CN108159400B - 人附睾蛋白4在制备NF-κB激动剂中的应用 - Google Patents
人附睾蛋白4在制备NF-κB激动剂中的应用 Download PDFInfo
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Abstract
本发明公开了一种人附睾蛋白4在制备NF‑κB激动剂中的应用。本发明提示缺氧可以诱导肾小管上皮细胞内源性HE4高表达，高表达的HE4诱导NF‑κb通路的激活进一步上调NF‑κb的下游分子TIMP1进而促进肾纤维化，这一发现为研究肾纤维化机制和抗纤维化的治疗提供了重要的启示。
Description
技术领域：
本发明属于生物医药领域，具体涉及到人附睾蛋白(Human epididymis protein4,HE4)蛋白在制备NF-κB激动剂中的应用。
背景技术：
肾间质纤维化是各种CKD引起的异常损伤修复反应，主要病理学表现是肌成纤维细胞增殖和大量细胞外基质(extracellular matrix，ECM)沉积构成的网状瘢痕结构，是过度修复的表现。肾纤维化是许多慢性肾脏疾病的进展到终末期的共同病理表现[1]，主要特征为ECM增厚，先于炎症或组织损伤[2]。ECM是组织结构和器官重塑的重要组成部分，含有重要的结构蛋白(如胶原蛋白(collagens)，纤连蛋白(fibronectin)和层粘连蛋白(laminins))，对纤维化疾病的发生发展有重要作用[3]。I型胶原(collagen I)作为ECM重要成分，其显著累积导致肾组织结构和功能的改变以及最终的器官衰竭[4]。此外，基底膜ECM的形成主要是IV型胶原蛋白(collagen IV)。在基底膜中，I型胶原的功能主要是促进TGF-β上调和上皮-间质转化(epithelial-mesenchymal transition，EMT)参与肾纤维化。在正常生理条件下，ECM的过度沉积可被基质金属蛋白酶(matrix metalloproteinases，MMPs)降解，MMPs可受其抑制剂-金属蛋白酶的组织抑制剂(tissue inhibitors ofmetalloproteinases，TIMPs)的调节。MMPs和TIMPs功能之间的良好平衡是维持ECM稳态的关键[5]。缺氧条件下培养的肾成纤维细胞能增加I型前胶原α1mRNA水平[6]。缺氧也可以改变体外Ⅳ型胶原降解酶(MMP2和MMP9)的表达和活性[7-10]。我们前期研究发现，缺氧可引起ECM尤其是胶原沉积，参与肾纤维化，但其分子机制尚未阐明。
HE4参与肾间质纤维化
HE4是由WFDC2基因编码的小分子分泌型蛋白，它是含有乳清蛋白(WAP)结构域的家族成员，通过WAP和4个重复的二硫化结构域发挥着抑制蛋白酶活性的功能[11-12]。并且，HE4在细胞的生长和分化中也发挥着重要作用[13]。有研究报道，在狗纤维化病变的肾脏组织中HE4基因表达的上调最为显著[14]，在小鼠的纤维化肾组织中也能检测到HE4表达的上调[15]。并且在人肾移植活检标本中发现HE4基因的转录水平与低肾小球滤过率(estimated glomerular filtration rate，eGFR)密切相关[16,17]。血清中HE4的浓度也被证实与系统性红斑狼疮发生狼疮性肾炎和CKD相关[18]，并在大样本队列中证实血HE4与肾脏疾病的纤维化程度和疾病严重程度相关[19]。我们的前期研究也证实，CKD患者肾脏组织病理HE4表达情况与肾间质纤维化程度成正相关[16]。LeBleu等人采用红色荧光标记α-SMA启动子的转基因小鼠，建立UUO模型，发现在小鼠的肌成纤维细胞中发现高表达的HE4蛋白，它可以通过抑制I型胶原蛋白的水解酶MMPs的活性，促进ECM的堆积及肾间质纤维化；并且在UUO模型和5/6肾切除的小鼠模型中，给予中和HE4的抗体可以逆转肾间质纤维化的进程[20]。但是，缺氧是如何诱导损伤肾小管上皮细胞分泌HE4参与肾间质纤维化的呢？高表达的HE4参与肾间质纤维化的分子机制是什么呢？这些问题都值得进一步研究探讨。
