發(fā)布時間：2018-08-30 17:16

【摘要】：目的：探索HO-1在激素非依賴性前列腺癌化療抵抗中的作用及其分子機(jī)制。 在西方發(fā)達(dá)國家，前列腺癌是最常見的腫瘤，居癌死因的第二位。幾乎所有的前列腺癌患者接受雄激素去勢治療后18-24個月均會發(fā)展為激素非依賴性前列腺癌，此時轉(zhuǎn)移率很高。雖然使用了各種抗癌藥物以緩解疼痛，但總體生存率沒有得到改善，并且對標(biāo)準(zhǔn)的放化療產(chǎn)生了耐藥性。化療抵抗是影響晚期前列腺癌患者生存和生活質(zhì)量的主要障礙。因此，探索激素非依賴性前列腺癌化療抵抗中分子靶點(diǎn)和發(fā)展全新的治療策略是至關(guān)重要的。 HO-1是一種細(xì)胞保護(hù)性酶類，是體內(nèi)抵抗氧化應(yīng)激主要的防御機(jī)制。其代謝產(chǎn)物有二價鐵，一氧化碳，膽紅素。多種刺激均可誘導(dǎo)HO-1生成，如低氧，重金屬，化療藥物以及氧化應(yīng)激。HO-1具有抗炎，抗氧化，抗凋亡等生理作用。最近，一些研究結(jié)果證實(shí)相比周圍正常組織，腫瘤組織中的內(nèi)源性HO-1表達(dá)增多，如鱗癌，黑色素瘤，惡性膠質(zhì)瘤，淋巴瘤等。同時，用siRNA敲低HO-1后可抑制膀胱癌的生長。上述結(jié)果均說明HO-1是促腫瘤因子。更為重要的是，HO-1可抑制化療藥物誘導(dǎo)的凋亡，，特異性抑制HO-1基因或抑制HO-1活性可增強(qiáng)腫瘤細(xì)胞對化療藥物的敏感性。在化療藥物引發(fā)的氧化應(yīng)激環(huán)境下，腫瘤細(xì)胞本身可能上調(diào)抗氧化酶系統(tǒng)，增強(qiáng)抗凋亡能力以抵御抗癌藥物引起的氧化應(yīng)激，這可能是產(chǎn)生化療抵抗的主要原因之一，但尚缺乏有力的證據(jù)。作為強(qiáng)大的抗氧化和抗凋亡酶，抑制HO-1自理論上可以增強(qiáng)化療藥物敏感性。然而，抑制HO-1增強(qiáng)化療藥物的敏感性的確切機(jī)制尚不完全明了。 因此本研究在以往實(shí)驗(yàn)基礎(chǔ)之上，以人前列腺癌標(biāo)本和PC-3細(xì)胞為實(shí)驗(yàn)對象，通過觀察人前列腺癌標(biāo)本探索HO-1是否與臨床前列腺癌病理特征間存在統(tǒng)計學(xué)關(guān)系及其與凋亡相關(guān)蛋白BCL-2和Bax間的關(guān)系;通過觀察HO-1是否影響B(tài)CL-2、Bax、caspase-3、caspase-9及細(xì)胞色素C的表達(dá)，并分析其表達(dá)與細(xì)胞凋亡的關(guān)系，闡明HO-1在激素非依賴性前列腺癌化療抵抗中的作用及其分子機(jī)制，為臨床選擇有效治療方法提供實(shí)驗(yàn)依據(jù)。 方法：常規(guī)培養(yǎng)人激素非依賴性前列腺癌細(xì)胞（PC-3細(xì)胞），分為正常組、Znpp組、Hemin組、多西他賽組、多西他賽+Znpp組、多西他賽+Hemin組。 1免疫組化染色分析HO-1、BCL-2、Bax蛋白的表達(dá)。 2MTT法分析各實(shí)驗(yàn)組細(xì)胞存活率。 3DCFH-DA法檢測活性氧物質(zhì)（ROS）含量。 4TUNEL法檢測細(xì)胞凋亡率。 5Western blot法檢測HO-1、Bcl-2、Bax、caspase-3、caspase-9及細(xì)胞色素C蛋白含量。 結(jié)果： 1HO-1免疫組化表達(dá)與前列腺癌臨床病理特點(diǎn)間的關(guān)系。 HO-1表達(dá)量與臨床分期，術(shù)前PSA，精囊侵犯，Gleason分級呈正相關(guān)。與患者年齡，淋巴結(jié)轉(zhuǎn)移，尿道侵犯無關(guān)。 2在人前列腺癌標(biāo)本中HO-1與凋亡相關(guān)蛋白Bcl-2和Bax免疫組化表達(dá)中的關(guān)系。 Bcl-2表達(dá)隨HO-1表達(dá)增高而增高，Bax則呈相反的趨勢。 3MTT法分析Hemin和Znpp對多西他賽誘導(dǎo)的PC-3細(xì)胞毒性的作用。 4抑制HO-1增強(qiáng)多西他賽誘導(dǎo)的PC-3細(xì)胞凋亡。 5多西他賽誘導(dǎo)ROS生成以及抑制HO-1可增強(qiáng)多西他賽誘導(dǎo)ROS生成。 6多西他賽誘導(dǎo)的ROS上調(diào)Bax，下調(diào)Bcl-2表達(dá)，而HO-1則可逆轉(zhuǎn)。 7抑制HO-1增強(qiáng)多西他賽誘導(dǎo)的線粒體凋亡途徑。 結(jié)論：HO-1在前列腺癌中呈高表達(dá)趨勢，其可能是通過上調(diào)Bcl-2表達(dá)和下調(diào)Bax表達(dá),抑制細(xì)胞凋亡而促進(jìn)前列腺癌的進(jìn)展。抑制HO-1可增強(qiáng)多西他賽誘導(dǎo)的凋亡，其中潛在的分子機(jī)制為依賴于ROS誘導(dǎo)的線粒體凋亡途徑。
[Abstract]:Objective: To explore the role and molecular mechanism of HO-1 in the chemoresistance of hormone independent prostate cancer.
