本文選題：TRPM8 + TRPA1��； 參考：《武漢大學(xué)》2014年博士論文

【摘要】：前列腺癌是泌尿外科最常見的惡性腫瘤之一,對男性健康威脅巨大。全球范圍內(nèi),前列腺癌的發(fā)病率占據(jù)男性所有惡性腫瘤發(fā)病率的第一位,高達28%,是導(dǎo)致男性癌癥死亡的第二大誘因(11%)。在不同的地區(qū)和種族中前列腺癌的發(fā)病率有所不同,例如在美國,前列腺癌已經(jīng)超過了肺癌,躍居腫瘤威脅男性健康的首位。在我國,最初前列腺癌發(fā)病率較低,但是隨著人民生活水平的不斷改善及因此導(dǎo)致的飲食結(jié)構(gòu)的變化、社會發(fā)展所致人口老齡化的加劇以及診療技術(shù)的不斷進步,前列腺癌的檢出率逐年增加,呈現(xiàn)出了驚人的升高趨勢。在前列腺癌早期即雄激素依賴階段,癌細(xì)胞的生存及增值依賴雄激素,所以抗雄激素治療可以顯著的抑制腫瘤的生長及發(fā)展,但是抗雄激素治療并不能阻止前列腺癌最終進展到雄激素非依賴階段。腫瘤的復(fù)發(fā)及廣泛轉(zhuǎn)移將最終導(dǎo)致前列腺癌患者的死亡,而復(fù)發(fā)和轉(zhuǎn)移同時也是治療的難點。在全球范圍內(nèi)還沒有有效的治療雄激素非依賴性前列腺癌的方法。 Ca2+作為細(xì)胞內(nèi)第二信使參與了細(xì)胞增殖和凋亡這兩個相互矛盾的過程,Ca2+平衡失調(diào)在腫瘤的發(fā)生、發(fā)展中起至關(guān)重要的作用,因而Ca2+通道具有非常高的潛能成為腫瘤治療的新靶點。瞬時受體電位(TRP)家族中具有Ca2+通透性的成員也因此受到了大家的廣泛關(guān)注,其中一個重要的成員就是trpm8最初被稱為trp-p8。trpm8的轉(zhuǎn)錄產(chǎn)物是陽離子通道蛋白TRPM8,該通道對Ca2+具有透過性的,可以被低溫和薄荷醇激活,參與細(xì)胞內(nèi)ca2+濃度的調(diào)控[5-7]。在正常組織中,只有感覺神經(jīng)和前列腺中可以檢測到大量的trpm8表達,因此它被認(rèn)為是具有前列腺特異性的基因。trpm8在前列腺癌中的表達較正常前列腺相比顯著增高,并且其表達水平與前列腺癌分級密切相關(guān),因此有研究者認(rèn)為trpm8基因具有作為一種診斷指標(biāo)和瘤標(biāo)的潛力。這種TRPM8的表達變化也許與細(xì)胞Ca2+平衡失調(diào)以及腫瘤的發(fā)生、發(fā)展有關(guān),但TRPM8的確切生理功能還不清楚。Henshall等人的研究顯示TRPM8的表達可能受雄激素的調(diào)控,因為抗雄激素治療能顯著的降低其在前列腺癌中的表達。而小片段干擾RNA(siRNA)沉默前列腺上皮細(xì)胞內(nèi)TRPM8表達的實驗則表明它的沉默能誘導(dǎo)細(xì)胞凋亡,這充分證明了TRPM8對維持前列腺上皮細(xì)胞內(nèi)Ca2+穩(wěn)態(tài)具有至關(guān)重要的作用,而且與siRNA效果相似,TRPM8通道阻斷劑也能誘導(dǎo)前列腺癌LNCaP細(xì)胞的凋亡,這些結(jié)果說明了TRPM8通道是前列腺上皮細(xì)胞存活所必需的。 薄荷醇(mentho1)是自然界的一種化合物,提取自植物薄荷。因為可以給人帶來涼爽的感覺,薄荷醇被廣泛的應(yīng)用于化妝品、調(diào)味品及制藥業(yè)。薄荷醇具有止癢、止痛、刺激和抗炎等作用,可用于治療頭痛、神經(jīng)痛、聲音嘶啞等,在非處方用藥中薄荷醇的使用濃度高達16%[15]。研究證實薄荷醇是TRPM8通道蛋白特異性的激動劑,TRPM8通道蛋白在感覺神經(jīng)中大量表達,可被低溫激活,激活后給人體帶來涼爽的感覺,至此薄荷醇誘導(dǎo)涼爽感覺的機制才得以揭示[16,17]。另外有報道稱薄荷醇可以誘導(dǎo)前列腺癌雄激素依賴性LNCaP細(xì)胞發(fā)生持續(xù)性的Ca2+內(nèi)流,從而導(dǎo)致細(xì)胞凋亡。近年來隨著研究的深入,有研究發(fā)現(xiàn)薄荷醇對于TRPA1通道具有雙峰作用,即在低濃度時激活該通道,而在高濃度時抑制該通道[18,19]。TRPA1是TRP家族中另一位冷感覺感受器,與TRPM8的同源性較低,對于該通道的研究主要集中于溫度感受,較少涉及腫瘤。 本課題擬在DU145細(xì)胞中討論TRPM8的功能；具體的方法是使用薄荷醇激動TRPM8通道,以此作為干預(yù)因素觀察DU145細(xì)胞的生長增殖能力、運動能力和凋亡的變化,從而初步探索TRPM8通道在雄激素非依賴性前列腺癌中的功能以及作為治療靶點的可能性,為開發(fā)雄激素非依賴性前列腺癌治療藥物以及基因治療前列腺癌提供理論基礎(chǔ)。 第一部分雄激素非依賴性前列腺癌DU145細(xì)胞中TRPM8和TRPAl的表達及活性的研究 目的：檢測TRPM8和TRIA1在雄激素非依賴性前列腺癌DU145細(xì)胞中是否表達,以及薄荷醇是否通過作用于TRPM8通道來影響DU145細(xì)胞的生物學(xué)行為。 方法：通過RT-PCR實驗、Western blot實驗和免疫組織化學(xué)實驗從不同水平例如mRNA水平、蛋白水平以及直觀觀察層面檢測TRPM8和TRPA1在DU145細(xì)胞中的表達；通過Ca2+成像實驗觀察薄荷醇能否誘導(dǎo)DU145細(xì)胞內(nèi)Ca2+濃度的變化,以此判斷DU145細(xì)胞中的TRPM8通道是否具有生物活性。 結(jié)果：RT-PCR結(jié)果顯示TRPM8在雄激素非依賴性前列腺癌DU145細(xì)胞中有表達,而且發(fā)現(xiàn)可被薄荷醇激活的TRPA1在DU145細(xì)胞中表達缺失。PCR產(chǎn)物純化后進行DNA序列測定,將測序所得結(jié)果運用BLAST與GenBank數(shù)據(jù)庫中發(fā)布序列進行序列分析,結(jié)果顯示高度相同。為進一步驗證PCR結(jié)果,我們采用Western blot和免疫組織化學(xué)法檢測了DU145細(xì)胞中TRPM8和TRPA1的蛋白表達,結(jié)果證實TRPM8在DU145細(xì)胞中有大量表達,而TRPA1也未檢測到表達。為了驗證DU145細(xì)胞中表達的TRPM8通道是否具有活性,我們采用熒光染色Ca2+成像法檢測薄荷醇對TRPM8通道的激活能力。結(jié)果顯示,在未添加薄荷醇干預(yù)時,DU145細(xì)胞中有極低量的熒光,隨著薄荷醇的滴入,細(xì)胞中熒光亮度急劇升高,具有典型的TRPM8通道激活曲線,由此可證明DU145細(xì)胞中確有TRPM8通道蛋白的表達,且具有生物活性；薄荷醇可激活TRPM8通道引起Ca2+內(nèi)流。 結(jié)論：雄激素非依賴性前列腺癌DU145細(xì)胞中有大量的具有生物活性的TRPM8通道蛋白表達,而可同被薄荷醇激活的TRPA1卻未被檢測到表達。薄荷醇可激活DU145細(xì)胞中的TRPM8通道誘導(dǎo)Ca2+內(nèi)流。第二部分薄荷醇激活TRPM8通道對雄激素非依賴性前列腺癌DU145細(xì)胞生物學(xué)行為影響的研究 目的：以薄荷醇作為干預(yù)因素,觀察TRPM8通道的激活對DU145細(xì)胞的增殖、遷移/侵襲和凋亡的產(chǎn)生何種影響,從而初步探索TRPM8通道在雄激素非依賴性前列腺癌中的功能以及作為治療靶點的可能性,期望以TRPM8為突破口找尋治療雄激素非依賴性前列腺癌的新途徑。 方法：采用MTT法檢測梯度濃度的薄荷醇對DU145細(xì)胞增殖能力的影響,篩選出合適的薄荷醇濃度以備后用。而對于薄荷醇對細(xì)胞周期以及遷移/侵襲的影響則分別采用流式細(xì)胞周期實驗、劃痕實驗和transwell實驗來檢測。Hochest33258染色及流式細(xì)胞凋亡實驗檢測薄荷醇對DU145細(xì)胞凋亡的影響。Western blot檢測周期相關(guān)蛋白Cdk2、Cdk4、Cdk6及遷移相關(guān)蛋白磷酸化FAK(FAK-pY-397). 結(jié)果：MTT實驗結(jié)果顯示,與未經(jīng)薄荷醇處理的細(xì)胞相比,不同濃度(25,50,75,100μM)薄荷醇處理組的細(xì)胞數(shù)量明顯下降。將未處理的細(xì)胞設(shè)定為100%,則處理組與其相比分別為100%±2.68%VS90.66%±6.60%,82.78%±7.24%,70.12%±9.96%,53.41%±6.45%,25μM組p0.05其余組p0.01。細(xì)胞經(jīng)TRPM8通道阻斷劑BCTC預(yù)處理20分鐘后,薄荷醇所致的細(xì)胞增殖抑制被部分恢復(fù)(p0.01)。