神经干细胞Ankrd17蛋白和MCMV嗜神经蛋白M122相互作用与胎脑发育异常机制
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摘要
【目的】
1.构建能表达小鼠Ankrd17基因的小发卡RNAs（shRNAs）慢病毒载体，为选择性沉默Ankrd17基因在神经干细胞（NSCs）中的表达奠定基础；
2.研究神经干细胞Ankrd17基因沉默后MCMV感染对其增殖、分化功能和凋亡的影响，为明确MCMV致神经系统损伤机制提供新的思路和依据；
3.研究神经干细胞Ankrd17基因沉默后MCMV感染对其Wnt信号通路相关分化基因表达的影响，以明确Ankrd17分子与MCMV嗜神经蛋白M122的相互作用与胎脑发育异常机制的关系。
【方法】
1.构建重组干扰序列载体质粒：将含Ankrd17基因siRNA序列的DNAoligo退火形成双链DNAoligo，再将该双链DNAoligo连接到BSAI酶切的pBSilence1.2线性化表达质粒载体上。将连接好的产物转入到制备好的细菌感受态细胞，通过对长出的克隆菌落进行提取质粒和SacI酶切和测序鉴定，即得到成功转染的菌株命名为pBSi/U6-Ankrd17；
2.构建重组慢病毒载体质粒：将正确克隆的质粒pBSi/U6-Ankrd17用Lipofectamine2000转染至小鼠源性的视网膜血管内皮细胞，经PCR鉴定筛选有效靶点。根据筛选的有效靶点基因序列设计合成shRNA-Ankrd17片段。通过将慢病毒载体质粒pLVX-shRNA2-m用PstI和BamHI进行双酶切反应后，将shRNA-Ankrd17的稀释退火产物与之连接，构建重组慢病毒载体质粒pLVX-shRNA-Ankrd17，通过DNA测序进行鉴定；
3.制备载体慢病毒：在慢病毒包装系统LentiviralPackingMixture的帮助下将pLVX-shRNA-Ankrd17载体共转染至293T细胞，收获并浓缩所得到的的慢病毒上清，即为最终的慢病毒颗粒（Lenti-Ankrd17shRNA）储存液，用逐孔稀释法测定病毒滴度；
4.检测RNAi效率：用Lenti-Ankrd17shRNA慢病毒液感染胎鼠神经干细胞，通过荧光显微镜观察荧光表达情况来判断最适感染条件，并通过Real-TimePCR检测Lenti-Ankrd17shRNA对NSCs中Ankrd17基因的干扰效率；
5.后续实验分组：①慢病毒干扰+MCMV感染组；②MCMV感染对照组；③慢病毒干扰对照组；④正常对照组；每组设3复孔；
6.用CCK-8法检测神经干细胞Ankrd17基因沉默后MCMV感染对其增殖的影响；
7.用流式细胞术检测神经干细胞Ankrd17基因沉默后MCMV感染对其分化的影响；
8.用PI染色法检测神经干细胞Ankrd17基因沉默后MCMV感染对其凋亡的影响；
9.用Real-TimePCR方法检测神经干细胞Ankrd17基因沉默后MCMV感染对其Wnt信号通路相关基因（wnt1,wnt3,wnt7a,ngn1,ngn2,c-myc和cyclinD1）表达的影响。
【结果】
1.重组干扰序列载体构建：测序结果表明，目的基因Ankrd17成功插入到载体质粒pBSilence1.2中，成功构建pBSi/U6-Ankrd17克隆载体，且与目的序列完全一致；
2.重组慢病毒载体质粒构建：PCR反应显示克隆正确的3个载体质粒均可扩增出Ankrd17基因产物，通过Real-TimePCR成功筛选出有效干扰靶点pBS1.2Ankrd17-1载体质粒；酶切及测序结果表明，成功构建重组慢病毒载体质粒pLVX-shRNA-Ankrd17，与目的序列完全一致；
3.载体慢病毒制备：成功制备出表达Ankrd17基因shRNA的慢病毒颗粒浓缩液，病毒滴度为1.0×108TU/mL；
4.选择性沉默效率：荧光显微镜下观察发现，NSCs在转染慢病毒颗粒5d后，在MOI=5并添加5μg/ml的polybrene的转染条件下转染效率最好，Real-TimePCR结果显示，与空病毒组和正常细胞对照组相比，pLVX-shRNA-Ankrd17-1可以明显降低NSCs中Ankrd17表达水平，其沉默效率达到67.3%；
5.细胞增殖：与MCMV感染对照组相比，慢病毒干扰+MCMV感染组的细胞增殖活性明显升高（p<0.05）；
6.细胞分化：分化培养3d后，慢病毒干扰组nestin的表达水平明显高于正常对照组（p<0.05），而GFAP和NSE的阳性比率亦明显低于正常对照组（p<0.05）。而慢病毒干扰+MCMV感染组在分化培养第3d时，其GFAP和NSE的阳性细胞率明显高于感染对照组（p<0.05）；
