本文選題：前列腺癌 + 冷凍消融��； 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的冷凍消融治療中晚期前列腺癌療效顯著,可提高患者局部控制率及生活質(zhì)量,已成為中晚期前列腺癌患者臨床主要治療手段,同時冷凍消融術(shù)后可誘導(dǎo)機(jī)體產(chǎn)生冷凍免疫反應(yīng),激活機(jī)體免疫系統(tǒng),清除體內(nèi)殘余腫瘤細(xì)胞,有助于防止腫瘤的轉(zhuǎn)移與復(fù)發(fā)。但是,冷凍免疫持續(xù)的時間及強(qiáng)度個體差異較大,同時體內(nèi)也存在許多免疫負(fù)向調(diào)節(jié)的細(xì)胞因子及免疫細(xì)胞,不同程度地影響或制約冷凍免疫效果。如何有效控制負(fù)向免疫調(diào)節(jié)因素,最大化發(fā)揮冷凍免疫的效果成為該領(lǐng)域研究的熱點。轉(zhuǎn)化生長因子β(transforming growth factorβ,TGF-β)是一種經(jīng)典的、在人體分布廣泛的細(xì)胞因子,可抑制多種免疫細(xì)胞的增殖與分化,具有較強(qiáng)免疫抑制作用。同時,髓源性抑制細(xì)胞(Myeloid derived suppressor cells,MDSCs)為一種具有較強(qiáng)負(fù)性免疫調(diào)節(jié)作用的細(xì)胞,其抑制活性受到TGF-β在內(nèi)的多種細(xì)胞因子調(diào)節(jié)。目前,許多基礎(chǔ)及臨床研究結(jié)果已經(jīng)證明,TGF-β阻斷可以延緩腫瘤的進(jìn)展,本課題組前期研究亦發(fā)現(xiàn)冷凍消融后TGF-β水平發(fā)生變化,推測TGF-β可能參與冷凍免疫調(diào)節(jié),阻斷TGF-β可能有助于改善冷凍免疫效果,亦有可能引起部分免疫調(diào)節(jié)細(xì)胞(如MDSCs)的變化。據(jù)此,我們推斷,TGF-β單抗阻斷與冷凍消融聯(lián)合治療可能增強(qiáng)冷凍免疫強(qiáng)度,并影響體內(nèi)MDSCs變化,進(jìn)而影響預(yù)后。據(jù)此,本研究采用細(xì)胞實驗及動物實驗,探究冷凍消融聯(lián)合TGF-β單抗阻斷治療對小鼠前列腺癌的療效及MDSCs的變化,為提高冷凍消融療效提供新的理論依據(jù)。材料和方法1.細(xì)胞實驗:將RM-1細(xì)胞分為四組:A組為對照組(對照組)。B組為單純冷凍消融組(冷凍組)。C組為單純TGF-β單抗阻斷(抗體組)。D組為冷凍消融聯(lián)合TGF-β單抗阻斷組(聯(lián)合組)。治療后用ELISA檢測細(xì)胞上清液中TGF-β含量,同時用Transwell及MTT檢驗不同治療對細(xì)胞功能學(xué)影響。2.動物實驗:構(gòu)建小鼠前列腺癌移植瘤模型,分組同細(xì)胞學(xué)實驗。小鼠腹股溝成瘤后,根據(jù)分組分別進(jìn)行不同治療,每隔三天測量腫瘤體積大小。另外,術(shù)后7天及14天處死小鼠,收集外周血、脾臟、肺組織、腫瘤組織標(biāo)本。HE染色觀察肺轉(zhuǎn)移情況,流式細(xì)胞術(shù)檢測脾臟淋巴細(xì)胞中MDSCs、CD4~+T、CD8~+T細(xì)胞以及骨髓中MDSCs的變化情況。LDH殺傷試驗檢測CTL細(xì)胞殺傷能力改變情況。ELISA檢測各組小鼠血清中TGF-β及IL-10的變化情況。免疫組化染色觀察腫瘤組織局部CD8~+T細(xì)胞及MDSCs變化情況。為觀察腫瘤局部侵襲及轉(zhuǎn)移能力改變情況,蛋白免疫印跡法(Western bolt)檢測不同治療后上皮間質(zhì)轉(zhuǎn)化相關(guān)蛋白N-cadherin,E-cadherin,Vimentin表達(dá)情況。結(jié)果:1.RM-1細(xì)胞經(jīng)過冷凍聯(lián)合TGF-β單抗阻斷治療后,細(xì)胞的增殖、遷移及侵襲能力明顯減弱。另外,與對照組比較冷凍組,抗體組以及聯(lián)合組三組細(xì)胞上清TGF-β含量減少。