本文選題：前列腺癌　切入點(diǎn)：上皮間質(zhì)轉(zhuǎn)化轉(zhuǎn)化　出處：《天津醫(yī)科大學(xué)》2014年碩士論文

【摘要】：在世界范圍內(nèi),前列腺癌的發(fā)病率在所有男性惡性腫瘤中位居第二位,死亡率在男性惡性腫瘤中位居第六位。前列腺癌發(fā)病率有明顯的地域和種族差異,發(fā)達(dá)國(guó)家發(fā)病率高于發(fā)展中國(guó)家。亞洲前列腺癌的發(fā)病率雖然遠(yuǎn)遠(yuǎn)低于歐美國(guó)家,但近年來(lái)呈上升趨勢(shì),且增長(zhǎng)比歐美發(fā)達(dá)國(guó)家更為迅速。早在1941年,雄激素剝奪治療(ADT)便作為前列腺癌的主要治療方式,通過(guò)阻礙雄激素與雄激素受體結(jié)合從而達(dá)到治療目的。但這種治療方式存在一個(gè)問(wèn)題：一部分經(jīng)過(guò)ADT治療的激素依賴性前列腺癌(ADPC)經(jīng)過(guò)1-2年的標(biāo)準(zhǔn)治療后轉(zhuǎn)變?yōu)槿?shì)抵抗性前列腺癌(CRPC)。在此過(guò)程中,研究發(fā)現(xiàn)腫瘤細(xì)胞的化療抵抗、轉(zhuǎn)移、侵襲及復(fù)發(fā)能力增強(qiáng),這可能與腫瘤干細(xì)胞(CSCs)增多和上皮-間質(zhì)轉(zhuǎn)化(EMT)的發(fā)生密切相關(guān)。然而有關(guān)此方面的證據(jù)仍較少。 第一部分ADT激活TGF-β1信號(hào)通路 目的：探討ADT與TGF-β1信號(hào)通路的關(guān)系 方法： 1.研究人群分為三組：ADPC、ADT和CRPC組。 2.免疫組化檢測(cè)上述三組中TGF-β1表達(dá)。 結(jié)果：三組結(jié)果比較,在CRPC組腫瘤標(biāo)本中,TGF-β1表達(dá)增高。 結(jié)論：ADPC經(jīng)ADT治療后轉(zhuǎn)變?yōu)镃RPC的過(guò)程中激活了TGF-β1信號(hào)通路。 第二部分TGF-β1誘導(dǎo)EMT發(fā)生的同時(shí)增加了CD44的表達(dá) 目的：研究TGF-β1對(duì)EMT和CD44的作用 方法：體外培養(yǎng)激素依賴性前列腺癌細(xì)胞株(LNCaP)和去勢(shì)抵抗性前列腺癌細(xì)胞株(CWR22RV1),經(jīng)TGF-β1(5ng/ml)分別刺激上述細(xì)胞后,應(yīng)用蛋白免疫印跡法(Western blot)分別檢測(cè)0、3、6、9小時(shí)CD44、p-Smad2和Smad2的表達(dá)水平；應(yīng)用Western blot分別檢測(cè)0、12、24、36、48小時(shí)E-cadherin、 vimentin的表達(dá)水平。 結(jié)果： 1.TGF-β1增加了CD44的表達(dá)。 2. TGF-β1對(duì)EMT的發(fā)生有促進(jìn)作用。 結(jié)論：TGF-β1誘導(dǎo)EMT發(fā)生的同時(shí)增加了CD44的表達(dá)。 第三部分TGF-β1通過(guò)調(diào)節(jié)CD44誘導(dǎo)EMT 目的：TGF-β1誘導(dǎo)EMT機(jī)制的研究 方法： 1.體外培養(yǎng)LNCaP和CWR22RV1細(xì)胞,將上述細(xì)胞分別分為四組。先將SB431542(TGF-β1受體抑制劑)加入第三、第四組,2小時(shí)后再向第二、第四組加入TGF-β1(5ng/ml),3小時(shí)后提取蛋白,應(yīng)用Western blot檢測(cè)四組中CD44、 p-Smad2和Smad2的表達(dá)水平。 2.體外培養(yǎng)LNCaP和CWR22RV1細(xì)胞,將上述細(xì)胞分別分為三組：對(duì)照組、實(shí)驗(yàn)組1、實(shí)驗(yàn)組2。向?qū)φ战M轉(zhuǎn)入sicontrol質(zhì)粒,向?qū)嶒?yàn)組1轉(zhuǎn)入siCD44質(zhì)粒處理24小時(shí),向?qū)嶒?yàn)組2轉(zhuǎn)入siCD44質(zhì)粒處理48小時(shí),分別提取蛋白,應(yīng)用Western blot檢測(cè)三組中CD44表達(dá)水平。 3.體外培養(yǎng)LNCaP和CWR22RV1細(xì)胞,將上述細(xì)胞分別分為四組。先將sicontrol質(zhì)粒轉(zhuǎn)入第一、第二組,將siCD44質(zhì)粒轉(zhuǎn)入第三、第四組,48小時(shí)后向第二、第四組加入TGF-β1(5ng/ml),24小時(shí)后分別提取蛋白,應(yīng)用Western blot分別檢測(cè)CD44、E-cadherin和Vimentin表達(dá)水平。 