发明内容：
本发明的目的是提供HE4蛋白(人附睾蛋白4)在制备NF-κB的激动剂中的应用。
本发明人研究发现缺氧诱导HK2细胞中HE4显著上调。另外，缺氧条件下HK2细胞HIF-1α高表达，过表达的HIF-1α通过结合HE4启动子直接转录调控HE4的表达。我们进一步以HE4为诱饵蛋白，利用Co-IP方法，结合质谱LC-MS/MS，从成纤维细胞中筛选出了与HE4作用较强的蛋白—IKBIP(inhibitor of NF-κB kinase interacting protein)。由于IKBIP可通过使IKBα(NF-κB inhibitor alpha)磷酸化入核，而且过表达的HE4可以促进P65的磷酸化，磷酸化的P65(PP65)与抑制蛋白IκB解离入核，因此共同激活NF-κB通路。而NF-κB通路在炎症细胞浸润、成纤维细胞活化、ECM分泌等多种促纤维化途径中均发挥关键作用。此外，我们还发现，HE4可以通过激活NF-κB信号通路来上调TIMP(金属蛋白酶的组织抑制剂)，抑制MMP2(Ⅳ型胶原降解酶)的活性，从而抑制ECM的降解。缺氧处理的肾小管上皮细胞和肌成纤维细胞共培养可以显著提高肌成纤维细胞的增殖，缺氧条件下HE4siRNA转染的肾小管上皮细胞与肌成纤维细胞共培养可以抑制肌成纤维细胞的增殖。进一步在体内实验中小鼠腹腔注射采用慢病毒载体构建的HE4siRNA后构建UUO模型，与对照组相比可逆转纤维化。这些数据表明，HE4是肾纤维化潜在的生物标志物和新的治疗靶标。
因此，本发明提供了人附睾蛋白4在制备NF-κB的激动剂中的应用。
本发明还提供了人附睾蛋白4在制备金属蛋白酶组织抑制剂的激动剂或者Ⅳ型胶原降解酶抑制剂中的应用。
本发明提示缺氧可以诱导肾小管上皮细胞内源性HE4高表达，高表达的HE4诱导NF-κb通路的激活进一步上调NF-κb的下游分子TIMP1进而促进肾纤维化，这一发现为研究肾纤维化机制和抗纤维化的治疗提供了重要的启示。
附图说明：
图1是体内、外实验证实缺氧诱导肾小管上皮细胞高表达HE4的图，其中(A)Western blot和qRT-PCR分析常氧(N)或缺氧(H)条件下HK2细胞HE4，胶原Ⅳ，胶原Ⅰ和α-SMA(间充质标记)在不同的时间的表达。直方图显示对于对照β-肌动蛋白校正的平均体积密度(n＝3)。(B)在动物模型(UUO)中HE4，collagenⅣ和collagenⅠ和α-SMA的表达分别为通过蛋白质印迹和qRT-PCR分析进行分析。(D)UUO模型组和假手术组小鼠肾组织HE4，胶原Ⅳ，胶原Ⅰ的免疫组织化学分析结果分析。放大倍数×400。
图2是Western blot图，其是HIF-1α过表达质粒转染HK2细胞，Western blot检测发现过表达的HIF-1α促进HE4，胶原IV，胶原Ⅰ和α-SMA表达；
图3是证明HIF-1α可以通过与HE4启动子结合直接诱导HE4的上调的图；
图4是利用质谱LC-MS/MS，结合Co-IP方法，从肾小管上皮细胞中筛选出了与HE4作用的筛选图；
图5是用Western印迹分析HE4，P65，磷酸化的P65(PP65)和NF-κb的相关活性图；
图6是证明HE4是NF-κB信号通路的激动剂，通过激活NF-κB信号通路参与肾纤维化的实验图；
图7是证明L-GFP-HE4注射入C57小鼠，构建UUO模型纤维化程度减轻的实验图。
具体实施方式：
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
实施例1：
一、材料和方法
