CN116987734A - 一种先兆子痫动物模型的构建方法及其应用 - Google Patents
一种先兆子痫动物模型的构建方法及其应用 Download PDFInfo
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Abstract
本发明涉及生物医药领域，特别涉及一种先兆子痫动物模型的构建方法及其应用，本申请利用基因技术对小鼠PTEN基因进行干扰，构建了过表达PTEN基因的腺病毒载体，并将该载体转化至小鼠中，构建先兆子痫小鼠动物模型，经验证，该过表达载体转入怀孕小鼠体内后，小鼠表现出了先兆子痫病症，该动物模型成功建立，该模型的建立成功证明PTEN对先兆子痫的促进作用，为后续在先兆子痫中针对PTEN的进一步体内外作用机制研究提供理论依据。同时，该动物模型可用于后续PTEN在先兆子痫发生发展机制研究，同时还可辅助寻找先兆子痫相关防治药物，并且进行药效评价能为先兆子痫提供有效治疗策略。
Description
【技术领域】
本发明涉及生物医药领域，特别涉及一种先兆子痫动物模型的构建方法及其应用。
【背景技术】
先兆子痫(PE)是一种严重的妊娠期疾病，每年影响全球约2万母亲和婴儿。其临床表现为妊娠20周后新发高血压和或伴有蛋白尿。重度先兆子痫可并发肾脏、心脏、肺、肝和神经系统功能障碍、血液学紊乱、胎儿生长受限、死产。目前，先兆子痫最明显的两种病理生理机制是血管生成平衡破坏和全身炎症反应增加。建立一种类似人类病理的先兆子痫动物模型是探讨先兆子痫发生机理和治疗方法的基础。
肿瘤抑制因子PTEN是一种双重蛋白质和脂质磷酸酶，参与细胞增殖，分化，代谢，凋亡和侵袭等细胞功能，其异位表达已被证明与滋养细胞疾病有关，如上皮样滋养细胞肿瘤、葡萄胎、早期自然流产和PE。它在细胞滋养细胞和合体滋养层中表达，在PE患者的胎盘中显著升高。体外实验表明LPS可以通过上调PTEN抑制滋养层细胞的侵袭，诱发先兆子痫。miR-221-3p可以通过下调PTEN促进滋养层细胞迁移、侵袭和EMT进展。然而，PTEN在体内对先兆子痫的作用还未探究。
由此可见，利用过表达PTEN建立先兆子痫动物模型，有助于揭示PTEN在先兆子痫发生发展中的重要作用，为先兆子痫的治疗提供潜在靶点以及提供一种新的先兆子痫造模方法。
【发明内容】
鉴于上述内容，为了提高对肌肉损伤修复的精确治疗效果，降低病人痛苦，减少使用物理性康复治疗手段，对肌肉损伤部位进行精准干预，能对针对患者的自体特异性进行治疗，精准用药，使肌肉损伤快速恢复，有必要进行本申请的研究。
为达到上述目的，本申请采用了如下技术方案：
本发明提供了一种先兆子痫动物模型的构建方法，所述方法为将过表达PTEN基因的重组载体转入小鼠体内即得先兆子痫动物模型，所述过表达PTEN基因的重组载体的构建方法为：采用引物对克隆PTEN基因，将克隆出来的基因片段连接到pDC316-CMV-MCS-EFla-copGFP载体中即得；
所述PTEN基因的CDS序列如序列表SEQ ID NO.1所示；
所述pDC316-CMV-MCS-EFla-copGFP载体的碱基序列如序列表SEQ ID NO.2所示；
所述引物对的上游引物序列如序列表SEQ ID NO.3所示；下游引物序列如序列表SEQ IDNO.4所示。
进一步的，所述重组载体转入小鼠体内的方法为尾部注射。
本发明还包括采用上述构建方法构建得到的先兆子痫动物模型在研究子痫前期疾病的发病过程及机制中的应用。
本发明还包括采用上述构建方法构建得到的先兆子痫动物模型在防治子痫前期疾病药物筛选和药效评价中的应用。
本发明具有以下有益效果：
