CN116059406A - 一种用于治疗糖尿病肾脏疾病的Bdh1基因药物 - Google Patents
一种用于治疗糖尿病肾脏疾病的Bdh1基因药物 Download PDFInfo
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Abstract
一种用于治疗糖尿病肾脏疾病的Bdh1基因药物,包括基因载体和Bdh1基因。采用血清型为AAV9的腺相关病毒基因递送载体与Bdh1基因结合,产生AAV9‑Bdh1‑GFP基因药物,所述AAV9‑Bdh1‑GFP基因药物通过乙酰乙酸‑琥珀酸‑延胡索酸代谢促进NRF2核易位并激活NRF2介导的抗氧化途径,NRF2的激活改善高糖和高脂诱导的糖脂毒性。且该基因药物能够在小鼠肾脏组织中成功表达BDH1,改善小鼠肾脏的炎症、纤维化和凋亡情况,在不依赖于血糖水平的情况下对糖尿病肾脏起到保护作用,达到防治糖尿病肾脏疾病的目的。
Description
技术领域
本发明涉及基因治疗领域,具体涉及一种用于治疗糖尿病肾脏疾病的Bdh1基因药物。
背景技术
糖尿病肾脏疾病(diabetic kidney disease,DKD)是由糖尿病所导致的慢性肾脏疾病,是糖尿病主要的微血管并发症之一,以持续性白蛋白尿和肾小球滤过率(eGFR)下降为特征。我国约20%-40%的糖尿病患者并发DKD,成为我国及大部分发达国家末期肾功能衰竭和死亡的重要原因,同时与心血管疾病的发病率和死亡率显著增加相关。目前,可采用以下方式进行干预和治疗DKD:营养限制、戒烟、运动、减重、服用西医口服药(如SGLT2抑制剂、DPP-4抑制剂和胰岛素促泌剂等)和胰岛素注射降糖等。然而,以上方式的依从性均较差,不能有效控制血糖而改善DKD,且西医口服药和胰岛素注射会引起低血糖诱发心梗和脑卒中等更严重的病情。目前尚无有效的防治手段来延缓DKD的进展,因此,致力于寻找防治DKD的方法是迫切需要的。
基因治疗(gene therapy)是指将外源正常基因导入靶细胞,以纠正或补偿缺陷和异常基因引起的疾病,以达到治疗目的。能实现治疗蛋白的长期表达和组织特异性表达,从根源上解决传统疗法存在的一系列问题。外源性遗传物质在人体细胞内表达需要进入细胞核,这需要通过载体来实现。目前针对基因治疗的载体一般分为病毒(主要包括慢病毒、腺病毒、逆转录病毒、腺相关病毒等)和非病毒载体(主要包括裸质粒DNA、脂质体、纳米载体等)。其中病毒载体是最主要的基因导入技术方式,是最常用的递送系统。
腺相关病毒(Adeno-Associated Virus,AAV)是安全级别最高RG1级的基因治疗载体,具有安全性高、免疫原性低、病毒滴度高、耐受性好等优点,肾脏研究中常用的AAV载体有AAV2、AAV4、AAV6、AAV8和AAV9等。由于高血糖与高血脂诱导糖尿病肾脏的Bdh1表达显著下调,因此,假设逆转疾病引起的基因表达失衡更能直接对肾脏起到保护作用。
发明内容
本发明通过AAV基因载体介导的Bdh1表达,改善了小鼠肾脏的炎症、纤维化和凋亡情况,逆转了高糖和高脂诱导的ROS过度产生。与依赖于降低血糖对肾脏起到保护作用的西医口服药相比,Bdh1可通过促进乙酰乙酸-琥珀酸-延胡索酸代谢间接调节核红细胞相关因子2(NRF2)介导的抗氧化途径,即在不依赖于血糖水平的情况下对糖尿病肾脏起到保护作用,能有效达到低副作用、防治DKD的目的。
本发明采用下述的技术方案:
一种用于治疗糖尿病肾脏疾病的Bdh1基因药物包括基因载体和Bdh1基因,所述Bdh1的序列如SEQ ID NO.1所示;所述Bdh1基因药物通过乙酰乙酸-琥珀酸-延胡索酸代谢促进NRF2核易位并激活NRF2介导的抗氧化途径,所述NRF2的激活改善高糖和高脂诱导的糖脂毒性。
