本文選題：低血清培養(yǎng)基 + 間充質(zhì)干細(xì)胞　； 參考：《中國協(xié)和醫(yī)科大學(xué)》2009年博士論文

【摘要】： 背景:間充質(zhì)干細(xì)胞(multipotent mesenchymal stem cells,MSC)是一種存在于多種組織中的具有自我更新和多向分化能力的多能干細(xì)胞。MSC具有易于分離和擴(kuò)增、多向分化、低免疫原性和免疫調(diào)節(jié)的特性,這些特性使它們成為組織工程和再生醫(yī)學(xué)的理想種子細(xì)胞。細(xì)胞治療中的一個重要問題是MSC替代來源的的可獲得性,而細(xì)胞治療的成功的決定性因素則是MSC的生物學(xué)特性。對MSC生物學(xué)特性的理解有利于充分的利用MSC的治療潛能,因此,MSC生物學(xué)特性的研究越來越引起人們的興趣。到目前為止,大多數(shù)的治療性應(yīng)用是以成體骨髓來源的MSC為研究對象,但是成體骨髓MSC存在骨髓體積有限,獲取標(biāo)本時需進(jìn)行侵襲性操作以及MSC的絕對數(shù)量和分化能力會隨著年齡增加而下降等不足。這就需要研究人員尋找替代的MSC細(xì)胞來源,同時,研制良好的細(xì)胞培養(yǎng)試劑為MSC的生長和擴(kuò)增提供適合的環(huán)境,從而滿足臨床治療和研究方面的需求。 目的:本研究旨在鑒定低血清濃度完全培養(yǎng)基培養(yǎng)的MSC的一般特性如形態(tài)、核型、增殖動力學(xué)、表型特點(diǎn)和分化潛能等生物學(xué)性狀,從而提供一種MSC的培養(yǎng)試劑并為MSC作為組織工程的種子細(xì)胞提供理論基礎(chǔ)。 方法:①應(yīng)用貼壁培養(yǎng)的方法從胎兒的骨髓和肺組織以及臍帶中分離獲得單個細(xì)胞,在低血清完全培養(yǎng)基中培養(yǎng),除去非貼壁的細(xì)胞、通過體外傳代培養(yǎng)使細(xì)胞純化。②測定培養(yǎng)的MSC的核型和生長曲線。③應(yīng)用流式細(xì)胞儀對培養(yǎng)的MSC進(jìn)行細(xì)胞周期分析。④運(yùn)用流式細(xì)胞技術(shù)檢測MSC的免疫表型和胚胎干細(xì)胞的全能標(biāo)志物。⑤在誘導(dǎo)體系中定向誘導(dǎo)MSC向脂肪細(xì)胞、成骨細(xì)胞和軟骨細(xì)胞分化,利用油紅0、茜素紅和阿爾新藍(lán)染色觀測脂滴形成、鈣鹽沉積和酸性粘多糖合成情況;利用流式細(xì)胞儀檢測系分化特異性標(biāo)志物過氧化物酶體增生物激活受體-γ(PPAR-γ)、骨鈣蛋白(osteocalcin)和Ⅱ型膠原的表達(dá)。⑥采用兩步誘導(dǎo)方案使骨髓和肺組織來源的MSC體外誘導(dǎo)分化為神經(jīng)細(xì)胞,利用激光共聚焦顯微鏡檢測誘導(dǎo)細(xì)胞中的神經(jīng)細(xì)胞標(biāo)志物微管相關(guān)蛋白2(microtubule- associated protein2,MAP2)、Nestin和神經(jīng)膠質(zhì)酸性蛋白(glial fibrillary acidic protein,GFAP)的表達(dá)。⑦采用兩步誘導(dǎo)方法使肺組織來源的MSC體外誘導(dǎo)分化為肝細(xì)胞,利用RT-PCR方法檢測誘導(dǎo)細(xì)胞中的肝細(xì)胞標(biāo)志物如甲胎蛋白(αFP)、白蛋白(albumin)和細(xì)胞角蛋白18(cytokeratin18,CK18)的表達(dá);利用糖原染色和培養(yǎng)上清中白蛋白的分泌水平對誘導(dǎo)后細(xì)胞進(jìn)行功能性檢測。 結(jié)果:①低血清濃度完全培養(yǎng)基培養(yǎng)的MSC貼壁生長和呈長梭形,細(xì)胞生長速度快,細(xì)胞融合時出現(xiàn)漩渦狀外觀。②MSC具有正常核型以及典型的“S”型生長曲線。③細(xì)胞周期表明大多數(shù)細(xì)胞處于G0/G1期,少部分的細(xì)胞處于活躍增殖期。④MSC高表達(dá)間充質(zhì)細(xì)胞標(biāo)志物如CD13,CD29,CD44,CD90,CD49e,CD105,CD166和HLA-Ⅰ,而不表達(dá)造血細(xì)胞和內(nèi)皮細(xì)胞的標(biāo)志物,如CD31、CD34、CD11b、CD45、CD117,CD133和HLA-Ⅱ。