Tankyrase2在放射性肺损伤中AEC和LFB凋亡调控中的作用研究

发布时间：2018-01-01 15:05

  本文关键词：Tankyrase2在放射性肺损伤中AEC和LFB凋亡调控中的作用研究　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： Tankyrase2 放射性肺损伤 细胞凋亡 肺泡上皮细胞 肺成纤维细胞

【摘要】：放射性肺损伤(radiation pulmonary injury,RPI)是胸部的肿瘤放射治疗、骨髓移植预处理等常见的并发症之一,也见于战时的核辐射及平时的核事故。RPI通常包括早期的放射性间质性肺炎(radiation interstitial pneumonitis,RIP)和晚期的放射性肺纤维化(radiation pulmonary fibrosis,RPF)两个阶段。有关文献报道常规放疗RPI的发病率约为30%,近年随着放疗方案的不断改进优化,RPI的发生率有所降低,但RPI一旦发生,将严重影响放疗后患者的生存质量,甚至危及其生命。RPI的发生也是核辐射和核事故救治的瓶颈问题之一。RPI机制研究有一定的进展,但仍远未阐明,也尚缺乏安全有效的防治药物,故对RPI机制进行深入研究,进而发现防治新靶点,对于提高临床胸部肿瘤放疗效果、改善放疗患者的生存质量以及提高放射损伤救治水平具有重要的理论和实际意义。RPI的发生具有多源性,且具有明显的种属和个体差异,DNA是γ射线损伤靶分子,细胞凋亡是DNA损伤结局之一,其在RPI过程中的起到重要作用。肺泡上皮细胞(alveolar epithelial cell,AEC)是RPI的主要的靶细胞,肺成纤维细胞(lung fibroblasts,LFB)是RPI发生发展的主要效应细胞。既往研究发现Tankyrase2在易/抗RPF小鼠RPI进程中存在表达差异,该分子作为端粒结合蛋白,其结构特点以及在细胞中分布的多样性使其在多种领域发挥相应的作用,通过调节端粒长度可以影响染色体的稳定性,而且其与细胞衰老、死亡和癌变密切相关;其结构域PARP是细胞凋亡和坏死的转换点。Tankyrase2在RPI过程中的作用未见文献报道。本文通过整体和细胞实验相结合,探讨Tankyrase2在RPI过程中AEC和LFB凋亡调控中的作用。材料和方法1.Tankyrase2及细胞凋亡与RPF种属差异的相关性研究:C57BL/6J和C3H/He N小鼠各60只,随机将其各分成对照组和照射组,采用60Coγ射线照射,全胸单次20Gy照射建立RPI模型。分别在照射后15min、1d、3d、7d、1m、3m取材,采用原位末端标记法、免疫印迹、实时荧光定量PCR等方法检测肺组织的细胞凋亡、DNA损伤、AIF、TNKS2、PARP表达,研究细胞DNA损伤和凋亡在RPI进展中的作用及其是否与RPF易发相关,并初步探讨Tankyrase2与小鼠RPI易发相关性。2.Tankyrase2在照射致AEC和LFB生物学行为改变中的作用研究:A549细胞和MRC-5细胞传代培养,60Coγ射线照射,剂量分别为5、10和20Gy,应用PARP抑制剂3-AB(浓度为5m M)。分别在照射后30min、1h、3h、6h、12、24h、48h和72h及3-AB作用后24h应用上述照射方法处理细胞,应用MTT法、γH2AX免疫荧光、AO/EB染色、实时荧光定量PCR、免疫印迹等方法检测细胞增殖活力、DNA损伤、细胞凋亡、AIF、TNKS2、PARP的表达。旨在阐明Tankrase2在照射所致AEC和LFB生物学行为改变中的作用。实验结果1.γ射线照射后C3H/He N小鼠和C57BL/6J小鼠肺组织中细胞凋亡和Tankyrase2表达存在差异1.1 C3H/He N小鼠和C57BL/6J小鼠照射后对肺组织中细胞凋亡存在差异:照射后1-3d C57BL/6J小鼠肺组织凋亡细胞显著多于C3H/He N小鼠,以肺泡上皮细胞为主;照射后1m即炎症期,两种小鼠细胞凋亡水平无明显差异,以上皮细胞、单核巨噬细胞为主;照射后3m即纤维化初期C57BL/6J小鼠肺组织凋亡细胞数显著少于C3H/He N小鼠,后者以巨噬细胞的凋亡为主。1.2 C3H/He N小鼠和C57BL/6J小鼠照射后肺组织中DNA损伤存在差异:照射后1-7d,C57BL/6J小鼠肺组织γH2AX表达显著高于C3H/He N小鼠高;照射后1m,两种小鼠肺组织γH2AX的表达无差异;照射后3m,C3H/He N小鼠肺组织中γH2AX的表达较C57BL/6J小鼠高。1.3 C3H/He N小鼠和C57BL/6J小鼠照射后肺组织中AIF表达存在差异:照射后7d、1m、3m,C3H/He N小鼠肺组织中AIF的表达较对照组显著增加,照射后1d、3d,AIF的表达较对照组无显著差异;照射后各时间点,C57BL/6J小鼠肺组织中AIF的表达较照射前显著增加;照射后1d、3d、1m,C3H/He N小鼠肺组织中AIF的表达较C57BL/6J小鼠低,照射后7d,C3H/He N小鼠肺组织中AIF的表达较C57BL/6J小鼠无显著差异,照射后3m,C3H/He N小鼠肺组织中AIF的表达较C57BL/6J小鼠高。1.4 C3H/He N小鼠和C57BL/6J小鼠照射后肺组织中TNKS2表达存在差异:(1)RNA水平,照射前,C3H/He N小鼠肺组织中TNKS2表达显著高于C57BL/6J小鼠,照射后,C3H/He N小鼠肺组织中TNKS2的表达高于C57BL/6J小鼠(除3d)。(2)蛋白水平,照射前,C3H/He N小鼠肺组织中Tankyrase2较C57BL/6J小鼠高,照射后C3H/He N小鼠TNKS2表达较C57BL/6J小鼠高(除1m)。1.5 C3H/He N小鼠和C57BL/6J小鼠照射后肺组织中PARP表达存在差异:对照组C3H/He N小鼠肺组织PARP表达较C57BL/6J小鼠低;照射后各个时间C57BL/6J小鼠肺组织中PARP表达均较C3H/He N小鼠高(除1d)。2.γ射线照射后AEC和LFB DNA损伤和凋亡的发生及Tankyrase2、PARP表达等存在差异2.1不同剂量γ射线照射后A549细胞和MRC-5细胞增殖活力的影响不同:5、10和20Gyγ射线照射后,三个剂量均抑制A549细胞增殖;照射后48和72h,10和20Gyγ射线抑制MRC-5细胞增殖,照射后72h,5Gyγ射线照射促进其增殖。2.2不同剂量γ射线照射后A549细胞和MRC-5细胞DNA损伤的影响不同:不同剂量γ射线照射后A549细胞均出现DNA损伤,照射后30min、1h、3h,DNA损伤细胞不断增加,剂量越高,DNA损伤细胞越多,照射后6h、12h阳性细胞逐渐减少,但仍较对照组多;不同剂量γ射线照射后MRC-5细胞只在部分时间点可见阳性细胞出现,及γH2AX的表达。2.3不同剂量γ射线照射后A549细胞和MRC-5细胞凋亡的影响不同:(1)20Gyγ射线照射后各时间点A549细胞凋亡细胞数目均较5和10Gyγ射线照射组多(除12h),10Gyγ射线照射后各时间点凋亡细胞数目均多于5Gyγ射线照射组(除3h)。