本文選題：胰腺癌 + 免疫治療　； 參考：《中國人民解放軍醫(yī)學院》2016年博士論文

【摘要】：研究背景：胰腺癌惡性程度高,預后極差,包括手術、化療和放療在內的腫瘤傳統(tǒng)治療手段在殺傷腫瘤的同時會激發(fā)機體炎癥反應,并對抗腫瘤免疫造成一定程度的抑制和損傷。流行學和分子生物學證據顯示胰腺癌的發(fā)生和發(fā)展與慢性炎癥密切相關。NLRP3炎性體是一種由NLRP3蛋白、銜接蛋白ASC和Caspase-1前體構成的蛋白復合物,是調節(jié)炎癥反應的重要平臺,在機體受到刺激的情況下,NLRP3蛋白能夠募集銜接蛋白ASC和Caspase-1前體,形成炎性體復合物,介導IL-1β和IL-18的成熟分泌,進而調控炎癥反應。研究證實IL-18可增強胰腺癌細胞侵襲能力.而IL-18升高則與胰腺癌預后不佳相關。由此推斷,NLRP3炎性體失調可能與胰腺癌的發(fā)生和發(fā)展相關,但目前尚無相關研究。免疫治療已經成為腫瘤治療的第四大手段,CIK細胞作為免疫治療的重要部分,已經在臨床治療中得到應用,但其在殺傷腫瘤細胞的同時可以引起細胞因子大量釋放,誘發(fā)細胞因子釋放綜合征。此外,免疫細胞的抗腫瘤免疫能力還受到NLRP3炎性體的抑制。因此,抑制NLRP3炎性體聯合免疫細胞治療,在抑制腫瘤相關炎性反應的同時重建并增強抗腫瘤免疫,可以實現協(xié)同抗腫瘤效果最大化。目的：研究NLRP3炎性體在胰腺癌中的表達情況及意義,建立免疫細胞的制備和檢測技術平臺,探討NLRP3炎性體調控聯合CIK細胞治療胰腺癌的可行性和有效性。方法：1)利用免疫組化檢測組織芯片中98例胰腺癌組織中NLRP3炎性體的表達,分析NLRP3炎性體各組分表達與胰腺癌患者臨床病理因素的相關性,進行生存分析研究NLRP3炎性體與胰腺癌預后的相關性。2)建立CIK制備和檢測技術平臺,取健康志愿者外周血,分離PBMC,培養(yǎng)擴增為CIK細胞，流式細胞術檢測PBMC和C1K細胞中活化T細胞和中央型記憶T細胞比例,并進行比較以評估CIK細胞的活化程度和抗腫瘤能力。3)采用Western Blot檢測人胰腺癌細胞株SW1990和PANC-1中NLRP3炎性體的表達,并檢測MNS對胰腺癌細胞株中NLRP3炎性體表達的影響。使用劃痕試驗、Transwell侵襲和遷移試驗以及CCK-8法評估抑制NLRP3炎性體對胰腺癌細胞侵襲轉移及增殖能力的影響。4)利用裸鼠左側腋下皮內注射人胰腺癌細胞株SW1990,建立BLAB/c裸鼠胰腺癌皮下瘤模型，，將24只荷瘤裸鼠以每組6只的數量隨機分為MNS(腹腔注射)組、CIK(尾靜脈注射)組、MNS(腹腔注射)聯合CIK(尾靜脈注射)組以及對照組(溶媒)，以腫瘤體積、瘤重以及抑瘤率為指標觀察各干預組對胰腺癌皮下瘤生長的影響。治療2周后處死裸鼠,取皮下瘤進行HE和免疫組化檢測，進一步分析抑制炎性體聯合CIK細胞對胰腺癌的治療效應及內在機制。結果：1)免疫組化結果顯示NLRP3炎性體各組分在胰腺癌組織中表達水平較癌旁組織明顯升高,且與胰腺癌臨床分期以及淋巴結侵犯相關(P0.05),此外NLRP3炎性體各組分異常表達均與胰腺癌預后相關,其中高表達NLRP3的患者的中位生存時間(12個月)明顯短于NLRP3低表達者(21個月,P0.01),高表達ASC(13個月 VS 18個月,P0.01)、高表達Caspase-1(13個月VS 17個月，P0.01)以及高表達IL-1β(13個月 VS 18個月,P0.01)也均與胰腺癌預后不佳有關。2)流式細胞術顯示CIK細胞中CD3+CD8+T細胞和CD3+CD56+T細胞比例分別高達71.31%±18.77%和34.62%±15.67%。相比PBMC,培養(yǎng)成熟的CIK細胞中活化T細胞和中央型記憶T細胞比例顯著升高(P0.05)。3)Western Blot檢測顯示人胰腺癌細胞株PANC-1和SW1990均表達NLRP3炎性體,此外MNS可以顯著抑制胰腺癌細胞株中NLRP3炎性體的表達(P0.05)：Transwe11試驗和劃痕實驗結果顯示抑制NLRP3炎性體組分后,胰腺癌細胞侵襲和遷移能力顯著降低(P0.05);CCK8實驗結果顯示抑制NLRP3炎性體能夠抑制胰腺癌細胞株的增殖,對CIK細胞增殖則無明顯影響。4)成功構建裸鼠胰腺癌皮下瘤模型,致瘤率100%,體內實驗顯示MNS腹腔注射聯合CIK細胞尾靜脈注射抗腫瘤效果最強,與對照組、CIK組和MNS組相比差異具有統(tǒng)計學意義(P0.05)。皮下瘤HE和免疫組化檢測顯示聯合治療組腫瘤呈現為片狀壞死,且NLRP3炎性體表達明顯下調。結論：1)胰腺癌組織和細胞株中高表達NLRP3炎性體。2)抑制NLRP3炎性體可以抑制胰腺癌的增殖和轉移,但對免疫細胞無顯著影響。3)抑制NLRP3炎性體聯合CIK細胞治療在在抑制腫瘤相關炎性反應的同時重建并增強抗腫瘤免疫,實現更強的聯合抗腫瘤效果。
[Abstract]:Background: pancreatic cancer has a high malignancy and a poor prognosis. Traditional treatments, including surgery, chemotherapy and radiotherapy, can stimulate the body's inflammatory response while killing the tumor, and cause a certain degree of inhibition and damage against tumor immunity. Epidemiology and molecular biological evidence show the occurrence and development and chronic of pancreatic cancer. Inflammation closely related.NLRP3 is a protein complex composed of NLRP3 protein, ASC and Caspase-1 precursor. It is an important platform for regulating inflammatory response. In the case of stimulation of the body, NLRP3 protein can collect ASC and Caspase-1 precursors, form inflammatory complex, mediate the maturity of IL-1 beta and IL-18. IL-18 can enhance the invasiveness of pancreatic cancer cells. The increase of IL-18 is associated with poor prognosis of pancreatic cancer. It