更重要的是,表達(dá)胚胎干細(xì)胞標(biāo)志物Oct4,Nanog,Sox2和SSEA-4,其中傳代到第25代(P25)的肺組織來源的MSC仍可保持免疫表型和胚胎干細(xì)胞標(biāo)志物表達(dá)的穩(wěn)定。⑤MSC經(jīng)誘導(dǎo)后,油紅0、茜素紅和阿爾新藍(lán)染色均為陽性,且高表達(dá)系分化特異性標(biāo)志物,說明其能夠分化為脂肪細(xì)胞、骨細(xì)胞和軟骨細(xì)胞,其中肺組織來源的MSC能保持其三系分化能力到P25。⑥神經(jīng)細(xì)胞標(biāo)志物在誘導(dǎo)細(xì)胞中的表達(dá)水平顯著高于其在對照細(xì)胞中的水平。⑦與對照細(xì)胞相比,肝細(xì)胞標(biāo)志物在誘導(dǎo)細(xì)胞中的表達(dá)上調(diào);誘導(dǎo)后細(xì)胞具有儲存糖原和分泌白蛋白的功能。 結(jié)論:①低血清濃度完全培養(yǎng)基是一種很好的培養(yǎng)MSC的細(xì)胞試劑。②利用該培養(yǎng)基培養(yǎng)的MSC具有較強(qiáng)增殖能力,可滿足組織工程種子細(xì)胞的需要。③利用該培養(yǎng)基培養(yǎng)的MSC可在體外定向分化為脂肪組織、骨組織和軟骨組織,而且肺組織來源的MSC能夠?qū)⑦@種多系分化潛能保持到P25,從而為進(jìn)一步構(gòu)建組織工程化骨或軟骨提供了充足的細(xì)胞來源。④利用該培養(yǎng)基培養(yǎng)的MSC表達(dá)胚胎干細(xì)胞的標(biāo)志物,而且能在體外分化為中胚層以外的細(xì)胞如外胚層的神經(jīng)細(xì)胞和/或內(nèi)胚層的肝細(xì)胞。⑤利用該培養(yǎng)基培養(yǎng)的MSC具有良好的生物學(xué)特性,可作為組織工程和再生醫(yī)學(xué)的優(yōu)秀種子細(xì)胞。 背景:間充質(zhì)干細(xì)胞(multipotent mesenchymal stem cells,MSC)是能分化為中胚層組織的多能干細(xì)胞,它可以從機(jī)體的各種組織中分離得到。MSC還因其對免疫細(xì)胞的免疫調(diào)節(jié)作用而備受關(guān)注,該調(diào)節(jié)作用能調(diào)節(jié)多種免疫效應(yīng)功能。最近,一個新的CD4+T細(xì)胞亞類被命名為Th17細(xì)胞,它特異性地優(yōu)先表達(dá)IL-17。Th17細(xì)胞被認(rèn)為是自身免疫性疾病和變態(tài)反應(yīng)性疾病發(fā)展過程和宿主對抗病原體過程中的重要效應(yīng)細(xì)胞。雖然MSC免疫調(diào)節(jié)功能的準(zhǔn)確機(jī)制還不是很清楚,但是MSC已被用于多種臨床試驗,旨在降低免疫介導(dǎo)的疾病的負(fù)擔(dān)。 目的:研究胎兒骨髓間充質(zhì)干細(xì)胞(FBM-MSC)對人T細(xì)胞的免疫調(diào)節(jié)作用。 方法:我們在存在或不存在FBM-MSC的情況下培養(yǎng)PBMC和CD4+T細(xì)胞,PBMC或CD4+T細(xì)胞與FBM-MSC的共培養(yǎng)被稱為共培養(yǎng)細(xì)胞。除非特別說明,培養(yǎng)基均包含PHA和rIL-2。我們采用實時定量PCR,ELISA和流式細(xì)胞分析等方法檢測:①正常人外周血單個核細(xì)胞(PBMC)和CD4+T細(xì)胞及其與FBM-MSC共培養(yǎng)中IL17的表達(dá)水平,觀察FBM-MSC對Th17細(xì)胞的調(diào)節(jié)作用;②添加外源性的IL-6,IL-6中和抗體,轉(zhuǎn)化生長因子-β1(TGF-β1)后IL17表達(dá)水平的改變,觀察IL-6通路在FBM-MSC調(diào)節(jié)IL17中的作用;③正常人PBMC和CD4+T細(xì)胞及其與FBM-MSC共培養(yǎng)中IL-1和IL-23在FBM-MSC調(diào)節(jié)IL17中的作用;④PBMC和CD4+T細(xì)胞及其與FBM-MSC共培養(yǎng)中IFN-γ的表達(dá)水平,研究FBM-MSC對Th1細(xì)胞的調(diào)節(jié)作用;⑤PBMC及其與FBM-MSC共培養(yǎng)中Foxp3的表達(dá)水平,初步研究FBM-MSC對Treg的調(diào)節(jié)作用。 