Prostate cancer is the most common cancer in western developed countries, ranking second in cancer deaths. Almost all prostate cancer patients develop hormone-independent prostate cancer 18-24 months after androgen castration, with a high metastasis rate. Although various anticancer drugs are used to relieve pain, the overall survival rate is not achieved. Chemotherapy resistance is a major obstacle to the survival and quality of life of patients with advanced prostate cancer. Therefore, it is essential to explore molecular targets and develop novel therapeutic strategies for hormone-independent prostate cancer chemotherapy resistance.
HO-1 is a kind of cell-protective enzymes, which is the main defense mechanism against oxidative stress in vivo. Its metabolites include divalent iron, carbon monoxide, bilirubin. A variety of stimuli can induce HO-1 production, such as hypoxia, heavy metals, chemotherapy drugs and oxidative stress. HO-1 has anti-inflammatory, anti-oxidative, anti-apoptotic and other physiological effects. Recently, some research results. It was confirmed that HO-1 expression was increased in tumor tissues, such as squamous cell carcinoma, melanoma, malignant glioma, lymphoma, and so on. Meanwhile, HO-1 knockdown by siRNA inhibited the growth of bladder cancer. Producing HO-1 gene or inhibiting the activity of HO-1 can enhance the sensitivity of tumor cells to chemotherapeutics. Under the oxidative stress environment induced by chemotherapeutics, tumor cells themselves may up-regulate the antioxidant enzyme system and enhance their anti-apoptosis ability to resist the oxidative stress induced by anticancer drugs. This may be one of the main reasons for chemotherapeutics resistance. Inhibiting HO-1, as a powerful antioxidant and antiapoptotic enzyme, can theoretically enhance chemosensitivity. However, the exact mechanism of inhibiting HO-1 to enhance chemosensitivity is not fully understood.
Therefore, based on previous experiments, this study explored whether HO-1 had statistical relationship with clinicopathological features of prostate cancer and apoptosis-related proteins BCL-2 and Bax by observing human prostate cancer specimens and PC-3 cells, and whether HO-1 affected BCL-2, Bax, caspase-Bax. 3. The expression of caspase-9 and cytochrome C, and the relationship between the expression of HO-1 and apoptosis were analyzed to elucidate the role of HO-1 in hormone-independent prostate cancer chemotherapeutic resistance and its molecular mechanism.
Methods: Human hormone-independent prostate cancer cells (PC-3) were cultured and divided into normal group, Znpp group, hemin group, docetaxel group, docetaxel + Znpp group and docetaxel + hemin group.
1 immunohistochemical staining was used to analyze the expression of HO-1, BCL-2 and Bax protein.
Cell viability was analyzed by 2MTT.
The content of reactive oxygen species (ROS) was detected by 3DCFH-DA.
Apoptosis rate was detected by 4TUNEL.
The contents of HO-1, Bcl-2, Bax, Caspase-3, caspase-9 and cytochrome C protein were detected by 5Western blot.
Result:
The relationship between the expression of 1HO-1 and clinicopathological features of prostate cancer.
HO-1 expression was positively correlated with clinical stage, preoperative PSA, seminal vesicle invasion and Gleason grade, but not with age, lymph node metastasis and urethral invasion.
2 the relationship between HO-1 and apoptosis related protein Bcl-2 and Bax expression in human prostate cancer specimens.
The expression of Bcl-2 increased with the increase of HO-1 expression, while Bax showed an opposite trend.
3MTT assay was used to analyze the effects of Hemin and Znpp on docetaxel induced PC-3 cell toxicity.
4 inhibition of HO-1 enhances docetaxel induced apoptosis in PC-3 cells.
5 docetaxel induced ROS production and inhibition of HO-1 could enhance docetaxel induced ROS production.
6 docetaxel induced ROS up-regulated Bax, down regulated Bcl-2 expression, while HO-1 reversed.
7 inhibition of HO-1 enhances docetaxel induced mitochondrial apoptosis pathway.
CONCLUSION: HO-1 is highly expressed in prostate cancer, which may promote the progression of prostate cancer by up-regulating the expression of Bcl-2 and down-regulating the expression of Bax. Inhibition of HO-1 may enhance the apoptosis induced by docetaxel. The underlying molecular mechanism is ROS-dependent mitochondrial apoptosis pathway.
【學(xué)位授予單位】：河北醫(yī)科大學(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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