流式細(xì)胞周期檢測結(jié)果顯示,與空白組相比,細(xì)胞經(jīng)100μM薄荷醇處理24h(p0.05)、48h和72h(p0.01)后,其處于Go/G1期的細(xì)胞量顯著增加(49.12%±1.92%VS61.71%±2.70%,77.65%±1.63%,71.81%±2.46%),然而流式細(xì)胞凋亡檢測及Hochest33258染色實驗提示,100μM薄荷醇處理并未引起細(xì)胞凋亡。劃痕實驗提示,將空白組的遷移率設(shè)定為100%,則薄荷醇處理24h和48h組與之相比分別為100%VS58.62%±11.55%和48.21%±11.11%。細(xì)胞經(jīng)TRPM8通道阻斷劑BCTC預(yù)處理20分鐘后,薄荷醇誘導(dǎo)的細(xì)胞遷移抑制被部分恢復(fù)(p0.05)。Transwell實驗顯示,薄荷醇抑制了細(xì)胞的侵襲能力,且Western blot檢測發(fā)現(xiàn)周期相關(guān)蛋白Cdk2、Cdk4、Cdk6及遷移相關(guān)蛋白磷酸化FAK表達量顯著下調(diào)。 結(jié)論：薄荷醇通過激活TRPM8通道抑制了DU145細(xì)胞的增殖和遷移、侵襲能力,但未誘導(dǎo)細(xì)胞凋亡。實驗結(jié)果證實了通過激活TRPM8通道治療雄激素非依賴性前列腺癌的可能性,為TRPM8靶向治療激素抵抗型前列腺癌提供了理論依據(jù)。
[Abstract]:Prostate cancer is one of the most common malignant tumors in the Department of Urology and has a great threat to male health. The incidence of prostate cancer is the first of all the male malignant tumors worldwide, up to 28%, which is the second major cause of male cancer death (11%). The incidence of prostate cancer in different regions and races has been found. Different, for example, in the United States, prostate cancer has exceeded lung cancer and is leaping cancer to the top of the male health. In China, the incidence of prostate cancer is low in the country, but with the improvement of the living standard of the people and the change of the diet structure, the aging of the population caused by the social development and the continuous progress of diagnosis and treatment technology. In the early stage of prostate cancer, the survival and increment of cancer cells depend on androgens, so anti androgen therapy can significantly inhibit the growth and development of cancer, but anti androgen therapy does not prevent the final progression of prostate cancer. The recurrence and extensive metastasis of the tumor will eventually lead to the death of the prostate cancer patients, and the recurrence and metastasis are also the difficulty of the treatment. There is no effective treatment for androgen independent prostate cancer worldwide.
Ca2+, as the second messenger within the cell, participates in the two contradictory processes of cell proliferation and apoptosis, and the imbalance of Ca2+ plays a vital role in the development of the tumor. Therefore, the Ca2+ channel has a very high potential to be a new target for cancer treatment. The members of the transient receptor potential (TRP) family with Ca2+ permeability are also the cause of the disease. It has attracted wide attention. One of the important members is that TRPM8, which was originally called trp-p8.trpm8, is a cationic channel protein TRPM8, which is transmissive to Ca2+, can be activated by low temperature and menthol, and participates in the regulation of the intracellular ca2+ concentration of [5-7]. in normal tissues, only sensory nerves and prostaglandin A large number of TRPM8 expressions can be detected in the glands, so it is believed that the expression of the prostate specific gene.Trpm8 is significantly higher in the prostate cancer than in the normal prostate, and the expression level is closely related to the classification of the prostate cancer. Therefore, some researchers believe that the TRPM8 gene is a diagnostic marker and a tumor marker. Potential. Changes in the expression of this TRPM8 may be related to the imbalance of Ca2+ balance and the development of tumor, but the exact physiological function of TRPM8 is not clear. The study of.Henshall et al. Shows that the expression of TRPM8 may be regulated by androgens, because anti androgen therapy can significantly reduce the expression of TRPM8 in prostate cancer. The effect of RNA (siRNA) silence on the expression of TRPM8 in the epithelial cells of the prostate showed that its silence could induce apoptosis, which fully demonstrated that TRPM8 played a crucial role in maintaining Ca2+ homeostasis in the prostate epithelial cells, and was similar to the effect of siRNA. The TRPM8 channel inhibitor could also induce the apoptosis of LNCaP cells in prostate cancer. These results indicate that TRPM8 channels are necessary for the survival of prostatic epithelial cells.
