【摘要】：目的:研究NOP16表達(dá)水平改變對前列腺癌細(xì)胞體外、體內(nèi)Docetaxel化療敏感性的影響,并初步探討其分子機(jī)制。方法:運(yùn)用RT-q PCR方法檢測NOP16在PCa和BPH組織中的表達(dá)量;利用RT-q PCR及Western Blot檢測NOP16在正常前列腺細(xì)胞系RWPE-1以及PCa細(xì)胞系LNCa P和PC3中的表達(dá)量;構(gòu)建慢病毒載體,將其與PC3細(xì)胞一起培養(yǎng)轉(zhuǎn)染PC3細(xì)胞,使NOP16短發(fā)卡RNA(sh RNA)在PC3細(xì)胞內(nèi)進(jìn)行表達(dá),經(jīng)藥物篩選及進(jìn)行穩(wěn)定轉(zhuǎn)染獲得穩(wěn)定NOP16低水平的PC3細(xì)胞株,并使用RT-q PCR、Western Blot驗證轉(zhuǎn)染后細(xì)胞中NOP16的表達(dá)量;使用cellcount法和CCK-8法檢測NOP16-KDPC3細(xì)胞增殖能力的變化,使用細(xì)胞遷移遷徙實(shí)驗技術(shù)(Transwell)檢測NOP16-KDPC3細(xì)胞遷徙能力,磷脂酰絲氨酸外翻分析(Annexin V)檢測NOP16-KDPC3細(xì)胞的凋亡的變化。使用不同濃度Docetaxel處理PC3細(xì)胞24小時、48小時后CCK-8法檢測細(xì)胞活性;使用亞致死量Docetaxel處理PC3-Vector、PC3-NOP16-KD細(xì)胞,使用CCK-8檢測細(xì)胞活性。使用PC3-Vector和PC3-NOP16-KD細(xì)胞做單克隆形成實(shí)驗并且應(yīng)用Docetaxel處理,檢測細(xì)胞增殖能力;培養(yǎng)PC3-Vector和PC3-NOP16-KD細(xì)胞并且應(yīng)用Docetaxel處理后做凋亡實(shí)驗,檢測細(xì)胞凋亡情況;培養(yǎng)PC3-Vector和PC3-NOP16-KD細(xì)胞并且應(yīng)用Docetaxel處理后做細(xì)胞周期實(shí)驗,檢測細(xì)胞增殖情況;培養(yǎng)PC3-Vector和PC3-NOP16-KD細(xì)胞并且應(yīng)用Docetaxel處理后做Transwell實(shí)驗,檢測細(xì)胞遷徙情況。使用Western Blot檢測PC3、PC3-Vector和PC3-NOP16-KD三種細(xì)胞分別被0nmol/L、5nmol/L、10nmol/L三種濃度的Docetaxel處理24小時后的Caspase3含量,檢測細(xì)胞凋亡。結(jié)果:NOP16在Pca組織中表達(dá)高于BPH組織;NOP16在前列腺癌細(xì)胞系LNCa P及PC3中表達(dá)高于正常前列腺細(xì)胞系RWPE-1,而在上述兩種不同的前列腺癌細(xì)胞系比較中沒有觀察到明顯的變化;使用生物工程技術(shù),利用慢病毒作為載體將sh RNA轉(zhuǎn)染進(jìn)入PC3細(xì)胞,將NOP16表達(dá)下調(diào)。可以通過PC3細(xì)胞建立PC3-NOP16-KD穩(wěn)定表達(dá)的細(xì)胞;通過特異性sh RNA的慢病毒載體可以下調(diào)內(nèi)源性NOP16水平,借此可出現(xiàn)細(xì)胞體外增殖能力下降的現(xiàn)象;通過PC3細(xì)胞的NOP16表達(dá)降低可以抑制細(xì)胞遷徙能力;觀察PC3-NOP16-KD細(xì)胞可以發(fā)現(xiàn)與正常PC3細(xì)胞相比其明顯易發(fā)生凋亡;觀察發(fā)現(xiàn)PC3-NOP16-KD細(xì)胞內(nèi)蛋白合成水平較低。PC3細(xì)胞經(jīng)過不同濃度Docetaxel處理后,細(xì)胞的活性降低,計算其半數(shù)致死量發(fā)現(xiàn)其中IC50約在30nmol/L(24h)、20nmol/L(48h)之間;使用亞致死量濃度的Docetaxel,分別暴露PC3-Vector、PC3-NOP16-KD細(xì)胞,結(jié)果顯示:隨Docetaxel濃度逐漸加大后細(xì)胞活性呈明顯下降,NOP16-KD細(xì)胞對Docetaxel的濃度更加敏感,提示抑制NOP16可以強(qiáng)化Docetaxel對前列腺癌細(xì)胞的抑制作用;NOP16-KD組細(xì)胞克隆形成數(shù)目顯著少于對照組,與對照組相比Docetaxel處理后NOP16-KD組細(xì)胞單克隆數(shù)明顯降低,提示NOP16可能通過參與細(xì)胞增殖能力抵抗Docetaxel對細(xì)胞的損傷作用;與對照組相比NOP16-KD組細(xì)胞凋亡比率明顯較高,與對照組相比Docetaxel處理后NOP16-KD組細(xì)胞凋亡同樣明顯升高,提示抑制NOP16表達(dá)可能提高PC3細(xì)胞的Docetaxel化療敏感性;細(xì)胞周期實(shí)驗顯示NOP16-KD組細(xì)胞增殖能力低于對照組;此外,Docetaxel處理后NOP16-KD組細(xì)胞增值能力同樣顯著低于對照組。提示NOP16可能通過參與細(xì)胞增殖能力抵抗Docetaxel對細(xì)胞的抑制作用;細(xì)胞遷徙實(shí)驗結(jié)果顯示NOP16-KD組細(xì)胞遷徙能力低于對照組,Docetaxel處理后NOP16-KD組細(xì)胞遷徙能力同樣顯著低于對照組,提示NOP16可能通過參與細(xì)胞遷徙能力抵抗Docetaxel對細(xì)胞的抑制作用;PC3-NOP16-KD組Caspase3含量大于對照組,此外經(jīng)過Docetaxel處理后Caspase3含量呈現(xiàn)Docetaxel劑量依賴性增加明顯大于對照組,說明NOP16表達(dá)異常與Docetaxel化療可以協(xié)同作用誘發(fā)凋亡。裸鼠成瘤實(shí)驗證實(shí)NOP16-KD細(xì)胞成瘤體積小于對照組,Docetaxel化療后NOP16-KD組小鼠腫瘤體積進(jìn)一步縮小,體內(nèi)實(shí)驗顯示NOP16能夠顯著提高Docetaxel化療敏感性。結(jié)論:NOP16異常表達(dá)可改變前列腺癌細(xì)胞的體外生物學(xué)行為,體外、體內(nèi)實(shí)驗顯示NOP16-KD可以提高Docetaxel化療敏感性。NOP16具有成為基因治療前列腺癌的潛在靶點(diǎn)。
