本文關(guān)鍵詞：研究BMP-2對(duì)骨髓間充質(zhì)干細(xì)胞促進(jìn)造血干細(xì)胞增殖的影響　出處：《四川醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 造血干細(xì)胞 間充質(zhì)干細(xì)胞 BMP-2 微龕 增殖

【摘要】：目的:生存在骨髓微環(huán)境即微龕(niche)中的造血干細(xì)胞(HSCs)為所有成熟血細(xì)胞的原始造血細(xì)胞,其具有高度自我更新能力與多向分化潛能。微龕極其復(fù)雜,由基質(zhì)細(xì)胞及其前體間充質(zhì)干細(xì)胞(MSCs)、細(xì)胞因子、細(xì)胞外基質(zhì)等組成,它們對(duì)于HSC生理功能的調(diào)控起著重要的作用。niche主要分為“成骨微龕”和“血管微龕”,前者主要維持HSC靜息狀態(tài),后者可以調(diào)節(jié)HSC增殖、分化、遷移等。骨形態(tài)發(fā)生蛋白(BMP)-2是轉(zhuǎn)化生長(zhǎng)因子(TGF-β)超家族中的一員,是一個(gè)多功能蛋白,作為重要的成骨因子可刺激MSC分化為成骨細(xì)胞,構(gòu)成“成骨niche”的主體結(jié)構(gòu),此外它還能促進(jìn)血管的形成,關(guān)于它在成骨niche中的研究較多,而其在血管niche方面的作用研究較少。BMP-2在造血調(diào)控中也起重要作用,關(guān)于其在胚胎期造血中的研究較多,而其在成人造血中的作用研究得較少。目前關(guān)于MSC促進(jìn)HSC增殖的研究較多,而關(guān)于BMP-2對(duì)MSC促進(jìn)HSC增殖的影響的研究較少。本研究通過transwell非接觸共培養(yǎng)CD34+細(xì)胞與MSC,并用BMP-2干預(yù),在第三天時(shí)檢測(cè)HSC的增殖指標(biāo),可以了解MSC和BMP-2對(duì)CD34+細(xì)胞增殖的影響以及BMP-2信號(hào)通路對(duì)MSC促進(jìn)HSC增殖的影響。這能為骨髓CD34+細(xì)胞體外擴(kuò)增的深入研究提供依據(jù),同時(shí)為我們下一步探討相關(guān)機(jī)制的研究奠定基礎(chǔ),且這有利于造血干細(xì)胞移植(HSCT)的發(fā)展及惡性血液病的治療。方法:①結(jié)合MSCs貼壁生長(zhǎng)的特性采用全骨髓法體外擴(kuò)增成人BMMSCs至P3,并用流式細(xì)胞術(shù)(FCM)鑒定MSCs的表面標(biāo)志及純度;②通過密度梯度離心法分離骨髓單個(gè)核細(xì)胞(MNCs),再用mini MACS磁珠分選儀從MNCs中分選成人骨髓CD34+細(xì)胞,并用FCM鑒定其純度;③利用Transwell小室(微網(wǎng)孔徑0.4um)非接觸共培養(yǎng)骨髓CD34+細(xì)胞與P3代的BMMSCs,并用BMP-2干預(yù),具體分組為:單純HSC組、HSC+BMP2組、HSC+MSC組、HSC+MSC+BMP2組。培養(yǎng)至72小時(shí)收集樣本;④用以下方法檢測(cè)HSC在不同組的增殖情況:計(jì)數(shù)板計(jì)數(shù)每組HSC的四個(gè)復(fù)孔的數(shù)目;TRIZOL法提取各組HSC的RNA后測(cè)定RNA濃度并計(jì)算各組HSC的RNA的含量;實(shí)時(shí)熒光定量PCR法檢測(cè)各組HSC的增殖基因Ki67的m RNA水平的表達(dá),PCR的Ct值利用2-△△Ct分析法進(jìn)行相對(duì)定量(RQ)分析;⑤統(tǒng)計(jì)學(xué)分析方法:所有統(tǒng)計(jì)學(xué)數(shù)據(jù)均用SPSS17.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析,兩兩比較用單因素方差分析,均以“均數(shù)±標(biāo)準(zhǔn)差(X±SD)”表示,當(dāng)P0.05時(shí)差異被認(rèn)為具有統(tǒng)計(jì)學(xué)意義。結(jié)果:①全骨髓法培養(yǎng)的BMMSCs成梭形,貼壁生長(zhǎng),排列成旋渦狀;流式細(xì)胞術(shù)檢測(cè)顯示傳至第3代的BMMSCs高表達(dá)CD105,而CD45、CD34陰性表達(dá),MSCs的純度為52.4%。②mini MACS磁珠分選出了較高純度的CD34+細(xì)胞,純度為81.9%。③各組HSC的計(jì)數(shù)(×104個(gè)):共培養(yǎng)組(3.03±0.50)、HSC+BMP2組(1.70±0.51)均高于HSC單純培養(yǎng)組(1.2±0.43);共培養(yǎng)+BMP2組(5.29±0.20)高于共培養(yǎng)組(3.03±0.50);P值均0.05。④各組HSC的RNA量(RNA濃度的單位為ng/ul,總RNA還需×50ul):HSC+BMP2組(138±3.464)及共培養(yǎng)組(163±3.559)高于HSC單純培養(yǎng)組(113±2.944);共培養(yǎng)+BMP2組(241±3.651)高于共培養(yǎng)組(163±3.559);P值均0.05。⑤熒光定量PCR法檢測(cè)HSC的Ki67的m RNA相對(duì)表達(dá)量,即RQ值:共培養(yǎng)組(3.27±0.08)及HSC+BMP2組(2.44±0.26)高于HSC單純培養(yǎng)組(1.00±0.13);共培養(yǎng)+BMP2組(5.08±0.20)高于共培養(yǎng)組(3.27±0.08);P值均0.05。結(jié)論:①利用全骨髓法成功培養(yǎng)出MSC;②應(yīng)用免疫磁珠分選法成功分選出HSC;③BMP-2可以促進(jìn)HSC增殖;④MSC可以促進(jìn)HSC增殖;⑤MSC與BMP-2具有協(xié)同效應(yīng),即BMP-2信號(hào)通路可以促進(jìn)MSC對(duì)HSC的增殖作用。
