有氧运动对肝原位移植瘤小鼠术后免疫功能和代谢组学影响的研究

发布时间：2018-05-04 17:24

本文选题：肝原位移植瘤 + 术后　；参考：《第二军医大学》2015年博士论文

【摘要】：原发性肝癌是医学临床常见的恶性肿瘤之一,病死率高且治疗效果欠佳,尚无确切有效的治愈措施。手术切除仍是目前其治疗的最有效手段之一,虽然相关的术后治疗方案的研究近年来获得很大的突破,然而其疗效依然不能令人满意。因此,对于肝癌大国,研究降低肝癌患者术后复发率和提高生存质量的术后康复方案有着巨大的社会意义和经济价值。现代医学认为术后复发与患者免疫功能有着高度相关性,已初步证实围手术期机体免疫状态及术后细胞免疫功能与其病情进展、复发、转移密切相关,所以寻找能够改善肝癌患者术后免疫状态的途径和方法是当前肝癌肿瘤康复领域的研究方向之一。中医自古提倡运动,针对肿瘤患者,更体现在“既病防变”和“愈后防复”,有太极拳、八段锦等运动疗法应用于该人群。现代医学推崇运动疗法作为肿瘤康复的主要治疗手段之一,明确了证实太极拳等运动疗法可提高肿瘤患者机体免疫系统的功能。虽然运动方法在肿瘤康复具有重要的地位和不可取代的作用,但是,包括美国最新版的《肿瘤幸存者运动锻炼指南》在内,目前尚无一套明确且规范的运动方案用于指导肝癌患者术后的康复,尤其表现在介入运动的时机和强度,缺乏该阶段运动与免疫状态相关的客观理论依据。基上所述,本研究第一部分拟构建肝原位移植瘤小鼠术后运动模型,探索合理的运动介入时机,并观察不同运动强度对小鼠模型代谢产物的影响；第二部分以脾脏T淋巴细胞为主要研究对象,探讨运动强度对小鼠模型T细胞免疫功能的影响；第三部分通过蛋白质印迹法研究运动对模型小鼠淋巴细胞功能的影响机制；第四部分运用代谢组学方法评估运动对肝原位移植瘤小鼠术后的影响,为下一步研究提供新思路。第一部分肝原位移植瘤小鼠术后运动模型的构建目的：全面探索肝原位移植瘤小鼠切除术后的状态,观察运动对小鼠术后状态的影响。方法：对肝原位移植瘤切除术后小鼠进行不同强度(中等强度：12m/min和高强度运动：18m/min)和不同运动时间(术后早期运动和术后后期运动)的干预,建立术后小鼠运动模型；从体重、自主活动度、血生化和肝功能等基础指标评价小鼠术后康复情况。结果：运动前后组间体重差别并无统计学意义；运动开始时间对小鼠自主活动度的影响具有差异,p0.05具有统计学意义；血生化和肝功能等指标,可见术后后期中等强度运动的模型小鼠血清中ALB为32.00±2.53g/1,术后后期高强度运动的模型小鼠血清中ALB为32.12±2.53 g/l,两者均高于术后后期正常运动模型小鼠血清中含量：26.44±4.10 g/l,且p0.05,差异具有统计学意义。术后后期中等强度运动的模型小鼠血清中TP为57.83±2.71 g/l,术后后期高强度运动的模型小鼠血清中TP为52.62±4.37g/l,两者均高于术后后期正常运动模型小鼠血清中含量：48.77±4.41g/l,且p0.05,差异具有统计学意义。结论：术后后期运动对模型小鼠整体状态的恢复效果最佳,模型建立成功；运动强度与肝功能等指标呈反比趋势。第二部分有氧运动对肝原位移植瘤小鼠术后免疫功能影响的实验研究目的：观察不同介入时间和强度的运动对模型小鼠免疫功能的影响。方法：1、用刀豆蛋白A对模型小鼠脾淋巴细胞进行刺激,用MTT法检测不同干预方法对小鼠T淋巴细胞增殖的影响。2、制备模型小鼠脾淋巴细胞悬液,磁珠分选T淋巴细胞,流式细胞仪检测CD69+、25+的表达率。3、制备模型小鼠脾淋巴细胞悬液,磁珠分选T淋巴细胞,ELISA法检测PMA加Ion诱导T细胞分泌IL-10, TNF-a等分泌。结果：1、介入时间与强度对模型小鼠淋巴细胞增殖的影响：对于肝原位移植瘤切除术后的模型小鼠,术后早期开始中等强度运动的小鼠淋巴细胞增殖能力为(227.42±0.62)%,术后早期开始高强度运动的小鼠淋巴细胞增殖能力为(188.38±0.23)%,两者均低于术后正常运动小鼠,即(348.03±0.89)%。但术后后期进行中等强度运动的小鼠淋巴细胞增殖能力为(233.77±0.30)%,与术后后期进行正常运动的小鼠淋巴细胞增殖能力(235.03±0.89)%相比,两者数值接近,且两者差异无统计学意义。术后后期进行高强度运动的小鼠淋巴细胞增殖能力为(329.12±0.43)%,优于术后后期进行正常运动的小鼠淋巴细胞增殖能力(235.03±0.89)%,但两者间差异无统计学意义。交互分析后显示运动开始时间和运动强度相结合对肝原位移植瘤切除术后模型小鼠脾淋巴细胞增殖能力的影响p0.05,统计学差异有意义。2、介入时间与强度对模型小鼠T淋巴细胞表面因子CD69+、CD25+的影响：对于肝原位移植瘤切除术后早期运动的模型小鼠,中等强度跑台运动干预的模型小鼠T淋巴细胞表面CD69+表达率为(36.54±0.46)%,高强度跑台运动干预的模型小鼠T淋巴细胞表面CD69+表达率为(38.72±0.20)%,两者T淋巴细胞表面CD69+表达率均低于正常运动小鼠的(53.38±0.25)%。在术后后期进行运动的小鼠中,高强度运动干预下的模型小鼠T淋巴细胞表面CD69+表达率为(40.31±0.33)%,中等强度运动干预下的模型小鼠T淋巴细胞表面CD69+表达率为(45.21±0.36)%,均大于正常运动后的模型小鼠T淋巴细胞表面CD69+表达率,即(37.15±0.18)%,且p0.05,差异具有统计学意义。对于肝原位移植瘤切除术后早期的模型小鼠,中等强度跑台运动干预的模型小鼠T淋巴细胞表面CD25+表达率为(37.82±0.26)%,高强度跑台运动干预的模型小鼠T淋巴细胞表面CD25+表达率为(41.63±0.37)%,两者T淋巴细胞表面CD25+表达率均低于正常运动小鼠的(45.65±0.22)%。在术后后期进行运动的小鼠中,中等强度运动干预下的模型小鼠T淋巴细胞表面CD25+表达率为(42.72±0.29)%,高强度运动干预下的模型小鼠T淋巴细胞表面CD25+表达率为(46.40±0.43)%,均大于正常运动后的模型小鼠T淋巴细胞表面CD25+表达率,即(41.63±0.37)%,且p0.05,差异具有统计学意义。3、介入时间与强度对模型小鼠T淋巴细胞分泌细胞因子的影响：对于肝原位移植瘤切除术后的模型小鼠,术后早期进行运动的小鼠,运动强度对其T淋巴细胞分泌TNF-a的影响无统计学差异。术后后期运动的小鼠,中等强度运动组与高强度运动组均与正常运动组具有较大差异,且p0.05,差异具有统计学意义。各组具体T淋巴细胞中TNF-a分别为：术后后期进行中等强度跑台运动的模型小鼠为188.74±96.10 pg/ml,术后后期进行高强度跑台运动的模型小鼠为205.93±110.04pg/ml,术后后期进行正常运动的模型小鼠为450.30±116.37 pg/ml. IL-10在强度上差别没有统计学意义,但进行交互分析后发现术后后期运动的小鼠,IL-10分泌量低于术后早期运动的小鼠。IL-10/TNF-α在术后后期进行运动的小鼠中,中等强度运动为10.68,高强度运动为9.21,均大于正常运动小鼠：5.72。IL-2在术后后期进行运动的小鼠中,中等强度运动组淋巴细胞中IL-2为748.22±354.11 pg/ml,小于正常运动组小鼠：1257.49±279.85 pg/ml。IL-6, IFN-γ各组小鼠间差异无统计学意义。结论：1、术后后期进行中或高强度运动对小鼠脾淋巴细胞增殖能力有促进作用,尤其高强度运动的效应最为明显。2、术后休息后期进行中或高强度运动对模型小鼠T淋巴细胞表面CD69+, CD25+表达均有积极作用,强度与表达率成正比。3、从淋巴细胞中TNF-α, IL-10等细胞因子分泌差别可见术后后期进行中或高强度运动可能可以抑制肝原位移植瘤小鼠术后的复发进程。第三部分有氧运动对肝原位移植瘤小鼠术后淋巴细胞功能的影响机制目的：本研究重点是不同介入时间和强度运动对肝原位移植瘤术后小鼠淋巴细胞功能的影响机制。方法：制备模型小鼠脾淋巴细胞悬液,提取蛋白后采用Wersten blot方法检测JNK, PARP, Caspase3, Bax, Bcl-2等蛋白。