本文關(guān)鍵詞：Tankyrase2在放射性肺損傷中AEC和LFB凋亡調(diào)控中的作用研究　出處：《安徽醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： Tankyrase2 放射性肺損傷 細(xì)胞凋亡 肺泡上皮細(xì)胞 肺成纖維細(xì)胞

【摘要】：放射性肺損傷(radiation pulmonary injury,RPI)是胸部的腫瘤放射治療、骨髓移植預(yù)處理等常見(jiàn)的并發(fā)癥之一,也見(jiàn)于戰(zhàn)時(shí)的核輻射及平時(shí)的核事故。RPI通常包括早期的放射性間質(zhì)性肺炎(radiation interstitial pneumonitis,RIP)和晚期的放射性肺纖維化(radiation pulmonary fibrosis,RPF)兩個(gè)階段。有關(guān)文獻(xiàn)報(bào)道常規(guī)放療RPI的發(fā)病率約為30%,近年隨著放療方案的不斷改進(jìn)優(yōu)化,RPI的發(fā)生率有所降低,但RPI一旦發(fā)生,將嚴(yán)重影響放療后患者的生存質(zhì)量,甚至危及其生命。RPI的發(fā)生也是核輻射和核事故救治的瓶頸問(wèn)題之一。RPI機(jī)制研究有一定的進(jìn)展,但仍遠(yuǎn)未闡明,也尚缺乏安全有效的防治藥物,故對(duì)RPI機(jī)制進(jìn)行深入研究,進(jìn)而發(fā)現(xiàn)防治新靶點(diǎn),對(duì)于提高臨床胸部腫瘤放療效果、改善放療患者的生存質(zhì)量以及提高放射損傷救治水平具有重要的理論和實(shí)際意義。RPI的發(fā)生具有多源性,且具有明顯的種屬和個(gè)體差異,DNA是γ射線損傷靶分子,細(xì)胞凋亡是DNA損傷結(jié)局之一,其在RPI過(guò)程中的起到重要作用。肺泡上皮細(xì)胞(alveolar epithelial cell,AEC)是RPI的主要的靶細(xì)胞,肺成纖維細(xì)胞(lung fibroblasts,LFB)是RPI發(fā)生發(fā)展的主要效應(yīng)細(xì)胞。既往研究發(fā)現(xiàn)Tankyrase2在易/抗RPF小鼠RPI進(jìn)程中存在表達(dá)差異,該分子作為端粒結(jié)合蛋白,其結(jié)構(gòu)特點(diǎn)以及在細(xì)胞中分布的多樣性使其在多種領(lǐng)域發(fā)揮相應(yīng)的作用,通過(guò)調(diào)節(jié)端粒長(zhǎng)度可以影響染色體的穩(wěn)定性,而且其與細(xì)胞衰老、死亡和癌變密切相關(guān);其結(jié)構(gòu)域PARP是細(xì)胞凋亡和壞死的轉(zhuǎn)換點(diǎn)。Tankyrase2在RPI過(guò)程中的作用未見(jiàn)文獻(xiàn)報(bào)道。本文通過(guò)整體和細(xì)胞實(shí)驗(yàn)相結(jié)合,探討Tankyrase2在RPI過(guò)程中AEC和LFB凋亡調(diào)控中的作用。材料和方法1.Tankyrase2及細(xì)胞凋亡與RPF種屬差異的相關(guān)性研究:C57BL/6J和C3H/He N小鼠各60只,隨機(jī)將其各分成對(duì)照組和照射組,采用60Coγ射線照射,全胸單次20Gy照射建立RPI模型。分別在照射后15min、1d、3d、7d、1m、3m取材,采用原位末端標(biāo)記法、免疫印跡、實(shí)時(shí)熒光定量PCR等方法檢測(cè)肺組織的細(xì)胞凋亡、DNA損傷、AIF、TNKS2、PARP表達(dá),研究細(xì)胞DNA損傷和凋亡在RPI進(jìn)展中的作用及其是否與RPF易發(fā)相關(guān),并初步探討Tankyrase2與小鼠RPI易發(fā)相關(guān)性。2.Tankyrase2在照射致AEC和LFB生物學(xué)行為改變中的作用研究:A549細(xì)胞和MRC-5細(xì)胞傳代培養(yǎng),60Coγ射線照射,劑量分別為5、10和20Gy,應(yīng)用PARP抑制劑3-AB(濃度為5m M)。分別在照射后30min、1h、3h、6h、12、24h、48h和72h及3-AB作用后24h應(yīng)用上述照射方法處理細(xì)胞,應(yīng)用MTT法、γH2AX免疫熒光、AO/EB染色、實(shí)時(shí)熒光定量PCR、免疫印跡等方法檢測(cè)細(xì)胞增殖活力、DNA損傷、細(xì)胞凋亡、AIF、TNKS2、PARP的表達(dá)。