發(fā)布時間：2018-05-04 17:24

  本文選題：肝原位移植瘤 + 術后��； 參考：《第二軍醫(yī)大學》2015年博士論文

【摘要】：原發(fā)性肝癌是醫(yī)學臨床常見的惡性腫瘤之一,病死率高且治療效果欠佳,尚無確切有效的治愈措施。手術切除仍是目前其治療的最有效手段之一,雖然相關的術后治療方案的研究近年來獲得很大的突破,然而其療效依然不能令人滿意。因此,對于肝癌大國,研究降低肝癌患者術后復發(fā)率和提高生存質(zhì)量的術后康復方案有著巨大的社會意義和經(jīng)濟價值�，F(xiàn)代醫(yī)學認為術后復發(fā)與患者免疫功能有著高度相關性,已初步證實圍手術期機體免疫狀態(tài)及術后細胞免疫功能與其病情進展、復發(fā)、轉(zhuǎn)移密切相關,所以尋找能夠改善肝癌患者術后免疫狀態(tài)的途徑和方法是當前肝癌腫瘤康復領域的研究方向之一。中醫(yī)自古提倡運動,針對腫瘤患者,更體現(xiàn)在“既病防變”和“愈后防復”,有太極拳、八段錦等運動療法應用于該人群�，F(xiàn)代醫(yī)學推崇運動療法作為腫瘤康復的主要治療手段之一,明確了證實太極拳等運動療法可提高腫瘤患者機體免疫系統(tǒng)的功能。雖然運動方法在腫瘤康復具有重要的地位和不可取代的作用,但是,包括美國最新版的《腫瘤幸存者運動鍛煉指南》在內(nèi),目前尚無一套明確且規(guī)范的運動方案用于指導肝癌患者術后的康復,尤其表現(xiàn)在介入運動的時機和強度,缺乏該階段運動與免疫狀態(tài)相關的客觀理論依據(jù)�；纤�,本研究第一部分擬構(gòu)建肝原位移植瘤小鼠術后運動模型,探索合理的運動介入時機,并觀察不同運動強度對小鼠模型代謝產(chǎn)物的影響；第二部分以脾臟T淋巴細胞為主要研究對象,探討運動強度對小鼠模型T細胞免疫功能的影響；第三部分通過蛋白質(zhì)印跡法研究運動對模型小鼠淋巴細胞功能的影響機制；第四部分運用代謝組學方法評估運動對肝原位移植瘤小鼠術后的影響,為下一步研究提供新思路。第一部分肝原位移植瘤小鼠術后運動模型的構(gòu)建目的：全面探索肝原位移植瘤小鼠切除術后的狀態(tài),觀察運動對小鼠術后狀態(tài)的影響。方法：對肝原位移植瘤切除術后小鼠進行不同強度(中等強度：12m/min和高強度運動：18m/min)和不同運動時間(術后早期運動和術后后期運動)的干預,建立術后小鼠運動模型；從體重、自主活動度、血生化和肝功能等基礎指標評價小鼠術后康復情況。結(jié)果：運動前后組間體重差別并無統(tǒng)計學意義；運動開始時間對小鼠自主活動度的影響具有差異,p0.05具有統(tǒng)計學意義；血生化和肝功能等指標,可見術后后期中等強度運動的模型小鼠血清中ALB為32.00±2.53g/1,術后后期高強度運動的模型小鼠血清中ALB為32.12±2.53 g/l,兩者均高于術后后期正常運動模型小鼠血清中含量：26.44±4.10 g/l,且p0.05,差異具有統(tǒng)計學意義。術后后期中等強度運動的模型小鼠血清中TP為57.83±2.71 g/l,術后后期高強度運動的模型小鼠血清中TP為52.62±4.37g/l,兩者均高于術后后期正常運動模型小鼠血清中含量：48.77±4.41g/l,且p0.05,差異具有統(tǒng)計學意義。結(jié)論：術后后期運動對模型小鼠整體狀態(tài)的恢復效果最佳,模型建立成功；運動強度與肝功能等指標呈反比趨勢。第二部分有氧運動對肝原位移植瘤小鼠術后免疫功能影響的實驗研究目的：觀察不同介入時間和強度的運動對模型小鼠免疫功能的影響。方法：1、用刀豆蛋白A對模型小鼠脾淋巴細胞進行刺激,用MTT法檢測不同干預方法對小鼠T淋巴細胞增殖的影響。