本文選題：小鼠間充質(zhì)干細(xì)胞株 + 胎盤(pán)源間充質(zhì)干細(xì)胞��； 參考：《蘇州大學(xué)》2009年碩士論文

【摘要】： 樹(shù)突狀細(xì)胞(dendritic cells, DCs)是功能最強(qiáng)大的抗原提呈細(xì)胞(antigen presenting cells, APCs)。由于表達(dá)豐富的協(xié)同刺激分子以及分泌多種具有免疫活性的細(xì)胞因子,DCs可以有效刺激初始T細(xì)胞活化。DCs的分化發(fā)育過(guò)程包括未成熟和成熟兩個(gè)階段,而DCs的生物學(xué)功能也與其成熟程度密切相關(guān)。在免疫穩(wěn)態(tài)時(shí),未成熟DCs(immature DCs, iDCs)通過(guò)不斷攝取死細(xì)胞相關(guān)產(chǎn)物維持細(xì)胞正常的更新?lián)Q代。當(dāng)缺乏炎癥因子時(shí),這些iDCs表達(dá)較低水平的協(xié)同刺激分子,從而誘導(dǎo)引流淋巴結(jié)內(nèi)T細(xì)胞無(wú)能、凋亡或調(diào)節(jié)性T細(xì)胞的產(chǎn)生。當(dāng)感染發(fā)生時(shí),iDCs在抗原和炎癥因子的刺激下成熟,進(jìn)入淋巴組織刺激初始或靜息T細(xì)胞活化,從而介導(dǎo)效應(yīng)T細(xì)胞應(yīng)答。總之,DCs的功能主要依賴(lài)于細(xì)胞活化成熟的程度,不同亞群的iDCs參與誘導(dǎo)和維持外周耐受,而最終發(fā)育成熟的DCs可有效地促進(jìn)效應(yīng)T細(xì)胞的產(chǎn)生。 由于基因缺損、移植等原因,DCs的成熟過(guò)程常常被打亂,從而可能產(chǎn)生自身免疫性疾病或移植物抗宿主病(graft versus host disease, GVHD)。有報(bào)道顯示,在系統(tǒng)性紅斑狼瘡(systemic lupus erythematosus, SLE)患者體內(nèi)存在IFN-α刺激DCs緩慢活化。另外,在小鼠疾病模型中應(yīng)用未活化DCs可顯著改善急性GVHD的惡性程度。因此,調(diào)節(jié)DCs的活化程度有可能成為治愈這些疾病的有效手段。 間充質(zhì)干細(xì)胞(mesenchymal stem cells, MSCs)具有強(qiáng)大的調(diào)節(jié)功能,在疾病治療方面的應(yīng)用前景讓人期待。目前研究發(fā)現(xiàn),MSCs不但可以抑制CD8+細(xì)胞毒性T細(xì)胞(cytotoxic T lymphocytes, CTLs)和自然殺傷(nature killer, NK)細(xì)胞的活化,也能抑制細(xì)胞或非特異性絲裂原介導(dǎo)的T細(xì)胞增殖。雖然MSCs對(duì)許多免疫細(xì)胞都具有直接的負(fù)調(diào)控效應(yīng),但MSCs對(duì)免疫應(yīng)答的起始過(guò)程——DCs的成熟——是否也具有免疫調(diào)節(jié)功能還不清楚。 本實(shí)驗(yàn)分為兩個(gè)部分,分別研究小鼠和人MSCs對(duì)DCs分化成熟的影響。在第一部分中,小鼠骨髓(bone marrow, BM)細(xì)胞經(jīng)mGM-CSF和mIL-4刺激5 d可得到iDCs,與小鼠間充質(zhì)干細(xì)胞株C3H10T1/2體外共培養(yǎng),同時(shí)加入LPS刺激4 d。結(jié)果發(fā)現(xiàn),對(duì)照組DCs懸浮生長(zhǎng)并大量聚集成團(tuán),細(xì)胞表面出現(xiàn)樹(shù)枝樣突起,呈典型的成熟DCs形態(tài)。而共培養(yǎng)組DCs散在分布,細(xì)胞呈圓形,無(wú)樹(shù)枝樣突起;細(xì)胞表型檢測(cè)發(fā)現(xiàn),共培養(yǎng)組DCs表面CD11c、MHC-Ⅱ、CD86、CD40的表達(dá)均受到抑制;共培養(yǎng)組DCs細(xì)胞數(shù)低于對(duì)照組,提示C3H10T1/2細(xì)胞還可以抑制DCs的擴(kuò)增;此外,共培養(yǎng)組DCs刺激脾細(xì)胞增殖的能力也明顯下降,且伴隨IFN-γ和IL-10分泌的減少。 在第二部分中,首先從人胎盤(pán)分離得到MSCs并進(jìn)行鑒定,隨后分別研究了胎盤(pán)源MSCs(placenta derived MSCs, PMSCs)對(duì)外周血(peripheral blood, PB)和臍血(umbilical cord blood, UCB)單核源DCs分化成熟的影響。胎盤(pán)組織經(jīng)Ⅳ型膠原酶消化、貼壁和傳代培養(yǎng)可獲得人PMSCs,運(yùn)用倒置顯微鏡和流式細(xì)胞儀進(jìn)行細(xì)胞形態(tài)和表型的鑒定,證實(shí)已成功培養(yǎng)得到MSCs。同時(shí)應(yīng)用hGM-CSF和hIL-4誘導(dǎo)獲得人PB和UCB單核源DCs,然后與PMSCs進(jìn)行共培養(yǎng)。結(jié)果顯示,即使在LPS刺激下,共培養(yǎng)組DCs表面CD80、CD86、CD83、CD40的表達(dá)仍受到明顯抑制,而CD14的表達(dá)則被上調(diào)。另外,PB單核源DCs刺激淋巴細(xì)胞增殖的能力也受到抑制。 綜上所述,本實(shí)驗(yàn)表明MSCs可以在細(xì)胞形態(tài)、表型、刺激淋巴細(xì)胞增殖以及細(xì)胞因子分泌四個(gè)方面抑制DCs的成熟。本結(jié)果為進(jìn)一步研究MSCs免疫調(diào)節(jié)DCs的分子機(jī)制提供了實(shí)驗(yàn)基礎(chǔ),也為臨床治療各種疾病帶來(lái)啟示和幫助。
[Abstract]:Dendritic cells (DCs) is the most powerful antigen presenting cell (antigen presenting cells, APCs). DCs can effectively stimulate the differentiation and development of the initial T cell activation.DCs, including the two stages of immature and mature stages due to the rich expression of synergistic stimulators and the secreting of many immune active cytokines. The biological function of DCs is closely related to its maturity. In the immune homeostasis, the immature DCs (immature DCs, iDCs) maintains normal cell regeneration through the continuous intake of dead cell related products. When lack of inflammatory factors, these iDCs express lower levels of synergistic stimulators, thus inducing T cells in the drainage lymph nodes. Incompetence, the production of apoptosis or regulatory T cells. When infection occurs, iDCs matures under the stimulation of antigen and inflammatory factors and enters the lymphoid tissue to stimulate initial or resting T cell activation, which mediates the response of the effect of T cells. In a word, the function of DCs is mainly dependent on the degree of cell activation and maturation, and the iDCs of different subgroups is involved in the induction and maintenance of the cells. Zhou tolerance, and the final mature DCs can effectively promote the production of T cells.
