CN115873943B - 骨形态发生蛋白2在子痫前期诊断、预防和治疗中的应用 - Google Patents
骨形态发生蛋白2在子痫前期诊断、预防和治疗中的应用 Download PDFInfo
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Abstract
本发明涉及生物医药技术领域,具体涉及骨形态发生蛋白2在子痫前期诊断、预防和治疗中的应用。本发明经过实验发现子痫患者胎盘BMP2mRNA水平和舒张压水平呈负相关,胎盘BMP2mRNA可以作为早期预测靶标。本发明证明BMP2能够增加人滋养层细胞的侵袭力和内皮特性获得,改善子痫前期早期的胎盘滋养细胞侵袭不足和血管重塑不良,挽救子痫前期表型,改善母胎结局,对子痫前期的预防和治疗具有重要价值和应用前景。
Description
技术领域
本发明涉及生物医药技术领域,具体涉及骨形态发生蛋白2在子痫前期诊断、预防和治疗中的应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
子痫前期(pre-eclampsia,PE)是常见的妊娠相关疾病,在妊娠女性中发病率为5-8%,以妊娠20周后新发的高血压(≥140/90mmHg)和蛋白尿(尿蛋白≥0.3g/24h)为特征,是全球孕产妇及围产儿患病和死亡的常见危险因素,严重威胁母胎安全。由于子痫前期的病因和发病机制复杂,目前在临床诊疗中除对症治疗外尚缺乏有效靶标治疗药物,一旦病情进展,除及时终止妊娠外,目前尚没有其他有效治疗措施。因此,深入阐明发病机制并筛选有效的治疗靶标对子痫前期临床干预尤为必要。
虽然子痫前期病因和发病机制不清楚,但是普遍认为是一种胎盘源性疾病,滋养层细胞的发育障碍与子痫前期的发生发展直接相关,其病理生理过程分为两个阶段。第一阶段为妊娠早期胎盘滋养细胞侵袭不足,导致子宫螺旋动脉血管重塑不良,继而胎盘血液灌注减少;第二阶段为胎盘氧化应激,释放抗血管生成因子至母体血液循环,造成母体全身性血管内皮功能和相应器官受损,引起妊娠晚期高血压、蛋白尿等子痫前期临床表型和胎儿宫内生长受限的出现。骨形态发生蛋白(BMPs)属于TGF-β超家族,参与发育、血管生成和生殖过程。目前,骨形态发生蛋白2(BMP2)被确定能够在体外促进滋养层细胞侵袭,然而BMP2对子痫前期的预防和治疗的作用鲜有报道,对BMP2体内作用的深入研究对子痫前期临床干预尤为必要。
发明内容
为了解决现有技术的不足,本发明的目的是提供骨形态发生蛋白2在子痫前期诊断、预防和治疗中的应用。子痫前期患者胎盘BMP2mRNA水平和舒张压呈负相关,提示BMP2在子痫前期中可能起保护作用,胎盘BMP2 mRNA水平具有诊断价值。BMP2能够降低子痫前期大鼠模型血压,提高胎盘和胎儿重量,改善胎盘发育,在子痫前期的预防和药物治疗中有广阔的应用前景。
为了实现上述目的,本发明是通过如下的技术方案来实现:
第一方面,本发明提供了检测胎盘BMP2 mRNA水平的试剂在制备子痫前期诊断产品中的应用。
第二方面,本发明提供了BMP2在制备治疗或预防子痫前期产品中的应用。
上述本发明的一种或多种技术方案取得的有益效果如下:
本发明经过实验发现子痫患者胎盘BMP2 mRNA水平和舒张压水平呈负相关,胎盘BMP2 mRNA可以作为早期预测靶标。
本发明证明BMP2能够增加人滋养层细胞的侵袭力和内皮特性获得,改善子痫前期早期的胎盘滋养细胞侵袭不足和血管重塑不良,挽救子痫前期表型,改善母胎结局,对子痫前期的预防和治疗具有重要价值和应用前景。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为实施例1中子痫前期胎盘BMP2 mRNA水平与患者舒张压(DBP)的Pearson相关性分析(N=27);
图2为实施例1中子痫前期组(N=27)和正常对照组(N=42)胎盘BMP2 mRNA水平,其中HP为正常对照组,PE为子痫前期组;
图3为实施例2中人滋养层细胞细胞分别用空白载体或BMP2(25ng/ml)处理36小时的细胞侵袭性分析图;左图显示代表性图像,右图显示定量结果;比例尺,200μm;
图4为人滋养层细胞细胞分别用空白载体或BMP2(25ng/ml)处理12小时的内皮样小管形成情况分析图;左图显示代表性图像,右图显示定量结果;比例尺,200μm;
图5为妊娠早期绒毛外植体分别用空白载体或BMP2(25ng/ml)处理24或48小时绒毛边缘细胞的迁移距离的测量和分析图;左图显示代表性图像,右图显示定量结果;比例尺,100μm;
