本文選題：MDSCs　切入點：bFGF　出處：《吉林大學(xué)》2009年碩士論文

【摘要】： 目前,MDSCs的研究存在許多熱點問題,體外細胞培養(yǎng)中的純化、擴增、定向分化等難題尚在探索中。人們在探討適宜的干細胞培養(yǎng)條件的同時,特別關(guān)注生物活性因子對干細胞增殖的影響。bFGF和CNTF作為生物活性因子的成員,因其生物活性的多效性而備受基礎(chǔ)與臨床研究的重視。 本文進行的有關(guān)研究,旨在進一步探討成體干細胞的培養(yǎng)條件,為有關(guān)研究與應(yīng)用奠定實驗基礎(chǔ)。本實驗通過酶消化法分離大鼠MDSCs,然后用密度梯度離心法和差速貼壁法純化獲取MDSCs,進行體外原代和傳代培養(yǎng)。并通過免疫細胞化學(xué)法(desmin、Sca-1)對所獲得的細胞進行鑒定。通過MTT比色法檢測不同濃度(6.25、12.50、25.00、50.00、100.00ng/ml)的bFGF和CNTF單獨應(yīng)用96h以及二者(100.0ng/ml)聯(lián)合作用24h、48h、72h和96h對MDSCs增殖的影響。MTT的OD值顯示: 1.與陰性對照組比較,除6.25(ng/ml)組外,12.50ng/ml至100.00ng/ml實驗組的MDSCs均有明顯的增殖(P0.05)。在6.25ng/ml至50.00ng/ml期間,MDSCs的增殖隨bFGF和CNTF濃度升高而顯著增強(P0.05)。 2.在6.25ng/ml至50.00ng/ml期間,bFGF與CNTF對MDSCs的促增殖作用沒有明顯差異(P0.05)。隨其濃度升高,在50.00ng/ml時,bFGF對MDSCs的促增殖作用明顯低于CNTF組,并存在顯著性差異(P0.05)。但50.00ng/ml組促增殖作用接近頂峰,與100.00ng/ml組比較,差異無統(tǒng)計學(xué)意義(P0.05)。 3.bFGF和CNTF的促增殖效應(yīng)隨培養(yǎng)時間的延長而逐漸明顯,但促增殖作用的顯效時間有明顯差異。與陰性對照組比較,出現(xiàn)顯著促增殖效應(yīng)的時間(P0.05)分別為:bFGF單獨作用96 h、CNTF單獨作用72h、二者聯(lián)合應(yīng)用48h。實驗組在24h內(nèi)盡管MDSCs數(shù)量增多,但沒有顯著性差異(P0.05)。 4.在48h-96h期間,bFGF和CNTF聯(lián)合組促進增殖的作用最強,并與其它實驗組間存在顯著差異(P0.05)。 本實驗提示:bFGF和CNTF均可促進體外培養(yǎng)的大鼠MDSCs增殖,而且具有協(xié)同增效作用。但是,bFGF和CNTF促進MDSCs增殖作用具有顯效時間的差異性,而且在12.5-50ng/ml期間,促MDSCs增殖作用還呈濃度依賴性。
[Abstract]:At present, there are many hot problems in the research of MDSCs, such as purification, amplification, directional differentiation and so on. The effects of bioactive factors on the proliferation of stem cells. BFGF and CNTF as members of bioactive factors have attracted much attention in basic and clinical studies because of their biological activity. The purpose of this study is to further explore the culture conditions of adult stem cells. In this experiment, MDSCs were isolated by enzyme digestion, then purified by density gradient centrifugation and differential adhesion method. MTT colorimetric assay was used to detect the effects of bFGF and CNTF at different concentrations on the proliferation of MDSCs for 96 h and 24 h, 48 h, 72 h and 96 h, respectively. 1.Compared with the negative control group, the proliferation of MDSCs in the 100.00ng/ml group was significantly increased with the increase of bFGF and CNTF concentrations, except for the 6.25ng / ml group, and the proliferation of P0.05G / ml in the 100.00ng/ml group was significantly increased with the increase of bFGF and CNTF concentration during the period of 6.25ng/ml to 50.00ng/ml. 2. There was no significant difference between CNTF and 6.25ng/ml in promoting the proliferation of MDSCs between 6.25ng/ml and 50.00ng/ml. With the increase of 6.25ng/ml concentration, the proliferation of MDSCs in 50.00ng/ml group was significantly lower than that in CNTF group, and there was significant difference (P 0.05). However, the proliferation effect of 50.00ng/ml group was near the peak. There was no significant difference compared with 100.00ng/ml group (P 0.05). The proliferative effect of 3.bFGF and CNTF was more and more obvious with the increase of culture time, but there was significant difference between the two groups. The time of significant proliferative effect (P0.05) was 96 h and 72 h, respectively. In the experimental group, the number of MDSCs increased, but there was no significant difference in the number of MDSCs in the experimental group (P 0.05). 4. During 48h-96h, the combination of bFGF and CNTF had the strongest effect on promoting proliferation, and there was a significant difference between the two groups and other experimental groups (P 0.05). The results suggest that both CNTF and BFGF can promote the proliferation of MDSCs in vitro and have synergistic effect. However, the effects of bFGF and CNTF on the proliferation of MDSCs have significant difference in time, and during the period of 12.5-50ng/ml, the effects of BFGF and CNTF on the proliferation of MDSCs were significantly different. The proliferative effect of MDSCs was also concentration-dependent.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：D919

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