7.细胞凋亡：MCMV感染组NSCs的凋亡率在感染后第5d明显高于正常对照组（p<0.05），慢病毒干扰组中在第3d起凋亡率明显高于正常对照组（p<0.05），而慢病毒干扰+MCMV感染组的细胞凋亡率始终明显高于感染对照组（p<0.05）。
8.Wnt基因表达：在慢病毒干扰组，Wnt1的表达量在第1d和第3d时明显低于正常对照组，而第5d时又显著升高（p<0.05），Wnt3和Wnt7a在分化培养第5d时高于正常对照组（p<0.05）；慢病毒干扰+MCMV感染组的Wnt1表达则呈逐渐上升趋势，在感染后第3d和第5d明显高于感染对照组（p<0.05），而Wnt3和Wnt7a的表达从感染后第1d到第5d始终高于感染对照组（p<0.05）。
9.Ngn基因表达：慢病毒干扰组的Ngn1表达始终低于正常对照组，Ngn2基因在第1d和第3d时表达水平稍低于正常对照组，第5d时则明显高于正常对照组（p<0.05）；而慢病毒干扰+MCMV感染组的Ngn1表达却明显高于感染对照组（p<0.05），其Ngn2表达亦始终高于感染对照组。
10.c-myc和cyclinD1基因表达：慢病毒干扰组c-myc基因的表达水平始终低于正常对照组，cyclinD1在第1d和第3d时表达量稍低于正常对照组，第5d时开始上升，明显高于正常对照组（p<0.05）；而在慢病毒干扰+MCMV感染组的c-myc表达在感染后第3d和第5d显著高于感染对照组（p<0.05），其cyclinD1基因除在第1d时稍高于感染对照组外，在感染后第3d和第5d均低于感染对照组。
【结论】
1.成功构建了小鼠Ankrd17基因shRNA慢病毒载体pLVX-shRNA-Ankrd17；该慢病毒载体可以有效沉默胎鼠NSCs的Ankrd17基因；
2.选择性沉默神经干细胞Ankrd17基因后，MCMV感染对NSCs的增殖活性和早期NSCs分化的抑制程度明显减弱，并导致NSCs的凋亡比率增高；
3.选择性沉默神经干细胞Ankrd17基因可部分减轻MCMV诱导的NSCsWnt信号通路分化相关基因wnt1，wnt3，wnt7a，ngn1和ngn2以及c-myc的表达异常，并下调cyclinD1表达水平；
4.MCMV嗜神经蛋白M122蛋白可通过与神经干细胞Ankrd17分子相互作用，干扰NSCs的Wnt信号通路分化相关基因表达进而抑制NSCs的增殖和分化，这可能是先天性CMV感染所致胎脑发育异常的重要机制之一。
【目的】
研究大蒜新素对人巨细胞病毒（HCMV）感染人胚肺成纤维（HELF）细胞后即刻早期基因、早期基因和晚期基因表达的影响，探讨和明确大蒜新素抗HCMV效应的机制。
【方法】
选择HCMV在增殖周期中主要表达即刻早期基因ul123和ul122（分别编码IE72和IE86蛋白），早期基因ul54（编码UL54蛋白）和晚期基因ul83（编码pp65蛋白）作为靶序列设计特异性引物，同时用GAPDH基因作为管家基因，建立实时荧光定量PCR系统来检测HCMVul122，ul123，ul54和ul83mRNA水平的时序性动态变化。实验分组：①大蒜新素处理HCMVAD169毒株（MOI=2.5）感染HELF细胞组（大蒜新素处理组），大蒜新素剂量为9.6μg/ml（2.28倍IC50浓度）；②HCMVAD169毒株（MOI=2.5）感染HELF细胞对照组（HCMV感染对照组）；③更昔洛韦（GCV）处理HCMVAD169毒株（MOI=2.5）感染HELF细胞对照组（GCV处理组），GCV剂量为2.3μg/ml（2.28倍IC50浓度）；正常HELF细胞为阴性对照。用实时荧光定量PCR方法检测各组细胞感染后0.5h、2h、4h、6h、12h和24h时间点HCMV的ul122、ul123、ul54和ul83基因在转录水平的动态变化。
【结果】
1.大蒜新素对HCMVul122基因表达的影响：HCMVAD169株ul122mRNA在感染后2h表达量已明显增高，随后逐渐增长。两种药物处理组ul122mRNA的表达量在感染后2h～24h均低于同时间点病毒感染细胞组，但GCV处理组在感染后0.5h～24h与病毒对照组水平相当（p>0.05）。大蒜新素对AD169ul122mRNA表达的抑制率在感染后0.5h～24h各时间点分别是21.3%，36.1%，40.8%，47.3%，60.2%和75.2%，抑制效应在感染后24h最强。
2.大蒜新素对HCMVul123基因表达的影响：HCMVul123mRNA的表达规律同ul122，在感染后2h表达量明显升高，之后持续增高。大蒜新素处理组ul123的表达量始终明显低于感染对照组（p<0.01），且大蒜新素对ul123mRNA表达的抑制程度随时间延长而增强，在感染后24h抑制率达到70.4%，而GCV组则与病毒感染组比较无明显差异（p>0.05）。
