麻黄碱对人支气管上皮细胞增殖及表达Eotaxin和IL-8的影响

发布时间：2019-05-30 04:30

【摘要】：目的：研究麻黄碱对体外培养的人支气管上皮细胞(16HBE)形态、增殖及表达嗜酸性粒细胞趋化因子(Eotaxin)、白细胞介素-8(IL-8)的影响，探讨麻黄治疗支气管哮喘的机制。方法：将16HBE细胞株传代培养，选择生长状态良好的第4、5代细胞用于后续实验：⑴将16HBE细胞分别接种于6孔板和96孔板并随机分为5组，分别用含不同浓度(0，150，，300，600，1200μg/mL)麻黄碱的培养基培养，培养24、48、72小时后，倒置相差显微镜下观察各组16HBE细胞形态及生长状况；CCK-8检测16HBE细胞增殖情况；⑵将16HBE细胞接种于6孔板并随机分为6组：①空白对照组（A组）：完全培养基培养18h；②麻黄碱组（B组）：含麻黄碱300μg/mL的完全培养基培养18h；③TNF-α组（C组）：含TNF-α20ng/mL的完全培养基培养18h；④麻黄碱+TNF-α组（D组）：含麻黄碱300μg/mL的完全培养基培养1h后加入100ng/mLTNF-α，共培养18h；⑤地塞米松+TNF-α组（E组）：含地塞米松1.4μg/mL的完全培养基培养1h后加入100ng/mL TNF-α，共培养18h；⑥麻黄碱+地塞米松+TNF-α组（F组）：含麻黄碱300μg/mL、地塞米松1.4μg/mL的完全培养基培养1h后加入100ng/mL TNF-α，共培养18h。D、E、F组TNF-α终浓度均为20ng/mL；⑶荧光定量PCR检测各组细胞Eotaxin、IL-8mRNA的表达；免疫荧光法观察各组细胞Eotaxin、IL-8蛋白的表达；双抗体夹心酶联免疫吸附法（ELISA）检测各组细胞上清液中Eotaxin、IL-8蛋白的浓度。结果：⑴不同浓度（150，300，600，1200μg/mL）的麻黄碱作用于16HBE细胞24h、48h、72小时后，其细胞形态和生长状况与不加麻黄碱（0μg/mL麻黄碱）组比较无明显差异；⑵CCK-8检测结果示：不同浓度麻黄碱作用于16HBE细胞24h，各组细胞的吸光度(OD)值分别为0.97±0.17，0.99±0.24，1.14±0.25，1.11±0.18，0.95±0.18，各组间比较差异无统计学意义（P=0.504）；作用48h各组细胞的OD值分别为1.09±0.43，1.16±0.99，1.16±0.87，1.18±0.56，1.11±0.30，各组间比较差异无统计学意义(P=0.282)；作用72h各组细胞的OD值分别为1.40±0.13，1.39±0.10，1.39±0.16，1.41±0.01，1.40±0.01，各组间比较差异无统计学意义（P=0.18）；⑶荧光定量PCR显示：A至F组细胞Eotaxin mRNA的表达量(RQ值)分别为0.99±0.01，0.98±0.02，3.03±0.32，1.82±0.23，1.78±0.02，0.97±0.01，各组间比较差异有统计学意义（P0.01）。与A组比较，C组的Eotaxin mRNA表达量明显升高，差异有统计学意义(P0.01)；与C组比较，D、E、F组的Eotaxin mRNA表达量明显降低，以F组降低最明显，差异均有统计学意义(P均0.01）。A至F组IL-8mRNA的RQ值分别为0.95±0.05,0.93±0.14,7.18±0.31,4.44±0.24，4.11±0.19，2.97±0.39，各组间比较差异有统计学意义(P0.01)。与A组比较，C组的IL-8mRNA表达量明显升高，差异有统计学意义(P0.01)。与C组比较，D、E、F组的IL-8mRNA表达量均明显降低，以F组降低最明显，差异均有统计学意义(P均0.01）；⑷免疫荧光显示：各组16HBE细胞均可见Eotaxin和IL-8表达，主要表达在细胞浆中，C组表达最强，D、E、F组表达均较C组弱，F组最弱；⑸ELISA检测结果显示：A至F组细胞上清液中Eotaxin的浓度(pg/mL)分别为1.72±0.16，1.60±0.17，4.05±0.20，3.70±0.21，3.33±0.33，2.76±0.15，各组间比较差异有统计学意义(P0.01)。与A组比较，C组Eotaxin浓度明显升高，差异有统计学意义(P0.01)。与C组比较，D、E、F组Eotaxin浓度均明显降低，F组最低，差异均有统计学意义(P均0.01)；A至F组细胞上清液中IL-8浓度(pg/mL)分别为17.45±2.32，18.78±1.05，27.24±1.91，23.62±3.40，19.25±2.36，14.12±3.14，各组间比较差异有统计学意义(P0.01)。与A组比较，C组IL-8浓度明显升高，差异有统计学意义（P0.01）。与C组比较，D、E、F组IL-8浓度均明显降低，F组最低，差异均有统计学意义（P分别0.05，0.01，0.01）。结论：⑴在一定浓度范围内，麻黄碱对体外培养的16HBE形态及增殖无影响；⑵麻黄碱能抑制前炎症因子TNF-α刺激16HBE表达及分泌Eotaxin和IL-8，这可能是中药麻黄治疗哮喘的机制之一；⑶麻黄碱与糖皮质激素抑制16HBE表达及分泌Eotaxin和IL-8具有协同作用。
[Abstract]:Objective: To study the effect of ephedrine on the morphology, proliferation and expression of eosinophil chemotactic factor (Eotaxin) and interleukin-8 (IL-8) in cultured human bronchial epithelial cells (16 HBE) in vitro, and to study the mechanism of Ephedra in the treatment of bronchial asthma. Methods:16 HBE cells were subcultured and the 4th and 5th generation cells with good growth status were selected for subsequent experiments. The 16 HBE cells were respectively inoculated in 6-well and 96-well plates and were randomly divided into 5 groups. The cells were cultured for 24,48 and 72 hours with the medium containing different concentrations (0,150,300,600,1200. mu.g/ mL) of ephedrine. The morphology and growth of 16 HBE cells in each group were observed under an inverted phase-contrast microscope; the proliferation of 16 HBE cells was detected by CCK-8;16 HBE cells were seeded in 6-well plates and were randomly divided into 6 groups: control blank control group (group A): full medium culture for 18 h; Ephedrine group (group B): culturing for 18 h with a total culture medium containing 300 & mu; g/ mL of ephedrine; culturing for 18 h in a complete culture medium containing TNF-20ng/ mL; and adding 100 ng/ ml of LTF-1 for 18 h after culturing for 1 hour with a total culture medium containing 300 & mu; g/ mL of ephedrine; Dexamethasone + TNF-1 (Group E): After 1 h of complete culture with dexamethasone 1.4. mu.g/ mL,100 ng/ mL of TNF-1 was added, co-cultured for 18 h; Ephedrine + dexamethasone + TNF-necrosis group (group