本文選題：瞬時(shí)受體電位通道7(TRPM7) + 前列腺癌(PCa)�。� 參考：《安徽醫(yī)科大學(xué)》2015年博士論文

【摘要】：前列腺癌(PCa)是老年男性最常見的惡性腫瘤,在美國已經(jīng)多年占據(jù)新發(fā)惡性腫瘤病例首位,嚴(yán)重危害到男性的健康。由于PCa在早期階段缺少特異性的臨床癥狀,極易與前列腺增生相混淆,患者在因癥狀就診時(shí),往往已經(jīng)進(jìn)入晚期甚至轉(zhuǎn)移。因此,早期診斷對(duì)于PCa治療時(shí)機(jī)的把握尤為重要。目前公認(rèn)PCa篩查標(biāo)準(zhǔn)仍然是直腸指診聯(lián)合血清前列腺特異抗原(prostate specific antigen, PS A)檢測(cè),但因特異性偏低,往往能導(dǎo)致過度診斷和過度治療而受到廣泛質(zhì)疑(1)。近年來的影像學(xué)發(fā)展日新月異,新技術(shù)不斷改進(jìn),如MRI波譜分析手段,仍然對(duì)PCa的診斷正確率提高仍十分有限。臨床PCa的診斷“金標(biāo)準(zhǔn)”還是前列腺穿刺活檢,但因其有創(chuàng)性、陽性率不高、較高感染率、以及潛在并發(fā)癥風(fēng)險(xiǎn),臨床仍不是滿意的PCa診斷方法,促使廣大的科研工作者及臨床醫(yī)生努力尋求、探索新的篩選標(biāo)準(zhǔn)、辦法。在PCa早期階段的治療以根治手術(shù)為主,其后伴隨病程進(jìn)展,可以有放療和內(nèi)分泌治療以及化療的推薦,盡管有多種治療方式層出不窮,尤其新的化療藥物和二線內(nèi)分泌治療藥物不斷推出,大大延長(zhǎng)了患者的生存時(shí)間并改善生活質(zhì)量,但患者仍會(huì)不可避免地進(jìn)入到去勢(shì)抵抗性前列腺癌(Castration-resistant prostate cancer, CRPC)階段,最終短期內(nèi)發(fā)生死亡。因此,對(duì)于PCa的發(fā)生和發(fā)展的機(jī)制上的研究是探索新型PCa腫瘤標(biāo)記物、前瞻性判斷治療以及靶向治療的必要途徑。在腫瘤的研究領(lǐng)域,離子通道的探索始終方興未艾,并發(fā)現(xiàn)了一系列參與腫瘤形成的離子通道。文獻(xiàn)報(bào)道,瞬時(shí)受體電位(transient receptor potential, TRP)作為新型的Ca2+通道,參與人體眾多生理和病理過程。近年來TRP家族受到廣泛重視,其家族成員之一的瞬時(shí)受體電位通道7(transient receptor potential melastatin 7, TRPM7)更是腫瘤研究的熱點(diǎn)。多項(xiàng)研究表明,該通道蛋白在人甲狀腺腫瘤、乳腺癌、胃癌、胰腺腫瘤、成視網(wǎng)膜細(xì)胞瘤、肺癌等組織或細(xì)胞上表達(dá)有顯著升高,已經(jīng)成為浸潤(rùn)性乳腺導(dǎo)管乳腺癌的預(yù)后指標(biāo),而具有靶向治療的意義。進(jìn)而,TRPM7是否也在PCa的發(fā)生發(fā)展中發(fā)揮作用,最近也有學(xué)者在嘗試探究。本研究旨在于研究PCaPC-3細(xì)胞中,TRPM7在其中的調(diào)控及其作用機(jī)制,觀察TRPM7對(duì)TRAIL誘導(dǎo)PC-3細(xì)胞凋亡的影響,并探討其可能的作用機(jī)制。主要研究?jī)?nèi)容概括如下：1. TRPM7在前列腺癌組織中的表達(dá)為了檢測(cè)PCa組織中TRPM7是否表達(dá),通過Western blot檢測(cè)PCa組織中TRPM7的蛋白表達(dá)情況。結(jié)果表明,癌變組織中TRPM7的蛋白表達(dá)明顯高于正常組織。2. TRPM7非特異性阻斷劑及TRPM7-siRNA對(duì)PC-3細(xì)胞中的TRPM7的抑制作用采用非特異性TRPM7阻斷劑Gd3+或2-APB,阻斷中TRPM7的表達(dá),通過RT-PCR、 Western blot檢測(cè)PC-3細(xì)胞TRPM7表達(dá)水平。結(jié)果發(fā)現(xiàn),與正常組相比,Gd3+或2-APB都能夠不同程度地降低PC-3細(xì)胞中T RPM7 mRNA和蛋白的表達(dá)。另一方面,利用LipofectamineTM2000將TRPM7-siRNA轉(zhuǎn)染到PC-3細(xì)胞中,同樣觀察到TRPM7 mRNA和蛋白在TRPM7-siRNA轉(zhuǎn)染PC-3細(xì)胞中的表達(dá)明顯下調(diào)。3. TRPM7對(duì)PC-3細(xì)胞凋亡的影響采用RT-PCR、Western blot和流式細(xì)胞儀檢測(cè)方法,檢測(cè)TRPM7非特異性抑制劑及TRPM7-siRNA 對(duì) PC-3細(xì)胞的凋亡的影響。結(jié)果發(fā)現(xiàn),非特異性阻斷劑Gd3+、2-APB 或 TRPM7-siRNA刺激PC-3細(xì)胞后,能顯著促進(jìn)其凋亡。另外,還發(fā)現(xiàn)DR4、DR5 和 cleaved Caspase-3蛋白明顯增加。4. TRAIL誘導(dǎo)的PC-3細(xì)胞凋亡后對(duì)TRPM7的影響采用RT-PCR、Western blot和流式細(xì)胞儀檢測(cè)方法,觀察TRAIL誘導(dǎo)PC-3細(xì)胞凋亡后,觀察TRPM7的影響。結(jié)果發(fā)現(xiàn),TRAIL組的PC-3細(xì)胞中TRPM7的mRNA和蛋白表達(dá)明顯高于正常組。表明TRAIL誘導(dǎo)后PC-3細(xì)胞中TRPM7表達(dá)水平上升。5. TRAIL誘導(dǎo)的PC-3細(xì)胞凋亡后,阻斷TRPM7對(duì)PC-3細(xì)胞凋亡的影響采用RT-PCR、Western blot和流式細(xì)胞儀檢測(cè)方法,觀察非特異性阻斷劑Gd3+、 2-APB 或 TRPM7-siRNA對(duì)TRAIL誘導(dǎo)PC-3細(xì)胞凋亡的影響。結(jié)果發(fā)現(xiàn),TRPM7非特異性抑制劑Gd3+、2-APB 及 TRPM7-siRNA,可以明顯促進(jìn)TRAIL誘導(dǎo)PC-3細(xì)胞的凋亡活動(dòng)。同時(shí),還發(fā)現(xiàn)沉默TRPM7后,PC-3細(xì)胞中DR4、DR5 和 cleaved Caspase-3凋亡蛋白明顯激活。6. TRPM7通過AKT通路對(duì)PC-3細(xì)胞凋亡的調(diào)節(jié)機(jī)制研究采用RT-PCR及Western blot的方法,檢測(cè)TRPM7對(duì)PC-3細(xì)胞中AKT信號(hào)通路的調(diào)控作用。結(jié)果發(fā)現(xiàn),沉默TRPM7后,磷酸化AKT水平降低,表明TRPM7沉默可以調(diào)節(jié)AKT信號(hào)通路。同時(shí), AKT下游蛋白P70S6K的磷酸化水平在TRPM7沉默后降低,進(jìn)一步提示TRPM7可以正向調(diào)控AKT信號(hào)通路。以上結(jié)果表明：下調(diào)TRPM7表達(dá)可以增加PC-3細(xì)胞的凋亡,其機(jī)制可能與AKT信號(hào)通路有關(guān),這將為PCa的研究與治療提供新的思路。