3.大蒜新素对HCMVul54基因表达的影响：ul54mRNA在感染后4h开始表达量明显增多（p<0.05），之后持续增加。两种药物处理组ul54mRNA表达量始终低于病毒对照组（p<0.05）。大蒜新素和GCV对ul54mRNA表达的抑制率在感染后24h分别为45.4%和27.2%。
4.大蒜新素对HCMVul83基因表达的影响：ul83mRNA在HCMV感染后6h开始表达明显增多（p<0.05），感染后12h～24h表达量持续上升。在感染后12h～24h两种药物处理组ul83mRNA表达量明显低于病毒对照组（p<0.05），大蒜新素和GCV对ul83mRNA表达的抑制率在感染后24h分别为45.9%和26.2%。
【结论】
1.大蒜新素可显著抑制HCMVAD169毒株IE基因（ul122和ul123）的转录过程，导致其mRNA表达明显降低，抑制率在感染后24h可达到75.2%和70.4%；对E基因（ul54）和L基因（ul83）转录水平亦有所抑制，抑制率在感染后24h分别为45.4%和45.9%，表明HCMV的IE基因可能是大蒜新素抗HCMV作用的主要环节。
2.GCV对HCMVAD169毒株IE基因没有明显的抑制作用，对ul54和ul83在感染后24h抑制率分别为27.2%和26.2%，表明GCV抗HCMV的作用机制主要在于抑制病毒DNA聚合酶。
3.大蒜新素和GCV对HCMVAD169相关基因的抑制效应不同与其作用位点不同有关。
Objectives:
1. To construct a lentiviral vector with shRNA targeting murine Ankrdl7gene for transfection.
2. To study the effect of MCMV infection on the proliferation, differentiation and apoptosis of neural stem cells (NSCs) after the Ankrdl7gene 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 Ankrdl7gene silencing of NSCs.
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 connection product to the prepared bacterial competent cells, the plasmids were extracted from the positive colonies, digested by Sacl and confirmed by DNA sequencing, then we obtain the successfully transfected strain which named pBSi/U6-Ankrdl7.
2. Construct the recombinant lentiviral vector plasmid:The correct plasmid pBSi/U6-Ankrdl7was transfected into the murine-derived retinal vascular endothelial cells by Lipofectamine2000, and then screened the effective target by Real-Time PCR. Then designed and synthesized the shRNA-Ankrdl7fragment 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-Ankrd17shRNA) or negative control shRNA (Lenti-NCshRNA), and the optimum conditions of infection was determined through the observation of fluorescence microscopy. And the Ankrdl7gene RNAi efficiency was determined through measuring Ankrdl7mRNA level by Real-Time PCR.
