本文選題：DD3基因 + 前列腺癌�。� 參考：《山東大學(xué)》2016年博士論文

【摘要】：背景前列腺癌(PCa)是男性泌尿生殖系統(tǒng)較常見(jiàn)的惡性腫瘤,在歐美國(guó)家,PCa發(fā)病率高居男性惡性腫瘤之首。由于我國(guó)人口老齡化以及診斷技術(shù)不斷提高,PCa的發(fā)病率以每年10%的速度攀升,嚴(yán)重威脅著中老年男性的健康。然而,PCa的發(fā)病機(jī)制十分復(fù)雜,PCa發(fā)生以及進(jìn)展過(guò)程中諸多影響因素尚未闡明,制約著PCa的有效診治。近年來(lái),前列腺癌抗原3(differential display code 3 for prostate cancer, DD3)與前列腺癌的關(guān)系越來(lái)越受到人們的重視。(1)長(zhǎng)鏈非編碼RNA與PCa長(zhǎng)鏈非編碼RNA(long non-coding RNA, lncRNA)是長(zhǎng)度超過(guò)200個(gè)核苷酸、并具有調(diào)控基因的表達(dá)作用的非編碼RNA。越來(lái)越多的研究表明,lncRNA參與細(xì)胞凋亡、腫瘤浸潤(rùn)和轉(zhuǎn)移等過(guò)程,在腫瘤的發(fā)生、發(fā)展過(guò)程中發(fā)揮著促癌或抑癌的作用；lncRNA還可以通過(guò)表觀遺傳調(diào)控的方式影響腫瘤細(xì)胞的生長(zhǎng),從而有希望成為新型腫瘤標(biāo)志物并成為腫瘤治療的靶點(diǎn),同時(shí)lncRNA在腫瘤診斷和腫瘤治療方面顯示出廣闊的臨床應(yīng)用前景。DD3是目前發(fā)現(xiàn)的一種PCa組織中高表達(dá)的一種lncRNA,與PCa關(guān)系密切,在對(duì)PCa的診斷效能上其敏感性和特異性均優(yōu)于傳統(tǒng)的PSA,有良好的臨床應(yīng)用前景。(2)DD3與PCaDD3基因是在1999年由Bussemakers等發(fā)現(xiàn)的一種前列腺癌特異性基因,位于人類染色體9q21～22。DD3基因不表達(dá)蛋白,但它與前列腺癌的發(fā)生密切相關(guān),它特異性的高表達(dá)于人類約95%的前列腺癌細(xì)胞,而在正常前列腺和良性前列腺增生細(xì)胞中僅僅少量表達(dá)或無(wú)表達(dá)。有研究發(fā)現(xiàn),DD3的基因表達(dá)用于鑒別前列腺癌和前列腺良性病變的準(zhǔn)確性可達(dá)100%。前列腺以外的惡性及良性病變則沒(méi)有DD3的表達(dá)。這說(shuō)明DD3基因是目前為止鑒別前列腺惡性腫瘤的最特異腫瘤標(biāo)記物。與PSA不同,DD3基因不受患者年齡、前列腺體積及其它如前列腺炎等前列腺疾病的影響。同時(shí)有研究發(fā)現(xiàn),DD3基因表達(dá)與前列腺癌的腫瘤體積、病理分期以及病理的Gleason評(píng)分呈明顯相關(guān),且在患者的腫瘤體積0.5mL和Gleason評(píng)分≥7的病例中,DD3基因表達(dá)呈明顯升高。在一項(xiàng)前列腺癌預(yù)后的多因素研究分析中,DD3基因被認(rèn)為可以成為前列腺癌預(yù)后分析的獨(dú)立預(yù)測(cè)因子。有研究表明,結(jié)合患者PSA以及Gleason評(píng)分,DD3預(yù)測(cè)前列腺癌是否存在腫瘤包膜外侵犯的準(zhǔn)確率可達(dá)90%。而對(duì)于Gleason評(píng)分較低、低PSA值和低DD3基因表達(dá)的前列腺癌患者,臨床上可以僅給予密切的監(jiān)測(cè)隨訪,等待觀察。PCa患者尿液中的細(xì)胞和細(xì)胞碎片能檢測(cè)出DD3的高表達(dá),劉光香等人運(yùn)用RT-PCR技術(shù)研究發(fā)現(xiàn),在臨床54例PCa患者中,其外周血DD3 mRNA表達(dá)陽(yáng)性者48例,另有23例前列腺增生患者和9例健康成年男性外周血未見(jiàn)DD3mRNA的陽(yáng)性表達(dá)[15]。這可能是前列腺癌患者血液中的淋巴細(xì)胞吞噬了前列腺癌細(xì)胞,從而血液中可檢測(cè)到DD3 mRNA的表達(dá)。DD3基因是長(zhǎng)鏈非編碼RNA,而目前多種證據(jù)表明lncRNA在多種腫瘤的生長(zhǎng)及侵襲轉(zhuǎn)移中發(fā)揮了非常重要的作用。