CN114807343A - 检测胰岛β细胞去分化的标志物的用途 - Google Patents
检测胰岛β细胞去分化的标志物的用途 Download PDFInfo
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Abstract
本发明公开了一种检测胰岛β细胞去分化的标志物的用途,所述标志物为miR‑483‑5p,其miRNA成熟体序列如SEQ ID NO:1或SEQ ID NO:2所示。miR‑483‑5p正/反向引物用于制备2型糖尿病β细胞去分化诊断试剂盒。本发明的miR‑483‑5p作为2型糖尿病早期β细胞去分化的标记物,可以敏感、准确的预警早期、低水平血糖的损害。
Description
技术领域
本发明属于生物医药领域,特别涉及检测β细胞去分化的标志物的用途。
背景技术
糖尿病是以持续高血糖为特征的一种慢性代谢性疾病,主要是由于体内胰岛素分泌绝对或相对不足或胰岛素抵抗而导致的以糖代谢紊乱为主的一种综合病症。根据发病原因和机制的不同,糖尿病可分为:1型糖尿病、2型糖尿病、妊娠期糖尿病和其他特殊类型的糖尿病。目前,2型糖尿病占糖尿病总数的95%以上。而且,全球2型糖尿病的患病人数不断攀升,其主要危害是可引起心、脑、肾、视网膜病变等一系列严重并发症。虽然2型糖尿病的发病机制目前尚未完全阐明,但诸多研究数据显示,胰岛β细胞功能障碍是2型糖尿病发生发展的中心环节之一。
目前,糖尿病的诊断主要是通过糖耐量实验。糖尿病诊断标准为,空腹血糖≥7.0mmol/L或饭后2小时≥11.1mmol/L。当糖尿病症状者血糖到达这个数据时就可以确诊。糖尿病最典型的临床表现为“三多一少”,“三多”是指多饮、多食、多尿,“一少”是指体重减少,但是当出现三多一少时,糖尿病的病情已经不轻了。大多数早期患者没有症状,血糖数值也没有达到糖尿病的诊断标准。可能在发病多年之后才诊断出患有糖尿病,而此时则已出现并发症,对身体的各种组织特别是心脑血管系统、神经系统、眼睛、肾脏已造成长期损害。因此,找到一种明确而有效的生物标志物来作为2型糖尿病的早期预警信号,从而早诊断、早干预、早治疗,对控制糖尿病的发生和加重具有重要意义。
诸多临床研究都表明,当初诊断2型糖尿病时,胰岛细胞功能已经丧失过半。随着病程进展,β细胞凋亡,功能衰退仍会进行性发展,患者最终需要胰岛素替代治疗。众多2型糖尿病患者尸检结果显示,胰岛β细胞总数只有正常水平的50%,研究者最初将其归咎于β细胞的大量凋亡。后续研究显示,虽然胰腺中胰岛素的免疫荧光阳性率大幅下降,但被检测到的细胞凋亡与功能减退程度却不呈正比。由此提示,胰岛β细胞总数的大量减少原因可能发生了去分化。Cinti等通过研究2型糖尿病病人捐献的胰腺组织,证明了人体中也存在胰岛β细胞去分化的现象,并且去分化的β细胞比例(31.9%)远高于正常人(8.7%)。因此,去分化是造成β细胞功能丧失的一个重要原因。β细胞在受到代谢应激性损伤后的首先表型为去分化而非凋亡,这一“自私”行为保证了其能够继续存活而不至于立即死亡。去分化很可能是细胞在机体对其需求增大时的一种应对方式,如果机体出现肥胖和胰岛素抵抗、炎症因子损伤等持续性刺激,这些细胞就只能离其成熟状态渐行渐远,最终丧失分泌功能而真正“凋亡”。
microRNA(miRNA)是一类高度保守的非编码小RNA,长度约为18-25个核苷酸。miRNA广泛存在于原核和真核生物体内,对细胞的增殖、分化、凋亡、胚胎的发育、器官的形成、内分泌的调控、疾病的发生、发展都起着重要的调节作用。miRNA通过靶向一个或多个基因的3’非编码区域,降解其mRNA或抑制其翻译的进行,从而达到对基因表达的调控作用。miRNA与肿瘤、心脑血管疾病、内分泌疾病等诸多疾病的发生密切相关。
发明内容
发明目的:本发明公开了用于检测胰岛β细胞去分化的miRNA分子标志物miR-483-5p的用途,其正/反向引物用于制备2型糖尿病β细胞去分化诊断试剂盒。
技术方案:检测胰岛β细胞去分化的标志物的用途,所述标志物为miR-483-5p,其miRNA成熟体序列如SEQ ID NO:1或SEQ ID NO:2所示;
