CD109在胰腺癌发生发展中的作用

发布时间：2018-03-22 16:35

本文选题：PRSS3　切入点：CD109　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景:胰腺癌因其发病隐匿而成为恶性程度最高的肿瘤之一,绝大多数的病人就诊时已经是局部侵袭或已发生转移,这就导致胰腺癌的平均存活期限是3~6个月,并且确诊胰腺癌后,与其他常见恶性肿瘤相比,其治疗方法有限。因此,寻找与胰腺癌相关的肿瘤标志物,为肿瘤治疗提供新的靶向,成为研究者们关注的热点。以往研究证实PRSS3的高表达与胰腺癌生存呈负相关,分泌到细胞外的PRSS3可裂解细胞膜蛋白CD109,但裂解产物在胰腺癌的发生发展中的作用机制尚未见报道。目的:本研究的目的是分析PRSS3裂解CD109蛋白的裂解位点及研究裂解产物在胰腺癌中的作用机制,寻找胰腺癌可能的治疗靶点,为胰腺癌靶向治疗奠定重要的分子理论基础。方法:用Western blotting方法检测8种人胰腺癌细胞系BxPC-3、PANC-1、Patu8988s、Miapaca-2、CFPAC-1、Capan-1、Suit-2、Patu8988t细胞中CD109蛋白表达水平;生物素标记的胰腺癌细胞Patu8988s与活性PRSS3孵育后,收集上清中脱落的tCD109片段,用nanoLC-MS/MS法分析裂解片段;根据CD109的裂解位点构建tCD109动物细胞分泌型表达载体pSec-tCD109;将pSec-tCD109转染到Patu8988t和Miapac-2细胞,采用MTT及Transwell方法观察tCD109对肿瘤细胞增殖、迁移和浸润的影响;应用免疫共沉淀分析tCD109与TGF-β受体的作用关系及其调节细胞生物学行为的信号通路。结果:1.在胰腺癌细胞中,BxPC-3、PANC-1、Patu8988s、Suit-2高表达CD109蛋白,而CFPAC-1、Capan-1、Miapaca-2、Patu8988t未检测出CD109蛋白表达或只检出低水平表达。2.在胰腺癌Patu8988s细胞中,PRSS3的表达能够裂解细胞膜表面CD109蛋白,裂解位点在R663-F664之间。3.蛋白裂解片段V22-R663即tCD109可促进胰腺癌细胞的增殖。MTT检测Patu8988t-tCD109组细胞数(1.967±0.056)明显高于Patu8988t-pcDNA3.1组(1.224±0.051)(p0.05)。同样,Miapac-2-tCD109组细胞数(2.266±0.027)也明显高于Miapac-2-pcDNA3.1组(1.409±0.06)(p0.05),4.tCD109具有促进胰腺癌细胞侵袭和转移能力。Transwell结果显示Patu8988t-tCD109穿过Matrigel及膜的数量(42.54±3.21)明显高于对照组(10.10±2.30)(p0.05)。5.tCD109与TGF-β1竞争性的结合TGF-β受体,从而抑制其下游信号传导。结论:1.PRSS3裂解CD109的产物,即功能活性片段tCD109可与TGF-β1竞争性的结合TGF-β受体,从而抑制其下游信号传导,促进胰腺癌细胞系的增殖、侵袭和转移能力。2.tCD109可能是胰腺癌靶向治疗的靶点,为胰腺癌靶向治疗奠定了重要的分子理论基础。
[Abstract]:Background: pancreatic cancer has become one of the most malignant tumors due to its occult incidence. Most of the patients had been locally invasive or metastasized at the time of visit, which resulted in the average survival period of pancreatic cancer being 3 ~ 6 months. After the diagnosis of pancreatic cancer, the treatment of pancreatic cancer is limited compared with other common malignancies. Therefore, the search for tumor markers associated with pancreatic cancer provides a new target for tumor therapy. Previous studies have shown that the high expression of PRSS3 is negatively related to the survival of pancreatic cancer. PRSS3 lytic cell membrane protein CD109 secreted into cells has not been reported. Objective: the purpose of this study was to analyze the cleavage site of PRSS3 CD109 protein and to study its cleavage. The mechanism of degradation products in pancreatic cancer, Methods: Western blotting method was used to detect the expression of CD109 protein in eight human pancreatic cancer cell lines BxPC-3PANC-1 and Patu8988sm. Biotin labeled pancreatic cancer cell line Patu8988s was incubated with active PRSS3. Then the tCD109 fragments were collected from the supernatant and analyzed by nanoLC-MS/MS method. The tCD109 animal cell secretory expression vector pSec-tCD109 was constructed according to the CD109 cleavage site. PSec-tCD109 was transfected into Patu8988t and Miapac-2 cells. MTT and Transwell were used to observe the effect of tCD109 on proliferation, migration and infiltration of tumor cells. The relationship between tCD109 and TGF- 尾 receptor and the signal pathway of regulating cell biological behavior were analyzed by immunoprecipitation. Results: 1. BxPC-3PANC-1 Patu8988s Suit-2 overexpressed CD109 protein in pancreatic cancer cells. However, the expression of CD109 protein was not detected or was only detected at a low level. In Patu8988s cells of pancreatic cancer, the expression of PRSS3 could break down the CD109 protein on the surface of the cell membrane. The number of cells in Patu8988t-tCD109 group was significantly higher than that in Patu8988t-pcDNA3.1 group (1.224 卤0.051). Similarly, the number of cells in Miapac-2-tCD109 group was 2.266 卤0.027) significantly higher than that in Miapac-2-pcDNA3.1 group (1.409 卤0.06p0.05p0.05t CD109). The ability of invasion and metastasis. Transwell results showed that the number of Patu8988t-tCD109 passing through Matrigel and membrane was 42.54 卤3.21), which was significantly higher than that in control group (10.10 卤2.30)(p0.05).5.tCD109 and TGF- 尾 1 competitive binding TGF- 尾 receptor). Conclusion: 1. The product of CD109, the functional active fragment tCD109, can competitively bind TGF- 尾 receptor with TGF- 尾 1, thus inhibiting its downstream signal transduction and promoting the proliferation of pancreatic cancer cell line. The ability of invasion and metastasis. 2. TCD109 may be the target of targeted therapy for pancreatic cancer, which provides an important molecular theoretical basis for targeted therapy of pancreatic cancer.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.9

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