1、本发明利用基因技术对小鼠PTEN基因进行干扰，构建了小鼠PTEN过表达的腺病毒。为先兆子痫领域过表达PTEN相关研究提供体内动物实验模型：一方面，目前在先兆子痫领域PTEN基因对先兆子痫作用的研究报道较少，本模型的建立成功证明PTEN对先兆子痫的促进作用，为后续在先兆子痫中针对PTEN的进一步体内外作用机制研究提供理论依据。另一方面，此动物模型的成功建立，可用于后续PTEN在先兆子痫发生发展机制研究，同时还可辅助寻找先兆子痫有效治疗策略。
【附图说明】
图1为pDC316-CMV-MCS-EFla-copGFP载体结构图；
图2为妊娠小鼠PTENmRNA的相对表达结果图；
图3为妊娠小鼠血压和尿蛋白的结果图；
图4为妊娠小鼠胎盘和子宫的HE染色结果图；
图5为妊娠小鼠的解剖结果图。
【具体实施方式】
下面结合附图和实施例和试验对本发明作进一步说明，本申请的具体实施例对本发明的技术方案做进一步的说明，但不应理解为对本发明的限制：
实施例1：
过表达PTEN腺病毒载体的构建：
①从NCBI网站上得到PTEN基因CDS区信息并设计引物克隆出PTEN基因；其中，PTEN基因CDS序列如序列表SEQ ID NO.1所示；采用引物对对PTEN基因进行扩增，引物对其上游引物序列为：5'-TGGATTCGACTTAGACTTGACCT-3'(SEQ ID NO.3)；下游引物序列为：5'-GGTGGGTTATGGTCTTCAAAAGG-3'(SEQ ID NO.4)；
②将步骤①的基因克隆至腺病毒系统穿梭载体pDC316-CMV-MCS-EFla-copGFP中，该载体购买于广州艾基生物技术有限公司，结构如图1所示，核酸序列如序列表SEQ IDNO.2所示；
③将步骤②携带目的基因片段的穿梭质粒线性化后与腺病毒大骨架质粒共转入在特定的大肠杆菌中进行同源重组。
④由于腺病毒骨架质粒是氨苄抗性，而与穿梭质粒同源重组后，氨苄抗性丢失，同时表达卡那霉素抗性，因此，通过抗性的变化可筛选出腺病毒载体骨架重组子，挑取重组子进行酶切鉴定，挑选酶切鉴定正确的克隆，进行下一步腺病毒载体的包装。将筛选到的重组腺病毒运用脂质体法(LipoFiter，Hanbio公司)转染到293细胞中，由于腺病毒载体基因组中的早期基因E1缺失，利用带有E1基因的293细胞作为包装细胞，通过倍比扩增，富集病毒颗粒并将其命名为Ad-PTEN载体。
以不含目的基因的载体pDC316-CMV-MCS-EFla-copGFP(Amp)为对照，并命名为Ad-NC。
小鼠的PTEN基因的过表达效果如图2所示，图2中A为Ad-PTEN实验组和Ad-NC对照组mRNA相对表达量的柱状图，B为Ad-PTEN实验组和Ad-NC对照组的荧光表达图，从B图可见，Ad-PTEN实验组的PTEN基因有明显条带，Ad-NC对照组无明显条带，从A图可见，实验组小鼠胎盘的PTEN的mRNA和蛋白水平较对照组明显升高(P<0.05)。
实施例2：
构建动物模型和验证：
选取8-12周龄SPF级雄性CD-1小鼠6只；8-12周龄SPF级雌性CD-1小鼠12只；将12只孕鼠随机分为2组：对照组、Ad-PTEN组，每组6只。对照组于妊娠第13天尾静脉注射1×109PFU/200ul的Ad-NC腺病毒；Ad-PTEN组于妊娠第13天尾静脉注射1×109PFU/200ul的Ad-PTEN腺病毒。
分别在妊娠第12、14、16、18天上午8：00～11：00，使用无创尾动脉血压测量仪测量孕鼠血压。每只孕鼠测量3次收缩压，并计算其均值作为此次的血压值。结果如图3所示，图3A图为妊娠小鼠血压的结果图，B图为妊娠小鼠尿蛋白的结果图；图中可见，于妊娠第13天开始给予相应药物后，与Ad-NC组相比，Ad-PTEN组尾动脉收缩压水平均显著升高(P＜0.0001)(图3A)。Ad-PTEN组出现孕鼠妊娠期高血压。