[Abstract]:Prostate cancer (PCa) is the most common malignant tumor in old men. It has been the first case of new malignant tumor in the United States for many years and seriously endangers the health of men. Because of the lack of specific clinical symptoms in the early stage, PCa is very easy to be confused with the hyperplasia of prostate. Therefore, early diagnosis is particularly important for the timing of PCa treatment. It is recognized that the PCa screening standard is still the detection of prostate specific antigen (PS A) of the combined rectal finger diagnosis (prostate), but it is widely questioned because of its low specificity, which often leads to excessive diagnosis and over treatment (1). With the development of new technology and new technology, such as MRI spectrum analysis, the diagnostic accuracy of PCa is still very limited. Clinical PCa diagnosis "gold standard" or prostate biopsy, but because of its invasive, low positive rate, high infection rate, and potential complications risk, clinical still is not satisfactory PCa diagnostic method, promoting the clinical The majority of researchers and clinicians seek to explore new screening criteria. In the early stages of the PCa treatment, radical surgery is the main procedure, followed by progress in the course of the disease, which can be recommended for radiotherapy, endocrine therapy and chemotherapy, although there are a variety of treatments, especially new chemotherapeutic drugs and second line endocrinology. The continuous release of therapeutic drugs greatly prolongs the patient's survival time and improves the quality of life, but the patient will still inevitably enter the Castration-resistant prostate cancer (CRPC) stage and eventually die in the short term. Therefore, research on the mechanism of the occurrence and development of PCa is to explore new P Ca tumor markers, prospective treatment and the necessary approach to target therapy. In the field of cancer research, the exploration of ion channels is always in the ascendant, and a series of ion channels involved in the formation of tumors have been discovered. It is reported that the transient receptor potential (transient receptor potential, TRP) is a new Ca2+ channel and participates in the human body. Many physiological and pathological processes. The TRP family has been widely recognized in recent years. The transient receptor potential channel 7 (transient receptor potential melastatin 7, TRPM7) is one of the hotspots of cancer research. A number of studies have shown that this channel protein is in human goiter, breast, gastric, pancreatic, and retinoblastoma. There is a significant increase in tissue or cell expression in lung cancer, which has become a prognostic indicator of invasive mammary ductal carcinoma and has the significance of targeting therapy. Further, TRPM7 has also played a role in the development of PCa. Recently, some scholars have been trying to explore. The purpose of this study is to study the regulation and regulation of TRPM7 in PCaPC-3 cells. Its mechanism is to observe the effect of TRPM7 on the apoptosis of PC-3 cells