Hepcidin和铁代谢在前列腺癌进展中的作用机制研究

发布时间：2018-06-26 09:13

本文选题：Hepcidin + 前列腺癌　；参考：《苏州大学》2014年博士论文

【摘要】：前列腺癌是男性常见的恶性肿瘤，在男性癌症死亡人数中居第二位。新的研究认为机体铁代谢在前列腺癌的生长，血管发生和转移中起着重要的作用。铁是人体维持机能必要的微量元素，广泛参与人体的生长代谢。很多肿瘤的发生发展对铁的需要增加，被认为是维持肿瘤细胞生长和发展的基本物质，有研究认为，通过调控铁代谢的变化，减少胞内铁的利用，可以影响肿瘤的进展，作为抗肿瘤治疗的方向。 Hepcidin由肝细胞合成和分泌，是已知的在机体铁代谢调节中起重要作用的核心分子，称之为铁调素。它通过减少网状内皮细胞系统对胞内游离铁的释放，同时通过减少十二指肠的铁吸收，负性调节铁代谢平衡，在机体铁代谢中起到核心调控作用。已有的研究通过一系列动物模型和已知的铁代谢异常患者探讨Hepcidin的作用机制，敲除Hepcidin基因的小鼠可出现铁超载，而过表达Hepcidin的小鼠以及过分泌Hepcidin的人群都则表现为缺铁性贫血。Hepcidin在肿瘤人群中的表达，对机体铁代谢的作用已引起很多学者的重视。有研究表明，在机体铁代谢中Hepcidin通过对其受体Ferroportin的调节，将细胞内游离铁转运至胞外，肿瘤细胞伴随低表达的Ferroportin产生额外的胞内游离铁，使得肿瘤细胞更具有侵袭性，，而Hepcidin在前列腺癌中的作用未见有详细论述。因此，我们研究Hepcidin的表达与前列腺癌的相关性，对前列腺癌细胞的Hepcidin进行干扰后对肿瘤细胞生物学功能的影响，以及Hepcidin调控前列腺癌细胞铁代谢的作用机制，对Hepcidin通过调控铁代谢对前列腺癌的进展产生影响进行探讨。研究分三部分：第一部分：Hepcidin的表达与前列腺癌的临床相关性研究目的：研究Hepcidin在前列腺癌患者中的表达变化以及与铁代谢的临床相关性。方法：采用酶联免疫法（ELISA）测定前列腺癌骨转移患者25例、前列腺癌无骨转移患者30例和普通对照组前列腺增生患者30例的血清Hepcidin、IL-6、sTfR和BMP6的表达，以及检测所有标本的血红蛋白（HB）；免疫组化检测组织标本的Hepcidin受体Ferroportin的表达，分析其与前列腺癌分级分期的相关性关系，研究Hepcidin与前列腺癌的临床关联。结果：前列腺癌骨转移患者的血清Hepcidin较前列腺癌无骨转移组、普通对照前列腺增生组有明显增高，有统计学差异（P0.05）；并且与IL-6、BMP6的表达呈正相关，与sTfR的表达呈负相关；前列腺癌组织中Hepcidin的受体Ferroportin表达较前列腺增生组织明显减少，染色浅，差异明显；Ferroportin的表达和前列腺癌的恶性程度呈反相关。结论：前列腺癌患者的Hepcidin表达增高，受体Ferroportin表达降低，可能与机体的炎症状态、骨转移的程度有关，Hepcidin可能作为临床判断前列腺癌的进展程度的指标，为前列腺癌的诊断提供新的方法。第二部分:Hepcidin调控前列腺癌细胞生物学功能的研究目的：探讨Hepcidin在前列腺癌细胞中的表达变化，以及Hepcidin对前列腺癌细胞增殖、迁移和凋亡等细胞生物学功能的影响。方法：测定Hepcidin在前列腺癌细胞中的表达；通过小干扰RNA的方法下调前列腺癌细胞株PC3、DU145中的Hepcidin，研究Si-Hepcidin前列腺癌细胞增殖、迁移、细胞周期、抗凋亡能力的表达变化情况；并将Si-Hepcidin前列腺癌细胞株PC3植入裸鼠体内形成前列腺癌细胞移植瘤，分析Si-Hepcidin前列腺癌细胞成瘤与对照组在体内的生长差异。结果：前列腺癌细胞的Hepcidin表达较正常前列腺细胞高，有统计学差异（P0.05）；Hepcidin干扰后的前列腺癌细胞株增殖、迁移、抗凋亡能力较空白对照组和阴性对照组明显减弱，有统计学差异（P0.05）；Si-Hepcidin的前列腺癌细胞在裸鼠体内成瘤体积较对照组小，有统计学差异（P0.05）。结论：Hepcidin在前列腺癌细胞中高表达；Si-Hepcidin对前列腺癌细胞的增殖、迁移、细胞周期、抗凋亡能力产生影响，并且引起前列腺癌细胞成瘤的改变，说明Hepcidin可能通过铁代谢的信号通路对肿瘤细胞的生长和侵袭产生调控，在前列腺癌的进展中起到重要作用。第三部分：外源性Hepcidin调控前列腺癌细胞铁代谢的分子机制研究目的：探讨Hepcidin与前列腺癌细胞中铁代谢信号通路分子表达变化的关系，通过外源性Hepcidin对前列腺癌细胞生物学功能产生的变化及铁代谢分子机制研究，说明Hepcidin通过调控细胞铁代谢对前列腺癌的进展产生影响。方法：体外培养前列腺癌细胞株PC3和Si-Hepcidin PC3，运用Real-Time PCR和Western-blot检测两组中铁代谢的膜转运蛋白Ferroportin的变化，免疫荧光法测定两组细胞的胞内铁的表达；Si-Hepcidin PC3加入外源性Hepcidin500nm，与Si-HepcidinPC3组比较观察Hepcidin对前列腺癌细胞增殖、迁移和抗凋亡能力的改变；Real-TimePCR和Western-blot检测两组中铁代谢膜转运蛋白Ferroportin的变化，免疫荧光法测定两组细胞的胞内铁的表达。结果：Si-Hepcidin PC3中Hepcidin受体Ferroportin的表达升高，胞内铁含量减少，与PC3组比较有统计学差异（P0.05）；加入外源性Hepcidin后，+Hepcidin PC3组的增殖、迁移和抗凋亡能力增强，与Si-Hepcidin PC3组比较有统计学差异（P0.05）；加入外源性Hepcidin后，+Hepcidin PC3组Ferroportin的表达较Si-Hepcidin PC3组降低，胞内铁含量增加，有统计学差异（P0.05）。结论：Hepcidin调控前列腺癌细胞铁代谢中膜转运蛋白Ferroportin和胞内游离铁的表达，对前列腺癌细胞的增殖、迁移和抗凋亡能力产生影响。Hepcidin可作为治疗前列腺癌进展的新靶点，通过Hepcidin调控前列腺癌的铁代谢，对前列腺癌治疗起到积极的临床意义。
