AQP1基因敲除鼠滋养细胞水通透性的变化研究

发布时间：2018-01-24 17:18

本文关键词： 水通道蛋白1 基因敲除鼠滋养细胞细胞培养水通道蛋白1 滋养细胞水通透性母胎液体平衡水通道蛋白1 滋养细胞划痕损伤迁移　出处：《广州医学院》2011年硕士论文　论文类型：学位论文

【摘要】：母胎液体平衡对整个妊娠过程至关重要,正常的羊水量也是胎儿活动、生长和发育所必需的。胎盘作为机体妊娠期间的临时器官,在母胎液体交换及营养物质代谢等过程中发挥重要作用。水通道蛋白(Aquaporins,AQPs)是一类对水具有高度选择性的糖蛋白,能够根据渗透压或流体静脉压介导水快速被动地跨生物膜转运。迄今为止,已发现13种水通道蛋白(AQP 0～12)存在于哺乳动物中,其表达、分布及相关功能研究也已被陆续报道。众多研究已证实多种水通道蛋白基因在人、鼠及羊等哺乳动物的胎盘、胎膜组织中分布表达,提示AQPs可能在妊娠生理,尤其是母胎液体平衡中发挥关键作用。课题组前期研究已证实AQP1基因敲除孕鼠的相关妊娠表型发生变化,提供了AQP1在妊娠生理及羊水平衡中的直接证据。本课题利用AQP1敲除小鼠模型建立了孕鼠胎盘滋养细胞体外培养系统,通过测试AQP1基因敲除型滋养细胞与野生型滋养细胞的水通透性差异,从功能方面提供了AQP1在母胎液体平衡中发挥作用的直接证据。另外,本课题对AQP1基因敲除型滋养细胞的迁移能力做了初步研究,为AQP1基因在产科相关疾病及妊娠滋养细胞疾病中的作用提供了理论基础。第一部分AQP1基因敲除鼠滋养细胞水通透性的变化研究第一章野生型及AQP1基因敲除型胎盘滋养细胞的培养及鉴定【研究目的】培养符合实验要求的野生型(wild type,AQP1+/+)及AQP1基因敲除型(AQP1-knockout, AQP1-/-)胎盘滋养细胞。【方法】成年健康野生型CD1小鼠(AQP1+/+)及AQP1基因敲除小鼠(AQP1+/+)分别按雌雄小鼠等量合笼交配,第二日检出阴道栓者记为妊娠第1天(1 gestational day, 1GD)。选取12GD孕鼠的胎盘组织,应用组织块培养法对AQP1+/+及AQP1-/-胎盘组织进行滋养细胞原代培养;胰蛋白酶消化法进行传代培养;应用滋养细胞内的特异细胞骨架蛋白细胞角蛋白7(CK-7)进行免疫化学染色细胞鉴定。【结果】组织块培养第2日细胞便开始从组织块边缘向周围生长,数日后细胞呈片状铺展生长;15日即可进行细胞传代;两组滋养细胞在肉眼可见的大小、形态及生长情况未见明显区别。细胞角蛋白7(CK-7)染色示细胞纯度达95%以上。【结论】成功建立了野生型和AQP1基因敲除型滋养细胞体外培养体系,以备本研究后续实验之用。第二章野生型及AQP1基因敲除型滋养细胞水通透性测定【研究目的】课题组前期研究显示AQP1基因敲除小鼠母胎液体平衡受损,本研究通过检测AQP1基因敲除型滋养细胞水通透性的变化,直接从功能上探讨AQP1在母胎液体平衡中的作用。【方法】 1.应用免疫荧光方法证实AQP1基因在滋养细胞的表达。 2.应用钙黄绿素荧光淬灭方法对成功培养的AQP1基因敲除型(AQP1-/-)与野生型(AQP1+/+)滋养细胞进行细胞质膜水通透性分析比较。【结果】 1.免疫荧光结果显示AQP1在野生型滋养细胞膜表达。 2.滋养细胞质膜水通透性分析显示:低渗液情况下, AQP1-/-滋养细胞水通透性显著低于AQP1+/+(P=0.009),仅为野生型孕鼠滋养细胞的57%;高渗液情况下,AQP1-/-滋养细胞水通透性显著低于AQP1+/+(P=0.036),其水通透性仅为野生型孕鼠滋养细胞的64%。【结论】研究结果显示AQP1基因敲除型滋养细胞水通透性显著低于野生型,从功能方面提供了AQP1在母胎液体平衡中发挥重要作用的直接证据。第二部分AQP1基因敲除型滋养细胞迁移变化的初步研究【研究目的】研究体外培养野生型及AQP1基因敲除型孕鼠胎盘滋养细胞划痕损伤后,细胞迁移行为的变化。【方法】利用传代培养的野生型及AQP1基因敲除型小鼠胎盘滋养细胞制作划痕损伤模型,观察划痕损伤后两组细胞的迁移情况变化。【结果】划痕损伤后,AQP1基因敲除型滋养细胞划痕处细胞迁移数明显低于野生型滋养细胞数量。【结论】 AQP1基因在滋养细胞迁移过程发挥重要作用,尤其是创伤后的迁移修复。
[Abstract]:The maternal fetal fluid balance is very important to the whole process of pregnancy, normal amniotic fluid volume and fetal activity, growth and development required. As the temporary organ placenta during pregnancy, play an important role in maternal fetal fluid exchange and metabolism of nutrients in the process.
Water channel protein (Aquaporins, AQPs) is a glycoprotein with high selectivity for water, according to the osmotic pressure or fluid venous pressure mediated passive water transport across biological membranes. So far, has found 13 kinds of water channel protein (AQP 0~12) exist in mammals, the expression, distribution and related research the function has also been reported. Many studies have confirmed that a variety of aquaporin genes in human, rat and sheep placental mammals, the expression and distribution of fetal membranes, suggesting that AQPs may be in the physiology of pregnancy, especially play a key role in maternal fetal fluid balance.
Our previous studies have demonstrated that AQP1 gene knockout phenotypes related to pregnancy in pregnant rats changes, provide direct evidence for AQP1 in pregnancy and amniotic fluid balance. This paper used AQP1 knockout mice model was established for pregnant rat placental trophoblast cells in vitro culture system, through the test of AQP1 gene knock in trophoblast cell water permeability difference with the wild type cell, from function provides direct evidence that AQP1 play a role in maternal fetal fluid balance. In addition, the subject of the AQP1 knockout trophoblast migration made a preliminary study, provides a theoretical basis for the role of AQP1 gene in obstetric disease and gestational trophoblastic disease.
Study on the change of water permeability in the trophoblast of AQP1 gene knockout mice
