本文選題：肥大細胞 + CD88��； 參考：《安徽醫(yī)科大學》2017年碩士論文

【摘要】：目的探討建立CD88表型肥大細胞的方法,研究融合蛋白C5a刺激肥大細胞脫顆粒情況,利用氯化鈷刺激肝實質(zhì)細胞,建立肝實質(zhì)細胞缺氧損傷模型,將脫顆粒的肥大細胞與肝實質(zhì)細胞AML12共培養(yǎng),觀察肥大細胞脫顆粒對受損肝實質(zhì)細胞的影響。方法 (1)提取骨髓源肥大細胞和皮膚源肥大細胞,流式鑒定肥大細胞表型以及甲苯胺藍法鑒定肥大細胞;(2)應用PMA或者離子霉素處理骨髓源肥大細胞和P815細胞后,流式鑒定其CD88表達情況,并尋找PMA和離子霉素刺激肥大細胞表達CD88的合適處理時間以及劑量;(3)蛋白免疫印跡法(Western blot)分析檢測肥大細胞CD88、β-actin蛋白的表達水平的影響;(4)應用C5a融合蛋白刺激CD88表型的肥大細胞,β-內(nèi)酰胺酶法檢測肥大細胞脫顆粒情況;(5)應用氯化鈷處理肝實質(zhì)細胞AML12建立肝缺氧模型;(6)將脫顆粒的肥大細胞與受損的肝實質(zhì)細胞AML12共培養(yǎng),觀察肥大細胞脫顆粒對受損肝實質(zhì)細胞的影響。結果(1)通過細胞因子誘導分化,一段時間后可以獲得大量高純度的肥大細胞,皮膚提取肥大細胞未能獲得肥大細胞;(2)骨髓源肥大細胞經(jīng)過PMA或者離子霉素刺激后,流式檢測其CD88表達明顯上升,在時間和劑量的對比試驗中發(fā)現(xiàn),PMA和離子霉素刺激肥大細胞表達CD88的最適時間為24h,最適宜濃度分別為50ng/ml和1000ng/ml,而P815肥大細胞株未出現(xiàn)明顯的CD88表達;(3)蛋白免疫印跡法結果提示:與未經(jīng)PMA或離子霉素刺激的肥大細胞相比,經(jīng)過刺激的肥大細胞,其CD88蛋白的表達明顯增加;(4)融合蛋白C5a刺激CD88表型肥大細胞后,應用β-內(nèi)酰胺酶法測肥大細胞脫顆粒發(fā)現(xiàn),與未加C5a的對照組相比,其脫顆粒明顯增加,且隨融合蛋白C5a加入濃度的增加而增加,呈現(xiàn)出濃度依賴性;(5)根據(jù)文獻報道,經(jīng)過預實驗,尋找合適的氯化鈷濃度來建立肝實質(zhì)細胞的缺氧損傷;(6)應用氯化鈷建立肝實質(zhì)細胞受損模型后發(fā)現(xiàn),在輕微的肝實質(zhì)細胞受損情況下,肥大細胞脫顆粒能夠刺激肝實質(zhì)細胞反應性的增生,而在重度肝實質(zhì)細胞受損的情況下,肥大細胞脫顆粒能夠進一步促進損傷的發(fā)生,發(fā)揮抑制肝實質(zhì)細胞增殖的作用。結論通過細胞因子誘導可以獲得大量高純度的肥大細胞,經(jīng)過PMA或離子霉素刺激后,其表面CD88表達明顯上升,經(jīng)C5a刺激后,肥大細胞脫顆粒并呈現(xiàn)濃度依賴性,脫顆粒的肥大細胞可以抑制重度受損的肝實質(zhì)細胞增殖。
[Abstract]:Objective to investigate the method of establishing CD88 phenotypic mast cells, to study the degranulation of mast cells stimulated by fusion protein C5a, and to establish a model of hypoxia-induced liver parenchymal cells injury by using cobalt chloride to stimulate liver parenchymal cells. The effects of degranulation of mast cells on injured hepatic parenchymal cells were observed by co-culture of degranulated mast cells and hepatic parenchymal cells (AML12). Methods (1) Bone marrow-derived mast cells and skin derived mast cells were extracted, mast cell phenotypes were identified by flow cytometry and mast cells were identified by toluidine blue, (2) bone marrow-derived mast cells and P815 cells were treated with PMA or ionomycin. The expression of CD88 was detected by flow cytometry. The appropriate treatment time and dose of PMA and ionomycin to stimulate the expression of CD88 in mast cells were found; (3) Western blot analysis was used to detect the expression level of CD88 and 尾 -actin in mast cells; (4) C5a fusion protein was used to stimulate the expression of CD88. Type A mast cells, 尾 -lactamases were used to detect degranulation of mast cells; (5) liver hypoxia model was established by cobalt chloride treatment of hepatic parenchymal cells; (6) degranulated mast cells were co-cultured with damaged hepatic parenchymal cells (AML12). To observe the effect of mast cell degranulation on injured hepatic parenchymal cells. Results (1) after inducing differentiation by cytokines, a large number of high purity mast cells could be obtained after a period of time, but the mast cells extracted from the skin could not obtain mast cells, (2) the bone marrow-derived mast cells were stimulated by PMA or ionomycin. The expression of CD88 was significantly increased by flow cytometry. The optimal time for CD88 expression of mast cells stimulated by PMA and ionomycin was 24 h, and the optimal concentration was 50ng/ml and 1000ng / ml, respectively, but no CD88 expression was found in P815 mast cell line; (3) Western blot. The results suggest that compared with mast cells without PMA or ionomycin stimulation, (4) after stimulation of CD88 phenotypic mast cells by fusion protein C5a, the degranulation of mast cells was detected by 尾 -lactamase method, and it was found that the degranulation of mast cells was significantly increased compared with the control group without C5a. The concentration of C5a protein increased in a concentration-dependent manner. (5) according to the literature, the concentration of the fusion protein C5a increased. (6) the liver parenchymal cell damage model was established by using cobalt chloride to establish the liver parenchymal cell damage model, and it was found that the liver parenchymal cells were damaged in the case of mild hepatic parenchymal cell damage. Mast cell degranulation can stimulate the reactive proliferation of hepatic parenchyma cells, but in the case of severe hepatic parenchymal cell injury, mast cell degranulation can further promote the occurrence of injury and play a role in inhibiting the proliferation of hepatic parenchymal cells. Conclusion A large number of high purity mast cells can be obtained by cytokine induction. After stimulation with PMA or ionomycin, the expression of CD88 on the surface of mast cells increased significantly. After C5a stimulation, mast cells were degranulated in a concentration-dependent manner. Degranulated mast cells inhibit the proliferation of severely damaged hepatic parenchymal cells.
【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】：2017
【分類號】：R657.3

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