1、细胞培养和实验条件人近端肾小管上皮细胞系(HK2)采用10％胎牛血清的F12培养基(Invitrogen，Carlsbad，CA)培养。正常条件下(21％O2,5％CO2,37℃)和低氧(1％O2，5％CO2，37℃)培养箱(Precision Scientific，Winchester，VA，USA)进行0,6,12,24,48和72小时培养。
2、动物模型C57BL/6J小鼠购自空军军医大学实验动物中心(中国西安)，重20-30g，雄性。通过阻断左侧输尿管构建UUO模型，后1周，2周，3周处死动物，立即取样，其中一部分用4％多聚甲醛固定，另一部分在-80℃保存待分析。所有小鼠的工作都是根据第四军医大学实验动物中心的动物管理和伦理委员会的指导方针进行的。
3、免疫组化和免疫荧光将石蜡包埋的组织样品切成约3-5μm厚，二甲苯脱蜡并用梯度酒精重新水化。3％氢过氧化30分钟阻断内源性过氧化物酶活性，柠檬酸盐缓冲液浸泡载玻片，并加热用于抗原热修复，用正常山羊血清封闭30分钟后，一抗4℃孵育过夜HE4(1:30，Abcam)，α-SMA(1：500，Abcam)，Ⅳ型胶原蛋白(collagenⅣ)(1:50，Sangon,shanghai)，Ⅰ型胶原(collagenⅠ)(1:50，Abcam)4℃孵育过夜。次日用抗鼠或抗兔IgG二抗封闭30分钟，用生物素-链霉亲和素HRP检测系统和二氨基联苯胺(DAB)(DAKO，日本东京)评估免疫组织化学染色。我们从三张幻灯片中随机选取5个相同大小的视野，计数阳性染色以分析结果。
免疫荧光分析，十二孔板中加入无菌玻璃盖玻片，将HK-2细胞接种在此盖玻片上。(1％O2，5％CO2，37℃)培养箱中培养HK-2细胞48h，常氧培养条件下(21％O2，5％CO2，37℃)。95％酒精固定细胞爬片15min，磷酸盐缓冲盐水洗3次，每次5min，0.5％Triton X-100磷酸盐缓冲液透化10min。用10％正常山羊或兔血清封闭1小时后，一抗HE4(1：80，Abcam)，α-SMA(1：200，Abcam)，collagenⅣ(1：100，上海生工生物技术有限公司)，collagenⅠ(1:50，上海生工生物技术有限公司)4℃过夜培养，然后第二抗体(FITC标记的山羊抗小鼠或抗-兔IgG)室温孵育1h，共聚焦激光显微镜观察结果。
4、蛋白制备和蛋白质印迹法
UUO模型(C57，雌性)(0.1g)肾组织和细胞(2×107)分别置于1.5ml Eppendorf管中，用200μl/管Tris-HCl，pH 8.0,150mmol/L NaCl，0.1％SDS，1％Nonidet P-40,0.5％脱氧胆酸钠，0.02％叠氮钠，100μg/ml苯基甲基磺酰氟，1μg/ml抑肽酶)处理5分钟，超声振荡匀浆，12000rpm 4℃离心15分钟，加入1/4体积的5×上样缓冲液，95℃加热10分钟。-20℃保存。进行蛋白质印迹，将蛋白在8％SDS-聚丙烯酰胺凝胶上电泳，然后转移到硝酸纤维素膜(Millipore，Bedford，MA)上。用5％Tris缓冲盐水(20mol/l Tris，0.15mol/l NaCl，pH7.0,0.1％吐温20)配置的无脂牛奶封闭后，第一抗体4℃过夜孵育：α-SMA(1：1000，Abcam，美国)，胶原蛋白Ⅳ(1：300，生工生物公司，中国)，Ⅰ型胶原(1：1000，生工生物公司，中国)，HE4(1：100，Abcam，美国)。次日用TBST洗涤膜5分钟三次，次日Tris缓冲盐水中洗涤膜3次每次15分钟，辣根过氧化物酶标记的抗兔或抗小鼠二抗(1：5000，ZSGB-BIO，中国)室温孵育1小时。增强化学发光系统(Amersham，Bioscience)检测结果。进行β-actin作为内参。
5、定量实时RT-PCR
