冷冻消融联合TGF-β单抗治疗小鼠前列腺癌及对MDSCs影响的实验研究

发布时间：2018-05-18 13:11

本文选题：前列腺癌 + 冷冻消融　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的冷冻消融治疗中晚期前列腺癌疗效显著,可提高患者局部控制率及生活质量,已成为中晚期前列腺癌患者临床主要治疗手段,同时冷冻消融术后可诱导机体产生冷冻免疫反应,激活机体免疫系统,清除体内残余肿瘤细胞,有助于防止肿瘤的转移与复发。但是,冷冻免疫持续的时间及强度个体差异较大,同时体内也存在许多免疫负向调节的细胞因子及免疫细胞,不同程度地影响或制约冷冻免疫效果。如何有效控制负向免疫调节因素,最大化发挥冷冻免疫的效果成为该领域研究的热点。转化生长因子β(transforming growth factorβ,TGF-β)是一种经典的、在人体分布广泛的细胞因子,可抑制多种免疫细胞的增殖与分化,具有较强免疫抑制作用。同时,髓源性抑制细胞(Myeloid derived suppressor cells,MDSCs)为一种具有较强负性免疫调节作用的细胞,其抑制活性受到TGF-β在内的多种细胞因子调节。目前,许多基础及临床研究结果已经证明,TGF-β阻断可以延缓肿瘤的进展,本课题组前期研究亦发现冷冻消融后TGF-β水平发生变化,推测TGF-β可能参与冷冻免疫调节,阻断TGF-β可能有助于改善冷冻免疫效果,亦有可能引起部分免疫调节细胞(如MDSCs)的变化。据此,我们推断,TGF-β单抗阻断与冷冻消融联合治疗可能增强冷冻免疫强度,并影响体内MDSCs变化,进而影响预后。据此,本研究采用细胞实验及动物实验,探究冷冻消融联合TGF-β单抗阻断治疗对小鼠前列腺癌的疗效及MDSCs的变化,为提高冷冻消融疗效提供新的理论依据。材料和方法1.细胞实验:将RM-1细胞分为四组:A组为对照组(对照组)。B组为单纯冷冻消融组(冷冻组)。C组为单纯TGF-β单抗阻断(抗体组)。D组为冷冻消融联合TGF-β单抗阻断组(联合组)。治疗后用ELISA检测细胞上清液中TGF-β含量,同时用Transwell及MTT检验不同治疗对细胞功能学影响。2.动物实验:构建小鼠前列腺癌移植瘤模型,分组同细胞学实验。小鼠腹股沟成瘤后,根据分组分别进行不同治疗,每隔三天测量肿瘤体积大小。另外,术后7天及14天处死小鼠,收集外周血、脾脏、肺组织、肿瘤组织标本。HE染色观察肺转移情况,流式细胞术检测脾脏淋巴细胞中MDSCs、CD4~+T、CD8~+T细胞以及骨髓中MDSCs的变化情况。LDH杀伤试验检测CTL细胞杀伤能力改变情况。ELISA检测各组小鼠血清中TGF-β及IL-10的变化情况。免疫组化染色观察肿瘤组织局部CD8~+T细胞及MDSCs变化情况。为观察肿瘤局部侵袭及转移能力改变情况,蛋白免疫印迹法(Western bolt)检测不同治疗后上皮间质转化相关蛋白N-cadherin,E-cadherin,Vimentin表达情况。结果:1.RM-1细胞经过冷冻联合TGF-β单抗阻断治疗后,细胞的增殖、迁移及侵袭能力明显减弱。另外,与对照组比较冷冻组,抗体组以及联合组三组细胞上清TGF-β含量减少。2.随时间推移,各组小鼠肿瘤体积逐渐增大。治疗后7天及14天,冷冻组、抗体组及联合组肿瘤体积均明显小于对照组。同时,与冷冻组及抗体组比较,联合组减瘤效果最明显(P0.001)。另外,联合治疗组肺转移发生率以及肺转移灶数量明显小于其余三组。3.经过不同治疗后,与对照组比较,其余三组荷瘤小鼠血清TGF-β及IL-10含量呈明显降低趋势,差异具有统计学意义(P0.05)。4.随时间推移,对照组及抗体组小鼠脾脏MDSCs含量呈逐渐升高趋势,而冷冻组与联合组脾脏MDSCs含量逐渐下降。治疗后7天及14天:冷冻组、抗体组及联合组小鼠脾脏MDSCs含量均低于对照组,仅冷冻组及联合组与对照组比较差异具有统计学意义(P0.05)。5.随时间推移,对照组小鼠骨髓MDSCs呈逐渐上升趋势,冷冻组及联合组呈逐渐下降趋势,抗体组成先下降后上升趋势。另外,其余三组小鼠脾脏MDSCs含量明显小于对照组。同时,联合组骨髓MDSCs含量低于冷冻组及抗体组,但仅与抗体组比较具有统计学差异(P=0.001、P=0.630)。6.随时间推移,联合组肿瘤组织中MDSCs含量呈逐渐降低趋势。与其他组比较MDSCs含量明显减少,差异具有统计学意义(P0.05)。7.随时间推移,对照组小鼠脾脏中CD4~+T及CD8~+T细胞含量逐渐减少,而冷冻组、抗体组及联合组呈上升趋势;与其他三组比较,联合组小鼠脾脏中CD4~+T及CD8~+T细胞在7天、14天均显著增加(P0.05)。8.随时间推移,对照组CTL细胞杀伤活性呈下降趋势,而冷冻组、抗体组及联合组逐渐上升;与其他三组比较,联合组在7天、14天CTL细胞杀伤活性均显著增加(P0.05)。9.冷冻消融联合TGF-β单抗阻断治疗可以抑制RM-1细胞EMT的发生。Western bolt检测相关蛋白,联合组N-cadherin,Vimentin等间质标志物表达比较对照组明显减弱,而上皮标志E-cadherin表达增加。10.骨髓、脾脏以及肿瘤局部MDSCs数量与外周血TGF-β分泌水平呈正相关(P0.001)。同时,肿瘤局部MDSCs数量与浸润CD8~+T细胞数量呈负相关(P0.001),与外周血IL-10水平呈正相关(P0.001)。结论:1.体外细胞实验证明冷冻消融联合TGF-β单抗阻断可以明显减弱RM-1前列腺癌细胞增殖,迁移及侵袭能力。2.动物实验显示冷冻消融联合TGF-β单抗阻断可以明显减小荷瘤小鼠肿瘤负荷,减少肺转移发生率及肺转移数量,同时降低外周血TGF-β及IL-10含量。3.冷冻消融联合TGF-β单抗阻断后,荷瘤小鼠骨髓、脾脏及肿瘤组织中MDSCs含量明显减少,且与外周血TGF-β含量呈正相关。同时,联合治疗可使脾脏中CD4~+T及CD8~+T细胞以及肿瘤局部CD8~+T细胞含量明显升高,提高CTL细胞杀伤能力,增强机体免疫能力。
[Abstract]:Objective cryosurgery is effective in the treatment of advanced prostate cancer. It can improve the local control rate and the quality of life. It has become the main treatment method for the patients with advanced prostate cancer. The cryopreservation can induce the body to produce the cryopreservation immune response, activate the immune system and remove the residual tumor cells in the body. However, there are many differences in the duration and intensity of cryosurgery, and there are many negative immunomodulatory cytokines and immune cells in the body, which affect or restrict the effect of cryosurgery in varying degrees. How to effectively control the negative immune modulation factor and maximize the effect of cryosurgery Transforming growth factor beta (TGF- beta) is a classic, widely distributed cytokine, which inhibits the proliferation and differentiation of many immune cells and has a strong immunosuppressive effect. At the same time, myeloid suppressor cells (Myeloid derived suppressor cells, MDSCs) are a kind of immunosuppressive agents. The inhibitory activity of the cells with