SEQ ID NO:1:5’-AAGACGGGAGGAAAGAAGGGAG-3’;
SEQ ID NO:2:5’-AAGACGGGAGAAGAGAAGGGAG-3’。
进一步地,一种检测2型糖尿病β细胞去分化的方法,步骤如下:
(1)提取血清/血浆总RNA及制备cDNA;
(2)运用基于实时荧光定量PCR的方法检测miR-483-5p水平,其中U6是内参;
(3)miRNA分子标志物的评估。
进一步地,步骤(2)实时荧光定量PCR检测根据染料法检测。实时荧光定量PCR的miR-483-5p正/反向引物如SEQ ID NO:3(人)和SEQ ID NO:4(小鼠)所示,U6正/反向引物如SEQ ID NO:5(人)和SEQ ID NO:6(小鼠)所示。检测2型糖尿病β细胞去分化的miRNA分子标志物miR-483-5p的正向引物和反向引物,用于制备2型糖尿病β细胞去分化诊断试剂盒。
SEQ ID NO:3:
F:5’-AAGACGGGAGGAAAGAAGGGAG-3’
R:5’-GTGCAGGGTCCGAGGTATTC-3’
SEQ ID NO:4:
F:5’-ACACTCCAGCTGGGAAGACGGGAGGAAAGAA-3’
R:5’-CTCAACTGGTGTCGTGGA-3’
SEQ ID NO:5:
F:5’-CGCTTCGGCAGCACATATACTA-3’
R:5’-CGCTTCACGAATTTGCGTGTCA-3’
SEQ ID NO:6:
F:5’-CTCGCTTCGGCAGCACA-3’
R:5’-AACGCTTCACGAATTTGCGTG-3’
进一步地,所述miR-483-5p的靶向基因序列为:
Pdx1 3′UTR 3-CCCCCTCCTCTTCCCCCTCCCTC-5;
MafA 3′UTR 3-CGAGGCTC-CTTCCCCTTCCGTC-5。
区别于传统生物标志物,miRNA可在血清/血浆中稳定存在,易于分离提取,不易受pH变化或反复冷冻的影响;并且血清/血浆的获得是微创的,仅需提供血样即可进行,无需其他组织样本,在很大程度上减轻患者的痛苦;最重要的是定量准确,将大大提高2型糖尿病β细胞去分化的特异度和灵敏度。该类小分子RNA生物标志物的成功开发有助于2型糖尿病的早期预警。miR-483-5p在2型糖尿病早期患者血清和β细胞中异常高表达,发现β细胞成熟标志物关键基因Pdx1及MafA表达下降。miR-483-5p抑制了维持β细胞特异性(β cellidentity)的转录因子Pdx1及MafA的mRNA和蛋白表达,而使得内分泌祖细胞标志物(Ngn3)及干细胞标志物(OCT4、Nanog)表达上调。过表达miR-483-5p促使胰岛β细胞发生去分化,β细胞丧失胰岛素分泌功能。因此,从血清/血浆中筛选到的miRNA作为2型糖尿病早期β细胞去分化的标记物,可以敏感、准确的预警早期、低水平血糖的损害。
有益效果:本发明的miR-483-5p作为2型糖尿病早期β细胞去分化的标记物,可以敏感、准确的预警早期、低水平血糖的损害。
附图说明
图1:miR-483-5p在2型糖尿病早期患者血清及胰岛中异常高表达:分别提取2型糖尿病患者血清及胰岛RNA,荧光定量PCR检测miR-483-5p的水平,发现同对照组相比,2型糖尿病患者血清(A)及胰岛(B)中miR-483-5p的表达显著升高,(n=10)(**P<0.01,***P<0.001);
图2:2型糖尿病小鼠血清中miR-483-5p的表达明显上调,并且在小鼠胰岛中,miR-483-5p的表达与Pdx1及MafA的表达呈负相关,而与Ngn3、Oct4和Nanog的表达呈正相关,(A)和(B)图为2型糖尿病小鼠及对照组血清(A)及原代胰岛(B)miR-483-5p的表达水平;(C)和(D)图为2型糖尿病小鼠及对照小鼠原代胰岛中Pdx1(C)及MafA(D)的表达水平,(E)-(G)图为2型糖尿病小鼠及对照小鼠原代胰岛中Ngn3(E)、Oct4(F)和Nanog(G)的表达水平,(n=10)(**P<0.01,***P<0.001,****P<0.0001);
