本文選題：SKP2前列腺癌　切入點(diǎn)：報(bào)告基因試驗(yàn)　出處：《大連醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景:異常的基因表達(dá)是腫瘤的一個(gè)重要標(biāo)志,通常驅(qū)動(dòng)著細(xì)胞增殖,分化和惡性腫瘤細(xì)胞的轉(zhuǎn)移。細(xì)胞周期失衡是腫瘤發(fā)生的重要因素。前列腺癌是公認(rèn)的男性發(fā)病率最高的非上皮源性惡性腫瘤之一,也是因癌癥導(dǎo)致死亡的重要因素。多項(xiàng)研究指出,與細(xì)胞周期調(diào)控密切相關(guān)的SKP2基因的異常表達(dá)在前列腺癌的發(fā)展中起到了重要的作用。在多種人類腫瘤中SKP2均呈現(xiàn)過表達(dá),其異位表達(dá)也會(huì)促進(jìn)腫瘤的發(fā)生。這些都提示SKP2的表達(dá)異常在惡性腫瘤的發(fā)展中具有重要的意義,而抑制SKP2基因的過表達(dá)顯然是惡性腫瘤治療中頗具前景的戰(zhàn)略。轉(zhuǎn)錄治療是基因治療的熱點(diǎn)內(nèi)容,通過直接干預(yù)轉(zhuǎn)錄過程對(duì)腫瘤細(xì)胞異常基因表達(dá)進(jìn)行糾正。隨著越來越多的治療癌癥的靶向基因得以確定,以及新藥研發(fā)技術(shù)的不斷提高,轉(zhuǎn)錄治療也得以迅速發(fā)展。如何在大量不同種類的化合物中篩選出有效的抗腫瘤藥物,是藥物研發(fā)的重要環(huán)節(jié)。報(bào)告基因試驗(yàn)的推廣使得藥物的高通量篩選(HTS)變得更便捷、經(jīng)濟(jì)和高效。然而,受到不完整的順式作用原件的調(diào)控以及表觀遺傳環(huán)境差異的影響,隨機(jī)插入的報(bào)告基因常常因位于側(cè)面的凝聚染色質(zhì)而表觀沉默,影響了傳統(tǒng)的報(bào)告基因試驗(yàn)的可靠性。考慮到這些問題,Yan等研發(fā)出一種基于內(nèi)部核糖體進(jìn)入位點(diǎn)原位報(bào)告基因試驗(yàn),報(bào)告基因可以被設(shè)計(jì)并選擇性地插入一段基因序列最接近內(nèi)源基因編碼區(qū)下游的位置,并在原有的轉(zhuǎn)錄調(diào)節(jié)機(jī)制下與內(nèi)源基因共同表達(dá)。這種新型的報(bào)告基因試驗(yàn)避免了傳統(tǒng)方法的不足,其有效性有待進(jìn)一步論證。目的:考慮到前列腺癌的高發(fā)病率、死亡率以及治療效果的不確切,以及SKP2基因異常表達(dá)在前列腺癌中的重要作用,我們期待能夠應(yīng)用Yan等研發(fā)的基于內(nèi)部核糖體進(jìn)入位點(diǎn)原位報(bào)告基因試驗(yàn),通過高通量篩選,找到能夠在轉(zhuǎn)錄水平抑制SKP2基因過表達(dá)的化合物,并通過后續(xù)實(shí)驗(yàn)驗(yàn)證其有效性并嘗試探尋其機(jī)制,以期為惡性腫瘤的研究和治療提供新的思路。方法:利用前列腺癌細(xì)胞DU145細(xì)胞系,通過Yan等研發(fā)的基于內(nèi)部核糖體進(jìn)入位點(diǎn)原位報(bào)告基因試驗(yàn)技術(shù),構(gòu)建共表達(dá)SKP2-FLuc DU145細(xì)胞。然后,利用上述重組細(xì)胞通過luciferase試驗(yàn)進(jìn)行HTS藥物篩選,我們共篩選了來自NCI化合物庫、She實(shí)驗(yàn)組和Chembridge化合物庫超過8000種化合物。利用前列腺癌細(xì)胞(DU145、PC3、22RV1和C4-2B細(xì)胞系),通過real-time PCR試驗(yàn)、Western blot試驗(yàn)和MTT試驗(yàn)等將篩選出的化合物做進(jìn)一步驗(yàn)證。結(jié)果:通過上述新型的報(bào)告基因試驗(yàn)技術(shù),我們成功通過HTS和luciferase試驗(yàn)初步篩選出10個(gè)化合物。將這些化合物進(jìn)一步做real-time PCR試驗(yàn),得到在轉(zhuǎn)錄水平抑制SKP2的化合物48X(NCI librarys)。通過多個(gè)前列腺癌細(xì)胞系Western blot試驗(yàn),我們進(jìn)一步證實(shí)該化合物對(duì)前列腺癌細(xì)胞SKP2具有抑制作用,并且該效應(yīng)與劑量和作用時(shí)間呈正相關(guān),并且在加藥后8小時(shí)即可有效抑制SKP2。MTT結(jié)果證實(shí),4組前列腺癌細(xì)胞系細(xì)胞在經(jīng)過48X化合物處理后,出現(xiàn)細(xì)胞增殖能力降低的功能性改變。結(jié)論:本研究進(jìn)一步證實(shí)了Yan等開發(fā)的基于內(nèi)部核糖體進(jìn)入位點(diǎn)原位報(bào)告基因試驗(yàn)技術(shù)的可行性和有效性。本研究所篩選出的48X(NCI librarys)化合物對(duì)前列腺癌細(xì)胞SKP2基因具有在轉(zhuǎn)錄水平上的抑制作用,并且對(duì)前列腺癌細(xì)胞具有功能性效應(yīng)(細(xì)胞增殖能力下降)。
[Abstract]:Background: abnormal gene expression is an important sign of the tumor, usually drives cell proliferation, differentiation and metastasis of malignant tumor cells. Cell cycle imbalance is an important factor in tumor. Prostate cancer is known as the male incidence of non epithelial malignant tumor with the highest, is also an important factor leading to death due to cancer. A number of studies have indicated that the abnormal expression of SKP2 gene is closely related with the regulation of the cell cycle in the development of prostate cancer plays an important role in a variety of human tumors. SKP2 showed the expression of the ectopic expression may promote the occurrence of tumors. These results suggest that abnormal expression of SKP2 plays an important role in the development of malignant tumor, inhibit the overexpression of the SKP2 gene is obviously the treatment of malignant tumor, promising strategy. Antiretroviral therapy is a hot topic in gene therapy, through direct intervention The process of transcription to correct abnormal gene expression of tumor cells with cancer treatment. More and more target genes to be determined, and the research and development of new drugs and technology continue to