17β-雌二醇对小鼠肺成纤维细胞的影响研究
发布时间：2018-08-29 09:52
【摘要】：目的观察17β-雌二醇（17β-E2）对小鼠肺成纤维细胞内小凹蛋白（Caveolin-1）的表达以及细胞外信号调节激酶（extracellular signal-regulated kinase，ERK）信号转导途径的影响改变，是否可以降低I、III型胶原蛋白的表达，探讨17β-E2抗肺纤维化的作用，为进一步的实验提供理论依据。 方法取液氮下冻存的小鼠成纤维细胞，37℃水浴复苏1min后接种于含体积分数为10%的血清、体积分数为1%双抗的RPMI-1640培养基中，于5%CO2、37℃培养箱中培养24h、3d、5d后，通过倒置相差显微镜观察小鼠成纤维细胞形态变化；用浓度为20mg/L、50mg/L和100mg/L的SiO2刺激小鼠肺泡巨噬细胞24h，产生的上清液经离心提取后-20℃环境备用；根据SiO2浓度、17β-E2浓度以及17β-E2的干预时间三因素三水平，运用正交设计分为9组进行MTT增殖实验，确定17β-E2最佳的作用浓度和时间；依据正交设计的结果将细胞进行分成5组进行处理，即空白对照组、SiO-82组、SiO2+10mol/L浓度的17β-E2组（低浓度17β-E2干预组）、SiO-2+107mol/L浓度的17β-E2组（中浓度17β-E2干预组）和SiO2+10-6mol/L浓度的17β-E2组（高浓度17β-E2干预组）；每组细胞处理后依次运用MTT法测定细胞增殖，流式细胞术测定细胞周期和细胞凋亡，激光共聚焦显微镜观察细胞内钙离子浓度变化和免疫组织化学法测量细胞内Caveolin-1、ERK、I、III型胶原蛋白表达情况。 结果1形态学变化：取刚复苏的小鼠成纤维细胞放置于倒置相差显微镜下，以倍数为10×10镜下观察发现，细胞多呈圆球形且高度折光，悬浮在培养液中。细胞约24h后开始贴壁，细胞多呈梭状，偶见三角形或多角形，约3d左右细胞可布满培养瓶70%到80%的面积，约5d左右可铺满整个培养瓶瓶壁，折光度明显降低，呈“铺路石”般的致密状，适时可以进行传代培养。2正交设计：正交设计的方差分析，结果显示：不同浓度的SiO2、17β-E2以及17β-E2的干预时间均对细胞增殖预实验影响差异有统计学意义（P0.05）；进一步对正交设计结果进行极差分析，其结果显示：对MTT细胞增殖实验的影响最大的是因素SiO2浓度，其次为17β-E2浓度，最低为17β-E2的干预时间，最佳配对因素SiO2、17β-E2的浓度和17β-E2干预时间分别为20mg/L、10-6mol/L和24h。3细胞增殖：与空白组对照组比较，在加入SiO2刺激得来的上清液后，SiO2组的细胞吸光度值明显升高（P0.05）；加入干预剂17β-E2后，干预组的吸光度值较SiO2组明显降低，且随着干预剂17β-E2浓度的增加，吸光度值越加降低（P0.05）。4细胞周期：与空白对照组比较，在加入SiO2刺激得来的上清液后，SiO2组G2和S期的细胞比例明显增加，G1期的细胞比例明显减少（P0.05）；使用干预剂17β-E2后，干预组G2和S期的细胞比例较SiO2组明显减少，，G1期的细胞比例明显增多（P0.05）；且随着17β-E2的浓度升高，G1期的细胞比例明显增多，G2和S期的细胞比例明显减少（P0.05）。5细胞凋亡：与正常对照组的细胞比较，SiO2组的小鼠肺成纤维细胞凋亡率显著降低（P0.05）；在加入17β-E2后，与SiO2组进行比较，处于凋亡晚期的细胞比例增多（P0.05），并随着雌二醇浓度的升高，凋亡细胞比例的增高越加明显（P0.05）。6免疫组织化学法：与空白对照组比较，SiO2组细胞内的ERK、I和III胶原表达大量增加（P0.05），在加入干预剂17β-E2后，与SiO2组进行比较，干预组细胞内的小凹蛋白表达增加，而ERK、I和III型胶原的表达在减少（P0.05）；且随着雌二醇浓度的增高，小凹蛋白的表达增加越明显， ERK、I和III型胶原的表达减少的也越明显（P0.05）。7激光光共聚焦：与空白对照组比较，SiO2组内细胞的荧光强度增高，呈强荧光的状态（P0.05）。加入干预剂17β-E2后，干预组内细胞的荧光强度较SiO2组明显减弱（P0.05）；随着17β-E2浓度的增高，与SiO2组比较，其细胞的荧光强度明显越弱，呈弱荧光表达（P0.05），但与空白对照组比较仍然高出（P0.05）。 结论117β-E2能通过阻滞小鼠肺成纤维细胞由G1期进入S期和G2期来抑制SiO2引起的细胞增殖，诱导其凋亡。217β-E2可诱导小凹蛋白表达，并抑制成纤维细胞内ERK蛋白、I、III型胶原的表达。317β-E2能抑制SiO2引起的小鼠肺成纤维细胞内钙离子浓度的升高，降低钙离子的浓度。
[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.
【学位授予单位】：河北联合大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965
本文编号：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.
【学位授予单位】：河北联合大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965
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