【作者】 李燕；

【导师】 药立波； 刘新平；

【作者基本信息】 第四军医大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 人NDRG2(N-myc down-stream regulated gene 2)基因为本研究小组首先发现并报道,该基因可能与细胞增殖和分化相关,但其具体的生物学功能有待进一步研究。我们课题组前期研究的结果对本课题的深入研究有重要的提示作用:RNA点杂交实验表明NDRG2在人的唾液腺组织中表达量极高;免疫组化结果显示NDRG2在小鼠的舌下腺导管上皮细胞特异性高表达;酵母双杂交实验寻找能与NDRG2相互作用的分子,最终筛选到12个有正确读框的的克隆,其中有3个克隆是Na~+/K~+-ATPaseβ1的肽段;生物信息学预测,在NDRG2上游调控区存在潜在的雌激素受体(ER)结合位点。唾液腺导管细胞的重要生物学功能是重吸收唾液中的Na~+和Cl-等电解质,从而参与调节唾液(正常唾液渗透压为血浆渗透压的1/9)的生成,而唾液进入导管后Na~+的重吸收依赖导管细胞腔侧分布的上皮细胞钠通道(epithelial sodium channel, ENaC)。有文献报道非洲蟾蜍卵母细胞中,NDRG2可以激活ENaC。Na~+/K~+-ATPaseβ1是Na~+/K~+-ATPase(又称为钠钾泵)的亚基,是Na~+/K~+-ATPase发挥泵功能不可缺少的组成成分。存在于唾液腺导管细胞基底侧的Na~+/K~+-ATPase,以主动耗能的方式将细胞中的Na~+泵到细胞外,形成一定的细胞内外Na~+浓度阶差,进而影响唾液腺导管细胞腔侧ENaC对从腺泡分泌进入导管的原始唾液中Na~+的重吸收。临床口腔干燥症的好发人群为绝经后女性,50%以上的绝经后女性有自觉口干症状,口干病人的唾液电解质成分与正常人差异很大,唾液渗透压和电解质浓度往往明显增高、唾液粘稠。先前的研究结果强烈提示NDRG2在唾液腺组织中发挥重要的功能,特别是与唾液的生成可能高度相关,本课题将对此进行探讨和验证。【目的】1、明确NDRG2在唾液腺组织中的分布和定位;2、研究NDRG2在唾液腺细胞中的功能与Na~+/K~+-ATPaseβ1的关联性;3、探讨NDRG2在人唾液腺细胞中是否受到雌激素的调控;4、整体水平分析雌激素与唾液形成之间的关系以及NDRG2在其中可能发挥的功能。【方法】1、通过免疫组织化学和间接免疫荧光的方法明确NDRG2在唾液腺组织的分布与定位,RT-PCR分析正常人颌下腺NDRG1、NDRG2、NDRG3和NDRG4的表达情况。2、建立单层唾液腺导管细胞离子转运模型,检测NDRG2对各种离子和水分子转运的影响;运用哺乳动物双杂交和免疫共沉淀实验验证NDRG2与Na~+/K~+-ATPaseβ1的相互作用;间接免疫荧光观察NDRG2与Na~+/K~+-ATPaseβ1在细胞内是否存在共定位;分析NDRG2对Na~+/K~+-ATPase酶活性的影响;Real time PCR和Western Blot检测NDRG2对Na~+/K~+-ATPaseβ1表达的影响。3、Real time PCR和Western Blot分析17-β-雌二醇(E2)对NDRG2表达水平的影响;运用双报告基因、ChIP和EMSA等基因转录调控实验明确NDRG2是否受E2-ER转录复合物的调控。4、建立去势雌性大鼠模型(去卵巢手术)模拟绝经后女性体内雌激素水平,检测大鼠唾液流量、唾液电解质成分及颌下腺NDRG2、Na~+/K~+-ATPase和ENaC表达水平的变化。【结果】1、NDRG2蛋白在唾液腺导管细胞胞浆中特异性高表达NDRG2蛋白主要表达于人三对大唾液腺(腮腺、颌下腺和舌下腺)以及大鼠的颌下腺的导管细胞胞浆,而腺泡细胞中表达很弱。RT-PCR结果表明正常人颌下腺组织NDRG2 mRNA表达相对高,NDRG1和NDRG3的表达相对较低,而NDRG4几乎不表达、2、NDRG2可以结合并稳定Na~+/K~+-ATPaseβ1蛋白在单层唾液腺导管细胞离子转运模型中,感染NDRG2腺病毒或转染针对NDRG2的干涉片段,特异性过表达或沉默NDRG2,结果唾液腺导管细胞基底侧与腔侧Na~+和Cl-浓度比值明显与NDRG2表达量成正比,而细胞基底侧与腔侧K~+和Ca2~+浓度比值没有明显的变化,基底侧和腔侧的液体体积均未出现显著改变。哺乳动物双杂交和免疫共沉淀结果进一步证实NDRG2和Na~+/K~+-ATPaseβ1可以相互作用。间接免疫荧光结果显示NDRG2和Na~+/K~+-ATPaseβ1在人永生化唾液腺导管细胞HSG的核周胞浆存在部分共定位。HSG细胞中相同总蛋白中Na~+/K~+-ATPase的活性与NDRG2的表达水平成正比,并且NDRG2可以稳定Na~+/K~+-ATPaseβ1蛋白,延长Na~+/K~+-ATPaseβ1蛋白的半衰期。3、NDRG2受E2-ERβ转录复合物的调控,是雌激素的靶基因E2可以时间和剂量依赖的方式上调NDRG2的表达,并且NDRG2 mRNA和蛋白水平的表达均有所增加。间接免疫荧光实验表明:ERα和ERβ均主要分布于人唾液腺组织的导管细胞中,ERβ表达强度明显高于ERα,ERβ和NDRG2均高表达于唾液腺导管细胞。ERβ可以剂量依赖的方式增加NDRG2上游调控区报告基因的活性,而ERα不能激活报告基因,ERβ的特异性激动剂DPN能够增强报告基因活性,而ERα的特异性激动剂PPT不能激活报告基因,运用ERβ配体结合区截短突变体或干涉ERβ的表达均能显著降低报告基因的活性,并且将潜在的ERE(estrogen response elements)进行点突变,报告基因的活性明显降低。ChIP和EMSA实验检测到外源性ERβ或内源性ERβ均能结合于NDRG2上游调控区预测的ERE。4、NDRG2参与去势雌性大鼠口腔干燥去卵巢大鼠手术后体内雌激素水平明显迅速下降,去势雌性大鼠逐渐出现毛发枯疏、骨质疏松等与体内雌激素水平相关的表征;有明显的口腔干燥症状,即唾液流量减少、饮水增多及唾液中Na~+和Cl-浓度增高。PCR结果显示去卵巢大鼠颌下腺组织中NDRG2和ENaCβ的mRNA水平明显减少,Na~+/K~+-ATPaseβ1mRNA水平无明显变化;免疫组化结果表明NDRG2、Na~+/K~+-ATPaseβ1和ENaCβ的蛋白含量均明显降低。【结论】本课题揭示了NDRG2是受雌激素调控的下游靶基因之一,初步证实E2-ER转录复合物-NDRG2-Na~+/K~+-ATPaseβ1通路的存在,并且这个通路可能参与临床绝经后女性口腔干燥症。更多还原

【Abstract】 NDRG2 (N-myc down-stream regulated gene 2), a novel gene which was discovered and reported firstly by our research group. Although NDRG2 is possibly related to cell proliferation and differentiation, the exactly biological function has not yet been completely elucidated. Our previous findings have showed some interesting phenomena and which have important implications for our future research: NDRG2 is detected highly expressed in human salivary gland tissue by RNA dot blot assay; and immunoreactivity could be seen mainly in duct cells of sublingual gland in mice by the