(2)5Gyγ射线照射后,各时间点A549细胞AIF的表达均高于对照组,10Gyγ射线照射后,各时间点AIF的表达也均高于对照组,且10Gyγ射线照射后各时间点AIF表达均高于5Gyγ射线照射组。(3)5Gy、10Gyγ射线照射后,应用抑制剂3-AB处理的A549细胞,其AIF的表达显著低于未处理组细胞。处理组细胞,10Gyγ射线照射后A549细胞的AIF表达较5Gyγ射线处理组高。(4)不同剂量γ射线照射后MRC-5细胞未检测到凋亡细胞的出现及AIF的表达。2.4不同剂量γ射线照射后A549细胞和MRC-5细胞TNKS2、PARP表达的影响不同:(1)RNA水平,5和10Gyγ射线照射后1-12h,A549细胞TNKS2表达较对照组低,照射后30min,A549细胞TNKS2表达显著高于对照组;蛋白水平,5Gyγ射线照射后30min、12h,Tankyrase2表达较对照组低,照射后1和3h,A549细胞的Tankyrase2表达较对照组高;10Gyγ射线照射后各时间点Tankyrase2表达较对照组高(除12h),照射后30min、1h、6h、24h,10Gyγ射线照射组的Tankyrase2表达较5Gy照射组高。(2)RNA水平,5Gyγ射线照射后各时间点,MRC-5细胞TNKS2表达较照射前高,10Gyγ射线照射后各时间点,MRC-5细胞TNKS2的表达较照射前高(除30min),5Gyγ射线照射后30min、1h、12h,MRC-5细胞TNKS2表达较10Gyγ射线照射组高,10Gyγ射线照射后3h,TNKS2的表达较5Gyγ射线照射组高;蛋白水平,5Gyγ射线照射后30min和24h,MRC-5细胞的Tankyrase2的表达较照射前低,照射后1-12h,MRC-5细胞的Tankyrase2的表达较照射前高,10Gyγ射线照射后各时间点Tankyrase2的表达较照射前高,10Gyγ射线照射后各时间点Tankyrase2的表达高于5Gyγ射线照射组(除1h、3h)。(3)5Gyγ射线照射后30min、3h、12h,A549细胞的PARP表达较对照组低,照射后1和6h,PARP表达较照射前高;10Gyγ射线照射后各时间点PARP表达较照射前高;10Gyγ射线照射后各时间点PARP表达较5Gyγ射线照射组高(除1h);5Gyγ射线照射后30min、3h和12h,MRC-5细胞的PARP的表达较照射前高;照射后24h,MRC-5细胞的PARP的表达较照射前低;10Gyγ射线照射后各时间点PARP的表达较照射前高;10Gyγ射线照射后6、12和24h,PARP的表达高于5Gyγ射线照射组。结论1.γ射线照射后C57BL/6J与C3H/He N小鼠肺组织不同细胞凋亡的差异为两种小鼠是否易发RPF的机制之一:γ射线照射后C57BL/6J小鼠易出现DNA损伤、肺泡上皮细胞等实质细胞凋亡,而巨噬细胞等致纤维化细胞不易凋亡,故C57BL/6J小鼠易发生RPF;反之,γ射线照射后C3H/He N小鼠肺组织肺泡上皮细胞较少发生凋亡,而巨噬细胞等致纤维化细胞通过凋亡清除,与该小鼠不易发生RPF相关。2.Tankyrase2于RPF是否易发呈负相关。3.AEC较LFB对γ射线更敏感,易出现DNA损伤及细胞凋亡;Tankyrase2对于照射所致AEC凋亡具有正调控作用,对于LFB则为负调控作用。
[Abstract]:Radiation pulmonary injury (radiation pulmonary injury, RPI) is the chest tumor radiotherapy, one of the common complications of bone marrow transplantation, but also in wartime and peacetime nuclear radiation nuclear accident.RPI usually includes early radiation interstitial pneumonia (radiation interstitial, pneumonitis, RIP) and late radiation pulmonary fibrosis (radiation pulmonary fibrosis, RPF) two phase. The relevant literature of conventional radiotherapy in the incidence of RPI is about 30%, in recent years, with the continuous optimization of treatment plan is improved, the incidence rate of RPI decreased, but once RPI occurs, will seriously affect the quality of life of patients after radiotherapy, one of bottleneck problems of nuclear radiation and nuclear accident occurred and treatment the.RPI mechanism and even endanger its life.RPI a certain progress, but is still far from clear, also lack of effective and safe drug prevention, so the mechanism of deep RPI In the study, and find new targets for prevention, to improve the clinical effect of thoracic radiotherapy, improve the quality of life of patients with radiotherapy and has important theoretical and practical significance to improve the level of treatment.RPI radiation injury with multiple source, and has obvious species and individual differences, DNA is a gamma ray damage target cells apoptosis is one of the DNA damage in the end, in the process of RPI play an important role. Alveolar epithelial cells (alveolar