【摘要】： 研究背景和目的人臍帶血間充質(zhì)干細(xì)胞(human umbilical cord bloodmesenchymal stem cells,hUCB-MSCs)具有其他組織來源的間充質(zhì)干細(xì)胞的生物學(xué)特性。目前的研究表明hUCB-MSCs含量較少,培養(yǎng)體系眾多,但培養(yǎng)效率較低。對UCB-MSCs體外向成肌細(xì)胞分化的研究不夠深入。因此,我們選擇hUCB-MSCs作為研究的種子細(xì)胞,優(yōu)化體外培養(yǎng)條件,探討其體外誘導(dǎo)成肌細(xì)胞的能力,為肌源性疾病的治療提供理想的種子細(xì)胞。 實(shí)驗(yàn)方法①hUCB-MNCs培養(yǎng)體系的建立:應(yīng)用淋巴細(xì)胞分離液法、羥乙基淀粉沉降法和羥乙基淀粉+淋巴細(xì)胞分離液兩步法三不同方法分別從臍帶血中獲得單個(gè)核細(xì)胞(mononuclear cells,MNCs),應(yīng)用L-DMEM、DMEM/F12和MesencultTM不同培養(yǎng)基,在不同胎牛血清(fetal bovine serum,FBS)濃度和細(xì)胞接種密度下,培養(yǎng)UCB-MNCs。比較所獲得的MNCs數(shù)量、原代培養(yǎng)時(shí)間和不同培養(yǎng)條件下hUCB-MSCs的生長情況。流式細(xì)胞儀對P3細(xì)胞進(jìn)行細(xì)胞表面標(biāo)記鑒定,并誘導(dǎo)成骨,觀察其生物學(xué)特征。②基于上一部分的UCB-MSCs培養(yǎng)體系,建立以5-氮雜胞苷(5-azacytidine,5-Aza)為誘導(dǎo)劑的誘導(dǎo)成肌分化體系:MTT法選擇5-Aza適宜的誘導(dǎo)濃度,對P3 hUCB-MNCs進(jìn)行體外向成肌細(xì)胞誘導(dǎo)分化,于誘導(dǎo)后第7d和第14d進(jìn)行MyoD1、myogenin和myosin heavy chain免疫熒光染色和Real-time PCR檢測。 實(shí)驗(yàn)結(jié)果建立了hUCB-MSCs穩(wěn)定有效的培養(yǎng)體系:應(yīng)用羥乙基淀粉+淋巴細(xì)胞分離液兩步法采集單個(gè)核細(xì)胞的數(shù)量較多(P＜0.01);10%FBS DMEM/F12和MesencultTM培養(yǎng)基,T25培養(yǎng)瓶中細(xì)胞接種密度為5×10~7時(shí)培養(yǎng)效率高于各對照組(P＜0.01);貼壁細(xì)胞表達(dá)CD29、CD105,不表達(dá)CD34、CD45、CD90、CD133;經(jīng)成骨誘導(dǎo)培養(yǎng)21d后,細(xì)胞Yon Kossa染色可見礦化結(jié)節(jié)。P3 UCB-MNCs經(jīng)5μmmol/L5-Aza誘導(dǎo)7d后,免疫熒光檢測僅表達(dá)MyoD1;當(dāng)誘導(dǎo)后14d時(shí),可見MyoD1和myogenin陽性表達(dá)。始終無myosin heavy chain的表達(dá)。Real-time PCR檢測顯示誘導(dǎo)后14d較7d,MyoD1和myogenin的相對濃度增高,而無myosin heavy chain的表達(dá)。另外,在未誘導(dǎo)條件下UCB-MNCs同樣能表達(dá)MyoD1。 實(shí)驗(yàn)結(jié)論臍帶血中存在一定數(shù)量的間充質(zhì)干細(xì)胞,通過改善hUCB-MSCs分離方法,優(yōu)化培養(yǎng)條件,提高了培養(yǎng)效率。UCB-MSCs不表達(dá)造血干細(xì)胞和內(nèi)皮細(xì)胞表面標(biāo)記,表達(dá)與骨髓間充質(zhì)干細(xì)胞表面標(biāo)記,并能夠誘導(dǎo)成骨,符合間充質(zhì)干細(xì)胞的特點(diǎn)。在成肌細(xì)胞分化中,hUCB-MSCs經(jīng)5μmmol/L5-Aza誘導(dǎo)后,表達(dá)成肌細(xì)胞分化過程中的調(diào)節(jié)因子,能夠向成肌細(xì)胞分化,而且在未誘導(dǎo)條件下能夠保持成肌細(xì)胞分化的潛能。
[Abstract]:The research background and the human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) have the biological characteristics of other tissue-derived mesenchymal stem cells. The present study shows that the content of hUCB-MSCs is less, the culture system is numerous, but the culture efficiency is low. The research on the differentiation of UCB-MSCs in vitro to myoblasts is not in-depth. Therefore, we selected hUCB-MSCs as the seed cells of the study, to optimize the in vitro culture conditions, to explore its ability to induce myoblasts in vitro, and to provide the ideal seed cells for the treatment of myogenic diseases. The establishment of hUCB-MNCs culture system was carried out by using a two-step method of lymphocyte separation, hydroxyethyl starch sedimentation and hydroxyethyl starch + lymphocyte separation. The UCB-M was cultured under different fetal bovine serum (FBS) concentrations and cell seeding density. NCs. Comparison of the number of MNCs, primary culture time and the generation of hUCB-MSCs under different culture conditions In that long case, the cell surface mark of the P3 cell was identified by flow cytometry, and the bone formation was induced and the organism was observe. Based on the UCB-MSCs culture system, the induction of 5-azacytidine (5-Aza) as an inducer was established. The appropriate induction concentration of 5-Aza was selected by the MTT method, and the differentiation of P3 hUCB-MNCs in vitro to myoblasts was carried out. oD1, mygenin, and myosin heavy chain immunofluorescence staining and Real-time PC R. The results of the experiment established a stable and effective culture system of hUCB-MSCs: the number of mononuclear cells was much higher than that of the two-step method with hydroxyethyl starch + lymphocyte separation (P <0.01);10% FBS DMEM/ F12 and Mesenu In the ltTM culture medium, the culture efficiency of the cells in the T25 culture flask was 5-10-7, and the culture efficiency was higher than that of the control group (P <0.01). The expression of CD29 and CD105 in the adherent cells did not express CD34, CD45, CD90 and CD133. After 21 days of bone-induced culture, the cell Yon Kossa was stained. Color-visible mineralized nodules. After 7 days of induction of P3 UCB-MNCs via 5. m mmol/ L 5-Aza, only MyoD1 was expressed by immunofluorescence test; when 14 d after induction, MyoD1 and myoge were visible. nin positive expression. No myosin heavy c at all times The expression of hain was detected by Real-time PCR and the relative concentrations of MyoD1 and mygenin increased compared to 7 d after induction, without myosin heavy c In addition, UCB-MNCs can also be used in uninduced conditions The expression of MyoD1. The experiment concluded that there was a certain number of mesenchymal stem cells in the cord blood, and by improving the method of separation of hUCB-MSCs, the tissue culture was optimized. the UCB-MSCs do not express the surface marks of the hematopoietic stem cells and the endothelial cells, and the expression is marked with the surface of the bone marrow mesenchymal stem cells and can induce the osteogenesis, In the differentiation of myoblasts, hUCB-MSCs were induced by 5. mu. mmol/ L 5-Aza, and the regulation factors in the differentiation of myoblasts can be expressed.
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R329

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