HK2细胞在低氧(1％O2，5％CO2，37℃)中培养6小时，12小时，24小时，48小时和72小时，对照细胞在常氧条件下(21％O2，5％CO2，37℃)培养相同时间。提取HK2细胞总RNA，使用PrimeScriptTM RT Master Mix(Takara，日本)将mRNA逆转录为cDNA，我们在实时PCR系统(ABI PRISMH 7700；Applied Biosystems，Foster，CA)上采用SYBR Green试剂盒(Takara，日本)用25μl反应体系进行qRT-PCR检测基因表达，步骤包含95℃5秒，60℃30秒和72℃1分钟的45个循环。使用2-ΔΔCT方法计算β-actin标准化的mRNA表达。所有PCR重复三次。引物序列在“表1”中列出。
表1
6、质粒构建体和转染
HIF-1α过表达质粒由上海吉凯公司(Genechem)制造，货号：GOSE65662(human)，让外源性的HIF-1α基因序列在细胞内得到表达，过表达的质粒是一过性表达，不整合到细胞染色体组，主要用于瞬时上调脊椎动物细胞中HIF-1α的表达。HIF-1αsiRNA质粒由上海吉凯公司制造，货号为GIEE80171(human)主要本产品针对HIF1A的shRNA编码克隆，可用于在脊椎动物细胞内瞬时或稳定表达，从而消减HIF-1α的表达，本产品已经过测序，并证实了插入的shRNA编码序列正确。HE4过表达质粒由赛戈生物公司构建，让外源性的HE4基因序列在细胞内得到表达，过表达的质粒是一过性表达，不整合到细胞染色体组，主要用于瞬时上调脊椎动物细胞中HE4的表达。HE4siRNA质粒由上海吉凯基因公司构建，货号：GIEE62418(human)，本产品包含3个针对HE4(基因为Wfdc2，NM_026323)的shRNA编码克隆，可用于在脊椎动物细胞内瞬时或稳定表达，从而消减HE4的表达，本产品已经过测序，并证实了插入的shRNA编码序列正确。
HK2细胞接种在6孔板常氧或缺氧培养，生长至50％-60％进行转染实验，转染前两个小时，更换无胎牛血清和抗生素的培养基，用Lipofectamine 2000进行转染(InvitrogenAB.,Lidinggo,Sweden)，细胞转染48小时后收获细胞进行下一步实验。
7、双荧光素酶报告基因测定
构建了HE4的野生型和突变型3'UTR载体，用pcDNA-HIF-1α(3.2μg，其是将HIF-1α转入pcDNA载体中)和/或pGL3-HE4(3.2μg，其是将HE4野生型启动子转入pGL3-Basic载体中)以及突变载体(3.2ug，其是将HE4突变型3'UTR启动子转入pGL3-Basic载体中)共转染细胞，使用Lipofectamine 2000(Invitrogen AB.,Lidinggo,Sweden)海肾荧光素酶载体PRL-TK(Promega，美国)作为转染效率的对照。使用双荧光素酶报告基因检测系统(Promega，美国)来测量荧光素酶活性。实验重复三次。
具体步骤如下：
1.HE4启动子基因扩增，纯化，酶切，连接及转化
1)设计巢式PCR引物，根据巢式PCR合成含有HE4启动子的DNA片段。引物详见表2-1，以HK-2细胞的基因组DNA为模板，扩增HE4的野生型启动子和突变型3'UTR启动子。
表2-1.引物设计表
| HE4pro-NestW-F1(扩增HE4的野生型启动子) | 5-GGACAAGGGTGAGATGAATGA-3 |
| HE4pro-NestW-R1(扩增HE4的野生型启动子) | 5-ATGCAGTGAGAATGAGGGCTA-3 |
| HE4pro-NestN-NheIF1(扩增HE4的突变型3'UTR启动子) | 5-CTAGCTAGCACCGCTGTGATGACCATCTT-3 |
| HE4pro-NestN-XhoIR1(扩增HE4的突变型3'UTR启动子) | 5-CCGCTCGAGTTCAAACCCTCAGCCTGTCA-3 |
2)1％凝胶电泳后，与紫外灯下切取目的片段，放入干净的1.5ml EP管中称重(若胶块超过300mg，应放入多个EP管中)