a strong negative immune regulation is regulated by a variety of cytokines, including TGF- beta. At present, many basic and clinical results have shown that TGF- beta blockage can delay the progression of the tumor. In our previous study, the changes in the level of TGF- beta after cryosurgery were also found. It is suggested that TGF- beta may be involved in the cold. Freezing immunoregulation, blocking TGF- beta may help to improve the effect of cryosurgery and may also cause changes in some immunoregulatory cells (such as MDSCs). Accordingly, we infer that the combined treatment of TGF- beta monoclonal antibody and cryosurgery may enhance the cryosurgery intensity and affect the changes in the body's MDSCs, and then affect the prognosis. Accordingly, this study adopted a detailed study. The effect of cryosurgery combined with TGF- beta McAb blocking therapy on the prostate cancer of mice and the changes of MDSCs were investigated in cell experiment and animal experiment. The 1. cell experiments of 1. cells were divided into four groups: the A group was the control group (the control group), the.B group was the pure cryosurgery group.C Group.D was the group of simple TGF- beta monoclonal antibody (antibody group) group.D and TGF- beta McAb blockage group (joint group). After treatment, the content of TGF- beta in the cell supernatant was detected by ELISA, and the effects of different treatments on the cell function of.2. animal experiment with different treatments were used to construct the mouse model of prostate cancer transplantation tumor, and the group was grouped with the cytology. After the mouse groin was tumorigenic, the tumor size was measured every three days, and the mice were killed every three days. In addition, the mice were killed at 7 and 14 days after the operation. The peripheral blood, spleen, lung tissue and tumor tissue were collected to observe the lung metastasis, and the flow cytometry was used to detect MDSCs, CD4~+T, CD8~+T cells and bone in the spleen lymphocytes. Change of MDSCs in the medullary,.LDH killing test was used to detect the change of CTL cell killing ability..ELISA was used to detect the changes of TGF- beta and IL-10 in the serum of each group. The changes of local CD8~+T cells and MDSCs in tumor tissues were observed by immunohistochemical staining. Tern bolt) detected the expression of epithelial mesenchymal transition related protein N-cadherin, E-cadherin, Vimentin after different treatments. Results: the proliferation, migration and invasion ability of 1.RM-1 cells were obviously weakened after freezing and TGF- beta monoclonal antibody blocking treatment. In addition, compared with the control group, the three groups of cell supernatant TGF were compared with the control group, the antibody group and the combined group. The tumor volume of mice in each group increased gradually as time went on. The volume of tumor in the frozen group, the antibody group and the combined group were significantly smaller than that of the control group 7 and 14 days after treatment. At the same time, the tumor reduction effect of the combined group and the antibody group was most obvious (P0.001). Besides, the incidence of lung metastasis and the number of lung metastases in the combined treatment group were also compared with the group of frozen group and antibody group (.2.). Compared with the other three groups of.3., the content of TGF- beta and IL-10 in the other three groups of tumor bearing mice was significantly lower than the control group. The difference was statistically significant (P0.05).4. with time, and the MDSCs content in the spleen of the control group and the antibody