發(fā)布時(shí)間：2018-11-25 12:24

【摘要】：目的：前列腺癌（Prostatic cancer,Pca）是西方國家最常見的男性惡性腫瘤，而近年我國該病發(fā)病率亦逐年上升，大部分患者最終會(huì)進(jìn)展為非雄激素依賴的Pca，但目前尚無公認(rèn)可以有效的治療非雄激素依賴Pca的手段。因此，，尋求可以有效治療非雄激素依賴前列腺癌的新的治療手段是目前臨床迫切需要解決的問題。三氧化二砷（Arsenic trioxide，As203，ATO)已被證實(shí)對(duì)于治療復(fù)發(fā)性或難治性急性早幼粒細(xì)胞白血�。ˋPL）具有良好療效。但某些二期臨床試驗(yàn)結(jié)果發(fā)現(xiàn)單獨(dú)用ATO治療一些實(shí)體瘤，如轉(zhuǎn)移性腎細(xì)胞癌或轉(zhuǎn)移性黑色素瘤，其療效相對(duì)有限。故尋找一種輔助藥物，與ATO聯(lián)合使用，使低濃度的ATO發(fā)揮高濃度的作用效果，并降低潛在的毒副作用，很有可能是一種改進(jìn)ATO治療某些實(shí)體腫瘤的可行有效的方法。Hedgehog（Hh）信號(hào)通路晚期前列腺癌中呈高表達(dá)。最近研究表明ATO能夠通過抑制Hh信號(hào)通路下游GLI蛋白而抑制尤文肉瘤和生神經(jīng)管細(xì)胞瘤的增殖；而Smo阻滯劑Cyclopamine（CYC）亦可通過對(duì)Hh信號(hào)通路的阻滯作用而抑制前列腺癌等腫瘤的生長。故本實(shí)驗(yàn)選取非雄激素依賴前列腺癌PC3細(xì)胞為研究對(duì)象，觀察低劑量三氧化二砷聯(lián)合Cyclopamine對(duì)PC3細(xì)胞的抑制作用及其可能的分子作用機(jī)制。 方法：1.運(yùn)用免疫熒光及DAPI染色鑒定PC3細(xì)胞及PC3細(xì)胞內(nèi)HH-GLI信號(hào)通路的表達(dá)情況。2.采用MTT比色法檢測不同濃度ATO及Cyclopamine單獨(dú)或聯(lián)合使用對(duì)不同的時(shí)間點(diǎn)PC3細(xì)胞生長的抑制。3.應(yīng)用裸鼠致瘤實(shí)驗(yàn)進(jìn)一步驗(yàn)證ATO聯(lián)合Cyclopamine對(duì)PC3細(xì)胞生長的抑制效果。4.Real-time PCR法檢測ATO聯(lián)合Cyclopamine對(duì)PC3細(xì)胞內(nèi)Hh信號(hào)通路中重要分子的mRNA的表達(dá)的影響。5.檢測ATO聯(lián)合Cyclopamine對(duì)PC3細(xì)胞內(nèi)細(xì)胞凋亡及細(xì)胞周期的影響。 結(jié)果：1.免疫熒光劑DAPI染色結(jié)果顯示腫瘤增殖抗原Ki67、前列腺特異性膜抗原PSAM及HH-GLI1信號(hào)通路中重要的核內(nèi)轉(zhuǎn)錄因子GLI1均在PC3細(xì)胞中呈高表達(dá)2.MTT結(jié)果顯示ATO或Cyclopamine單獨(dú)使用均能抑制PC3細(xì)胞的生長，呈現(xiàn)明顯的劑量及時(shí)間依賴性；二者聯(lián)合使用則優(yōu)于單劑量、甚至二倍劑量的ATO或Cyclopamine，經(jīng)Chou’s公式分析二者可顯著協(xié)同抑制PC3細(xì)胞生長。3.動(dòng)物實(shí)驗(yàn)進(jìn)一步顯示ATO聯(lián)合Cyclopamine使用較單劑量組更能明顯抑制PC3細(xì)胞移植瘤的生長(p 0.05)。4.Real-time PCR分析結(jié)果顯示ATO聯(lián)合Cyclopamine使用較單劑量組更明顯下調(diào)Hh信號(hào)通路中GLI1蛋白mRNA的表達(dá)(p 0.05)。5.流式細(xì)胞術(shù)檢測結(jié)果顯示，ATO聯(lián)合Cyclopamine使用具有輕微誘導(dǎo)凋亡作用，且較單劑量組能更明顯阻滯PC3細(xì)胞于S期(p 0.01)。 結(jié)論：低劑量砷聯(lián)合Cyclopamine能協(xié)同抑制非雄激素依賴前列腺癌PC3細(xì)胞的生長，其機(jī)制與二者聯(lián)合使用可更明顯下調(diào)PC3細(xì)胞中Hh信號(hào)通路中GLI1蛋白、改變細(xì)胞周期及輕微誘導(dǎo)凋亡作用有關(guān)。故低劑量砷聯(lián)合Cyclopamine有可能成為潛在的治療非雄激素依賴前列腺癌的有效治療方式。
[Abstract]:Objective: prostate cancer (Prostatic cancer,Pca) is the most common male malignant tumor in western countries. In recent years, the incidence of prostate cancer in China has been increasing year by year. Most of the patients will eventually develop into androgen dependent Pca,. However, there is no recognized effective treatment of non-androgen-dependent Pca. Therefore, it is urgent to seek a new treatment for androgen-dependent prostate cancer. Arsenic trioxide (Arsenic trioxide,As203,ATO) has been proved to be effective in the treatment of recurrent or refractory acute promyelocytic leukemia (APL). However, some secondary clinical trials showed that the efficacy of ATO alone in the treatment of some solid tumors, such as metastatic renal cell carcinoma or metastatic melanoma, was relatively limited. Therefore, to find a kind of auxiliary drug, combined with ATO, so that low concentration of ATO can play a high concentration effect, and reduce the potential side effects. It may be a feasible and effective method to improve ATO in the treatment of some