1、一d一目 录摘 要1Abstract4缩略词表7前 言8日U 舌一8实验材料10实验方法19实验结果3 1讨论43结论46参考文献47综j查5 1硕士在读期间发表论文57致谢58一6一辛伐他汀改善辐射诱导的小鼠组织损伤摘要研究目的：辐射经常用于癌症治疗以达到控制肿瘤的发展，大约50的癌症病人接受放射治疗。尽管放疗在抑制肿瘤细胞增殖和促进其凋亡方面有很好的效果，但是对正常组织的毒性仍然是阻碍其治疗癌症最重要的因素。放疗的副作用主要是导致急性辐射综合症，包括胃肠道和造血系统两大方面。普遍认为，辐射诱导的细胞凋亡，氧化应激和炎症也会引起组织损伤。最近在辐射引起正常组织损伤方面，最受关注的是辐射诱导产
2、生的内皮损伤。内皮功能障碍在辐射反应的发展过程中似乎扮演着越来越重要的角色。甲基戊二酰辅酶A还原酶抑制剂，即他汀类药物，广泛应用于临床以降低血清胆固醇水平和减少心脏发病率和死亡率。然而除了众所周知的降低胆固醇作用，他汀类药物还有独立于降脂作用之外的多效性生物作用。其中包括改善内皮功能、降低氧化应激、抑制炎症、调节免疫系统等。此外，已经发现辛伐他汀能够动员内皮祖细胞(EPC)，内皮祖细胞是一群能自我更新、增殖、分化为内皮细胞的多能祖细胞，具有维持血管内皮完整性、参与修复损伤血管和促进血管再生的作用，对局部缺血后的修复具有重要作用。本课题，我们假设：辛伐他汀能够提高60Co丫射线辐射后小鼠的生存率
3、，并研究了辛伐他汀辐射保护作用的可能机制。研究方法：C57小鼠接受不同剂量的辛伐他汀(2，10，20 mgkgd)灌胃给药两周，然后以60Co丫射线8Gy致死剂量辐射，在辐射后30天的观察期内记录小鼠的生存率。鉴于小鼠辐射后6天开始出现死亡，C57小鼠在高剂量的辛伐他汀(20 mgkgd)灌胃两周后，接受4Gy剂量的辐射，7天后处死小鼠，观察小鼠肠组织和骨组织的病理形态变化，同时测定这两种组织内细胞凋亡情况；取血后测定小鼠外周血血象的变化；检测胸腺和脾脏组织中超氧化物歧化酶(SOD)活性和丙二醛(MDA)水平；利用WestemBlot的方法检测小鼠胸腺组织中相关凋亡蛋白的表达。第二军医大学硕士
4、学位论文研究结果：1辛伐他汀显著提高了8 Gy致死剂量辐射后小鼠的生存率。辐射模型组小鼠的生存期均未超过11天。同模型组相比，辛伐他汀低剂量、中剂量组(2，10 mgkgd)的生存率并没有得到明显的改善。辛伐他汀高剂量组(20 mgkgd)小鼠的生存率明显提高了35。2辛伐他汀改善辐射诱导的肠组织损伤。病理检查发现，同正常组相比，小鼠在4 Gy辐射后第7天空肠结构的完整性被破坏，平均绒毛高度显著减少。同模型组相比高剂量辛伐他汀组能够保护肠结构的完整性，且增加平均绒毛的高度。原位末端标记(TUNEL)法检测分析辐射诱导肠组织小肠绒毛细胞大量凋亡，提前给予辛伐他汀后小鼠肠组织细胞的凋亡得到明显的抑
5、制。3辛伐他汀改善辐射诱导的骨组织损伤。通过检测股骨骨髓的病理学变化和细胞凋亡情况发现：同正常组相比，辐射组的骨髓没有被造血细胞充满，存在着大面积的空隙，辛伐他汀治疗组小鼠骨髓造血功能得到明显的恢复。辐射组骨髓内凋亡细胞的数量明显高于正常组，辛伐他汀治疗组能够明显抑制骨髓细胞的凋亡。4辛伐他汀增加红细胞和血红蛋白的数量。辐射后第7天检Nd,鼠外周血血象的变化包括白细胞、红细胞、血小板、血红蛋白。发现辐射能够降低这些血细胞的数目，产生明显的辐射后血细胞变化。辛伐他汀治疗组能够显著地加速红细胞和血红蛋白的恢复，但是不能改变白细胞和血小板的数量。此外，同正常组和模型组相比，辛伐他汀治疗组都能够提高辐
6、射后外周血内皮祖细胞的水平。5辛伐他汀提高超氧化物歧化酶活性和降低丙二醛含量。同模型组相比，辛伐他汀治疗组明显增加了胸腺和脾脏组织中超氧化物歧化酶(SOD)的活性；同时，小鼠胸腺和脾脏组织中丙二醛(MDA)的含量在辛伐他汀处理后，相比于辐射模型组也得到了明显的降低。6辛伐他汀下调胸腺组织内p53蛋白的表达。在辐射后第1、3、7天分别提取胸腺并通过Westemblot的方法检测组织内凋亡相关蛋白的表达。结果显示：同正常组相比，辐射会引起p53及磷酸化p53表达增加，辛伐他汀处理后可以抑制p53的过表达及其磷酸化。辛伐他汀并不改变p38蛋白的表达水平。辛伐他汀改善辐射诱导的小鼠组织损伤结论：辛伐他
7、汀能够提高小鼠辐射后的生存率和减轻辐射导致的组织损伤。辛伐他汀能够抑制辐射引起的凋亡，其机制可能与下调p53以及磷酸化p53蛋白相关。关键词：辛伐他汀，辐射，生存率，辐射保护第二军医大学硕士学位论文AbstractBackgrounds and 0bjectives：Radiation is frequently used in cancer therapy to achievelocal tumor contr01About half of people with cancer are treated、析th radiation therapyDespite of inducing anti
8、proliferative and cellkilling effects in tumor tissue，normal tissuetoxicity remains the single most important obstacle to uncomplicated cancer cure，nletoxicity of radiation is associated with induction of acute radiation syndromes involvingthe gastrointestinal tract(GI)and hematopoietic system(HP)It
9、 is also believed that cellapoptosis，oxidative stress and inflammation induced by radiation contribute to tissuedamageRecently,radiation-induced endothelial injury has been the subject ofconsiderable interest relative to radiation toxicities in normal tissueEndothelialdysfunction appears to play an
10、increasingly important role in the development of radiationresponse13The 3-hydroxy一3-methylglutaryl coenzyme A(HMG-CoA)reductaseinhibitors，also known as statins，are widely used in the clinic for lowering serumcholester01 and decreasing cardiac morbidity and mortalityHowever,beyond theirwell-known ch