The first chapter of culture and identification of wild and AQP1 gene knockout placental trophoblast cells
[purpose]
Wild type (AQP1+/+) and AQP1 gene knockout (AQP1-knockout, AQP1-/-) placental trophoblast cells were cultured in accordance with the requirements of the experiment.
[method]
Healthy adult wild type CD1 mice (AQP1+/+) and AQP1 knockout mice (AQP1+/+) respectively in male and female mice mated with second days, detection of vaginal suppository records is the first day of pregnancy (1 gestational, day, 1GD). Select the 12GD placenta of pregnant rats, using tissue culture method of AQP1+/+ and AQP1-/- in placenta the organization of primary cultured trophoblast cells; trypsin digestion were cultured; cell specific cytoskeletal protein in trophoblast cell keratin 7 (CK-7) immunohistochemical staining were identified.
[results]
Second cell tissue culture began to grow from the edge to the surrounding tissue, the number of days after cell growth 15, patchy spreading; cells were passaged; trophoblast cells in the two groups of visible size, morphology and growth had no obvious difference. The expression of cytokeratin 7 (CK-7) staining showed that the cell purity was more than 95%.
[Conclusion]
The in vitro culture system of wild and AQP1 gene knockout trophoblastic cells was successfully established in order to prepare for the follow-up experiment of this study.
Determination of water permeability in the second chapter of wild type and AQP1 gene knockout trophoblast
[purpose]
Our previous study showed that AQP1 gene knockout mice impaired maternal fetal fluid balance, this study through the detection of AQP1 knock out type cell water permeability, the direct role of AQP1 in maternal fetal fluid balance from the function.
[method]
1. the expression of AQP1 gene in trophoblastic cells was confirmed by immunofluorescence.
2., we used calcein fluorescence quenching method to analyze the plasma membrane permeability of AQP1 gene knockout (AQP1-/-) and wild type (AQP1+/+) trophoblast cells.
[results]
1. immunofluorescence results showed that AQP1 was expressed in the membrane of the wild type trophoblastic cell.
2. trophoblast cell plasma membrane water permeability analysis showed that low permeability solution, water permeability of AQP1-/- in trophoblast cells was significantly lower than that of AQP1+/+ (P=0.009), only wild type trophoblast cells in pregnant rats 57%; hypertonic solution, water permeability of AQP1-/- in trophoblast cells was significantly lower than that of AQP1+/+ (P= 0.036), the water permeability of trophoblast cells was only wild type of pregnant rats 64%.
[Conclusion]
The results showed that AQP1 knockout trophoblast cell water permeability were significantly lower than the wild type, the function provides direct evidence that AQP1 play an important role in maternal fetal fluid balance.
A preliminary study on the migration and changes of AQP1 gene knockout trophoblastic cells in the second part
[purpose]
To study the change of cell migration behavior after the scratch injury of placental trophoblast in the wild and AQP1 gene knockout mice in vitro.
[method]
A scratch injury model was established by subculture of wild type and AQP1 knockout mouse placenta trophoblast cells. The migration of two groups of cells after scratching injury was observed.
[results]
After scratch injury, the number of cell migration in the scratch cells of AQP1 gene knockout cells was significantly lower than that of the wild type trophoblastic cells.
[Conclusion]
AQP1 gene plays an important role in the migration of trophoblastic cells, especially after traumatic migration and repair.

【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

【参考文献】