DD3作為一種不表達(dá)蛋白的lncRNA,在前列腺癌的發(fā)生發(fā)展中的具體作用以及DD3對(duì)前列腺癌增殖、凋亡、轉(zhuǎn)移和侵襲能力的影響目前仍不明確,有待進(jìn)一步研究。目的通過(guò)細(xì)胞學(xué)、分子生物學(xué)及動(dòng)物模型構(gòu)建等技術(shù)從體內(nèi)和體外兩方面系統(tǒng)的探討DD3基因?qū)τ谇傲邢侔┥L(zhǎng)、凋亡、轉(zhuǎn)移和侵襲能力的影響,為我們進(jìn)一步研究DD3基因在前列腺癌發(fā)生、發(fā)展中的作用及其機(jī)制打下良好的基礎(chǔ),為前列腺癌的臨床診療提供新的線索和方向。方法我們采用在體外培養(yǎng)前列腺癌研究中較常用的LNCaP細(xì)胞株和PC3細(xì)胞株。然后采用實(shí)時(shí)定量PCR技術(shù)測(cè)定LNCaP細(xì)胞株與PC3細(xì)胞株的DD3基因表達(dá)水平,并通過(guò)小RNA干擾技術(shù)(siRNA)和基因過(guò)表達(dá)技術(shù)構(gòu)建DD3敲減質(zhì)粒和DD3過(guò)表達(dá)質(zhì)粒及其對(duì)應(yīng)的對(duì)照質(zhì)粒,慢病毒包裝質(zhì)粒后,通過(guò)細(xì)胞轉(zhuǎn)染建立DD3低表達(dá)的LNCaPDD3-細(xì)胞株和DD3高表達(dá)的PC3DD+細(xì)胞株及兩者的對(duì)照組細(xì)胞株LNCaPNC和PC3Ctrl。然后采用細(xì)胞計(jì)數(shù)法和MTT方法檢測(cè)所構(gòu)建的前列腺癌細(xì)胞株LNCaPDD3-、LNCaPNC、PC3DD+和PC3Ctrl的細(xì)胞增殖能力,從而確定DD3對(duì)前列腺癌細(xì)胞的增殖能力的影響；用ABL-N誘導(dǎo)前列腺癌細(xì)胞株LNCaPDD3+、LNCaPNC、PC3DD+和PC3Ctrl凋亡后,運(yùn)用Annexin V+PI雙染法檢測(cè)各組細(xì)胞株的早、中晚期的凋亡情況,并確定DD3基因在前列腺癌細(xì)胞凋亡中的作用；運(yùn)用劃痕實(shí)驗(yàn)和Transwell小室進(jìn)一步檢測(cè)上述各前列腺癌細(xì)胞株的遷移以及侵襲能力,確定DD3基因在前列腺癌細(xì)胞遷移、侵襲中的作用。在體內(nèi)實(shí)驗(yàn)中,我采用裸鼠體內(nèi)成瘤實(shí)驗(yàn)檢測(cè)上述各組細(xì)胞株在體內(nèi)形成的種植瘤的體積、重量,然后將前列腺癌瘤體石蠟包埋、切片,并利用免疫組化染色法檢測(cè)瘤體組織細(xì)胞中Ki67蛋白的表達(dá)水平；并利用TUNEL法檢測(cè)前列腺癌腫瘤組織細(xì)胞的凋亡水平；進(jìn)一步研究DD3在體內(nèi)對(duì)前列腺癌細(xì)胞增殖及凋亡的影響。結(jié)果1.q-PCR檢測(cè)結(jié)果表明,用RNAi技術(shù)特異性的沉默LNCaP細(xì)胞內(nèi)的DD3的表達(dá)后,與對(duì)照組LNCaPNC細(xì)胞相比,干擾后LNCaPDD3-細(xì)胞內(nèi)DD3的表達(dá)顯著降低(P0.01)；而過(guò)表達(dá)DD3后的PC3DD3+細(xì)胞內(nèi)的DD3的表達(dá)水平顯著高于對(duì)照組PC3Ctrl細(xì)胞(P0.01)。證明LNCaPDD3-和PC3DD+細(xì)胞株建立成功。2.細(xì)胞計(jì)數(shù)法和MTT方法檢測(cè)LNCaPDD3-和PC3DD+細(xì)胞相對(duì)于其對(duì)照細(xì)胞LNCaPNC和PC3Ctrl的增殖能力,結(jié)果表明,DD3低表達(dá)的LNCaPDD3-細(xì)胞的增殖速度明顯低于其對(duì)照組LNCaPNC細(xì)胞(P0.05)；而DD3高表達(dá)PC3DD3+細(xì)胞的增殖速度明顯高于其對(duì)照組pC3Ctrl細(xì)胞(P0.05)。3. Annexin V+PI雙染法檢測(cè)前列腺癌各細(xì)胞株凋亡情況的結(jié)果顯示：DD3低表達(dá)的LNCaPDD3-細(xì)胞株的抗凋亡能力顯著低于對(duì)照組LNCaPNC細(xì)胞(P0.01)；而DD3高表達(dá)的PC3DD3+細(xì)胞的抗凋亡能力明顯高于對(duì)照組PC3Ctrl細(xì)胞(P0.01)。4.