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

【摘要】：目的探討建立CD88表型肥大細(xì)胞的方法,研究融合蛋白C5a刺激肥大細(xì)胞脫顆粒情況,利用氯化鈷刺激肝實(shí)質(zhì)細(xì)胞,建立肝實(shí)質(zhì)細(xì)胞缺氧損傷模型,將脫顆粒的肥大細(xì)胞與肝實(shí)質(zhì)細(xì)胞AML12共培養(yǎng),觀察肥大細(xì)胞脫顆粒對(duì)受損肝實(shí)質(zhì)細(xì)胞的影響。方法 (1)提取骨髓源肥大細(xì)胞和皮膚源肥大細(xì)胞,流式鑒定肥大細(xì)胞表型以及甲苯胺藍(lán)法鑒定肥大細(xì)胞;(2)應(yīng)用PMA或者離子霉素處理骨髓源肥大細(xì)胞和P815細(xì)胞后,流式鑒定其CD88表達(dá)情況,并尋找PMA和離子霉素刺激肥大細(xì)胞表達(dá)CD88的合適處理時(shí)間以及劑量;(3)蛋白免疫印跡法(Western blot)分析檢測(cè)肥大細(xì)胞CD88、β-actin蛋白的表達(dá)水平的影響;(4)應(yīng)用C5a融合蛋白刺激CD88表型的肥大細(xì)胞,β-內(nèi)酰胺酶法檢測(cè)肥大細(xì)胞脫顆粒情況;(5)應(yīng)用氯化鈷處理肝實(shí)質(zhì)細(xì)胞AML12建立肝缺氧模型;(6)將脫顆粒的肥大細(xì)胞與受損的肝實(shí)質(zhì)細(xì)胞AML12共培養(yǎng),觀察肥大細(xì)胞脫顆粒對(duì)受損肝實(shí)質(zhì)細(xì)胞的影響。結(jié)果(1)通過(guò)細(xì)胞因子誘導(dǎo)分化,一段時(shí)間后可以獲得大量高純度的肥大細(xì)胞,皮膚提取肥大細(xì)胞未能獲得肥大細(xì)胞;(2)骨髓源肥大細(xì)胞經(jīng)過(guò)PMA或者離子霉素刺激后,流式檢測(cè)其CD88表達(dá)明顯上升,在時(shí)間和劑量的對(duì)比試驗(yàn)中發(fā)現(xiàn),PMA和離子霉素刺激肥大細(xì)胞表達(dá)CD88的最適時(shí)間為24h,最適宜濃度分別為50ng/ml和1000ng/ml,而P815肥大細(xì)胞株未出現(xiàn)明顯的CD88表達(dá);(3)蛋白免疫印跡法結(jié)果提示:與未經(jīng)PMA或離子霉素刺激的肥大細(xì)胞相比,經(jīng)過(guò)刺激的肥大細(xì)胞,其CD88蛋白的表達(dá)明顯增加;(4)融合蛋白C5a刺激CD88表型肥大細(xì)胞后,應(yīng)用β-內(nèi)酰胺酶法測(cè)肥大細(xì)胞脫顆粒發(fā)現(xiàn),與未加C5a的對(duì)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(xiàn)報(bào)道,經(jīng)過(guò)預(yù)實(shí)驗(yàn),尋找合適的氯化鈷濃度來(lái)建立肝實(shí)質(zhì)細(xì)胞的缺氧損傷;(6)應(yīng)用氯化鈷建立肝實(shí)質(zhì)細(xì)胞受損模型后發(fā)現(xiàn),在輕微的肝實(shí)質(zhì)細(xì)胞受損情況下,肥大細(xì)胞脫顆粒能夠刺激肝實(shí)質(zhì)細(xì)胞反應(yīng)性的增生,而在重度肝實(shí)質(zhì)細(xì)胞受損的情況下,肥大細(xì)胞脫顆粒能夠進(jìn)一步促進(jìn)損傷的發(fā)生,發(fā)揮抑制肝實(shí)質(zhì)細(xì)胞增殖的作用。結(jié)論通過(guò)細(xì)胞因子誘導(dǎo)可以獲得大量高純度的肥大細(xì)胞,經(jīng)過(guò)PMA或離子霉素刺激后,其表面CD88表達(dá)明顯上升,經(jīng)C5a刺激后,肥大細(xì)胞脫顆粒并呈現(xiàn)濃度依賴性,脫顆粒的肥大細(xì)胞可以抑制重度受損的肝實(shí)質(zhì)細(xì)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(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R657.3

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