本文選題：前列腺癌 + 鈉鉀泵�。� 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景前列腺癌占西方發(fā)達(dá)國(guó)家男性惡性腫瘤發(fā)病率首位,近年來(lái)在中國(guó)的發(fā)病率也逐年上升。大部分的前列腺癌屬于相對(duì)惰性的腫瘤,然而部分前列腺癌進(jìn)展迅速,并很快發(fā)生轉(zhuǎn)移威脅病人生命,如何區(qū)分這部分進(jìn)展迅速的病人,研究前列腺癌增殖進(jìn)展的分子機(jī)制,對(duì)提高前列腺癌病人的生存率意義重大。早期研究者將NKAIN2命名為T細(xì)胞淋巴瘤斷裂位點(diǎn)相關(guān)靶點(diǎn)1(T-cell lymphoma breakpoint associated target 1(TCBA1)),基于能與鈉鉀泵β1 亞基相互作用(Na,K-ATPase)而被命名為鈉鉀泵相互作用因子2,屬于NKAIN跨膜蛋白超家族的一員。課題組前期通過高通量SNP芯片分析發(fā)現(xiàn)NKAIN2在前列腺癌標(biāo)本中發(fā)生斷裂及缺失,推測(cè)NKAIN2可能是抑癌基因。進(jìn)一步通過分析大量前列腺癌樣本,發(fā)現(xiàn)前列腺癌中,尤其是中國(guó)的病例中,NKAIN2經(jīng)常通過缺失及斷裂發(fā)生失活,并且通過體外細(xì)胞實(shí)驗(yàn)證明NKAIN2能夠抑制腫瘤細(xì)胞的增殖、遷移和侵襲,并促進(jìn)腫瘤細(xì)胞凋亡。鈉鉀泵在腫瘤中的作用已經(jīng)引起了廣泛關(guān)注,很多研究發(fā)現(xiàn)其在多種腫瘤中活性增強(qiáng),而鈉鉀泵抑制劑如強(qiáng)心苷等,已經(jīng)被證實(shí)對(duì)多種腫瘤包括前列腺癌有抑制作用,并且有些抑制劑已經(jīng)進(jìn)入臨床試驗(yàn)。課題組前期發(fā)現(xiàn)NKAIN2在前列腺癌中發(fā)揮抑癌作用,而作為鈉鉀泵相互作用因子,我們推測(cè)其可能通過抑制鈉鉀泵發(fā)揮抑癌作用,并且在細(xì)胞水平上進(jìn)行探索和驗(yàn)證。方法基于以上認(rèn)識(shí),本研究首先通過western blotting及鈉鉀泵活性試劑盒檢測(cè)前列腺癌細(xì)胞系中鈉鉀泵α1和β1亞基的表達(dá)及鈉鉀泵活性,并通過免疫組化在前列腺癌組織標(biāo)本中檢測(cè)鈉鉀泵α1和β1亞基的表達(dá);運(yùn)用免疫共沉淀驗(yàn)證了 NKAIN2與鈉鉀泵β1亞基的相互作用;運(yùn)用CRISPR/CAS9基因敲除技術(shù),構(gòu)建Cas9/SgRNA質(zhì)粒慢病毒載體轉(zhuǎn)染22RV1細(xì)胞系(NKAIN2高表達(dá)),并通過Western blotting和Cruiser酶切敲除及突變檢測(cè)試劑盒檢測(cè)敲除效率;進(jìn)一步通過裸鼠成瘤實(shí)驗(yàn)在體內(nèi)驗(yàn)證NKAIN2的抑癌作用,同時(shí)通過Western blotting檢測(cè)敲除NKAIN2后PI3K/Akt通路激活情況;接著在過表達(dá)、沉默NKAIN2的前列腺癌細(xì)胞系22RV1、PC-3中通過Western blotting及無(wú)機(jī)磷試劑盒檢測(cè)鈉鉀泵β1亞基的表達(dá)及鈉鉀泵活性,分析NKAIN2對(duì)鈉鉀泵的影響;并在前列腺癌細(xì)胞系22RV1、PC-3中沉默鈉鉀泵β1亞基,分析鈉鉀泵對(duì)前列腺癌生物學(xué)功能的影響。結(jié)果1.本研究通過western blotting檢測(cè)發(fā)現(xiàn)前列腺癌細(xì)胞系中鈉鉀泵α1和β1亞基的表達(dá)較前列腺永生化上皮細(xì)胞系增高;并通過免疫組化檢測(cè)了 30對(duì)前列腺癌標(biāo)本,發(fā)現(xiàn)鈉鉀泵α1和β1亞基在前列腺癌組織中較癌旁組織表達(dá)增高;鈉鉀泵活性檢測(cè)試劑盒同樣發(fā)現(xiàn)前列腺癌細(xì)胞系鈉鉀泵活性較前列腺永生化上皮細(xì)胞系增強(qiáng)。2.Western blotting和Cruiser酶檢測(cè)結(jié)果鑒定22RV1 NKAIN2敲除細(xì)胞株構(gòu)建成功,進(jìn)一步裸鼠成瘤實(shí)驗(yàn)提示NKAIN2敲除細(xì)胞系成瘤體積明顯較對(duì)照組增大,并且發(fā)現(xiàn)敲除NKAIN2后激活PI3K/Akt通路。3.在PC-3及22RV1細(xì)胞中,過表達(dá)NKAIN2能抑制鈉鉀泵β1亞基的表達(dá),相反沉默NKAIN2能增強(qiáng)鈉鉀泵β1亞基的表達(dá)。4.在PC-3及22RV1細(xì)胞中,過表達(dá)NKAIN2能抑制鈉鉀泵活性,相反沉默NKAIN2能增強(qiáng)鈉鉀泵的活性。5.在PC-3及22RV1細(xì)胞中,沉默鈉鉀泵β1亞基的表達(dá),抑制細(xì)胞增殖、遷移和侵襲,促進(jìn)細(xì)胞凋亡。結(jié)論本研究通過體內(nèi)實(shí)驗(yàn)證實(shí)NKAIN2對(duì)前列腺癌生長(zhǎng)有抑制作用,而鈉鉀泵α1和β1亞基的表達(dá)在前列腺癌細(xì)胞系及標(biāo)本中增高,鈉鉀泵活性在前列腺癌細(xì)胞系同樣增強(qiáng)。NKAIN2可能通過抑制鈉鉀泵的表達(dá)及活性,進(jìn)一步抑制前列腺癌的增殖和進(jìn)展。這一發(fā)現(xiàn)可能為前列腺癌診斷治療和預(yù)后評(píng)估提供新的理論依據(jù)和研究方向。