epithelial cell, AEC) are the major target cells of RPI lung fibroblast cells (lung, fibroblasts, LFB) is the main effect in the development of RPI cells. Previous studies have found that Tankyrase2 / anti RPF mouse RPI is differentially expressed in the process, as the molecular telomere binding protein, its structure and distribution in cell diversity which play important roles in many fields, through the regulation of telomere length Can affect the stability of the chromosomes, and the cell senescence, death and carcinogenesis is closely related to its structure; PARP domain is the conversion of apoptosis and necrosis of.Tankyrase2 function has not been reported in the literature in the process of RPI. By combining the whole and cell experiment, investigate the role of the Tankyrase2 in the process of RPI AEC and LFB apoptosis materials and methods. Correlation between 1.Tankyrase2 and apoptosis and RPF species differences: C57BL/6J and C3H/He N mice were 60, were randomly divided into control group and irradiation group, using 60Co gamma rays, RPI model of full chest single 20Gy radiation. After irradiation respectively in 15min, 1D, 3D, 7d 1m, 3M, were using the TUNEL method, Western blotting, apoptosis in lung tissue were determined by real-time fluorescence quantitative PCR methods such as AIF, TNKS2, DNA damage, PARP expression, and apoptosis of DNA cell injury in the progression of RPI's loss And whether RPF is prone to, and to investigate the correlation between RPI Tankyrase2 and mice prone to.2.Tankyrase2 induced effects on behavior of AEC and LFB in the biological changes in the irradiation: passage A549 cells and MRC-5 cell culture, 60Co gamma irradiation doses were 5,10 and 20Gy, application of PARP inhibitor 3-AB (the concentration of 5m M) respectively. In irradiated 30min, 1H, 3h, 6h, 12,24h, 24h using the irradiation method treated cells 48h and 72h and 3-AB, using MTT method, gamma H2AX immunofluorescence, AO/EB staining, real-time quantitative PCR and Western blot method to detect cell proliferation, DNA damage, cell apoptosis, AIF TNKS2, the expression of PARP in Tankrase2. To elucidate the irradiation induced AEC and the biological behavior of LFB change in the role. The experimental results of 1. Tankyrase2 cell apoptosis and expression of gamma ray irradiated C3H/He N mice and C57BL/6J mice lung tissue difference of 1.1 C3H /He N and C57BL/6J mice after irradiation are different on the apoptosis of lung tissue cells in the lung tissue of C57BL/6J mice: 1-3D cell apoptosis after irradiation significantly more than C3H/He N mice to alveolar epithelial cells; after irradiation 1m inflammation, two mice apoptosis level had no obvious difference in epithelial cells, monocytes and macrophages. 