本文選題：轉(zhuǎn)化生長因子-β激活激酶 + 前列腺癌��； 參考：《蘭州大學(xué)》2014年碩士論文

【摘要】：研究背景 局限性前列腺癌的五年生存率為100%,但是進(jìn)展期的前列腺癌的五年生存率大約為31%。大多數(shù)患者采用內(nèi)分泌治療24月內(nèi),。絕大多數(shù)患者晚期前列腺癌又會發(fā)生骨轉(zhuǎn)移,且骨轉(zhuǎn)移事件一旦發(fā)生將會直接危害患者生活質(zhì)量和生命。 目前,前列腺癌唯一可以治愈的治療手段為前列腺癌根治術(shù)。而不能行前列腺癌根治術(shù)的患者,目前多會全雄激素阻斷治療,繼而大約70%的患者會出現(xiàn)骨轉(zhuǎn)移現(xiàn)象。因此急需開發(fā)一種新的有效的治療方法,來增加CRPC患者的生存時間、降低骨轉(zhuǎn)移相關(guān)并發(fā)癥對的發(fā)生率。 前列腺癌的發(fā)生、發(fā)展和基因突變及多種細(xì)胞因子的失衡有關(guān)。進(jìn)展期前列腺癌可以高表達(dá)多種和細(xì)胞生長、侵襲、遷移及成瘤性等相關(guān)的生長因子,TGF-岱是其中的一種。TGF-β在前列腺癌的發(fā)展中表現(xiàn)為雙重作用,在腫瘤細(xì)胞的早期主要起到抑制腫瘤細(xì)胞生長的作用,但是在進(jìn)展期腫瘤中TGF-β的作用轉(zhuǎn)變?yōu)榇龠M(jìn)腫瘤細(xì)胞的侵襲性和至瘤性,高表達(dá)的TGF-β可以誘導(dǎo)溶骨細(xì)胞介導(dǎo)的骨質(zhì)破壞。經(jīng)典的TGF-β信號通路為smad信號通路,但是non-smad信號通路在腫瘤骨轉(zhuǎn)移中的研究的也比較多。Non-samd信號通路中的轉(zhuǎn)化生長因子β蛋白激活激酶1(TGF-β activated protein kinase1, TAK1)對乳腺癌的骨轉(zhuǎn)移起著重要作用。 因此本研究通過siRNA沉默人前列腺癌DU145細(xì)胞系TAK1基因的表達(dá),初步研究敲除TAK1基因?qū)θ饲傲邢侔〥U145細(xì)胞增殖、侵襲、遷移、凋亡及藥物敏感性性的變化及作用機(jī)制。在前列腺癌發(fā)展、轉(zhuǎn)移、耐藥等方面,為深入研究TAK1基因的作用機(jī)制奠定基礎(chǔ)。 研究目的 研究TAK1siRNA對目標(biāo)基因表達(dá)的干擾作用,檢測沉默TAK1表達(dá)對前列腺癌DU145細(xì)胞增殖、侵襲、遷移、凋亡及藥物敏感性的影響及相關(guān)蛋白的表達(dá)情況,并初步探討其作用機(jī)制。 研究方法 1.應(yīng)用siRNA靶向沉默前列腺癌DU145細(xì)胞的TAK1基因的表達(dá),qRT-PCR檢測TAK1mRNA的表達(dá)情況,計算TAK1siRNA的基因敲除效率。 2.應(yīng)用蛋白免疫印跡方法檢測TAK1siRNA轉(zhuǎn)染前后TAK1蛋白表達(dá)的變化 3.成功沉默TAK1基因表達(dá)后,利用繪制的生長曲線檢測沉默TAK1基因?qū)η傲邢侔┘?xì)胞增殖能力影響。 4.應(yīng)用Transwell小室和劃痕實驗來分析TAK1siRNA轉(zhuǎn)染前后前列腺癌細(xì)胞DU145侵襲和遷徙能力的變化。 5.利用骨髓誘導(dǎo)細(xì)胞外基質(zhì)(BM-ECM)和transwell小室(8.0μm和0.4μm孔徑)構(gòu)建體外骨轉(zhuǎn)移模型,觀察BM-ECM對前列腺癌細(xì)胞的生長促進(jìn)作用和趨化作用。 6.在TAK1siRNA轉(zhuǎn)染前后,利用流式細(xì)胞儀檢測人前列腺癌細(xì)胞凋亡情況的變化。 7.用MTT法檢測TAK1siRNA轉(zhuǎn)染前后前列腺癌DU145細(xì)胞對三種化療藥物(多西紫杉醇(docetaxel)、奧沙利鉑(L-OHP)及5-氟尿嘧啶(5-FU))的藥物敏感性的變化。 8.在TAK1siRNA轉(zhuǎn)染前后,通過實時熒光定量PCR和Western blot檢測前列腺癌細(xì)胞表皮生長因子受體(EGFR)、凋亡抑制基因(Bcl-2)、環(huán)氧合酶-2(COX-2、β-連環(huán)蛋白(β-catenin)、MMP-2、9(基質(zhì)金屬蛋白酶-2、9)及氨基末端激酶(JNK)等基因在轉(zhuǎn)錄和翻譯水平的變化。 研究結(jié)果 1.利用脂質(zhì)體轉(zhuǎn)染TAK1siRNA可以高效的敲除TAK1基因的表達(dá),RT-PCR和western blot分析提示,TAK1siRNA可以顯著抑制TAK1基因的轉(zhuǎn)錄與翻譯,差異有統(tǒng)計學(xué)意義(P0.05)。 2.細(xì)胞生長曲線提示利用TAK1siRNA敲除TAKl基因后,前列腺癌DU145細(xì)胞的增殖能力被減弱,從第四天開始差異有統(tǒng)計學(xué)意義(P0.05) 3.利用transwell和劃痕實驗檢測侵襲和遷移能力,TAK1siRNA轉(zhuǎn)染后前列腺癌DU145細(xì)胞的侵襲和遷移能力均明顯下降,差異有統(tǒng)計學(xué)意義(P0.05)。 4.流式細(xì)胞儀檢測轉(zhuǎn)染前后前列腺癌細(xì)胞凋亡率分別為4.13%士0.11%和13.21%士1.41%,敲除TAKl基因表達(dá)可以明顯增加前列腺癌細(xì)胞的凋亡率,兩組之間比較,差異有統(tǒng)計學(xué)意義(P0.05)。 5.骨髓誘導(dǎo)細(xì)胞外基質(zhì)(BM-ECM)對前列腺癌DU145細(xì)胞具有促進(jìn)生長、侵襲和遷移作用,差異有統(tǒng)計學(xué)意義(P0.05)。 