CN111265656A - Dio2在制备预测或治疗宫腔粘连的药物中的应用 - Google Patents
Dio2在制备预测或治疗宫腔粘连的药物中的应用 Download PDFInfo
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Abstract
本发明公开了DIO2在制备预测或治疗宫腔粘连的药物中的应用。本发明发现宫腔粘连患者，子宫内膜中DIO2表达降低，尤其以上皮细胞减少最为显著。体外结果显示，DIO2可以通过将甲状腺素T4转换为T3而有效逆转子宫内膜上皮细胞的EMT，抑制上皮细胞的纤维化。因此，DIO2在宫腔粘连诊断和治疗方面可能发挥重要作用，可以用于宫腔粘连的临床诊断及制备治疗宫腔粘连及其他与子宫纤维化相关疾病的药物。
Description
技术领域
本发明属于生物医药领域，涉及DIO2在制备预测或治疗宫腔粘连的药物中的应用。
背景技术
宫腔粘连(intrauterine adhesion,IUA)是多种因素引起的子宫内膜严重损伤，致使子宫内壁间粘连，宫腔全部或部分封闭，构成内膜的上皮与间质细胞被纤维化的间质和增殖分化障碍的上皮代替，子宫内膜对性激素刺激失去反应。IUA是继发性不孕的最常见病因，在不孕妇女中占比高达25-30％，在我国，随着各种宫腔手术特别是人流刮宫术数量的上升，宫腔粘连及子宫内膜纤维化的发生率明显增高。目前主要治疗方法是宫腔镜下粘连分离手术，对重度广泛内膜损伤效果不佳，其术后粘连复发率高达62.5％。因此亟需寻找一种有效的IUA的预测及治疗方法。
DIO2是一种将甲状腺素T4转化为活性甲状腺素T3的酶，在特发性肺纤维化患者的肺中，DIO2的活性和表达水平显著高于对照人群，并且与疾病程度严重相关，但是，DIO2与宫腔粘连是否存在相关性，是否影响子宫内膜纤维化等研究未见报道。
发明内容
本发明的目的是提供DIO2在制备治疗宫腔粘连药物中的应用。
本发明的另一目的是提供DIO2在制备宫腔粘连诊断试剂中的应用。
本发明的目的通过以下技术方案实现：
DIO2在制备治疗子宫纤维化相关疾病的药物中的应用。
DIO2在制备治疗宫腔粘连的药物中的应用。
DIO2作为检测靶点在制备宫腔粘连预测或辅助诊断试剂中的应用。
按照美国生殖协会(AFS)1988年分类标准，宫腔粘连患者分为轻、中、重度，依据子宫内膜纤维化占宫腔面积(1＝<1/3，2＝1/3-2/3,4＝>2/3)、粘连程度(1＝薄膜样，2＝薄膜与致密之间，4＝致密粘连)及月经量(0＝正常，2＝月经微量，4＝闭经)评分，1-4分为轻度，5-8分为中度。9-12分为重度。不同程度的宫腔粘连患者子宫内膜中DIO2的表达水平有差异，因此可根据DIO2表达量来达到宫腔粘连患者分型的目的。
检测DIO2的试剂在制备宫腔粘连辅助诊断试剂中的应用。
所述的检测DIO2的试剂优选检测DIO2表达量的试剂。
所述的检测DIO2表达量的试剂为DIO2基因的特异性引物(如PCR引物或qPCR引物)或者检测DIO2蛋白的抗体。
本发明中所述的宫腔粘连指由于子宫内膜基底层损伤，功能层再生修复障碍，形成以内膜纤维化为特征的子宫壁间粘连。
本发明中所述的子宫内膜纤维化引起的疾病指宫腔粘连或子宫内膜疤痕化以及由此导致的子宫性不孕、反复流产及胎盘植入等。
有益效果：
我们发现宫腔粘连患者，子宫内膜中DIO2表达显著降低，尤其以上皮细胞减少最为显著。而且体外结果显示，DIO2可以通过将甲状腺素T4转换为T3而有效逆转子宫内膜上皮细胞向间质转化(EMT)，抑制上皮细胞的纤维化。因此，DIO2在宫腔粘连诊断及治疗方面可能发挥重要作用，可以用于制备治疗宫腔粘连辅助诊断、治疗或者制备治疗其他与子宫纤维化相关的疾病的药物。
附图说明
图1、A：qPCR分析子宫内膜组织中DIO2的表达量；B：免疫组化分析子宫内膜组织中DIO2的表达量；C：正常及重度宫腔粘连患者子宫内膜组织中DIO2表达量的ROC曲线；
图2、A：DIO2、T4对细胞纤维化相关基因表达的影响；B：T3对细胞纤维化相关基因表达的影响；
具体实施方式
实施例1DIO2在正常及重度宫腔粘连患者子宫内膜组织中的表达
1、材料，试剂，设备
1.1人子宫内膜组织来源
收集36例正常子宫内膜组织以及36例重度宫腔粘连综合征患者子宫内膜组织，获取子宫内膜组织时，确保内膜组织获取阶段全部统一为增殖晚期阶段。所有参与者均签署了纸质版的知情同意书，并经过南京鼓楼医院伦理委员会批准。
1.2主要试剂
TRNzol(天根)、反转录试剂(雅酶)、SYBR Ggreen(雅酶)、氯仿、异丙醇、二甲苯、无水乙醇、95％、80％、75％乙醇、3％双氧水、柠檬酸修复液、DIO2引物序列(Forward primer5’-3’ACTCGGTCATTCTGCTCAAG(SEQ ID NO.1)Reverse primer 5’-3’ATTGCCACTGTTGTCACCTC(SEQ ID NO.2))、DIO2抗体(abcam)、DAB、苏木素。