11、olesterol-lowering activity,statins also possess pleiotropic biological effectsindependent of their beneficial effects on blood cholesterollevelsSuch pleiotropic effectsinclude improving endothelial function，decreasing oxidative stress and inflammation，andregulating immune systemIn addition，simvasta
12、tin has also been found to mobilizeendothelial progenitor cells(EPCs)，a heterogeneous subpopulation of bone marrowmononuclear cells that have an enhanced potential to differentiate into endothelial cellsfrom the bone marrow and accelerate vascular structure formationIn the current study,wetested our
13、 hypothesis that simvastatin was able to decrease the mortality in a 60CoT-radiation-induced mice model，and investigated the radioprotective effect of simvastatinand its potential mechanismsMethods：Mice were treated intragastrically with simvastatin 2，1 0，20 mgkgd for 2weeks and suffered an 8-Gy 1et
14、hal dose of radiation after the final administrationThesurvival rate was calculatedat the end of a 3 0一day observing periodMice were radiatedwith a dose of 4 Gy after 20 mgkgd simvastatin treatment for 2 weeksSeven days later,the mice were sacrificedMorphological changes of j ejunum and bone marrow
15、wereobserved and apoptotic cells of both tissues were determinedPefipheral blood cells werecounted，and superoxide dismutase(SOD)activity and malondialdehyde(MDA)level intissues of both thymus and spleen were measuredThe expression of apoptosisrelatedproteins in thymus were analysed by Western blot-d
16、辛伐他汀改善辐射诱导的小鼠组织损伤Results：1 Simvastatin increased the 30day survival rate of mice after 8 Gy radiationA1l of themice in control group did not survive longer than 1 1 days after 8 Gy radiationPretreatment with simvastatin 2，1 0 mgkgd for consecutive 14 days did not reducethe mortality when compared wi
17、th the control groupHowever,simvastatin 20mgkgd pretreatment significantly increased the 30-day survival rate to 352Simvastatin attenuated radiation-induced jejunum injuryHistopathologic examinationfound that the integrity of jejunum structure Was damaged and the mean villi heightwas significantly d
18、ecreased in mice 7 days after 4 Gy radiation when compared withthe control onesSimvastatin 20 mgkgd pretreatment for 1 4 days preserved theintegrity ofj ejunum structureand increased the mean villi height when compared withthe untreated radiation groupTUNEL staining showed that 4 Gy radiation induce
19、dlarger number of apoptotic cells per villi in the jejunum tissues of mice compared withthe control onesHowever,simvastatin pre-administration dramatically inhibitedradiation-induced apoptosis3Simvastatin improved radiationinduced bone marrow damageThe long-termrecovery of the hematopoietic system i
20、s relative to replenishment of functionalhematopoietic stem cells and progenitor cellsThus，histological changes andapoptotic cells of the femoral marrow were examinedThe marrow of the radiatedgroup Was not replete with hematopoietic cells as compared to the femoral marrowfrom the control mice，but re