結(jié)果:實時定量PCR,ELISA和流式細(xì)胞分析等結(jié)果表明:①在含有或不含有PHA和rIL-2刺激的情況下,IL-17在PBMC和CD4+T細(xì)胞中的分子和細(xì)胞表達(dá)水平均顯著低于其在共培養(yǎng)中的水平(p＜0.05);②添加外源性的IL-6能增加IL-17的表達(dá)水平,而添加外源性的IL-6中和抗體則會顯著的降低IL-17的表達(dá)水平,添加外源性的TGFβ1會導(dǎo)致IL-17表達(dá)水平的降低;③IL-1在CD4+T細(xì)胞培養(yǎng)上清中的濃度低于其在共培養(yǎng)中的濃度(p＜0.05),而IL-23在PBMC中的轉(zhuǎn)錄水平與其在共培養(yǎng)中的水平無明顯差異(p＞0.05),IL-23在CD4+T細(xì)胞中的蛋白分泌水平與其在共培養(yǎng)中的水平亦無明顯差異(p＞0.05);④Th1細(xì)胞產(chǎn)生的IFN-γ在PBMC和CD4+T細(xì)胞中的mRNA和蛋白表達(dá)水平顯著低于其在共培養(yǎng)中的水平(p＜0.05);⑤Foxp3在PBMC中的mRNA表達(dá)水平低于其在共培養(yǎng)中的水平(p＜0.05)。 結(jié)論:FBM-MSC對人Th17細(xì)胞具有正性調(diào)節(jié)作用。IL-6是該調(diào)節(jié)作用的機(jī)制之一,IL-1亦可能是其中的一個機(jī)制,而IL-23似乎在我們研究的調(diào)節(jié)作用中不發(fā)揮作用。另外,FBM-MSC對人Th1細(xì)胞具有抑制作用并且能抑制Th1細(xì)胞產(chǎn)生IFN-γ,而對Treg細(xì)胞具有促進(jìn)作用。
[Abstract]:Background: multipotent mesenchymal stem cells (MSC) is a kind of pluripotent stem cell with self renewal and multidirectional differentiation in a variety of tissues..MSC has the characteristics of easy separation and amplification, multidifferentiation, low immunogenicity and immune regulation. These properties make them a tissue engineering and regenerative medicine. One of the important problems in cell therapy is the availability of MSC alternative sources, and the decisive factor in the success of the cell therapy is the biological characteristics of MSC. The understanding of the biological characteristics of MSC is beneficial to the full utilization of the therapeutic potential of MSC. Therefore, the study of the biological characteristics of MSC is becoming more and more popular. So far, most of the therapeutic applications are based on the MSC of adult bone marrow origin, but adult bone marrow MSC has a limited volume of bone marrow, the need for invasive operations to obtain specimens and the decline of the absolute number and differentiation ability of MSC as the age increases. This requires researchers to find alternative MSC. Cell source, meanwhile, develop good cell culture reagents to provide suitable environment for MSC growth and expansion, so as to meet the needs of clinical treatment and research.
