| Objective:Ischemic disease, such as ischemic stroke, ischemic cardiomyopathy, avascularnecrosis of the femoral head, ischemic bowel disease etc. seriously endangers people’shealth. Incidence of this disease is increasing year by year and patients tend to be youngerdue to continuous development of society and economy, changes of people’s diet pattern,high-fat diet, increased pressure of life and work, aging of society, making ischemicdisease becoming a social problem perplexing public. Ischemic disease decreases qualityof life and brings a heavy burden to family, society and their county because of chroniccourse, hogenesis of the disease and high rate in death and disability.Recently, patients’ symptom could be improved by thrombolytic drugs likeanti-freezing, interventional procedures and so on. However, satisfactory effect is hard to achieved when the all the vessels or vessels with a diameter below2mm are involved.Moreover, operation treatment brings risks such as reperfusion injury, complications,expensive cost, restenosis. Therefore, it is crucial to find an effective, simple, and feasibletreatment.Hockel et al first reported application of remedial vessel-newborn treatment, whichperformed treatment via activation of reproduction of local vessels and rebuilt of injurytissues. This treatment is becoming more and more important in fields of ischemic diseaseand injury recovery. In our previous research, we found that HGF promoted healing ofsurface in burning wound and restoration of wounds; played a protecting role in vesselsand muscles of ischemic models; meanwhile, HGF upregulated mRNA levels of Notch1and Dll4. In this study, we focus on the regulation of Ad-HGF to hUCMSCs onreproduction, mitotic cycle, tube structure and migration via Notch1/Dll4signalingpathway. Meanwhile, alterations in mRNA or protein levels of Notch1/Dll4signalingpathway and vessel markers were detected. These data demonstrate the associationbetween HGF and hUCMSCs signaling pathway and reveal that Ad-HGF may promotedifferentiation to vessels therefore provide a new understanding to further research andclinical treatment.Part1. Separation, culture, identification and differentiation of human umbilicalcord mesenchymal stem cellsMethods: Get primary hUCMSCs from mature newborn umbilical cord with tissueadherent assay. Cell phenotype and mitotic cycle were detected with flow cytometry.Potent of differentiation was identified by evaluation of differentiation to neuro or adiposeResults: Primary hUCMSCs were successfully separated and cultured. Using flowcytometry, we found that cells expressed mesenchymal cellular markers CD44（96.73%）,CD105（96.10%） and integrin receptor CD29（99.53%）; low expessions in hematopoieticmarkers CD34（0.8%） and CD45（1.91%） or human leukocyte antigen HLA-DR （1.41%）were according to specific cell phenotype in hUCMSCs. Most hUCMSCs were in G0/G1.Neuron cells were identified, as the masculine finding in Nestin Immunohistochemistryassay. Meanwhile, adipose were identified, as vacuolar lipid droplets were detected and the masculine finding in oil red O stain.Conclusion: Multi-differentiation potential hUCMSCs were successfully separatedand cultured therefore could be used for further researches.Part2. Development and identification of human Notch1receptor adenovirusMethods: Designed and synthesized shRNA sequence according to mRNA sequenceof Notch1（NM017617） from Gene Bank and attached this sequence to pGenesil-1.1plasmid for enzyme digestion and order-checking. Transfect Notch1-shRNA expressioncassettes to adenovirus expression vector pGsadeno via homologous recombination invitro. Obtained recombine adenovirus by liposome transfection of HEK293after enzymedigestion was linearized. Transfect hUCMSCs by adenovirus and detected cell morphaand expression of GFP. Extracted total RNA and protein, then detected Notch1on mRNAlevel and protein level respectively by RT-PCR and western blot.Results: Enzyme digestion and order-checking showed that adenovirus plasmid wassuccessfully developed. After transfection of hUCMSCs, expressions of