【摘要】：目的研究PTN(Pleiotrophin,多效生長因子)在胰腺癌細(xì)胞株Miapaca-2和Capan-1中的表達(dá)情況,利用免疫熒光技術(shù)、Q-PCR、Western blot法檢測PTN mRNA和蛋白的表達(dá),從而篩選較穩(wěn)定的高表達(dá)細(xì)胞系,為后期建立小鼠原位胰腺癌模型并通過干擾原位胰腺癌中PTN蛋白的表達(dá),進(jìn)一步驗證PTN與其受體結(jié)合是否激活相關(guān)信號傳導(dǎo)通路在胰腺癌神經(jīng)浸潤發(fā)生中的作用提供科學(xué)依據(jù)。PTN可作為潛在的藥物作用靶點,進(jìn)一步對相關(guān)信號通路進(jìn)行干預(yù),抑或體外合成相應(yīng)的PTN抑制劑,從而達(dá)到下調(diào)PTN或完全抑制的目的,阻止腫瘤的進(jìn)一步生長及胰腺癌神經(jīng)浸潤的發(fā)生,為胰腺癌的早期診斷、分期及治療尋找新途徑提供科學(xué)依據(jù)。方法用DMEM及1640培養(yǎng)液對Miapaca-2和Capan-1細(xì)胞進(jìn)行傳代培養(yǎng),取指數(shù)生長期細(xì)胞,利用Q-PCR法和Western blot方法檢測PTN m RNA和蛋白在胰腺癌細(xì)胞株中的表達(dá),并采用免疫熒光化學(xué)法檢測PTN在胰腺癌細(xì)胞中的表達(dá)位置。結(jié)果Q-PCR結(jié)果顯示,PTN mRNA在MIA Pa Ca-2細(xì)胞中表達(dá)明顯較高,且與Capan-1細(xì)胞相比,MIA Pa Ca-2中PTN m RNA表達(dá)量為Capan-1的1.193倍。Western blot結(jié)果顯示:目的蛋白PTN的分子量大小約為19KDa,內(nèi)參GAPDH蛋白大小為36KDa。然后應(yīng)用Image J軟件進(jìn)行半定量分析,結(jié)果發(fā)現(xiàn),PTN蛋白在MIA PaCa-2和Capan-1細(xì)胞均有表達(dá),且與Capan-1相比,MIA Pa Ca-2細(xì)胞系中PTN蛋白的表達(dá)更加明顯。免疫熒光結(jié)果顯示PTN的陽性表達(dá)呈紅色顆粒,主要定位于胞漿,部分胞核亦可見陽性著色。隨后我們將Q-PCR、Western blot、免疫熒光結(jié)果進(jìn)行對比,結(jié)果表明胰腺癌細(xì)胞中PTN在mRNA及蛋白水平表達(dá)具有一致性。結(jié)論Mia Pa Ca-2細(xì)胞中PTN mRNA和蛋白均呈更高表達(dá),主要定位于胞漿中,因此,該細(xì)胞可作為良好的研究工具,為進(jìn)一步建立小鼠原位胰腺癌模型,以研究PTN與受體結(jié)合是否促進(jìn)胰腺癌神經(jīng)浸潤的發(fā)生提供依據(jù)。
[Abstract]:Objective to study the expression of PTN (Pleiotrophin, multipotent growth factor (PTN (Pleiotrophin,) in pancreatic cancer cell line Miapaca-2 and Capan-1, and to detect the expression of PTN mRNA and protein by Q-PCR Western blot. Thus, a stable high expression cell line was screened to establish a mouse model of pancreatic cancer in situ and to interfere with the expression of PTN protein in pancreatic carcinoma in situ. To further verify whether the binding of PTN to its receptor can activate the role of related signal transduction pathway in the neurogenesis of pancreatic cancer, PTN can be used as a potential drug target to further interfere with the related signal pathway. Or the synthesis of corresponding PTN inhibitors in vitro, so as to down-regulate PTN or complete inhibition, prevent the further growth of tumors and the occurrence of pancreatic cancer neural infiltration, which is the early diagnosis of pancreatic cancer. Staging and treatment to find new ways to provide scientific basis. Methods Miapaca-2 and Capan-1 cells were subcultured with DMEM and 1640 medium. The expression of PTN m RNA and protein in pancreatic cancer cell lines was detected by Q-PCR and Western blot methods. The expression of PTN in pancreatic cancer cells was detected by immunofluorescence method. Results Q-PCR showed that the expression of, PTN mRNA in MIA Pa Ca-2 cells was significantly higher than that in Capan-1 cells. The expression of PTN m RNA in MIA Pa Ca-2 was 1.193 times of that of Capan-1. The results showed that the molecular weight of the target protein PTN was about 19K Daa and the internal reference GAPDH protein size was 36KDa. Then the semi-quantitative analysis was carried out by Image J software. The results showed that PTN protein was expressed in both MIA PaCa-2 and Capan-1 cells, and the expression of PTN protein in, MIA Pa Ca-2 cell line was more obvious than that in Capan-1 cell line. The results of immunofluorescence showed that the positive expression of PTN was red granules, mainly located in the cytoplasm, and the positive staining was also seen in some nuclei. The results showed that the expression of PTN in pancreatic cancer cells was consistent at the level of mRNA and protein. Conclusion the expression of PTN mRNA and protein in Mia Pa Ca-2 cells is higher, which is mainly located in the cytoplasm. Therefore, this cell can be used as a good tool for the establishment of in situ pancreatic cancer model in mice. To study whether the binding of PTN to receptor can promote the neurogenesis of pancreatic cancer.
【學(xué)位授予單位】：河南科技大學(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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