is concluded that the dysregulation of NLRP3 may be associated with the occurrence and development of pancreatic cancer, but there is no related research. Immunotherapy has become the fourth major means of cancer treatment, CIK thin. As an important part of immunotherapy, cell has been used in clinical treatment, but it can cause cytokine release and induce cytokine release syndrome at the same time of killing tumor cells. In addition, the anti-tumor immunity ability of immune cells is also inhibited by NLRP3 inflammatory body. Therefore, inhibition of NLRP3 inflammatory body combined with immunization. Cell therapy, while reconstructing and enhancing anti-tumor immunity, can maximize the synergistic anti-tumor effect. Objective: To study the expression and significance of NLRP3 inflammatory body in pancreatic cancer, to establish an immune cell preparation and detection technology platform, and to explore the regulation of NLRP3 inflammatory body combined with CIK cells for the treatment of the pancreas The feasibility and effectiveness of cancer. Methods: 1) the expression of NLRP3 inflammatory body in 98 cases of pancreatic cancer tissues was detected by immunohistochemistry. The correlation between the expression of NLRP3 inflammatory components and the clinicopathological factors of pancreatic cancer patients was analyzed, and the survival analysis was carried out to study the correlation between the NLRP3 inflammatory body and the prognosis of pancreatic cancer. The CIK preparation and the establishment of CIK were established. The testing technology platform was used to take the peripheral blood of healthy volunteers, isolate PBMC and develop CIK cells. Flow cytometry was used to detect the proportion of activated T cells and central memory T cells in PBMC and C1K cells, and to compare the activation degree of CIK cells and the ability to resist tumor.3). Western Blot was used to detect the SW1990 and PANC-1 of human pancreatic cancer cell lines. The expression of NLRP3 inflammatory body and the effect of MNS on the expression of NLRP3 inflammatory body in pancreatic cancer cell lines. Using scratch test, Transwell invasion and migration test, and CCK-8 method to evaluate the effect of NLRP3 on the invasion, metastasis and proliferation of pancreatic cancer cells,.4) by injecting human pancreatic cancer cell line SW1 in the left subaxillary skin of nude mice,.4. 990, a model of subcutaneous tumor of BLAB/c nude mice was established, and 24 tumor bearing nude mice were randomly divided into MNS (intraperitoneal injection) group, CIK (tail vein injection) group, MNS (intraperitoneal injection) combined with CIK (caudal vein injection) group and control group (dissolvent). The tumor volume, tumor weight and tumor suppressor rate were used to observe the subcutaneous of pancreatic cancer in the intervention groups. The effect of tumor growth. After 2 weeks of treatment, the nude mice were killed and the subcutaneous tumor was examined by