本文選題：胚腎　切入點：輸尿管芽　出處：《復旦大學》2014年碩士論文

【摘要】：第一部分E13.5大鼠胚胎腎臟體外發(fā)育模型的建立目的：分離E13.5大鼠胚胎后腎(Metanephric kidneys),采用二維培養(yǎng)法體外培養(yǎng)并進行鑒定,建立大鼠胚胎腎臟體外發(fā)育模型。方法：選取性成熟SD雄性大鼠與雌性大鼠數(shù)只進行配種,獲得E13.5天孕鼠。用戊巴比妥麻醉并脫頸處死孕鼠,75%酒精浸泡消毒,在無菌條件下解剖獲得E13.5胚胎腎臟。培養(yǎng)時,將胚腎轉(zhuǎn)移至Transwell filter半透膜上,在6孔板中加入1ml DMEM/F-12培養(yǎng)液(含10%胎牛血清和1%雙抗),于二氧化碳培養(yǎng)箱(37。C、5%C02)中連續(xù)培養(yǎng)3-5天。用倒置解剖顯微鏡拍照記錄發(fā)育情況。用免疫熒光染色法對培養(yǎng)5天的胚腎進行標志物染色并采用激光共聚焦顯微鏡拍照觀察。對培養(yǎng)5天的胚腎進行石蠟包埋、切片以及HE染色,觀察胚腎發(fā)育的結構變化。結果：1.E13.5胚腎貼Transwell filter半透膜生長,組織面積逐漸增大,輸尿管芽分枝逐漸增多,后腎間充質(zhì)逐漸發(fā)生分化：2.免疫熒光染色結果顯示,胚腎輸尿管芽(E-cadherin標記)分枝隨著時間的增加逐漸增多,培養(yǎng)5天后輸尿管芽成樹枝狀展開,周圍分布著正在形成中的腎小球(Wt-1標記)；3.HE染色顯示培養(yǎng)5d后胚腎中出現(xiàn)大量正在形成的腎小管、腎小球以及集合管樣結構。結論：體外二維培養(yǎng)法能夠連續(xù)培養(yǎng)E13.5大鼠胚腎至少5天,胚腎具有輸尿管芽分枝和腎小球結構,表明發(fā)育模型基本構建成功,滿足體外實驗研究的要求。第二部分低氧微環(huán)境對大鼠胚腎體外發(fā)育表型的影響目的：觀察不同氧分壓低氧微環(huán)境下大鼠胚腎發(fā)育表型的改變。方法：體外分離E13.5大鼠胚胎腎臟,將同一窩胚腎進行隨機分為實驗組和對照組,每組各8-10個腎臟。實驗組通過向培養(yǎng)箱中注入氮氣(N2)制造不同氧分壓(1%-5%02)的低氧條件,模擬發(fā)育時期宮內(nèi)生理性低氧微環(huán)境,對照組胚腎則于常規(guī)氧分壓(21%02)條件下培養(yǎng)。運用免疫熒光染色技術對腎臟發(fā)育進行染色標記(E-cadherin標記輸尿管芽,Wt-1標記發(fā)育中的腎小球),運用激光共聚焦顯微鏡進行拍照記錄。統(tǒng)計并比較兩組胚腎發(fā)育形態(tài)上的差異。結果：1.極度低氧(1%02)條件下輸尿管芽分枝和腎小球發(fā)育幾乎停滯,胚腎發(fā)育受到顯著抑制。2.適度低氧(3%02以及5%02)條件下體外連續(xù)培養(yǎng)3d,胚腎發(fā)育均受顯著抑制,表現(xiàn)為實驗組輸尿管芽分枝和發(fā)育中的腎小球數(shù)量較對照組顯著減少(P0.05)。3.適度低氧(5%02)條件下延長培養(yǎng)時間至5d,與對照組相比輸尿管芽分枝數(shù)和發(fā)育中的腎小球數(shù)目仍顯著減少(P0.05)。結論：1%-5%02低氧環(huán)境能夠顯著抑制體外培養(yǎng)的大鼠胚腎的發(fā)育,減少輸尿管芽分枝和腎小球形成數(shù)目。第三部分低氧微環(huán)境影響大鼠胚腎發(fā)育的機制研究目的：探索低氧微環(huán)境影響體外培養(yǎng)的大鼠胚腎發(fā)育的可能機制。方法：體外分離E13.5大鼠胚腎,將同一窩胚腎隨機分為實驗組和對照組,每組各8-10個腎臟。實驗組選取5%02作為低氧條件模擬宮內(nèi)低氧微環(huán)境,將胚腎置于5%02低氧環(huán)境培養(yǎng),對照組胚腎置于21%02常氧環(huán)境培養(yǎng)。運用免疫熒光染色技術標記胚腎輸尿管芽分枝(anti-E-cadherin)、腎小球(anti-Wt-1)發(fā)育以及后腎間充質(zhì)多能干細胞(anti-Six2)。運用EdU(胸腺嘧啶核苷類似物)摻入法檢測培養(yǎng)3天后兩組胚腎細胞增殖情況,運用TUNEL(末端脫氧核苷酸轉(zhuǎn)移酶)法檢測培養(yǎng)3天后兩組胚腎細胞凋亡情況。采用實時定量熒光PCR儀檢測相關基因相對的表達RQ值(RQ值=2-△△cT),并進行統(tǒng)計分析。所用照片均采用AZ100宏觀激光共聚焦顯微鏡進行拍攝,運用美國NCBI公司Image J軟件進行熒光半定量分析,統(tǒng)計并比較實驗組和對照組差異。結果：1.與常氧組胚腎相比,低氧組大鼠胚腎細胞增殖受到顯著抑制,但細胞凋亡顯著減輕(均P0.05)。2.低氧組大鼠胚腎后腎間充質(zhì)多能干細胞標志分子Six2表達較常氧組顯著增多(P0.05)。3.與常氧組相比,低氧組大鼠胚腎基因表達發(fā)生顯著變化,其中代表著后腎間充質(zhì)向上皮轉(zhuǎn)分化的關鍵調(diào)控因子Wnt9b和Wnt4表達顯著減少,代表足細胞和腎小管終末細胞的標志分子Nsph2和Aqp1表達顯著減少(均P0.05)；后腎間充質(zhì)干細胞進行自我更新的關鍵調(diào)控因子Six2和維持其生存的關鍵因子Wt-1及Pax2表達均顯著增多(P0.05)。結論：低氧顯著抑制體外培養(yǎng)條件下大鼠胚腎細胞增殖和細胞凋亡,下調(diào)后腎間充質(zhì)分化相關基因表達,上調(diào)后腎間充質(zhì)Six2+多能干細胞生存及自我更新關鍵因子表達,進而可能參與后腎間充質(zhì)多能干細胞池細胞數(shù)目的維持。
[Abstract]:The first part: to establish models of the development of embryonic kidney in vitro E13.5 rats of rat embryos after separation of E13.5 (Metanephric kidneys), using a two-dimensional culture method in vitro culture and identification, establishment of embryonic development in vitro kidney rats. Methods: adult SD male rats and female rats were mated for only a few, E13.5 day. Pregnant rats were anaesthetized with pentobarbital and sacrificed rats, 75% alcohol immersion disinfection, under sterile conditions to obtain E13.5 embryonic kidney. Anatomical culture, the embryo kidney is transferred to Transwell filter in 6 well plate semipermeable membrane, adding 1ml