5. The experimental groups:①Lenti-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 Ankrdl7gene silencing of NSCs.
7. Flow cytometry was employed to test the changes of MCMV infection on NSCs and its differentiated cells after the Ankrdl7gene silencing of NSCs.
8. PI staining was used to measure MCMV infection on apoptosis of NSCs after the Ankrdl7gene 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 Ankrdl7gene silencing of NSCs.
Results:
1. The construction of recombinant plasmid of interference sequence:DNA sequencing showed that the target gene Ankrd17was 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 Ankrdl7products, 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-Ankrd17shRN A was1.0×108TU/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 Ankrdl7gene 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 Ankrd17-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 Ankrd17-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 Ankrd17-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 cyclinD1was 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 Ankrd17-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 cyclinD1was significantly lower than the infected control group from3d to5d post infection (p<0.05).
Conclusion:
1. Successfully constructed the lentiviral vector with shRNA targeting mouse Ankrd17gene (pLVX-shRNA-Ankrd17); and the Ankrdl7-shRNA can effectively silence the NSCs Ankrd17gene.
2. When the Ankrd17gene 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:wnt1, wnt3, wnt7a, ngnl, ngn2and c-myc, and down-regulate the expression of cyclinD1, 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 congenital CMV infection.
Objective
To 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.
Methods
We choose the main immediate early genes ul123and ul122(who coding IE72and IE86proteins respectively), the iconic early genes ul54(coding UL54protein) and the iconic late genes ul83(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, ul54and ul83mRNA 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 AD169strain 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.
Results
1. 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 ul54gene of HCMV:The expression level of ul54mRNA was significantly increased from4h post-infection (p<0.05), then kept increasing. The expression of ul54mRNA 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 ul54mRNA were45.4%and27.2%at24h post-infection.
