【作者】 杨轶凡；

【导师】 孔祥波；

【作者基本信息】 吉林大学 ， 外科学， 2007， 博士

【摘要】 水通道蛋白(aquaporin, AQP)是细胞对水跨膜转运的通道蛋白,目前研究表明AQP在肿瘤的生长、侵袭和转移中具有重要作用,应用AQP抑制剂可以抑制肿瘤血管生成,控制肿瘤的侵袭和转移。为明确水通道蛋白1(aquaporin1, AQP1)、水通道蛋白2(aquaporin2, AQP2)及水通道蛋白3(aquaporin3, AQP3)在肾透明细胞癌组织中的表达及分布,并探讨其在肾透明细胞癌发病和免疫治疗中的意义,本文首先应用免疫组织化学技术检测AQP1、AQP2及AQP3在裸鼠肾透明细胞癌转移瘤中的表达及分布,结果表明AQP1、AQP2及AQP3在肿瘤细胞和新生血管内皮均有表达,但表达强度有所差别。然后应用逆转录-聚合酶链反应、免疫印迹法及免疫组织化学技术检测AQP1、AQP2及AQP3在肾透明细胞癌及癌旁组织中的表达及分布,结果表明AQP1主要表达于癌旁组织的近曲小管、肾小球毛细血管内皮和肾癌组织中的新生血管内皮及肿瘤细胞;AQP2主要表达于癌旁组织的集合管,肾癌组织中没有表达;AQP3主要表达于癌旁组织的远曲小管、集合管、肾小球毛细血管内皮细胞和癌组织中的新生血管内皮及肿瘤细胞。本文首次证实肾透明细胞癌中AQP1、AQP3表达水平较癌旁肾小球毛细血管组织增多,较癌旁肾小管组织降低。AQP1及AQP3分布于肾透明细胞癌组织不同部位,可以增加肿瘤细胞和新生血管内皮对水的通透性运输,促进肿瘤血管生成和肿瘤细胞生长,增强肿瘤的侵袭和转移能力。该研究达到国内领先水平。更多还原