F): containing 300. m u.g/ mL of ephedrine, The expression of Eotaxin and IL-8 in each group was observed by fluorescence quantitative PCR, and the expression of Eotaxin and IL-8 in each group was observed by fluorescence quantitative PCR. The concentration of Eotaxin and IL-8 in the supernatant of each group was detected by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Results: Ephedrine of different concentrations (150,300,600,1200 & mu; g/ mL) acted on 16HBE cells for 24 h,48 h and 72 h, and the cell morphology and growth were not significantly different from that of Ephedrine (0. mu.g/ mL ephedrine) group; the results of the detection of CCK-8 showed that the different concentration of ephedrine on the 16HBE cells for 24 h, The OD values of the cells were 0.97, 0.17, 0.99, 0.24, 1.14, 0.25, 1.11, 0.18, 0.95 and 0.18, respectively. The OD values of the cells in each group were 1.09, 0.43, 1.16, 0.99, 1.16, 0.87, 1.18, 0.56, 1.11 and 0.30, respectively (P = 0.282). The OD values of the cells were 1.40, 0.13, 1.39, 0.10, 1.39, 0.16, 1.41, 0.01, 1.40 and 0.01, respectively, and the difference between the groups was not statistically significant (P = 0.18). The quantitative PCR showed that the expression of Eotaxin mRNA in group A to F (RQ value) was 0.99, 0.01, 0.98, 0.02, 3.03, 0.32, 1.82, 0.23, 1.78, 0.02, 0.97 and 0.01, respectively. There was a significant difference between the groups (P0.01). Compared with group A, the expression of Eotaxin mRNA in group C was significantly higher, and the difference was significant (P0.01); in comparison with group C, the expression of Eotaxin mRNA in group D, E and F decreased significantly, and in group F, the difference was statistically significant (P <0.01). The RQ values of IL-8 mRNA in group A to F were 0.95, 0.05, 0.93, 0.14, 7.18, 0.31, 4.44, 0.24, 4.11, 0.19, 2.97 and 0.39, respectively. Compared with group A, the expression of IL-8 mRNA in group C was significantly higher and the difference was significant (P0.01). Compared with group C, the expression of IL-8 mRNA in group D, E and F decreased significantly, and in group F, the expression of IL-8 in group D, E and F was significantly lower, and the difference was statistically significant (P <0.01). The expression of Eotaxin and IL-8 in each group of 16HBE cells showed that the expression of Eotaxin and IL-8 in each group was mainly expressed in the cytoplasm, and the expression of D and E in group C was the strongest. The results showed that the concentration of Eotaxin (pg/ mL) in the supernatant of A to F group was 1.72, 0.16, 1.60, 0.17, 4.05, 0.20, 3.70, 0.21, 3.33, 0.33, 2.76 and 0.15 respectively (P0.01). Compared with group A, the concentration of Eotaxin in group C was significantly higher and the difference was statistically significant (P0.01). Compared with group C, the concentrations of Eotaxin in group D, E and F decreased significantly (P <0.01). The concentration of IL-8 in the supernatant of group A to F (pg/ mL) was 17.45, 2.32, 18.78, 1.05, 27.24, 1.91, 23.62, 3.40, 19.25, 2.36, 14.12 and 3.14, respectively. There was a significant difference between the groups (P0.01). Compared with group A, the concentration of IL-8 in group C was significantly higher and the difference was significant (P0.01). The levels of IL-8 in group D, E and F were significantly lower in group C than in group C (P 0.05, 0.01, 0.01 respectively). Conclusion: Ephedrine can inhibit the expression and secretion of Eotaxin and IL-8, which may be one of the mechanisms of Chinese ephedra in the treatment of asthma. Ephedrine and Glucocorticoid inhibit the expression of 16 HBE and the secretion of Eotaxin and IL-8.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R562.25

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