进一步的,所述基因载体为腺相关病毒。
进一步的,所述腺相关病毒载体血清型为AAV1、AAV2、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9中的一种。
进一步的,所述腺相关病毒载体血清型为AAV9,所述基因药物为AAV9-Bdh1-GFP。
进一步的,所述Bdh1基因药物上的Bdh1基因插入在载体AAV9上的启动子CAG后。
进一步的,所述Bdh1基因药物给药方式为静脉注射给药。
Bdh1基因药物在制备治疗糖尿病肾脏疾病药物中的用途。
本发明的有益效果是:
与依赖于降低血糖对肾脏起到保护作用的西医口服药相比,Bdh1基因药物在不依赖于血糖水平的情况下对糖尿病肾脏起到保护作用,使Bdh1在肾脏有效表达,安全性高,副作用低,提高患者的生活质量,有利于大规模生产,更能有效达到并防治DKD的目的。
附图说明
图1:免疫组织化学染色检测DKD患者肾脏组织内BDH1蛋白的表达水平
图2:免疫组织荧光染色检测DKD患者肾脏组织内BDH1蛋白的表达水平
图3:表达Bdh1的腺相关病毒载体图谱
图4:采用AAV9-Bdh1-GFP在小鼠体内表达Bdh1的实验策略
图5:AAV9-Bdh1-GFP和Vector注射对小鼠体重和血糖的影响结果
图6:AAV9-Bdh1-GFP和Vector注射后小鼠尿ACR水平结果
图7:AAV9-Bdh1-GFP介导的GFP在肾脏的表达效果
图8:Western Blot检测小鼠肾脏内BDH1蛋白的表达水平及BDH1蛋白相对表达量
图9:ELISA、琥珀酸和延胡索酸检测试剂盒检测小鼠肾脏中β-OHB、AcAc、琥珀酸和延胡索酸的水平结果
图10:小鼠肾脏组织中NRF2蛋白在细胞核内的表达情况
图11:H&E染色分析小鼠肾脏组织结构形态结果
图12:Masson染色分析小鼠肾脏组织纤维化水平及纤维化定量结果
图13:免疫组织化学染色分析小鼠肾脏组织内炎症因子IL-1β的表达水平
图14:Tunel染色分析小鼠肾脏组织凋亡情况及凋亡细胞定量结果
图15:高糖或高脂处理HK2细胞后Bdh1的表达水平
图16:高糖或高脂处理HK2细胞后BDH1蛋白的表达情况及BDH1蛋白含量的定量结果
图17:pCMV3-BDH1-Flag在HK2细胞内的转染效率
图18:HK2细胞内过表达BDH1并予以高糖或高脂刺激后ROS的水平
图19:HK2细胞内过表达BDH1并予以高糖或高脂刺激后炎症因子IL-1β的表达情况及IL-1β蛋白含量的定量结果
图20:HK2细胞内过表达BDH1并予以高糖或高脂刺激后细胞上清中炎症因子IL-1β和IL-18的水平
图21:HK2细胞内过表达BDH1并予以高糖或高脂刺激后细胞核内NRF2蛋白的表达水平及NRF2蛋白含量的定量结果
图22:过表达BDH1并予以高糖或高脂刺激后HK2细胞内AcAc、琥珀酸和延胡索酸的水平
图23:Bdh1延缓DKD进展的分子机制示意图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。下述实施例中所用实验方法如无特殊说明,均为常规方法;所用材料、试剂等,如无特殊说明,均可从商业途径得到。
正常肾脏样本收集自接受肿瘤肾切除术的无糖尿病或肾脏疾病的个体,DKD肾脏组织收集自DKD患者进行肾脏穿刺的个体。将收集的临床肾脏标本制作石蜡切片,进行免疫组织化学(图1)和免疫组织荧光染色(图2)。与正常对照相比,BDH1蛋白在糖尿病肾脏疾病患者的肾脏组织中表达明显减少。
