17β-雌二醇對小鼠肺成纖維細胞的影響研究
發(fā)布時間：2018-08-29 09:52
【摘要】：目的觀察17β-雌二醇（17β-E2）對小鼠肺成纖維細胞內(nèi)小凹蛋白（Caveolin-1）的表達以及細胞外信號調(diào)節(jié)激酶（extracellular signal-regulated kinase，ERK）信號轉(zhuǎn)導途徑的影響改變，是否可以降低I、III型膠原蛋白的表達，探討17β-E2抗肺纖維化的作用，為進一步的實驗提供理論依據(jù)。 方法取液氮下凍存的小鼠成纖維細胞，37℃水浴復蘇1min后接種于含體積分數(shù)為10%的血清、體積分數(shù)為1%雙抗的RPMI-1640培養(yǎng)基中，于5%CO2、37℃培養(yǎng)箱中培養(yǎng)24h、3d、5d后，通過倒置相差顯微鏡觀察小鼠成纖維細胞形態(tài)變化；用濃度為20mg/L、50mg/L和100mg/L的SiO2刺激小鼠肺泡巨噬細胞24h，產(chǎn)生的上清液經(jīng)離心提取后-20℃環(huán)境備用；根據(jù)SiO2濃度、17β-E2濃度以及17β-E2的干預時間三因素三水平，運用正交設計分為9組進行MTT增殖實驗，確定17β-E2最佳的作用濃度和時間；依據(jù)正交設計的結(jié)果將細胞進行分成5組進行處理，即空白對照組、SiO-82組、SiO2+10mol/L濃度的17β-E2組（低濃度17β-E2干預組）、SiO-2+107mol/L濃度的17β-E2組（中濃度17β-E2干預組）和SiO2+10-6mol/L濃度的17β-E2組（高濃度17β-E2干預組）；每組細胞處理后依次運用MTT法測定細胞增殖，流式細胞術測定細胞周期和細胞凋亡，激光共聚焦顯微鏡觀察細胞內(nèi)鈣離子濃度變化和免疫組織化學法測量細胞內(nèi)Caveolin-1、ERK、I、III型膠原蛋白表達情況。 結(jié)果1形態(tài)學變化：取剛復蘇的小鼠成纖維細胞放置于倒置相差顯微鏡下，以倍數(shù)為10×10鏡下觀察發(fā)現(xiàn)，細胞多呈圓球形且高度折光，懸浮在培養(yǎng)液中。細胞約24h后開始貼壁，細胞多呈梭狀，偶見三角形或多角形，約3d左右細胞可布滿培養(yǎng)瓶70%到80%的面積，約5d左右可鋪滿整個培養(yǎng)瓶瓶壁，折光度明顯降低，呈“鋪路石”般的致密狀，適時可以進行傳代培養(yǎng)。2正交設計：正交設計的方差分析，結(jié)果顯示：不同濃度的SiO2、17β-E2以及17β-E2的干預時間均對細胞增殖預實驗影響差異有統(tǒng)計學意義（P0.05）；進一步對正交設計結(jié)果進行極差分析，其結(jié)果顯示：對MTT細胞增殖實驗的影響最大的是因素SiO2濃度，其次為17β-E2濃度，最低為17β-E2的干預時間，最佳配對因素SiO2、17β-E2的濃度和17β-E2干預時間分別為20mg/L、10-6mol/L和24h。3細胞增殖：與空白組對照組比較，在加入SiO2刺激得來的上清液后，SiO2組的細胞吸光度值明顯升高（P0.05）；加入干預劑17β-E2后，干預組的吸光度值較SiO2組明顯降低，且隨著干預劑17β-E2濃度的增加，吸光度值越加降低（P0.05）。4細胞周期：與空白對照組比較，在加入SiO2刺激得來的上清液后，SiO2組G2和S期的細胞比例明顯增加，G1期的細胞比例明顯減少（P0.05）；使用干預劑17β-E2后，干預組G2和S期的細胞比例較SiO2組明顯減少，，G1期的細胞比例明顯增多（P0.05）；且隨著17β-E2的濃度升高，G1期的細胞比例明顯增多，G2和S期的細胞比例明顯減少（P0.05）。5細胞凋亡：與正常對照組的細胞比較，SiO2組的小鼠肺成纖維細胞凋亡率顯著降低（P0.05）；在加入17β-E2后，與SiO2組進行比較，處于凋亡晚期的細胞比例增多（P0.05），并隨著雌二醇濃度的升高，凋亡細胞比例的增高越加明顯（P0.05）。6免疫組織化學法：與空白對照組比較，SiO2組細胞內(nèi)的ERK、I和III膠原表達大量增加（P0.05），在加入干預劑17β-E2后，與SiO2組進行比較，干預組細胞內(nèi)的小凹蛋白表達增加，而ERK、I和III型膠原的表達在減少（P0.05）；且隨著雌二醇濃度的增高，小凹蛋白的表達增加越明顯， ERK、I和III型膠原的表達減少的也越明顯（P0.05）。