本文選題：同種異體 + 間充質(zhì)干細(xì)胞�。� 參考：《遼寧醫(yī)學(xué)院》2012年碩士論文

【摘要】：目的 探討大鼠BMSCs、ADSCs、PMSCs誘導(dǎo)成骨后在體外環(huán)境下的抗原性差異。 方法 8w齡SPF級(jí)近交系Wistar大鼠，取材獲得三種間充質(zhì)干細(xì)胞：BMSCs、ADSCs、PMSCs，對上述細(xì)胞進(jìn)行分離培養(yǎng)，噻唑藍(lán)（MTT）法記錄細(xì)胞生長曲線；取第3~5代細(xì)胞采用CD分子表面標(biāo)志物鑒定細(xì)胞并定向誘導(dǎo)為成骨樣細(xì)胞；堿性磷酸酶染色、鈣結(jié)節(jié)染色檢測細(xì)胞成骨分化能力；應(yīng)用Transwell共培養(yǎng)體系檢測成骨誘導(dǎo)后的三種細(xì)胞對同種異體SPF級(jí)近交系WKY大鼠脾淋巴細(xì)胞增殖的影響；采用流式細(xì)胞儀檢測淋巴細(xì)胞CD4+、CD8+的陽性率；采用免疫酶聯(lián)反應(yīng)檢測TGF-β1、IL-2的含量。 結(jié)果 ①細(xì)胞形態(tài)差別：BMSCs細(xì)胞較細(xì)長，ADSCs細(xì)胞趨向于圓形，PMSCs細(xì)胞趨向于多角。②生長曲線分析：PMSCs潛伏期短，于傳代后即進(jìn)入一個(gè)相對快速的對數(shù)增殖，4-5d后進(jìn)入平臺(tái)期；而BMSCs與ADSCs增殖則相對滯后，對數(shù)增殖集中于3-5d，第六日后細(xì)胞進(jìn)入平臺(tái)期。三種細(xì)胞均可通過體外擴(kuò)增方法進(jìn)行大量培養(yǎng)。成骨誘導(dǎo)后，三種細(xì)胞的生長曲線分析：三種細(xì)胞潛伏期為1d， PMSCs于誘導(dǎo)后3-4d進(jìn)入平臺(tái)期；BMSCs和ADSCs于誘導(dǎo)后4-5d進(jìn)入平臺(tái)期。③P5代（成骨誘導(dǎo)前）CD分子表面標(biāo)志物鑒定：PMSCs細(xì)胞CD29陽性率85%，，CD34陽性率為1.33%；BMSCs細(xì)胞CD29陽性率為90%，CD34陽性率為0.97%；ADSCs細(xì)胞CD29陽性率為80%，CD34陽性率為0.99%。④三種細(xì)胞經(jīng)過茜素紅染色后均可見紫紅色鈣結(jié)節(jié)，提示三種細(xì)胞均可以向成骨樣細(xì)胞方向誘導(dǎo)；三種細(xì)胞經(jīng)過堿性磷酸酶染色顯示深淺不一咖啡色顆粒，Kaplow評(píng)級(jí)：PMSCs（4分）＞BMSCs（3分）＞ADSCs（2分）。⑤在體外與同種異體淋巴細(xì)胞共培養(yǎng)試驗(yàn)中，MTT檢測顯示三種細(xì)胞都表現(xiàn)出抑制淋巴細(xì)胞增殖的作用，抑制淋巴細(xì)胞增殖的作用與誘導(dǎo)時(shí)間正相關(guān)。單因素方差分析：三種細(xì)胞誘導(dǎo)前后及三者之間橫向比較免疫源性均無統(tǒng)計(jì)學(xué)差異。流式細(xì)胞儀檢測共培養(yǎng)后的CD4+、CD8+淋巴細(xì)胞陽性率均降低。免疫酶聯(lián)反應(yīng)檢測共培養(yǎng)后上清液中IL-2表達(dá)量均降低；TGF-β1表達(dá)量均增加。 結(jié)論 體外環(huán)境下，三種間充質(zhì)干細(xì)胞成骨誘導(dǎo)前后及三者之間橫向比較免疫源性沒有差異。
[Abstract]:Objective to investigate the difference of antigenicity of rat BMSCsADP PMSCs induced by osteogenesis in vitro. Methods three kinds of mesenchymal stem cells (BMSCs) were obtained from 8 w SPF inbred strain Wistar rats. The above cells were isolated and cultured. The cell growth curve was recorded by thiazolyl methylene tetrachloride (MTT) method. The cells were identified by CD molecular surface markers and induced into osteoblast-like cells, alkaline phosphatase staining and calcium nodule staining were used to detect the osteogenic differentiation ability of the cells. Transwell co-culture system was used to detect the effect of three kinds of osteoblast induced cells on the proliferation of splenic lymphocytes of SPF class inbred rat, and the positive rate of CD4 / CD8 was detected by flow cytometry. The content of TGF- 尾 _ 1 and IL-2 was detected by immuno-enzyme-linked reaction. Results (1) the differentiation of cell morphology and the tendency of ADSCs to round PMSCs tended to be polygonal. The growth curve analysis showed that the incubation period of BMSCs was shorter than that of BMSCs. After passage, it entered a relatively rapid logarithmic proliferation for 4-5 days, but BMSCs and ADSCs were relatively delayed in proliferation, the logarithmic proliferation was concentrated at 3-5 days, and the cells entered the platform phase after the sixth day. All three kinds of cells can be cultured in large quantities by in vitro amplification. Growth curve analysis of three kinds of cells after osteogenic induction: the incubation period of three kinds of cells was 1 day, PMSCs entered the plateau stage 3-4 days after induction; BMSCs and ADSCs entered the platform.3P5 passage 4 to 5 days after induction. (CD29 positive rate of BMSCs and ADSCs was 1.33 CD34 positive rate of BMSCs cells was 1.33 and CD34 positive rate of BMSCs cells was 0.97% before osteogenesis induction. The CD29 positive rate of ADSCs cells was 80 and CD34 positive rate was 0.99.4 all three kinds of cells could be induced to osteoblast by alizarin red staining. The results of alkaline phosphatase staining showed that the three kinds of cells showed different shades of brown granules: PMSCs (4 points) > BMSCs (3 points) > ADSCs (2 points). In vitro co-culture test with allogeneic lymphocytes, MTT assay showed that all three kinds of cells were present. To inhibit the proliferation of lymphocytes, The inhibition of lymphocyte proliferation was positively correlated with the induction time. Univariate ANOVA: there was no significant difference in immunogenicity between the three kinds of cells before and after induction. The positive rate of CD 4 and CD 8 lymphocytes in co culture was decreased by flow cytometry. The expression of IL-2 in supernatant was decreased by immunoenzyme linked reaction (Elisa) and the expression of TGF- 尾 1 in supernatant was increased. Conclusion in vitro, there is no difference in immunogenicity between the three mesenchymal stem cells before and after osteogenesis.
【學(xué)位授予單位】：遼寧醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392.12

【參考文獻(xiàn)】

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1 黃謙;吳濤;梁杰;楊科躍;金丹;;全骨髓培養(yǎng)法和密度梯度離心法分離hBMSCs的比較研究[J];中國修復(fù)重建外科雜志;2009年11期

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