劃痕實(shí)驗(yàn)和Transwell小室檢測(cè)DD3基因?qū)η傲邢侔┘?xì)胞的遷移、侵襲能力的結(jié)果發(fā)現(xiàn)：DD3低表達(dá)的LNCaPDD3-細(xì)胞的遷移和侵襲能力明顯低于對(duì)照組LNCaPNc細(xì)胞(P0.05)；而DD3高表達(dá)的PC3DD3+細(xì)胞的遷移和侵襲能力明顯高于對(duì)照組PC3Ctrl細(xì)胞(P0.05)。5.測(cè)量裸鼠成瘤實(shí)驗(yàn)所獲得的LNCaPDD3-與LNCaPNC,PC3DD+與pC3Ctrl種植瘤的體積和重量發(fā)現(xiàn)DD3低表達(dá)的LNCaPDD3-組種植瘤體積和瘤重均小于對(duì)照組LNCaPNC(P0.01);而DD3基因高表達(dá)的PC3DD3+組種植腫瘤的體積和瘤體重量均大于對(duì)照組PC3Ctrl(P0.01)。對(duì)瘤體組織采用TUNEL法檢測(cè)組織細(xì)胞凋亡水平和免疫組化染色方法檢查細(xì)胞增殖標(biāo)志物Ki67蛋白表達(dá)水平的結(jié)果發(fā)現(xiàn)DD3低表達(dá)的LNCaPDD3-組種植腫瘤中組織細(xì)胞的凋亡水平明顯高于對(duì)照組LNCaPNC(P0.05);而腫瘤組織細(xì)胞的增殖能力也明顯小于對(duì)照組LNCaPNC(P0.05)；DD3高表達(dá)的PC3DD3+組腫瘤組織細(xì)胞的凋亡水平明顯低于對(duì)照組PC3Ctrl(P0.05);而腫瘤組織細(xì)胞的增殖能力明顯大于對(duì)照組PC3Ctrl(P0.05)。結(jié)論(1)DD3基因參與了前列腺癌細(xì)胞體外生長(zhǎng)的調(diào)控,可促進(jìn)前列腺癌細(xì)胞在體外的增殖、遷移和侵襲能力,能提高前列腺癌細(xì)胞體外抗凋亡能力。(2)DD3參與調(diào)控前列腺癌細(xì)胞的體內(nèi)成瘤能力,可以促進(jìn)前列腺癌細(xì)胞在體內(nèi)的增殖能力和提高癌細(xì)胞在體內(nèi)的抗凋亡能力。(3)進(jìn)一步深入研究DD3在前列腺癌細(xì)胞中的作用機(jī)制可為前列腺癌的早期診斷和研制新的靶向治療藥物提供重要幫助。
[Abstract]:Background Prostate cancer ( PCa ) is a common malignant tumor in male urinary system . In European and American countries , the incidence of PCa is higher than that of male malignant tumor . In recent years , the incidence rate of PCa is increasing at 10 % per year , which severely threatens the health of middle - aged and old men .
DD3 gene is a kind of prostate cancer specific gene which is highly expressed in human chromosome 9q21 - 22 . DD3 gene is not expressed in human chromosome 9q21 - 22 . DD3 gene is not expressed in human chromosome 9q21 - 22.DD3 . The effect of DD3 gene on the growth , apoptosis , metastasis and invasion of prostate cancer cells was investigated by means of cell counting and MTT assay .
The apoptosis of prostate cancer cell line LNCaPDD3 + , LNCaPNC , PC3DD + and PC3Ctrl was induced by ABL - N , and the apoptosis of early and middle and late stages of each group was detected by annexin V + PI double staining method , and the role of DD3 gene in the apoptosis of prostate cancer cells was determined .
