The Study Of The Interaction Of Ankrd17Protein Of Neural Stem Cells With MCMV Neurotropic Protein M122on The Mechanism Of Abnormal Development Of The Fetal Brain

Objectives:1. To construct a lentiviral vector with shRNA targeting murine Ankrd17gene for transfection.2. To study the effect of MCMV infection on the proliferation, differentiation and apoptosis of neural stem cells (NSCs) after the Ankrd17gene silencing of NSCs.3. To explore the influence of MCMV infection on the expression of the differentiation related genes in the Wnt signaling pathway of NSCs after the Ankrd17gene silencing ofNSCs.Methods:1. Construct the recombinant plasmid of interference sequence:Ankrdl7gene specific DNA oligo nucleotides were annealed to form a double-stranded DNA oligo, they were connected to the pBSilencel.2linearized expression vector which digested by the BSAI. Then transferred the ligated product to prepared bacterial competent cells, the plasmids were extracted from the positive colonies, digested by SacI and confirmed by DNA sequencing, then we obtain the successfully transfected strain which named pBSi/U6-Ankrd17.2. Construct the recombinant lentiviral vector plasmid:The correct plasmid pBSi/U6-Ankrdl7was transfected to the murine-derived retinal vascular endothelial cells by Lipofectamine2000, then screening the effective target by Real-Time PCR. Then designed and synthesized the shRNA-Ankrd17fragment according to the effective target. After the lentivirus vector plasmid pLVX-shRNA2-m was digested by PstI and BamHI enzymes, connected the annealing products shRNA-Ankrdl7to it, then the lentivirus vector plasmid pLVX-shRNA-Ankrdl7was constructed, and verified by DNA sequencing.3. Preparate the lentiviral particles:In the help of the lentiviral packaging system-Lentiviral Packing Mixture, we transfected the pLVX-shRNA-Ankrdl7lentivirus vector into293T cells, then the lentivirus supermatant were harvested and concentrated, and we obtained the final lentiviral particles(Lenti-Ankrdl7shRNA). The viral titer was determined using the hole-by-hole dilution method.4. Detect RNAi efficiency:The neural stem cells were transfected by lentiviral particles expressing Ankrd17shRNA (Lenti-Ankrdl7shRNA) or negative control shRNA (Lenti-NCshRNA), and the optimum conditions of infection was determined through the observation of fluorescence microscopy. And the Ankrd17gene RNAi efficiency was determined through measuring Ankrd17mRNA level by Real-Time PCR.5. The experimental groups:①enti-Ankrd17shRNA and MCMV infection group;②MCMV infection control group;③Lenti-Ankrd17shRNA control group;④Normal control group; each group has three repeats.6. CCK-8method was used to detect MCMV infection on the proliferation of NSCs after the Ankrd17gene silencing of NSCs.7. Flow cytometry was employed to test the changes of MCMV infection on NSCs and its differentiated cells after the Ankrd17gene silencing of NSCs.8. PI staining was used to measure MCMV infection on apoptosis of NSCs after the Ankrd17gene silencing of NSCs.9. Real-Time PCR method was used to determine the influence of MCMV infection on the expression level of related genes’in Wnt signaling pathway after the Ankrd17gene silencing of NSCs.Results:1. The construction of recombinant plasmid of interference sequence:DNA sequencing showed that the target gene Ankrdl7was successfully inserted into the plasmid vector pBSilencel.2, and the pBSi/U6-Ankrdl7cloning vector was successfully constructed.2. The construction the recombinant lentiviral vector plasmid:PCR results showed that all the three plasmid vectors can be amplified the Ankrd17products, and the most effective interference target was successfully screened out by Real-Time PCR. The restriction enzyme digestion and sequencing results showed that we have successfully constructed the recombinant lentiviral plasmid vector-pLVX-shRNA-Ankrd17.3. The preparation of lentiviral particles:The titer of recombinant lentiviral particles of Lenti-Ankrd17shRNA was1.0x108TU/ml.4. Detection of RNAi efficiency:Five days after NSCs were transfected with lentiviral