宫内生理性低氧微环境对哺乳动物胚胎早期肾脏发育的影响

发布时间：2018-04-04 09:05

本文选题：胚肾　切入点：输尿管芽　出处：《复旦大学》2014年硕士论文

【摘要】：第一部分E13.5大鼠胚胎肾脏体外发育模型的建立目的：分离E13.5大鼠胚胎后肾(Metanephric kidneys),采用二维培养法体外培养并进行鉴定,建立大鼠胚胎肾脏体外发育模型。方法：选取性成熟SD雄性大鼠与雌性大鼠数只进行配种,获得E13.5天孕鼠。用戊巴比妥麻醉并脱颈处死孕鼠,75%酒精浸泡消毒,在无菌条件下解剖获得E13.5胚胎肾脏。培养时,将胚肾转移至Transwell filter半透膜上,在6孔板中加入1ml DMEM/F-12培养液(含10%胎牛血清和1%双抗),于二氧化碳培养箱(37。C、5%C02)中连续培养3-5天。用倒置解剖显微镜拍照记录发育情况。用免疫荧光染色法对培养5天的胚肾进行标志物染色并采用激光共聚焦显微镜拍照观察。对培养5天的胚肾进行石蜡包埋、切片以及HE染色,观察胚肾发育的结构变化。结果：1.E13.5胚肾贴Transwell filter半透膜生长,组织面积逐渐增大,输尿管芽分枝逐渐增多,后肾间充质逐渐发生分化：2.免疫荧光染色结果显示,胚肾输尿管芽(E-cadherin标记)分枝随着时间的增加逐渐增多,培养5天后输尿管芽成树枝状展开,周围分布着正在形成中的肾小球(Wt-1标记)；3.HE染色显示培养5d后胚肾中出现大量正在形成的肾小管、肾小球以及集合管样结构。结论：体外二维培养法能够连续培养E13.5大鼠胚肾至少5天,胚肾具有输尿管芽分枝和肾小球结构,表明发育模型基本构建成功,满足体外实验研究的要求。第二部分低氧微环境对大鼠胚肾体外发育表型的影响目的：观察不同氧分压低氧微环境下大鼠胚肾发育表型的改变。方法：体外分离E13.5大鼠胚胎肾脏,将同一窝胚肾进行随机分为实验组和对照组,每组各8-10个肾脏。实验组通过向培养箱中注入氮气(N2)制造不同氧分压(1%-5%02)的低氧条件,模拟发育时期宫内生理性低氧微环境,对照组胚肾则于常规氧分压(21%02)条件下培养。运用免疫荧光染色技术对肾脏发育进行染色标记(E-cadherin标记输尿管芽,Wt-1标记发育中的肾小球),运用激光共聚焦显微镜进行拍照记录。统计并比较两组胚肾发育形态上的差异。结果：1.极度低氧(1%02)条件下输尿管芽分枝和肾小球发育几乎停滞,胚肾发育受到显著抑制。2.适度低氧(3%02以及5%02)条件下体外连续培养3d,胚肾发育均受显著抑制,表现为实验组输尿管芽分枝和发育中的肾小球数量较对照组显著减少(P0.05)。3.适度低氧(5%02)条件下延长培养时间至5d,与对照组相比输尿管芽分枝数和发育中的肾小球数目仍显著减少(P0.05)。结论：1%-5%02低氧环境能够显著抑制体外培养的大鼠胚肾的发育,减少输尿管芽分枝和肾小球形成数目。第三部分低氧微环境影响大鼠胚肾发育的机制研究目的：探索低氧微环境影响体外培养的大鼠胚肾发育的可能机制。方法：体外分离E13.5大鼠胚肾,将同一窝胚肾随机分为实验组和对照组,每组各8-10个肾脏。实验组选取5%02作为低氧条件模拟宫内低氧微环境,将胚肾置于5%02低氧环境培养,对照组胚肾置于21%02常氧环境培养。运用免疫荧光染色技术标记胚肾输尿管芽分枝(anti-E-cadherin)、肾小球(anti-Wt-1)发育以及后肾间充质多能干细胞(anti-Six2)。运用EdU(胸腺嘧啶核苷类似物)掺入法检测培养3天后两组胚肾细胞增殖情况,运用TUNEL(末端脱氧核苷酸转移酶)法检测培养3天后两组胚肾细胞凋亡情况。采用实时定量荧光PCR仪检测相关基因相对的表达RQ值(RQ值=2-△△cT),并进行统计分析。所用照片均采用AZ100宏观激光共聚焦显微镜进行拍摄,运用美国NCBI公司Image J软件进行荧光半定量分析,统计并比较实验组和对照组差异。结果：1.与常氧组胚肾相比,低氧组大鼠胚肾细胞增殖受到显著抑制,但细胞凋亡显著减轻(均P0.05)。2.低氧组大鼠胚肾后肾间充质多能干细胞标志分子Six2表达较常氧组显著增多(P0.05)。3.与常氧组相比,低氧组大鼠胚肾基因表达发生显著变化,其中代表着后肾间充质向上皮转分化的关键调控因子Wnt9b和Wnt4表达显著减少,代表足细胞和肾小管终末细胞的标志分子Nsph2和Aqp1表达显著减少(均P0.05)；后肾间充质干细胞进行自我更新的关键调控因子Six2和维持其生存的关键因子Wt-1及Pax2表达均显著增多(P0.05)。结论：低氧显著抑制体外培养条件下大鼠胚肾细胞增殖和细胞凋亡,下调后肾间充质分化相关基因表达,上调后肾间充质Six2+多能干细胞生存及自我更新关键因子表达,进而可能参与后肾间充质多能干细胞池细胞数目的维持。
[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.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692

【共引文献】