The maturation process of DCs is often disrupted because of genetic defect and transplantation, which may lead to autoimmune disease or graft versus host disease (GVHD). It is reported that IFN- alpha stimulates DCs slowly in patients with systemic lupus erythematosus (systemic lupus erythematosus, SLE). The use of unactivated DCs in the mouse disease model can significantly improve the degree of malignancy of acute GVHD. Therefore, the regulation of the activation of DCs may be an effective means to cure these diseases.
Mesenchymal stem cells (MSCs) has a strong regulatory function and is expected to be expected in the treatment of disease. At present, MSCs can not only inhibit the activation of CD8+ cell toxic T cells (cytotoxic T lymphocytes, CTLs) and natural killer (nature), but also inhibit cell or nonspecific cells. Heterosexual mitogen mediated proliferation of T cells. Although MSCs has a direct negative regulatory effect on many immune cells, MSCs is not clear about the initiation of the immune response, the maturation of DCs, whether or not it has the function of immunomodulatory.
The experiment was divided into two parts. In the first part, the mice bone marrow (bone marrow, BM) cells stimulated 5 d by mGM-CSF and mIL-4, and the mice were co cultured with the mouse mesenchymal stem cell line C3H10T1/2 in vitro and added LPS stimulation 4 d.. The control group was suspended and grown in suspension. A large number of clustered clusters, the surface of the cell appeared on the surface of the cell, showing a typical mature DCs form. While the co culture group DCs scattered in the distribution, the cells were round and no dendritic. The cell phenotype detection showed that the expression of DCs surface CD11c, MHC- II, CD86 and CD40 in co culture group were suppressed, and the number of DCs cells in co culture group was lower than that of the control group, suggesting C3H10T1/2 finer. The cells also inhibited the amplification of DCs. In addition, the ability of DCs to stimulate splenocytes proliferation in the co culture group decreased significantly, and the secretion of IFN- and IL-10 decreased.
In the second part, MSCs was first isolated from human placenta and identified. Then, the effects of placental source MSCs (placenta derived MSCs, PMSCs) on the differentiation and maturation of peripheral blood (peripheral blood, PB) and umbilical cord blood (umbilical cord blood) were studied. PMSCs was obtained by using inverted microscope and flow cytometry to identify the cell morphology and phenotype. It was confirmed that MSCs. was successfully cultured with hGM-CSF and hIL-4 to induce PB and UCB mononuclear DCs, and then co culture with PMSCs. The results showed that the DCs surface of the co culture group was CD80, CD86, and, even under LPS stimulation. The expression of CD14 was upregulated, while the expression of PB was up-regulated. In addition, the ability of monocyte derived DCs to stimulate lymphocyte proliferation was also inhibited.
To sum up, this experiment shows that MSCs can inhibit the maturation of DCs in the four aspects of cell morphology, phenotype, stimulating lymphocyte proliferation and cytokine secretion. This result provides an experimental basis for further study of the molecular mechanism of MSCs immunomodulatory DCs, and also provides inspiration and help for the clinical treatment of various diseases.

【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(lèi)號(hào)】：R392.1

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