǎng)、逐步純化,流式細(xì)胞儀檢測(cè)細(xì)胞表面標(biāo)志,并通過條件培養(yǎng)基定向誘導(dǎo),最后根據(jù)形態(tài)學(xué)、生物學(xué)特征、細(xì)胞表面標(biāo)志以及分化潛能鑒定hAMSCs純度。 2、建立雙向MLR體系,加入絲裂霉素處理過的不同比例的hAMSCs,用3H摻入法測(cè)定淋巴細(xì)胞增殖率并用ELISA法測(cè)定混合培養(yǎng)上清液中白細(xì)胞介素-2(IL-2)和干擾素-γ(IFN-γ)的含量,觀察hAMSCs對(duì)淋巴細(xì)胞增殖和相關(guān)細(xì)胞因子的影響。 研究結(jié)果: 1、從羊膜中分離的細(xì)胞接種24小時(shí)后便有少量的細(xì)胞貼壁,經(jīng)1周后逐漸形成扁平單層細(xì)胞,呈漩渦狀生長(zhǎng)或成簇生長(zhǎng),隨著細(xì)胞密度的增加,胞體變得細(xì)長(zhǎng),形態(tài)類似成纖維細(xì)胞。培養(yǎng)的細(xì)胞可以穩(wěn)定生長(zhǎng)傳代,而且體外培養(yǎng)10代以后,細(xì)胞的增殖速度無(wú)明顯減慢。 2、hAMSCs的原代細(xì)胞在接種后2~8天后進(jìn)入對(duì)數(shù)生長(zhǎng)期,相差顯微鏡下觀察細(xì)胞突起向周圍伸展,出現(xiàn)兩個(gè)核細(xì)胞分裂相的hAMSCs多見,細(xì)胞密度增大,彼此相連;11~14天進(jìn)入平臺(tái)期,hAMSCs鋪滿瓶底,細(xì)胞擴(kuò)增趨緩,原代培養(yǎng)結(jié)束。hAMSCs傳代后P3代和P15代的生長(zhǎng)曲線顯示,隨著hAMSCs的逐漸純化,細(xì)胞倍增速度基本穩(wěn)定。傳代后6~12小時(shí),相差顯微鏡下即可觀察到細(xì)胞貼壁;2~4天后進(jìn)入到對(duì)數(shù)生長(zhǎng)期,擴(kuò)增速度明顯快于原代培養(yǎng);6~7天即可鋪滿培養(yǎng)板底。 3、hAMSCs表達(dá)有BMSCs相似的表面標(biāo)志,流式細(xì)胞儀檢測(cè)顯示,hAMSCs表達(dá)CD29、CD44和CD105,不表達(dá)CD34、CD45、HLA-DR和CD106。 4、hAMSCs向神經(jīng)細(xì)胞誘導(dǎo)24h后,細(xì)胞形態(tài)明顯改變,胞體回縮,胞核部分折光性增強(qiáng),出現(xiàn)類似于樹突及軸突樣結(jié)構(gòu),染色可見兔抗人神經(jīng)元特異性烯醇化酶(NSE)和兔抗人膠質(zhì)纖維酸性蛋白(GFAP)陽(yáng)性。hAMSCs向成骨細(xì)胞誘導(dǎo)分化2周后,細(xì)胞形態(tài)由紡錘形轉(zhuǎn)變?yōu)槎嘟切?染色可見I型膠原陽(yáng)性;3～4周后可見鈣結(jié)節(jié)形成。 5、用3H-TdR摻入法對(duì)淋巴細(xì)胞增殖進(jìn)行檢測(cè)。hAMSCs以不同的hAMSCs /外周血單個(gè)核細(xì)胞(PBMC)比例加入MLR體系共同培養(yǎng)后,各組cpm值均較培養(yǎng)前明顯下降(P均0.05);hAMSCs /PBMC比例為1?5和1?10的兩組,其增殖抑制率均明顯強(qiáng)于1?500組(P均0.05)。 6、hAMSCs與未經(jīng)活化的淋巴細(xì)胞混合培養(yǎng)( PBMC1+hAMSCs組、PBMC2+hAMSCs組)時(shí),培養(yǎng)上清中的IL-2、IFN-γ水平與單獨(dú)淋巴細(xì)胞培養(yǎng)(PBMC1組或PBMC2組)上清中的水平無(wú)明顯變化(P均0.05);hAMSCs加入MLR體系(PBMC1+ PBMC2+hAMSCs組)中培養(yǎng)時(shí),細(xì)胞因子IL-2和IFN-γ的分泌均明顯低于單純MLR體系(PBMC1+PBMC2組)中的量(P0.05)。 研究結(jié)論: 1、體外可以分離培養(yǎng)hAMSCs,hAMSCs具有與BMSCs相似的細(xì)胞生物學(xué)特性以及多向分化潛能。 2、hAMSCs可以抑制MLR體系中淋巴細(xì)胞的增殖,呈劑量依賴性;并可下調(diào)MLR體系中IL-2和IFN-γ水平,對(duì)同種異體免疫反應(yīng)具有負(fù)調(diào)節(jié)作用,為日后臨床聯(lián)合應(yīng)用MSCs和造血干細(xì)胞移植以預(yù)防和治療移植物抗宿主病(GVHD)提供理論基礎(chǔ)。 3、hAMSCs可以作為間充質(zhì)干細(xì)胞(MSCs)基礎(chǔ)及臨床應(yīng)用研究的全新來(lái)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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

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