2.隨時間推移,各組小鼠腫瘤體積逐漸增大。治療后7天及14天,冷凍組、抗體組及聯(lián)合組腫瘤體積均明顯小于對照組。同時,與冷凍組及抗體組比較,聯(lián)合組減瘤效果最明顯(P0.001)。另外,聯(lián)合治療組肺轉(zhuǎn)移發(fā)生率以及肺轉(zhuǎn)移灶數(shù)量明顯小于其余三組。3.經(jīng)過不同治療后,與對照組比較,其余三組荷瘤小鼠血清TGF-β及IL-10含量呈明顯降低趨勢,差異具有統(tǒng)計學(xué)意義(P0.05)。4.隨時間推移,對照組及抗體組小鼠脾臟MDSCs含量呈逐漸升高趨勢,而冷凍組與聯(lián)合組脾臟MDSCs含量逐漸下降。治療后7天及14天:冷凍組、抗體組及聯(lián)合組小鼠脾臟MDSCs含量均低于對照組,僅冷凍組及聯(lián)合組與對照組比較差異具有統(tǒng)計學(xué)意義(P0.05)。5.隨時間推移,對照組小鼠骨髓MDSCs呈逐漸上升趨勢,冷凍組及聯(lián)合組呈逐漸下降趨勢,抗體組成先下降后上升趨勢。另外,其余三組小鼠脾臟MDSCs含量明顯小于對照組。同時,聯(lián)合組骨髓MDSCs含量低于冷凍組及抗體組,但僅與抗體組比較具有統(tǒng)計學(xué)差異(P=0.001、P=0.630)。6.隨時間推移,聯(lián)合組腫瘤組織中MDSCs含量呈逐漸降低趨勢。與其他組比較MDSCs含量明顯減少,差異具有統(tǒng)計學(xué)意義(P0.05)。7.隨時間推移,對照組小鼠脾臟中CD4~+T及CD8~+T細(xì)胞含量逐漸減少,而冷凍組、抗體組及聯(lián)合組呈上升趨勢;與其他三組比較,聯(lián)合組小鼠脾臟中CD4~+T及CD8~+T細(xì)胞在7天、14天均顯著增加(P0.05)。8.隨時間推移,對照組CTL細(xì)胞殺傷活性呈下降趨勢,而冷凍組、抗體組及聯(lián)合組逐漸上升;與其他三組比較,聯(lián)合組在7天、14天CTL細(xì)胞殺傷活性均顯著增加(P0.05)。9.冷凍消融聯(lián)合TGF-β單抗阻斷治療可以抑制RM-1細(xì)胞EMT的發(fā)生。Western bolt檢測相關(guān)蛋白,聯(lián)合組N-cadherin,Vimentin等間質(zhì)標(biāo)志物表達(dá)比較對照組明顯減弱,而上皮標(biāo)志E-cadherin表達(dá)增加。10.骨髓、脾臟以及腫瘤局部MDSCs數(shù)量與外周血TGF-β分泌水平呈正相關(guān)(P0.001)。同時,腫瘤局部MDSCs數(shù)量與浸潤C(jī)D8~+T細(xì)胞數(shù)量呈負(fù)相關(guān)(P0.001),與外周血IL-10水平呈正相關(guān)(P0.001)。結(jié)論:1.體外細(xì)胞實驗證明冷凍消融聯(lián)合TGF-β單抗阻斷可以明顯減弱RM-1前列腺癌細(xì)胞增殖,遷移及侵襲能力。2.動物實驗顯示冷凍消融聯(lián)合TGF-β單抗阻斷可以明顯減小荷瘤小鼠腫瘤負(fù)荷,減少肺轉(zhuǎn)移發(fā)生率及肺轉(zhuǎn)移數(shù)量,同時降低外周血TGF-β及IL-10含量。3.冷凍消融聯(lián)合TGF-β單抗阻斷后,荷瘤小鼠骨髓、脾臟及腫瘤組織中MDSCs含量明顯減少,且與外周血TGF-β含量呈正相關(guān)。同時,聯(lián)合治療可使脾臟中CD4~+T及CD8~+T細(xì)胞以及腫瘤局部CD8~+T細(xì)胞含量明顯升高,提高CTL細(xì)胞殺傷能力,增強(qiáng)機(jī)體免疫能力。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.25

【參考文獻(xiàn)】

相關(guān)會議論文 前1條

1 郭志;王海濤;邢文閣;劉方;李保國;于海鵬;李勇;;直腸超聲引導(dǎo)經(jīng)皮氬氦冷凍治療中晚期前列腺癌42例[A];第十五屆全國泌尿外科學(xué)術(shù)會議論文集[C];2008年

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