本发明所表达Bdh1的腺相关病毒载体AAV9图谱如图3所示,在启动子CAG后插入Bdh1靶点序列(序列如SEQ ID NO.1所示);载体上携带有绿色荧光蛋白(GFP),可通过检测荧光信号来评判感染的效果。利用小鼠对AAV9-Bdh1-GFP治疗糖尿病肾脏疾病的效果进行体内研究,从江苏集萃药康生物科技有限公司(中国江苏)购买了5周龄的m/m(n=4)和db/db(n=8)雄鼠。从南通特洛菲饲料科技有限公司(中国江苏)购买含60%的高脂肪饮食(HFD)和对照饮食。所有动物实验均在如下条件进行:室温23±1℃,相对湿度60%±10%,在单独通风IVC笼子中交替进行12h明暗循环。对于AAV9介导的Bdh1在小鼠肾脏的表达,从北京合生基因科技有限公司(中国北京)合成pAAV9-ITR-CAG-Bdh1-IRES-EGFP-WPRE-Sv40polyA-ITR(AAV9-Bdh1-GFP),将100μLAAV9-Bdh1-GFP(3.40E+12vg/mL)或阴性对照Vector(1.90E+13vg/mL)通过db/db及m/m小鼠尾静脉进行注射,每周记录小鼠的体重和血糖,检测尿ACR,肾脏中β-羟基丁酸(β-OHB)-乙酰乙酸(AcAc)-琥珀酸-延胡索酸的含量,NRF2蛋白的表达及肾脏炎症、纤维化和凋亡等。
本发明采用AAV9-Bdh1-GFP在小鼠体内表达Bdh1的实验策略如图4所示,将小鼠适应性喂养一周后通过小鼠尾静脉将AAV9-Bdh1-GFP或Vector注射入体内,喂养至17周时麻醉处死小鼠后收取标本进行检测。从AAV9-Bdh1-GFP和Vector注射开始每周记录小鼠体重和血糖,结果表明AAV9-Bdh1-GFP注射不影响小鼠的体重和血糖(图5)。注射Vector组的db/db小鼠尿白蛋白/肌酐(ACR)比值较正常小鼠明显升高,而注射AAV9-Bdh1-GFP组小鼠的尿ACR得到改善(图6)。收集小鼠肾脏组织制作冰冻切片于荧光显微镜下检测GFP的荧光强度,可以看到AAV9-Bdh1-GFP载体介导的GFP在肾脏组织中成功表达(图7)。接着通过WesternBlot观察到AAV9-Bdh1-GFP注射小鼠肾脏中BDH1表达比Vector注射小鼠增加(图8)。在进一步的组织学分析(H&E)染色中,可以看到注射Vector的db/db小鼠的肾脏中部分肾小管出现萎缩变形,肾小球固缩,而注射AAV9-Bdh1-GFP的db/db小鼠的肾脏形态结构相较于Vector组有明显改善(图11)。此外,AAV9-Bdh1-GFP注射也显著减少了DKD病理相关的纤维化、炎症和凋亡(图12-14)。作为BDH1的底物,β-OHB在注射AAV9-Bdh1-GFP的db/db小鼠肾脏中的水平降低,注射Vector的db/db小鼠肾脏中的AcAc、琥珀酸和延胡索酸水平降低,而通过AAV9-Bdh1-GFP的注射得到逆转(图9)。最后通过免疫荧光我们发现AAV9-Bdh1-GFP注射促进了db/db小鼠肾脏中NRF2的核易位(图10)。这些发现共同为Bdh1作为DKD治疗靶点的应用前景提供了有力支持。
采用HK2细胞(人肾皮质近曲小管上皮细胞)对pCMV3-BDH1-Flag治疗糖尿病肾脏疾病的效果进行体外实验研究,HK2细胞在含有10%胎牛血清的DMEM/F12培养基中培养,并补充1%青霉素-链霉素。将HK2细胞在37℃下用5%CO2培养至60-70%汇合,将细胞暴露于正常对照、高糖或棕榈酸48小时。从北京义翘神州科技股份有限公司(中国北京)合成BDH1过表达质粒pCMV3-BDH1-Flag(Flag-BDH1)和载体质粒pCMV3,并用Lipofectamine 3000(Invitrogen)转染到HK2细胞中,检测细胞内ROS的水平,NRF2蛋白的表达以及AcAc-琥珀酸-延胡索酸的含量。高糖(HG)和高脂(PA)模拟的2型糖尿病肾脏微环境可以诱导Bdh1的mRNA和蛋白表达显著下调(图15-16)。