TGF-β1通过调节CD44的表达促进前列腺癌上皮间质转化和转移

发布时间：2018-04-04 15:16

本文选题：前列腺癌　切入点：上皮间质转化转化　出处：《天津医科大学》2014年硕士论文

【摘要】：在世界范围内,前列腺癌的发病率在所有男性恶性肿瘤中位居第二位,死亡率在男性恶性肿瘤中位居第六位。前列腺癌发病率有明显的地域和种族差异,发达国家发病率高于发展中国家。亚洲前列腺癌的发病率虽然远远低于欧美国家,但近年来呈上升趋势,且增长比欧美发达国家更为迅速。早在1941年,雄激素剥夺治疗(ADT)便作为前列腺癌的主要治疗方式,通过阻碍雄激素与雄激素受体结合从而达到治疗目的。但这种治疗方式存在一个问题：一部分经过ADT治疗的激素依赖性前列腺癌(ADPC)经过1-2年的标准治疗后转变为去势抵抗性前列腺癌(CRPC)。在此过程中,研究发现肿瘤细胞的化疗抵抗、转移、侵袭及复发能力增强,这可能与肿瘤干细胞(CSCs)增多和上皮-间质转化(EMT)的发生密切相关。然而有关此方面的证据仍较少。第一部分ADT激活TGF-β1信号通路目的：探讨ADT与TGF-β1信号通路的关系方法： 1.研究人群分为三组：ADPC、ADT和CRPC组。 2.免疫组化检测上述三组中TGF-β1表达。结果：三组结果比较,在CRPC组肿瘤标本中,TGF-β1表达增高。结论：ADPC经ADT治疗后转变为CRPC的过程中激活了TGF-β1信号通路。第二部分TGF-β1诱导EMT发生的同时增加了CD44的表达目的：研究TGF-β1对EMT和CD44的作用方法：体外培养激素依赖性前列腺癌细胞株(LNCaP)和去势抵抗性前列腺癌细胞株(CWR22RV1),经TGF-β1(5ng/ml)分别刺激上述细胞后,应用蛋白免疫印迹法(Western blot)分别检测0、3、6、9小时CD44、p-Smad2和Smad2的表达水平；应用Western blot分别检测0、12、24、36、48小时E-cadherin、 vimentin的表达水平。结果： 1.TGF-β1增加了CD44的表达。 2. TGF-β1对EMT的发生有促进作用。结论：TGF-β1诱导EMT发生的同时增加了CD44的表达。第三部分TGF-β1通过调节CD44诱导EMT 目的：TGF-β1诱导EMT机制的研究方法： 1.体外培养LNCaP和CWR22RV1细胞,将上述细胞分别分为四组。先将SB431542(TGF-β1受体抑制剂)加入第三、第四组,2小时后再向第二、第四组加入TGF-β1(5ng/ml),3小时后提取蛋白,应用Western blot检测四组中CD44、 p-Smad2和Smad2的表达水平。 2.体外培养LNCaP和CWR22RV1细胞,将上述细胞分别分为三组：对照组、实验组1、实验组2。向对照组转入sicontrol质粒,向实验组1转入siCD44质粒处理24小时,向实验组2转入siCD44质粒处理48小时,分别提取蛋白,应用Western blot检测三组中CD44表达水平。 3.体外培养LNCaP和CWR22RV1细胞,将上述细胞分别分为四组。先将sicontrol质粒转入第一、第二组,将siCD44质粒转入第三、第四组,48小时后向第二、第四组加入TGF-β1(5ng/ml),24小时后分别提取蛋白,应用Western blot分别检测CD44、E-cadherin和Vimentin表达水平。结果： 1.在LNCaP和CWR22RV1细胞中,单加TGF-β1组与对照组比较CD44表达水平明显增高；单加SB431542组和SB431542、TGF-β1两者都加组分别与对照组比较,CD44表达水平无明显改变。 2.在LNCaP和CWR22RV1细胞中,转染质粒后CD44表达水平由高到低依次为转入sicontrol质粒组、转入siCD44质粒处理24小时组、转入siCD44质粒处理48小时组。 3.在LNCaP和CWR22RV1细胞中,单加TGF-β1组与对照组比较,CD44和Vimentin表达水平增高,E-cadherin表达水平降低；转入siCD44质粒组和转入siCD44质粒、TGF-β1两者都加组分别与对照组比较,CD44、Vimentin表达水平降低、E-cadherin表达水平增高。结论：TGF-β1通过上调CD44表达水平诱导EMT的发生。第四部分前列腺癌鼠模型建立和体内靶向抑制CD44阻碍前列腺癌转移目的：建立前列腺癌鼠模型并体内研究靶向抑制CD44与前列腺癌转移的关系。方法： 1.体外培养LNCaP和CWR22RV1细胞,用盐霉素(salinomycin)分别处理0、24、48小时后分别提取蛋白,应用Western blot检测CD44表达水平。 2.建立前列腺癌鼠模型,随机分为实验组(腹腔注射盐霉素)和对照组(腹腔注射玉米油)。取原位瘤及转移灶,比较两组间转移情况。 3.免疫组化检测上述两组CD44、E-cadherin和Vimentin表达水平。结果： 1.盐霉素抑制CD44表达。 2.实验组转移灶数量明显少于对照组。 3.实验组CD44、Vimentin表达水平低于对照组,E-cadherin表达水平高于对照组。结论：体内靶向抑制CD44有效阻碍前列腺癌转移和EMT的发生。
[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 .

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25

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