結(jié)果： 1.在LNCaP和CWR22RV1細(xì)胞中,單加TGF-β1組與對(duì)照組比較CD44表達(dá)水平明顯增高；單加SB431542組和SB431542、TGF-β1兩者都加組分別與對(duì)照組比較,CD44表達(dá)水平無(wú)明顯改變。 2.在LNCaP和CWR22RV1細(xì)胞中,轉(zhuǎn)染質(zhì)粒后CD44表達(dá)水平由高到低依次為轉(zhuǎn)入sicontrol質(zhì)粒組、轉(zhuǎn)入siCD44質(zhì)粒處理24小時(shí)組、轉(zhuǎn)入siCD44質(zhì)粒處理48小時(shí)組。 3.在LNCaP和CWR22RV1細(xì)胞中,單加TGF-β1組與對(duì)照組比較,CD44和Vimentin表達(dá)水平增高,E-cadherin表達(dá)水平降低；轉(zhuǎn)入siCD44質(zhì)粒組和轉(zhuǎn)入siCD44質(zhì)粒、TGF-β1兩者都加組分別與對(duì)照組比較,CD44、Vimentin表達(dá)水平降低、E-cadherin表達(dá)水平增高。 結(jié)論：TGF-β1通過(guò)上調(diào)CD44表達(dá)水平誘導(dǎo)EMT的發(fā)生。 第四部分前列腺癌鼠模型建立和體內(nèi)靶向抑制CD44阻礙前列腺癌轉(zhuǎn)移 目的：建立前列腺癌鼠模型并體內(nèi)研究靶向抑制CD44與前列腺癌轉(zhuǎn)移的關(guān)系。 方法： 1.體外培養(yǎng)LNCaP和CWR22RV1細(xì)胞,用鹽霉素(salinomycin)分別處理0、24、48小時(shí)后分別提取蛋白,應(yīng)用Western blot檢測(cè)CD44表達(dá)水平。 2.建立前列腺癌鼠模型,隨機(jī)分為實(shí)驗(yàn)組(腹腔注射鹽霉素)和對(duì)照組(腹腔注射玉米油)。取原位瘤及轉(zhuǎn)移灶,比較兩組間轉(zhuǎn)移情況。 3.免疫組化檢測(cè)上述兩組CD44、E-cadherin和Vimentin表達(dá)水平。 結(jié)果： 1.鹽霉素抑制CD44表達(dá)。 2.實(shí)驗(yàn)組轉(zhuǎn)移灶數(shù)量明顯少于對(duì)照組。 3.實(shí)驗(yàn)組CD44、Vimentin表達(dá)水平低于對(duì)照組,E-cadherin表達(dá)水平高于對(duì)照組。 結(jié)論：體內(nèi)靶向抑制CD44有效阻礙前列腺癌轉(zhuǎn)移和EMT的發(fā)生。
[Abstract]:In the world , the incidence of prostate cancer is the second among all male malignancies , with mortality in the sixth place among men with malignant tumors . The incidence of prostate cancer is significantly higher than in developing countries . In recent years , the incidence of prostate cancer has been higher than in developing countries . In recent years , androgen deprivation therapy ( ADT ) has been a major treatment modality for prostate cancer . In 1941 , androgen deprivation therapy ( ADT ) is a major treatment modality for prostate cancer . In 1941 , androgen deprivation therapy ( ADT ) is a major treatment modality for prostate cancer . This may be closely related to the increase in tumor cells ( CSCs ) and the occurrence of epithelial - mesenchymal transition ( EMT ) . However , there is still less evidence in this regard .