[Abstract]:Objective: To study the effect of NOP16 expression on the sensitivity of prostate cancer cells to Docetaxel chemotherapy in vitro and in vivo, and to explore its molecular mechanism.Methods: The expression of NOP16 in PCa and BPH tissues was detected by RT-q PCR, and the expression of NOP16 in normal prostate cell line RWPE-1 and PCa cell line L was detected by RT-q PCR and Western Blot. The expression of NOP16 short hairpin RNA (sh RNA) in PC3 cells was obtained by drug screening and stable transfection, and the expression of NOP16 in PC3 cells was verified by RT-q PCR and Western Blot. Cell count assay and CCK-8 assay were used to detect the proliferation of NOP16-KDPC3 cells, cell migration assay (Transwell) was used to detect the migration ability of NOP16-KDPC3 cells, and phosphatidylserine valgus assay (Annexin V) was used to detect the apoptosis of NOP16-KDPC3 cells. Cell viability was measured by CCK-8 assay hours later; PC3-Vector and PC3-NOP16-KD cells were treated with sublethal dose of Docetaxel, and the cell viability was detected by CCK-8 assay. Monoclonal formation experiments were performed with PC3-Vector and PC3-NOP16-KD cells, and cell proliferation was detected by Docetaxel treatment; PC3-Vector and PC3-NOP16-KD cells were cultured with Docetax. Cell proliferation was detected by cell cycle assay after treatment with Docetaxel; PC3-Vector and PC3-NOP16-KD cells were cultured with Docetaxel; PC3-Vector and PC3-NOP16-KD cells were cultured with Docetaxel and Transwell assay was performed after treatment with Docetaxel. The expression of NOP16 in PC3, PC3-Vector and PC3-NOP16-KD cell lines LNCa P and PC3-NOP16-KD was higher than that in normal prostate cell lines RWPE-1 and NOP16 was higher than that in Pca and PC3-NOP16-KD cell lines RWPE-1. No significant changes were observed in the two different prostate cancer cell lines mentioned above; sh RNA was transfected into PC3 cells using lentiviruses as vectors by biotechnology and NOP16 expression was down-regulated. PC3-NOP16-KD stably expressed cells could be established by PC3 cells; and sh RNA-specific lentiviral vectors could be down-regulated. The level of endogenous NOP16 could induce the decrease of cell proliferation in vitro; the decrease of NOP16 expression in PC3 cells could inhibit cell migration; the apoptosis of PC3-NOP16-KD cells could be observed in comparison with normal PC3 cells; the protein synthesis level of PC3-NOP16-KD cells was lower than that of normal PC3 cells. The activity of PC3-Vector and PC3-NOP16-KD cells decreased after treatment with different concentrations of Docetaxel, and