particles, we found the best transfection conditions were multiplicity of infection (MOI) was5, and added5μg/ml polybrene. The Real-Time PCR results suggested that when compared with the NC-shRNA group and the normal cell group, pLVX-shRNA-Ankrdl7-1can be significantly reduced the expression levels of Ankrd17gene in NSCs, and the silencing efficiency was reached67.3%.5. Cell proliferation:The cell proliferative activity in Ankrdl7-shRNA group infected by MCMV was significantly higher than infected control group (p<0.05).6. Cell differentiation:The rate of nestin positive cells in Ankrdl7-shRNA group was significantly higher than the normal control group and the rates of GFAP and NSE positive cells were significantly lower than the control group at the third days during differentiation culture (p<0.05). But the rates of GFAP and NSE positive cells in Ankrdl7-shRNA group infected by MCMV were significantly higher than the infected control group at the third days during differentiation culture (p<0.05).7. Cell apoptosis:The apoptosis rate in the MCMV infected group was significantly higher than the normal control group at5d post infection (p<0.05), and the apoptosis rate in Ankrdl7-shRNA group was significantly higher than the normal control group from3d to5d (p<0.05), while the apoptosis rates in Ankrdl7-shRNA group infected by MCMV were always significantly higher than the infected control group (p<0.05).8. The expression of Wnt genes:In the Ankrdl7-shRNA group, the expression level of Wntl gene was significantly lower than normal control group from1d to3d, then obviously increased and exceeded it at5d (p<0.05); while the expression levels of Wnt3and Wnt7a were significantly higher than normal control group at5d during differentiation culture (p<0.05). But in the MCMV infected Ankrdl7-shRNA group, the level of Wntl was increased gradually, and significantly higher than the infected control group from3d to5d post infection (p<0.05); and the levels of Wnt3and Wnt7a were always significantly higher than the infected control group (p<0.05).9. The expression of Ngn genes:In the Ankrdl7-shRNA group, the expression level of Ngnl was always lower than normal control group; while the level of Ngn2was slightly lower than the normal control group from1d to3d, then rose and obviously higher than the normal control group at5d (p<0.05). But in the MCMV infected Ankrdl7-shRNA group, the level of Ngnl was always significantly higher than the infected control group (p<0.05); and the expression level of Ngn2was also higher than the infected control group;10. The expression of c-myc and cyclinD1genes:In the Ankrd17-shRNA group, the expression level c-myc was always lower than normal control group; while the level cyclinDl was slightly lower than the normal control group from1d to3d, then rose and obviously higher than the normal control group at5d (p<0.05). In the MCMV infected Ankrdl7-shRNA group, the level of c-myc was significantly higher than the infected control group from3d to5d post infection (p<0.05); while the level of cyclinDl was significantly lower than the infected control group from3d to5d post infection (p<0.05).Conclusion:1. Successfully constructed the lenti viral vector with shRNA targeting mouse Ankrd17gene (pLVX-shRNA-Ankrdl7); and the Ankrdl7-shRNA can effectively silence the NSCs Ankrdl7gene.2. When the Ankrdl7gene of NSCs was silenced by Ankrdl7-shRNA, the inhibitory degree of MCMV against the proliferation and early differentiation of NSCs was significantly weakened, but led the apoptosis ratio of NSCs increased.3. The silencing of Ankrd17gene of NSCs could partly decrease the abnormal expression of the differentiation related genes of Wnt signal pathway:wntl, wnt3, wnt7a, ngn1, ngn2and c-myc, and down-regulate the expression of cyclinDl, which were induced by MCMV.4. The neurotropic protein M122of MCMV could be interact with the Ankrd17protein of neural stem cells to interfere the expression of the differentiation related genes of Wnt signal pathway, and then inhibit the proliferation and differentiation of