Effects Of Pacenta-Specific 8 (PLAC8) On Early Embryonic Development And Embryo Implantation

Objective(1)PLAC8 in different species plays an important role in embryonic development,and previous studies have found that in the early human embryo PLAC8 protein expression in various periods,but specific role during human embryonic development remains unknown,so we slow virus PLAC8 interference carrier is built with clinical abandoned early embryo,PLAC8 affecting people in embryonic development.Furthermore,the mechanism of PLAC8 on human embryonic stem cells was discussed at the cellular level.(2)The previous study found that PLAC8 was also expressed in the process of human blastocyst implantation in vitro,which was related to the implantation status of embryos.The expression of PLAC8 was high under the condition of good implantation status.This study discussed the specific influence of PLAC8 on the embryo implantation process.MethodsPart 1 we created sh RNA lentiviral vectors that could be transcribed to interfere with PLAC8 m RNA expression to down-regulate PLAC8 expression.The optimal titer was determined by infecting mouse embryos.The lentivirus vector was used to infect clinically discarded early 3PN embryos,and the effect on the embryo cleavage rate was observed.Furthermore,the mechanism of PLAC8 on human embryonic stem cells(h ESCs)was discussed at the cellular level.h ESCs cells were infected by lentivirus,and the efficiency of PLAC8 interference group and empty vector group was detected by real-time fluorescent quantitative PCR(q PCR)and Western Blot(WB).q PCR was used to detect the relative expression of proliferation-related genes in human embryonic stem cells transfected with lentivirus.Western Blot was used to analyze the expression of marker proteins in cell proliferation.FCM(flow cytometry)was further used to detect the effect of PLAC8 interference on human embryonic stem cell h ESCs apoptosis.Part 2 In this experiment,we first constructed the lentivirus vector of PLAC8 RNAi.Mouse embryos were obtained by in vitro fertilization,and the mouse embryos developed to the blastocyst stage were collected.Set the lentivirus titer to 1×104TU/ml,1×10~5TU/ml,1×10~6TU/ml,1×10~7TU/ml,1×10~8TU/ml transfected blastocyst,to determine the optimal lentivirus titer for blastocyst infection;At last,the lentivirus-transfected blastocyst was transplanted into the pseudopregnant female mouse using mouse embryo uterus transplantation technology,and the embryo implantation in the uterus was observed after 2.5 days.ResultsPart 1 Successfully constructed the lentivirus interference system of PLAC8;Lentivirus can successfully infect mouse embryos and early human embryos.1×107pfu/ml was used to induce PLAC8 RNAi lentivirus vector to infect 3PN embryos on the 3rd day.The cleavage rate of embryos in the knockdown group(6.5)was significantly lower than that in the negative control group(10.57±1.87).The rate of embryo stop development in the knockout group(86%)was significantly higher than that in the negative control group(53%).Lentivirus-interfering vector was successfully transfected into human embryonic stem cell h ESCs.Compared with the negative control group,the proliferation-related genes CCND1 and Ki67 in the knockdown group were down-regulated.Meanwhile,cyclin D1 and PCNA proteins were down-regulated in the knockdown group compared with the negative control group.The apoptosis rate of FCM in the treatment group was higher than that of the control group.Part 2 Blastocyst was successfully collected after in vitro fertilization.It was found that the titer of lentivirus was the most suitable to infect blastocyst at 1×10~7pfu/ml.After successfully transplanting the infected blastocyst into the mouse uterus,it was found that the blastocyst implantation rate in PLAC8 group was lower than that in the negative control group after 2.5 days.Conclusion1. For the first time,lentivirus vector PLAC8 RNAi was constructed to infect early-developing human embryos.2. It has been proved that PLAC8 affects the development of early embryos,and knocking down the expression of PLAC8 gene in early embryos reduces the cleavage rate of embryos.3. Cell experiment further verified that the expression of PLAC8 gene knocked down human embryonic stem cell could reduce the expression of cell proliferation and promote cell apoptosis.4. PLAC8 gene expression in knockdown blastocysts led to a decrease in embryo implantation.