【摘要】：目的:觀察梯度濃度胸腺素β4(Thymosin-β4)對大鼠骨髓源性內皮祖細胞(EPCs)生物學行為的影響,為改善EPCs移植治療中樞神經(jīng)系統(tǒng)損傷這一策略的有效性,推動EPCs移植為主體的治療手段在臨床中的應用提供更為廣泛的思路。方法:SPF級SD大鼠90~100 g,雌雄不拘,采用密度梯度離心法分離大鼠骨髓源性單個核細胞,接種于鼠纖維連接蛋白(FN)預先包被過夜的24孔培養(yǎng)板中,以含有10%胎牛血清(FBS)的EGM-2完全培養(yǎng)基培養(yǎng),隔日換液,7d后行細胞形態(tài)學及免疫細胞化學雙抗體熒光檢測(CD133及VEGFR-2),激光共聚焦顯微鏡觀察并記錄拍照,同時行Dil標記的乙�；兔芏戎鞍准爱惲蚯杷釤晒馑厍G豆凝集素-1(Dil-ac-LDL及FITC-UEA-1)熒光檢測吞噬實驗,激光共聚焦顯微鏡觀察并記錄拍照,進一步鑒定目的細胞為內皮祖細胞。設置梯度濃度的Tβ4(1 000ng/ml、100 ng/ml、10 ng/ml、1 ng/ml)條件培養(yǎng)基分別處理EPCs,分別采用CCK-8細胞增殖實驗、粘附能力實驗以及Transwell細胞遷移能力實驗以觀察EPCs的增殖能力、粘附能力及遷移能力的變化。同時在統(tǒng)一濃度(1 000 ng/ml)下干預不同時間(24 h、48 h、72 h)后以上述同樣的方法分別進行細胞功能學實驗以觀察EPCs的增殖能力及遷移能力的變化。結果:Tβ4能夠顯著增強體外培養(yǎng)EPCs的增殖、遷移和黏附能力,并且呈現(xiàn)一定的劑量效應關系,在1 000 ng/ml條件培養(yǎng)基培養(yǎng)時其增殖、遷移及黏附能力均得到顯著增強(與對照組相比,P0.05)。在1 000 ng/ml條件培養(yǎng)基培養(yǎng)時隨著干預時間的逐漸推移,各時間點的EPCs增殖能力均顯著增強且優(yōu)于空白對照組(P0.05);黏附能力在48 h達到最大效應(與對照組相比,P0.05)。結論:Tβ4可顯著增強體外培養(yǎng)的大鼠骨髓源性EPCs的生物學活性,并且呈現(xiàn)一定的劑量效應關系,1 000 ng/ml Tβ4條件培養(yǎng)基下預處理48 h可獲得最優(yōu)生物學效應。
[Abstract]:Aim: to observe the effect of gradient concentration of thymosin 尾 4 (Thymosin- 尾 4) on the biological behavior of rat bone marrow-derived endothelial progenitor cells (EPCs) in order to improve the efficacy of EPCs transplantation in the treatment of central nervous system injury. Promoting the application of EPCs transplantation as the main therapeutic means in clinical practice provides a more extensive way of thinking. Methods: bone marrow-derived mononuclear cells of SPF grade SD rats were isolated by density gradient centrifugation and inoculated into a 24 well culture plate coated with rat fibronectin (FN) for the night. The cells were cultured on EGM-2 medium containing 10% fetal bovine serum (FBS) and changed into solution every other day. After 7 days, cell morphology and immunocytochemical double antibody fluorescence detection (CD133 and VEGFR-2) were detected. Laser confocal microscopy was used to observe and record the pictures. Dil labeled low density lipoprotein and fluorescein thiocyanate agglutinin 1 (Dil-ac-LDL and FITC-UEA-1) were used to detect phagocytosis, and laser confocal microscopy was used to record and photograph the phagocytosis. The target cells were identified as endothelial progenitor cells. EPCs, was treated with T 尾 _ 4 (1 000 ng 路ml ~ (-1) ng/ml,10 ng/ml,1 ng/ml) condition medium with gradient concentration. CCK-8 cell proliferation test was used. The ability of proliferation, adhesion and migration of EPCs were observed by adhesion assay and migration ability test of Transwell cells. At the same time, the cell function of EPCs was observed by the same method after intervention at different time (24 h, 48 h, 72 h) at the same concentration (1 000 ng/ml) to observe the changes of proliferation and migration ability of EPCs. Results: t 尾 4 significantly enhanced the proliferation, migration and adhesion of EPCs cultured in vitro, and showed a dose-effect relationship. Migration and adhesion were significantly increased (compared with the control group, P0.05). When cultured on 1 000 ng/ml conditioned medium, the proliferative ability of EPCs at each time point was significantly enhanced and better than that of the blank control group (P0.05). The adhesion ability reached the maximum effect at 48 h (compared with the control group, P0.05). Conclusion: t 尾 4 can significantly enhance the biological activity of rat bone marrow derived EPCs cultured in vitro, and has a dose-effect relationship. The optimal biological effect can be obtained by preconditioning on 1 000 ng/ml T 尾 4 medium for 48 h.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R651.2

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