immunohistochemistry; yeast two-hybrid approach was used to screen the proteins which can interact with NDRG2, and of these 12 final positive clones, 3 fragments encoded Na~+/K~+-ATPaseβ1; Bioinformatics analysis revealed that there exist potential binding sites of ER (estrogen receptor) in the regulatory region of NDRG2.The main function of salivary duct cells is to reabsorb Na~+ and Cl- from saliva, and involved in the forming of normal saliva (low osmotic pressure), while the reabsorption of Na~+ depend on the epithelial sodium channel (ENaC). It has been reported that NDRG2 can activate ENAC in Xenopus laevis Oocytes. Na~+/K~+-ATPaseβ1 is a subunit of Na~+/K~+-ATPase (the other name is Na~+/K~+-pump), which is the indispensable part of the pump function. Na~+/K~+-ATPase locate on the basolateral side of salivary duct cells can pump Na~+ outside by expending energy, and this can form the Na~+ concentration difference, then effect the absorption of Na~+ on luminal side by ENaC from saliva which is excreted from acinus to duct. Oral dryness usually occurs in post-menopause female, more than 50% post-menopause female have conscious oral dryness, and the electrolyte ingredients of oral dryness patients have great discrepancy compared with normal persons, osmotic pressure and electrolyte concentration increase obviously and the saliva become very viscous.The foregoing research suggest intensively that NDRG2 play a very important role in salivary tissue, especially closely related with the forming of saliva, so more investigation and verification will be done in the present research.【Objectives】(1) To clear the distribution and localization of NDRG2 in salivary gland;(2) to investigate the function of NDRG2 in salivary gland and the correlation with Na~+/K~+-ATPaseβ1; (3) to probe whether NDRG2 was regulated by estrogen in transcription level; (4) to further analyze the relationship between estrogen and saliva forming, and the possible roles of NDRG2 involved in it.【Methods】(1) Immunohistochemistry and Immunofluorescence were used to detect the distribution and localization of NDRG2 in salivary gland tissue, and RT-PCR was applied to analyze the expression of NDRG1, NDRG2, NDRG3 and NDRG4 in human normal submaxillary gland. (2) Set up the salivary duct cell monolayer ion transport model to detect the effects of NDRG2 on the ions and H2O transport; the physical interaction between NDRG2 and Na~+/K~+-ATPaseβ1 were further detected with mammalian two-hybrid and co-immunoprecipitation; immunofluorescence was used to observe to the possible co-localization between NDRG2 and Na~+/K~+-ATPaseβ1; analyze the effect of NDRG2 to the activity of Na~+/K~+-ATPase; Real time PCR and Western Blot were applied to detect the effects of NDRG2 on the expression and function of Na~+/K~+-ATPaseβ1. (3) Real time PCR and Western Blot were used to detect the effect of 17β-estradiol (E2) on NDRG2 expression; and we assessed that whether NDRG2 was transcription regulated by E2-NDRG2 transcription complex by the dual report luciferase assay, ChIP and EMSA. (4) Ovariectomized rats were used to mimic estrogen level of the post-menopause female, and the variations of saliva flow, saliva electrolyte ingredients and related molecules NDRG2, Na~+/K~+-ATPase and ENaC expression in submaxillary gland were observed after ovariectomy.