因此,接下来在HK2细胞中将BDH1过表达(图17),通过DCFH-DA探针检测发现BDH1过表达后明显抑制了由HG或PA诱导的细胞内ROS的产生(图18)和IL-1β蛋白的表达(图19-20)。HG或PA的刺激抑制了NRF2的核易位,而将BDH1过表达后能够明显增加细胞核内NRF2蛋白的水平(图21)。与小鼠体内实验观察到的一样,HG或PA刺激的HK2细胞中AcAc、琥珀酸和延胡索酸水平降低,而通过过表达BDH1得到逆转(图22)。图23展示由Bdh1介导的βOHB代谢,通过AcAc-琥珀酸-延胡索酸代谢途径促进NRF2进入细胞核发挥抗氧化的作用,从而延缓DKD进展的分子机制示意图。
实验例一
β-OHB检测(F9242-A,上海泛柯实业有限公司):(1)肾脏组织样本处理:用预冷的PBS(0.01M,pH=7.4)冲洗组织,去除残留血液,每个样本称取10mg组织加入200μLPBS(含1%PMSF)中进行匀浆并超声破碎。后将匀浆液于5000×g离心10分钟,取上清检测。(2)按照试剂盒说明书稀释标准品。(3)加样:分别设空白孔、标准孔、待测样品孔。在酶标包被板上标准品准确加样50μl,待测样品孔中先加样品稀释液40μl,然后再加待测样品10μl。(4)温育:用封板膜封板后置37℃温育30分钟。(5)洗涤:小心揭掉封板膜,弃去液体,甩干,每孔加满1X洗涤液,静置30秒后弃去,如此重复5次,拍干。(6)加酶:每孔加入酶标试剂50μl,空白孔除外。(7)温育:操作同(4)。(8)洗涤:操作同(5)。(9)显色:每孔先加入显色剂A50μl,再加入显色剂B50μl,轻轻震荡混匀,37℃避光显色10分钟。(10)终止:每孔加终止液50μl,终止反应。(11)测定:以空白孔调零,450nm波长依序测量各孔的吸光度(OD值)。
实验例二
AcAc检测(JL15388,上海江莱生物科技有限公司;2M-KMLJM220724m,南京卡米洛生物工程有限公司):(1)样本处理:肾脏组织:同β-OHB检测。HK2细胞:收集已经完成干预的HK-2细胞,每1×106个细胞加入100μL预冷PBS用超声波破碎仪将细胞完全破碎后,5000×g离心10分钟,取上清即可检测。(2)从室温平衡60分钟后的铝箔袋中取出所需板条,空白孔中加50μL样本稀释液,标准品孔各加50μL不同浓度的标准品,样本孔中加50μL待测样本。(3)每孔加入100μL辣根过氧化物酶(HRP)标记的检测抗体,用封板膜封闭反应孔,37℃孵育60分钟。(4)弃去液体,包上吸水纸用力拍干,用排枪加350μL洗涤液到各孔中,静置1分钟弃去液体,吸水纸上拍干,如此重复操作5次。(7)每孔加入底物A、B各50μL,37℃避光孵育15分钟。(8)每孔加入50μL终止液,15分钟内,在450nm波长处测定各孔OD值。(9)根据标准曲线计算出各样本中AcAc含量后进行统计学分析。
实验例三
琥珀酸检测(MAK335,Sigma-Aldrich Co.LLC):(1)试剂准备:所有试剂使用前达到室温。将10μL 20mmol/L的标准品加入190μL的超纯水中吹打混匀,稀释成1mmol/L的标准品备用。每一反应孔配制检测试剂混合物工作液:85μL检测缓冲液+1μL酶混合物+1μL辅被作用物+1μL PEP溶液+1μL染料试剂,现配现用;(2)样品准备:肾脏组织:同β-OHB检测。HK2细胞:收集已经完成干预的HK-2细胞,每1×106个细胞加入100μL超纯水快速进行超声裂解,再14000转离心5分钟得到上清液备用;(3)加样:在96孔板中一组样品设置3个孔,每孔加20μL样品,再将20μL超纯水加入另一孔作为空白对照。一组样品的一孔内加5μL标准品,另两个复孔以及空白对照孔加5μL超纯水。再在每一孔中加入反应工作液80μL,轻轻摇晃孔板混匀;(4)孵育:在室温下孵育30分钟;(5)检测:检测570nm波长下的OD值;(6)计算琥珀酸浓度:琥珀酸浓度(μmol/L)=((样品OD值-空白孔OD值)/(标准品OD值-样品OD值))*250。