图3:miR-483-5p靶向抑制Pdx1及MafA的mRNA和蛋白表达:(A)图为miR-483-5p以Pdx1及MafA的mRNA为靶点的结合序列对照图(miR-483-5p调控位点预测图);(B)图为荧光素酶双报告基因系统检测miR-483-5p直接与Pdx1及MafA的3′UTR区相互作用情况;(C)图为通过实时定量PCR检测转染miR-483-5p模拟物/抑制物48h后,miR-483-5p的表达情况;(D)和(E)图为通过实时定量PCR及Western Blot检测转染miR-483-5p模拟物48h后,Pdx1及MafA的mRNA(D)和蛋白(E)表达变化情况;(F)和(G)图为通过实时定量PCR及Western Blot检测转染miR-483-5p抑制物48h后,Pdx1及MafA的mRNA(F)和蛋白(G)表达变化情况,(n=2-3;*P<0.05,**P<0.01,***P<0.001);
图4:过表达miR-483-5p促使了β细胞去分化:将合成的miR-483-5p模拟物转染MIN6细胞,48h后进行定量PCR分析,检测β细胞特异性标志分子Pdx1、MafA、Insulin 1和Insulin 2(A)及去分化标志分子Ngn3、Oct4、Nanog(B)的表达,(n=2-3;*P<0.05,**P<0.01)。
具体实施方式
实施例1 2型糖尿病人血清及胰岛中miR-483-5p的检测。
1)血清的制备:
A:抽取每一位实验对象3ml外周静脉血入分离胶+促凝剂真空采血管,常温或于37℃水浴箱中静置30-60min;
B:室温或者4℃下离心1500g,持续5-10min,离心完毕后分三层,上层淡黄色清亮血清层,中间分离胶层,最底层为暗红色血细胞层;移至超净台进一步操作,从而避免唾液、外界酶等污染标本。
C:将取出的血清转移至无酶的Epp管中,12000g离心5min,进一步弃去残留的细胞或细胞碎片,离心温度为4℃;移至新的无酶1.5ml冻存管中,-80℃保存备用。
2)原代胰岛的提取:
2型糖尿病患者及正常对照组的原代胰岛由天津医科大学王树森教授馈赠,在此不做描述。
3)从血清或胰岛中提取RNA
A:取出-80℃冻存样本置冰上融化,吸取200μl加入到600μl Trizol中,于振荡器上充分混匀,所得到的匀浆于室温(15-30℃)静置10min,使核酸蛋白复合物充分分离;
B:加入1/5体积的氯仿,即120μl氯仿,剧烈漩涡混匀15sec,并于室温下静置10min;
C:将离心机调制4℃,12000g离心15min,可见样品分三层:上层为无色水相(RNA溶解于其中),中间层为红色层(DNA溶解于其中),下层则为有机层(为蛋白质等有机物质)(离心结束后取样时不可晃动或颠倒管子,防止离心后三层再次混合);
D:用200μl移液器吸取上层水相(注意避免吸取中间层)移入新的1.5ml无酶Epp管中,并加入等体积的异丙醇,充分混匀后,室温下静置25min;
E:静置结束后于4℃,12000g离心10min,弃去上清。
F:加入事先配好的75%乙醇600μl,4℃,7500g离心5min。弃去上清,室温晾干约5min,加入20μl RNAase-free ddH2O,轻轻吹打混匀,充分溶解RNA;
4)RNA浓度和纯度检测
取提取的RNA 1μl,打开紫外分光光度计,获取在260nm和280nm波长下的吸光度,计算二者比值,若260/280 0D值处于1.8-2.1范围,则RNA浓度大约在25-50ng/μl,则可以进行下一步实验。
5)cDNA的合成及实时荧光定量PCR
使用TOYOBO公司生产的ReverTra Ace-α-逆转录试剂盒进行逆转录,合成cDNA。
A:逆转录:体系20μl,所有操作均在冰上进行。取一0.2ml RNAase-free离心管,加入以下试剂:
逆转录反应体系
B:逆转录程序
37℃60min;
85℃5min;
4℃60min。
