【作者】 魏巍；

【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(aquaporins,AQPs)是对水专一的通道蛋白,普遍存在于动、植物及微生物中。它所介导的自由水快速被动的跨生物膜转运,是水进出细胞的主要途径。到目前为止已克隆的哺乳动物水通道蛋白家族有13个成员(AQP0～AQP12),其基因结构、基因表达调控、染色体定位、蛋白质结构、组织分布和生理功能均得到了较为深入的研究。多年研究表明,为满足快速增殖、分裂和侵袭转移的需要,肿瘤细胞一系列酶的活性和表达发生改变,细胞基本结构成分如蛋白质、脂类和核酸的合成加强,包括核酸合成的基本原料—核苷酸的生物合成明显加强,以至蛋白表达谱系或蛋白活性都有改变。同时癌细胞向周围基质侵袭和进出血管(淋巴管)时需要其体积和细胞形态的改变。癌细胞的所有这些生命活动都离不开水的微环境和参与,癌细胞比正常细胞更需要水分子的快速跨膜转运。但AQPs功能对肿瘤的影响至今尚未有系统研究。本实验通过S-P免疫组织化学方法、RT-PCR及Western blot方法,对AQP1、AQP2及AQP3在正常膀胱组织及膀胱癌组织的表达进行检测,我们在检测正常膀胱组织中的AQP时以腺性膀胱炎组织作为对照,结果证实AQP1主要定位于膀胱粘膜下微血管和小动脉的内皮细胞,AQP2和AQP3定位于膀胱粘膜的上皮细胞的细胞膜及胞质中。腺性膀胱炎组织中AQP1和AQP2的表达较正常组织弱,有显著性差异(P﹤0.01),而AQP3则基本无表达。再以正常膀胱组织作为对照组研究AQP1、AQP2及AQP3在膀胱癌组织的表达情况,结果AQP1、AQP2及AQP3在膀胱癌组织中的表达均高于正常组织(P﹤0.01)有显著差异,说明水通道蛋白在肿瘤的增殖过程中起一定作用。为进一步证实此观点我们设计了膀胱癌T24细胞培养增殖抑制实验,我们利用AQP抗体来破坏AQP蛋白的功能,从而达到抑制AQPs蛋白的作用,用MTT法检测其抑制率,实验结果表明浓度比为1:1和1:10的AQP2及AQP3抗体对膀胱癌T24细胞抑制率为23.47%和16.89%、35.63%和33.51,说明高浓度的AQP2和AQP3抗体对膀胱癌T24细胞的增殖有一定抑制作用。而AQP1抗体对膀胱癌T24细胞的增殖无明显抑制作用,因为在膀胱癌T24细胞RT-PCR实验中AQP1mRNA无阳性条带,说明膀胱癌T24细胞无AQP1蛋白的表达。本实验证实AQP1、AQP2及AQP3在正常膀胱组织及膀胱癌组织中均有表达,且在肿瘤组织中的表达增高,通过抑制高表达的水通道蛋白的功能可以达到抑制肿瘤细胞增殖的效果,这为今后研究治疗膀胱肿瘤的药物提供一种新思路。更多还原

【Abstract】 Aquaporins(AQPs) is the specific water channel protein ,it resides in animal、plants and microorganism generally. It is the main way that wanter is transported through biomembrane by aquaporins.As yet there are thirteen isforms in mammal cloned by scientist(AQP0~AQP12),their genetic structure、gene expression and regulation、chromosomal localization、protein structure、tussue deposition and physicalogical function have been deeply reached in last decade.Many years study have indicated that the tumor cell has changed a series of enzymetic active and expression for the requirement of the tumor cell proliferation、cleavage、invasion and metastatic, the synthesis of tumor cell elementary structure for instance protein、lipide and nucleic acid was strengthened,even the synthesis of the ribbonucleotide was strengthened also.The protein expression lineage or the protein active was changed.The invasion to interstitial substance and through blood vessel(lympgatic vessel) need chang the volume and shape of the tumor cell. The vital movement of tumor cell need water microenvironment and participation.The tumor cell need more water than normal cell. The elementary biology characteristic of malignant tumor is the infinite proliferation and disdifferentiation of malignant tumor.The tumor cell need more water than normal cell for their proliferation、cleavage、invasion and metastatic.Majority tumor tissue have high tissue space liquid pressure and capillary osmosis.Now a great quantity research have confirmed that AQP high expressed in many kinds tumor tissue、cell line and capillary endothelial cell. So many researchers think that AQPs has relation with the high vascular permeability in tumor tissue. Utilizing the AQPs suppressive agent could significantly degrade tumor metastasize. This illustrate that AQPs can promote tumor metastasis.New viewpoint considered AQPs possess carcinogenesis characteristics. The cDNA of AQPs ectopic expression coule cause many phenoltypic change including cell proliferation et al.The bladder tumor is exogenous noumenon tumor, tumor tissue grew fast, the proliferation and metastasis need a lot of water to metabolism, AQPs participate this process. But now there are a few reports about this, my experiments detect AQPs in normal bladder tissue and bladder tumor tissue by S-P immunohistochemistry、RT-PCR and western blot methods,and suppress the tumor cell proliferation through inhibiting AQPs in the next experiment. Offering a new ways to cure bladder tumor.Method Detect the expression of AQP1、AQP2 and AQP3 in fifty normal bladder tissues by S-P immunohistochemical method and RT-PCR method. Use cystitis glandularis as control group.Next trial using S-P immunohistochemical method、RT-PCR method and Western blot method detect AQP1、AQP2 and AQP3 in bladder tissue. Use normal bladder tissue as control group. Mensurate gray scale of S-P dyeing result, and calculate the immunohistochemical positive exponent through the formula: immunohistochemical positive exponent = exponent area/detecting tissue area×positive gray scale. Use its average scale as positive cell expression rate. The last trial we use the antibody of AQP1、AQP2 and AQP3 to inhibit bladder cancer T24 cell, and use MTT method calculate cell inhibition rate.Results AQP1 expresses in mucous membrane and capillary endothe -liocyte of urinary bladder,AQP2 and AQP3 express in cellular membrane and periplast. The positive exponent of AQP1、AQP2 and AQP3 in bladder tumor was higher than in normal bladder tissue (P﹤0.01). The inhibiting rate of ratio 1:1,1:10 of AQP2 antibody is 23.47% and 16.89% to bladder cancer T24 cell. The inhibiting rate of ratio 1:1,1:10 of AQP3 antibody is 35.63% and 33.51 to bladder cancer T24 cell. The AQP1 antibody had no effect on the bladder cancer T24 cell because AQP1 didn’t express in T24 cell.Conclusions AQP1 expresses in mucous membrane and capillary endotheliocyte of urinary bladder,AQP2 and AQP3 express in cellular membrane and periplast; the expression of AQP1、AQP2 and AQP3 in bladder tumor was higher than in normal bladder tissue (P﹤0.01); inhibiting AQP2 and AQP3 could reduce bladder cancer T24 cell proliferation.更多还原