旨在闡明Tankrase2在照射所致AEC和LFB生物學(xué)行為改變中的作用。實(shí)驗(yàn)結(jié)果1.γ射線照射后C3H/He N小鼠和C57BL/6J小鼠肺組織中細(xì)胞凋亡和Tankyrase2表達(dá)存在差異1.1 C3H/He N小鼠和C57BL/6J小鼠照射后對(duì)肺組織中細(xì)胞凋亡存在差異:照射后1-3d C57BL/6J小鼠肺組織凋亡細(xì)胞顯著多于C3H/He N小鼠,以肺泡上皮細(xì)胞為主;照射后1m即炎癥期,兩種小鼠細(xì)胞凋亡水平無(wú)明顯差異,以上皮細(xì)胞、單核巨噬細(xì)胞為主;照射后3m即纖維化初期C57BL/6J小鼠肺組織凋亡細(xì)胞數(shù)顯著少于C3H/He N小鼠,后者以巨噬細(xì)胞的凋亡為主。1.2 C3H/He N小鼠和C57BL/6J小鼠照射后肺組織中DNA損傷存在差異:照射后1-7d,C57BL/6J小鼠肺組織γH2AX表達(dá)顯著高于C3H/He N小鼠高;照射后1m,兩種小鼠肺組織γH2AX的表達(dá)無(wú)差異;照射后3m,C3H/He N小鼠肺組織中γH2AX的表達(dá)較C57BL/6J小鼠高。1.3 C3H/He N小鼠和C57BL/6J小鼠照射后肺組織中AIF表達(dá)存在差異:照射后7d、1m、3m,C3H/He N小鼠肺組織中AIF的表達(dá)較對(duì)照組顯著增加,照射后1d、3d,AIF的表達(dá)較對(duì)照組無(wú)顯著差異;照射后各時(shí)間點(diǎn),C57BL/6J小鼠肺組織中AIF的表達(dá)較照射前顯著增加;照射后1d、3d、1m,C3H/He N小鼠肺組織中AIF的表達(dá)較C57BL/6J小鼠低,照射后7d,C3H/He N小鼠肺組織中AIF的表達(dá)較C57BL/6J小鼠無(wú)顯著差異,照射后3m,C3H/He N小鼠肺組織中AIF的表達(dá)較C57BL/6J小鼠高。1.4 C3H/He N小鼠和C57BL/6J小鼠照射后肺組織中TNKS2表達(dá)存在差異:(1)RNA水平,照射前,C3H/He N小鼠肺組織中TNKS2表達(dá)顯著高于C57BL/6J小鼠,照射后,C3H/He N小鼠肺組織中TNKS2的表達(dá)高于C57BL/6J小鼠(除3d)。(2)蛋白水平,照射前,C3H/He N小鼠肺組織中Tankyrase2較C57BL/6J小鼠高,照射后C3H/He N小鼠TNKS2表達(dá)較C57BL/6J小鼠高(除1m)。1.5 C3H/He N小鼠和C57BL/6J小鼠照射后肺組織中PARP表達(dá)存在差異:對(duì)照組C3H/He N小鼠肺組織PARP表達(dá)較C57BL/6J小鼠低;照射后各個(gè)時(shí)間C57BL/6J小鼠肺組織中PARP表達(dá)均較C3H/He N小鼠高(除1d)。2.γ射線照射后AEC和LFB DNA損傷和凋亡的發(fā)生及Tankyrase2、PARP表達(dá)等存在差異2.1不同劑量γ射線照射后A549細(xì)胞和MRC-5細(xì)胞增殖活力的影響不同:5、10和20Gyγ射線照射后,三個(gè)劑量均抑制A549細(xì)胞增殖;照射后48和72h,10和20Gyγ射線抑制MRC-5細(xì)胞增殖,照射后72h,5Gyγ射線照射促進(jìn)其增殖。2.2不同劑量γ射線照射后A549細(xì)胞和MRC-5細(xì)胞DNA損傷的影響不同:不同劑量γ射線照射后A549細(xì)胞均出現(xiàn)DNA損傷,照射后30min、1h、3h,DNA損傷細(xì)胞不斷增加,劑量越高,DNA損傷細(xì)胞越多,照射后6h、12h陽(yáng)性細(xì)胞逐漸減少,但仍較對(duì)照組多;不同劑量γ射線照射后MRC-5細(xì)胞只在部分時(shí)間點(diǎn)可見(jiàn)陽(yáng)性細(xì)胞出現(xiàn),及γH2AX的表達(dá)。2.3不同劑量γ射線照射后A549細(xì)胞和MRC-5細(xì)胞凋亡的影響不同:(1)20Gyγ射線照射后各時(shí)間點(diǎn)A549細(xì)胞凋亡細(xì)胞數(shù)目均較5和10Gyγ射線照射組多(除12h),10Gyγ射線照射后各時(shí)間點(diǎn)凋亡細(xì)胞數(shù)目均多于5Gyγ射線照射組(除3h)。