Menthol (mentho1) is a natural compound extracted from plant menthol. Because it can bring a cool feeling to people, menthol is widely used in cosmetics, condiments and pharmaceuticals. Menthol has antipruritic, analgesic, irritation and anti-inflammatory effects, which can be used to treat headache, neuralgia, hoarseness and so on. It is thinner in non prescription drugs. The use of the concentration of alcohols up to 16%[15]. confirmed that menthol is a specific excitant of TRPM8 channel protein. The TRPM8 channel protein is expressed in a large number of sensory nerves, which can be activated at low temperature and activate to bring cool feeling to the human body. At this point, the mechanism of inducing cool sensation by menthol can reveal [16,17]. in addition to menthol. The prostatic cancer androgen dependent LNCaP cells can induce persistent Ca2+ inflow and lead to cell apoptosis. In recent years, some studies have shown that menthol has the effect on the TRPA1 channel in Shuangfeng, that is to activate the channel at low concentration, and the inhibition of the channel [18,19].TRPA1 at the high concentration is the other one in the TRP family. The location of cold sensation receptors is lower than that of TRPM8. The study of this channel mainly focuses on temperature perception and less on tumors.
The purpose of this study is to discuss the function of TRPM8 in DU145 cells. The specific method is to use menthol to stimulate the TRPM8 channel as an intervention factor to observe the growth and proliferation of DU145 cells, exercise ability and apoptosis, so as to explore the function of TRPM8 channel in androgen independent prostate cancer and to be a therapeutic target. It provides a theoretical basis for the development of androgen independent prostate cancer drugs and gene therapy for prostate cancer.
Part one: expression and activity of TRPM8 and TRPAl in androgen independent prostate cancer DU145 cells
Objective: to detect the expression of TRPM8 and TRIA1 in androgen independent prostate cancer DU145 cells and whether menthol affects the biological behavior of DU145 cells by acting on the TRPM8 channel.
Methods: the expression of TRPM8 and TRPA1 in DU145 cells was detected at different levels, such as mRNA level, protein level and visual observation at different levels, such as mRNA level, protein level and visual observation at different levels, such as the level of mRNA, protein level and visual observation, and the changes in the concentration of Ca2+ in DU145 cells were observed by the Ca2+ imaging experiment to judge the DU145 cells by the RT-PCR experiment and the immunohistochemical experiment. Whether the TRPM8 channel has biological activity.
Results: RT-PCR results showed that TRPM8 was expressed in androgen independent prostate cancer DU145 cells, and it was found that the deletion of.PCR products expressed by menthol activated TRPA1 in DU145 cells was purified to carry out DNA sequencing. The sequencing results were analyzed with the sequence of BLAST and GenBank data base. In order to further verify the PCR results, we detected the protein expression of TRPM8 and TRPA1 in DU145 cells by using Western blot and immunohistochemistry. The results confirmed that TRPM8 was expressed in DU145 cells, and TRPA1 was not detected. In order to verify whether TRPM8 channels expressed in DU145 cells were active, we picked up the activity of TRPM8 in the DU145 cells. The activation ability of menthol to TRPM8 channel was detected by fluorescein Ca2+ imaging. The results showed that there was a very low fluorescence in DU145 cells without menthol intervention. With the drop of menthol, the luminosity of the cells increased sharply, with a typical TRPM8 channel activation curve, which proved that there was a TRPM8 channel in DU145 cells. Protein expression and bioactivity; menthol can activate TRPM8 channel to cause Ca2+ influx.