[Abstract]:Background prostate cancer accounts for the highest incidence of male malignant tumors in developed countries in western countries. In recent years, the incidence of prostate cancer in China has also increased year by year. Most of the prostate cancer is a relatively inert tumor. However, some prostate cancers are progressing rapidly, and the metastasis threatens the patient's life quickly, and how to distinguish this part of the rapid progress of the patients, The molecular mechanism of the progression of prostate cancer is of great significance for improving the survival rate of prostate cancer patients. Early researchers named NKAIN2 as the target of T cell lymphoma at the target point 1 (T-cell lymphoma breakpoint associated target 1 (TCBA1)), based on the ability to interact with the sodium and potassium pump beta 1 subunit (Na, K-ATPase) and be named sodium potassium The pump interaction factor 2 is a member of the NKAIN transmembrane protein superfamily. In the earlier period, we found that the NKAIN2 was broken and missing in the specimens of prostate cancer by high throughput SNP chip analysis, and that NKAIN2 might be a tumor suppressor gene. Further analysis of a large number of prostate cancer samples, especially in Chinese cases, N KAIN2 is often inactivated by deletion and fracture, and it has been proved that NKAIN2 can inhibit the proliferation, migration and invasion of tumor cells and promote tumor cell apoptosis through in vitro cell experiment. The role of sodium potassium pump in tumor has attracted wide attention. Many studies have found that the activity of the sodium potassium pump is enhanced in many kinds of tumors, and the sodium potassium pump inhibitors such as Strong glycosides have been proved to have inhibitory effects on a variety of tumors, including prostate cancer, and some of the inhibitors have already entered clinical trials. We have found that NKAIN2 plays a role in inhibiting cancer in prostate cancer. As a sodium potassium pump interaction factor, we speculate that it may inhibit cancer by inhibiting the sodium potassium pump and is fine. In this study, the expression of sodium potassium pump alpha 1 and beta 1 subunits in prostate cancer cell lines and the activity of sodium potassium pump were detected by Western blotting and sodium potassium pump active kits. The interaction between NKAIN2 and sodium potassium pump beta 1 subunit was verified by immunoprecipitation; CRISPR/CAS9 gene knockout technique was used to construct the Cas9/SgRNA plasmid lentivirus vector transfected to 22RV1 cell line (NKAIN2 high expression), and the knockout efficiency was detected by Western blotting and Cruiser enzyme knockout and mutation detection kit, and the nude mice were further formed into a tumor. The experiment was used to verify the tumor suppressor effect of NKAIN2 in vivo, and the activation of PI3K/Akt pathway after knockout NKAIN2 was detected by Western blotting; then the prostate cancer cell line of NKAIN2 was expressed in 22RV1, and the expression