实验例四
延胡索酸检测(MAK060,Sigma-Aldrich Co.LLC):(1)标准品稀释:延胡索酸检测缓冲液在使用前达到室温。将10μL 0.1mol/L的延胡索酸标准品加入990μL检测缓冲液里面混匀,稀释成1mol/L的标准品溶液。依次加入0、5、10、15、20和25μL的1mol/L标准品溶液到96孔板中,每孔分别含标准品0、5、10、15、20、25nmole。再向每孔中加入检测缓冲液定容至50μL,每个浓度设置两个复孔;(2)样品准备:肾脏组织:同β-OHB检测。HK2细胞:收集已经完成干预的HK-2细胞,每1×106个细胞加入100μL检测缓冲液快速进行超声裂解,再13000xg离心10分钟得到上清液,将50μL样品加入96孔板中,每组设置三个复孔;(3)加入主反应物:每孔加入检测缓冲液90μL,显影剂8μL,酶混合物2μL,在水平摇床上充分混匀,然后在室温下避光孵育30分钟;(4)测定:测定450nm波长下的OD值;(5)根据标准品算出的标准曲线,算出待测样品物质的量,再根据所加样品的体积以及延胡索酸的分子量(116.07g/mole)计算出延胡索酸的摩尔浓度。
实验例五
Western Blot实验:肾脏组织:按照10mg+100μLRIPA裂解液(含1%PMSF)(P0013B,上海碧云天生物技术有限公司)进行组织匀浆,12000g离心10分钟,收取上清加入5X上样缓冲液,100℃加热10分钟使蛋白变性。HK2细胞:取出干预完成的细胞,使用冷PBS清洗三遍,后用RIPA裂解液(含1%PMSF)裂解细胞30分钟,12000g离心10分钟,收上清加入5X凝胶上样缓冲液,100℃加热10分钟使蛋白变性。使用10%或12%SDS-PAGE胶电泳后PVDF膜转膜,5%BSA室温封闭1小时,一抗4℃冰箱摇晃过夜孵育,一抗货号分别为:BDH1(ab193156,Abcam,Cambridge,MA,USA),IL-1β(#12242,Cell Signaling Technology,USA),NRF2(sc518033,Santa Cruz Biotech,CA,USA),Tubulin(AF0001,碧云天,中国),Histone H3(AF0009,碧云天,中国),Flag(AF5051,碧云天,中国).次日用PBST洗膜三遍后换对应种属的二抗,室温1小时,PBST再洗膜后显影,将膜至于显影液中(Millipore Corporation),然后控干放入发光检测机器中曝光。条带强度使用ImageJ software软件定量。
实验例六
实时荧光定量PCR实验:实验使用TRIzol Reagent(Invitrogen,Carlsbad,CA,USA)提
取HK2细胞的总RNA。使用20μl反转录体系(FSQ-201,TOYOBO,CO.,LTD)进行反转录合成cDNA,使用SYBR GreenMix(QIAGEN,German)进行荧光定量PCR,仪器型号为Analytikjena qTOWER 3G real-time PCR system(JENA,German)。Bdh1基因的相对表达量由其和β-actin基因的CT值的比值计算得出。
实验例七
