益气活血方对大鼠放射性肺损伤敏感基因表达的影响
发布时间：2019-05-10 13:58
【摘要】：用基因表达谱分析大鼠放射性肺损伤的差异表达基因,找出放射性肺损伤的敏感基因；用益气活血方对被射线照射的大鼠进行灌胃,观察益气活血方对大鼠放射性肺损伤敏感基因mRNA及蛋白表达的影响,以期从转录及翻译层面分析该药的作用机制,为该药在临床上推广应用提供理论依据。 健康成年雌性SD大鼠90只,随机分为单纯照射组(模型组)30只、照射加中药组(中药组)30只、空白对照组(对照组)30只。模型组及中药组大鼠均采用6MV-X直线加速器照射其右肺,照射前使用7%的水合氯醛(5·kg-1)腹腔注射麻醉,俯卧于平台,模拟机下定位,铅块遮挡左肺及纵隔,照射野面积3×3cm,每次5Gy,每周一次,总量30Gy。对照组大鼠只进行麻醉,不予照射。于模型组及中药组大鼠受照当天开始,中药组每次灌胃益气活血方剂1ml,2次/日；模型组及对照组每次灌胃蒸馏水1m1,2次/日。三组大鼠分别于照射后第4,8,12,16,20周末随机抽取6只,处死并取其右肺组织保存。取部分第4,12周末模型组受照肺组织及对照组正常肺组织,提取其总RNA,并交由华大基因公司做数字化基因表达谱分析。其余肺组织分别用RT-PCR的方法检测MMP-2, MMP-9, TIMP-1, TIMP-2mRNA表达在个时间点的表达,用免疫印迹法(Western-blotting)及免疫组化法检测MMP-2, MMP-9, TIMP-1, TIMP-2在各个时间点的表达,实验数据采用SPSS.17.0做统计分析。 基因表达谱结果提示MMP-2, MMP-9, TIMP-1, TIMP-2基因或可能为放射性肺损伤发生的敏感基因。RT-PCR, Western-blotting,免疫组化结果提示MMP-2, MMP-9mRNA及蛋白表达于照射后第4周至20周出现一个先增高后下降的趋势,MMP-2mRNA及蛋白表达在第12周达高峰,而MMP-9mRNA及蛋白表达在16周时达到高峰。TIMP-1, TIMP-2mRNA及蛋白表达在照射后第4周至20周期间出现逐渐升高的趋势。中药组各mRNA及蛋白表达趋势与模型组相似,但在不同时间点有不同变化。 我们发现MMP-2, MMP-9, TIMP-1, TIMP-2基因可能为放射性肺损伤发生的敏感基因。益气活血方在放射性肺损伤的各个时间段,从转录及翻译层面对MMP-2/TIMP-2, MMP-9/TIMP-1的表达进行调整,使MMPs/TIMPs趋于平衡,从而减轻放射性肺损伤,这可能是该药防治放射性肺损伤的分子生物学机制。
[Abstract]:The differentially expressed genes of radiation lung injury in rats were analyzed by gene expression profile, and the sensitive genes of radiation lung injury were found out. The rats irradiated by radiation were given intragastrically with Yiqi Huoxue recipe to observe the effect of Yiqi Huoxue recipe on the expression of sensitive gene mRNA and protein in rats with radiation lung injury, in order to analyze the mechanism of action of Yiqi Huoxue recipe from the aspects of transcription and translation. It provides a theoretical basis for the popularization and application of the drug in clinic. 90 healthy adult female SD rats were randomly divided into three groups: simple irradiation group (model group, n = 30), irradiation plus traditional Chinese medicine group (n = 30) and blank control group (control group, n = 30). Both the model group and the traditional Chinese medicine group were irradiated with 6MV-X linear accelerator. Before irradiation, 7% chloral hydrate (5 kg-1) was injected into the abdominal cavity for anesthesia, prone to the platform, located under the simulator, and the left lung and mediastinum were blocked by lead block. The area of irradiation field is 3 脳 3cm, 5 Gym each time, once a week, the total amount is 30g. The rats in the control group were anesthetized without irradiation. From the day the rats in the model group and the traditional Chinese medicine group were exposed to irradiation, the rats in the traditional Chinese medicine group were perfused with 1ml of Yiqi Huoxue recipe twice a day, and the model group and the control group were given distilled water 1ml twice a day at a time. At the end of the 20th week, 6 rats in the three groups were randomly selected and sacrificed and preserved in their right lung tissue. The total RNA, was extracted from the exposed lung tissue of the model group and the normal lung tissue of the control group at the end of the 4th and 12th week, and the digital gene expression profile was analyzed by Huada Gene Company. The expression of MMP-2, MMP-9, TIMP-1, TIMP-2mRNA in other lung tissues was detected by RT-PCR at a time point, and MMP-2, MMP-9, TIMP-1, was detected by immunoblotting (Western-blotting) and immunohistochemistry. The expression of TIMP-2 at each time point was analyzed by SPSS.17.0. The results of gene expression profile suggest that MMP-2, MMP-9, TIMP-1, TIMP-2 gene or sensitive gene may be caused by radiation lung injury. RT-PCR, Western-blotting, Immunohistochemical results suggest MMP-2, The expression of MMP-9mRNA and protein increased at first and then decreased at the 4th to 20th week after irradiation. The expression of MMP-2mRNA and protein reached the peak at the 12th week, while the expression of MMP-9mRNA and protein reached the peak at the 16th week. The expression of TIMP-2mRNA and protein increased gradually from the 4th week to the 20th week after irradiation. The expression trend of mRNA and protein in traditional Chinese medicine group was similar to that in model group, but there were different changes at different time points. We found that MMP-2, MMP-9, TIMP-1, TIMP-2 gene may be a sensitive gene for radiation lung injury. Yiqi Huoxue recipe adjusts the expression of MMP-2/TIMP-2, MMP-9/TIMP-1 from the aspects of transcription and translation in all time periods of radiation lung injury, so that MMPs/TIMPs tends to be balanced, so as to reduce radiation lung injury. This may be the molecular biological mechanism of the drug in the prevention and treatment of radiation-induced lung injury.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R563