[Abstract]:Prostate cancer is the most common malignant tumor in men and the second in the number of male cancer deaths. The new study suggests that iron metabolism plays an important role in the growth of prostate cancer, angiogenesis and metastasis. Iron is a necessary trace element for the maintenance of human body and is widely involved in the growth and metabolism of human body. Many tumors are developed and developed. The increase in iron needs is considered to be the basic substance to maintain the growth and development of tumor cells. Studies have suggested that the reduction in the use of intracellular iron by regulating changes in iron metabolism can affect the progression of tumors as a direction for antitumor treatment.
Hepcidin is synthesized and secreted by hepatocytes. It is known as the core molecule that plays an important role in the regulation of iron metabolism. It is called ferretin. It reduces the release of intracellular free iron by the reticuloendothelial cell system and reduces iron metabolism by reducing the iron absorption of the duodenum, and plays a core role in the iron metabolism of the body. Control. Previous studies have explored the mechanism of Hepcidin's action through a series of animal models and known patients with abnormal iron metabolism. The mice that knock out the Hepcidin gene can be overloaded with iron, and those who overexpress Hepcidin and those who have secreted Hepcidin are expressed as the expression of iron deficient blood poor.Hepcidin in the tumor population. The role of body iron metabolism has attracted many scholars' attention. Some studies have shown that in the body iron metabolism, Hepcidin transtransport the intracellular free iron to the extracellular by regulating its receptor Ferroportin, and the tumor cells accompany the low expression of Ferroportin to produce extra intracellular free iron, making the tumor cells more invasive, while Hepcidin is in the front. The role of adenocarcinoma in the adenocarcinoma has not been discussed in detail.
Therefore, we study the correlation between the expression of Hepcidin and prostate cancer, the influence of Hepcidin on the prostate cancer cells and the biological function of the tumor cells, and the mechanism of Hepcidin regulation of the iron metabolism in the prostate cancer cells, and explore the effect of Hepcidin on the progress of the prostate cancer by regulating iron metabolism. It is divided into three parts:
Part one: the correlation between the expression of Hepcidin and prostate cancer.
Objective: To study the expression of Hepcidin in patients with prostate cancer and the clinical correlation with iron metabolism. Methods: the expression of serum Hepcidin, IL-6, sTfR and BMP6 in 25 patients with prostate cancer, 30 cases of prostate cancer without bone metastases and 30 patients with benign prostatic hyperplasia by enzyme linked immunosorbent assay (ELISA). And the detection of hemoglobin (HB) of all specimens; immunohistochemical detection of the expression of Hepcidin receptor Ferroportin in tissue specimens, analysis of the correlation with the classification and staging of prostate cancer, and to study the clinical association of Hepcidin with prostate cancer.