First part ADT activates TGF - 尾1 signaling pathway

Objective : To investigate the relationship between ADT and TGF - 尾1 signal pathway

Method :

1 . The study population was divided into three groups : ADPC , ADT , and group of patients .

2 . The expression of TGF - 尾1 was detected by immunohistochemistry .

Results : Compared with the three groups , TGF - 尾1 expression was increased in the tumor specimens of the patients with the tumor .

Conclusion : The TGF - 尾1 signal pathway is activated in ADPC after ADT treatment .

The second part TGF - 尾1 induces EMT and increases CD44 expression .

Objective : To study the effect of TGF - 尾1 on EMT and CD44

Methods : The expression levels of CD44 , p - Smad2 and Smad2 were detected by Western blot after stimulated with TGF - 尾1 ( 5ng / ml ) .
The levels of E - cadherin and vimentin were detected by Western blot in 0,12,24,36,48 hours respectively .

Results :

1 . TGF - 尾1 increases CD44 expression .

2 . TGF - 尾1 contributes to EMT .

Conclusion : TGF - 尾1 induced EMT increased the expression of CD44 at the same time .

The third part TGF - 尾1 regulates CD44 - induced EMT .

Objective : To study the mechanism of TGF - 尾1 induced EMT .

Method :

1 . The cells were cultured in vitro , and the cells were divided into four groups . The expression levels of CD44 , p - Smad2 and Smad2 in four groups were detected by Western blot after adding TGF - 尾1 ( 5ng / ml ) to the second and fourth groups after two hours . The expression of CD44 , p - Smad2 and Smad2 in four groups was detected by Western blot .

2 . The cells were cultured in vitro , and the cells were divided into three groups : the control group , the experimental group 1 , the experimental group 2 , the control group transferred to sicCD44 plasmid , transferred to the siCD44 plasmid for 24 hours in the experimental group , then transferred to the siCD44 plasmid for 48 hours in the experimental group , and the protein was extracted respectively , and the expression level of CD44 in the three groups was detected by Western blot .

3 . The cells were cultured in vitro , and the cells were divided into four groups . The expression level of CD44 , E - cadherin and Vimentin was detected by Western blot after the expression of siCD44 plasmid was transferred to the first and second groups . TGF - 尾1 ( 5ng / ml ) was added to the second and fourth groups after 48 hours .

Results :

1 . Compared with the control group , the expression level of CD44 was significantly higher in the LNAs and CWR22RV1 cells than in the control group .
Compared with control group , the expression level of CD44 was not changed .

2 . The expression level of CD44 after transfection was transferred from high to low into the sichuplasmid group and transferred to the siCD44 plasmid for 24 hours after transfection into the siCD44 plasmid , and then transferred to the siCD44 plasmid for 48 hours .

3 . The expression level of CD44 and Vimentin was increased and the level of E - cadherin was decreased in LNAs and CWR22RV1 cells compared with the control group .
The expression level of CD44 and Vimentin was decreased and the level of E - cadherin was increased .

Conclusion : TGF - 尾1 induced EMT by up - regulation of CD44 expression level .

The fourth part of prostate cancer murine model establishment and in vivo targeted inhibition of CD44 inhibits prostate cancer metastasis

Objective : To establish a murine model of prostate cancer and to study the relationship between CD44 and prostate cancer metastasis in vivo .

Method :

1 . The cells were cultured in vitro and treated with salinomycin for 0,24 and 48 hours respectively . The expression level of CD44 was detected by Western blot .

2 . A murine model of prostate cancer was established , which was divided into two groups : experimental group ( intraperitoneal injection of salt omycin ) and control group ( intraperitoneal injection of corn oil ) .

3 . The expression levels of CD44 , E - cadherin and vimentin were detected by immunohistochemistry .

Results :

1 . Salomycin inhibits CD44 expression .

2 . The number of foci in the experimental group was significantly lower than that of the control group .

3 . The expression level of CD44 and Vimentin in the experimental group was lower than that of the control group , and the level of E - cadherin was higher than that of the control group .

Conclusion : In vivo targeted inhibition of CD44 effectively inhibited the metastasis of prostate cancer and EMT .

【學(xué)位授予單位】：天津醫(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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