用过表达DNFGFR2方法构建肺发育缺陷猪模型

发布时间：2019-01-28 20:58

【摘要】：器官移植延长了许多患者的寿命,提高了他们的生活质量,但是供体器官短缺严重制约着器官移植的发展,异种器官移植是解决这一难题的好方法。目前许多实验室致力于异种移植实验,包括肺移植,他们大多以猪肺为供体,狒狒替代人类作为受体,但在移植过程中存在免疫排斥等问题。本研究希望获得一个肺缺陷的猪模型,用病人的iPSCs注射进猪囊胚中,补偿性的填补肺发育微环境,产生一个完全的病人iPSCs来源的肺。肺发育开始时,从胚胎原肠的上皮细胞中伸出两个肺芽,两个肺芽反复分支形成各级气管,这种分支发育模式称为分支形态发生。FGFR2的一个亚型FGFR2b参与调控肺芽上皮分支形态发生。为了在肺分支形态发生过程中阻断FGFR2b功能,影响肺发育而不影响其他胚胎组织。本试验用人源SFTPC启动子在转基因猪的肺泡上皮细胞中表达显性失活FGFR2c和FGFR2b,以及表达可溶性显性失活的FGFR2b-HFc,阻断气管分支和上皮分化,获得肺发育缺陷的转基因猪。研究目的通过阻断FGFR2b下游信号构建肺缺陷猪模型。研究方法1.设计和构建三种表达显性失活FGFR2的重组质粒,分别表达FGFR2c的胞外区和跨膜区(DNFGFR2c)和FGFR2b的胞外区和跨膜区(DNFGFR2b),以及表达FGFR2b胞外区和IgG的重链恒定区的重组蛋白(DNFGFR2b-HFc)。2.用细胞核转染法将构建好的DNFGFR2c、DNFGFR2b和)NFGFR2b-HFc重组质粒分别转染入猪原代成纤维细胞,经Puromycin筛选形成单细胞克隆,挑取细胞克隆并PCR鉴定。3.借助体细胞核移植方法,以转基因细胞作为体细胞核移植供体,以去核的卵母细胞为受体,经电融合形成囊胚,存活的囊胚移植到代孕母猪体内,怀孕母猪生出克隆猪。4.对获得的克隆小猪进行基因型和表型鉴定,用基因组PCR扩增方式进行基因型鉴定；基因芯片和RT-PCR方法检测nRNA水平表达；组织学观察肺发育情况；免疫组织化学检测肺间质和实质发育。实验结果应用体细胞核移植技术获得三种克隆猪,基因型鉴定显示所有克隆猪均为转基因阳性,肺组织mRNA检测也为阳性。DNFGFR2c受体猪怀孕4头,其中1头受体猪E43剖出3只发育正常胎儿。另外3头受体猪足日分娩出7头胎儿,其中4头死胎,2头出生后立即死亡,还有1头出生后一个月死亡。E43和新生DNFGFR2c转基因猪的肺均未表现为明显发育滞后,新生DNFGFR2c转基因猪的围产期死亡率高(6/7)。DNFGFR2b受体猪怀孕3头,1头受体猪E43剖出3只发育正常胎儿,1头受体猪E110剖出3头发育正常胎儿,另1头受体猪足日分娩出2头死胎。E43DNFGFR2b转基因猪,肺叶以间质较多,肺气管分支较少,E110 DNFGFR2b转基因猪肺泡偏大,肺泡隔薄,肺泡隔胶原纤维较少,肺发育稍微滞后。DNFGFR2b-HFc受体猪怀孕2头,1头怀孕受体猪E43剖出6只正常胎儿；另1头受体猪E110剖出6头发育正常胎儿。E43 DNFGFR2b-HFc转基因猪,肺叶以间质为主,实质少,肺气管分支较少,肺远端间皮下间质中未见细支气管。E110DNFGFR2b转基因猪肺体积小,肺泡结构不典型,肺泡小,肺泡隔较厚,肺泡隔中胶原纤维较多,肺发育严重滞后。结论肺上皮细胞过表达DNFGFR2c、DNFGFR2b和DNFGFR2b-HFc能够部分阻断FGFs和FGFR2b信号通路,猪肺发育缺陷,导致死胎现象。DNFGFR2c转基因猪肺发育稍微滞后于野生型猪；DNFGFR2b转基因猪肺泡偏大,肺泡隔薄,肺泡隔胶原纤维较少；DNFGFR2b-HFc转基因猪肺远小于野生型猪,实质区偏小,肺泡隔较厚,肺泡小,间质异常发育,肺间质和肺泡隔中胶原纤维远多于野生型。
[Abstract]:Organ transplantation prolongs the service life of many patients, improves their quality of life, but the shortage of donor organs seriously restricts the development of organ transplantation, and xenotransplantation is a good way to solve this problem. At present, many laboratories are committed to xenotransplantation, including lung transplantation, most of which are the donor of the pig's lung and the donor to replace the human as the receptor, but there are problems such as immune rejection in the process of transplantation. The study was intended to obtain a model of a lung defect, injected into the porcine blastula with the patient's iPSCs, with a compensatory filling of the lung development microenvironment, resulting in a complete patient's iSCs-derived lung. At the beginning of the development of the lung, two pulmonary buds are extended from the epithelial cells of the primary intestine of the embryo, and the two pulmonary bud branches repeatedly branch to form all levels of the trachea, and the branch development mode is called the branch form. One subtype of FGFR2, FGFR2, was involved in the control of the branch morphology of the pulmonary bud. in ord to block that function of the FGFR2 b during the pulmonary branch morphogenesis, the development of the lung is affect without affecting other embryonic tissue. This test uses the SFTPC promoter to express the dominant loss of the FGFR2 and the FGFR2 in the alveolar epithelial cells of the transgenic pig, as well as the expression of the soluble dominant-lost FGFR2-HFc, blocking the branch of the trachea and the epithelial differentiation, and obtaining the transgenic pig of the lung development defect. The purpose of this study was to construct a model of lung defect by blocking the downstream signal of FGFR2. Study Method 1. Three recombinant plasmids expressing a dominant loss of FGFR2 were designed and constructed to express the extracellular region of FGFR2 and the extracellular region and transmembrane region of the