7激光光共聚焦：與空白對照組比較，SiO2組內(nèi)細胞的熒光強度增高，呈強熒光的狀態(tài)（P0.05）。加入干預劑17β-E2后，干預組內(nèi)細胞的熒光強度較SiO2組明顯減弱（P0.05）；隨著17β-E2濃度的增高，與SiO2組比較，其細胞的熒光強度明顯越弱，呈弱熒光表達（P0.05），但與空白對照組比較仍然高出（P0.05）。 結(jié)論117β-E2能通過阻滯小鼠肺成纖維細胞由G1期進入S期和G2期來抑制SiO2引起的細胞增殖，誘導其凋亡。217β-E2可誘導小凹蛋白表達，并抑制成纖維細胞內(nèi)ERK蛋白、I、III型膠原的表達。317β-E2能抑制SiO2引起的小鼠肺成纖維細胞內(nèi)鈣離子濃度的升高，降低鈣離子的濃度。
[Abstract]:Objective To observe the effect of 17 beta-estradiol (17 beta-E2) on the expression of Caveolin-1 and extracellular signal-regulated kinase (ERK) signal transduction pathway in mouse lung fibroblasts, and to explore the anti-pulmonary fibrosis effect of 17 beta-E2. Further experiments provide theoretical basis.
Methods Mouse fibroblasts frozen in liquid nitrogen were inoculated in RPMI-1640 medium containing 10% serum and 1% bi-antibody after resuscitation at 37 C for 1 minute. After 24 hours, 3 days and 5 days in 5% CO2 and 37 C incubator, the morphological changes of mouse fibroblasts were observed by inverted phase contrast microscope. L and 100mg/L SiO2 stimulated alveolar macrophages for 24h, and the supernatant was extracted by centrifugation at - 20 C. According to the concentration of SiO2, the concentration of 17beta-E2 and the intervening time of 17beta-E2, the MTT proliferation experiment was divided into 9 groups by orthogonal design to determine the best concentration and time of 17beta-E2. Results The cells were divided into five groups: control group, SiO-82 group, SiO 2+10mol/L 17beta-E2 group (low concentration 17beta-E2 intervention group), SiO-2+107mol/L 17beta-E2 group (medium concentration 17beta-E2 intervention group) and SiO 2+10-6mol/L 17beta-E2 group (high concentration 17beta-E2 intervention group). Cell proliferation was measured by T method, cell cycle and apoptosis were measured by flow cytometry, intracellular Caveolin-1, ERK, I, III collagen protein expression was measured by confocal laser microscopy and immunohistochemistry.