group increased gradually, while the freezing group and the combined group of spleen MDSCs were gradually increased. After 7 and 14 days after treatment, the content of MDSCs in the cryotherapy group, the antibody group and the combined group of mice was lower than the control group. The difference of the freezing group and the combined group with the control group was statistically significant (P0.05).5. with the time, the MDSCs of the bone marrow in the control group was gradually rising, and the freezing group and the combined group showed a gradual decline trend. In addition, the content of MDSCs in the spleen of the other three groups was significantly lower than that of the control group. At the same time, the content of bone marrow MDSCs in the combined group was lower than that of the cryopreservation group and the antibody group, but only with the antibody group, there was a statistical difference (P=0.001, P=0.630).6. with time, and the content of MDSCs in the tumor tissue of the combined group decreased gradually. Compared with the other groups, the MDSCs content was significantly reduced, the difference was statistically significant (P0.05).7. with time, and the content of CD4~+T and CD8~+T cells in the spleen of the mice in the control group decreased gradually, while the freezing group, the antibody group and the combined group showed an upward trend. Compared with the other three groups, the CD4~+T and CD8~+T cells in the spleen of the combined group were 7 days and 14 days. With the increase of (P0.05).8., the cytotoxicity of CTL cells in the control group decreased, while the freezing group, the antibody group and the combined group increased gradually. Compared with the other three groups, the combined group was significantly increased in the 7 day and 14 days of the CTL cell killing activity (P0.05.9. cryosurgery combined with TGF- beta monoclonal antibody blocking therapy can inhibit the EMT.West of RM-1 cells. " Ern bolt detection of related proteins, the expression of N-cadherin, Vimentin and other interstitial markers in the combined group was significantly lower than that in the control group, while the expression of E-cadherin in the epithelial markers increased.10. bone marrow, the number of MDSCs in the spleen and tumor was positively correlated with the level of TGF- beta in peripheral blood (P0.001). Meanwhile, the number of local MDSCs and the number of CD8~+T cells infiltrated in the tumor. Negative correlation (P0.001) was positively correlated with the level of peripheral blood IL-10 (P0.001). Conclusion: 1. in vitro cell test demonstrated that cryosurgery combined with TGF- beta monoclonal antibody could significantly weaken the proliferation of RM-1 prostate cancer cells, migration and invasion ability in.2. animal experiments showed that cryosurgery combined with TGF- beta monoclonal antibody could significantly reduce tumor bearing tumor bearing mice. The content of MDSCs in the bone marrow, spleen and tumor tissues of the tumor bearing mice decreased significantly after the reduction of TGF- beta and IL-10 content in peripheral blood.3. and the blockage of TGF- beta monoclonal antibody, and the content of TGF- beta in the peripheral blood was positively correlated with the content of TGF- beta in the peripheral blood. Meanwhile, combined treatment could make the spleen CD4~+T and CD8~+T cells in the spleen. The content of CD8~+T cells in local tumor increased significantly, which increased the killing ability of CTL cells and enhanced the immune function of the body.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.25

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