2、制備模型小鼠脾淋巴細胞懸液,磁珠分選T淋巴細胞,流式細胞儀檢測CD69+、25+的表達率。3、制備模型小鼠脾淋巴細胞懸液,磁珠分選T淋巴細胞,ELISA法檢測PMA加Ion誘導T細胞分泌IL-10, TNF-a等分泌。結(jié)果：1、介入時間與強度對模型小鼠淋巴細胞增殖的影響：對于肝原位移植瘤切除術后的模型小鼠,術后早期開始中等強度運動的小鼠淋巴細胞增殖能力為(227.42±0.62)%,術后早期開始高強度運動的小鼠淋巴細胞增殖能力為(188.38±0.23)%,兩者均低于術后正常運動小鼠,即(348.03±0.89)%。但術后后期進行中等強度運動的小鼠淋巴細胞增殖能力為(233.77±0.30)%,與術后后期進行正常運動的小鼠淋巴細胞增殖能力(235.03±0.89)%相比,兩者數(shù)值接近,且兩者差異無統(tǒng)計學意義。術后后期進行高強度運動的小鼠淋巴細胞增殖能力為(329.12±0.43)%,優(yōu)于術后后期進行正常運動的小鼠淋巴細胞增殖能力(235.03±0.89)%,但兩者間差異無統(tǒng)計學意義。交互分析后顯示運動開始時間和運動強度相結(jié)合對肝原位移植瘤切除術后模型小鼠脾淋巴細胞增殖能力的影響p0.05,統(tǒng)計學差異有意義。2、介入時間與強度對模型小鼠T淋巴細胞表面因子CD69+、CD25+的影響：對于肝原位移植瘤切除術后早期運動的模型小鼠,中等強度跑臺運動干預的模型小鼠T淋巴細胞表面CD69+表達率為(36.54±0.46)%,高強度跑臺運動干預的模型小鼠T淋巴細胞表面CD69+表達率為(38.72±0.20)%,兩者T淋巴細胞表面CD69+表達率均低于正常運動小鼠的(53.38±0.25)%。在術后后期進行運動的小鼠中,高強度運動干預下的模型小鼠T淋巴細胞表面CD69+表達率為(40.31±0.33)%,中等強度運動干預下的模型小鼠T淋巴細胞表面CD69+表達率為(45.21±0.36)%,均大于正常運動后的模型小鼠T淋巴細胞表面CD69+表達率,即(37.15±0.18)%,且p0.05,差異具有統(tǒng)計學意義。對于肝原位移植瘤切除術后早期的模型小鼠,中等強度跑臺運動干預的模型小鼠T淋巴細胞表面CD25+表達率為(37.82±0.26)%,高強度跑臺運動干預的模型小鼠T淋巴細胞表面CD25+表達率為(41.63±0.37)%,兩者T淋巴細胞表面CD25+表達率均低于正常運動小鼠的(45.65±0.22)%。在術后后期進行運動的小鼠中,中等強度運動干預下的模型小鼠T淋巴細胞表面CD25+表達率為(42.72±0.29)%,高強度運動干預下的模型小鼠T淋巴細胞表面CD25+表達率為(46.40±0.43)%,均大于正常運動后的模型小鼠T淋巴細胞表面CD25+表達率,即(41.63±0.37)%,且p0.05,差異具有統(tǒng)計學意義。3、介入時間與強度對模型小鼠T淋巴細胞分泌細胞因子的影響：對于肝原位移植瘤切除術后的模型小鼠,術后早期進行運動的小鼠,運動強度對其T淋巴細胞分泌TNF-a的影響無統(tǒng)計學差異。術后后期運動的小鼠,中等強度運動組與高強度運動組均與正常運動組具有較大差異,且p0.05,差異具有統(tǒng)計學意義。各組具體T淋巴細胞中TNF-a分別為：術后后期進行中等強度跑臺運動的模型小鼠為188.74±96.10 pg/ml,術后后期進行高強度跑臺運動的模型小鼠為205.93±110.04pg/ml,術后后期進行正常運動的模型小鼠為450.30±116.37 pg/ml. IL-10在強度上差別沒有統(tǒng)計學意義,但進行交互分析后發(fā)現(xiàn)術后后期運動的小鼠,IL-10分泌量低于術后早期運動的小鼠。IL-10/TNF-α在術后后期進行運動的小鼠中,中等強度運動為10.68,高強度運動為9.21,均大于正常運動小鼠：5.72。