1.3主要仪器
qPCR仪(Roche)、PCR仪(ABI)、烘箱、显微镜(Leica)。
1.4主要方法
1.4.1组织RNA提取及实时荧光定量PCR
采用Trizol法提取总RNA。取1ug RNA反转录后得到cDNA，用适量体积的RNase-free水稀释后，SYBR Green方法进行荧光定量PCR检测，不同目标基因的表达量采用18S作为内参标准的ΔΔCT值做统计。
1.4.2子宫内膜组织免疫组化
1.60℃恒温箱烤片60min，二甲苯处理3次，每次5min，梯度酒精(100％乙醇5min，95％乙醇5min，80％乙醇5min，75％乙醇5min)处理后，水冲洗适当时间，3％H2O2浸泡15min(去除内源性过氧化物酶)，水冲洗适当时间；柠檬酸修复后一抗37℃孵育2h，PBST冲洗3遍，每次5min，二抗孵育8min，PBST冲洗3遍，每次5min。DAB显色，苏木素染核，封片后显微镜观察并拍照。
2、结果
重度宫腔粘连患者子宫内膜组织中，DIO2表达显著减少，上皮细胞中的表达下降最为显著(图1)。
实施例2DIO2对子宫内膜上皮细胞纤维化的影响
1、材料，试剂，设备
1.1主要试剂
I型胶原酶(Sigma)、透明质酸酶(Sigma)、DNAase(Roche)、上皮细胞培养基(Gibco)、DMEM/F12培基(Wisent Inc)、血清(Gibco)、Trizol(Invitrogen)。TGFβ1(PeproTech)、甲状腺素T4/T3(MCE)、FN/N-cad/E-cad/a-SMA/DIO2抗体(abcam)、电泳液、转膜液、脱脂牛奶(bio-rad)、TBST
1.2主要仪器
细胞培养箱、摇床、PVDF硝酸纤维素膜、双垂直电泳槽、凝胶成像分析系统
1.3主要方法
1.3.1原代子宫内膜上皮细胞分离培养
将内膜组织放置在新的60mm培养皿中，剪碎组织至无肉眼可见块状，加入配置好的消化液，吹打混匀，37℃培养箱中放置3min；取出培养皿，镜下观察组织消化情况；吹打消化后的组织，加DMEM/F12完培终止消化，将组织悬液滴加到40μm筛子里。筛子里剩余的组织转移至干净的60mm培养皿中，加入DMEM/F12培基，吹打混匀后将悬液滴加到100μm筛子里，获得上皮细胞，重复消化3-5遍，24h后细胞换液，细胞长至合适密度时予以TGFβ1(10ng/ml)处理24h后加入甲状腺素T4(100nM)/转染DIO2/转染DIO2+T4/加入甲状腺素T3(20nM/100nM)处理48h后提取蛋白。
1.3.2WB
弃去培基，用预冷的PBS洗3-4遍，加适量细胞裂解液，放至冰上裂解30min，吸至EP管中离心15min取上清，测蛋白浓度，按上样量30μg计算体积，加loadingbuffer，99℃金属水浴锅加热10-15min，10％胶跑至溴酚蓝到底，转膜1-2h，5％牛奶封闭1h；一抗4℃孵育过夜，TBST洗5min，洗3遍，二抗室温孵育1h，TBST洗5min，洗3遍；曝光。
2、结果
TGF-β处理24h后E-cad下降，N-cad、FN、a-SMA表达增加，单独T3处理或者DIO2与T4共同处理后，E-cad表达增加，N-cad、FN、a-SMA表达下降，表明DIO2可以将甲状腺素T4转换为T3，逆转子宫内膜上皮细胞EMT及抑制纤维化过程。
序列表
<110> 南京鼓楼医院
<120> DIO2 在制备预测或治疗宫腔粘连的药物中的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
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<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
attgccactg ttgtcacctc 20
Claims (6)
1.DIO2作为检测靶点在制备宫腔粘连预测或辅助诊断试剂中的应用。
2.DIO2在制备治疗子宫纤维化引起的疾病的药物中的应用。
3.根据权利要求2所述的应用，其特征在于DIO2在制备治疗宫腔粘连的药物中的应用。
4.检测DIO2的试剂在制备宫腔粘连预测或辅助诊断试剂中的应用。
5.根据权利要求4所述的应用，其特征在于所述的检测DIO2的试剂为检测DIO2表达量的试剂。
6.根据权利要求5所述的应用，其特征在于所述的检测DIO2表达量的试剂为DIO2基因的特异性引物或者检测DIO2蛋白的抗体。
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