CN110542759B - Gmfb作为糖尿病肾病的生物标记物的应用 - Google Patents

Gmfb作为糖尿病肾病的生物标记物的应用 Download PDF

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CN110542759B
CN110542759B CN201910269196.7A CN201910269196A CN110542759B CN 110542759 B CN110542759 B CN 110542759B CN 201910269196 A CN201910269196 A CN 201910269196A CN 110542759 B CN110542759 B CN 110542759B
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吕立夏
徐国彤
朱彤
龚浩宇
邱天羽
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Abstract

本发明提供了一种GMFB作为糖尿病肾病的生物标记物的应用。本发明实验发现:GMFB在早期糖尿病大鼠的肾小管上皮细胞中表达,而在正常大鼠的肾小管上皮细胞中不表达。相比正常STZ诱导2周的TIDM大鼠,GMFB敲除的STZ诱导2周的T1DM大鼠在链脲霉素腹腔注射诱导下的肾病早期相关标记物Kim‑1、MCP‑1、IL‑1beta的mRNA表达量显著降低,说明敲除GMFB能在早期阻断糖尿病肾病的发病,避免糖尿病肾病产生。

Description

GMFB作为糖尿病肾病的生物标记物的应用
技术领域
本发明属于生物检测及生物医药技术领域,具体涉及GMFB作为糖尿病肾病的生物标记物的应用。
背景技术
随着我国经济的高速发展和老龄化进程的加速,糖尿病(diabetes milletus,DM)的患病率呈快速上升趋势,成为继心脑血管疾病、肿瘤之后另一个严重危害人民健康的重要慢性非传染性疾病。世界卫生组织推测,在 2025年全球糖尿病患者将达到3亿。糖尿病肾病是糖尿病病人最重要的合并症之一,其在我国的发病率呈上升趋势,目前已成为终末期肾脏病的第二位原因,仅次于各种肾小球肾炎。糖尿病肾病存在复杂的代谢紊乱,一旦发展到终末期肾脏病,往往比其他肾脏疾病的治疗更加棘手,因此及时防治对于延缓糖尿病肾病的意义重大。糖尿病肾病的病因和发病机制不清。目前认为系多因素参与,在一定的遗传背景以及部分危险因素的共同作用下致病。 Kim-1(van Timmeren M M,Mc V D H,BaillyV,et al.Tubular kidney injury molecule-1(KIM-1)in human renal disease.Journalof Pathology,2007,212(2):209-17.)、MCP-1(Haller H,Bertram A,Nadrowitz F, MenneJ.Monocyte chemoattractant protein-1 and the kidney.Curr Opin NephrolHypertens.2016;25(1):42-9.)、IL-1b(Moreno JA,Gomez-Guerrero C,Mas S, etal.Targeting inflammation in diabetic nephropathy:a tale of hope.Expert OpinInvestig Drugs.2018;27(11):917-930..)被认为是糖尿病肾病早期相关标记物,在糖尿病的发生发展中发挥着直接作用。
胶质细胞成熟因子beta(glia maturation factor beta,GMFB)最早是从牛脑分离纯化的17kd酸性胞浆蛋白,进化上高度保守,在中枢神经系统主要由星形胶质细胞产生,对脑组织生长、分化和再生有重要的作用,其表达在发育期上调,成年明显降低。新近研究显示GMFB是一种促炎因子,与人中枢神经系统退行性疾病密切相关,如阿茨海默病和帕金森病;GMFB基因敲除小鼠能够抵抗实验性自身免疫性脑炎和MPTP的毒性。