(2)5Gyγ射線照射后,各時(shí)間點(diǎn)A549細(xì)胞AIF的表達(dá)均高于對(duì)照組,10Gyγ射線照射后,各時(shí)間點(diǎn)AIF的表達(dá)也均高于對(duì)照組,且10Gyγ射線照射后各時(shí)間點(diǎn)AIF表達(dá)均高于5Gyγ射線照射組。(3)5Gy、10Gyγ射線照射后,應(yīng)用抑制劑3-AB處理的A549細(xì)胞,其AIF的表達(dá)顯著低于未處理組細(xì)胞。處理組細(xì)胞,10Gyγ射線照射后A549細(xì)胞的AIF表達(dá)較5Gyγ射線處理組高。(4)不同劑量γ射線照射后MRC-5細(xì)胞未檢測(cè)到凋亡細(xì)胞的出現(xiàn)及AIF的表達(dá)。2.4不同劑量γ射線照射后A549細(xì)胞和MRC-5細(xì)胞TNKS2、PARP表達(dá)的影響不同:(1)RNA水平,5和10Gyγ射線照射后1-12h,A549細(xì)胞TNKS2表達(dá)較對(duì)照組低,照射后30min,A549細(xì)胞TNKS2表達(dá)顯著高于對(duì)照組;蛋白水平,5Gyγ射線照射后30min、12h,Tankyrase2表達(dá)較對(duì)照組低,照射后1和3h,A549細(xì)胞的Tankyrase2表達(dá)較對(duì)照組高;10Gyγ射線照射后各時(shí)間點(diǎn)Tankyrase2表達(dá)較對(duì)照組高(除12h),照射后30min、1h、6h、24h,10Gyγ射線照射組的Tankyrase2表達(dá)較5Gy照射組高。(2)RNA水平,5Gyγ射線照射后各時(shí)間點(diǎn),MRC-5細(xì)胞TNKS2表達(dá)較照射前高,10Gyγ射線照射后各時(shí)間點(diǎn),MRC-5細(xì)胞TNKS2的表達(dá)較照射前高(除30min),5Gyγ射線照射后30min、1h、12h,MRC-5細(xì)胞TNKS2表達(dá)較10Gyγ射線照射組高,10Gyγ射線照射后3h,TNKS2的表達(dá)較5Gyγ射線照射組高;蛋白水平,5Gyγ射線照射后30min和24h,MRC-5細(xì)胞的Tankyrase2的表達(dá)較照射前低,照射后1-12h,MRC-5細(xì)胞的Tankyrase2的表達(dá)較照射前高,10Gyγ射線照射后各時(shí)間點(diǎn)Tankyrase2的表達(dá)較照射前高,10Gyγ射線照射后各時(shí)間點(diǎn)Tankyrase2的表達(dá)高于5Gyγ射線照射組(除1h、3h)。(3)5Gyγ射線照射后30min、3h、12h,A549細(xì)胞的PARP表達(dá)較對(duì)照組低,照射后1和6h,PARP表達(dá)較照射前高;10Gyγ射線照射后各時(shí)間點(diǎn)PARP表達(dá)較照射前高;10Gyγ射線照射后各時(shí)間點(diǎn)PARP表達(dá)較5Gyγ射線照射組高(除1h);5Gyγ射線照射后30min、3h和12h,MRC-5細(xì)胞的PARP的表達(dá)較照射前高;照射后24h,MRC-5細(xì)胞的PARP的表達(dá)較照射前低;10Gyγ射線照射后各時(shí)間點(diǎn)PARP的表達(dá)較照射前高;10Gyγ射線照射后6、12和24h,PARP的表達(dá)高于5Gyγ射線照射組。結(jié)論1.γ射線照射后C57BL/6J與C3H/He N小鼠肺組織不同細(xì)胞凋亡的差異為兩種小鼠是否易發(fā)RPF的機(jī)制之一:γ射線照射后C57BL/6J小鼠易出現(xiàn)DNA損傷、肺泡上皮細(xì)胞等實(shí)質(zhì)細(xì)胞凋亡,而巨噬細(xì)胞等致纖維化細(xì)胞不易凋亡,故C57BL/6J小鼠易發(fā)生RPF;反之,γ射線照射后C3H/He N小鼠肺組織肺泡上皮細(xì)胞較少發(fā)生凋亡,而巨噬細(xì)胞等致纖維化細(xì)胞通過(guò)凋亡清除,與該小鼠不易發(fā)生RPF相關(guān)。2.Tankyrase2于RPF是否易發(fā)呈負(fù)相關(guān)。3.AEC較LFB對(duì)γ射線更敏感,易出現(xiàn)DNA損傷及細(xì)胞凋亡;Tankyrase2對(duì)于照射所致AEC凋亡具有正調(diào)控作用,對(duì)于LFB則為負(fù)調(diào)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〃杈鵑珮?shù)海?

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