4. The effect of allitridin on the ul83gene of HCMV: The expression of ul83mRNA started increased significantly at6h post-infection(p<0.05), and kept increasing at12h24h post-infection. The expression of ul83RNA 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 ul83mRNA were45.9%and26.2%at24h post-infection.
Conclusion
1. 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 (ul54) and late gene (ul83) 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 ul54and ul83genes 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.
1.构建能表达小鼠Ankrd17基因的小发卡RNAs（shRNAs）慢病毒载体，为选择性沉默Ankrd17基因在神经干细胞（NSCs）中的表达奠定基础；
2.研究神经干细胞Ankrd17基因沉默后MCMV感染对其增殖、分化功能和凋亡的影响，为明确MCMV致神经系统损伤机制提供新的思路和依据；
3.研究神经干细胞Ankrd17基因沉默后MCMV感染对其Wnt信号通路相关分化基因表达的影响，以明确Ankrd17分子与MCMV嗜神经蛋白M122的相互作用与胎脑发育异常机制的关系。
【方法】
1.构建重组干扰序列载体质粒：将含Ankrd17基因siRNA序列的DNAoligo退火形成双链DNAoligo，再将该双链DNAoligo连接到BSAI酶切的pBSilence1.2线性化表达质粒载体上。将连接好的产物转入到制备好的细菌感受态细胞，通过对长出的克隆菌落进行提取质粒和SacI酶切和测序鉴定，即得到成功转染的菌株命名为pBSi/U6-Ankrd17；
2.构建重组慢病毒载体质粒：将正确克隆的质粒pBSi/U6-Ankrd17用Lipofectamine2000转染至小鼠源性的视网膜血管内皮细胞，经PCR鉴定筛选有效靶点。根据筛选的有效靶点基因序列设计合成shRNA-Ankrd17片段。通过将慢病毒载体质粒pLVX-shRNA2-m用PstI和BamHI进行双酶切反应后，将shRNA-Ankrd17的稀释退火产物与之连接，构建重组慢病毒载体质粒pLVX-shRNA-Ankrd17，通过DNA测序进行鉴定；
3.制备载体慢病毒：在慢病毒包装系统LentiviralPackingMixture的帮助下将pLVX-shRNA-Ankrd17载体共转染至293T细胞，收获并浓缩所得到的的慢病毒上清，即为最终的慢病毒颗粒（Lenti-Ankrd17shRNA）储存液，用逐孔稀释法测定病毒滴度；
4.检测RNAi效率：用Lenti-Ankrd17shRNA慢病毒液感染胎鼠神经干细胞，通过荧光显微镜观察荧光表达情况来判断最适感染条件，并通过Real-TimePCR检测Lenti-Ankrd17shRNA对NSCs中Ankrd17基因的干扰效率；
5.后续实验分组：①慢病毒干扰+MCMV感染组；②MCMV感染对照组；③慢病毒干扰对照组；④正常对照组；每组设3复孔；
6.用CCK-8法检测神经干细胞Ankrd17基因沉默后MCMV感染对其增殖的影响；
7.用流式细胞术检测神经干细胞Ankrd17基因沉默后MCMV感染对其分化的影响；
8.用PI染色法检测神经干细胞Ankrd17基因沉默后MCMV感染对其凋亡的影响；
9.用Real-TimePCR方法检测神经干细胞Ankrd17基因沉默后MCMV感染对其Wnt信号通路相关基因（wnt1,wnt3,wnt7a,ngn1,ngn2,c-myc和cyclinD1）表达的影响。
【结果】
1.重组干扰序列载体构建：测序结果表明，目的基因Ankrd17成功插入到载体质粒pBSilence1.2中，成功构建pBSi/U6-Ankrd17克隆载体，且与目的序列完全一致；
2.重组慢病毒载体质粒构建：PCR反应显示克隆正确的3个载体质粒均可扩增出Ankrd17基因产物，通过Real-TimePCR成功筛选出有效干扰靶点pBS1.2Ankrd17-1载体质粒；酶切及测序结果表明，成功构建重组慢病毒载体质粒pLVX-shRNA-Ankrd17，与目的序列完全一致；
3.载体慢病毒制备：成功制备出表达Ankrd17基因shRNA的慢病毒颗粒浓缩液，病毒滴度为1.0×108TU/mL；
4.选择性沉默效率：荧光显微镜下观察发现，NSCs在转染慢病毒颗粒5d后，在MOI=5并添加5μg/ml的polybrene的转染条件下转染效率最好，Real-TimePCR结果显示，与空病毒组和正常细胞对照组相比，pLVX-shRNA-Ankrd17-1可以明显降低NSCs中Ankrd17表达水平，其沉默效率达到67.3%；
5.细胞增殖：与MCMV感染对照组相比，慢病毒干扰+MCMV感染组的细胞增殖活性明显升高（p<0.05）；
6.细胞分化：分化培养3d后，慢病毒干扰组nestin的表达水平明显高于正常对照组（p<0.05），而GFAP和NSE的阳性比率亦明显低于正常对照组（p<0.05）。而慢病毒干扰+MCMV感染组在分化培养第3d时，其GFAP和NSE的阳性细胞率明显高于感染对照组（p<0.05）；
7.细胞凋亡：MCMV感染组NSCs的凋亡率在感染后第5d明显高于正常对照组（p<0.05），慢病毒干扰组中在第3d起凋亡率明显高于正常对照组（p<0.05），而慢病毒干扰+MCMV感染组的细胞凋亡率始终明显高于感染对照组（p<0.05）。
