本文選題：PRSS3　切入點：CD109　出處：《鄭州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景:胰腺癌因其發(fā)病隱匿而成為惡性程度最高的腫瘤之一,絕大多數(shù)的病人就診時已經(jīng)是局部侵襲或已發(fā)生轉(zhuǎn)移,這就導(dǎo)致胰腺癌的平均存活期限是3~6個月,并且確診胰腺癌后,與其他常見惡性腫瘤相比,其治療方法有限。因此,尋找與胰腺癌相關(guān)的腫瘤標(biāo)志物,為腫瘤治療提供新的靶向,成為研究者們關(guān)注的熱點。以往研究證實PRSS3的高表達(dá)與胰腺癌生存呈負(fù)相關(guān),分泌到細(xì)胞外的PRSS3可裂解細(xì)胞膜蛋白CD109,但裂解產(chǎn)物在胰腺癌的發(fā)生發(fā)展中的作用機(jī)制尚未見報道。目的:本研究的目的是分析PRSS3裂解CD109蛋白的裂解位點及研究裂解產(chǎn)物在胰腺癌中的作用機(jī)制,尋找胰腺癌可能的治療靶點,為胰腺癌靶向治療奠定重要的分子理論基礎(chǔ)。方法:用Western blotting方法檢測8種人胰腺癌細(xì)胞系BxPC-3、PANC-1、Patu8988s、Miapaca-2、CFPAC-1、Capan-1、Suit-2、Patu8988t細(xì)胞中CD109蛋白表達(dá)水平;生物素標(biāo)記的胰腺癌細(xì)胞Patu8988s與活性PRSS3孵育后,收集上清中脫落的tCD109片段,用nanoLC-MS/MS法分析裂解片段;根據(jù)CD109的裂解位點構(gòu)建tCD109動物細(xì)胞分泌型表達(dá)載體pSec-tCD109;將pSec-tCD109轉(zhuǎn)染到Patu8988t和Miapac-2細(xì)胞,采用MTT及Transwell方法觀察tCD109對腫瘤細(xì)胞增殖、遷移和浸潤的影響;應(yīng)用免疫共沉淀分析tCD109與TGF-β受體的作用關(guān)系及其調(diào)節(jié)細(xì)胞生物學(xué)行為的信號通路。結(jié)果:1.在胰腺癌細(xì)胞中,BxPC-3、PANC-1、Patu8988s、Suit-2高表達(dá)CD109蛋白,而CFPAC-1、Capan-1、Miapaca-2、Patu8988t未檢測出CD109蛋白表達(dá)或只檢出低水平表達(dá)。2.在胰腺癌Patu8988s細(xì)胞中,PRSS3的表達(dá)能夠裂解細(xì)胞膜表面CD109蛋白,裂解位點在R663-F664之間。3.蛋白裂解片段V22-R663即tCD109可促進(jìn)胰腺癌細(xì)胞的增殖。MTT檢測Patu8988t-tCD109組細(xì)胞數(shù)(1.967±0.056)明顯高于Patu8988t-pcDNA3.1組(1.224±0.051)(p0.05)。同樣,Miapac-2-tCD109組細(xì)胞數(shù)(2.266±0.027)也明顯高于Miapac-2-pcDNA3.1組(1.409±0.06)(p0.05),4.tCD109具有促進(jìn)胰腺癌細(xì)胞侵襲和轉(zhuǎn)移能力。Transwell結(jié)果顯示Patu8988t-tCD109穿過Matrigel及膜的數(shù)量(42.54±3.21)明顯高于對照組(10.10±2.30)(p0.05)。5.tCD109與TGF-β1競爭性的結(jié)合TGF-β受體,從而抑制其下游信號傳導(dǎo)。結(jié)論:1.PRSS3裂解CD109的產(chǎn)物,即功能活性片段tCD109可與TGF-β1競爭性的結(jié)合TGF-β受體,從而抑制其下游信號傳導(dǎo),促進(jìn)胰腺癌細(xì)胞系的增殖、侵襲和轉(zhuǎn)移能力。2.tCD109可能是胰腺癌靶向治療的靶點,為胰腺癌靶向治療奠定了重要的分子理論基礎(chǔ)。
[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 鹵0.051). Similarly, the number of cells in Miapac-2-tCD109 group was 2.266 鹵0.027) significantly higher than that in Miapac-2-pcDNA3.1 group (1.409 鹵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 鹵3.21), which was significantly higher than that in control group (10.10 鹵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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.9

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本文編號：1649479