用過表達(dá)DNFGFR2方法構(gòu)建肺發(fā)育缺陷豬模型
發(fā)布時間：2019-01-28 20:58
【摘要】：器官移植延長了許多患者的壽命,提高了他們的生活質(zhì)量,但是供體器官短缺嚴(yán)重制約著器官移植的發(fā)展,異種器官移植是解決這一難題的好方法。目前許多實驗室致力于異種移植實驗,包括肺移植,他們大多以豬肺為供體,狒狒替代人類作為受體,但在移植過程中存在免疫排斥等問題。本研究希望獲得一個肺缺陷的豬模型,用病人的iPSCs注射進(jìn)豬囊胚中,補(bǔ)償性的填補(bǔ)肺發(fā)育微環(huán)境,產(chǎn)生一個完全的病人iPSCs來源的肺。肺發(fā)育開始時,從胚胎原腸的上皮細(xì)胞中伸出兩個肺芽,兩個肺芽反復(fù)分支形成各級氣管,這種分支發(fā)育模式稱為分支形態(tài)發(fā)生。FGFR2的一個亞型FGFR2b參與調(diào)控肺芽上皮分支形態(tài)發(fā)生。為了在肺分支形態(tài)發(fā)生過程中阻斷FGFR2b功能,影響肺發(fā)育而不影響其他胚胎組織。本試驗用人源SFTPC啟動子在轉(zhuǎn)基因豬的肺泡上皮細(xì)胞中表達(dá)顯性失活FGFR2c和FGFR2b,以及表達(dá)可溶性顯性失活的FGFR2b-HFc,阻斷氣管分支和上皮分化,獲得肺發(fā)育缺陷的轉(zhuǎn)基因豬。研究目的通過阻斷FGFR2b下游信號構(gòu)建肺缺陷豬模型。研究方法1.設(shè)計和構(gòu)建三種表達(dá)顯性失活FGFR2的重組質(zhì)粒,分別表達(dá)FGFR2c的胞外區(qū)和跨膜區(qū)(DNFGFR2c)和FGFR2b的胞外區(qū)和跨膜區(qū)(DNFGFR2b),以及表達(dá)FGFR2b胞外區(qū)和IgG的重鏈恒定區(qū)的重組蛋白(DNFGFR2b-HFc)。2.用細(xì)胞核轉(zhuǎn)染法將構(gòu)建好的DNFGFR2c、DNFGFR2b和)NFGFR2b-HFc重組質(zhì)粒分別轉(zhuǎn)染入豬原代成纖維細(xì)胞,經(jīng)Puromycin篩選形成單細(xì)胞克隆,挑取細(xì)胞克隆并PCR鑒定。3.借助體細(xì)胞核移植方法,以轉(zhuǎn)基因細(xì)胞作為體細(xì)胞核移植供體,以去核的卵母細(xì)胞為受體,經(jīng)電融合形成囊胚,存活的囊胚移植到代孕母豬體內(nèi),懷孕母豬生出克隆豬。4.對獲得的克隆小豬進(jìn)行基因型和表型鑒定,用基因組PCR擴(kuò)增方式進(jìn)行基因型鑒定；基因芯片和RT-PCR方法檢測nRNA水平表達(dá)；組織學(xué)觀察肺發(fā)育情況；免疫組織化學(xué)檢測肺間質(zhì)和實質(zhì)發(fā)育。實驗結(jié)果應(yīng)用體細(xì)胞核移植技術(shù)獲得三種克隆豬,基因型鑒定顯示所有克隆豬均為轉(zhuǎn)基因陽性,肺組織mRNA檢測也為陽性。DNFGFR2c受體豬懷孕4頭,其中1頭受體豬E43剖出3只發(fā)育正常胎兒。另外3頭受體豬足日分娩出7頭胎兒,其中4頭死胎,2頭出生后立即死亡,還有1頭出生后一個月死亡。E43和新生DNFGFR2c轉(zhuǎn)基因豬的肺均未表現(xiàn)為明顯發(fā)育滯后,新生DNFGFR2c轉(zhuǎn)基因豬的圍產(chǎn)期死亡率高(6/7)。DNFGFR2b受體豬懷孕3頭,1頭受體豬E43剖出3只發(fā)育正常胎兒,1頭受體豬E110剖出3頭發(fā)育正常胎兒,另1頭受體豬足日分娩出2頭死胎。E43DNFGFR2b轉(zhuǎn)基因豬,肺葉以間質(zhì)較多,肺氣管分支較少,E110 DNFGFR2b轉(zhuǎn)基因豬肺泡偏大,肺泡隔薄,肺泡隔膠原纖維較少,肺發(fā)育稍微滯后。DNFGFR2b-HFc受體豬懷孕2頭,1頭懷孕受體豬E43剖出6只正常胎兒；另1頭受體豬E110剖出6頭發(fā)育正常胎兒。E43 DNFGFR2b-HFc轉(zhuǎn)基因豬,肺葉以間質(zhì)為主,實質(zhì)少,肺氣管分支較少,肺遠(yuǎn)端間皮下間質(zhì)中未見細(xì)支氣管。E110DNFGFR2b轉(zhuǎn)基因豬肺體積小,肺泡結(jié)構(gòu)不典型,肺泡小,肺泡隔較厚,肺泡隔中膠原纖維較多,肺發(fā)育嚴(yán)重滯后。結(jié)論肺上皮細(xì)胞過表達(dá)DNFGFR2c、DNFGFR2b和DNFGFR2b-HFc能夠部分阻斷FGFs和FGFR2b信號通路,豬肺發(fā)育缺陷,導(dǎo)致死胎現(xiàn)象。DNFGFR2c轉(zhuǎn)基因豬肺發(fā)育稍微滯后于野生型豬；DNFGFR2b轉(zhuǎn)基因豬肺泡偏大,肺泡隔薄,肺泡隔膠原纖維較少；DNFGFR2b-HFc轉(zhuǎn)基因豬肺遠(yuǎn)小于野生型豬,實質(zhì)區(qū)偏小,肺泡隔較厚,肺泡小,間質(zhì)異常發(fā)育,肺間質(zhì)和肺泡隔中膠原纖維遠(yuǎn)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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R-332;R655.3
[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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R-332;R655.3
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