【Results】1. NDRG2 protein is specific highly expressed in the duct cell cytoplasm of salivary glandNDRG2 immunoreactivity could be seen mainly in duct cell cytoplasm of human three pairs of main salivary glands (parotid, submaxillary gland and sublingual gland) and rat submaxillary gland, while a very weak stain in acinus cells. The result of RT-PCR showed NDRG2 mRNA expression is high in human salivary gland, while the expression of NDRG1 and NDRG3 are relatively low, and NDRG4 is hardly expressed.2. NDRG2 binds and stabilizes Na~+/K~+-ATPaseβ1 proteinIn salivary duct cell monolayer ion transport model, NDRG2 were over expressed or silenced in HSG cells by using adenovirus or siRNA and the ratios of basolateral Na~+ concentration to apical Na~+ concentration is directly proportional to the NDRG2 protein level. And Cl- has the similar change as Na~+, while K~+ and Ca2~+ haven’t obviously changes with NDRG2 expression change, meanwhile, the medium volume of both sides was not detected pronounced difference. The NDRG2-Na~+/K~+-ATPaseβ1 direct physical interaction was confirmed by mammalian two-hybrid and co-immunoprecipitation. NDRG2 and Na~+/K~+-ATPaseβ1 could co-localized in the perinuclear region of the cytoplasm in human immortalization salivary duct cell line—HSG. And we found Na~+/K~+-ATPase activity is directly proportional to NDRG2 protein level in the same total protein in HSG cells; and NDRG2 could increase the Na~+/K~+-ATPaseβ1 stability, and extend Na~+/K~+-ATPaseβ1 protein half-life.3. NDRG2 is regulated by E2-ERβtranscription complex, and NDRG2 is the target gene of estrogenE2 up-regulated NDRG2 expression in both mRNA and protein levels by time and dose dependent manners. Indirect immunofluorescence showed that ERαand ERβboth distributed in duct cells of salivary gland tissue, but the protein of ERβwas expressed much more than ERα, moreover, ERβand NDRG2 were both highly expressed in duct cells; ERβcould increase the activity of NDRG2 regulatory region report gene in a dose dependent manner, while ERαcouldn’t activate report gene, and ERβspecific agonist DPN could activate NDRG2 report gene, while ERαspecific agonist PPT hadn’t the same effect. ERβligand binding domain deletion mutant or silencing the expression of ERβby siRNA could both reduce the activity of NDRG2 report gene, and point mutation in potential ERE (estrogen response elements) reduced report gene remarkable. ChIP and EMSA assays showed exogenous or endogenous ERβcould bind with predicted ERE in NDRG2 regulatory region.4. NDRG2 involves in the oral dryness of ovariectomized rats The serum estradiol decreased quickly in ovariectomized rats, and ovariectomized rats appeared sparse hair and osteoporosis related with low estradiol level and showed apparent oral dryness symptoms including saliva flow diminishing, water intake increasing and saliva electrolyte ingredients Na~+ and Cl- concentration increasing. The protein level of NDRG2, Na~+/K~+-ATPaseβ1 and ENaCβin submaxillary gland were obviously decreased in ovariectomized rats as compared with control and sham operation animals, although the mRNA of Na~+/K~+-ATPaseβ1 wasn’t changed remarkably.【Conclusions】We identified NDRG2 is one target gene of estrogen and provided the preliminary evidence that there exists the E2-ER-NDRG2-Na~+/K~+-ATPaseβ1 pathway and which is probably associated with post-menopause women oral dryness.更多还原