结果：Western blot方法检测到,较术后正常运动小鼠磷酸化JNK表达,术后早期中等强度运动降低小鼠脾淋巴细胞磷酸化JNK的表达；较同时间段术后正常运动小鼠PARP的表达,术后早期中等强度运动升高小鼠淋巴细胞PARP的表达,后期高强度运动也对]PARP的升高有促进作用。较术后正常运动小鼠capase3的表达,术后早期大强度运动升高caspase3的表达。结论：运动可能通过抑制了线粒体介导的凋亡途径影响小鼠淋巴细胞凋亡,说明运动后有一定的抗凋亡效果。第四部分肝原位移植瘤小鼠术后运动模型代谢组学的研究目的：本研究重点是不同运动形式对荷瘤术后小鼠血清代谢谱变化的影响,旨在从代谢组学角度开展有氧运动对肝癌术后免疫功能恢复(康复)干预效果的观察,为肝癌术后患者开展运动康复指导做出有益探索,促进术后患者快速康复研究提供新思路和新途径。方法：制备移植性荷瘤小鼠术后模型,采用超高效液相色谱与质谱联用(UPLC-QTo F-/MS)研究平台利用偏最小二乘乘判别分析法比较术后不同运动量组和对照组代谢物谱差异。通过变量重要性指标和t检验选取潜在的生物标志物,结合标准品对照和数据库检索对潜在生物标志物进行分析和鉴定。结果：术后早期高中强度运动组与正常运动组代谢差异主要为能量代谢；术后晚期运动组与对照组小鼠相比,中强度运动组和高强度运动组小鼠代谢组学分析结果显示低强度运动组小鼠血清中LPE(溶血性磷脂酰乙醇胺),LPC(溶血性磷脂酰胆碱),Pyruvate(丙酮酸),Indoxyl (吲哚酚),Indoxyl sulfuric acid(吲哚硫酸),PE(16:0/0:0), PE(18:0/0:0)磷脂酰乙醇胺等代谢物质含量下降。高强度运动组小鼠血清中Serotonin(5-羟色胺),Tyramine(酪酸),Succinic acid(琥珀酸)含量升高,Indoxyl(吲哚酚),PE(18：0/0：0)(磷脂酰乙醇胺)含量下降。结论：有氧运动可能通过降低术后应激导致的磷脂酶活性异常升高,降低溶血性磷脂含量,从而抑制其引发的线粒体途径细胞凋亡。有氧运动干预可以在降低术后应激反应程度,改善小鼠能量代谢和情绪活动,提高小鼠运动和自主活动能力,增加进食量,改善肾功能等多方面促进肝癌术后小鼠的快速恢复,具有较高的临床借鉴意义,值得进一步完善和研究。
[Abstract]:Primary liver cancer is one of the most common malignant tumors in medical clinic, with high mortality and poor treatment effect. There is no effective and effective cure. Surgical resection is still one of the most effective methods for the treatment of primary liver cancer. Although the related treatment scheme has been made a great breakthrough in recent years, the curative effect is still not satisfactory. Therefore, for the great country of liver cancer, it is of great social significance and economic value to study the postoperative rehabilitation program to reduce the recurrence rate and improve the quality of life of the patients with liver cancer. Its progression, recurrence and metastasis are closely related, so finding ways and methods to improve the immune status of the patients with liver cancer is one of the research directions in the field of cancer rehabilitation. The method is used in the population. Modern medicine advocates sports therapy as one of the main treatments for cancer rehabilitation. It is clear that exercise therapy such as Taijiquan can improve the function of the immune system of the tumor patients. Although the exercise method has important status and undesirable role in the cancer recovery, it includes the latest edition of the United States. There is no clear and standardized exercise program to guide the postoperative rehabilitation of patients with liver cancer, especially in the time and intensity of intervention, and the lack of objective theoretical basis for the correlation between the movement and the immune state. In the second part, the effect of exercise intensity on the T cell immune function of mouse model was studied in the second part, and the third part was studied by Western blot. The effect mechanism of lymphocyte function in model mice; the fourth part uses metabonomics to evaluate the effect of exercise on liver orthotopic xenografts in mice, and provide new ideas for the next step. The effect of exercise on the postoperative state of mice. Methods: the mice were treated with different intensities (moderate intensity: 12m/min and high intensity exercise: 18m/min) and the intervention of different exercise time (early postoperative movement and postoperatively and postoperatively). Results: the postoperative recovery of mice was evaluated by biochemical and hepatic functions. Results: there was no significant difference in weight difference between the groups before and after exercise, and the effect of exercise start time on the degree of autonomic