ELISA检测炎症因子IL-1β和IL-18的表达:(1)将已完成干预的各组HK-2细胞培养液收集于离心管,2000转/分离心20分钟,收集上清液按组分装标记,存于-20℃冰箱备用;(2)根据说明书稀释标准品;(3)加样:设置空白、标准品及待测样品孔,各设立3个复孔。空白孔不加样品及酶标试剂,标准孔加不同浓度标准品50μL,待测样品孔每孔加入样本50μL,盖上封板膜,轻轻振荡混匀,37℃温育30分钟;(4)配液:30×浓缩洗涤液用蒸馏水稀释成1×洗涤液备用;(5)洗涤:揭掉封板膜弃去液体,每孔加满1×洗涤液,静置30s后倒掉,重复操作5次,拍干;(6)加酶温育:空白孔不加酶标试剂,其余各孔均加50μL酶标试剂,用封板膜封板后于37℃孵箱中温育30分钟;(7)洗涤:操作步骤同(5);(8)显色:每孔先加50μL显色剂A,再加50μL显色剂B,轻轻摇晃混匀,37℃避光显色15分钟;(9)终止:每孔加50μL终止液以终止反应;(10)测定:在终止反应15分钟内,在酶标仪上检测450nm波长下各孔的吸光度(OD值)。(11)各孔OD值减去空白孔OD值后,以标准品的浓度为横坐标,OD值为纵坐标,计算出标准曲线,再以调整的样品OD值在标准曲线上计算出相应的样品浓度。
实验例八
DCFH-DA荧光探针检测ROS:(1)HK-2细胞准备:按照需要准备HK2细胞接种于6孔板中,在计划的时间加入刺激因素干预后进行探针装载。(2)装载探针:吸去6孔板中原有培养基,无菌PBS漂洗3次,每孔细胞加入1mL用基础培养基以1:1000稀释的DCFH-DA(S0033S,碧云天,中国),轻轻摇晃均匀,放回37℃,5%CO2培养箱内孵育20分钟,操作过程注意避光。(3)清洗细胞并采集图像:取出6孔板,用负压吸引器去掉含DCFH-DA探针的培养基,每孔用基础培养基轻轻摇晃洗涤3次,充分去除未进入细胞内的DCFH-DA,操作过程注意避光。于30分钟内在荧光显微镜下观察并采集图像。
实验例九
肾脏组织H&E染色:(1)包埋肾脏组织:取小鼠1/2肾脏组织放入4%多聚甲醛中固定24小时后进行脱水,然后浸石蜡并包埋,包埋后放置冰箱冷却后可放置室温长期保存。(2)组织切片:在切片机上切出4μm厚的肾脏组织,粘附于载玻片上制作组织切片,37℃干燥过夜。(3)脱腊、水化:100%二甲苯溶液中溶解石蜡10分钟,共4次;再用梯度乙醇溶液(100%、95%、90%、80%)进行水化各10分钟,最后自来水冲洗。(4)染色:冲洗好的片子甩干水分依次用苏木素染色5分钟,1%盐酸酒精分化30秒,碳酸锂返蓝2分钟,细胞质用伊红染10分钟,每步操作之间均需自来水冲洗并尽量甩干水分。(5)脱水、透明:将染色完成的片子依次放于梯度酒精溶液(80%、90%、95%、100%)中各浸泡20秒,再置于100%酒精溶液中充分脱水5分钟;100%二甲苯溶液中透明10分钟,共2次。(6)封片、镜检:用中性树胶封片,晾干半天即可显微镜下观察并拍照。
实验例十
肾脏组织Masson染色:(1)肾脏组织切片制备同H&E染色。(2)脱蜡、水化:100%二甲苯溶液浸泡切片15分钟,共2次;95%、70%、30%梯度酒精溶液中浸泡2分钟;蒸馏水浸洗2分钟后放入30-40℃温热水中再漂洗2次(30-60秒)。(3)染色:染色前需先用蒸馏水润湿切片30-60秒;接着用R1染液染细胞核60秒,R2染液染细胞浆60秒,R1与R2染色后都用冲洗液冲净切片,再用R3分色液分色8分钟后直接用R4蓝色染液复染5分钟,再无水乙醇冲净。(4)封片、镜检:片子晾干后,滴加10μL无毒环保封固剂,小心盖上盖玻片,晾干后即可镜检。
实验例十一
肾脏组织Tunel荧光染色:(1)肾脏组织切片脱蜡前全部操作同H&E染色。(2)将vial1与vial2按照1:9比列混匀为Tunel反应混合物备用。(3)脱蜡完成的组织切片用PBS清洗两次后,将样品周围擦干。(4)每张切片滴加50μL Tunel反应混合物于样品之上。(5)37℃环境中孵育60分钟,注意避光和保湿,PBS清洗3次。(6)片子稍晾干即可封片拍照,注意避光。
实验例十二