8.Wnt基因表达：在慢病毒干扰组，Wnt1的表达量在第1d和第3d时明显低于正常对照组，而第5d时又显著升高（p<0.05），Wnt3和Wnt7a在分化培养第5d时高于正常对照组（p<0.05）；慢病毒干扰+MCMV感染组的Wnt1表达则呈逐渐上升趋势，在感染后第3d和第5d明显高于感染对照组（p<0.05），而Wnt3和Wnt7a的表达从感染后第1d到第5d始终高于感染对照组（p<0.05）。
9.Ngn基因表达：慢病毒干扰组的Ngn1表达始终低于正常对照组，Ngn2基因在第1d和第3d时表达水平稍低于正常对照组，第5d时则明显高于正常对照组（p<0.05）；而慢病毒干扰+MCMV感染组的Ngn1表达却明显高于感染对照组（p<0.05），其Ngn2表达亦始终高于感染对照组。
10.c-myc和cyclinD1基因表达：慢病毒干扰组c-myc基因的表达水平始终低于正常对照组，cyclinD1在第1d和第3d时表达量稍低于正常对照组，第5d时开始上升，明显高于正常对照组（p<0.05）；而在慢病毒干扰+MCMV感染组的c-myc表达在感染后第3d和第5d显著高于感染对照组（p<0.05），其cyclinD1基因除在第1d时稍高于感染对照组外，在感染后第3d和第5d均低于感染对照组。
【结论】
1.成功构建了小鼠Ankrd17基因shRNA慢病毒载体pLVX-shRNA-Ankrd17；该慢病毒载体可以有效沉默胎鼠NSCs的Ankrd17基因；
2.选择性沉默神经干细胞Ankrd17基因后，MCMV感染对NSCs的增殖活性和早期NSCs分化的抑制程度明显减弱，并导致NSCs的凋亡比率增高；
3.选择性沉默神经干细胞Ankrd17基因可部分减轻MCMV诱导的NSCsWnt信号通路分化相关基因wnt1，wnt3，wnt7a，ngn1和ngn2以及c-myc的表达异常，并下调cyclinD1表达水平；
4.MCMV嗜神经蛋白M122蛋白可通过与神经干细胞Ankrd17分子相互作用，干扰NSCs的Wnt信号通路分化相关基因表达进而抑制NSCs的增殖和分化，这可能是先天性CMV感染所致胎脑发育异常的重要机制之一。
【目的】
研究大蒜新素对人巨细胞病毒（HCMV）感染人胚肺成纤维（HELF）细胞后即刻早期基因、早期基因和晚期基因表达的影响，探讨和明确大蒜新素抗HCMV效应的机制。
【方法】
选择HCMV在增殖周期中主要表达即刻早期基因ul123和ul122（分别编码IE72和IE86蛋白），早期基因ul54（编码UL54蛋白）和晚期基因ul83（编码pp65蛋白）作为靶序列设计特异性引物，同时用GAPDH基因作为管家基因，建立实时荧光定量PCR系统来检测HCMVul122，ul123，ul54和ul83mRNA水平的时序性动态变化。实验分组：①大蒜新素处理HCMVAD169毒株（MOI=2.5）感染HELF细胞组（大蒜新素处理组），大蒜新素剂量为9.6μg/ml（2.28倍IC50浓度）；②HCMVAD169毒株（MOI=2.5）感染HELF细胞对照组（HCMV感染对照组）；③更昔洛韦（GCV）处理HCMVAD169毒株（MOI=2.5）感染HELF细胞对照组（GCV处理组），GCV剂量为2.3μg/ml（2.28倍IC50浓度）；正常HELF细胞为阴性对照。用实时荧光定量PCR方法检测各组细胞感染后0.5h、2h、4h、6h、12h和24h时间点HCMV的ul122、ul123、ul54和ul83基因在转录水平的动态变化。
【结果】
1.大蒜新素对HCMVul122基因表达的影响：HCMVAD169株ul122mRNA在感染后2h表达量已明显增高，随后逐渐增长。两种药物处理组ul122mRNA的表达量在感染后2h～24h均低于同时间点病毒感染细胞组，但GCV处理组在感染后0.5h～24h与病毒对照组水平相当（p>0.05）。大蒜新素对AD169ul122mRNA表达的抑制率在感染后0.5h～24h各时间点分别是21.3%，36.1%，40.8%，47.3%，60.2%和75.2%，抑制效应在感染后24h最强。
2.大蒜新素对HCMVul123基因表达的影响：HCMVul123mRNA的表达规律同ul122，在感染后2h表达量明显升高，之后持续增高。大蒜新素处理组ul123的表达量始终明显低于感染对照组（p<0.01），且大蒜新素对ul123mRNA表达的抑制程度随时间延长而增强，在感染后24h抑制率达到70.4%，而GCV组则与病毒感染组比较无明显差异（p>0.05）。
3.大蒜新素对HCMVul54基因表达的影响：ul54mRNA在感染后4h开始表达量明显增多（p<0.05），之后持续增加。两种药物处理组ul54mRNA表达量始终低于病毒对照组（p<0.05）。大蒜新素和GCV对ul54mRNA表达的抑制率在感染后24h分别为45.4%和27.2%。
4.大蒜新素对HCMVul83基因表达的影响：ul83mRNA在HCMV感染后6h开始表达明显增多（p<0.05），感染后12h～24h表达量持续上升。在感染后12h～24h两种药物处理组ul83mRNA表达量明显低于病毒对照组（p<0.05），大蒜新素和GCV对ul83mRNA表达的抑制率在感染后24h分别为45.9%和26.2%。
【结论】
1.大蒜新素可显著抑制HCMVAD169毒株IE基因（ul122和ul123）的转录过程，导致其mRNA表达明显降低，抑制率在感染后24h可达到75.2%和70.4%；对E基因（ul54）和L基因（ul83）转录水平亦有所抑制，抑制率在感染后24h分别为45.4%和45.9%，表明HCMV的IE基因可能是大蒜新素抗HCMV作用的主要环节。
2.GCV对HCMVAD169毒株IE基因没有明显的抑制作用，对ul54和ul83在感染后24h抑制率分别为27.2%和26.2%，表明GCV抗HCMV的作用机制主要在于抑制病毒DNA聚合酶。
3.大蒜新素和GCV对HCMVAD169相关基因的抑制效应不同与其作用位点不同有关。
Objectives:
