本文選題：前列腺癌 + 細(xì)胞膜ATP5B　； 參考：《吉林大學(xué)》2017年碩士論文

【摘要】：前列腺癌(Prostate cancer,PCa)是男性泌尿生殖系統(tǒng)常見的惡性腫瘤之一,發(fā)生轉(zhuǎn)移后治療難度加大,且預(yù)后不佳。因此,闡明PCa轉(zhuǎn)移機(jī)制、識別高轉(zhuǎn)移潛能前列腺癌的潛在診療靶點(diǎn)尤為重要。在本課題組前期工作中,我們選用前列腺正常上皮細(xì)胞,前列腺癌細(xì)胞PC-3和前列腺癌高轉(zhuǎn)移細(xì)胞PC-3M通過噬菌體七肽庫進(jìn)行差減篩選,得到了只與PC-3M特異性結(jié)合的短肽B04,并且證實(shí)B04能夠有效抑制PC-3M細(xì)胞的增殖、轉(zhuǎn)移。隨后,通過蛋白質(zhì)組學(xué)等方法檢測到了短肽B04的受體蛋白為PC-3M細(xì)胞膜上的ATP5B,并驗(yàn)證了ATP5B在PC-3M細(xì)胞的細(xì)胞膜異位表達(dá)。ATP5B通常表達(dá)于線粒體內(nèi)膜,在某些細(xì)胞中可以異位表達(dá)于細(xì)胞膜,其功能并未明確。異位表達(dá)于PC-3M膜表面的ATP5B的功能有待鑒定,因此在本實(shí)驗(yàn)中,我們旨在發(fā)現(xiàn)和探究ATP5B在細(xì)胞膜異位表達(dá)對人前列腺癌細(xì)胞侵襲轉(zhuǎn)移能力的影響及其機(jī)制。實(shí)驗(yàn)結(jié)果:1.ATP5B在前列腺癌及癌旁組織的表達(dá)及定位通過免疫組化染色法檢測98例前列腺癌組織芯片中ATP5B蛋白的表達(dá)情況,前列腺組織芯片包括58例前列腺癌組織、37例前列腺癌旁組織和3例正常前列腺組織。并對ATP5B在細(xì)胞膜異位表達(dá)情況進(jìn)行分析,結(jié)果顯示:(1)ATP5B在前列腺癌組織中可出現(xiàn)腫瘤細(xì)胞膜的異位表達(dá);且細(xì)胞膜異位表達(dá)陽性率在浸潤擴(kuò)散的前列腺癌組織中明顯增高(p0.05)。(2)ATP5B在細(xì)胞膜異位表達(dá)陽性率在Gleason≥8的前列腺癌組織中明顯增高(p0.05)。2.ATP5B在細(xì)胞膜異位表達(dá)對前列腺癌細(xì)胞侵襲轉(zhuǎn)移能力的影響⑴細(xì)胞膜ATP5B過表達(dá)與抑制表達(dá)的模型建立分別用膽固醇和白皮杉醇處理PC-3M細(xì)胞,采用Western blot、實(shí)時定量PCR、ATP水平檢測等技術(shù)從蛋白、基因和功能水平探究膽固醇和白皮杉醇對于PC-3M細(xì)胞膜ATP5B的表達(dá)是否有影響。結(jié)果顯示:(1)蛋白水平:膽固醇處理后PC-3M的線粒體蛋白ATP5B降低,膜蛋白ATP5B增加,總蛋白ATP5B無明顯變化;白皮杉醇處理后PC-3M的線粒體蛋白ATP5B無明顯變化,膜蛋白ATP5B降低,總蛋白ATP5B降低,(2)基因水平,膽固醇處理PC-3M后,ATP5B m RNA相對表達(dá)量無明顯變化;白皮杉醇處理PC-3M后,ATP5B m RNA相對表達(dá)量降低。(3)功能水平,膽固醇促進(jìn)PC-3M細(xì)胞外ATP生成,白皮杉醇抑制細(xì)胞外ATP生成。以上結(jié)果提示,膽固醇促使ATP5B由線粒體異位至細(xì)胞膜,增加細(xì)胞膜ATP5B的表達(dá);而白皮杉醇可以抑制細(xì)胞膜ATP5B的表達(dá)。⑵ATP5B在細(xì)胞膜異位表達(dá)促進(jìn)前列腺癌侵襲轉(zhuǎn)移的體內(nèi)外研究采用細(xì)胞劃痕實(shí)驗(yàn)、Transwell小室遷移/侵襲實(shí)驗(yàn)來檢測改變ATP5B在細(xì)胞膜異位表達(dá)量后,PC-3M的侵襲轉(zhuǎn)移功能在體外的改變。結(jié)果顯示:膽固醇處理PC-3M后,ATP5B在細(xì)胞膜異位表達(dá)量增高,其體外遷移、侵襲均提高(p0.05);細(xì)胞膜ATP5B抑制劑白皮杉醇處理PC-3M后,其體外遷移、侵襲均明顯下降(p0.05)。隨后采用人前列腺癌雞胚絨毛尿囊膜轉(zhuǎn)移模型檢測抑制細(xì)胞膜ATP5B表達(dá)前后PC-3M半體內(nèi)轉(zhuǎn)移能力變化。結(jié)果顯示:抑制PC-3M細(xì)胞膜ATP5B,其轉(zhuǎn)移能力明顯下降(p0.05)。以上結(jié)果提示ATP5B在細(xì)胞膜異位表達(dá)可促進(jìn)前列腺癌侵襲轉(zhuǎn)移。⑶ATP5B在細(xì)胞膜的異位表達(dá)促進(jìn)前列腺癌侵襲轉(zhuǎn)移機(jī)制初步探索采用Westren Blot、PCR檢測改變ATP5B在細(xì)胞膜異位表達(dá)量后,c-Myc、VEGFA的蛋白以及m RNA的變化。結(jié)果顯示:ATP5B在PC-3M細(xì)胞膜異位表達(dá)量的改變會引起c-Myc、VEGFA蛋白及轉(zhuǎn)錄水平的變化,并且改變ATP5B在細(xì)胞膜的異位表達(dá)量后其體外誘導(dǎo)HUVEC細(xì)胞管型形成的能力也隨之發(fā)生變化。推測:ATP5B在細(xì)胞膜異位表達(dá)可能通過激活c-Myc原癌基因并上調(diào)VEGFA來促進(jìn)前列腺癌的侵襲轉(zhuǎn)移。以上實(shí)驗(yàn)結(jié)果說明了ATP5B在高級別前列腺癌和浸潤擴(kuò)散的前列腺癌組織的細(xì)胞膜上高表達(dá);可分別通過膽固醇和白皮杉醇處理PC-3M細(xì)胞來構(gòu)建細(xì)胞膜ATP5B過表達(dá)和抑制表達(dá)模型;ATP5B在細(xì)胞膜異位表達(dá)促進(jìn)前列腺癌細(xì)胞體內(nèi)外侵襲轉(zhuǎn)移,其機(jī)制可能是通過激活C-Myc原癌基因并上調(diào)VEGFA來促進(jìn)前列腺癌的侵襲轉(zhuǎn)移。以上結(jié)果也進(jìn)一步說明ATP5B在細(xì)胞膜異位表達(dá)可促進(jìn)前列腺癌細(xì)胞的侵襲轉(zhuǎn)移,有望成為前列腺癌轉(zhuǎn)移的檢測指標(biāo)或治療靶點(diǎn)。