Objective: the purpose of this study was to identify the general characteristics of MSC with low serum concentration, such as morphology, karyotype, proliferation kinetics, phenotypic characteristics and differentiation potential, so as to provide a MSC culture reagent and provide a theoretical basis for MSC as a seed cell of tissue engineering.
Methods: (1) single cells were isolated from the bone marrow, lung tissue and umbilical cord of the fetus by using the method of adherent culture. The cells were cultured in the low serum complete medium, removed non adherent cells and purified by external generation and culture. (2) the karyotype and growth curve of the cultured MSC were measured. (3) the flow cytometry was applied to the culture of the cultured MSC. Cell cycle analysis. (4) using flow cytometry to detect the immunophenotype of MSC and the omnipotent marker of embryonic stem cells. (5) induced MSC to differentiate into adipocytes, osteoblasts and chondrocytes in the induction system, and use oil red 0, alizarin red and alcian blue staining to observe lipid droplets, calcium salt deposition and acid mucopolysaccharide synthesis. The expression of peroxisome activation receptor - gamma (PPAR- gamma), osteocalcin (osteocalcin) and type II collagen were detected by flow cytometry (FCM). 6. The two step induction scheme was used to induce the bone marrow and lung tissue derived MSC into neural cells in vitro, and the laser confocal microscopy was used to detect the induced refinement. The expression of microtubule related protein 2 (microtubule- associated protein2, MAP2), Nestin and glial acidic protein (glial fibrillary acidic protein, GFAP) in the cell. (7) the two step induction method was used to induce the lung tissue source to differentiate into liver cells in vitro, and the hepatocytes were detected by the RT-PCR method. The expression of cell markers such as alpha fetoprotein (alpha FP), albumin (albumin) and cytokeratin 18 (cytokeratin18, CK18), and the functional detection of the induced cells by glycogen staining and the secretion of albumin in the supernatant.
Results: (1) the MSC adherent growth and long spindle shape in the low serum concentration culture medium, the cell growth speed and the whirlpool appearance in the cell fusion. (2) MSC has normal karyotype and the typical "S" type growth curve. (3) cell cycle indicates that most cells are in G0/G1 stage, and few cells are in active proliferation period. (4) MSC High expression of mesenchymal cell markers such as CD13, CD29, CD44, CD90, CD49e, CD105, CD166 and HLA- I, but not the markers of hematopoietic and endothelial cells, such as CD31, CD34, CD11b, CD45, etc. MSC can still maintain the stability of the immunophenotype and the expression of the markers of embryonic stem cells. 5. After induction, the staining of oil red 0, alizarin red and Alicia blue were both positive, and the high expression line was differentiated to differentiate into adipocytes, bone cells and chondrocytes, and the MSC of the lung tissue derived from the lung tissue could maintain its differentiation. The expression level of the nerve cell markers in the induced cells was significantly higher than that in the control cells. The expression of hepatocyte markers was up regulated in the induced cells compared with the control cells, and the cells had the function of storing glycogen and secreting albumin compared with the control cells.
