【摘要】： 目的 探討影響人臍血間充質(zhì)干細胞(UCB-MSCs)成功分離培養(yǎng)的相關因素及其生物學特性,并對其向成骨、成脂肪細胞誘導分化的能力進行研究。 方法 (1)分別從不同胎齡(≥40周、37周和≤32周)、臍血中單個核細胞(MNCs)的數(shù)量(≥2.5×10~9/L、＜2.5×10~9/L)、不同細胞接種密度(1×10~4 cells/ml、1×10~6 cells/ml、1×10~8 cells/ml)、不同胎牛血清(FBS)濃度(5%、10%、15%、20%)以及培養(yǎng)瓶是否被FBS包被等方面對UCB-MSCs的分離培養(yǎng)成功率進行分析比較;通過倒置相差顯微鏡及透射電鏡觀察細胞的形態(tài)、生長增殖情況及其超微結構;并描繪細胞生長曲線;計算并分析比較不同胎齡組UCB-MSCs的集落形成能力和細胞倍增時間差異;用流式細胞儀對細胞表面標志物進行檢測。 (2)分別用成骨及成脂誘導液對UCB-MSCs進行誘導,通過茜素紅染色、堿性磷酸酶染色及活性測定等檢測UCB-MSCs向成骨細胞誘導分化的能力;通過油紅O染色檢測其向脂肪細胞誘導分化的能力。 結果 (1)UCB-MSCs的分離培養(yǎng)成功率為58.3%,且不同胎齡組培養(yǎng)成功率隨胎齡的增高而降低,差異有統(tǒng)計學意義(x~2=9.87,P=0.007);臍血中MNCs含量在2.5×10~9/L以上者培養(yǎng)成功率高達76.9%,與MNCs含量低于2.5×10~9/L組(36.4%)比較,差異有統(tǒng)計學意義(x~2=8.07,P=0.005);相同容量的臍血中MNCs與胎齡的相關性分析顯示,兩者呈負相關(n=20,r=-0.95,P＜0.01);在接種密度低于1×10~8 cells/ml組中,原代及傳代培養(yǎng)的MSCs生長及擴增情況均不如1×10~8 cells/ml組;FBS濃度為5%組與10%、15%、20%組相比,MSCs貼壁速度略慢,但MSCs的純度較高,破骨樣細胞含量明顯要少,后三組之間未見明顯差別,且5%FBS濃度組細胞傳代速度與其他三組相比無明顯差別;用FBS對培養(yǎng)瓶進行包被后,UCB-MSCs不僅在原代和傳代后的純度增加了,而且其擴增能力也明顯快于未包被組。倒置相差顯微鏡下觀察UCB-MSCs貼壁生長,呈成纖維細胞樣外觀,細胞呈螺旋狀排列;透射電鏡觀察UCB-MSCs細胞核大,胞核比例大,細胞器少,為低分化細胞;原代及傳代培養(yǎng)的UCB-MSCs生長曲線均呈S型,第3、5代細胞增殖能力最強;3個不同胎齡組的UCB-MSCs集落形成能力存在統(tǒng)計學差異(F=8.53,P＜0.05),低胎齡組集落形成能力大于高胎齡組;不同胎齡的UCB-MSCs細胞群體倍增時間比較差異無統(tǒng)計學意義(F=2.68,P＞0.05);流式細胞儀檢測結果顯示,UCB-MSCs均穩(wěn)定的表達與MSCs相關的表面抗原標志物CD29、CD44和CD90等,不表達造血標志CD34和CD45。 (2) UCB-MSCs成骨誘導后ALP活性逐漸增強,3周時誘導細胞ALP染色強陽性、茜素紅染色可檢測到大量鈣化基質(zhì)的形成;成脂誘導3周時油紅O染色可檢測到胞質(zhì)中脂滴的形成。 結論 (1)不同胎齡的臍血中均含有MSCs,其分離培養(yǎng)成功率受多種因素的影響;通過選擇較低胎齡的胎兒、采集足夠量的臍血、以較高的細胞密度接種、培養(yǎng)基中添加較低濃度的FBS、并將培養(yǎng)瓶預先用FBS進行包被、且在細胞培養(yǎng)的適當時機進行換液和傳代等,能在體外建立穩(wěn)定的UCB-MSCs培養(yǎng)體系。 (2) UCB-MSCs的形態(tài)特征、生長增殖特點及細胞表面標志物等生物學特性與骨髓MSCs相似,具有強大的生長增殖與自我更新能力。 (3) UCB-MSCs在體外誘導條件下可以向成骨細胞及脂肪細胞等間質(zhì)組織細胞定向分化,為其在臨床上的應用奠定了理論基礎。
[Abstract]:Purpose The related factors and their biological characteristics of the successful isolation and culture of human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) were discussed. line-and-study Methods (1) The number of mononuclear cells (MNCs) in the umbilicus was 10-4 cells/ ml,1-10-6 cells/ ml,1-10,10-4 cells/ ml,1-10-6 cells/ ml,10-9/ L,10-9/ L,10-9/ L,10-9/ L,10-9/ L, respectively. The success rate of the separation and culture of UCB-MSCs was analyzed by means of inverted phase contrast microscope and transmission electron microscope. The cell growth curve, colony-forming ability and cell doubling time difference of UCB-MSCs in different gestational age groups were calculated and analyzed, and flow cytometry was used. The cell surface markers were tested. (2) UCB-MSCs were induced by bone and fat-forming induction solution, and the ability of UCB-MSCs to induce differentiation into osteoblasts was detected by the staining of fluorescein red, alkaline phosphatase staining and activity determination. detection Results (1) The success rate of the separation and culture of UCB-MSCs was 58.3%, and the success rate of the different gestational age groups decreased with the increase of the gestational age, the difference was statistically significant (x ~ 2 = 9.87, P = 0.007), and the content of MNCs in the umbilical