Investigating The Anti-aging Effect And Mechanism Of FOXQ1 Overexpression On The Human Umbilical Cord Mesenchymal Stem Cells

Stem cells are kind of cells with abilities of self-renewal and potential multiple differentiation,and the study of stem cell translational medicine has been in Alzheimer's disease,Parkinson's disease,etc.Meanwhile,the research field has achieved fruitful results.The umbilical cord mesenchymal stem cells(hUC-MSCs)are convenient,low immunogenicity,and with differentiation ability and so on,and those advantages has become an important research object in translational research,but hUC-MSCs in vitro,with mechanical damage in operation inevitably,chemical injury.Then,the cell proliferation and differentiation ability also decreased,the most important is the self rewal of stem cells decreased.This has become a serious limitation to clinical application of key points.FOXQ1 is a significant transcriptional regulator in the Forkhead Box Family(FOX).In recent researches,FOXQ1 was found to be highly expressed in various tumor tissues,and the expression of FOXQ1 was positively correlated with the malignant degree of the patients.At the same time,studies in many tumor cell lines show that FOXQ1 can regulate cell proliferation,cell cycle,cell migration and so forth,and then regulate the occurrence and metastasis of tumor.In vitro experiments showed that the proliferation ability of tumor cells enhanced or weaked by overexpression or knockdown of FOXQ1.These results suggest that FOXQ1 is an important factor in tumor proliferation and metastasis,which it may be a gene target for future oncology research.In view of the special effect of FOXQ1,through FOXQ1 overexpression of hUC-MSCs,whether it has the function of delay the senescence of hUC-MSCs and promote the proliferation and cause canceration to become the key,meanwhile,this is first time to investgate FOXQ1 in the somatic cell and the mechanism of FOXQ1 in hUC-MSCs is still uncertain,which need to do further experiments.ObjectivesThrough lentivirus mediated,which make FOXQ1 overexpression in hUC-MSCs,in the in vitro and in vivo conditions,to explore whether overexpression of FOXQ1 can promote the proliferation and aging of hUC-MSCs,as well as the intrinsic mechanism of regulation of FOXQ1 in hUC-MSCs.Method 1.Construct FOXQ1 overexpression lentiviral vector and test its expression effect in hUC-MSCs.Firstly,Isolation of umbilical cord.Secondly,identification of hUC-MSCs surface antigen markers by flow cytometry.Afterthat amplification of CDS sequences of FOXQ1,to inserted sequences into the lentiviral vector EcoR I and Xba I restriction sites,packaging,extraction and purification of virus liquid,then infect and establish stable hUC-MSCs of FOXQ1 overexpression cell line through puromycin screening.Difference of FOXQ1 expression between over expression group and control group was detected by western blot and qPCR.2.Research on the effect of FOXQ1 overexpression on cell proliferation,cell senescene and so forth in vitro experiments.Firstly,to detect the expression of FOXQ1 in different passages hUC-MSCs.Then,the experimental cells were divided into P3-hUC-MSCs,P15-hUC-MSCs,P15-Vector-hUC-MSCs(virus vector group),P15-FOXQ1-hUC-MSCs(overexpression group).The cell proliferation was detected by CCK-8,and cell senescence level was detected by SA-?-gal.To detect senescence related genes(P16,P21,P53,SIRT1,PCNA)expression by western blot and qPCR,and to detect the changes of cell cycle by flow cytometry,and cell apoptosis was identified by fluorescence assay.Finally,to detect different levels of SASP(mainly detected IL-6,IL-8 expression level)before and after adding SIRT1 inhibitor EX527 by qPCR.3.In vivo experiment,using the effect that hUC-MSCs of FOXQ1 overexpression treat with APP+ mice,to reflect on changes of the survival and proliferation of hUC-MSCs after overexpression.Firstly,the experimental group were divided into 6 groups,namely WT group(C57BL/6 mice,non treatment negative control group),APP+ treatment(non positive treatment