DMEM/F-12 medium (containing 10% fetal bovine serum and 1% double antibody) in co2incubator, (37.C, 5%C02) in continuous culture for 3-5 days. Using an inverted dissecting microscope photographed development. By immunofluorescence staining of cultured embryonic kidney 5 days of marker staining with confocal laser Microscope observation. The cultured embryonic kidney 5 days were embedded in paraffin, sliced and stained with HE, observe the structural changes of embryonic kidney development. Results: 1.E13.5 embryo kidney paste Transwell filter membrane growth, tissue area increases gradually, the ureteric bud branching increased gradually, metanephric mesenchymal differentiation gradually occurred: 2. immunofluorescence staining the results showed that the embryo kidney ureteric bud (E-cadherin tag) branch gradually increased with the increase of time, after 5 days of culture the ureteric bud into dendritic expansion around the distribution is formed in the glomerulus (Wt-1 mark); 3.HE staining showed a large number of emerging renal tubule after 5D cultured embryonic kidney, glomerular and collecting tube like structure. Conclusion: the in vitro culture method to two-dimensional continuous culture of embryonic kidney of E13.5 rat embryo kidney has at least 5 days, ureteric bud branching and glomerular structure, shows that the development model of successful construction of full Experimental study on foot in vitro requirements. The second part phenotypic effects of hypoxia on embryonic kidney in vitro development of rats Objective: To observe the effects of different oxygen partial pressure hypoxia change of rat embryo kidney growth phenotype microenvironment. Methods: isolated E13.5 rat embryonic kidney, with a nest of embryonic kidney were randomly divided into experimental group and the control group, each group had 8-10 kidneys. The experimental group by injecting nitrogen into the incubator (N2) manufacturing different oxygen partial pressure (1%-5%02) in the hypoxic condition, simulation during the development of fetal physiological hypoxia microenvironment, embryo kidney in control group conventional oxygen pressure (21% to 02) under the condition of using immune culture. Fluorescence staining staining of kidney development (E-cadherin labeled the ureteric bud, Wt-1 marker in the development of glomerular, photographed) using confocal laser scanning microscopy. The statistics and compare the two groups of embryonic kidney development morphological differences. Results: 1. The degree of hypoxia (1%02) under the condition of ureteric bud branching and glomerular development is almost stagnant, embryo kidney growth was significantly inhibited by.2. hypoxia (3%02 and 5%02) during continuous 3D culture, embryo kidney growth was inhibited, the experimental group showed the number of branches and ureteric bud development in glomeruli was significantly reduced than the control group (P0.05).3. hypoxia (5%02) prolonged incubation time to 5D conditions, compared with the control group, the number of glomerular ureteric bud branches and development is significantly