Results: the serum Hepcidin of the patients with bone metastasis of prostate cancer was higher than that of prostate cancer without bone metastasis, and there was a significant increase in the normal control prostatic hyperplasia group (P0.05), and it was positively correlated with the expression of IL-6, BMP6, and negative correlation with the expression of sTfR; the expression of Hepcidin receptor Ferroportin in prostate cancer tissue was more than that of the prostate. There was a significant decrease in the number of tissues and a slight difference in staining. The expression of Ferroportin was negatively correlated with the malignancy of prostate cancer.
Conclusion: the expression of Hepcidin in the patients with prostate cancer is higher and the expression of receptor Ferroportin is reduced. It may be related to the inflammatory state of the body and the degree of bone metastasis. Hepcidin may be used as an indicator of the progression of prostate cancer, which provides a new method for the diagnosis of prostate cancer.
The second part: Hepcidin regulates the biological function of prostate cancer cells.
Objective: To investigate the changes in the expression of Hepcidin in prostate cancer cells and the effect of Hepcidin on the cell biological functions such as proliferation, migration and apoptosis of prostate cancer cells.
Methods: the expression of Hepcidin in prostate cancer cells was measured. The expression of prostate cancer cell line PC3 and Hepcidin in DU145 was downregulated by small interference RNA, and the expression of the proliferation, migration, cell cycle, anti apoptosis ability of Si-Hepcidin prostate cancer cells was studied, and Si-Hepcidin prostate cancer cell line PC3 was implanted in the nude mice. A prostate cancer cell transplant tumor was formed to analyze the growth difference between Si-Hepcidin prostate cancer cells and the control group in vivo.
Results: the expression of Hepcidin in prostate cancer cells was higher than that of normal prostate cells (P0.05). The proliferation, migration and anti apoptosis ability of prostate cancer cell lines after Hepcidin interference were significantly lower than those in the blank control group and negative control group (P0.05), and the prostate cancer cells of Si-Hepcidin were formed in nude mice. The tumor volume is smaller than that of the control group (P0.05). Conclusion: Hepcidin is highly expressed in the prostate cancer cells. Si-Hepcidin affects the proliferation, migration, cell cycle, anti apoptotic capacity of prostate cancer cells, and causes changes in the tumor cell formation of prostate cancer cells, indicating that Hepcidin may be through the signal pathway of iron metabolism to the tumor. Regulation of cell growth and invasion plays an important role in the progression of prostate cancer.
The third part: molecular mechanism of exogenous Hepcidin regulating iron metabolism in prostate cancer cells.
Objective: To investigate the relationship between Hepcidin and the changes in the expression of iron metabolism signaling pathway in prostate cancer cells. Through the study of the changes in biological function of prostate cancer cells and the molecular mechanism of iron metabolism by exogenous Hepcidin, the effect of Hepcidin on the progression of prostatic adenocarcinoma by regulating cell iron metabolism is investigated.
Methods: PC3 and Si-Hepcidin PC3 of prostate cancer cell lines were cultured in vitro. The changes of membrane transporter Ferroportin in two groups of iron metabolism were detected by Real-Time PCR and Western-blot. The expression of intracellular iron in two groups of cells was measured by immunofluorescence; Si-Hepcidin PC3 was added to exogenous Hepcidin500nm, and Hepci was compared with Si-HepcidinPC3 group. The proliferation, migration and resistance to apoptosis of prostate cancer cells were changed by DIN; changes in Real-TimePCR and Western-blot were detected in two groups of iron metabolic membrane transporter Ferroportin, and the expression of intracellular iron in two groups of cells was measured by immunofluorescence.
Results: the expression of Hepcidin receptor Ferroportin in Si-Hepcidin PC3 increased and the intracellular iron content decreased, and there was a statistical difference between PC3 group and PC3 group (P0.05). After adding exogenous Hepcidin, the proliferation, migration and anti apoptotic ability of +Hepcidin PC3 group were enhanced, and there was a statistical difference from Si-Hepcidin PC3 group (P0.05). The expression of Ferroportin in +Hepcidin PC3 group was lower than that in Si-Hepcidin PC3 group, and there was a significant difference in intracellular iron content (P0.05).
Conclusion: Hepcidin regulates the expression of membrane transporter Ferroportin and intracellular free iron in the iron metabolism of prostate cancer cells. The effect of.Hepcidin on the proliferation, migration and resistance to apoptosis of prostate cancer cells can be used as a new target for the treatment of prostate cancer. It can be used to regulate the iron metabolism of prostate cancer and to treat the prostate cancer by Hepcidin. To the positive clinical significance.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.25

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