收集孕鼠妊娠第17天8：00～妊娠18第天8：00的尿液，记录尿液体积，3000rpm 4℃离心10min,取上清，保存于-80℃。使用ELISA试剂盒测量测定尿蛋白含量。结果如图3B所示，与Ad-NC组组相比，Ad-PTEN组的尿蛋白含量显著升高(P＜0.05，图3B)。由此说明，PTEN基因的过表达成功诱导了小鼠先兆子痫。
解剖实验：
采用异氟烷吸入麻醉并脱颈处死孕鼠后，剖腹取胎，使用生理盐水冲洗胎鼠及胎盘上的血迹。用纱布吸干水分，记录胎鼠数量和胎鼠重量，观察胎鼠及胎盘有无畸形和其他异常情况，并进行拍照。结果如图5所示，各组胎鼠的数量和重量无统计学差异(图5B、5C)。但与RAd-NC组相比，Ad-PTEN组的妊娠小鼠出现死胎(图5A)。由此说明PTEN基因过表达后诱导的先兆子痫模型会影响胚胎的发育。
将胎盘和子宫使用4％多聚甲醛固定后进行石蜡包埋固定并进行HE染色。H&E染色结果显示(图4)：图中可见，与Ad-NC组相比，Ad-PTEN组的胎盘组织迷路结构紊乱，可见大量血窦不规则扩张，血窦内红细胞不均匀分布，少量血窦内较多红细胞，大量血窦内未见红细胞，滋养层细胞大量增生，并见较多滋养层细胞钙化；Ad-PTEN组子宫的壁蜕膜绒毛样结构增粗，上皮增厚，固有层伴较多粒细胞浸润，累及内膜上皮，少量内膜上皮细胞肿胀，胞浆疏松或呈空泡状，固有层局部水肿。提示PTEN过表达诱导的先兆子痫模型出现了胎盘功能障碍。
实施例3：
可利用实施例2构建得到的先兆子痫小鼠动物模型，筛选防治子痫前期疾病药物，并且进行相应的药效评价。
综上所述，本申请利用基因技术对小鼠PTEN基因进行干扰，构建了过表达PTEN基因的腺病毒载体，并将该载体转化至小鼠中，构建先兆子痫小鼠动物模型，经验证，该过表达载体转入怀孕小鼠体内后，小鼠表现出了先兆子痫病症，该动物模型成功建立，该模型的建立成功证明PTEN对先兆子痫的促进作用，为后续在先兆子痫中针对PTEN的进一步体内外作用机制研究提供理论依据。同时，该动物模型可用于后续PTEN在先兆子痫发生发展机制研究，同时还可辅助寻找先兆子痫相关防治药物，并且进行药效评价能为先兆子痫提供有效治疗策略。
上述说明是针对本发明较佳可行实施例的详细说明，但实施例并非用以限定本发明的专利申请范围，凡本发明所提示的技术精神下所完成的同等变化或修饰变更，均应属于本发明所涵盖专利范围。
Claims (4)
1.一种先兆子痫动物模型的构建方法，其特征在于，所述方法为将过表达PTEN基因的重组载体转入小鼠体内即得先兆子痫动物模型，所述过表达PTEN基因的重组载体的构建方法为：采用引物对克隆PTEN基因，将克隆出来的基因片段连接到pDC316-CMV-MCS-EFla-copGFP载体中即得；
所述PTEN基因的CDS序列如序列表SEQ ID NO.1所示；
所述pDC316-CMV-MCS-EFla-copGFP载体的碱基序列如序列表SEQ ID NO.2所示；
所述引物对的上游引物序列如序列表SEQ ID NO.3所示；下游引物序列如序列表SEQID NO.4所示。
2.根据权利要求1所述的一种先兆子痫动物模型的构建方法，其特征在于，所述重组载体转入小鼠体内的方法为尾部注射。
3.如权利要求1或2所述构建方法构建得到的先兆子痫动物模型在研究子痫前期疾病的发病过程及机制中的应用。
4.如权利要求1或2所述构建方法构建得到的先兆子痫动物模型在防治子痫前期疾病药物筛选和药效评价中的应用。
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