【摘要】：目的：研究小膠質(zhì)細胞不同程度低氧下TrKA的表達及梓醇對其影響。 方法：以BV2細胞株代表小膠質(zhì)細胞，在不同程度低氧及有無梓醇的情況下對其培養(yǎng)。應用間接免疫熒光細胞化學法、流式細胞術及Western-blot法分別對TrKA蛋白在小膠質(zhì)細胞的表達進行定位、定量及定性研究。免疫熒光法中FITC標記的綠色熒光代表TrKA。流式細胞術用百分率來表示TrKA陽性細胞比例。Western-blot法用灰度值來表示TrKA及pTrKA的表達量（灰度值與蛋白表達量成正比）。 結果： 1.小膠質(zhì)細胞在常氧培養(yǎng)下其膜上存在TrKA表達，低氧培養(yǎng)1.5hTrKA表達增加，而低氧培養(yǎng)14h時TrKA表達則降低。加入梓醇后使各低氧培養(yǎng)組的TrKA表達有所增加。 2.小膠質(zhì)細胞在常氧培養(yǎng)下TrKA陽性細胞比例為82.20±2.34%。低氧1.5h、4h后陽性細胞比例明顯增多（P0.05），分別可達87.27±0.42%、89.07±2.57%。隨著低氧時間延長，TrKA陽性細胞比例顯著下降（P0.05），低氧8h和14h分別為77.57±0.96%、73.13±2.37%。各低氧培養(yǎng)的小膠質(zhì)細胞加入梓醇后，TrKA陽性細胞比例均有明顯增加（P0.05），分別為90.63±1.17%、95.87±0.42%、84.07±2.60%、78.73±0.71%。 3.小膠質(zhì)細胞在常氧培養(yǎng)下有少量pTrKA表達，灰度值是0.94±0.02。低氧1.5h、4h pTrKA明顯增加（P0.01），其灰度值分別為1.94±0.73、4.58±0.44。隨著低氧時間延長，pTrKA表達降低，，低氧8h和14h灰度值分別為0.25±0.03、0.17±0.01，較常氧培養(yǎng)下降明顯（P0.05）。各低氧培養(yǎng)組加入梓醇后pTrKA的表達均有所升高（P0.05）,灰度值分別為2.18±0.57、8.62±0.53、1.15±0.05、1.37±0.24。 結論： 1.在小膠質(zhì)細胞膜上存在TrKA蛋白，在正常情況下有少量磷酸化表達。 2. TrKA蛋白表達量及其磷酸化水平與缺氧時間有關，低氧時間較短時（4h），TrKA及pTrKA增多，隨著低氧時間的延長，TrKA及pTrKA逐漸下降。 3.低氧條件下，梓醇可促進小膠質(zhì)細胞TrKA及pTrKA的表達。
[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 鹵2.34. The percentage of positive cells increased significantly (P0.05) after 1.5 h hypoxia, and reached 87.27 鹵0.42% (89.07 鹵2.57) respectively. The percentage of TrKA positive cells decreased significantly with the prolongation of hypoxic time (P0.05), and was 77.57 鹵0.96 鹵73.13 鹵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 鹵1.170.87 鹵0.42 and 84.07 鹵2.6078.73 鹵0.73 鹵0.73 respectively. There was a little pTrKA expression in microglia cultured in normoxic culture, and the gray value was 0.94 鹵0.02. The value of pTrKA increased significantly (P0.01) after 1.5 h hypoxia, and its gray value was 1.94 鹵0.73 鹵4.58 鹵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 鹵0.03 and 0.17 鹵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 鹵0.57 鹵0.57 鹵0.53 鹵1.15 鹵0.05 鹵1.37 鹵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.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R329.2

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