【摘要】：用基因表達(dá)譜分析大鼠放射性肺損傷的差異表達(dá)基因,找出放射性肺損傷的敏感基因；用益氣活血方對(duì)被射線(xiàn)照射的大鼠進(jìn)行灌胃,觀察益氣活血方對(duì)大鼠放射性肺損傷敏感基因mRNA及蛋白表達(dá)的影響,以期從轉(zhuǎn)錄及翻譯層面分析該藥的作用機(jī)制,為該藥在臨床上推廣應(yīng)用提供理論依據(jù)。 健康成年雌性SD大鼠90只,隨機(jī)分為單純照射組(模型組)30只、照射加中藥組(中藥組)30只、空白對(duì)照組(對(duì)照組)30只。模型組及中藥組大鼠均采用6MV-X直線(xiàn)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,俯臥于平臺(tái),模擬機(jī)下定位,鉛塊遮擋左肺及縱隔,照射野面積3×3cm,每次5Gy,每周一次,總量30Gy。對(duì)照組大鼠只進(jìn)行麻醉,不予照射。于模型組及中藥組大鼠受照當(dāng)天開(kāi)始,中藥組每次灌胃益氣活血方劑1ml,2次/日；模型組及對(duì)照組每次灌胃蒸餾水1m1,2次/日。三組大鼠分別于照射后第4,8,12,16,20周末隨機(jī)抽取6只,處死并取其右肺組織保存。取部分第4,12周末模型組受照肺組織及對(duì)照組正常肺組織,提取其總RNA,并交由華大基因公司做數(shù)字化基因表達(dá)譜分析。其余肺組織分別用RT-PCR的方法檢測(cè)MMP-2, MMP-9, TIMP-1, TIMP-2mRNA表達(dá)在個(gè)時(shí)間點(diǎn)的表達(dá),用免疫印跡法(Western-blotting)及免疫組化法檢測(cè)MMP-2, MMP-9, TIMP-1, TIMP-2在各個(gè)時(shí)間點(diǎn)的表達(dá),實(shí)驗(yàn)數(shù)據(jù)采用SPSS.17.0做統(tǒng)計(jì)分析。 基因表達(dá)譜結(jié)果提示MMP-2, MMP-9, TIMP-1, TIMP-2基因或可能為放射性肺損傷發(fā)生的敏感基因。RT-PCR, Western-blotting,免疫組化結(jié)果提示MMP-2, MMP-9mRNA及蛋白表達(dá)于照射后第4周至20周出現(xiàn)一個(gè)先增高后下降的趨勢(shì),MMP-2mRNA及蛋白表達(dá)在第12周達(dá)高峰,而MMP-9mRNA及蛋白表達(dá)在16周時(shí)達(dá)到高峰。TIMP-1, TIMP-2mRNA及蛋白表達(dá)在照射后第4周至20周期間出現(xiàn)逐漸升高的趨勢(shì)。中藥組各mRNA及蛋白表達(dá)趨勢(shì)與模型組相似,但在不同時(shí)間點(diǎn)有不同變化。 我們發(fā)現(xiàn)MMP-2, MMP-9, TIMP-1, TIMP-2基因可能為放射性肺損傷發(fā)生的敏感基因。益氣活血方在放射性肺損傷的各個(gè)時(shí)間段,從轉(zhuǎn)錄及翻譯層面對(duì)MMP-2/TIMP-2, MMP-9/TIMP-1的表達(dá)進(jìn)行調(diào)整,使MMPs/TIMPs趨于平衡,從而減輕放射性肺損傷,這可能是該藥防治放射性肺損傷的分子生物學(xué)機(jī)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.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R563

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