1. To construct a lentiviral vector with shRNA targeting murine Ankrdl7gene for transfection.
2. To study the effect of MCMV infection on the proliferation, differentiation and apoptosis of neural stem cells (NSCs) after the Ankrdl7gene 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 Ankrdl7gene silencing of NSCs.
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 connection product to the prepared bacterial competent cells, the plasmids were extracted from the positive colonies, digested by Sacl and confirmed by DNA sequencing, then we obtain the successfully transfected strain which named pBSi/U6-Ankrdl7.
2. Construct the recombinant lentiviral vector plasmid:The correct plasmid pBSi/U6-Ankrdl7was transfected into the murine-derived retinal vascular endothelial cells by Lipofectamine2000, and then screened the effective target by Real-Time PCR. Then designed and synthesized the shRNA-Ankrdl7fragment 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-Ankrd17shRNA) or negative control shRNA (Lenti-NCshRNA), and the optimum conditions of infection was determined through the observation of fluorescence microscopy. And the Ankrdl7gene RNAi efficiency was determined through measuring Ankrdl7mRNA level by Real-Time PCR.
5. The experimental groups:①Lenti-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 Ankrdl7gene silencing of NSCs.
7. Flow cytometry was employed to test the changes of MCMV infection on NSCs and its differentiated cells after the Ankrdl7gene silencing of NSCs.
8. PI staining was used to measure MCMV infection on apoptosis of NSCs after the Ankrdl7gene 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 Ankrdl7gene silencing of NSCs.
Results:
1. The construction of recombinant plasmid of interference sequence:DNA sequencing showed that the target gene Ankrd17was 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 Ankrdl7products, 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-Ankrd17shRN A was1.0×108TU/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 Ankrdl7gene 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 Ankrd17-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 Ankrd17-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 Ankrd17-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 cyclinD1was 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 Ankrd17-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 cyclinD1was significantly lower than the infected control group from3d to5d post infection (p<0.05).
Conclusion:
1. Successfully constructed the lentiviral vector with shRNA targeting mouse Ankrd17gene (pLVX-shRNA-Ankrd17); and the Ankrdl7-shRNA can effectively silence the NSCs Ankrd17gene.
2. When the Ankrd17gene 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:wnt1, wnt3, wnt7a, ngnl, ngn2and c-myc, and down-regulate the expression of cyclinD1, 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 congenital CMV infection.
Objective
To 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.
Methods
We choose the main immediate early genes ul123and ul122(who coding IE72and IE86proteins respectively), the iconic early genes ul54(coding UL54protein) and the iconic late genes ul83(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, ul54and ul83mRNA 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 AD169strain 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.
Results
1. 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 ul54gene of HCMV:The expression level of ul54mRNA was significantly increased from4h post-infection (p<0.05), then kept increasing. The expression of ul54mRNA 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 ul54mRNA were45.4%and27.2%at24h post-infection.
4. The effect of allitridin on the ul83gene of HCMV: The expression of ul83mRNA started increased significantly at6h post-infection(p<0.05), and kept increasing at12h24h post-infection. The expression of ul83RNA 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 ul83mRNA were45.9%and26.2%at24h post-infection.
Conclusion
1. 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 (ul54) and late gene (ul83) 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 ul54and ul83genes 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.
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