麻黃堿對人支氣管上皮細(xì)胞增殖及表達(dá)Eotaxin和IL-8的影響
發(fā)布時間：2019-05-30 04:30
【摘要】：目的：研究麻黃堿對體外培養(yǎng)的人支氣管上皮細(xì)胞(16HBE)形態(tài)、增殖及表達(dá)嗜酸性粒細(xì)胞趨化因子(Eotaxin)、白細(xì)胞介素-8(IL-8)的影響，探討麻黃治療支氣管哮喘的機(jī)制。方法：將16HBE細(xì)胞株傳代培養(yǎng)，選擇生長狀態(tài)良好的第4、5代細(xì)胞用于后續(xù)實(shí)驗：⑴將16HBE細(xì)胞分別接種于6孔板和96孔板并隨機(jī)分為5組，分別用含不同濃度(0，150，，300，600，1200μg/mL)麻黃堿的培養(yǎng)基培養(yǎng)，培養(yǎng)24、48、72小時后，倒置相差顯微鏡下觀察各組16HBE細(xì)胞形態(tài)及生長狀況；CCK-8檢測16HBE細(xì)胞增殖情況；⑵將16HBE細(xì)胞接種于6孔板并隨機(jī)分為6組：①空白對照組（A組）：完全培養(yǎng)基培養(yǎng)18h；②麻黃堿組（B組）：含麻黃堿300μg/mL的完全培養(yǎng)基培養(yǎng)18h；③TNF-α組（C組）：含TNF-α20ng/mL的完全培養(yǎng)基培養(yǎng)18h；④麻黃堿+TNF-α組（D組）：含麻黃堿300μg/mL的完全培養(yǎng)基培養(yǎng)1h后加入100ng/mLTNF-α，共培養(yǎng)18h；⑤地塞米松+TNF-α組（E組）：含地塞米松1.4μg/mL的完全培養(yǎng)基培養(yǎng)1h后加入100ng/mL TNF-α，共培養(yǎng)18h；⑥麻黃堿+地塞米松+TNF-α組（F組）：含麻黃堿300μg/mL、地塞米松1.4μg/mL的完全培養(yǎng)基培養(yǎng)1h后加入100ng/mL TNF-α，共培養(yǎng)18h。D、E、F組TNF-α終濃度均為20ng/mL；⑶熒光定量PCR檢測各組細(xì)胞Eotaxin、IL-8mRNA的表達(dá)；免疫熒光法觀察各組細(xì)胞Eotaxin、IL-8蛋白的表達(dá)；雙抗體夾心酶聯(lián)免疫吸附法（ELISA）檢測各組細(xì)胞上清液中Eotaxin、IL-8蛋白的濃度。結(jié)果：⑴不同濃度（150，300，600，1200μg/mL）的麻黃堿作用于16HBE細(xì)胞24h、48h、72小時后，其細(xì)胞形態(tài)和生長狀況與不加麻黃堿（0μg/mL麻黃堿）組比較無明顯差異；⑵CCK-8檢測結(jié)果示：不同濃度麻黃堿作用于16HBE細(xì)胞24h，各組細(xì)胞的吸光度(OD)值分別為0.97±0.17，0.99±0.24，1.14±0.25，1.11±0.18，0.95±0.18，各組間比較差異無統(tǒng)計學(xué)意義（P=0.504）；作用48h各組細(xì)胞的OD值分別為1.09±0.43，1.16±0.99，1.16±0.87，1.18±0.56，1.11±0.30，各組間比較差異無統(tǒng)計學(xué)意義(P=0.282)；作用72h各組細(xì)胞的OD值分別為1.40±0.13，1.39±0.10，1.39±0.16，1.41±0.01，1.40±0.01，各組間比較差異無統(tǒng)計學(xué)意義（P=0.18）；⑶熒光定量PCR顯示：A至F組細(xì)胞Eotaxin mRNA的表達(dá)量(RQ值)分別為0.99±0.01，0.98±0.02，3.03±0.32，1.82±0.23，1.78±0.02，0.97±0.01，各組間比較差異有統(tǒng)計學(xué)意義（P0.01）。與A組比較，C組的Eotaxin mRNA表達(dá)量明顯升高，差異有統(tǒng)計學(xué)意義(P0.01)；與C組比較，D、E、F組的Eotaxin mRNA表達(dá)量明顯降低，以F組降低最明顯，差異均有統(tǒng)計學(xué)意義(P均0.01）。A至F組IL-8mRNA的RQ值分別為0.95±0.05,0.93±0.14,7.18±0.31,4.44±0.24，4.11±0.19，2.97±0.39，各組間比較差異有統(tǒng)計學(xué)意義(P0.01)。與A組比較，C組的IL-8mRNA表達(dá)量明顯升高，差異有統(tǒng)計學(xué)意義(P0.01)。與C組比較，D、E、F組的IL-8mRNA表達(dá)量均明顯降低，以F組降低最明顯，差異均有統(tǒng)計學(xué)意義(P均0.01）；⑷免疫熒光顯示：各組16HBE細(xì)胞均可見Eotaxin和IL-8表達(dá)，主要表達(dá)在細(xì)胞漿中，C組表達(dá)最強(qiáng)，D、E、F組表達(dá)均較C組弱，F(xiàn)組最弱；⑸ELISA檢測結(jié)果顯示：A至F組細(xì)胞上清液中Eotaxin的濃度(pg/mL)分別為1.72±0.16，1.60±0.17，4.05±0.20，3.70±0.21，3.33±0.33，2.76±0.15，各組間比較差異有統(tǒng)計學(xué)意義(P0.01)。與A組比較，C組Eotaxin濃度明顯升高，差異有統(tǒng)計學(xué)意義(P0.01)。與C組比較，D、E、F組Eotaxin濃度均明顯降低，F(xiàn)組最低，差異均有統(tǒng)計學(xué)意義(P均0.01)；A至F組細(xì)胞上清液中IL-8濃度(pg/mL)分別為17.45±2.32，18.78±1.05，27.24±1.91，23.62±3.40，19.25±2.36，14.12±3.14，各組間比較差異有統(tǒng)計學(xué)意義(P0.01)。與A組比較，C組IL-8濃度明顯升高，差異有統(tǒng)計學(xué)意義（P0.01）。與C組比較，D、E、F組IL-8濃度均明顯降低，F(xiàn)組最低，差異均有統(tǒng)計學(xué)意義（P分別0.05，0.01，0.01）。結(jié)論：⑴在一定濃度范圍內(nèi)，麻黃堿對體外培養(yǎng)的16HBE形態(tài)及增殖無影響；⑵麻黃堿能抑制前炎癥因子TNF-α刺激16HBE表達(dá)及分泌Eotaxin和IL-8，這可能是中藥麻黃治療哮喘的機(jī)制之一；⑶麻黃堿與糖皮質(zhì)激素抑制16HBE表達(dá)及分泌Eotaxin和IL-8具有協(xié)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.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R562.25
本文編號：2488524
[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.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R562.25
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