activity in mice was different, and P0.05 had statistical significance. The indexes of blood biochemistry and liver function showed that the model of moderate intensity movement after the post operation was small. The ALB in the rat serum was 32 + 2.53g/1, and the ALB in the serum of the model mice with high intensity after the post operation was 32.12 + 2.53 g/l, both were higher than that in the sera of the normal model mice after the operation: 26.44 + 4.10 g/l, and P0.05, the difference was statistically significant. The TP in the model mice with moderate intensity after the post operation was 57.83 + 2.71. G/l, the TP in the serum of the model mice with high intensity exercise after the operation was 52.62 + 4.37g/l, both were higher than that in the sera of the normal model mice after the post operation: 48.77 + 4.41g/l, and P0.05, the difference was statistically significant. Conclusion: after the post operation, the recovery of the whole state of the model mice was the best, the model was established successfully; The experimental study on the effect of the second part of aerobic exercise on the immune function of the liver in situ transplanted tumor in mice was to observe the effect of different intervention time and intensity on the immune function of the model mice. Methods: 1, the spleen lymphocyte of model mice was stimulated with A, and MTT The effect of different intervention methods on the proliferation of T lymphocyte in mice was detected by.2. The splenic lymphocyte suspension was prepared in the model mice, the magnetic beads were selected to separate the T lymphocyte, the flow cytometry was used to detect the CD69+, the expression rate of 25+ was.3. The splenic lymphocyte suspension was prepared in the model mice, the magnetic beads were selected to separate the T lymphocytes, and the ELISA method detected PMA plus Ion to induce T cell secretory IL-10. Results: 1, the effect of intervention time and intensity on lymphocyte proliferation in model mice: the lymphocyte proliferation ability of mice with moderate intensity movement in early post operation was (227.42 + 0.62)%, and the proliferation ability of mice with high intensity exercise early after operation was (18). 8.38 + 0.23)%, both of them were lower than those of normal exercise mice after operation, that is (348.03 + 0.89)%, but the lymphocyte proliferation ability of mice with moderate intensity after operation was (233.77 + 0.30)%, compared with the lymphocyte proliferation ability (235.03 + 0.89)% (235.03 + 0.89)% of normal exercise after the post operation, and the difference was not statistically significant. The proliferation ability of mice with high intensity exercise after operation was (329.12 + 0.43)%, which was better than that of normal exercise (235.03 + 0.89)% (235.03 + 0.89)%, but there was no difference between them. The effect of P0.05 on the proliferation of spleen lymphocyte in model mice after resection, the statistical difference was significant.2, the effect of intervention time and intensity on the T lymphocyte surface factor CD69+, CD25+ in model mice: the model mice of early movement after liver transplantation tumor resection, and the model mice T lymphocyte intervened by medium intensity treadmill exercise The expression rate of the surface CD69+ was (36.54 + 0.46)%. The expression rate of CD69+ expression on the T lymphocyte surface of the model mice was (38.72 + 0.20)%, and the CD69+ expression rate of the T lymphocyte