抑制NLRP3炎性体联合CIK细胞治疗胰腺癌的实验研究

发布时间：2018-05-24 18:51

本文选题：胰腺癌 + 免疫治疗　；参考：《中国人民解放军医学院》2016年博士论文

【摘要】：研究背景：胰腺癌恶性程度高,预后极差,包括手术、化疗和放疗在内的肿瘤传统治疗手段在杀伤肿瘤的同时会激发机体炎症反应,并对抗肿瘤免疫造成一定程度的抑制和损伤。流行学和分子生物学证据显示胰腺癌的发生和发展与慢性炎症密切相关。NLRP3炎性体是一种由NLRP3蛋白、衔接蛋白ASC和Caspase-1前体构成的蛋白复合物,是调节炎症反应的重要平台,在机体受到刺激的情况下,NLRP3蛋白能够募集衔接蛋白ASC和Caspase-1前体,形成炎性体复合物,介导IL-1β和IL-18的成熟分泌,进而调控炎症反应。研究证实IL-18可增强胰腺癌细胞侵袭能力.而IL-18升高则与胰腺癌预后不佳相关。由此推断,NLRP3炎性体失调可能与胰腺癌的发生和发展相关,但目前尚无相关研究。免疫治疗已经成为肿瘤治疗的第四大手段,CIK细胞作为免疫治疗的重要部分,已经在临床治疗中得到应用,但其在杀伤肿瘤细胞的同时可以引起细胞因子大量释放,诱发细胞因子释放综合征。此外,免疫细胞的抗肿瘤免疫能力还受到NLRP3炎性体的抑制。因此,抑制NLRP3炎性体联合免疫细胞治疗,在抑制肿瘤相关炎性反应的同时重建并增强抗肿瘤免疫,可以实现协同抗肿瘤效果最大化。目的：研究NLRP3炎性体在胰腺癌中的表达情况及意义,建立免疫细胞的制备和检测技术平台,探讨NLRP3炎性体调控联合CIK细胞治疗胰腺癌的可行性和有效性。方法：1)利用免疫组化检测组织芯片中98例胰腺癌组织中NLRP3炎性体的表达,分析NLRP3炎性体各组分表达与胰腺癌患者临床病理因素的相关性,进行生存分析研究NLRP3炎性体与胰腺癌预后的相关性。2)建立CIK制备和检测技术平台,取健康志愿者外周血,分离PBMC,培养扩增为CIK细胞，流式细胞术检测PBMC和C1K细胞中活化T细胞和中央型记忆T细胞比例,并进行比较以评估CIK细胞的活化程度和抗肿瘤能力。3)采用Western Blot检测人胰腺癌细胞株SW1990和PANC-1中NLRP3炎性体的表达,并检测MNS对胰腺癌细胞株中NLRP3炎性体表达的影响。使用划痕试验、Transwell侵袭和迁移试验以及CCK-8法评估抑制NLRP3炎性体对胰腺癌细胞侵袭转移及增殖能力的影响。4)利用裸鼠左侧腋下皮内注射人胰腺癌细胞株SW1990,建立BLAB/c裸鼠胰腺癌皮下瘤模型，，将24只荷瘤裸鼠以每组6只的数量随机分为MNS(腹腔注射)组、CIK(尾静脉注射)组、MNS(腹腔注射)联合CIK(尾静脉注射)组以及对照组(溶媒)，以肿瘤体积、瘤重以及抑瘤率为指标观察各干预组对胰腺癌皮下瘤生长的影响。治疗2周后处死裸鼠,取皮下瘤进行HE和免疫组化检测，进一步分析抑制炎性体联合CIK细胞对胰腺癌的治疗效应及内在机制。结果：1)免疫组化结果显示NLRP3炎性体各组分在胰腺癌组织中表达水平较癌旁组织明显升高,且与胰腺癌临床分期以及淋巴结侵犯相关(P0.05),此外NLRP3炎性体各组分异常表达均与胰腺癌预后相关,其中高表达NLRP3的患者的中位生存时间(12个月)明显短于NLRP3低表达者(21个月,P0.01),高表达ASC(13个月 VS 18个月,P0.01)、高表达Caspase-1(13个月VS 17个月，P0.01)以及高表达IL-1β(13个月 VS 18个月,P0.01)也均与胰腺癌预后不佳有关。2)流式细胞术显示CIK细胞中CD3+CD8+T细胞和CD3+CD56+T细胞比例分别高达71.31%±18.77%和34.62%±15.67%。相比PBMC,培养成熟的CIK细胞中活化T细胞和中央型记忆T细胞比例显著升高(P0.05)。3)Western Blot检测显示人胰腺癌细胞株PANC-1和SW1990均表达NLRP3炎性体,此外MNS可以显著抑制胰腺癌细胞株中NLRP3炎性体的表达(P0.05)：Transwe11试验和划痕实验结果显示抑制NLRP3炎性体组分后,胰腺癌细胞侵袭和迁移能力显著降低(P0.05);CCK8实验结果显示抑制NLRP3炎性体能够抑制胰腺癌细胞株的增殖,对CIK细胞增殖则无明显影响。4)成功构建裸鼠胰腺癌皮下瘤模型,致瘤率100%,体内实验显示MNS腹腔注射联合CIK细胞尾静脉注射抗肿瘤效果最强,与对照组、CIK组和MNS组相比差异具有统计学意义(P0.05)。皮下瘤HE和免疫组化检测显示联合治疗组肿瘤呈现为片状坏死,且NLRP3炎性体表达明显下调。结论：1)胰腺癌组织和细胞株中高表达NLRP3炎性体。2)抑制NLRP3炎性体可以抑制胰腺癌的增殖和转移,但对免疫细胞无显著影响。3)抑制NLRP3炎性体联合CIK细胞治疗在在抑制肿瘤相关炎性反应的同时重建并增强抗肿瘤免疫,实现更强的联合抗肿瘤效果。
[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.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.9

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