transmembrane region (DNFGFR2 c) and FGFR2, respectively, and the recombinant protein (DNFGFR2-HFc) expressing the heavy chain constant region of the extracellular domain of FGFR2 and the IgG. The constructed DNFR2c, DNFGFR2 and NFGFR2b-HFc recombinant plasmids were transfected into the primary fibroblast of the pig respectively by using the nuclear transfection method, and the single cell clones were formed by Puromycin screening, and the cell clones were selected and identified by PCR. In the method of somatic cell nuclear transfer, the transgenic cells are used as the donor for somatic cell nuclear transfer, and the enucleated oocytes are the receptors, and the blastocysts are formed by electrofusion, and the surviving blastocysts are transplanted to the pregnant sows, and the pregnant sows generate the cloned pigs. Genotyping and phenotypic identification of the obtained cloned piglets were carried out by PCR. The expression of nRNA was detected by gene chip and RT-PCR. The results of the experiment showed that all the cloned pigs were transgenic and the mRNA of lung tissue was positive. The DNFGFR2 c receptor pig is a 4-head pregnant, with one of the 1 receptor pigs E43 cut out of 3 normal fetuses. A total of 7 fetuses were born on the 3-head pig's foot, of which 4 were dead, 2 were immediately after birth and one month after the first birth. The lung of the transgenic pigs of the E43 and the newly-born DNFGFR2 c had no obvious developmental delay, and the perinatal mortality of the newly-born DNFGFR2 c transgenic pigs was high (6/ 7). The DNFR2 b receptor pig was pregnant with 3 heads, and the 1-head pig E43 cut out 3 normal fetuses, and the 1-head pig E110 cut 3 heads to develop normal fetuses. The other 1 recipient pig was given two dead fetuses on the day. E43DNFGFR2 b transgenic pigs, the lung lobes are more interstitial, the branch of the lung trachea is less, the E110 DNFGFR2 b transgenic pig has large alveolar bone, the alveolar septum is thin, the alveolar septal collagen fiber is small, and the development of the lung is slightly delayed. The DNFGFR2 b-HFc receptor pig was pregnant for 2, and one of the 1 pregnant receptor pigs E43 had six normal fetuses, and the other 1 recipient pig E110 had six developed normal fetuses. E43 DNFGFR2 b-HFc transgenic pig, the lung lobe is mainly of the stroma, the substance is small, the branch of the pulmonary trachea is small, and the subdermal matrix of the lung far end is not found in the bronchia. The lung volume of E110DNFFGFR2 transgenic pig is small, the alveolar structure is not typical, the alveoli are small, the alveolar septum is thicker, the collagen fibers in the alveolar septum are more, and the lung development is severely delayed. Conclusion The overexpression of DNFR2c, DNFGFR2 and DNFR2b-HFc in the lung epithelial cells can partially block the signaling pathway of the FGFs and FGFR2, and the development of the lung of the pig leads to the phenomenon of stillbirth. The lung development of the DNFGFR2-c transgenic pig is slightly delayed than that of the wild-type pig, the lung of the DNFGFR2-b transgenic pig is large, the alveolar septum is thin, and the alveolar septal collagen fiber is less; the DNFGFR2-HFc transgenic pig lung is far smaller than that of the wild-type pig, the substantial area is small, the alveolar septum is thick, the alveoli are small, the interstitial is abnormal, The collagen fibers in the interstitial and alveolar septum of the lung are far more than that of the wild type.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R-332;R655.3

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