solid tumors. Hedgehog (Hh) signaling pathway is highly expressed in advanced prostate cancer. Recent studies have shown that ATO can inhibit the proliferation of Ewing's sarcoma and neurotuboma by inhibiting the downstream GLI protein of Hh signaling pathway. Smo blocker Cyclopamine (CYC) can inhibit the growth of prostate cancer and other tumors by blocking Hh signaling pathway. Therefore, the inhibitory effect of low dose arsenic trioxide combined with Cyclopamine on PC3 cells and its possible molecular mechanism were observed in non-androgen dependent prostate cancer (PC3) cells. Method 1: 1. Immunofluorescence and DAPI staining were used to identify the expression of HH-GLI signal pathway in PC3 cells and PC3 cells. 2. MTT colorimetric assay was used to detect the inhibitory effects of different concentrations of ATO and Cyclopamine on the growth of PC3 cells at different time points. The inhibitory effect of ATO combined with Cyclopamine on the growth of PC3 cells was further verified by tumorigenic assay in nude mice. The effect of ATO combined with Cyclopamine on the expression of mRNA in Hh signaling pathway in PC3 cells was detected by 4.Real-time PCR assay. 5. The effects of ATO combined with Cyclopamine on apoptosis and cell cycle of PC3 cells were detected. Results: 1. Immunofluorescence DAPI staining of tumor proliferative antigen Ki67, Both prostate-specific membrane antigen (PSAM) and GLI1, an important intracellular transcription factor in HH-GLI1 signaling pathway, were highly expressed in PC3 cells. The results showed that ATO or Cyclopamine alone could inhibit the growth of PC3 cells. There were obvious dose-dependent and time-dependent. The combination of the two groups was superior to the single dose, even the double dose of ATO or Cyclopamine, could significantly inhibit the growth of PC3 cells by Chou's formula analysis. Animal experiments further showed that ATO combined with Cyclopamine could significantly inhibit the growth of PC3 cell xenografts compared with single dose group (p0.05). 4.Real-time PCR analysis showed that ATO combined with Cyclopamine was more down-regulated than that of single dose group. Expression of GLI1 protein mRNA in Hh signaling pathway (p0.05). The results of flow cytometry showed that ATO combined with Cyclopamine could induce apoptosis slightly and block PC3 cells in S phase more obviously than single dose group (p0.01). Conclusion: low dose arsenic combined with Cyclopamine can synergistically inhibit the growth of PC3 cells with non-androgen dependent prostate cancer, and the combination of both can down-regulate the GLI1 protein in Hh signaling pathway in PC3 cells. Changes in cell cycle and slight induction of apoptosis are involved. Therefore, low dose arsenic combined with Cyclopamine may be a potential therapy for androgen-dependent prostate cancer.
【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】：2014
【分類號(hào)】：R737.25

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相關(guān)期刊論文 前2條

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