本文選題：臍帶 + 基質(zhì)干細(xì)胞　； 參考：《第三軍醫(yī)大學(xué)》2009年碩士論文

【摘要】： 目的:1.建立分離培養(yǎng)臍帶基質(zhì)干細(xì)胞(umbilical cord matrix stem cells , UC-MSCs)的方法,了解其生物學(xué)特性及分化潛能。2.探討體外微環(huán)境對UC-MSCs向生殖細(xì)胞分化的影響。3.觀察UC-MSCs移植后在小鼠早衰卵巢中的分布及生長,探討體內(nèi)微環(huán)境對UC-MSCs分化的影響。 方法:1.無菌條件下取正常足月順產(chǎn)新生兒的臍帶,用Ⅰ型膠原酶消化,分離單個核細(xì)胞,DMEM培養(yǎng),獲得貼壁細(xì)胞,監(jiān)測細(xì)胞生長曲線、流式細(xì)胞儀檢測其免疫表型。采用不同條件培養(yǎng)基誘導(dǎo)細(xì)胞向成脂、成骨、成肌方向分化2.采用兩種方法對UC-MSCs進(jìn)行誘導(dǎo)分化,觀察體外微環(huán)境對UC-MSCs分化的影響。①類胚體誘導(dǎo):將UC-MSCs進(jìn)行懸滴培養(yǎng),使其形成類胚體(embryonic body,EB),進(jìn)而采用將EB與人或小鼠卵巢顆粒細(xì)胞共培養(yǎng)、卵泡液條件培養(yǎng)基培養(yǎng)等方法,體外誘導(dǎo)UC-MSCs向早期生殖細(xì)胞分化。通過RT-PCR方法檢測特異性基因的表達(dá),流式細(xì)胞術(shù)和免疫組化檢測其免疫表型,觀察是否有生殖系特異性標(biāo)記物的出現(xiàn)。②單層細(xì)胞誘導(dǎo):采用卵泡液作為條件培養(yǎng)基,體外誘導(dǎo)UC-MSCs向生殖細(xì)胞分化。3.通過60COγ射線照射處理建立卵巢早衰(Premature ovarian failure , POF)動物模型,慢病毒介導(dǎo)綠色熒光蛋白(green fluorescent protein ,GFP)轉(zhuǎn)染UC-MSCs,經(jīng)尾靜脈移植于POF小鼠體內(nèi)。在不同時間點取材,通過共聚焦顯微鏡和熒光原位雜交(Fluorescence in situ hybridization ,FISH)方法檢測移植細(xì)胞在小鼠體內(nèi)的分布和生長情況;通過計數(shù)非閉鎖始基卵泡數(shù)目和測定雌激素水平等方法觀察卵巢的損傷修復(fù)情況。 結(jié)果:1.分離的臍帶單個核細(xì)胞培養(yǎng)后呈紡錘體樣,體外增殖達(dá)10代以上,P3代細(xì)胞中80以上處于G0/ G1期,約20%的細(xì)胞處于S + G2 +M期。其分子免疫表型為:CD44、CD73、CD90、CD105陽性,CD31、CD34、CD45、HLA-DR陰性,與骨髓間充質(zhì)干細(xì)胞的免疫表型相似。在不同的誘導(dǎo)條件下,UC-MSCs可向成脂、成骨、成肌方向分化。 2.誘導(dǎo)分化培養(yǎng)結(jié)果①將UC-MSCs進(jìn)行懸滴培養(yǎng),其能夠形成EB。②RT-PCR結(jié)果發(fā)現(xiàn): EB形成后5天表達(dá)生殖系標(biāo)記物Oct-4、Ifitm-3、stella、vasa、DAZL,作為對照的UC-MSCs僅表達(dá)Oct-4、Ifitm-3、stella。③流式細(xì)胞術(shù)檢測結(jié)果:EB形成5天后,其中SSEA-1陽性細(xì)胞占15.61%。④免疫組化檢測結(jié)果:形成后5天的EB與人或小鼠的卵巢顆粒細(xì)胞共培養(yǎng),10天后生殖系標(biāo)記物stella、vasa、SCP3、GDF-9陽性表達(dá),而顆粒細(xì)胞及采用卵泡液培養(yǎng)的EB均無表達(dá)。⑤單層細(xì)胞培養(yǎng)誘導(dǎo):UC-MSCs用含5%卵泡液培養(yǎng)基誘導(dǎo)20天后,細(xì)胞卷曲形成類組織樣團(tuán)塊,團(tuán)塊周邊不斷有懸浮細(xì)胞生成并隨之脫落,但未檢測到生殖系特異性標(biāo)記物的表達(dá)。3.①60COγ射線處理后病理切片提示:小鼠短期內(nèi)即出現(xiàn)卵巢儲備下降、卵泡閉鎖、卵巢萎縮等POF樣病變,心臟、肝臟、脾臟、肺臟、腎臟、腸道、大腦、骨髓等組織未見明顯病理損傷,說明模型構(gòu)建成功。②慢病毒介導(dǎo)GFP轉(zhuǎn)染UC-MSCs的效率達(dá)80%以上;UC-MSCs經(jīng)尾靜脈移植后,在受損小鼠卵巢中可發(fā)現(xiàn)大量GFP陽性細(xì)胞存活并生長,但在心臟、肝臟、脾臟、肺臟、腎臟、腸道、大腦、骨髓等組織中均未發(fā)現(xiàn)GFP陽性細(xì)胞存在。③卵泡計數(shù)和雌激素水平均提示干細(xì)胞移植組小鼠卵巢損傷修復(fù)情況比對照組小鼠修復(fù)情況好。 結(jié)論:1.人UC-MSCs可在體外培養(yǎng)、擴(kuò)增,細(xì)胞免疫表型與BM-MSCs相似,具有成脂、成骨、成肌等多向分化潛能,是一種理想的成體干細(xì)胞來源。2.UC-MSC體外懸滴培養(yǎng)可形成EB,與人或小鼠卵巢顆粒細(xì)胞共培養(yǎng)后均表達(dá)生殖系特異性標(biāo)記物,提示體外微環(huán)境對UC-MSCs向生殖細(xì)胞方向分化發(fā)揮十分重要的作用,初步表明UC-MSCs具有向早期生殖細(xì)胞分化的潛能。3.UC-MSCs移植后能夠遷移至功能早衰卵巢并存活生長,卵泡計數(shù)和E2水平提示UC-MSCs可能具有卵巢損傷修復(fù)作用,表明體內(nèi)微環(huán)境對UC-MSCs的的分化也發(fā)揮重要作用。
