【作者】 陈俊；

【导师】 胡晓勇；

【作者基本信息】 上海交通大学 ， 外科学（泌尿外）， 2018， 硕士

【摘要】 研究目的:1、研究组胺信号通路相关分子-组氨酸脱羧酶(HDC)和组胺受体分子在前列腺癌中表达情况及意义;2、研究组胺H3受体(H3R)对前列腺癌细胞增殖、转移和凋亡的影响及机制。研究方法:1、应用免疫组织化学染色法检测前列腺癌组织以及正常前列腺组织组氨酸脱羧酶表达水平;2、运用RT-PCR及western blot检测组氨酸脱羧酶和组胺受体在前列腺癌细胞中的表达;3、应用免疫组织化学方法检测前列腺癌组织和正常前列腺组织H3R的表达情况;4、使用特异性H3R的激动剂、拮抗剂来调节H3R的功能或者构建干扰组胺H3受体表达的载体,通过瞬时转染法在内源性H3R高表达的细胞株下调H3R的表达;5、进行体外侵袭实验和迁移实验,检测H3R对前列腺癌细胞侵袭力和迁移力的影响;6、利用CCK-8细胞增殖实验检测H3R对前列腺癌细胞增殖的影响;7、利用流式细胞技术,采取细胞凋亡检测试剂盒检测H3R对前列腺癌细胞凋亡的影响;8、利用western blot检测抑制H3R后凋亡相关蛋白的表达情况和下游可能发生变化的蛋白情况;9、通过裸鼠皮下成瘤实验观察H3R对前列腺癌细胞成瘤能力的影响。研究结果:1、免疫组织化学染色结果表明,前列腺癌组织中的组氨酸脱羧酶染色的强度明显高于正常的前列腺组织(P<0.05);2、m RNA和蛋白水平检测结果提示,前列腺癌细胞株(PC-3、LNCa P、22RV1及C4-2)组氨酸脱羧酶和4种组胺受体的表达水平同样明显高于对照细胞株RWPE-1,其中以H3R升高最显著(P<0.05);3、免疫组化结果显示前列腺癌组织中H3R染色强度明显高于正常前列腺组织,Gleason评分高的组织比Gleason评分低的组织升高更显著(P<0.05);4、体外迁移和侵袭实验表明干扰H3R的功能之后,前列腺癌细胞的迁移和侵袭能力均下降;5、体外细胞增殖实验表明,干扰H3R的功能之后,前列腺癌细胞的增殖能力明显下降(P<0.05);6、流式检查结果显示,干扰H3R的功能之后,前列腺癌细胞的凋亡明显增强;7、Western blot检测发现,干扰H3R的功能之后,凋亡相关蛋白BAX明显增加,抗凋亡蛋白BCL2明显降低,AR表达明显降低;8、裸鼠皮下成瘤实验显示,干扰H3R的功能之后,前列腺癌细胞的成瘤能力及肿瘤生长能力明显降低(P<0.05)。结论:1、前列腺癌中HDC和4种组胺受体表达明显上调(P<0.05);2、调节组胺受体H3R可影响雄激素受体AR的表达;3、组胺受体H3R信号通路对前列腺癌细胞的增殖、转移和凋亡有重要影响。综上,H3R在前列腺癌中可能起到促癌的作用,因此干扰前列腺癌中H3R的功能可能成为一种新的治疗策略。更多还原

【Abstract】 Objective: 1.To study the expression of histamine signal related molecules-histidine decarboxylase and histamine receptor in prostate cancer;2.To investigate the effect of histamine h3 receptor(H3R)on the proliferation,metastasis and apoptosis of prostate cancer cells and its mechanism.Methods: 1.The expression of histidine decarboxylase in prostate cancer tissues and normal prostatic tissues was detected by immunohistochemistry;2.The expressions of histidine decarboxylase and histamine receptor in prostate cancer cells(PC-3,LNCa P,22RV1 and C4-2 cells)and normal epithelial cell line RWPE-1 were detected by RT-PCR and western blot;3.The expression of H3 R was detected by immunohistochemistry in prostate cancer tissues and normal prostate tissues;4.We using specific H3 R agonists,antagonists to regulate the function of H3 R or constructing a vector that interferes with the histamine H3 receptor expression to down-regulating H3 R expression in endogenous H3R-overexpressing cell lines;5.In vitro,migration and invasion experiments were performed to detect the influence of H3 R on migration and invasion ability of prostate cancer cells;6.The effect of H3 R on the proliferation of prostate cancer cells was detected by CCK-8 assay;7.We using flow cytometry and apoptosis detection kit to detect the effect of H3 R on the apoptosis of prostate cancer cells;8.Western blot was used to detect the apoptosis-related protein and may change downstream proteins;9.We using xenografts model in nude mice to investigate the effect of H3 R in prostate cancer cells’ proliferation.Results: 1.The results of immunohistochemistry showed that the intensity of histidine decarboxylase staining in prostate cancer tissues was significantly higher than that in normal prostate tissues(P<0.05);2.The m RNA and protein levels of histidine decarboxylase and histamine receptor in prostate cancer cell lines(PC-3,LNCa P,22RV1 and C4-2)was also significantly higher than that in the control cell line RWPE-1,and H3 R is highest(P<0.05);3.Immunohistochemically results showed that the intensity of H3 R staining in prostate cancer was significantly higher than that in normal prostate tissue and it is higher in the tissues with high Gleason score than tissues with relative lower Gleason score(P<0.05);4.In vitro migration and invasion experiments showed that the ability of invasion and migration of prostate cancer cells decreased after the function of H3 R was inhibited(P<0.05);5.In vitro cell proliferation experiments showed that the interference of H3 R function can significantly decreased the cell proliferation of prostate cancer(P<0.05);6.Flow cytometry showed that interference with the function of H3 R can significantly increase the apoptosis of prostate cancer cells;7.Western blot experiment found that after the function of H3 R was disrupted,the apoptosis-associated protein BAX was significantly increased,the anti-apoptotic protein BCL2 was significantly decreased,and the expression of AR was significantly decreased;8.Nude mice xenografts tumor model showed that interference with the function of H3 R could restrains the growth of prostate cancer cells(P<0.05).Conclusions: 1.Histamine receptors is over-expressed in prostate cancer;2.Regulating the histamine receptor H3 R can affect the expression of androgen receptor;3.Histamine receptor H3 R can regulate the proliferation,metastasis and apoptosis of prostate cancer cells.In summary,H3 R may play a role in the proliferation,migration and invasion of prostate cancer cells,therefore,inhibition the function of H3 R in prostate cancer may be a new therapeutic strategy.更多还原