Dual-fluorescence Imaging Of Blood Vessels And Lymphatic Vessels In Prox1-GFP/Flt1-DsRed Transgenic Mice

Angiogenesis plays crucial roles in various condition.We bred Prox1-GFP with Flt1-Ds Red mice,got the Prox1-GFP/Flt1-Ds Red mice,so that lymphatic endothelial cells and vascular endothelial cells express GFP and Ds Red respectively.By using fluorescence microscope,we could observe vascular and lymphatic vessels sprouting,development and regression intensively.We bred PGFD mice with Cdh5-cre/ERT2 and VEGFR-2lox.With deleting VEGR-2 in vascular endothelial cells conditionally.,there is decline of lymphangiogenesis.The formation of PGFD mice will provide new method for study of angiogenesis and lymphangiogenesis.Purpose:Formation of PGFD mice,verify vascular endothelial and lymphatic endothelial cells expressing specific fluorescence,by means of IHC.Imaging harvested organs and tissues of PGFD mice by confocal microscope and two-photon microscope,so we could investigate the structure of blood vessels and lymphatic vessels.To induce angiogenesis and lymphangiogenesis,we take advantage of corneal micro-pocket assay.By deletion of VEGFR-2 from PGFD Cdh5-cre/ERT2 VEGFR-2Lox mice,we could observe differences after deletion of VEGFR-2 and investigate the mechanism of VEGFR-2 in angiogenesis and lymphangiogenesis.Materials an methods:1.By breeding Prox1-GFP and Flt1-Ds Red mice,got PGFD(Prox1-GFP/ Flt1-Ds Red)mice.To verify Ds Red expression correspondence with vascular endothelial cell and GFP and GFP expression correspondence with lymphatic endothelial cell by IHC.2.Harvested postnatal day 2 mice's organs and tissues,observed the characteristic structure of blood vessels and lymphatic vessels in these organs and tissues,then rebuilt the three-dimensional images.3.Implanting the growth factors into corneal stroma,by corneal micro-pocket,inducing angiogenesis and lymphangiogenesis,imaging the full process in intensively.Bred PGFD mice with other conditional knockout mice,got PGFD Cdh5-cre/ERT2 VEGFR-2Lox mice.Result:1.PGFD(Prox1-GFP/Flt1-Ds Red)mice were successfully set up,and it was confirmed by IHC.2.Laser confocal microscopy and two-photon microscopy were used to image various organs of PGFD mice on day 2 after birth.The structural characteristics of blood vessels and lymphatic vessels of various organs or tissues were found.3.VEGF-A and VEGF-C were implanted into the corneal stroma of PGFD mice by corneal micro-pocket technique to induce angiogenesis and lymphangiogenesis.The formation of blood and lymphatic vessels was observed by Axio Zoom in vivo.PGFD mice were crossed with other transgenic mice to obtain PGFD Cdh5-cre/ERT2 VEGFR-2Lox mice,and the effects of deletion of VEGFR-2 in the blood vessels.It was found that intravascular deletion of VEGFR-2 resulted in a decrease in VEGF-A-induced angiogenesis and no clear growth in the lymphatic vessels.It also causes no growth of VEGF-C-induced lymphatic vessels.Conclusion and Innovation:1.Successfully established PGFD(Prox1-GFP/Flt1-Ds Re)fluorescent protein expression mice.2.By establishing PGFD mice,it is possible to better observe the structural characteristics of blood vessels and lymphatic vessels of various organs.3.With corneal micro-pocket assay,the corneal stroma was implanted in VEGF-A and VEGF-C to induce the growth of blood and lymphatic vessels.4.PGFD mice were crossed with other transgenic mice to obtain PGFD Cdh5-cre/ERT2 VEGFR-2Lox mice,and the effects of knocking out genes encoding VEGFR-2 in blood vessels on vascular and lymphangiogenesis were observed.