发明内容
有鉴于背景技术中的技术现状,本发明的目的在于提供一种GMFB作为糖尿病肾病的生物标记物的应用。
本发明提供了一种GMFB作为糖尿病肾病的生物标记物的应用。
本发明提供了GMFB作为生物标记物在制备糖尿病肾病早期诊断及病程进展的试剂或试剂盒中的应用。
本发明提供了检测GMFB表达量的试剂在制备糖尿病肾病早期诊断及病程进展的试剂或试剂盒中的应用。
本发明提供了GMFB干扰剂在制备预防、改善或治疗糖尿病肾病的药物中的应用,所述GMFB干扰剂为干扰GMFB活性或下调GMFB表达的物质。
有益效果:本发明提供了GMFB作为糖尿病肾病的生物标记物的应用。本发明研究发现:GMFB在早期糖尿病大鼠的肾小管上皮细胞中表达,而在正常大鼠的肾小管上皮细胞中不表达。相比正常STZ诱导2周的TIDM大鼠,GMFB敲除的STZ诱导2周的T1DM大鼠在链脲霉素腹腔注射诱导下的肾病早期相关标记物Kim-1、MCP-1、IL-1beta的mRNA表达量显著降低,说明敲除GMFB能在早期阻断糖尿病肾病的发病,避免糖尿病肾病产生。
附图说明
图1为本发明实施例2所述Kim-1的表达量变化情况;
图2为本发明实施例2所述MCP-1的表达量变化情况;
图3为本发明实施例2所述IL-1β的表达量变化情况;
图4为本发明实施例3所述显微镜图。
具体实施方式
本发明提供了一种GMFB作为糖尿病肾病的生物标记物的应用。优选的,所述应用包括以GMFB作为生物标记物制备糖尿病肾病早期诊断及病程进展的试剂或试剂盒。本发明在I型糖尿病发病1周的肾脏能检测GMFB 表达显著增高,早于MCP-1、IL-1beta和kim-1的表达;可以作为早期糖尿病肾病的标志物。
本发明提供了检测GMFB表达量的试剂在制备糖尿病肾病早期诊断及病程进展的试剂或试剂盒中的应用。在本发明中,所述检测GMFB表达量的试剂优选包括检测肾脏GMFBmRNA表达量的试剂。鉴于GMFB表达量与糖尿病肾病早期诊断及病程进展的关系,将检测GMFB表达量的试剂应用于糖尿病肾病早期诊断及病程进展的试剂或试剂盒的制备具有良好的应用前景。
本发明还提供了GMFB干扰剂在制备预防、改善或治疗糖尿病肾病的药物中的应用。在本发明中,所述GMFB干扰剂为干扰GMFB活性或下调 GMFB表达的物质。鉴于GMFB表达量与糖尿病肾病早期诊断及病程进展的关系,将GMFB干扰剂应用于预防、改善或治疗糖尿病肾病的药物具有广阔的前景。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
糖尿病大鼠制备:采用雄性SD大鼠和GMFB敲除SD大鼠,160-180g。实验前先将大鼠饥饿24小时。单次腹腔内注射STZ(60mg/kg体重)来诱发DM,正常对照组腹腔注射等体积的柠檬酸溶液;24小时后断尾取血测血糖,血糖值低于250mg/dL的大鼠补充注射STZ。连续3天测血糖。将血糖连续3天超过250mg/dL的大鼠确定为DM大鼠(血糖低于250mg/dL的大鼠将被排除)。
实施例2
(1)链脲霉素腹腔注射野生型SD大鼠和GMFB敲除大鼠后,采用实施例1所述方法制备糖尿病大鼠模型。
(2)在STZ造模后2周,分别收集野生型SD大鼠和GMFB敲除大鼠的肾脏样本到1mlTrizol,装在1.5ml管中,抽提总RNA。经过逆转录和实时定量PCR分析,采用半定量的方法计算肾病早期相关标记物的表达量,分析表达量变化情况。主要步骤如下:
①向Trizol收集的样品中加入200ul(五分之一)体积的氯仿,剧烈混匀后,以12000rpm的转速4℃离心10min。
②离心后,将上清转移到新的离心管中,注意不要取到中间的蛋白层,加入等体积的异丙醇。
③12000rpm的转速,4℃离心10min,弃去上清,将沉淀用75%的乙醇洗1~2次。
④将沉淀室温干燥后,用适量的20ul DEPC水溶解。
⑤Nanodrop定量,计算反转录所需的体积。
⑥RNA反转录:
20ul的体系,1000ng的RNA,取4ul的Takara的逆转录试剂supermix,再加ddH2O补到20ul.