of sodium potassium pump beta 1 subunit and sodium potassium pump activity were detected by Western blotting and inorganic phosphorus kit in PC-3, and the NKAIN2 pair was analyzed. The effect of sodium potassium pump, and the effect of sodium potassium pump beta 1 subunit in prostate cancer cell line 22RV1, PC-3, analysis of the effect of sodium potassium pump on the biological function of prostate cancer. Results 1. this study found that the expression of sodium potassium pump alpha 1 and beta 1 subunits in prostate cancer cell lines increased by Western blotting detection; and the results were higher than that of the prostatic immortalized epithelial cell line. 30 pairs of prostate cancer specimens were detected by immunohistochemistry. The expression of sodium potassium pump alpha 1 and beta 1 subunits in the prostate cancer tissues was higher than that of the para cancerous tissue. The sodium potassium pump activity detection kit also found that the activity of sodium potassium pump in the prostate cancer cell line was more than that of the enhanced.2.Western blotting and Cruiser enzyme of the prostatic immortalized epithelial cell line. The 2RV1 NKAIN2 knockout cell line was successfully constructed. Further tumor formation experiments in nude mice suggest that the tumor volume of NKAIN2 knockout cell line is obviously larger than that of the control group, and the expression of NKAIN2 can inhibit the expression of the sodium potassium pump beta 1 subunit in the PC-3 and 22RV1 cells after the knockout of NKAIN2 after knockout NKAIN2, and the reverse silencing NKAIN2 can enhance the sodium potassium pump beta 1 subunit. The expression of.4. in PC-3 and 22RV1 cells inhibits the activity of sodium and potassium pumps by overexpression of NKAIN2. On the contrary, silent NKAIN2 can enhance the activity.5. of the sodium potassium pump in PC-3 and 22RV1 cells, silence the expression of the sodium potassium pump beta 1 subunit, inhibit cell proliferation, migration and invasion, and promote cell apoptosis. Conclusion this study confirmed the actual NKAIN2 against the prostate in vivo. The expression of the sodium potassium pump alpha 1 and beta 1 subunits increased in the prostate cancer cell lines and specimens. The sodium potassium pump activity in the prostate cancer cell line also enhanced.NKAIN2 may further inhibit the proliferation and progression of prostate cancer by inhibiting the expression and activity of the sodium potassium pump. This discovery may be a diagnostic treatment for prostate cancer. And provide a new theoretical basis and research direction for prognosis evaluation.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.25

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本文編號(hào)：1884110