图6为动物模型处理流程:用过表达sFlt-1或对照Fc的腺病毒于G8通过尾静脉注射大鼠体内,于G10-G13日给予尾静脉注射重组BMP2蛋白(10μg/kg)或PBS,G19处死取材;
图7中A为G7-G19,每三天测量记录平均动脉压数据;B为G7-G19,每三天测量记录平均收缩压数据;
图8中A为G19子鼠与胎盘的代表图像(N=7);B为大鼠胎盘HE染色图:上图显示代表性图像,其中LZ为迷路区,JZ为连接区,右图为统计结果;比例尺,2毫米,(N=9);C为在G19测定各组(N=44)子鼠重量结果图。D为在G19测定各组(N=44)胎盘重量结果图。定量结果以均数±标准差表示,统计方法为方差分析;*p<0.05,**p<0.01,***p<0.001;ns,没有显著性差异;
具体实施方式
本发明的第一种典型实施方式,检测胎盘BMP2 mRNA水平的试剂在制备子痫前期诊断产品中的应用。
该实施方式的一种或多种实施例中,所述产品包括引物、探针、芯片、核酸膜条、制剂或试剂盒。
本发明的第二种典型实施方式,BMP2在制备治疗或预防子痫前期产品中的应用。
该实施方式的一种或多种实施例中,所述产品的作用包括:
增加人滋养层细胞的侵袭力和内皮特性获得;
改善子痫前期早期的胎盘滋养细胞侵袭不足和血管重塑不良。
该实施方式的一种或多种实施例中,所述产品包括药物、食品、保健品。
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案,以下将结合具体的实施例与对比例详细说明本发明的技术方案。
实施例1
子痫前期胎盘BMP2 mRNA水平与患者舒张压(DBP)的Pearson相关性分析和子痫前期组(N=27)和正常对照组(N=42)胎盘BMP2mRNA水平检测
采集行剖宫产手术的子痫前期孕妇和健康孕妇的胎盘组织样本,生理盐水冲洗,液氮速冻然后转移-80度长期保存。本实施例中的研究由山东大学齐鲁医学院伦理委员会批准,遵守赫尔辛基宣言并获得患者知情同意。
(1)组织RNA提取
称取等量的组织样本然后加入300ul TRIzol,在组织研磨仪上进行研磨,再加入700μl TRIzol补齐混匀,静置裂解10分钟。4℃,12000g离心10min,吸取上清转移到新的EP管中。加入200μl氯仿震荡15s,静置5分钟,然后4℃,12000g离心15min,取最上层水相转移到新的EP管中,加入等体积异丙醇,混匀,室温静置10min。4℃,12000g离心15min,弃上清,加入75%乙醇沉淀RNA。4℃,8000g离心10min。用20-50ul DEPC水溶解RNA,Nanodrop one测定浓度和纯度。
(2)逆转录PCR
使用1μg RNA作为反应模板,根据RNA浓度计算反应体系1中RNA和DEPC水的体积,反应体系1的组成如表1所示,PCR仪设置参数为:42℃,2min;16℃。
表1反应体系1组成表
配置反应体系2进行逆转录,如反应体系2如表2所示,PCR仪设置参数为37℃,15min;85℃,5S;16℃。
表2反应体系2组成表
(3)定量PCR
使用TB green试剂盒进行实时荧光定量PCR反应,反应体系如表3所示,PCR仪设置参数如表4所示。
表3定量PCR反应体系组成表
表4PCR仪定量PCR设置参数
对血压和胎盘BMP2 mRNA表达量进行Pearson相关性分析,结果如图1所示,子痫前期患者胎盘BMP2 mRNA水平和舒张压呈负相关,提示BMP2在子痫中可能起保护作用。检测子痫前期和正常对照胎盘BMP2 mRNA表达量,结果如图2所示,子痫前期胎盘BMP2 mRNA水平显著升,表明BMP2 mRNA具有诊断价值,是子痫前期诊断的潜在靶标。
实施例2
人重组BMP2促进滋养层细胞侵袭和内皮特性获得
(1)用人重组BMP2蛋白(25ng/ml)(R&D,#355-BM-010)或空白载体(对照组,记为Ctrl)处理人滋养层细胞不同待用。
(2)用空白载体或BMP2(25ng/ml)预处理6小时,然后消化后重悬在含0.1%(vol/vol)胎牛血清的DMEM培养基中,取250μl含8x104个细胞的细胞悬液接种在Transwell小室中(Transwell小室已提前用40μl浓度为1mg/ml的基质胶包被,Transwell小室孔径为8μm)。Transwell小室下层加入750μl含10%(vol/vol)胎牛血清的DMEM培养基。将接种后的细胞在37℃孵育36h,将膜上部未侵袭的细胞擦拭干净,将膜下部细胞用冷甲醇(-20℃)固定并风干。细胞核用Hoechst33258染色,Olympus倒置显微镜成像,Image-J软件分析两组侵袭细胞数。如图3所示,BMP2培养可以增强细胞的侵袭性。