反转录程序:37℃15min,85℃5sec,然后可于-20℃冰箱保存备用。
⑦定量PCR
A:将RNA反转录后得到的cDNA 10倍稀释作为为模板,设计引物如表1。
表1扩增用引物列表
Figure BDA0002017822180000041
Figure BDA0002017822180000051
B:利用天根公司的SYBR Green实时荧光定量PCR检测试剂盒。
q-PCR的体系为20ul:2ul的cDNA模板,1ul的引物,10ul的2×PCR Mix, 7ul的ddH2O,检测目的基因的表达量。
PCR扩增条件如下:94℃变性10min,进入循环(95℃5sec,60℃60sec),一共40个循环,并收集溶解曲线。
C:数据分析,采用
Figure BDA0002017822180000052
法进行处理,以Gapdh做为内参。
实验结果如图1~3所示。其中,图1为Kim-1的表达量变化情况;图2 为MCP-1的表达量变化情况;图3为IL-1β的表达量变化情况。由图1~3 可以看出,用STZ腹腔注射制备的一型糖尿病大鼠,GMFB基因敲除大鼠大鼠肾脏在糖尿病发病两周Kim-1(附图1)、MCP-1(附图2)、IL-1beta (附图3)表达与正常大鼠制备的两周糖尿病模型肾脏表达有显著性降低,提示GMFB敲除抑制糖尿病肾脏炎症因子的表达。
实施例3
GMFB在早期糖尿病性肾脏组织的表达
(一)肾脏冰冻切片的制备
1.在STZ腹腔注射SD大鼠建模成功后,在糖尿病发病1周取肾脏组织;
2.固定:置于4%多聚甲醛固定48小时;
3.脱水:用10%、20%和30%的蔗糖过夜脱水;
5.包埋:用组织包埋液OCT包埋4℃平衡过夜;
6.液氮速冻:将肾脏用液氮快速冷冻,冷冻之前使得肾脏位置尽量垂直居中,-80℃保存冰冻样品;
7.切片:用冰切机按8μm的厚度进行连续切片,将切片放在-80℃保存,使用前吹干。
(二)肾脏冰冻切片免疫荧光
1.切片准备:取(一)中所得的肾脏冰冻切片样品进行免疫荧光染色检测;
2.烤片:50℃烤片半小时;
3.固定:4%PFA固定10分钟,再用PBS清洗三次,每次5分钟;
4.透膜:用0.25%Triton-X100透膜15min,PBS洗3次,每次5min;
5.封闭:5%马血清室温封闭1小时;
6.一抗:加5%马血清稀释的相应一抗GMFB抗体,置于湿盒内防止干燥,4℃孵育过夜;
7.二抗:PBS洗3次,每次5min;加5%马血清稀释的FITC标记的与一抗同种属来源的二抗,置于湿盒内,避光37℃孵育1小时;
8.DAPI染核:PBS洗3次,每次5min;用0.5μg/mlDAPI染细胞核1min, PBS洗3次,每次5min;
9.封片:用荧光封片剂封片;
10.拍照观察:在显微镜下拍照观察,结果见图4。
图4为1周和2周的肾脏冰冻切片免疫荧光照片,其中蓝色为DPAI染核,红色为Cy3标记的二抗(抗GMFB抗体)。由图4可知:在糖尿病发病1周,GMFB免疫荧光与正常对照组相比增强,发病2周GMFB比发病一周免疫荧光强度更强。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
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Claims (1)

1.GMFB干扰剂在制备预防或治疗早期糖尿病肾病的药物中的应用,其特征在于,所述GMFB干扰剂为干扰GMFB活性或下调GMFB表达的物质。
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CN105154527A (zh) * 2015-07-21 2015-12-16 同济大学 Gmfb的应用、gmfb干扰剂及gmfb干扰剂的应用
CN108939066A (zh) * 2018-07-13 2018-12-07 同济大学 Gmfb抗体作为制备糖尿病视网膜病治疗药物的应用

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