C:将逆转录的cDNA放入-80℃存放或加入RNAase-free H2O稀释至100μl进行下一步。
D:PCR反应体系10μl
实时荧光定量PCR反应体系
E:将上述体系加入八联管中,按下一个程序进行PCR扩增:
实时荧光定量PCR反应程序
每个样本设定三个副孔,分别记录内参及样本反应的CT值。
6)数据统计学分析
两组血清样本miRNA表达量的比值使用2-ΔΔCt法进行计算,其中ΔΔCt=[CT1(miRNA)-CT1(内参)]-[CT2(miRNA)-CT2(内参)],CT(miRNA)是样本miR-483-5p扩增的CT值,CT(内参)是样本内参基因扩增的CT值,CTl是2型糖尿病组样本扩增的CT值,CT2是健康对照组扩增的CT值。本实验所有数据均采用均值±标准差(±s)表示,组间差异性分析采用t检验,以P<0.05作为统计学差异参考标准。
结果如图1所示,miR-483-5p在2型糖尿病人血清(图1A)及原代胰岛(图1B)中的表达水平显著升高,具有显著差异(**P<0.01,***P<0.001)。这提示miR-483-5p可能参与了2型糖尿病的发生发展过程。
实施例2 2型糖尿病小鼠血清中miR-483-5p的表达明显上调,并且在小鼠胰岛中,miR-483-5p的表达与Pdx1及MafA的表达呈负相关,而与Ngn3、Oct4和Nanog的表达呈正相关。
本实施例中血清的制备,血清RNA的提取及实时荧光定量PCR的详细步骤参考实施例1,在此仅详述小鼠胰岛的提取及PCR检测。
1)小鼠原代胰岛的提取:
A:分离步骤:小鼠禁食过夜,用3.5%(w/v)水合氯醛进行腹腔注射麻醉;固定动物于手术台上;打开腹腔和胸腔并暴露心脏;找到胆总管在十二指肠开口处,带线结扎;剪破右心耳放血;找到肝总管与胆总管汇合处,准备插管;用静脉输液管插管(插管之前要排气),注入胶原酶V(1.5-2mL/只小鼠),可见胰腺充盈呈透明水泡状;将充盈的胰腺组织沿肠管剪切剥离下来,迅速放在冰上预冷的50mL灭菌的塑料离心管中(每管可放入2只小鼠胰腺);向50mL离心管中加入2-5mL的胶原酶V用于外消化,37℃水浴静置消化28min;将50mL离心管自水浴中取出,立即置于冰上终止消化;涡旋震荡3×5sec,直至组织破碎呈泥沙状;加入2倍体积的含10%FBS的HBSS(冰上预冷),混匀,进一步终止消化,用30目不锈钢筛子网过滤,除去未消化完全的组织块;4℃、350g离心(加速度:9,减速度:9)2min;弃上清,加入冰HBSS重悬细胞沉淀,4℃,350g离心(加速度:9;减速度:9)2min;弃上清,加入5mLHistopaque-1077重悬细胞沉淀,转移至10mL玻璃离心管中;小心用巴式管沿玻璃离心管管壁缓慢加入5mL HBSS,保持HBSS与Histopaque-1077之间的分层;4℃,500g离心(加速度:9;减速度:1)20min;离心后,胰岛位于HBSS与Histopaque-1077之间的夹层中,将此夹层用200μL移液器转移至预先加好含血清HBSS的6孔板中,在体式显微镜下用10μL移液器挑拣完整的胰岛。
B:纯度鉴定:将100mg胰岛双硫腙(DTZ)溶于30mL DMSO中,加入500μL 25%氨水,充分溶解,过滤除去不溶物,分装到EP管中,-20℃保存作为储存液;将所得胰岛用生理盐水洗涤两次后,加1mL生理盐水及10μL双硫腙储存液,37℃孵育30min,倒置显微镜下观察细胞着色情况,胰岛经DTZ染色后呈现猩红色,鉴定胰岛纯度>90%。
2)小鼠原代胰岛RNA的提取
RNA提取及逆转录使用的Epp管和移液器吸头均为Axygen公司生产的无RNA酶产品。塑料容器、玻璃器皿均经0.1%DEPC水处理以灭活RNA酶,高压蒸汽灭菌后烘干待用。75%乙醇的配置和RNA的溶解所使用的超纯水也均经0.1%DEPC处理。RNA提取过程中所有离心步骤均在4℃下进行。提取及逆转录操作全程均需佩戴口罩。具体步骤如下(以3.5Gm皿为例):
A:弃上清,用1ml PBS洗一遍,加入1ml Trizol后用移液器反复吹吸几次,室温下静置5min;