6.轉(zhuǎn)染TAK1siRNA后,前列腺癌DU145對多西他賽紫杉醇、L-OHP、5-FU的IC50顯著低于陰性對照組,TAK1基因沉默后前列腺癌DU145細(xì)胞對化療藥物的敏感性明顯增加,差異有統(tǒng)計學(xué)意義(P0.05)；對照組之間三種藥物的IC50之間IC50接近,差異沒有統(tǒng)計學(xué)意義(P0.05)。 7.轉(zhuǎn)染48h時,siRNA-TAK1組的COX-2、Bcl-2、JNK、β-Catenin, MMP2及MMP9的mRNA和蛋白相對表達(dá)量均低于negative control組及control組的表達(dá)量,差異有統(tǒng)計學(xué)意義(P0.05),而control組與negative control組之間,前列腺癌細(xì)胞中上述基因表達(dá)量差異無統(tǒng)計學(xué)意義P0.05) 結(jié)論： 沉默前列腺癌DU145細(xì)胞TAK1基因的表達(dá)可以降低凋亡相關(guān)蛋白Bcl-2、JNK,耐藥基因環(huán)氧合酶-2蛋白,表皮生長因子受體等的表達(dá),從而促進(jìn)細(xì)胞凋亡,抑制細(xì)胞生長,增加對化療藥物的敏感性；同時可以下調(diào)MMP2,MMP9表達(dá)可以降低前列腺癌DU145細(xì)胞的侵襲及遷移能力。TAKl基因可能是前列腺癌細(xì)胞信號轉(zhuǎn)導(dǎo)網(wǎng)絡(luò)中的一個關(guān)鍵性靶點。
[Abstract]:Research background
The five year survival rate of localized prostate cancer is 100%, but the five year survival rate of advanced prostate cancer is about 31%. in most patients with endocrine therapy for 24 months. Most patients with advanced prostate cancer will have bone metastases, and the occurrence of bone metastases will directly harm the quality of life and life of the patients.
At present, the only cure for prostate cancer is radical prostatectomy. But patients who do not undergo radical prostatectomy have more androgen blockade, and then about 70% of the patients have bone metastases. Therefore, a new and effective treatment method is urgently needed to increase the survival time of CRPC patients. The incidence of complications associated with bone metastases.
The development of prostate cancer is related to gene mutation and the imbalance of various cytokines. Progressing stage prostate cancer can express a variety of growth factors, such as cell growth, invasion, migration and tumorigenicity. TGF- Dai is one of the.TGF- beta in the development of prostate cancer, which is a double role in the early stage of cancer cells. It plays a role in inhibiting the growth of tumor cells, but the role of TGF- beta in advanced tumors is to promote the invasiveness and tumorigenicity of tumor cells. The high expression of TGF- beta can induce osteoblast mediated bone destruction. The classic TGF- beta signaling pathway is the Smad signaling pathway, but it is the research of the non-Smad signaling pathway in the metastasis of tumor bone. In addition, the TGF beta protein activated kinase 1 (TGF- beta activated protein kinase1, TAK1) in multiple.Non-samd signaling pathways plays an important role in bone metastasis of breast cancer.