HE and immunohistochemistry. The therapeutic effect and internal mechanism of the inhibition of inflammatory body combined with CIK cells to pancreatic cancer were further analyzed. Results: 1) the immunohistochemical results showed that the expression level of NLRP3 inflammatory components in the pancreatic cancer group was significantly higher than that in the paracancerous tissue. The clinical staging of pancreatic cancer and lymph node invasion (P0.05) were associated with the prognosis of pancreatic cancer. The median survival time (12 months) of the patients with high expression of NLRP3 (21 months, P0.01), high expression of ASC (13 months VS, 18 months, P0.01), and high expression of Caspase-1 (13) were all associated with the prognosis of pancreatic cancer. VS 17 months, P0.01) and high expression of IL-1 beta (13 months VS and 18 months, P0.01) were also associated with poor prognosis of pancreatic cancer.2) flow cytometry showed that the proportion of CD3+CD8+T cells and CD3+CD56+T cells in CIK cells was up to 71.31% + 18.77% and 34.62% + 15.67%. compared to PBMC. A significant increase in cell proportion (P0.05).3) Western Blot detection showed that human pancreatic cancer cell lines, PANC-1 and SW1990, expressed NLRP3 inflammatory bodies. In addition, MNS could significantly inhibit the expression of NLRP3 inflammatory bodies in pancreatic cancer cell lines (P0.05):Transwe11 test and scratch test results showed invasion and migration of pancreatic cancer cells after inhibition of the NLRP3 inflammatory components. The results showed a significant decrease in ability (P0.05); the results of CCK8 test showed that inhibition of NLRP3 inflammation could inhibit the proliferation of pancreatic cancer cell lines, and had no obvious influence on the proliferation of CIK cells. The model of subcutaneous tumor of pancreatic cancer in nude mice was successfully constructed, the tumor rate was 100%. In vivo experiments showed that MNS intraperitoneal injection of CIK cells combined with CIK cells had the strongest anti tumor effect, and compared with the control. The difference between group CIK and group MNS was statistically significant (P0.05). Subcutaneous tumor HE and immunohistochemistry showed that the tumor in the combined treatment group was flaky necrosis and the expression of NLRP3 inflammatory body was obviously down-regulated. Conclusion: 1) the high expression of NLRP3 inflammatory.2 in pancreatic cancer tissues and cell lines can inhibit the proliferation of pancreatic cancer and inhibit the proliferation of pancreatic cancer. Metastasis, but there is no significant effect on immune cells,.3) inhibition of NLRP3 inflammatory body combined with CIK cell therapy in the suppression of tumor related inflammatory responses while reconstructing and enhancing anti-tumor immunity to achieve a stronger combined antitumor effect.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.9

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