B:将皿中的Trizol转移至1.5ml Epp管中,加入0.2ml氯仿,上下振摇15s,室温静置3min;
C:4℃、12000g离心15min;
D:将上层水相转移至一个新的1.5ml EP管中,加入等体积的异丙醇,混匀,室温放置10min;
E:4℃、12000g离心10min;
F:弃上清,加入1ml预冷的75%乙醇,重悬并洗涤RNA沉淀;
G:4℃、7500g离心5min;
H:小心弃去上清,室温干燥5min,加入适量无RNA酶的超纯水溶解RNA沉淀,沉淀溶解后将RNA至于冰上;
I:用NanoDrop微量核酸检测仪进行RNA纯度分析,并检测RNA浓度,合格的RNA样品用于进一步实验。
3)cDNA的合成及实时荧光定量PCR
分别将实施例1的逆转录体系中miRNA颈环反向引物替换为Oligo(dT)(注:针对具有polyA尾的成熟mRNA);将实施例1的PCR体系中上下游引物替换为各个检测基因的mRNA引物(序列如表1所示)。
表1
结果见图2所示,同对照组相比,2型糖尿病小鼠血清中miR-483-5p的表达明显上调(图2A),并且2型糖尿病小鼠胰岛中miR-483-5p与Pdxl、MafA的表达呈负相关(图2B-D),而与Ngn3、Oct4和Nanog的表达呈正相关(图2E-G)。
实施例3 miR-483-5p通过靶向Pdx1及MafA,抑制其mRNA和蛋白表达
为了探究miR-483-5p促使胰岛β细胞去分化,使β细胞丧失分泌胰岛素功能的可能机制,寻找了在胰岛β细胞中miR-483-5p调控的靶点。
1)生物信息学筛选miR-483-5p的下游靶基因
A:通过DIANALAB、miRDB、miRanda和PicTar网站查找miR-483-5p的靶基因,并依据各基因功能(与胰岛β细胞功能相关)筛选出候选靶基因。
B:从众多miR-483-5p的潜在靶基因中筛选出在糖尿病中参与调控胰岛素合成和分泌的候选靶基因Pdx1和MafA,其3’UTR上分别存在14个与miR-483-5p种子序列完全互补配对的碱基。
2)双荧光素酶报告基因系统检测miR-483-5p与Pdx1和MafA的相互作用
A:根据预测的Pdx1、MafA与miR-483-5p的碱基互补配对区域序列设计并合成野生型及突变型Pdx1、MafA的3’UTR序列。其中所使用的酶切位点为Not I及Xho I,合成序列时加入相应酶切位点及保护碱基,具体序列如下表2所示。
表2
B:分别将合成的野生型/突变型Pdx1和MafA的两条单链退火形成互补双链,经酶切、连接、转化、筛选等一系列分子克隆技术,将野生型/突变型靶基因靶位点(target)序列克隆到psiCHECK-2载体(市购,本实验室保存)上的海肾荧光素酶(Renilla luciferase)之后,构建野生型/突变型靶基因Pdx1和MafA的3’UTR报告载体。由于psiCHECK-2载体同时表达萤火虫荧光素酶(Firefly luciferase)基因,因此将萤火虫荧光素酶的表达作为转染的内参。
C:将报告载体(野生型/突变型)与miR-483-5p的模拟物或者阴性对照模拟物共转染MIN6细胞,转染后48h裂解细胞,检测荧光信号的强度。其中,miR-483-5p的模拟物及阴性对照模拟物序列如下表3所示。
表3
结果如图3A和图3B所示,转染miR-483-5p的模拟物后,Pdx1和MafA报告基因表达量分别下调了51.3%和48.8%,这种抑制作用可通过靶位点的突变得到回复。
3)转染miR-483-5p模拟物或抑制物48h后,检测miR-483-5p的表达效率
根据转染试剂盒的说明步骤,运用miR-483-5p的模拟物/抑制物(序列见表4)或者阴性对照模拟物/抑制物转染MIN6细胞,48h后根据实施例3的实验步骤收取细胞RNA,采用实时荧光定量PCR(real-time PCR)的方法进行定量检测。
表4
结果如图3C所示,与阴性对照模拟物相比,转染miR-483-5p模拟物48h后,miR-483-5p的表达量显著上调,而转染miR-483-5p抑制物后,其表达量有明显下调,说明转染效率较高。