reduced (P0.05). Conclusion: 1%-5%02 can significantly inhibit hypoxia in cultured rat embryonic kidney development, reduce the number of ureteric bud branching and glomerular formation. The third part hypoxia rats mechanism of embryonic kidney development objective: To explore the effects of hypoxia in cultured rat embryonic kidney development and the possible mechanism. Methods: in vitro isolation E13.5 Embryo kidney of rats, with a nest of embryonic kidney were randomly divided into experimental group and control group, each group had 8-10 kidneys. Experimental group selected 5%02 as hypoxia simulated intrauterine hypoxia microenvironment, the embryo kidney in 5%02 hypoxia training, control group 21%02 in embryonic kidney normoxic environment using immunofluorescence culture. Labeling embryonic kidney ureteric bud branching (anti-E-cadherin) (anti-Wt-1), glomerular development and metanephric mesenchymal stem cells (anti-Six2). Using EdU (thymidine analogue) incorporation assay after 3 days of culture the proliferation of two groups of embryonic kidney cell situation by TUNEL (terminal deoxynucleotidyl transferase) assay after 3 days of culture of two groups of embryonic kidney cells apoptosis. The expression of the RQ value of real time fluorescence quantitative PCR instrument was used to detect genes related to relative (RQ = =2-, Delta cT) and statistical analysis. The photos were treated by AZ100 laser scanning confocal microscope in the macro For shooting, fluorescence semi quantitative analysis using the United States NCBI company Image J software. The difference between the experimental group and control group and statistics. Results: 1. compared with normoxia group embryo kidney, renal cell proliferation of embryonic rats in hypoxia group was significantly inhibited, but significantly reduced the apoptosis of embryonic kidney (P0.05).2. hypoxia group after the rat renal mesenchymal stem cell marker Six2 expression compared with normoxia group (P0.05.3.) increased significantly compared with normoxia group, hypoxia group rats the expression of embryonic kidney gene changed significantly, which represents the key regulation of metanephric mesenchymal transdifferentiation to epithelial factor Wnt9b and Wnt4 expression were significantly decreased, on behalf of podocytes and renal tubular cell end marker Nsph2 and Aqp1 expression were significantly decreased (P0.05); the key regulatory factor Six2 after renal mesenchymal stem cells to self renew and maintain its survival is the key factor Wt-1 and Pax2 expression were significantly increased Many (P0.05). Conclusion: hypoxia significantly inhibited the in vitro rat embryo kidney cell proliferation and apoptosis, downregulation of metanephric mesenchymal differentiation related gene expression and upregulation of metanephric mesenchymal stem cells Six2+ survival and self-renewal key factor expression in metanephric mesenchymal stem cells cell pool the number of possible and maintain.

【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R692

【共引文獻】

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