3M after irradiation; early fibrosis of lung tissue in C57BL/6J mice was significantly less than the number of apoptotic cells in C3H/He N mice, differences in lung tissue of the macrophage apoptosis is the main.1.2 C3H/He N and C57BL/6J mice after irradiation of DNA damage after irradiation of 1-7d, C57BL/6J expression in lung tissue of mice was significantly higher than that of C3H/He gamma H2AX N mice after irradiation; 1m no, two differentially expressed in mouse lung tissue gamma H2AX; after 3M irradiation, the expression of C3H/He in lung tissues in N mice compared with C57BL/6J mice high gamma H2AX.1.3 C3H/He N and C57BL/6J mice The difference of AIF expression in lung tissue after irradiation: after 7d irradiation, 1M, 3M, C3H/He expression in lung tissue of N mice in AIF increased significantly compared with the control group, after 1D irradiation, 3D, AIF expression compared with the control group had no significant difference; different time points after irradiation, lung tissue C57BL/6J AIF expression in mice compared with before irradiation increased significantly after irradiation; 1D, 3D, 1M, C3H/He expression in lung tissue of N mice in AIF compared with C57BL/6J mice, 7d after irradiation, compared with the C57BL/6J mice, there was no significant difference between the expression of C3H/He in lung tissue of N mice in AIF, 3M after irradiation, the difference of TNKS2 expression of C3H/He expression in lung tissue of N mice in AIF the.1.4 C3H/He N higher than those of C57BL/6J mice and C57BL/6J mice after irradiation in lung tissue: (1) RNA level before irradiation, C3H/He in lung tissue of N mice in the expression of TNKS2 was significantly higher than that of C57BL/6J mice after irradiation, TNKS2, C3H/He in lung tissue of N mice in the high expression in C57BL/6J mice (except 3D) (2). Protein Before irradiation, high Tankyrase2 level, compared to C57BL/6J mice C3H/He lung tissue in N mice, C57BL/6J mice with high C3H/He expression in N mice after TNKS2 irradiation (except 1m) expression of PARP in lung tissue of.1.5 C3H/He N in mice and C57BL/6J mice after irradiation in mice: the control group C3H/He N PARP expression in lung tissue of C57BL/6J mice was low; lung the C57BL/6J mice each time after irradiation, the expression of PARP in N were higher than C3H/He (except 1D) and Tankyrase2.2. after irradiation, AEC and LFB DNA damage and apoptosis, the expression of PARP, there are significant differences in 2.1 different doses of gamma ray irradiation after A549 cells and MRC-5 cell proliferation activity was different: 5,10 and 20Gy after irradiation, three doses were inhibited the proliferation of A549 cells after irradiation; 48 and 72h, 10 and 20Gy gamma rays inhibit the proliferation of MRC-5 cells after 72h irradiation, gamma ray irradiation 5Gy promote the proliferation of.2.2 different doses of gamma irradiation The damage effect of A549 cells and MRC-5 cells of different DNA: different doses of gamma ray irradiated A549 cells showed DNA damage after irradiation, 30min, 1H, 3h, DNA cell damage increased, the higher the dose, DNA damage and the number of cells after 6h irradiation, 12h positive cells