surface was lower than that of the normal mice (53.38 + 0.25)%. In the mice carried out after the operation, the model mice under the high intensity exercise intervention were T drenched. The expression rate of CD69+ on the surface of the cell was (40.31 + 0.33)%. The expression rate of CD69+ expression on the surface of T lymphocyte on the model mice was (45.21 + 0.36)%, which was greater than that of the normal exercise model mice, that is (37.15 + 0.18)%, and P0.05, and the difference was statistically significant. In the early post operation model mice, the CD25+ expression rate of T lymphocyte on the T lymphocyte surface was (37.82 + 0.26)% and the CD25+ expression rate was (41.63 + 0.37)% in the model mice with high intensity treadmill exercise intervention, and the expression rate of T lymphocyte surface CD25+ was lower than that of normal mice (45.65 + 0.22). The CD25+ expression rate of T lymphocyte on the T lymphocyte of model mice under moderate intensity exercise was (42.72 + 0.29)%, and the expression rate of CD25+ expression on the T lymphocyte surface of the model mice under high intensity exercise intervention was (46.40 + 0.43)%, which was greater than the CD25+ expression rate of the T lymphocyte surface after normal exercise. That is (41.63 + 0.37)%, and P0.05, the difference has statistical significance.3, the effect of intervention time and intensity on the secretion of cytokine in the T lymphocyte of model mice: the effect of exercise intensity on the secretion of TNF-a in the T lymphocyte of mice after the resection of hepatoma after the operation, the effect of exercise intensity on the secretion of TNF-a is not statistically significant. In the later exercise mice, the moderate intensity exercise group and the high intensity exercise group were all significantly different from the normal exercise group, and the difference was statistically significant. The TNF-a of the specific T lymphocytes in each group was 188.74 + 96.10 pg/ml in the later stage of the middle intensity running stage, and the high intensity running platform after the post operation was carried out. The model mice of exercise were 205.93 + 110.04pg/ml, and there was no statistical difference in the intensity of 450.30 + 116.37 pg/ml. IL-10 in the normal exercise model mice after the postoperatively. But after the interaction analysis, the mice with postoperatively later movement were found to be less IL-10 than the early postoperatively,.IL-10/TNF- alpha in the later period of the operation. In the exercise mice, the moderate intensity exercise was 10.68 and the high intensity exercise was 9.21, which were all larger than the normal exercise mice. The IL-2 of the lymphocyte in the middle intensity exercise group was 748.22 + 354.11 pg/ml in the middle intensity exercise group, less than the normal exercise group: 1257.49 + 279.85 pg/ml.IL-6, and the difference between the IFN- gamma mice in each group. There is no statistical significance. Conclusion: 1, middle or high intensity exercise can promote the proliferation of spleen lymphocyte in postoperatively and later stage, especially the effect of high intensity exercise is most obvious.2. The expression of CD69 + and CD25+ on the surface of T lymphocyte in the model mice has positive effect on the expression of CD69 + and CD25+ on the surface of the model mice after the post operation. The rate is proportional to.3, the difference in the secretion of TNF- alpha, IL-10 and other cytokines in