control group),P3(P3 stem cells treatment group),P15(P15 stem cells treatment group),P15-Vector(P15 lentivirus vector stem cells treatment group).P15-FOXQ1(P15 stem cells FOXQ1 ovexpression treatment group).Detection of APP gene expression in APP+ transgenic mice by PCR.There were 10 mice a group,with a total of 60 mice.After a month of stem cell injection treatment,their changes of learning and memory tested by morris water maze test.After the mice were killed,serum was taken to detect SOD.The expression of MAB1281 in hippocampus of mice was detected by immunohistochemistry.The expression of AD pathogenic proteins,such as,tau,p-tau(ser396)and p-tau(ser235),and senescence associated proteins p16,p21,p53,SIRT1,SIRT2,PCNA,which were detected by western blot in hippocampus area.Results1.Antigens of cells were detected by flow cytometry,and results accord with hUC-MSCs.Which are positive for CD29(99.8%),CD90(99.5%),HLA-ABC(99.6%)and negative for CD34(99.2%),CD45(99.9%),HLA-DR(99.6%).FOXQ1 overexpression recombinant plasmid was constructed successfully,and its infection was effective in protein and mRNA level(P<0.01).2.The results of western blot and SA-beta-gal showed that the expression of FOXQ1 decreased with the aging of stem cells.CCK-8 results showed that overexpression of FOXQ1 group's cell proliferation was significantly increased compared with the vector group(P<0.05),SA-?-gal staining showed that the expression of the group positive rate decreased significantly compared with the vector group(P<0.01).In the protein level,with the FOXQ1 overexpression,the expression of P16,P21,P53 were decreased,and the expression of SIRT1,PCNA were increased.Meanwhile,the mRNA level presents the same trend.After FOXQ1 overexpression,the proliferation ability of hUC-MSCs was enhanced,but it did not reach the ability of unlimited proliferation,and the cells still grew with the increase of passages.The number of apoptotic cells decreased obviously.The migration ability were significantly enhanced by FOXQ1 overexpression.In the SASP study,the expression of IL-6(P<0.01)and IL-8(P<0.05)in FOXQ1 over expression group were lower than that vector group.When the FOXQ1 overexpression group was added with SIRT1 inhibitor EX527,the SASP expression level was increased(P<0.05),and the expression level of SASP was close to that of vector group.3.The results of Morris water maze showed that the learning and memory ability of mice was significantly improved,and the escape latency,the times of crossing platform,and the residence time of platform quadrant were significantly improved(P<0.05)in the FOXQ1 over expression group.The results of MAB1281 IHC staining showed that the positive staining rate of FOXQ1 overexpression group was significantly increased(P<0.05).The level of SOD was significantly higher(P<0.05)in serum.In the hippocampus of mice,the results of western blot showed that the over expression group was significantly lower than that of the control group in the expression of P21,P53(P<0.05),and the difference of P16 was not significant between the two groups.The expression of SIRT1,SIRT2 and PCNA was significantly higher than that in control group(P<0.05).AD pathogenicity related protein,tau protein was not significantly different,but in the expression of phosphorylated tau protein p-tau(ser396),p-tau(ser235)(P<0.05),FOXQ1 overexpression group was significantly lower than that of the control group.Conclusion1.The antigens of separated cell accord with h UC-MSCs.Overexpression lentivirus system can effectively enhance expression of FOXQ1 on the hUC-MSCs in the protein and mRNA levels.2.After FOXQ1 overexpression,the abilities of proliferation,migration and antisenescene significantly promote in hUC-MSCs in vitro,and the cells did not become cancerous;what is more,FOXQ1 regulates the SASP through SIRT1,and then regulates cell senescence.3.FOXQ1 can promote hUC-MSCs migration,survival in brain of mouse in vivo.It was proved that the anti-aging ability of hUC-MSCs can enhanced by FOXQ1 overexpression indirectly.