blood was 2. The results showed that the culture success rate was 76.9%, the content of MNCs was lower than that of 2.5-10-9/ L (36.4%), the difference was statistically significant (x ~ 2 = 8.07, P = 0.005), and the correlation between MNCs and gestational age in the umbilical blood of the same volume showed negative correlation (n = 20, r =-0.95, P <0.01). In the group of 10 ~ 8 cells/ ml, the growth and amplification of MSCs in primary and subcultured cells were less than that of 1-10 ~ 8 cells/ ml, and the concentration of the FBS was 5% and 10%,15% and 20%, and the adherent rate of MSCs was slightly slower, but the purity of MSCs was higher. The content of osteoclast-like cells was significantly lower, and there was no significant difference between the three groups, and the cell passage speed of 5% FBS group was not significantly different from the other three groups; after the culture flask was coated with FBS, the UCB-MSCs were not only after primary and passage The purity of UCB-MSCs was increased, and the amplification ability of UCB-MSCs was significantly faster than that of uncoated group. UCB-MSCs were observed under the inverted phase-contrast microscope. The appearance of the fibroblast-like appearance and the cell-like appearance were observed by transmission electron microscope. The nucleus of UCB-MSCs was observed by transmission electron microscope. The growth curve of UCB-MSCs was the strongest in the third and fifth generation, and the colony-forming ability of the three different gestational age groups was higher than that of the high-gestational age group. The UCB-MSCs of different gestational age The difference of population doubling time was not significant (F = 2.68, P> 0.05). The results of flow cytometry showed that the expression of UCB-MSCs was stable to the surface antigen markers CD29 and CD44 associated with MSCs. And CD90 and the like, and the hematopoietic marker CD34 and the CD45 are not expressed. (2) the ALP activity of the UCB-MSCs is gradually enhanced after the osteogenesis induction, and the ALP staining of the induction cells is strongly positive at 3 weeks, and a large amount of the calcified matrix can be detected by the fluorescein red staining. to form Conclusion (1) The successful rate of separation and culture of MSCs in different gestational age is affected by many factors. And the culture flask is pre-coated with FBS and cultured in cell culture. A stable UCB-MSCs culture system can be established in vitro. (2) The morphological characteristics, growth and proliferation characteristics of UCB-MSCs and the growth and proliferation characteristics of UCB-MSCs can be established in vitro. The biological characteristics of the cell surface markers are similar to that of the bone marrow MSCs, and have strong growth and proliferation and self-renewal capacity. (3) UCB-MSCs are induced in vitro
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R329

【引證文獻】

相關碩士學位論文 前1條

1 葉興德;CRIF1在骨髓間充質(zhì)干細胞輻射氧化應激中的作用及其機制初步研究[D];第三軍醫(yī)大學;2013年

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