[Abstract]:Prostate cancer (PCa) is one of the common malignant tumors in the male genitourinary system. The treatment is difficult and the prognosis is not good after the metastasis. Therefore, it is very important to clarify the mechanism of PCa transfer and identify the potential diagnostic target of high metastatic potential prostate cancer. In our early work, we chose normal epithelium of the prostate. Cells, prostate cancer cell PC-3 and high metastatic prostate cancer cell PC-3M were screened by phage seven peptide library, and a short peptide B04 was obtained only with PC-3M specific binding, and it was proved that B04 could effectively inhibit the proliferation and metastasis of PC-3M cells. Then, the receptor protein of short peptide B04 was detected by proteomics as PC-3M. ATP5B on the cell membrane, and verifies that the ectopic expression of ATP5B in the cell membrane of PC-3M cells is usually expressed in the mitochondrial membrane, which can be expressed ectopic to the cell membrane in some cells. The function of the.ATP5B is not clear. The function of ATP5B expressed on the surface of the PC-3M membrane needs to be identified. In this experiment, we aim to discover and explore ATP5B The effect of ectopic expression of cell membrane on the invasion and metastasis of human prostate cancer cells and its mechanism. Experimental results: the expression and localization of 1.ATP5B in prostate cancer and para cancer tissue were detected by immunohistochemical staining in 98 cases of prostate cancer tissue microarray, and 58 cases of prostate cancer tissue were included in the anterior gland tissue chip. The heterotopic expression of ATP5B in the cell membrane was analyzed in 37 para cancer tissues and 3 normal prostate tissues. The results showed that (1) the ectopic expression of the tumor cell membrane could be found in the prostate cancer tissue (1) and the positive rate of ectopic expression of the cell membrane was significantly increased in the infiltrating and diffusing prostate cancer tissues (P0.05). (2) ATP5B was ATP5B in the prostate cancer tissue. The positive rate of ectopic expression of cell membrane increased significantly in the prostate cancer tissues of Gleason > 8 (P0.05). The effect of ectopic expression of.2.ATP5B on the invasion and metastasis of prostate cancer cells (1): the ATP5B overexpression of cell membrane and the model of inhibiting expression of the cell membrane, PC-3M cells were treated with cholesterol and paclitaxel respectively, and Western blot was used in real time. Quantitative PCR, ATP level detection and other techniques explored whether cholesterol and paclitaxel had an effect on the expression of ATP5B in the PC-3M cell membrane from protein, gene and functional levels. The results showed: (1) protein level: the mitochondrial protein ATP5B decreased in PC-3M after cholesterol treatment, the membrane protein ATP5B increased, and the total protein ATP5B had no obvious changes; after the treatment of paclitaxel The mitochondrial protein ATP5B of PC-3M had no obvious changes, the membrane protein ATP5B decreased, the total protein ATP5B decreased, and (2) the relative expression of ATP5B m RNA was not obviously changed after PC-3M treatment of PC-3M, and the relative expression of ATP5B m RNA decreased after the treatment of PC-3M. (3) the function level, the