IL-2在術后后期進行運動的小鼠中,中等強度運動組淋巴細胞中IL-2為748.22±354.11 pg/ml,小于正常運動組小鼠：1257.49±279.85 pg/ml。IL-6, IFN-γ各組小鼠間差異無統(tǒng)計學意義。結(jié)論：1、術后后期進行中或高強度運動對小鼠脾淋巴細胞增殖能力有促進作用,尤其高強度運動的效應最為明顯。2、術后休息后期進行中或高強度運動對模型小鼠T淋巴細胞表面CD69+, CD25+表達均有積極作用,強度與表達率成正比。3、從淋巴細胞中TNF-α, IL-10等細胞因子分泌差別可見術后后期進行中或高強度運動可能可以抑制肝原位移植瘤小鼠術后的復發(fā)進程。第三部分有氧運動對肝原位移植瘤小鼠術后淋巴細胞功能的影響機制目的：本研究重點是不同介入時間和強度運動對肝原位移植瘤術后小鼠淋巴細胞功能的影響機制。方法：制備模型小鼠脾淋巴細胞懸液,提取蛋白后采用Wersten blot方法檢測JNK, PARP, Caspase3, Bax, Bcl-2等蛋白。結(jié)果：Western blot方法檢測到,較術后正常運動小鼠磷酸化JNK表達,術后早期中等強度運動降低小鼠脾淋巴細胞磷酸化JNK的表達；較同時間段術后正常運動小鼠PARP的表達,術后早期中等強度運動升高小鼠淋巴細胞PARP的表達,后期高強度運動也對]PARP的升高有促進作用。較術后正常運動小鼠capase3的表達,術后早期大強度運動升高caspase3的表達。結(jié)論：運動可能通過抑制了線粒體介導的凋亡途徑影響小鼠淋巴細胞凋亡,說明運動后有一定的抗凋亡效果。第四部分肝原位移植瘤小鼠術后運動模型代謝組學的研究目的：本研究重點是不同運動形式對荷瘤術后小鼠血清代謝譜變化的影響,旨在從代謝組學角度開展有氧運動對肝癌術后免疫功能恢復(康復)干預效果的觀察,為肝癌術后患者開展運動康復指導做出有益探索,促進術后患者快速康復研究提供新思路和新途徑。方法：制備移植性荷瘤小鼠術后模型,采用超高效液相色譜與質(zhì)譜聯(lián)用(UPLC-QTo F-/MS)研究平臺利用偏最小二乘乘判別分析法比較術后不同運動量組和對照組代謝物譜差異。通過變量重要性指標和t檢驗選取潛在的生物標志物,結(jié)合標準品對照和數(shù)據(jù)庫檢索對潛在生物標志物進行分析和鑒定。結(jié)果：術后早期高中強度運動組與正常運動組代謝差異主要為能量代謝；術后晚期運動組與對照組小鼠相比,中強度運動組和高強度運動組小鼠代謝組學分析結(jié)果顯示低強度運動組小鼠血清中LPE(溶血性磷脂酰乙醇胺),LPC(溶血性磷脂酰膽堿),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)磷脂酰乙醇胺等代謝物質(zhì)含量下降。高強度運動組小鼠血清中Serotonin(5-羥色胺),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。結(jié)論：有氧運動可能通過降低術后應激導致的磷脂酶活性異常升高,降低溶血性磷脂含量,從而抑制其引發(fā)的線粒體途徑細胞凋亡。有氧運動干預可以在降低術后應激反應程度,改善小鼠能量代謝和情緒活動,提高小鼠運動和自主活動能力,增加進食量,改善腎功能等多方面促進肝癌術后小鼠的快速恢復,具有較高的臨床借鑒意義,值得進一步完善和研究。
[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).

【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R735.7

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