the half lethal dose of IC50 was found to be between 30 nmol/L (24 h) and 20 nmol/L (48 h). PC3-Vector and PC3-NOP16-KD cells were exposed to sublethal concentration of Docetaxel, respectively. Cells were more sensitive to the concentration of Docetaxel, suggesting that inhibiting NOP16 could enhance the inhibitory effect of Docetaxel on prostate cancer cells; the number of cell clones in NOP16-KD group was significantly less than that in control group, and the number of monoclonal cells in NOP16-KD group was significantly lower than that in control group, suggesting that NOP16 might participate in cell proliferation by inhibiting the concentration of Docetaxel. Compared with the control group, the apoptosis rate of NOP16-KD group was significantly higher, and the apoptosis rate of NOP16-KD group was also significantly higher, suggesting that inhibiting the expression of NOP16 might increase the sensitivity of PC3 cells to Docetaxel chemotherapy. In addition, the proliferation of NOP16-KD cells after Docetaxel treatment was also significantly lower than that of the control group. NOP16 may be involved in the ability of cell proliferation to resist the inhibition of Docetaxel on cells. Cell migration test showed that NOP16-KD cells migration ability was lower than that of the control group, and NOP16-KD cells were fine after Docetaxel treatment. Cell migration ability was also significantly lower than that of the control group, suggesting that NOP16 might be able to resist the inhibition of Docetaxel by participating in cell migration ability; the content of Caspase 3 in PC3-NOP16-KD group was higher than that of the control group; moreover, the content of Caspase 3 in Docetaxel-treated group was significantly higher than that in the control group, suggesting that the expression of NOP16 was different. The tumor-forming volume of NOP16-KD cells in nude mice was smaller than that of the control group. The tumor volume of NOP16-KD group was further reduced after Docetaxel chemotherapy. In vivo experiments showed that NOP16 could significantly increase the sensitivity of Docetaxel chemotherapy. Conclusion: The abnormal expression of NOP16 could change prostate cancer cells. Biological behavior in vitro and in vivo experiments have shown that NOP16-KD can increase the sensitivity of Docetaxel to chemotherapy. NOP16 may be a potential target for gene therapy of prostate cancer.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R737.25

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9 王世超;干擾EGFR對胃癌AGS細(xì)胞腫瘤生物學(xué)行為及化療敏感性的影響[D];鄭州大學(xué);2016年

10 趙倩;局部晚期非小細(xì)胞肺癌患者血清miR-23a表達(dá)與放化療敏感性相關(guān)研究[D];濟(jì)南大學(xué);2016年

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