3)切碎胶块，按照1mg＝1ul计算胶块体积，1％凝胶按照体积比1:3加入溶解液Buffer GM。
4)室温下充分溶解胶块，期间不停震荡促进胶块溶解。
5)待胶块完全溶解后，将溶液移入Spin Column中，12000rpm/min离心1min，弃滤液。
6)重复步骤5)
7)将700ul Buffer WB加入Spin Column中，室温12000rpm/min离心1min，弃滤液。
8)重复步骤7)
9)12000rp/min 1min空离一次，彻底取去除Spin Column中残留的液体，室温静置5min。
10)将30μl去离子水加入到Spin Column中，室温静置1min。
11)室温12000g/min离心2min，洗脱DNA。紫外分光光度计测量DNA片段浓度
12)回收DNA片段酶切，纯化。酶切体系详见表2-2
表2-2DNA片段、质粒酶切体系
13)DNA片段与pGL3-Basic载体按照载体摩尔:目的片段摩尔＝1：3-1：10的比例确立连接体系，将体系放入金属浴中，金属浴16h。具体连接体系详见表2-3
表2-3连接体系
DH-5α感受态细胞转化，挑取多个单克隆，摇菌，提取质粒。
2.质粒的酶切鉴定
1)DH-5α感受态细胞转化，挑取多个单克隆，摇菌，提取质粒。
2)质粒酶切鉴定，纯化。酶切体系详见表2-4
表2-4质粒酶切鉴定酶切体系
3)1％凝胶电泳酶切产物
4)凝胶电泳结果观察
3.重组质粒转染HK2细胞及其萤光素酶活性的测定。
1)HK2细胞常规复苏，传代。待其长至10cm培养皿底部80％时，接种于六孔板用含10％胎牛血清的F12培养基在37℃、5％CO2条件下，在细胞培养箱中培养。
2)至细胞融合为50-80％时，更换为无胎牛血清的高糖培养基，饥饿两小时后，使用Lipofectamine 2000转染试剂进行转染，构建好的PGL3-HE4报告载体分别与HIF-1α过表达质粒、TK质粒共转染HK2细胞。阳性对照组为包含CMV启动子的报告载体与HIF-1α过表达质粒、TK质粒共转染HK2细胞。阴性对照组为HIF-1α过表达质粒、PGL3-BASIC空载体与TK质粒共转染HK2细胞。
3)8小时后更换为含10％胎牛血清的F12培养基，继续培养48小时。
4)按照Dual-Luciferase Reporter Assay System(E1901)双荧光素酶报告系统说明书步骤收集细胞。
5)于Modulus Single Tube Luminometer Fluorometer and Absorbance Reader上读取荧光素酶检测数值。
8、染色质免疫沉淀分析
通过ChIP实验验证HIF-1α结合在HE4启动子。将HK-2细胞用1％多聚甲醛固定，并使用F550微尖细胞超声仪(Fisher Scientific，Waltham，美国)剪切来自分离的细胞核的染色质。离心后，将含有剪切染色质的上清液与抗HIF-1α抗体或对照IgG温育。然后在过夜孵育之前加入蛋白A-琼脂糖珠，随后洗脱免疫复合物。接下来用RNase和蛋白酶K处理复合物，并用苯酚/氯仿然后用氯仿萃取。DNA沉淀，洗涤，干燥，重新悬浮于水中，并通过PCR或qRT-PCR分析。表1中列出了PCR分析的引物和扩增子。
9、慢病毒构建体
GFP-HE4siRNA(Lv-GFP-HE4)或GFP作为对照(Lv-GFP-N，其不含有HE4的空载体)的慢病毒载体，均由上海吉凯公司构建。用阳离子脂质复合物方法(Lipofectamine 2000；Invitrogen；Thermo)将重组慢病毒载体和包装质粒(pHelper 1.0，包括gag/pol和pHelper2.0，包括病毒G)共转染293T细胞，培养24小时和48小时后，收集富含慢病毒颗粒的上清液，通过0.45μm过滤器(Millipore)过滤。最后用Lenti-PacTM慢病毒浓缩液(广州复能基因有限公司)浓缩病毒，测定效价。最终滴度为2×109TU/ml。