Notch1wasdetected with RT-PCR and Western Blot, resulting in significant inhibitions on mRNAlevel（silencing efficiency68.04%, P＜0.05） and protein level（silencing efficiency80.32%,P＜0.05）.Conclusion: Recombine adenovirus rAd5-Notch1-shRNA was successfullydeveloped, with a high potent to transfect hUCMSCs and an effect of Notch1silencing.Part3. Ad-HGF promotes differentiation of hUCMSCs to vascular endothelial cell invitroMethods: This study was performed on hUCMSCs,5groups were involved:adenovirus group （Ad-GFP group）, Ad-GFP-Notch1-shRNA group （Ad-shRNA group）,Ad-GFP-NICD group （Ad-NICD group）, Ad-HGF group and control group. Firstly, wedetected survival rates of each group by MTT test to decide the most appropriate infectionMOI. Then we observed cell morpha with microscope and detected expression of HGF insupernatant solution with ELISA. Detected OD490nmvalues with MTT and established cellgrowth curve. Detected status of mitotic cycle in each group with flow cytometry and thenevaluated potents to adherence, migration and differentiation to vessels in vitro. Detected genes Notch1, Dll4and Hey-2in Notch pathway and EphB4as vein marker and ephrin B2as artery marker. Meanwhile, detected protein levels of Notch1, EphB4and ephrin B2with western blot.Results: The best MOI for Ad-GFP was100and for Ad-NICD group,Ad-shRNAgroup and Ad-HGF group was50. Observations from microscope showed that all groupsexpressed increased GFP as culture time extending except Ad-HGF group. In Ad-GFP andAd-shRNA groups, cell morphas were not observed significant difference from controlgroup; in Ad-NICD group, cell reproduction was speeding therefore several incompletetube structures were detected; in Ad-HGF group, cell grew in distribution and cellreproduction was speeding at early stage and then a lot excretion vesicles were observed.Findings from ELISA showed that after48h culture, HGF in supernatant solutionincreased significantly （P＜0.05） in Ad-HGF group compared with control group andreached the peak value562.97±27.09pg/mL after96h culture. Cell production werespeeding in Ad-NICD and Ad-HGF groups and significant differences （P＜0.05） weredetected in48,72,96h; however, cell production was inhibited in Ad-shRNAgroup with asignificant difference（P＜0.05） in72h. Detection of mitotic cycle by flow cytometryshowed that proportion of cells in S stage increased （36.02%） in Ad-NICD group, thesecells were active in reproduction and possessed a high potent to reproduction. Celladherent assay showed that the rates of adherents were higher than control group inAd-HGF group at12,16,20h and in Ad-NICD group at20h （P＜0.05）; and the rate waslower than control group in Ad-shRNAgroup at8,12,16h （P＜0.05）. Transwell test foundthat cell migration were promoted in Ad-HGF group and Ad-NICD group whereas wasinhibited in Ad-shRNA group （P＜0.05）. Differentiation test showed that only inAd-NICD group and Ad-HGF group significant tube structures were found. By RT-PCR,mRNA levels of Notch1,Dll4,Hey-2in Ad-HGF group and Ad-NICD group were higherthan other groups （P＜0.05）, while in Ad-shRNA group was lower than other groups （P＜0.05）, no statistically significant difference was detected between Ad-GFP group andcontrol group, Ad-HGF group and Ad-NICD group （P>0.05）. No statistically significantdifference was detected in vein marker gene EphB4in Ad-GFP group,Ad-HGF group and Ad-shRNA group （P>0.05） but in Ad-NICD group it increased compared with controlgroup（P<0.05）. No statistically significant difference was detected in artery marker geneephrin B2among Ad-GFP group,Ad-shRNA group,control group and between Ad-HGFgroup and Ad-NICD group （P>0.05） whereas in Ad-HGF group and Ad-NICD group itincreased compared with other groups （P<0.05）. Western blot showed that Notch1andephrin B2expressed a high levels in Ad-HGF group and Ad-NICD group whereas a lowlevel in Ad-shRNAgroup with statistically significant difference （P<0.05）.Conclusion: HGF promotes differentiation of hUCMSCs to vessel via activation inNotch1/Dll4signaling pathway. |
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