Conclusion: a large number of bioactive TRPM8 channel proteins are expressed in androgen independent prostate cancer DU145 cells, but the expression of TRPA1, which is activated by menthol, is not detected. Menthol activates the TRPM8 channel in DU145 cells to induce the Ca2+ inflow. The second part of the TRPM8 channel activates the TRPM8 channel and is not dependent on the androgen. Study on the biological behavior of prostate cancer DU145 cells
Objective: To observe the effect of TRPM8 channel activation on the proliferation, migration / invasion and apoptosis of DU145 cells by using menthol as an intervention factor, so as to explore the function of TRPM8 channel in androgen independent prostate cancer and the possibility of as a target for treatment. It is expected to search for the treatment of androgen by TRPM8 as a breakthrough. A new way to rely on prostate cancer.
Methods: the effect of the gradient concentration of menthol on the proliferation of DU145 cells was detected by MTT method, and a suitable concentration of menthol was screened for subsequent use. The effects of menthol on cell cycle and migration / invasion were measured by flow cytometry, scratch test and Transwell test to detect.Hochest33258 staining and flow. The effect of menthol on apoptosis of DU145 cells by apoptosis assay,.Western blot detection of cycle related protein Cdk2, Cdk4, Cdk6 and phosphorylation of migration related proteins FAK (FAK-pY-397).
Results: the results of MTT test showed that the number of cells in the menthol treatment group with different concentrations (25,50,75100 mu M) decreased significantly compared with those without menthol treatment. The untreated cells were set to 100%, and the treatment group was 100% + 2.68%VS90.66% + 6.60%, 82.78% + 7.24%, 70.12% + 9.96%, 53.41% + 6.45%, 25 mu M P0.05, respectively. The cell proliferation inhibition induced by menthol was partially restored after 20 minutes of preconditioning with TRPM8 channel blocker BCTC for 20 minutes (P0.01). Flow cytometry showed that cell volume in Go/G1 phase increased significantly after 24h (P0.05), 48h and 72h (P0.01) treated with 100 mu M menthol (49.12% + 1.92%VS). 61.71% + 2.70%, 77.65% + 1.63%, 71.81% + 2.46%), however, flow cytometry and Hochest33258 staining showed that 100 - M menthol treatment did not cause cell apoptosis. The scratch test suggested that the mobility of the blank group was set to 100%, then the menthol treatment 24h and 48h group were 100%VS58.62% + 11.55% and 48.21% + 11.11%., respectively. After the cells were pretreated with TRPM8 channel blocker BCTC for 20 minutes, the cell migration inhibition induced by menthol was partially restored (P0.05).Transwell experiment showed that menthol inhibited the cell invasiveness, and the Western blot detection found that the cycle related protein Cdk2, Cdk4, Cdk6 and ex migration related protein phosphorylation FAK expression decreased significantly.
Conclusion: Menthol inhibits the proliferation, migration and invasion of DU145 cells by activating the TRPM8 channel, but does not induce apoptosis. The experimental results confirm the possibility of the treatment of androgen independent prostate cancer by activating the TRPM8 channel, which provides a theoretical basis for the targeted treatment of hormone resistant prostate cancer by TRPM8.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R737.25