本文编号：2473697
[Abstract]:The differentially expressed genes of radiation lung injury in rats were analyzed by gene expression profile, and the sensitive genes of radiation lung injury were found out. The rats irradiated by radiation were given intragastrically with Yiqi Huoxue recipe to observe the effect of Yiqi Huoxue recipe on the expression of sensitive gene mRNA and protein in rats with radiation lung injury, in order to analyze the mechanism of action of Yiqi Huoxue recipe from the aspects of transcription and translation. It provides a theoretical basis for the popularization and application of the drug in clinic. 90 healthy adult female SD rats were randomly divided into three groups: simple irradiation group (model group, n = 30), irradiation plus traditional Chinese medicine group (n = 30) and blank control group (control group, n = 30). Both the model group and the traditional Chinese medicine group were irradiated with 6MV-X linear accelerator. Before irradiation, 7% chloral hydrate (5 kg-1) was injected into the abdominal cavity for anesthesia, prone to the platform, located under the simulator, and the left lung and mediastinum were blocked by lead block. The area of irradiation field is 3 脳 3cm, 5 Gym each time, once a week, the total amount is 30g. The rats in the control group were anesthetized without irradiation. From the day the rats in the model group and the traditional Chinese medicine group were exposed to irradiation, the rats in the traditional Chinese medicine group were perfused with 1ml of Yiqi Huoxue recipe twice a day, and the model group and the control group were given distilled water 1ml twice a day at a time. At the end of the 20th week, 6 rats in the three groups were randomly selected and sacrificed and preserved in their right lung tissue. The total RNA, was extracted from the exposed lung tissue of the model group and the normal lung tissue of the control group at the end of the 4th and 12th week, and the digital gene expression profile was analyzed by Huada Gene Company. The expression of MMP-2, MMP-9, TIMP-1, TIMP-2mRNA in other lung tissues was detected by RT-PCR at a time point, and MMP-2, MMP-9, TIMP-1, was detected by immunoblotting (Western-blotting) and immunohistochemistry. The expression of TIMP-2 at each time point was analyzed by SPSS.17.0. The results of gene expression profile suggest that MMP-2, MMP-9, TIMP-1, TIMP-2 gene or sensitive gene may be caused by radiation lung injury. RT-PCR, Western-blotting, Immunohistochemical results suggest MMP-2, The expression of MMP-9mRNA and protein increased at first and then decreased at the 4th to 20th week after irradiation. The expression of MMP-2mRNA and protein reached the peak at the 12th week, while the expression of MMP-9mRNA and protein reached the peak at the 16th week. The expression of TIMP-2mRNA and protein increased gradually from the 4th week to the 20th week after irradiation. The expression trend of mRNA and protein in traditional Chinese medicine group was similar to that in model group, but there were different changes at different time points. We found that MMP-2, MMP-9, TIMP-1, TIMP-2 gene may be a sensitive gene for radiation lung injury. Yiqi Huoxue recipe adjusts the expression of MMP-2/TIMP-2, MMP-9/TIMP-1 from the aspects of transcription and translation in all time periods of radiation lung injury, so that MMPs/TIMPs tends to be balanced, so as to reduce radiation lung injury. This may be the molecular biological mechanism of the drug in the prevention and treatment of radiation-induced lung injury.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R563
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