4)实时荧光定量PCR及免疫印迹检测(Western Blot)miR-483-5p对内源靶基因的作用
A:根据Pdx1和MafA序列设计引物,引物序列如表1所示。
B:用Lipo2000分别转染50nM miR-483-5p模拟物/阴性对照模拟物及miR-483-5p抑制物/阴性对照抑制物;转染48h后用Trizol提RNA及蛋白;提取的RNA经逆转录后用SYBRgreen的方法进行荧光定量PCR检测Pdx1和MafA的表达;提取的蛋白经Western Blot检测转染miR-483-5p的模拟物及抑制物后,Pdx1和MafA蛋白水平的变化情况。
结果如图3D-3G所示,转染miR-483-5p模拟物能有效抑制Pdx1和MafA的表达;相反地,转染miR-483-5p抑制物能促进Pdx1和MafA的表达。
实施例4 miR-483-5p对胰岛β细胞去分化的影响
为了进一步研究miR-483-5p对胰岛β细胞去分化的影响,采用qRT-PCR(实时荧光定量,Real-time PCR)方法检测了转染miR-483-5p模拟物的MIN6细胞中胰岛β细胞标志性分子Pdx1、MafA、Insulin 1和Insulin 2的mRNA(信使核糖核酸)水平,同时分析了胰岛β细胞去分化标志分子Ngn3、OCT4和Nanog的mRNA表达。Pdx1、MafA、Ngn3、OCT4和Nanog的引物序列见表1,剩余分子的引物序列见表5。实时荧光定量PCR的具体步骤见实施例1所述的方法。不同的是分别将实施例1的逆转录体系中miRNA颈环反向引物替换为Oligo(dT)(注:针对具有polyA尾的成熟mRNA);将实施例1的PCR体系中上下游引物替换为各个检测基因的mRNA引物(序列如表1和表5所示)。
表5
结果见图4所示,MIN6细胞转染miR-483-5p模拟物后,明显抑制了β细胞特异性标志分子(如:Pdx1、MafA、Insulin 1和Insulin 2)的表达(图4A),却显著促使了β细胞去分化标志分子Ngn3、OCT4和Nanog的表达(图4B)。
综上,本发明发现,在2型糖尿病人及2型糖尿病小鼠血清中miR-483-5p的表达明显升高。过表达miR-483-5p促使胰岛β细胞去分化,丧失分泌胰岛素功能。
序列表
<110> 南京医科大学
<120> 检测胰岛β细胞去分化的标志物的用途
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
aagacgggag gaaagaaggg ag 22
Claims (6)
1.一种检测胰岛β细胞去分化的标志物的用途,所述标志物为miR-483-5p,其miRNA成熟体序列如SEQ ID NO:1或SEQ ID NO:2所示。
2.根据权利要求1所述的用途,其特征在于:检测胰岛β细胞去分化的方法,包括如下步骤:
(1)提取血清/血浆总RNA及制备cDNA;
(2)运用基于实时荧光定量PCR的方法检测miR-483-5D水平,U6是内参;
(3)miRNA分子标志物的评估。
3.根据权利要求3所述的用途,其特征在于:步骤(2)所述的实时荧光定量PCR检测采用染料法检测。
4.根据权利要求3所述的用途,其特征在于:步骤(2)中实时荧光定量PCR的miR-483-5p正/反向引物如SEQ ID NO:3和SEQ ID NO:4所示,U6正/反向引物如SEQ ID NO:5和SEQ IDN0:6所示。
5.根据权利要求4所述的用途,其特征在于:所述miR-483-5p正/反向引物用于制备2型糖尿病β细胞去分化诊断试剂盒。
6.根据权利要求1所述的用途,其特征在于:所述miR-483-5p的靶向基因序列为:
Pdx1 3′ UTR 3-CCCCCTCCTCTTCCCCCTCCCTC-5;
MafA 3′ UTR 3-CGAGGCTC-CTTCCCCTTCCGTC-5。
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