[Abstract]:Objective: to survive in the bone marrow microenvironment micro niche (niche) in hematopoietic stem cells (HSCs) as primitive hematopoietic cells all mature blood cells, which have high self-renewal capacity and multilineage differentiation potential. The micro niche is extremely complex, and its precursors by stromal cell derived mesenchymal stem cells (MSCs), cytokine extracellular matrix, etc., for they regulate physiological functions of HSC plays an important role for.Niche is mainly divided into "bone micro niche" and "micro vascular niche", the former mainly maintain HSC resting state, which can regulate HSC proliferation, differentiation, migration, bone morphogenetic protein (BMP) -2 transformation growth factor (TGF-) superfamily, is a multifunctional protein, as an important factor of bone can stimulate the differentiation of MSC into osteoblasts, constitute the main structure of bone niche, it can also promote the formation of blood vessels, about it in the bone in niche More research, and study the role of niche in vascular less.BMP-2 in hematopoietic regulation play an important role in embryonic hematopoiesis on its research and its role in adult hematopoiesis was less studied. More research on MSC currently promote the proliferation of HSC, and about BMP-2 of MSC effect the proliferation of HSC is less. This study by Transwell co culture CD34+ cells and MSC, and treated with BMP-2 at third days to detect the proliferation of HSC index, can understand the impact of MSC and BMP-2 on CD34+ cell proliferation and BMP-2 signaling pathway on MSC promote proliferation of HSC. This can provide the basis for further research for the amplification of bone marrow CD34+ cells in vitro, and to explore the related mechanisms of the study laid the foundation for our next step, and this is conducive to hematopoietic stem cell transplantation (HSCT) treatment and development of malignant hematological diseases. Methods: 1. Amplification of adult BMMSCs to P3 binding characteristics of MSCs cultured in vitro by the whole bone marrow method, and flow cytometry (FCM) identification of surface markers MSCs and purity; the separation of bone marrow mononuclear cells by density gradient centrifugation (MNCs), and mini MACS beads sorter sorting from MNCs adult bone marrow CD34+ cells the purity, and identified by FCM; using Transwell chamber (micro aperture 0.4um) bone marrow CD34+ cells and P3 generation BMMSCs non-contact co culture, and treated with BMP-2 specific groups: simple HSC group, HSC+BMP2 group, HSC+MSC group, HSC+MSC+ BMP2 group. Training 72 hours to collect samples with the following; detection of HSC in the proliferation of different groups: four holes the number count of each HSC group; group HSC RNA TRIZOL extraction method for determination of RNA concentration and HSC content were calculated by RNA; the proliferation was detected by HSC based real-time fluorescence quantitative PCR method 鍥燢i67鐨刴 RNA姘村鉤鐨勮〃杈，

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