[Abstract]:Objective: 1. to establish a method of isolation and culture of umbilical cord matrix stem cells (UC-MSCs), and to understand its biological characteristics and differentiation potential.2. to explore the effect of.2. on the differentiation of UC-MSCs to germ cells in vitro..3. observation of the distribution and growth of UC-MSCs after UC-MSCs transplantation in the premature ovarian failure of mice, and the study of the microenvironment in vivo to UC. The effect of -MSCs differentiation.
Methods: 1. the umbilical cord of normal full-term newborns was taken under aseptic condition. The mononuclear cells were digested with type I collagenase, and the mononuclear cells were isolated and cultured in DMEM. The adherent cells were obtained. The cell growth curve was monitored. The immunophenotype was detected by flow cytometry. Two methods were used to induce the cells to be fat, osteogenic and myogenic differentiation by different conditions. UC-MSCs was induced and differentiated, and the effect of microenvironment in vitro on UC-MSCs differentiation was observed. (1) embryoid body induction: UC-MSCs was cultured to form embryo like body (embryonic body, EB), and the methods of co culture of EB with human or mouse ovarian granulosa cells and culture medium culture of follicle liquid were used to induce early reproduction of UC-MSCs to early reproduction. Cell differentiation. The expression of specific genes was detected by RT-PCR, flow cytometry and immunohistochemistry were used to detect its immunophenotype, and the appearance of specific markers in the reproductive system was observed. 2 monolayer cells were induced: using follicular fluid as the conditioned medium, and inducing the differentiation of UC-MSCs into the germ cell differentiation.3. by 60CO gamma ray irradiation in vitro The animal model of Premature ovarian failure (POF) was established. The lentivirus mediated green fluorescent protein (green fluorescent protein, GFP) transfected UC-MSCs and transplanted into POF mice through the tail vein. The methods were obtained at different time points, through confocal microscopy and fluorescein in situ hybridization (Fluorescence in) method. The distribution and growth of the transplanted cells in the mice were detected, and the repair of the ovarian injury was observed by counting the number of non atresia base follicles and determining the level of estrogen.