gradually decreased, but still higher than the normal control group; after irradiation of MRC-5 cells only appeared in some time points visible positive cells, the apoptosis of A549 cells and MRC-5 cells and the expression of H2AX.2.3 of different doses of gamma ray irradiation after different: (1) each time point A549 cell apoptosis cell number 20Gy after irradiation were 5 and 10Gy gamma rays the irradiation group (except 12h), 10Gy ray irradiation at each time point after the number of apoptotic cells were more than 5Gy gamma ray irradiation group (except 3H). (2) 5Gy after irradiation, the expression of A549 at each time point AIF cells were higher than the control group, 10Gy after irradiation, each time point AIF The expression of 10Gy was higher than that of control group, and after irradiation, the expression of AIF was higher than that of 5Gy gamma ray irradiation group. (3) 5Gy, 10Gy after irradiation, application of inhibitors of 3-AB treated A549 cells, the expression of AIF was significantly lower than that of untreated cells. Treatment group cells, 10Gy gamma ray irradiation after A549 cell AIF expression compared with the 5Gy gamma ray treatment group. (4) after irradiation of MRC-5 cells was not detected in apoptotic cells and the expression of AIF.2.4 after irradiation of A549 cells and MRC-5 TNKS2 cells, PARP expression is different: (1) the level of RNA, and 5 10Gy after irradiation, 1-12h, expression of A549 cells in TNKS2 group was lower than the control group, after 30min irradiation, A549 cells TNKS2 expression was significantly higher than the control group; the protein level, 5Gy after irradiation, 30min, 12h, Tankyrase2 expression was lower than that in control group, 1 and 3h after irradiation, the expression of Tankyrase2 was on A549 cells Control group; the expression of Tankyrase2 was higher than that in control group 10Gy after irradiation (except 12h), after 30min irradiation, 1H, 6h, 24h, 10Gy gamma ray irradiation group Tankyrase2 expression than the 5Gy irradiation group. (2) the level of RNA, 5Gy after irradiation at different time points, the expression of MRC-5 compared with TNKS2 cells before irradiation, 10Gy after irradiation at different time points, the expression of MRC-5 cell TNKS2 was high before irradiation (except 30min), 5Gy after irradiation, 30min, 1H, 12h, MRC-5 cells TNKS2 expression than the 10Gy gamma ray irradiation group, 10Gy after irradiation by 3H, the expression of TNKS2 was 5Gy gamma ray irradiation group; protein level of 5Gy after irradiation, 30min and 24h, the expression of MRC-5 cell Tankyrase2 than before irradiation and low expression of MRC-5 cells after 1-12h irradiation, Tankyrase2 than 10Gy before irradiation, gamma ray irradiation after the expression of Tankyrase2 at each time point compared to the irradiation before the high gamma ray 10Gy at each time point after Tankyr irradiation ase2鐨勮〃杈鹃珮浜，

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