本文關(guān)鍵詞： 水通道蛋白1 基因敲除鼠 滋養(yǎng)細(xì)胞 細(xì)胞培養(yǎng) 水通道蛋白1 滋養(yǎng)細(xì)胞 水通透性 母胎液體平衡 水通道蛋白1 滋養(yǎng)細(xì)胞 劃痕損傷 遷移　出處：《廣州醫(yī)學(xué)院》2011年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：母胎液體平衡對(duì)整個(gè)妊娠過(guò)程至關(guān)重要,正常的羊水量也是胎兒活動(dòng)、生長(zhǎng)和發(fā)育所必需的。胎盤(pán)作為機(jī)體妊娠期間的臨時(shí)器官,在母胎液體交換及營(yíng)養(yǎng)物質(zhì)代謝等過(guò)程中發(fā)揮重要作用。 水通道蛋白(Aquaporins,AQPs)是一類(lèi)對(duì)水具有高度選擇性的糖蛋白,能夠根據(jù)滲透壓或流體靜脈壓介導(dǎo)水快速被動(dòng)地跨生物膜轉(zhuǎn)運(yùn)。迄今為止,已發(fā)現(xiàn)13種水通道蛋白(AQP 0～12)存在于哺乳動(dòng)物中,其表達(dá)、分布及相關(guān)功能研究也已被陸續(xù)報(bào)道。眾多研究已證實(shí)多種水通道蛋白基因在人、鼠及羊等哺乳動(dòng)物的胎盤(pán)、胎膜組織中分布表達(dá),提示AQPs可能在妊娠生理,尤其是母胎液體平衡中發(fā)揮關(guān)鍵作用。 課題組前期研究已證實(shí)AQP1基因敲除孕鼠的相關(guān)妊娠表型發(fā)生變化,提供了AQP1在妊娠生理及羊水平衡中的直接證據(jù)。本課題利用AQP1敲除小鼠模型建立了孕鼠胎盤(pán)滋養(yǎng)細(xì)胞體外培養(yǎng)系統(tǒng),通過(guò)測(cè)試AQP1基因敲除型滋養(yǎng)細(xì)胞與野生型滋養(yǎng)細(xì)胞的水通透性差異,從功能方面提供了AQP1在母胎液體平衡中發(fā)揮作用的直接證據(jù)。另外,本課題對(duì)AQP1基因敲除型滋養(yǎng)細(xì)胞的遷移能力做了初步研究,為AQP1基因在產(chǎn)科相關(guān)疾病及妊娠滋養(yǎng)細(xì)胞疾病中的作用提供了理論基礎(chǔ)。 第一部分AQP1基因敲除鼠滋養(yǎng)細(xì)胞水通透性的變化研究 第一章野生型及AQP1基因敲除型胎盤(pán)滋養(yǎng)細(xì)胞的培養(yǎng)及鑒定 【研究目的】 培養(yǎng)符合實(shí)驗(yàn)要求的野生型(wild type,AQP1+/+)及AQP1基因敲除型(AQP1-knockout, AQP1-/-)胎盤(pán)滋養(yǎng)細(xì)胞。 【方法】 成年健康野生型CD1小鼠(AQP1+/+)及AQP1基因敲除小鼠(AQP1+/+)分別按雌雄小鼠等量合籠交配,第二日檢出陰道栓者記為妊娠第1天(1 gestational day, 1GD)。選取12GD孕鼠的胎盤(pán)組織,應(yīng)用組織塊培養(yǎng)法對(duì)AQP1+/+及AQP1-/-胎盤(pán)組織進(jìn)行滋養(yǎng)細(xì)胞原代培養(yǎng);胰蛋白酶消化法進(jìn)行傳代培養(yǎng);應(yīng)用滋養(yǎng)細(xì)胞內(nèi)的特異細(xì)胞骨架蛋白細(xì)胞角蛋白7(CK-7)進(jìn)行免疫化學(xué)染色細(xì)胞鑒定。 【結(jié)果】 組織塊培養(yǎng)第2日細(xì)胞便開(kāi)始從組織塊邊緣向周?chē)L(zhǎng),數(shù)日后細(xì)胞呈片狀鋪展生長(zhǎng);15日即可進(jìn)行細(xì)胞傳代;兩組滋養(yǎng)細(xì)胞在肉眼可見(jiàn)的大小、形態(tài)及生長(zhǎng)情況未見(jiàn)明顯區(qū)別。細(xì)胞角蛋白7(CK-7)染色示細(xì)胞純度達(dá)95%以上。 【結(jié)論】 成功建立了野生型和AQP1基因敲除型滋養(yǎng)細(xì)胞體外培養(yǎng)體系,以備本研究后續(xù)實(shí)驗(yàn)之用。 第二章野生型及AQP1基因敲除型滋養(yǎng)細(xì)胞水通透性測(cè)定 【研究目的】 課題組前期研究顯示AQP1基因敲除小鼠母胎液體平衡受損,本研究通過(guò)檢測(cè)AQP1基因敲除型滋養(yǎng)細(xì)胞水通透性的變化,直接從功能上探討AQP1在母胎液體平衡中的作用。 【方法】 1.應(yīng)用免疫熒光方法證實(shí)AQP1基因在滋養(yǎng)細(xì)胞的表達(dá)。 2.應(yīng)用鈣黃綠素?zé)晒獯銣绶椒▽?duì)成功培養(yǎng)的AQP1基因敲除型(AQP1-/-)與野生型(AQP1+/+)滋養(yǎng)細(xì)胞進(jìn)行細(xì)胞質(zhì)膜水通透性分析比較。 【結(jié)果】 1.免疫熒光結(jié)果顯示AQP1在野生型滋養(yǎng)細(xì)胞膜表達(dá)。 2.滋養(yǎng)細(xì)胞質(zhì)膜水通透性分析顯示:低滲液情況下, AQP1-/-滋養(yǎng)細(xì)胞水通透性顯著低于AQP1+/+(P=0.009),僅為野生型孕鼠滋養(yǎng)細(xì)胞的57%;高滲液情況下,AQP1-/-滋養(yǎng)細(xì)胞水通透性顯著低于AQP1+/+(P=0.036),其水通透性僅為野生型孕鼠滋養(yǎng)細(xì)胞的64%。 【結(jié)論】 研究結(jié)果顯示AQP1基因敲除型滋養(yǎng)細(xì)胞水通透性顯著低于野生型,從功能方面提供了AQP1在母胎液體平衡中發(fā)揮重要作用的直接證據(jù)。 第二部分AQP1基因敲除型滋養(yǎng)細(xì)胞遷移變化的初步研究 【研究目的】 研究體外培養(yǎng)野生型及AQP1基因敲除型孕鼠胎盤(pán)滋養(yǎng)細(xì)胞劃痕損傷后,細(xì)胞遷移行為的變化。 【方法】 利用傳代培養(yǎng)的野生型及AQP1基因敲除型小鼠胎盤(pán)滋養(yǎng)細(xì)胞制作劃痕損傷模型,觀察劃痕損傷后兩組細(xì)胞的遷移情況變化。 【結(jié)果】 劃痕損傷后,AQP1基因敲除型滋養(yǎng)細(xì)胞劃痕處細(xì)胞遷移數(shù)明顯低于野生型滋養(yǎng)細(xì)胞數(shù)量。 【結(jié)論】 AQP1基因在滋養(yǎng)細(xì)胞遷移過(guò)程發(fā)揮重要作用,尤其是創(chuàng)傷后的遷移修復(fù)。
[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.

【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R346

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本文編號(hào)：1460571