小胶质细胞不同程度低氧下TrKA的表达及梓醇对其影响

发布时间：2018-07-21 17:27

【摘要】：目的：研究小胶质细胞不同程度低氧下TrKA的表达及梓醇对其影响。 方法：以BV2细胞株代表小胶质细胞，在不同程度低氧及有无梓醇的情况下对其培养。应用间接免疫荧光细胞化学法、流式细胞术及Western-blot法分别对TrKA蛋白在小胶质细胞的表达进行定位、定量及定性研究。免疫荧光法中FITC标记的绿色荧光代表TrKA。流式细胞术用百分率来表示TrKA阳性细胞比例。Western-blot法用灰度值来表示TrKA及pTrKA的表达量（灰度值与蛋白表达量成正比）。 结果： 1.小胶质细胞在常氧培养下其膜上存在TrKA表达，低氧培养1.5hTrKA表达增加，而低氧培养14h时TrKA表达则降低。加入梓醇后使各低氧培养组的TrKA表达有所增加。 2.小胶质细胞在常氧培养下TrKA阳性细胞比例为82.20±2.34%。低氧1.5h、4h后阳性细胞比例明显增多（P0.05），分别可达87.27±0.42%、89.07±2.57%。随着低氧时间延长，TrKA阳性细胞比例显著下降（P0.05），低氧8h和14h分别为77.57±0.96%、73.13±2.37%。各低氧培养的小胶质细胞加入梓醇后，TrKA阳性细胞比例均有明显增加（P0.05），分别为90.63±1.17%、95.87±0.42%、84.07±2.60%、78.73±0.71%。 3.小胶质细胞在常氧培养下有少量pTrKA表达，灰度值是0.94±0.02。低氧1.5h、4h pTrKA明显增加（P0.01），其灰度值分别为1.94±0.73、4.58±0.44。随着低氧时间延长，pTrKA表达降低，，低氧8h和14h灰度值分别为0.25±0.03、0.17±0.01，较常氧培养下降明显（P0.05）。各低氧培养组加入梓醇后pTrKA的表达均有所升高（P0.05）,灰度值分别为2.18±0.57、8.62±0.53、1.15±0.05、1.37±0.24。 结论： 1.在小胶质细胞膜上存在TrKA蛋白，在正常情况下有少量磷酸化表达。 2. TrKA蛋白表达量及其磷酸化水平与缺氧时间有关，低氧时间较短时（4h），TrKA及pTrKA增多，随着低氧时间的延长，TrKA及pTrKA逐渐下降。 3.低氧条件下，梓醇可促进小胶质细胞TrKA及pTrKA的表达。
[Abstract]:Aim: to study the expression of TrKA in microglia at different degrees of hypoxia and the effect of catalpol on it. Methods: BV2 cell line was used to represent microglia and cultured under different degrees of hypoxia and catalpol. The expression of TrKA protein in microglia was detected by indirect immunofluorescence cytochemistry, flow cytometry and Western-blot. In immunofluorescence method, the FITC labeled green fluorescence represents TrKA. The percentage of TrKA positive cells was expressed by flow cytometry. Western-blot was used to indicate the expression of TrKA and pTrKA by gray value (the gray value was directly proportional to the protein expression). Results: 1. The expression of TrKA on the membrane of microglia cultured in normoxic culture was increased at 1.5 h after hypoxia, but decreased at 14 h after hypoxia. After addition of catalpol, the expression of TrKA was increased in all hypoxic culture groups. 2. 2. The percentage of Trka positive cells in microglia cultured in normoxic culture was 82.20 卤2.34. The percentage of positive cells increased significantly (P0.05) after 1.5 h hypoxia, and reached 87.27 卤0.42% (89.07 卤2.57) respectively. The percentage of TrKA positive cells decreased significantly with the prolongation of hypoxic time (P0.05), and was 77.57 卤0.96 卤73.13 卤2.37 at 8 h and 14 h, respectively. The percentage of TrKA positive cells in microglia cultured with catalpol increased significantly (P 0.05), which were 90.63 卤1.170.87 卤0.42 and 84.07 卤2.6078.73 卤0.73 卤0.73 respectively. There was a little pTrKA expression in microglia cultured in normoxic culture, and the gray value was 0.94 卤0.02. The value of pTrKA increased significantly (P0.01) after 1.5 h hypoxia, and its gray value was 1.94 卤0.73 卤4.58 卤0.44, respectively. With the prolongation of hypoxia time, the expression of pTrKA decreased, the gray values of hypoxia at 8 h and 14 h were 0.25 卤0.03 and 0.17 卤0.01, respectively, which were significantly lower than those in normal oxygen culture (P0.05). The expression of pTrKA was increased in all hypoxic culture groups after addition of catalpol (P0.05), with a gray value of 2.18 卤0.57 卤0.57 卤0.53 卤1.15 卤0.05 卤1.37 卤0.24, respectively. Conclusion: 1. There is TrKA protein on microglia cell membrane and a small amount of phosphorylated expression in normal condition. 2. 2. The expression of TrKA protein and its phosphorylation level were related to the hypoxia time. The levels of TrKA and pTrKA increased at a shorter hypoxia time (4 h), and decreased gradually with the prolongation of hypoxia time. 3. Catalpol could promote the expression of TrKA and pTrKA in microglia under hypoxia.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R329.2

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