Therefore, through the expression of TAK1 gene in the DU145 cell line of siRNA silent human prostate cancer, this study preliminarily studies the changes in the proliferation, invasion, migration, apoptosis and drug sensitivity of the TAK1 gene in human prostate cancer cells, and the mechanism of the development, metastasis, and drug resistance of prostate cancer, which lays a foundation for the research of the mechanism of the TAK1 gene in the development of prostate cancer. Set the foundation.
research objective
To investigate the effect of TAK1siRNA on the expression of target gene, and to detect the effect of silent TAK1 expression on the proliferation, invasion, migration, apoptosis and drug sensitivity of DU145 cells in prostate cancer and the expression of related proteins, and to explore the mechanism of its action.
research method
1. siRNA was used to silence the expression of TAK1 gene in prostate cancer DU145 cells, qRT-PCR was used to detect the expression of TAK1mRNA, and the gene knockout efficiency of TAK1siRNA was calculated.
2. protein immunoblotting was used to detect the expression of TAK1 protein before and after TAK1siRNA transfection.
3. after successful silencing of TAK1 gene expression, the growth curve was used to detect the effect of silencing TAK1 gene on the proliferation of prostate cancer cells.
4. Transwell chamber and scratch test were used to analyze the changes of invasion and migration ability of prostate cancer cell DU145 before and after TAK1siRNA transfection.
5. bone marrow induced extracellular matrix (BM-ECM) and Transwell compartment (8 m and 0.4 micron m aperture) were used to construct an in vitro bone metastasis model to observe the growth and chemotaxis effect of BM-ECM on the growth of prostate cancer cells.
6. before and after TAK1siRNA transfection, the apoptosis of human prostate cancer cells was detected by flow cytometry.
7. the changes in susceptibility to three chemotherapeutic drugs (docetaxel (docetaxel), Asha Leigh Per (L-OHP) and 5- fluorouracil (5-FU) were detected by MTT method before and after TAK1siRNA transfection.
8. before and after TAK1siRNA transfection, the expression of epidermal growth factor receptor (EGFR), apoptosis suppressor gene (Bcl-2), COX-2, beta catenin (beta -catenin), MMP-2,9 (-2,9) and amino terminal kinase (JNK) were detected by real-time fluorescence quantitative PCR and Western blot in the transcription and translation levels. Change.
Research results
1. transfection of TAK1siRNA with liposomes can efficiently knock out the expression of TAK1 gene. RT-PCR and Western blot analysis suggest that TAK1siRNA can significantly inhibit the transcription and translation of the TAK1 gene, and the difference is statistically significant (P0.05).
The 2. cell growth curve suggested that the proliferation ability of DU145 cells in prostate cancer was weakened after TAK1siRNA knockout TAKl gene, and the difference was statistically significant from the beginning of the fourth day (P0.05).
3. the invasion and migration ability was detected by Transwell and scratch test. The invasion and migration ability of DU145 cells in prostate cancer were significantly decreased after TAK1siRNA transfection, and the difference was statistically significant (P0.05).
4. flow cytometry was used to detect the apoptosis rate of prostate cancer cells before and after transfection. The apoptosis rate of prostate cancer cells was significantly increased by knockout TAKl gene expression, and the difference between the two groups was statistically significant (P0.05).
5. bone marrow induced extracellular matrix (BM-ECM) promoted the growth, invasion and migration of prostate cancer DU145 cells, and the difference was statistically significant (P0.05).
After 6. transfection of TAK1siRNA, the IC50 of prostate cancer DU145 to docetaxel paclitaxel, L-OHP, 5-FU was significantly lower than that in the negative control group. The sensitivity of DU145 cells to chemotherapeutic drugs was significantly increased after the TAK1 gene silencing, and the difference was statistically significant (P0.05); the IC50 of the three drugs between the control groups was close to the IC50, and the difference was not statistically significant. Meaning (P0.05).
The expression of mRNA and protein in COX-2, Bcl-2, JNK, beta -Catenin, MMP2 and MMP9 in group siRNA-TAK1 was lower than that of negative control and control groups when transfected with 48h, and there was a significant difference between the negative control group and the control group. There was no significant difference in the expression of the above gene expression in the Prost adenocarcinoma cells between the group and the group. 5)
Conclusion:
The expression of TAK1 gene of silent prostate cancer DU145 cells can reduce the expression of apoptosis related protein Bcl-2, JNK, drug resistant gene cyclooxygenase -2 protein, epidermal growth factor receptor and so on, thus promoting apoptosis, inhibiting cell growth, increasing sensitivity to chemotherapeutic drugs, decreasing MMP2, MMP9 expression can reduce the prostate cancer DU14. 5 the invasion and migration ability of.TAKl cells may be a key target in prostate cancer cell signal transduction network.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.25

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