NSCs, which may be the mechnism of abnormal development of the fetal brain which caused by congetial CMV infection. ObjectiveTo discuss the expression of the immediate-early, early and late genes of human cytomegalovirus(HCMV) when infected human embryonic lung fibroblasts cell in vitro, and the effect of alltridin on HCMV related genes, to explore the mechanism of allitridin against HCMV.MethodsWe choose the main immediate early genes ul123and ul122(who coding IE72and IE86proteins respectively), the iconic early genes u154(coding UL54protein) and the iconic late genes u183(coding pp65protein) of HCMV as the target sequence to design the specific primers, to establish a real-time fluorescent quantitative PCR system to detect the dynamic change of the ul122, ul123, u154and u183mRNA of HCMV, and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as a housekeeper.The experimental groups:①HCMV AD169strain infected HELF cells (MOI=2.5) treated with allitridin;②HCMV ADI69strain infected HELF cells (MOI=2.5);③HCMV AD169strain infected HELF cells (MOI=2.5) treated with ganciclovir. All cultures were harvested at0.5h,2h,4h,6h,12h and24h post-infection separately, for SYBR Green real-time PCR analysis.Results1. The effect of allitridin on the ul122gene of HCMV:The expression level of HCMV ul122gene has been significantly increased at2h post-infection, and then gradually increased. The expression of the ul122gene both in allitridin-treated and GCV-treated groups were lower than virus infected groups during2h-24h post-infection, but there were no significant difference in virus infected groups and GCV-treated groups at0.5h-6h post-infection(p>0.05). The inhibitory rates of allitridin to ul122mRNA were21.3%,36.1%,40.8%,47.3%,60.2%and75.2%at0.5h-24h post-infection respectively, and the inhibiting effect were highest at24h post-infection.2. The effect of allitridin on the ul123gene of HCMV:The expression pattern of HCMV ul123gene was similar to the ul122gene; it was increased dramatically at2h post-infection, then continued to increase. The expression of ul123mRNA in allitridin-treated groups were markedly lower than virus infected groups (p<0.01), and the inhibitory rate of allitridin to ul123mRNA reached to70.4%at24h post-infection, but there were no significant difference between GCV-treated groups and virus infected groups(p>0.05).3. The effect of allitridin on the u154gene of HCMV:The expression level of u154mRNA was significantly increased from4h post-infection (p<0.05), then kept increasing. The expression of u154mRNA in the two drugs-treated groups were lower than the virus infected groups at all time points (p<0.05). The inhibitory rates of allitridin and GCV to u154mRNA were45.4%and27.2%at24h post-infection.4. The effect of allitridin on the u183gene of HCMV:The expression of u183mRNA started increased significantly at6h post-infection(p<0.05), and kept increasing at12h--24h post-infection. The expression of u183RNA in the two drugs-treated groups were obviously lower than virus infected groups during12h-24h post-infection (p<0.05), and the inhibitory rates of allitridin and GCV to u183mRNA were45.9%and26.2%at24h post-infection.Conclusion1. Allitridin can effectively suppress the transcription of immediate early genes (ul122and ul123) of HCMV, lead to the expression of mRNA significantly lowered, and the inhibitory rates were up to75.2%and70.4%at24h post-infection respectively. Allitridin was able to suppress the expression of early gene (u154) and late gene (u183) too; the inhibitory rates were45.4%and45.9%at24h post-infection respectively. This indicated that the IE genes of HCMV may be the key target of allitridin against HCMV.2. There was no obvious inhibition of GCV against the IE genes of HCMV AD169strain, and the inhibitory rates of GCV to the u154and u183genes were27.2%and26.2%respectively. This indicated that the main mechanism of GCV against HCMV lies in the inhibiting the DNA polymerase of the virus.3. The different inhibitory effects of allitridin and GCV against the HCMV AD169were related to the different acting site.