具体步骤如下：
A.慢病毒的包装
1)HEK293T细胞常规复苏传代，接种于6孔板，待细胞融合至6孔板底部70％时，可用于下一步实验。
2)弃去6孔板中培养基，加入1ml/孔的无FBS的opti-MEM培养基，饥饿2小时处理。
3)配制转染混合液，将10μl脂质体混合于250μlopti-MEM培养基中，室温静置10min。
4)配制转染混合液，将含有HE4鼠源siRNA的目的载体与慢病毒骨架蛋白质粒help1和help 2按照4:3:2比例混合，室温静置10min。
5)将3)和4)混匀，室温静置20min。
6)将混合液加入6孔板中后，孵育24小时收集细胞上清液并换液。
7)48小时后收集细胞上清液，保存至-80℃冰箱，择日进行慢病毒浓缩。
B.慢病毒的浓缩
1)将收集的细胞上清液用0.45μm的滤膜过滤掉细胞碎片。
2)按照慢病毒上清液：浓缩试剂体积＝5：1的比例加入浓缩试剂体积，4℃孵育过夜。
3)第二天，混合液在4℃条件下以3500g/min离心25min。
4)离心后弃掉上清液(注意：避免吸取下层沉淀)
5)按照病毒上清液体积，加入1/10的新鲜无菌1×PBS，轻柔重悬病毒沉淀。
6)量取少量测定病毒滴度，其余分装保存于-80℃冰箱。
说明：HE4鼠源siRNA由上海吉凯公司提供，具体货号为：GIEE83211(mouse)
10、病毒注入敲低HE4在UUO模型中表达
根据Nakamura等描述了小鼠的体内病毒转导的原理[27]。将C57雄性小鼠麻醉，在左肾下极与长轴平行进针入，小心推向上极，当针缓慢移除时，注射过滤纯化的慢病毒LV-GFP-HE4或对照(Lv-GFP-N)50μl。假手术组(sham)组仅注射生理盐水，不阻塞输尿管；对照组注射慢病毒对照，注射7天后阻塞左侧输尿管构建UUO模型。Lv-GFP-HE4组注射Lv-GFP-HE4，注射后第7天梗阻左侧输尿管，14天取肾脏。将组织用4％多聚甲醛固定进行组织化学染色，而另一些在-80℃保存以获得组织蛋白。
二、统计分析
数据以平均值±SEM表示。实验组和对照组使用t检验进行比较。多组数据间的差异采用SPSS19.0单因素方差分析，p<0.05为差异有统计学意义。
三、结果
1.体内、外实验证实缺氧诱导肾小管上皮细胞高表达HE4
为了确定缺氧是否影响HE4的表达，将HK2细胞置于低氧(1％O2，5％CO2，37℃)培养箱中6,12,24,48和72小时或者常氧条件下(21％O2，5％CO2，37℃)培养。蛋白免疫印迹实验显示HE4表达增加并具有时间依赖性，胶原蛋白I，胶原蛋白Ⅳ和α-SMA的表达也随着缺氧时间的增加而增加(图1A)。通过qRT-PCR验证差异表达的mRNAs，与western blot相一致，随着缺氧时间的增加，HE4和纤维化因子的表达增加(图1A右)，免疫荧光实验发现在缺氧条件下48小时HE4，α-SMA，I型胶原，IV型胶原的表达较0小时有显著增加(图1C)。进一步构建UUO模型，Western blot结果显示，与对照组相比，UUO模型组肾脏中HE4和纤维化因子随着纤维化程度的增加，表达显著升高(图1B)。RT-PCR分析也证实了相同的结果(图1B右)。同时，免疫组织化学染色显示UUO肾脏中肾小管细胞胞质中HE4表达增加模型中，在模型中观察到最高的表达3周(图1D)。
2、HIF-1α可以通过与HE4启动子结合直接诱导HE4的上调
HIF-1α过表达质粒和靶向序列的siRNA(small interfering RNA，siRNA)，分别转染HK2细胞。其中HIF-1α过表达质粒转染HK2细胞，Western blot检测发现过表达的HIF-1α促进HE4，胶原IV，胶原Ⅰ和α-SMA表达(图2)。