In vivo experiments , the volume and weight of the tumor cells formed in the above - mentioned groups of cell lines were detected by the experiments in nude mice , then paraffin of prostate cancer tumor was embedded and sectioned , and the expression level of Ki - 67 protein in tumor tissue cells was detected by immunohistochemistry staining .
TUNEL method was used to detect the apoptosis level of prostate cancer tissue cells .
Results 1.q - PCR assay showed that the expression of DD3 in LNCAPDD3 - cells was significantly lower than that of control group ( P0.01 ) .
The expression level of DD3 in PC3DD3 + cells after overexpression of DD3 was significantly higher than that of control group PC3Ctrl ( P0.01 ) . The proliferation ability of LNCaPDD3 - and PC3DD + cells was demonstrated by MTT assay .
The proliferation rate of PC3DD3 + cells was significantly higher than that in control group ( P0.05 ) . The results showed that the anti - apoptotic ability of the LNCaPDD3 - cell line with low DD3 expression was significantly lower than that of the control group ( P0.01 ) .
The anti - apoptotic ability of DD3 + cells was significantly higher than that of control group PC3Ctrl ( P0.01 ) . 4 . The migration and invasion ability of DD3 gene to prostate cancer cells was significantly lower than that in control group ( P0.05 ) .
Compared with control group , the tumor volume and tumor weight of LNCaPDD3 - group were significantly higher than those in control group ( P 0.01 ) , and the tumor volume and tumor weight of PC3DD3 + group were significantly higher than those in control group ( P 0.01 ) .
Conclusion ( 1 ) DD3 gene is involved in the regulation of prostate cancer cell proliferation , migration and invasion ability , and can promote the proliferation , migration and invasion ability of prostate cancer cells in vitro .

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.25

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