Results 1. Morphological changes: Freshly resuscitated mouse fibroblasts were placed under an inverted phase contrast microscope. The cells were mostly spherical and highly refractive, suspended in the culture medium. After about 24 hours, the cells began to adhere to the wall. The cells were mostly spindle-shaped, occasionally triangular or polygonal. The cells could be cultured around 3 days. Orthogonal design: analysis of variance of orthogonal design showed that different concentrations of SiO2, 17beta-E2 and the intervening time of 17beta-E2 all affected the cell proliferation pre-experiment. The difference was statistically significant (P 0.05); furthermore, the results of orthogonal design showed that the greatest influence on MTT cell proliferation was the concentration of SiO2, the next was the concentration of 17 beta-E2, the lowest was the intervening time of 17 beta-E2, and the best matching factors were the concentration of SiO2, 17 beta-E2 and the intervening time of 17 beta-E2 were 20 mg/L, 10-6, respectively. Mol/L and 24 h.3 cell proliferation: Compared with the control group, the absorbance of SiO2-stimulated supernatant increased significantly (P 0.05); the absorbance of SiO2-treated group was significantly lower than that of SiO2-treated group after the addition of 17 beta-E2, and the absorbance decreased with the increase of the concentration of 17 beta-E2 (P 0.05). Cell cycle: Compared with the blank control group, the proportion of G2 and S phase cells in SiO2 group increased significantly, and the proportion of G1 phase cells decreased significantly (P When the concentration of beta-E2 increased, the proportion of cells in G1 phase increased, and the proportion of cells in G2 and S phase decreased significantly (P 0.05). 5 cell apoptosis: Compared with the normal control group, the apoptosis rate of lung fibroblasts in SiO2 group decreased significantly (P 0.05); after the addition of 17 beta-E2, the proportion of cells in advanced stage of apoptosis increased compared with SiO2 group (P 0.05). With the increase of estradiol concentration, the proportion of apoptotic cells increased significantly (P 0.05). 6 Immunohistochemical method: Compared with the blank control group, the expression of ERK, I and III collagen in SiO2 group increased significantly (P 0.05). After adding 17 beta-E2, compared with SiO2 group, the expression of pituitary protein in the intervention group increased, while E The expression of RK, I and III collagen was decreased (P 0.05), and the expression of pit protein increased with the increase of estradiol concentration, and the expression of ERK, I and III collagen decreased significantly (P 0.05). 7 Laser confocal imaging: Compared with the blank control group, the fluorescence intensity of SiO2 cells increased, showing a strong fluorescence state (P 0.05). The fluorescence intensity of the cells in the intervention group was significantly lower than that in the SiO2 group (P 0.05), and the fluorescence intensity of the cells in the intervention group was significantly weaker than that in the SiO2 group (P 0.05), but still higher than that in the control group (P 0.05).
Conclusion 117 beta-E2 can inhibit the proliferation and apoptosis of mouse lung fibroblasts induced by SiO2 by blocking G1 phase into S phase and G2 phase. 217 beta-E2 can induce the expression of pit protein, and inhibit the expression of ERK protein, I, III collagen in fibroblasts. 317 beta-E2 can inhibit the calcium concentration in mouse lung fibroblasts induced by SiO2. Increase in calcium concentration.
【學位授予單位】：河北聯(lián)合大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R965
本文編號：2210841
[Abstract]:Objective To observe the effect of 17 beta-estradiol (17 beta-E2) on the expression of Caveolin-1 and extracellular signal-regulated kinase (ERK) signal transduction pathway in mouse lung fibroblasts, and to explore the anti-pulmonary fibrosis effect of 17 beta-E2. Further experiments provide theoretical basis.