Western blot检测发现HIF-1αsiRNA(pSilencer HIF-1α)降低HE4，胶原IV，胶原Ⅰ和α-SMA表达(图3A)。此外，我们利用萤光素酶报告基因实验检测HIF-1α对HE4基因(编码HE4)的转录调控。将HE4野生型启动子和两个HE4启动子缺失突变体(命名为M1-M2)克隆到pGL3-碱性荧光素酶载体中，检测HIF-1α对荧光素酶活性的影响。如图3B所示，与对照相比，用HE4野生型启动子载体转染的细胞中萤光素酶活性显着增加(p<0.01)。与野生型载体转染细胞相比，含有HE4启动子的HRE1(-736bp至-741bp)突变区荧光素酶活性无显著变化。相比之下，仅含有HRE2(-115bp到-121bp)的HE4启动子导致萤光素酶活性与对照活性相比显着增加。这些数据表明HIF-1α能诱导HE4在蛋白和mRNA水平的高表达。这种缺氧诱导的HE4表达与HIF-1α的转录有关，HE4的HRE2可能是HIF-1α的主要结合位点。另外，如图3C所示，ChIP实验分析发现(-115bp到-121bp)位点的204bp的显性条带。其他可能的结合位点没有明显的条带，对照IgG免疫沉淀，这些结果证实近端HRE2是HIF-1α结合HE4启动子的主要结合位点(图3C)。
3、HE4是NF-κB信号通路的激动剂，通过激活NF-κB信号通路参与肾纤维化
我们进一步以HE4为诱饵蛋白，利用质谱LC-MS/MS，结合Co-IP方法，从肾小管上皮细胞中筛选出了与HE4作用较强的蛋白—IKBIP(inhibitor of NF-κB kinaseinteracting protein)(图4)。查阅文献发现，IKBIP可通过使IKBα(NF-κB inhibitoralpha)磷酸化入核激活NF-κB通路，而NF-κB通路在炎症细胞浸润、成纤维细胞活化、ECM分泌等多种促纤维化途径中均发挥关键作用。
缺氧条件下的HE4过表达质粒转染HK2细胞48小时以后，然后用Western印迹分析HE4，P65，磷酸化的P65(PP65)和NF-κb的相关活性。发现HE4促进P65的磷酸化，并且增加NF-κb通路的活性(图5)。
进一步HE4siRNA转染HK2细胞48小时，然后用Western印迹分析HE4，P65，磷酸化的P65(PP65)和NF-κb下游靶基因TIMP1的表达。如图6A所示，在缺氧条件下，转染HE4siRNA后HE4，PP65和TIMP1表达下调，而对照组P65表达无明显变化。然后HE4过表达质粒(pcDNA-HE4)转染HK2细胞48小时后，用NF-κb通路抑制剂(JSH-23)(150mg/ml)处理HK2细胞24小时。我们发现用NF-κb信号抑制剂处理后，相比单独转染HE4组，胶原Ⅳ，胶原Ⅰ，α-SMA和TIMP1表达下调，MMP2表达上调(图6B)。因此证实HE4参与肾纤维化是通过激活NF-κb发挥作用的，HE4是NF-κb通路的激动剂。
4、L-GFP-HE4注射入C57小鼠，构建UUO模型，小鼠纤维化程度减轻
为了证实HE4在肾纤维化中的作用，将Lv-GFP-HE4注射到小鼠肾皮质，注射Lv-GFP-N组为对照组，7天后构建UUO。注射了Lv-GFP-HE4的小鼠相比对照组显示出纤维化进展的减缓。Masson染色显著减少，I型胶原含量，Ⅳ型胶原含量和α-SMA含量降低(图7B)。蛋白质印迹法检测胶原蛋白I，胶原蛋白Ⅳ，α-SMA的表达，也观察到相应蛋白表达与对照组有显着降低(图7A)。
综上所述，我们的研究结果提示缺氧可以诱导肾小管上皮细胞内源性HE4高表达，高表达的HE4诱导NF-κb通路的激活进一步上调NF-κb的下游分子TIMP1进而促进肾纤维化，这一发现为研究肾纤维化机制和抗纤维化的治疗提供了重要的启示。
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1.人附睾蛋白4在制备NF-κB的激动剂中的应用。
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