Conclusion: (1) the full medium of low serum concentration is a good cell reagent for the cultivation of MSC. (2) the MSC has strong proliferation ability and can meet the needs of tissue engineering seed cells. (3) the MSC can be differentiated into adipose tissue, bone tissue and cartilage tissue, and lung tissue in vitro. The source of MSC can maintain this multilineage differentiation potential to P25, thus providing a sufficient cell source for further construction of tissue engineered bone or cartilage. (4) using the culture medium MSC to express the markers of embryonic stem cells and to differentiate into neurons and / or endoderm cells outside the mesoderm in vitro, such as the ectoderm and / or the inner embryo. The MSC cultured in this medium has good biological characteristics and can be used as an excellent seed cell for tissue engineering and regenerative medicine.
Background: multipotent mesenchymal stem cells (MSC) is a pluripotent stem cell capable of differentiating into mesoderm tissue. It can be isolated from various tissues of the body and can be isolated from the body..MSC is also concerned because of its immunomodulatory effect on immune cells. This regulation can regulate a variety of immune effects. Recently, a new CD4+ is used. The subclass of T cells is named Th17 cell, and its specific priority expression of IL-17.Th17 cells is considered to be an important effector cell in the process of autoimmune and allergic disease and the host against pathogens. Although the exact mechanism of MSC immunoregulation is not clear, MSC has been used in a variety of clinical trials. The experiment was designed to reduce the burden of immune mediated diseases.
Objective: To study the immunomodulatory effect of fetal bone marrow mesenchymal stem cells (FBM-MSC) on human T cells.
Methods: PBMC and CD4+T cells were cultured in the presence or absence of FBM-MSC. Co culture of PBMC or CD4+T cells with FBM-MSC was called co culture cells. Unless specifically explained, the medium contained PHA and rIL-2.. We used real-time quantitative PCR, ELISA, and flow cytometry to detect: (1) normal human peripheral blood mononuclear cells ( PBMC) and CD4+T cells and the expression level of IL17 in co culture with FBM-MSC, and observe the regulatory effect of FBM-MSC on Th17 cells; (2) adding exogenous IL-6, IL-6 neutralizing antibodies, and transforming growth factor beta 1 (TGF- beta 1) to the expression level of IL17, and observing the role of IL-6 pathway in FBM-MSC regulation. The role of IL-1 and IL-23 in FBM-MSC regulation of IL17 in co culture with FBM-MSC; (4) PBMC and CD4+T cells and the expression level of IFN- gamma in co culture with FBM-MSC, and to study the regulation of FBM-MSC on Th1 cells.
Results: the results of real-time quantitative PCR, ELISA and flow cytometry showed that: (1) the expression level of IL-17 in PBMC and CD4+T cells was significantly lower than that in co culture (P < 0.05) in both PBMC and CD4+T cells with or without PHA and rIL-2 stimulation, and the addition of exogenous IL-6 could increase the expression level of IL-17. The IL-6 neutralization antibody of the source significantly reduced the expression level of IL-17, and the addition of exogenous TGF beta 1 could lead to the decrease of IL-17 expression level; (3) the concentration of IL-1 in the culture supernatant of CD4+T cells was lower than that in co culture (P < 0.05), and the level of IL-23 in PBMC was not significantly different from that in co culture (P >) 0.05) the protein secreting level of IL-23 in CD4+T cells was not significantly different from that in co culture (P > 0.05); (4) the level of mRNA and protein expression of IFN- gamma produced by Th1 cells in PBMC and CD4+T cells was significantly lower than that in co culture (P < 0.05); (5) Foxp3 in PBMC was lower than in co culture. The level (P < 0.05).
Conclusion: FBM-MSC has positive regulatory effect on human Th17 cells, and.IL-6 is one of the mechanisms of this regulation. IL-1 may also be one of the mechanisms, and IL-23 seems to not play a role in our study. In addition, FBM-MSC has a inhibitory effect on human Th1 cells and can inhibit the production of IFN- gamma in Th1 cells, and to Treg cells. It has a promoting effect.
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R392;R329.2

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