Results: 1. the 1. isolated umbilical cord mononuclear cells showed spindle like, in vitro proliferation of more than 10 generations, more than 80 of P3 cells were in G0/ G1 phase, and about 20% of the cells were in the S + G2 +M phase. The immunophenotype of the cells was CD44, CD73, CD90, CD105 positive, CD31, CD34, and negative, similar to the immunophenotype of bone marrow mesenchymal stem cells. Under the same induction conditions, UC-MSCs can differentiate into adipocytes, osteoblasts and myoblasts.
2. induced differentiation and culture results (1) the suspension culture of UC-MSCs was carried out, and it could form EB. (RT-PCR). The results showed that the reproductive system markers were expressed as Oct-4, Ifitm-3, Stella, Vasa, DAZL 5 days after the formation of EB, and the UC-MSCs only expressed Oct-4, Ifitm-3, and flow cytometry: 5 days later, the positive cells accounted for 15.6. 1%. (4) immunohistochemical detection results: 5 days after formation of EB and human or mouse ovarian granulosa cells co culture, 10 days later, the reproductive markers Stella, Vasa, SCP3, GDF-9 positive expression, but the granular cells and the follicle culture of EB were not expressed. 5 monolayer culture induction: UC-MSCs with 5% follicle culture medium induction 20 days, cell volume In the form of tissue like mass, there were suspending cells in the periphery of the group. But the expression of the specific markers of the reproductive system was not detected. The pathological sections of the.3. (60CO) gamma ray treatment suggested that the mice had POF like lesions, such as ovarian reserve, follicle atresia, ovarian atrophy, and the heart, liver, spleen, lungs and kidneys in the short term. There was no obvious pathological damage in the intestine, brain, bone marrow and other tissues, indicating the success of the model construction. (2) the efficiency of GFP transfected by lentivirus was more than 80%. After the transplantation of the tail vein, UC-MSCs could find a large number of GFP positive cells in the injured mouse's ovary to survive and grow, but in the heart, liver, spleen, lungs, kidney, intestines, brain, bone marrow, and so on GFP positive cells were not found in the tissue. The follicle count and estrogen level all suggest that the repair of ovarian injury in the stem cell transplantation group is better than that of the control group.
Conclusion: 1. human UC-MSCs can be cultured and amplified in vitro, and the cell immunophenotype is similar to BM-MSCs. It has multipotential differentiation potential, such as fat formation, osteogenesis and myoblast. It is an ideal adult stem cell source.2.UC-MSC in vitro suspension culture can form EB, and the reproductive system specific markers are expressed after CO culture with human or mouse ovarian granulosa cells. The external microenvironment plays an important role in the direction differentiation of UC-MSCs to the germ cells. It is preliminarily indicated that UC-MSCs has the potential to differentiate into early germ cells..3.UC-MSCs can migrate to the function of premature ovarian failure and survive. The follicle count and the level of E2 suggest that UC-MSCs may have the repair effect of ovarian injury, indicating the microloop in the body. It also plays an important role in the differentiation of UC-MSCs.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R329

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 李惠敏;李海波;李紅;;間質(zhì)干細(xì)胞移植治療卵巢早衰的研究進(jìn)展[J];現(xiàn)代婦產(chǎn)科進(jìn)展;2013年03期

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本文編號：2001784