21、covery of marrow hematopoiesis Was evident in thesimvastatintreated groupThe number of apoptotic cells from isolated femoralmarrow was larger in the radiated group than in the control，and simvastatinpretreatment significantly inhibited apoptosis4Simvastatin increased the number of RBC and EPCsThe nu
22、mber of WBC，RBC，PLTand HGB in the peripheral blood was measured 7 days after radiationIt was foundthat radiation reduced the number of all these cell types，resulting in a significantradiation decreasePretreatment of simvastatin significantly accelerated the recoveryof RBC and HGB，but did not change
23、the cell number of WBC and PLTIn addition，the number of EPCs from peripheral blood Was determined to find out whetherimprovement of myelosuppression benefited peripheral progenitor cellsInterestingly,it Was found that simvastatin pretreatment significantly increased the EPCs numberwhen compared eith
24、er to the control or radiated group5Simvastatin increased SOD activity and decreased MDA levelSOD activity,determined 7 days after radiation，was significantly lower in the thymus of the radiatedgroup than the contr01Twoweek pretreatment of simvastatin obviously elevated theSOD activity as compared t
25、o the radiated groupContrast to the result of SOD activity5一in the thymus，MDA level was significantly reduced after simvastatin pretreatment ascompared to the radiated miceConsistent with the result of thymus，changes of SODactivity and MDA were similar in the spleen6Simvastatin down-regulated p53 ex
26、pression，but not p3 8 in thymusThe expression ofapoptosis-related proteinsin thymus were analysed by Western blot on day 1，3，7Western blots revealed that p53 was significantly increased in thymus of irradiatedmice when compared to the control，and reduced in simvastatin treated micesimvastatin treatm
27、ent reversed the changeIt was suggested that simvastatin improvedradiation-induced tissue damage through inhibition of p53 and p-p53Conclusion：Simvastatin enhanced the survival rate and ameliorated tissues damage inradiation-induced miceThe radioprotective effect of simvastatin was possibly related
28、toinhibition ofp53 and p-p53 expressionKEY WORDS：Simvastatin，radiation，survival rate，radioprotection一6辛伐他汀改善辐射诱导的小鼠组织损伤英文缩写 英文名缩略词表姆 Ammonium PersulfateHMGCoAEPCsGIHPH&ETdTdUTPPODDABWBCRBCHGBPLTBSACMCDMS0M199FBSPBSROSSODMDAMVASPCThe 3-hydroxy一3-methylglutaryl coenzyme AEndothelial Progenitor CellsGa
29、strointestinalHematopoieticHematoxyllin and EosinTerminal Deoxynucleotidyl Transferase2-Deoxyuridine 5-TriphosphatePeroxidaseDiaminobenzidinewhite blood cellsred blood cellshemoglobinsplateletsBovine Serum AlbuminSodium Carboxy Methyl CelluloseDimethyl SulfoxideMedium 199Fetal Bovine SerumPhosphate Buffered SalineReactive Oxygen SpeciesSuperoxide DismutaseMalondialdehydMevalonic AcidSmooth Muscle Progenitor Cells中文名过硫酸铵甲基戊二酰还原酶内皮祖细胞胃肠造血苏木素伊红末端转移酶三磷酸脱氧尿甘过氧化物酶二氨基联苯胺白细胞红细胞血红蛋白血小板牛血清白蛋白羧甲基纤维素钠二甲基亚砜M199培养基胎牛血清磷酸缓冲液活性氧超氧化物歧化酶丙二醛甲羟戊酸亚滑肌祖细胞7一