improve, antiretroviral therapy is also developing rapidly. How to select the effective anti-tumor drugs in a large number of different types of compounds, is an important link in drug development. Promotion report gene test allows high throughput drug screening (HTS) has become more convenient, economical and efficient. However, affected by the incomplete cis acting original epigenetic regulation and environmental differences, often because of the random insertion of reporter gene is located on the side of the condensed chromatin and epigenetic silencing, affects the reliability of reporter gene the traditional test. Considering these problems, Yan developed an internal ribosome entry site based on in situ test report gene, the reporter gene can be designed and Selectively inserted a gene sequence of endogenous gene encoding region closest to the downstream position of the mechanism and regulation of endogenous gene transcription in the original expression. This new type of reporter gene assay to avoid the shortcomings of traditional methods, its effectiveness should be further argued. Objective: Considering prostate cancer high incidence and mortality the treatment effect is not exact, an important role in prostate cancer and abnormal expression of SKP2 gene, we look forward to the application of Yan based on the development of the internal ribosome entry site reporter gene by in situ test, high-throughput screening, found to inhibit SKP2 gene expression of the compounds at the transcriptional level, and through subsequent experiments to verify its effectiveness and try to explore its mechanism, to provide new ideas for research and treatment for malignant tumors. Methods: the prostate cancer cell line DU145, via Y An based on the development of the internal ribosome entry site reporter gene in situ test technique, construct the co expression of SKP2-FLuc DU145 cells. Then, the HTS drug screening by luciferase test using the recombinant cells, we were selected from the NCI compound library, experimental group She and Chembridge compound library of more than 8000 kinds of compounds. The prostate cancer cells (DU145. PC3,22RV1 and C4-2B cell lines by real-time PCR), Western test, blot test and MTT test will be screened compounds for further verification. Results: by the new report by test technology, we successfully passed the HTS and luciferase test results showed that 10 compounds. These compounds will further make the real-time PCR test, get inhibition of SKP2 at the transcriptional level of compound 48X (NCI librarys). Through a number of prostate cancer cell line Western blot test, we enter a Further confirmed that the compounds have inhibitory effects on prostate cancer SKP2 cells, and the positive effect and dose and time related, and confirmed in 8 hours after dosing can effectively inhibit the SKP2.MTT results, the prostate cancer cell line 48X cells in the 4 groups after treatment of functional compounds, reduce the cell proliferation. Conclusion: This study further confirmed that the Yan based on the development of the internal ribosome entry site reporter gene assay technology feasibility and effectiveness. This study selected 48X (NCI librarys) compounds have inhibitory effect on the transcription level of SKP2 gene in prostate cancer cells, and has the functional effects on prostate cancer cells (the cells decreased the proliferation ability).

【學(xué)位授予單位】：大連醫(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)】：R73-3

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本文編號(hào)：1567268