the lymphocyte can be seen in the middle or high intensity movement after the operation may inhibit the recurrence process of the hepatodisplace transplanted mice after operation. Third the mechanism of the effect of aerobic exercise on the lymphocyte function after the orthotopic liver transplantation in mice: the focus of this study is The mechanism of the influence of different intervention time and intensity exercise on the lymphocyte function of mice after orthotopic liver transplantation. Methods: to prepare the model mice splenic lymphocyte suspension and extract the protein by Wersten blot method to detect JNK, PARP, Caspase3, Bax, Bcl-2 and other proteins. Results: Western blot method was detected, compared with normal exercise mice after operation. The expression of phosphorylated JNK and early moderate intensity exercise decreased the expression of phosphorylated JNK in the spleen lymphocytes of mice after the operation, and the expression of PARP in normal mice after the same period of operation, the expression of PARP in the lymphocytes of mice at the early stage after the operation, and the high intensity exercise in the later period also promoted the increase of PARP, compared with the normal exercise after the operation. The expression of capase3 in mice and high intensity exercise increased Caspase3 expression in early post operation. Conclusion: exercise may affect the apoptosis of mouse lymphocyte by inhibiting the apoptosis pathway mediated by mitochondria, indicating the effect of anti apoptosis after exercise. Fourth This study focuses on the effect of different forms of exercise on the changes of serum metabolic profiles in mice after tumor bearing surgery. The purpose of this study is to observe the effect of aerobic exercise on the recovery of immune function after hepatoma (Kang Fu) from the metabonomics point of view. The purpose of this study is to make useful exploration for the guidance of sports Kang Fu in postoperative patients with liver cancer, and to promote the rapid Kang Fu study of postoperative patients. New ideas and new ways. Methods: to prepare the transplanted tumor bearing mice after the operation model, using the ultra high performance liquid chromatography and mass spectrometry (UPLC-QTo F-/MS) research platform using partial least squares multiplicative discriminant analysis to compare the difference of metabolite spectrum between the different exercise groups and the control group after the operation. Through the variable importance index and the t test, the potential students were selected. Results: the metabolic differences between the early senior high school intensity exercise group and the normal exercise group were mainly energy metabolism, and the postoperatively advanced sports group was compared with the control group, and the middle intensity exercise group and the high intensity exercise group were metabolomics. The results showed that the serum levels of LPE (hemolytic phosphatidylethanolamine), LPC (hemolytic phosphatidylcholine), Pyruvate (pyruvic acid), Indoxyl (Yin Duofen), Indoxyl sulfuric acid (indole sulfuric acid), PE (16:0/0:0), PE (18:0/0:0) phosphatidylethanolamine, etc. decreased in low intensity exercise mice. Tonin (5- HT), Tyramine (butyric acid), Succinic acid (succinic acid) content increased, Indoxyl (Yin Duofen), PE (18:0/0:0) (phosphatidyl b).

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7

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