cholesterol promoted the formation of extracellular matrix, and the inhibition of taxol inhibition. These results suggest that cholesterol promotes ATP5B from mitochondrial to cell membrane and increases the expression of ATP5B in cell membrane, while paclitaxel can inhibit the expression of ATP5B in cell membrane. (2) ATP5B in cell membrane ectopic expression to promote the invasion and metastasis of prostate cancer in vivo and in vitro study by cell scratch test, Transwell cell migration / invasiveness experiment was used to detect the changes in the invasion and transfer function of PC-3M after ATP5B's ectopic expression of cell membrane. The results showed that after cholesterol treatment PC-3M, the ectopic expression of ATP5B increased in cell membrane, and its invasion in vitro increased (P0.05). After the treatment of PC-3M, the ATP5B inhibitor of the cell membrane was migrated and attacked in vitro. Significantly decreased (P0.05). Then the transfer ability of PC-3M in the cell membrane was detected by the chick chorioallantoic membrane transfer model of human prostate cancer. The results showed that the metastasis ability of PC-3M cell membrane was significantly decreased (P0.05). The results suggested that the ectopic expression of ATP5B in the cell membrane could promote the invasion of prostate cancer. Metastasis. Heterotopic expression of ATP5B in cell membrane promotes the mechanism of invasion and metastasis of prostate cancer by using Westren Blot. PCR changes the changes in c-Myc, VEGFA protein and m RNA changes after ATP5B in cell membrane ectopic expression. The results show that ATP5B in PC-3M cell membrane ectopic expression can cause c-Myc, protein and transcriptional water The ability to induce the formation of HUVEC cells in vitro after ATP5B's ectopic expression in the cell membrane also changes. It is speculated that the ectopic expression of ATP5B in the cell membrane may promote the invasion and migration of the prostate cancer by activating the c-Myc proto oncogene and up regulation of VEGFA. These results indicate that ATP5B is at a high level. The cell membrane of prostate cancer and infiltrating prostate cancer tissue is highly expressed, and PC-3M cells can be treated with cholesterol and paclitaxel to construct the ATP5B overexpression and inhibition expression model of the cell membrane, and the ectopic expression of ATP5B in the cell membrane promotes invasion and metastasis of the prostate cancer cells in vivo and in vivo. The mechanism may be by activating the primary cancer of C-Myc. The gene and up regulation of VEGFA can promote the invasion and metastasis of prostate cancer. The above results also suggest that ATP5B can promote the invasion and metastasis of prostate cancer cells by ectopic expression of the cell membrane. It is expected to be a detection target or therapeutic target for the metastasis of prostate cancer.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.25

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