| Part1:Isolation, cultivation and detection of Notch signaling molecules in bone marrow-derived mesenchymal stem cells of C57BL/c miceObjective:To isolate and cultivate of the bone marrow-derived mesenchymal stem cells (BMSCs) in C57BL/c mice in vitro. Moreover, the expressing molecules of Notch signaling pathway were examined in BMSCs.Methods:Bone marrow mononuclear cells of C57BL/c mice were isolated with density gradient centrifugation and adherence screening method. Cells were purified and expanded through passaging in time. Then test the surface antigens of the third passage cells with flow cytometry method to confirm if they were BMSCs. These cells were further identified by the potentials in differentiating into osteoblasts, adipocytes and endothelial. Reverse transcription (RT)-PCR and Western Blot were used to detect the molecules of Notch signaling in the stem cells.Results:(1) A small amount of short spindle-shaped, star-shaped cells were adherent within24hours after isolation. Cells could be amplified to the density of106within2weeks. The third passage cells appearance of fibroblast spindle-shaped and grew fast.(2) Flow cytometry showed the third passage cells strongly express mesenchymal stem cells surface antigen CD29and CD90, but hardly expression of CD31, CD34and CD45, the specific antigen of endothelial or haematopoietic were dectected on these stem cells.(3)The third passage cells could differentiate into osteoblasts, adipocytes, and endothelial cells.(4) RT-PCR and Western Blot detected the expression of the Notch signaling molecules in the cells, such as Notch-1, Notch-2, Notch-4,D11-1, D11-4, Jag-1, Hes-1and Hey-1.Conclusion:The density gradient centrifugation and adherent culture can obtain high purified BMSCs of mice. The passaged cells grow fast and proliferate well, and posse multipotent properties. Interestingly, the stem cells expressed Notch signaling molecules, such as Notch-1, Notch-2, Notch-4, Dll-1, D11-4, to Jag-1, of Hes-1and Hey-1. Part2:miR-1promotes the differentiation of BMSCs into cardiomyocytes-like cells via down-regulation of D11-1-Hes-1/Notch pathwayObjective:(1) To investigate the expression of Notch signaling, Nkx2.5, GATA-4, cTnT and CX43in miR-1-transfected BMSCs on the day of1,7and14.(2) To examine the expression of downstream target molecular of Notch signaling, Nkx2.5, GATA-4, cTnT and CX43in BMSCs with Dll-1-silenced on the day of1,7and14.(3) To identify the expression of Nkx2.5, GATA-4, cTnT and CX43in BMSCs with Hes-1-silenced by day1,7and14.Methods:(1) Using pAJ-U6-shRNA-CMV-Puro/GFP recombinant lentiviral vector encoding miR-1transfected BMSCs, then investigated the expression of Notch signaling, Nkx2.5, GATA-4, cTnT and CX43by RT-qPCR and Western Blot assay on days1,7and14.(2) BMSCs were infected with lentiviral constructs encoding short hairpin RNAs (shRNAs) to silence D11-1gene, then identify the expression of downstream target molecular of Notch signaling, Nkx2.5, GATA-4, cTnT and CX43by RT-qPCR and Western Blot assay by day1,7and14.(3) Recombinant lentivirus-Hes-1-shRNA vector to transfect BMSCs to silence Hes-1gene, then the expression of Nkx2.5、GATA-4, cTnT and CX43were examined by RT-qPCR and Western Blot assay on days1,7and14.Results:(1) The expression of Dll-1, Hes-1was down-regulated significantly in BMSCs over-expressing miR-1by day7and14. Nkx2.5and GATA-4were dectected on day7, and the expression decreased by day14. Strikingly, the expression of cTnT and CX43were dectected on day7, and increased by day14.(2)The expression of Hes-1is significantly decreased in BMSCs infected with Dll-1-shRNA on days7and14. And the trends of expression of Nkx2.5, GATA-4, cTnT and CX43were similar to that of miR-1-transfected cells.(3) Knockdown of the Notch downstream target gene-Hes-1, also leads to the similar effects on cell lineage decisions as knockdown of D11-1.Conclusion:miR-1promotes the differentiation of BMSCs into cardiomyocyte-like cells via down-regulation of D11-1, which further influence Hes-1, the downstream molecules of Notch. Part3:Investigation of miR-1targeting the3’-UTR of D11-1Objective:To comfirm if D11-1is the direct target gene of miR-1.Methods:Wild type and mutant D11-13’-UTR gene sequence, which were amplified by PCR, were connected to double endonuclease digestion PsiCHECKTM-2vector respectively and transformed into competent cells. Then examined positive clones by plasmid restriction enzyme digestion and sequenced. After miR-1and plasmid had been cotransfected into293T cell, collected the cell cracking cytosol and examined