【Abstract】 Background : Aquaporins (AQPs) are a family of water-selective transporting glycoproteins that have a wide increase plasma membrane water permeability in secretory and adsorptive cells, and in those cells which the rapid regulated water movement is required.Up to now,there are 13 subtypes of AQPs have been found in mammals among which 8 were found in kidney.They play an important role in water reabsorption and cell secretion. Recent research showed that AQPs contribute to the growth,invation and metastasis of carcinomas.Some studies proofed that AQPs- inhibitor might inhibit the growth,invation and metastasis of carcinomas. Tumor of kidney has significant place in neoplasms of urinary system and male reproductive system. Most of tumor of kidney is renal carcinoma, especially clear cell carcinoma of kidney take the most. However, only a limited amount of information is available concerning the distribution of AQPs in tumor of kidney, so studying this mechanism could help us understanding more about the progression of clear cell carcinoma growth, invasion and metastasis.Objective:To detect expression level and distribution of aquaporin 1(AQP1), aquaporin 2(AQP2)and aquaporin 3 (AQP3)in clear cellcarcinoma and try to find out the significance of AQP1,AQP2 and AQP3 in the invasion of kidney carcinoma.Method:1. Six cases of metastatic tumor tissues were obtained from nude mice. The slides were detected by immunohistochemical staining for observing the expression of AQP1, AQP2 and AQP3 in kidney clear cell metastatic tumor tissue.2. Twenty cases of kidney clear cell carcinoma tissues and tissues beside tumors were acquired immediately after surgery. The expression and distribution of AQP1 ,AQP2 and AQP3 in above tissues were examined by reverse transcriptase polymerase chain reaction(RT-PCR) , western blot analysis and immunohistochemical technique(IHC).Result: 1. All of AQP1, AQP2 and AQP3 expressed on metastatic tumor tissues of kidney clear cell carcinoma. They expressed mainly on the capillary endothelia and neoplastic cell of tumor tissues. The expression levels of AQP1 and AQP3 on tumor tissues is dramatically higher than that of AQP2.2. Both AQP1 and AQP3 expressed on human kidney clear cell carcinoma tissues and tissues beside tumors. AQP2 expressed only on tissues beside tumors. The distribution and expression level of AQP1, AQP2 or AQP3 are different. IHC FingdingsThe AQP1 expressed mainly on the neoplastic cell, new vessels of tumors, capillary endothelium and proximal convoluted tubule of tissues beside tumors. The AQP2 expressed mainly on the collecting tube of tissues beside tumors. The AQP3 expressed mainly on the neoplastic cell, new vessels of tumors, capillary endothelium and renal tubule of tissues beside tumors.A semiquantitative assessment of the distribution of AQP1 and AQP3 showed that expression level in tumor tissues is remarkably stronger compared with capillary endothelium tissues beside tumors (P<0.05).However, the expression level of AQP1 and AQP3 in tumor tissues is weaker than renal tubule tissues beside tumors (P<0.05). RT-PCRAQP1 and AQP3 cDNA were expressed in kidney clear cell carcinoma and tissues beside tumor, while AQP2 expressed only in collectingduct beside tumor. AQP1 or AQP2 cDNA expression level in tumor tissues is remarkably weaker compared with that in renel cortex beside tumors (P<0.05) and stronger than that in renal medulla.(P<0.05).Western Blot AnalysisAQP1 and AQP3 were expressed in kidney clear cell carcinoma and tissues beside tumor, while AQP2 expressed only in tissues beside tumor.AQP1 or AQP3 expression is remarkably depressed in clear cell carcinoma tissues compared with that of renal cortex beside tumor (P<0.05) and ascended compared with that of renal medulla beside tumor(P<0.05).Discussion: Genesis, invasion and metastasis of tumor is a highly complicate process. This process is related with the trait and the surrounding environment of tumor tissue. Among this course, water metabolism plays an important role. To satisfy the fast proliferation, splitting, malignant tumors have the most actively metabolism, and tumor cells need more water to transport through the membrane than normal cells. So aquaporins are very important to the survival of tumor cells. Recent studies have approved that many tumor tissues, cell and capillary endothelia expressed aquaporins higher or lower than normal tissues. AQPs may be responsible for the high vascular permeability and interstitial fluid pressure in tumors. AQPs may also play a key role in tumor angiogenesis and may be involved in the genesis, development, invasion and metastasis.Though AQP1, AQP2 and AQP3 all have function to transport water, but the distribution and expression of them in kidney tissue, and the function in microenvironment are different. The distribution and expression of them in kidney clear cell tumor tissues have not been studied systematically.This study approved that the expression of AQP1 in tissue beside tumor was located on the capillary endothelia and proximal convoluted tubule as well as on the neoplastic cell and new capillary endothelium of tumors; the expression of AQP2 was located on the the collecting tube in tissue beside tumor; the expression of AQP3 in tissue beside tumor was located on the capillary endothelia and distal convoluted tubule as well as on the neoplastic cell and new capillary endothelium of tumors. This can transport water fast through capillary endothelia cells to satisfy the need of tumor cells.This study has also shown that a great deal of AQP1 and AQP3 expressed on the capillary endothelia cells and immature neonatal blood vessel of tumor. AQP1 or AQP3 expression level in kidney clear cell carcinoma tissue are remarkably stronger compared with capillary endothelia tissue beside tumors. Our result suggested that overexpression of AQP1 or AQP3 contributed to the hyperpermeability of tumor vessels in kidney clear cell carcinoma tissues. Our study also indicated that AQP1 or AQP3 may contributed to the angiogenesis and may be involved in the genesis,development,invasion and metastasis of kidney clear cell tumor.The expression and distribution of AQP1 and AQP3 on kidney clear cell carcinoma can ensure tumor cells to get more water with less energy and accelerate genesis,development , invasion and metastasis progression of tumors. Some studies showed that inhibitor to aim directly at AQPs can inhibit the growth and angiogenesis of tumors with the decrease of AQPs expression level. RNAi at AQPs can bring the same results.these suggest us inhibitor or RNAi at AQP1orAQP3 might be effective way of kidney clear cell carcinoma immunotherapy.But the detailed mechanism of it should be studied in continued works.Conclusion:This study confirmed first time that both AQP1 and AQP3 expressed on kidney clear cell carcinoma tissues and tissues beside tumor, but the distribution of AQP1 and AQP3 on tissues beside tumor was different,and AQP1 or AQP3 expression level in tumor tissues was remarkably stronger than that in capillary endothelia tissues beside tumors. Inhibition of the expression of AQP1 or AQP3 may suppress the process of the tumor angiogenesis, invasion and metastases.更多还原