免疫组织化学染色实验:(1)肾脏组织切片脱蜡和水化操作同H&E染色。(2)自来水充分水洗切片并用蒸馏水浸泡。(3)将切片于沸腾的修复液中加热3分钟后中取出自然冷却至室温。(4)PBS浸洗一次,用3%H2O2进行10分钟孵育,PBS浸洗2分钟/3次。(5)滴加50μL10%山羊血清于肾脏切片上,37℃封闭60分钟。(6)IL-1β(#12242,Cell SignalingTechnology,USA)和BDH1(ab193156,Abcam,Cambridge,MA,USA)用封闭液1:100稀释后加到切片上,放于湿盒中于4℃冰箱过夜,次日PBS浸洗2分钟/3次。(7)选择相应种属的生物素标记二抗,用PBS以1:200比例稀释,滴加50μL于肾脏组织上室温孵育1小时,PBS浸洗2分钟/3次。(8)用PBS以1:200比例稀释辣根酶标记链霉卵白素后加到肾脏组织上,37℃孵育20分钟,PBS浸洗2分钟/3次。(9)在显微镜下观察DAB显色情况,当肾脏组织出现棕色时,迅速放入自来水中终止显色。(10)甩干切片上的水分,用苏木素复染1分钟→自来水洗→1%盐酸酒精3秒→自来水洗→碳酸锂饱和液2分钟→自来水洗→脱水(80%酒精2min→90%酒精2min→95%酒精2min→100酒精2min→100酒精5min)。(11)用中性树胶封片,晾干半天即可显微镜下拍照。
实验例十三
免疫组织荧光染色实验:第(1)-(5)步同免疫组织化学染色。(6)NRF2(sc518033,Santa Cruz Biotech,CA,USA)和BDH1(ab193156,Abcam,Cambridge,MA,USA)用封闭液1:100稀释后加到切片上,放于避光湿盒中于4℃冰箱过夜,次日PBS浸洗2分钟/3次。(7)直接孵育相应种属荧光二抗,用PBS以1:200比例稀释,室温避光环境下孵育1小时,PBS浸洗2分钟/3次。(9)用PBS以1:500比例稀释DAPI用于细胞核染色,室温避光孵育10分钟后,用PBS浸洗2分钟/3次。(10)加上适量抗荧光淬灭剂,用盖玻片小心盖住,即可拍照分析。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (7)
1.一种用于治疗糖尿病肾脏疾病的Bdh1基因药物,其特征在于,所述Bdh1基因药物包括基因载体和Bdh1基因,所述Bdh1的序列如SEQ ID NO.1所示;所述Bdh1基因药物通过乙酰乙酸-琥珀酸-延胡索酸代谢促进NRF2核易位并激活NRF2介导的抗氧化途径,所述NRF2的激活改善高糖和高脂诱导的糖脂毒性。
2.根据权利要求1所述的一种用于治疗糖尿病肾脏疾病的Bdh1基因药物,其特征在于,所述基因载体为腺相关病毒。
3.根据权利要求2所述的一种用于治疗糖尿病肾脏疾病的Bdh1基因药物,其特征在于,所述腺相关病毒载体血清型为AAV1、AAV2、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9中的一种。
4.根据权利要求2所述的一种用于治疗糖尿病肾脏疾病的Bdh1基因药物,其特征在于,所述腺相关病毒载体血清型为AAV9,所述基因药物为AAV9-Bdh1-GFP。
5.根据权利要求1所述的一种用于治疗糖尿病肾脏疾病的Bdh1基因药物,其特征在于,所述Bdh1基因药物上的Bdh1基因插入在载体AAV9上的启动子CAG后。
6.根据权利要求1所述一种用于治疗糖尿病肾脏疾病的Bdh1基因药物,其特征在于,所述Bdh1基因药物给药方式为静脉注射给药。
7.权利要求1-6任意一项所述的Bdh1基因药物在制备治疗糖尿病肾脏疾病中的用途。
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