whether Dll-1was the direct target gene of miR-1through Dual-Luciferase Reporter Assay System.Results:MiR-1significantly inhibits the activity of luciferase compared with blank group (P<0.01) in the plasmid groups encoding Dll-13’-UTR. The same result was dectected in miR-1group compared to the negative control group (P<0.01). This indicated that miR-1can combine with the3’-UTR of D11-1, thereby inhibiting the activity of luciferase. In the experimental groups of plasmid encoding mutDll-13’-UTR, there was no statistical difference between miR-1group and blank group or negative control group, indicating that miR-1can not target the mutDll-13’-UTR to inhibit the activity of luciferase.Conclusion:Dll-1is the target gene of miR-1. Part4:Effects of BMSCs transfected with miR-1on repairment of infarct zone in acute myocardial infarction model of mouseObjective:To investigate the effects of transplantation with BMSCs over-expressing miR-1on myocardial repairment in acute myocardial infarction model of mouse.Methods:After evaluating the cardiac function by echocardiography, acute myocardial infarction was induced in mice by permanent ligation of the left anterior descending coronary artery. A total of80mice were randomly divided into4groups:(1) PBS group, the animals were transplanted with PBS (n=20);(2) BMSCs group, the animals were transplanted with BMSCs (without gene or Lentiviral vector, n=20);(3) BMSCsnull group, the animals were transplanted with BMSCs transfected with lentiviral vectors encoding eGFP gene (n=20);(4) BMSCs miR-1group:the animals were transplanted with BMSCs over-expression of miR-1(n=20). All the animals received exogenous transplantation of cells or PBS by day7after myocardial infarction (concentration of1×106cells/100ul, amount of25ul, injected at multiple points).4weeks after transplantation, echocardiography was performed to evaluate the cardiac function, subsequently, the cardiac sample was obtained and both infarction area and regional wall thickness were measured. Immuno-histochemistry was applied to evaluate the positive expression of CX43, cTnT, a-SMA, Desmin and Vimentin. Also, laser scanning co-focal microscope was used to observe the survival and differentiation of transplanted BMSCs. The expression of molecules of Notch signal in infarction area was detected by Western-blot.Results:(1)After transplantation for4weeks, a greater improvement in cardiac function was obtained in the cells implantation groups than that in the PBS group (P<0.05or P<0.01). The improvement in cardiac function was greater in BMSCsmiR-1group than in BMSCs or BMSCsnull group (all P<0.05), but there was no significant difference between BMSCs group and BMSCsnull group (P>0.05).(2)Compared with PBS group, the infarct size significantly decreased in the cells implantation groups (P<0.05or P<0.01). The decrease of infarct size was greater in BMSCsmiR-1group than in BMSCs or BMSCsnull group (all P<0.05). But there was no significant difference in infarct size between BMSCs group and BMSCsnull group (P>0.05).(3)Compared with the PBS group, the thickness of the infarct-zone was significantly thicker in these three cells implantation groups (P<0.05or P<0.01). Of note, the increase in thickness of the infarct-zone in the BMSCsmiR-1group was more significant than that in BMSCs or BMSCsnull group (all P<0.05). But there was no significant difference in thickness of the infarct-zone between BMSCs group and BMSCsnull group (P>0.05).(4)The immuno-histochemistry and western-blot results showed that the expression of CX43, cTnT, a-SMA, Desmin and Vimentin were all up-regulated in the infracted area in cells implantation groups than that of the PBS group (P<0.05). Of note, these markers are higher in BMSCsmiR-1group than in other cell transplantation groups (all P<0.05), whereas western-blot showed D11-1and Hes-1levels were decreased more in the infracted area (P<0.05or P<0.01).(5)The survival number of transplanted cells and the differentiation rates into myofibroblast was significantly higher in BMSCmiR-1group than in the BMSCsnull groups (P <0.05).Conclusion:Our findings suggest that (1) BMSCs modified with miR-1could remarkably promote the survival and differentiation of the stem cells into myofibroblast in the infracted area, decreased infarct size, thicker infarct area and improving the cardiac function;(2) Strinkingly, miR-1could attenuate the the expression of D11-1and Hes-1of the infracted area in vivo, which strongly support our previous experiment. |
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