Effects Of AuNPs On Angiogenesis Of Chicken Embryo Based On A Shell-less Culture System

The formation of new blood vessels is crucial for the growth and persistence of primary tumors or cancers.The induction of angiogenesis is prior to the formation of malignant tumors.The enhancement of vascularization is related to the invasion of tumors.Therefore,the development of inhibitors which interfere with the growth of tumor blood vessels is expected to provide new strategies for controlling the tumor growth.AuNPs is considered to be an ideal drug carrier for the treatment of cancer based on their characteristics,such as good biocompatibility,easy synthesis,and lower toxicity.In addition,AuNPs had functions of regulating the angiogenesis.However,these studies were mainly conducted on cultured cells,and they could not simulate the angiogenesis occurred in vivo,and it could not be observed and tracked in real time by using the vascular models which were established by experimental animals.CAM?Chorioallantoic membrane?,a highly vascularized tissue,is required for the development of poultry embryos.It can truly simulate the development of blood vessels in vivo,and is an important model for the study of angiogenesis in vivo,however,the presence of eggshell limits the visualization and real-time tracking of blood vessels.Therefore,the conditions of shell-less culture for chicken embryos were optimized in this study to establish the shell-less culture system by screening the pre-incubation time,culture medium and ventilation time,etc.The effects of AuNPs on vascular growth and development were investigated using CAM vessel model of chicken embryo in shell-less culture.Part 1 Establishment of shell-less culture system for chicken embryoIn order to solve the problems such as difficulty in onset of development at the early stage and hypoxia of chicken embryos cultured in shell-less culture system,the effects of different pre-incubation times on the rate of egg transfer successfully and survival rate of chicken embryo,as well as the expressions of hypoxic inducing factor-1??HIF-1??mRNA and protein were investigated to optimize the conditions of shell-less culture of chicken embryo.The development of cardiovascular system of chicken embryo and the growth and development of chick after hatching were evaluated.The objective of this part was to establish an efficient and stable shell-less culture system.The results were as following:?1?When eggs were pre-incubated at37.8?for 48 h,the integrity of embryo and rate of egg transferred successfully were significantly higher than that of the treatment group with pre-incubation at 37.8?for72 h?P<0.01?,the survival rate of chicken embryos pre-incubated for 48 h was significantly higher than that of the other three treatment groups within 5 days after egg transferring?P<0.01?.?2?The mortality rate of chicken embryo perforated aeration on day 8 was significantly lower than that of other aeration groups?P<0.01?,the expression of HIF-1?mRNA and protein in the un-aerated chicken embryo at day8 were significantly higher than that at day 7,and the expression of HIF-1?mRNA and protein significantly decreased after ventilation?P<0.01?.?3?There was no significant difference in the morphology of heart and the expression of GATA 4 and FGF mRNA and protein between the shell-less culture system and the normal hatching?P>0.05?.?4?The hatch rate of chicken embryo cultured in shell-less culture on day 21 after being pre-incubation at 37.8?for 48 h and aeration on day 8was 47.67±1.05%.?5?There was no significant difference in morphology and weight of hatched chickens between shell-less culture system and normal incubation?P>0.05?,and the chicken obtained from shell-less culture system had normal reproductive functions.Thus,pre-incubation at 37.8?for 48 h and ventilation at day 8 were recommend to construct a shell-less culture system for chicken embryoPart 2 Effects of AuNPs on CAM angiogenesis of chicken embryo in a shell-less culture systemEffects of 10 ng/?L,25 ng/?L,and 50 ng/?L AuNPs on CAM vessels of chicken embryo at day 7,day 8,and day 9 were explored by evaluation of vascular development and the expression of VEGF and FGF.The results were as following:?1?Before day 9,the CAM vessels of chicken embryo in the shell-less culture system had obvious branches and moderate distribution density,which is easier to be observed.?2? When the CAM was treated with 50 ng/?L AuNPs for 4 days,then the survival rates of embryo at day 7,day 8,and day 9 were significantly lower than that of other groups?P<0.01?.?3?There was no obvious effect on CAM vessels of embryo at day 7,day 8,and day 9 when the CAM treated with 10 ng/?L AuNPs.After being treated with 25 ng/?L and 50 ng/?L AuNPs for 48 h,the distribution of CAM blood vessels became sparse,and the color of blood vessel became lighter.When CAM of chicken embryo at day 8 was treated with 50 ng/?L AuNPs for 72 h,the blood vessel of CAM were discolored and dried,and the chicken embryo died.?4?When the CAM of chicken embryos at day 8 and day 9 were treated with 25 ng/?L AuNPs for 48 h,the expression of VEGF mRNA was significantly lower than that of control group?P<0.05?.No significantly difference in the expression of VEGF mRNA of CAM of embryos at the same time period was observed between 50 ng/?L AuNP treatment group and control group after treat for 24 h?P>0.05?.After the CAM of chicken embryo at day 8 was treated with 25 ng/?L AuNPs for 48 h,the expression of FGF mRNA was significantly lower than that of the control group?P<0.01?.?5?When the CAM of chicken embryo at day 8 was treated with 10 ng/?L,25 ng/?L,and 50 ng/?L AuNPs for 24 h,there was no significant difference in the expression of VEGFR2 and FGFR?P>0.05?,the expression of VEGFR2 protein decreased when the CAM was treated with 25 ng/?L and 50 ng/?L AuNPs for 48 h.The expression of FGFR protein decreased when the CAM were treated with 50 ng/?L AuNPs for 48 h.After being treated with 25 ng/?L AuNPs for 72 h,the expressions of VEGFR2 proteins in CAM were significantly down-regulated?P<0.01?,and the expressions of FGFR proteins were down-regulated?P<0.05?,and 50 ng/?L AuNPs could significantly down-regulate the expression of these two kinds of proteins?P<0.01?.50 ng/?L AuNPs had a greater toxicity to the development of chicken embryo,resulting in a high mortality rate.Compared with day 7 and day 9,the CAM of chicken embryo at day 8 had a stronger response to the treatment of AuNPs,and after being treated with AuNPs for more than 48 h,the generation of CAM vessels were inhibited.It is appropriate to treat the CAM of chicken embryo at day 8 with 25 ng/?L AuNPs for 48h to inhibit the growth and development of CAM.Conclusions:The chicken embryo shell-less culture system was established under the condition of pre-incubation for 48 h and ventilation on the 8^th day.Ventilation on the 8^th day could effectively reduce the expression of HIF-1?and relieve the hypoxia of chicken embryo.The hatching rate of the chicken embryo shell-less culture system was about 47.67%±1.05%,and the speed of growth and reproductive performance of the shell-less cultured chickens were not different from that of the normal hatching,moreover,this system supported the normal development of the cardiovascular system of chicken embryo.The CAM of chicken embryo culture without shell was used as the vascular model.25 ng/?L AuNPs could safely and effectively inhibit the formation of CAM blood vessels,down-regulate the expression of VEGF and FGF mRNA,reduce the expression of VEGFR2 and FGFR protein,and ensure the normal development of chicken embryo,which laid a foundation for the understanding of angiogenesis mechanisms and the development of new anti-vascular treatment strategies for cancer.