Methods Mouse fibroblasts frozen in liquid nitrogen were inoculated in RPMI-1640 medium containing 10% serum and 1% bi-antibody after resuscitation at 37 C for 1 minute. After 24 hours, 3 days and 5 days in 5% CO2 and 37 C incubator, the morphological changes of mouse fibroblasts were observed by inverted phase contrast microscope. L and 100mg/L SiO2 stimulated alveolar macrophages for 24h, and the supernatant was extracted by centrifugation at - 20 C. According to the concentration of SiO2, the concentration of 17beta-E2 and the intervening time of 17beta-E2, the MTT proliferation experiment was divided into 9 groups by orthogonal design to determine the best concentration and time of 17beta-E2. Results The cells were divided into five groups: control group, SiO-82 group, SiO 2+10mol/L 17beta-E2 group (low concentration 17beta-E2 intervention group), SiO-2+107mol/L 17beta-E2 group (medium concentration 17beta-E2 intervention group) and SiO 2+10-6mol/L 17beta-E2 group (high concentration 17beta-E2 intervention group). Cell proliferation was measured by T method, cell cycle and apoptosis were measured by flow cytometry, intracellular Caveolin-1, ERK, I, III collagen protein expression was measured by confocal laser microscopy and immunohistochemistry.
Results 1. Morphological changes: Freshly resuscitated mouse fibroblasts were placed under an inverted phase contrast microscope. The cells were mostly spherical and highly refractive, suspended in the culture medium. After about 24 hours, the cells began to adhere to the wall. The cells were mostly spindle-shaped, occasionally triangular or polygonal. The cells could be cultured around 3 days. Orthogonal design: analysis of variance of orthogonal design showed that different concentrations of SiO2, 17beta-E2 and the intervening time of 17beta-E2 all affected the cell proliferation pre-experiment. The difference was statistically significant (P 0.05); furthermore, the results of orthogonal design showed that the greatest influence on MTT cell proliferation was the concentration of SiO2, the next was the concentration of 17 beta-E2, the lowest was the intervening time of 17 beta-E2, and the best matching factors were the concentration of SiO2, 17 beta-E2 and the intervening time of 17 beta-E2 were 20 mg/L, 10-6, respectively. Mol/L and 24 h.3 cell proliferation: Compared with the control group, the absorbance of SiO2-stimulated supernatant increased significantly (P 0.05); the absorbance of SiO2-treated group was significantly lower than that of SiO2-treated group after the addition of 17 beta-E2, and the absorbance decreased with the increase of the concentration of 17 beta-E2 (P 0.05). Cell cycle: Compared with the blank control group, the proportion of G2 and S phase cells in SiO2 group increased significantly, and the proportion of G1 phase cells decreased significantly (P When the concentration of beta-E2 increased, the proportion of cells in G1 phase increased, and the proportion of cells in G2 and S phase decreased significantly (P 0.05). 5 cell apoptosis: Compared with the normal control group, the apoptosis rate of lung fibroblasts in SiO2 group decreased significantly (P 0.05); after the addition of 17 beta-E2, the proportion of cells in advanced stage of apoptosis increased compared with SiO2 group (P 0.05). With the increase of estradiol concentration, the proportion of apoptotic cells increased significantly (P 0.05). 6 Immunohistochemical method: Compared with the blank control group, the expression of ERK, I and III collagen in SiO2 group increased significantly (P 0.05). After adding 17 beta-E2, compared with SiO2 group, the expression of pituitary protein in the intervention group increased, while E The expression of RK, I and III collagen was decreased (P 0.05), and the expression of pit protein increased with the increase of estradiol concentration, and the expression of ERK, I and III collagen decreased significantly (P 0.05). 7 Laser confocal imaging: Compared with the blank control group, the fluorescence intensity of SiO2 cells increased, showing a strong fluorescence state (P 0.05). The fluorescence intensity of the cells in the intervention group was significantly lower than that in the SiO2 group (P 0.05), and the fluorescence intensity of the cells in the intervention group was significantly weaker than that in the SiO2 group (P 0.05), but still higher than that in the control group (P 0.05).
Conclusion 117 beta-E2 can inhibit the proliferation and apoptosis of mouse lung fibroblasts induced by SiO2 by blocking G1 phase into S phase and G2 phase. 217 beta-E2 can induce the expression of pit protein, and inhibit the expression of ERK protein, I, III collagen in fibroblasts. 317 beta-E2 can inhibit the calcium concentration in mouse lung fibroblasts induced by SiO2. Increase in calcium concentration.
【學位授予單位】：河北聯(lián)合大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R965
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