【作者】 王敏；

【导师】 韩春杨； 刘善斋；

【作者基本信息】 安徽农业大学 ， 兽医硕士（专业学位）， 2024， 硕士

【摘要】 兽医针灸疗法具有治病广泛、疗效迅速、操作安全、便于推广等特点,在临床应用中有许多独到之处,越来越受到国内外兽医工作者的重视。对于癌症患者来说,针灸被广泛用作辅助疗法,增强免疫功能,减少放化疗的副作用。然而,在针灸调节肿瘤免疫微环境方面的研究较少。本研究探究针刺对荷瘤小鼠肿瘤免疫微环境的影响,并为针刺在临床上辅助抗肿瘤药物治疗肿瘤疾病提供试验依据。方法:试验选取6周龄无特定病原体(SPF)级雄性C57BL/6小鼠10只,建立小鼠B16F10肿瘤模型,将其随机分为2组(n=5),即对照组和针刺组。针刺组小鼠每天白针针刺大椎、膈俞、肾俞和足三里穴,留针6 min,连续7 d。对照组同针刺组每天同步抓取、固定和假针刺。观察各组小鼠的生长情况,试验结束后采集小鼠脾脏、引流淋巴结和肿瘤组织。采用流式细胞术检测肿瘤、脾脏以及引流淋巴结(Tumor draining lymph node,TDLN)中淋巴细胞亚群及其表面的程序性死亡蛋白1(Programmed cell death 1,PD-1)、T细胞免疫球蛋白粘蛋白3(T cell immunoglobulin mucin 3,TIM3)等表达水平。选取10只雄性6周龄SPF级C57BL/6小鼠,小鼠MC38肿瘤模型建立成功后将其随机分为2组(n=5)即对照组和针刺组。针刺持续14 d,其余操作步骤和检测方法同B16F10小鼠组。结果:B16F10组小鼠和MC38组小鼠肿瘤生长变化在组间无显著性(P>0.05),在治疗结束后采集小鼠瘤重变化在组间无显著性(P>0.05)。流式细胞术检测B16F10荷瘤小鼠针刺组较对照组肿瘤浸润细胞(Tumor-infiltrating lymphocytes,TILs)中CD8~+T细胞表面的PD-1表达水平极显著降低(P<0.01),PD-1 TIM3共表达水平显著降低(P<0.05),Tex(Tim3~+TCF1~-)表达水平显著降低(P<0.05);肿瘤浸润细胞中NK细胞表面的PD-1表达水平显著降低(P<0.05);脾脏中的CD8~+T百分比极显著升高(P<0.001)。MC38荷瘤针刺组小鼠较对照组肿瘤浸润细胞中NK细胞中的肿瘤坏死因子α(Tumor necrosis factor-α,TNF-α)表达显著升高(P<0.05),NKT细胞中的Tex表达水平极显著降低(P<0.001),CD8~+T细胞表面的PD-1表达水平极显著降低(P<0.001),PD-1 TIM3共表达水平极显著降低(P<0.001);引流淋巴结中的颗粒酶B(Granzyme B,GZMB)和Tpex表达水平分别是显著升高(P<0.05)和极显著升高(P<0.01)。结论:针刺大椎、膈俞、肾俞和足三里穴可以降低肿瘤浸润细胞中CD8~+T细胞表面的PD-1、PD-1 TIM3共表达水平,改善荷瘤小鼠肿瘤免疫微环境。更多还原

【Abstract】 Veterinary acupuncture and moxibustion therapy has the characteristics of wide treatment,rapid curative effect,safe operation and easy popularization.It has many unique features in clinical application,and has been paid more and more attention by veterinarians at home and abroad.For cancer patients,acupuncture is widely used as an complementary therapy to boost the immune system and reduce the side effects of chemotherapy and radiotherapy.However,there is less research on acupuncture regulation of the immune microenvironment in tumor.This research investigated the impact of acupuncture on the tumor immune microenvironment in tumor-bearing mice,and it provided an experimental basis for the use of acupuncture as an adjunct to anti-tumor drugs in the clinical treatment of tumor.Methods:Ten male C57BL/6 mice without specific pathogen（SPF）grade aged 6 to 8 weeks were selected to establish the B16F10 tumor model,and were randomly divided into2 groups namely control group and acupuncture group（n=5）.In the acupuncture group,the mice were acupunctured at Dazhui,Geshu,Shenshu and Zusanli acupoints once a day for a week,and the needles were retained for 6 minutes.The control group mice was grasped and restrained synchronously with the acupuncture group mice each day.The control group was treated with mimetic acupuncture with shallow needle.The tumor growth of mice in each group was observed.Spleen,lymph nodes and tumor tissues were collected after the experiment.Flow cytometry was used to detect lymphocyte subpopulation and its expression levels of PD-1 and TIM3 in tumor,spleen and tumor draining lymph nodes.Ten male 6-8 week-old C57BL/6 mice SPF grade were selected and randomly divided into2 groups,namely control group and acupuncture group（n=5）,after the successful establishment of the MC38 tumor model.The remaining procedures and tests were the same as in the B16F10 mouse group.Results:There was no significant change in tumor growth in B16F10 group and MC38group,and no significant change in tumor weight after treatment.Flow cytometry detected that B16F10 tumour-bearing mice expressed the level of PD-1 on the surface of CD8⁺T cells in tumor infiltrating cells in the acupuncture group was highly significantly lower than that it expressed in the control group（P<0.01）.The coexpression level of PD-1TIM3 was significantly decreased（P<0.05）,and the expression level of Tex(Tim3⁺TCF1^-)was significantly decreased（P<0.05）.The expression level of PD-1 on the surface of NK cells in tumor infiltrating cells was significantly decreased（P<0.05）.The percentage of CD8⁺T in spleen was highly significantly increased（P<0.001）.In MC38 tumor-bearing mice,compared with the control group,the tumor cells expression of TNF-αin NK cells was significantly increased（P<0.05）and Tex level in NKT cells was highly significantly decreased（P<0.001）and the expression level of PD-1 and coexpression level of PD-1TIM3 on the surface of CD8⁺T cells were highly significantly decreased（P<0.001）.The expression levels of GZMB and Tpex in the lymph nodes were respectively significantly increased（P<0.05）and highly significantly increased（P<0.01）.Conclusion:Acupuncture at Dazhui,Geshu,Shenshu and Zusanli acupoints can reduce the level of PD-1 and PD-1⁺Tim3⁺on the surface of CD8⁺T cells in tumor infiltrating cells,and benefit the tumor immune microenvironment of tumour-bearing mice.更多还原