induced by TRAIL and explore its possible mechanism. The main contents are summarized as follows: 1. TRPM7 expression in the prostate cancer tissue was used to detect the expression of TRPM7 in PCa tissue and the protein expression of TRPM7 in PCa group by Western blot. The results showed that the cancer was cancerous. The protein expression of TRPM7 in the tissue was significantly higher than that of normal tissue.2. TRPM7 non specific blocker and TRPM7-siRNA in the inhibition of TRPM7 in PC-3 cells by using non-specific TRPM7 blocker Gd3+ or 2-APB, blocking the expression of TRPM7, and detecting the expression level by RT-PCR, and found that it was compared with the normal group. Both 3+ or 2-APB can reduce the expression of T RPM7 mRNA and protein in PC-3 cells to varying degrees. On the other hand, TRPM7-siRNA is transfected into PC-3 cells by LipofectamineTM2000, and the expression of TRPM7 mRNA and protein in TRPM7-siRNA cells is obviously down regulated. Tern blot and flow cytometry were used to detect the effect of TRPM7 non specific inhibitors and TRPM7-siRNA on the apoptosis of PC-3 cells. The results showed that the non specific blocker Gd3+, 2-APB or TRPM7-siRNA stimulated the PC-3 cells to promote apoptosis significantly. Furthermore, the DR4, DR5, and cleaved proteins were significantly increased. The effect of induced PC-3 cell apoptosis on TRPM7 was detected by RT-PCR, Western blot and flow cytometry. The effect of TRPM7 was observed after TRAIL induced apoptosis of PC-3 cells. The results showed that TRPM7 mRNA and protein expression in PC-3 cells of TRAIL group was obviously higher than that of normal group. After the apoptosis of PC-3 cells induced by.5. TRAIL, the effect of blocking TRPM7 on the apoptosis of PC-3 cells was detected by RT-PCR, Western blot and flow cytometry. The effect of the non specific blocker Gd3+, 2-APB, or TRPM7-siRNA on the apoptosis of the TRAIL induced cells was observed. The apoptotic activity of PC-3 cells was obviously promoted by TRAIL. Meanwhile, after the silence of TRPM7, the apoptotic proteins of DR4, DR5 and cleaved Caspase-3 in PC-3 cells obviously activated the regulation mechanism of.6. TRPM7 through AKT pathway. The results showed that after the silence of TRPM7, the level of phosphorylated AKT decreased, indicating that the TRPM7 silence could regulate the AKT signaling pathway. At the same time, the phosphorylation level of the downstream protein P70S6K of AKT decreased after TRPM7 silencing, further suggesting that TRPM7 could positively regulate the AKT signaling pathway. The above results showed that the downregulation of TRPM7 expression could increase the decline of PC-3 cells. Its mechanism may be related to the AKT signaling pathway, which will provide new ideas for the research and treatment of PCa.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R737.25

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 杜靈彬;李輝章;毛偉敏;余傳定;汪祥輝;;2000-2009年浙江省腫瘤登記地區(qū)前列腺癌發(fā)病和死亡分析[J];中華泌尿外科雜志;2014年10期

2 蔡龍俊;張古田;;前列腺癌冷凍治療的研究進(jìn)展[J];東南大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2014年02期

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本文編號(hào)：1981012