(3)10mg/mL的基质胶与含0.1%(vol/vol)胎牛血清DMEM培养基1:1(vol/vol)稀释,在96孔板的每孔中加入50μL稀释后的基质胶,37℃孵育2小时固化。将已经过空白载体或BMP2(25ng/ml)预处理6小时的细胞分别消化,然后重悬于含空白载体或BMP2的0.1%胎牛血清DMEM培养液中,将50μL含3×104个细胞的细胞悬液接种于基质胶中,37℃孵育12小时。采用Olympus倒置显微镜拍摄,观察小管形成,并采用ImageJ软件测量分析。如图4所示,BMP2处理可以促进内皮样小管形成。
(4)经山东大学齐鲁医学院伦理委员会批准和患者知情同意,采集因计划外妊娠行清宫术的健康孕妇的早期妊娠胎盘绒毛,并进行外植体培养。用眼科剪从绒毛尖端切下2-3毫米的小绒毛,并种植到基质胶包被的24孔培养皿中,在10% FBS DMEM培养基,37℃,3%O2/5% CO2/92%N2的培养环境中培养。选择成功锚定在基质胶上的绒毛进行后续实验。用人BMP2蛋白(25ng/ml)(R&D,#355-BM-010)或载体(PBS)进行处理,倒置显微镜连续两天拍照记录滋养层细胞生长和从绒毛边缘向远端迁移的情况,并使用ImageJ软件进行测量绒毛外生面积。如图5所示,BMP2处理后绒毛外生面积得到提高,促进了绒毛边缘细胞向远端迁移,迁移距离提高。
BMP2处理后相较对照组可显著增加人滋养层细胞的侵袭力和内皮特性获得,BMP2后能够促进外植体的滋养层细胞的迁移。综上所述,BMP2处理可能通过促进帮助滋养层细胞侵袭过程来改善子痫前期早期的滋养细胞侵袭不足和血管重塑不良,继而改善子痫前期的发展。
实施例3
重组BMP2可以改善子痫前期大鼠模型的母胎结局
(1)子痫前期大鼠模型的建立
本实施例中所用大鼠为Sprague-Dawley大鼠,购自北京维通利华实验动物公司,所有动物均按照国际动物护理指南进行护理,并获得山东大学动物护理与研究委员会的伦理批准。
将9周龄的雌性SD大鼠(200-220克)与年龄、重量相近的雄性SD大鼠进行交配,妊娠第1天(G1)定义为交配的第二天早上雌鼠阴道检出阴栓。孕大鼠随机分为Ad Fc+PBS(n=3)、Ad Fc+BMP2(n=3)、Ad Flt1+PBS(n=3)和Ad Flt1+BMP2(n=3)4组。根据之前的研究构建过表达sFlt-1或对照Fc腺病毒(吉凯生物),在G8(孕中期的早期)通过尾静脉注射1*109PFU腺病毒(Ad Fc或Ad Flt1),于G10-G13每天尾静脉注射重组BMP2蛋白(10μg/kg/天)(R&D,#355-BM-010)或载体(PBS)。
(2)大鼠血压测定
通过tail-cuff plethysmography(MRBP,IITC)测量收缩压(SBP)和平均血压(MBP)。在正式测量开始之前的3天,每天使用带尾袖的约束器固定大鼠30min进行适应训练以适应测量血压操作。适应测量操作后进行正式测量血压:用约束器固定大鼠,放置于恒温32℃的环境中平静15-20min,然后连续测量5次,间隔1分钟。若需要再次测量,须让大鼠休息5分钟方可再次测量。按照此方法,在G7-G19每三天测量一次血压。
(3)尿蛋白和肌酐检测
在G7、G13和G19采集随机尿,用大鼠尿蛋白ELISA试剂盒(ab108789,Abcam)和肌酐测定试剂盒(ab65340,Abcam)测量尿蛋白和肌酐。
(4)胎盘和子鼠取材
于G19将大鼠麻醉,采集血液标本,打开腹腔取出子宫,分离子鼠和胎盘。测量胎盘和子鼠重量,放置于白纸上相机拍照。然后留取部分胎盘固定于4%多聚甲醛中,用于后续苏木素-伊红染色(HE)染色。
如图7所示,注射sFlt-1过表达腺病毒的两组与注射对照Fc病毒的两组相比,血压明显升高,胎盘和子鼠重量下降,胎盘迷路/连接区比例降低,证明胎盘发育存在异常,母体血压升高,子鼠有宫内生长受限,符合子痫前期的表型,模型建立成功。如图8所示,在G10-13即妊娠中期连续四天尾静脉注射重组蛋白BMP2后,在模型组中,有BMP2干预的组与只有PBS载体干预的组相比,血压明显降低,胎盘和子鼠重量增加,胎盘迷路/连接区比例升高,挽救了子痫前期的表型,改善了母胎结局。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (1)
1.BMP2在制备治疗子痫前期产品中的应用,其特征在于,所述产品的作用包括:
增加人滋养层细胞的侵袭力和内皮特性获得;
改善子痫前期早期的胎盘滋养细胞侵袭不足和血管重塑不良;
所述产品为药物。
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