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3 梁本恒;譚穎堯;;藥物異構(gòu)體分離研究——薄荷醇與異薄荷醇的分離[J];廣東醫(yī)藥學(xué)院學(xué)報;1989年01期

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7 許衛(wèi)銘,王暉,馮小龍;不同濃度薄荷醇對甲硝唑透皮吸收的影響[J];中藥藥理與臨床;2001年03期

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9 曾常青;鄧金梅;陳家發(fā);;毛細(xì)管氣相色譜法測定復(fù)方硼酸散中薄荷醇和苯酚的含量[J];現(xiàn)代食品與藥品雜志;2007年03期

10 王暉，許衛(wèi)銘，王宗銳;薄荷醇對柴胡鎮(zhèn)痛成分表觀生物利用度的影響[J];中成藥;1996年06期

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4 林建;朱立麒;秦濤;庾慶華;楊倩;;DMSO和薄荷醇增強脂質(zhì)體介導(dǎo)的Bcap-37細(xì)胞轉(zhuǎn)染效率的研究[A];中國畜牧獸醫(yī)學(xué)會動物解剖學(xué)及組織胚胎學(xué)分會第十七次學(xué)術(shù)研討會論文集（下）[C];2012年

5 蔣木庚;楊紅;劉合群;周晨芳;;天然旋光性中華薄荷醇的研究[A];江蘇省農(nóng)藥學(xué)術(shù)研討會論文集[C];1997年

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2 周s，

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