| Part one Isolation,Purification and Identification of Bone Marrow Mesenchymal Stem CellsObjective: Bone marrow mesenchymal stem cells(BMSCs)are regarded as a sort of pluripotent stem cells.It has obvious advantages compared with other stem cells on account of its convenient to acquire,liable to culture and proliferate in vitro,the potential of multilineage differentiation and autologous transplantation without immunological rejection.By far,it has been the ideal seed cells in tissue engineering field.Therefore,in this experiment,SD rat BMSCs were isolated,amplified,purified and subcultivation.Then we characterized BMSCs in three aspects.Methods: The required bone marrow for the study was harvested from the long bone of 15 cases of three-week-old SD rats by whole bone marrow culture method.SD rat BMSCs were isolated,amplified by serial subcultivation and purified by digestion time control and subcultivation.We characterized BMSCs in three aspects:1 The cultured cells were observed,using inverted phase contrast microscope.2 The expression of CD90,CD29 and CD45 was detected,using flow cytometry.3 The multilineage differentiation potential was detected by adipogenic,osteoblasts and chondrogenic differentiation in vitro.Results:1 Primary passage of BMSCs had small cell nuclear and began to attach when cultured for 12 hours: part of the cell bodies spread and clung to the growth plane of the culture flasks.After 4 days,primary cells proliferated rapidly and protuberances with varying size and length were stretched out from the cell bodies.After 7 days,cells were mainly short shuttle-shaped,oval and irregularly-shaped ones were also visible,which were similar to fibroblasts in appearance.After passage,hematopoietic stem cells which grew suspendedly could be thrown away gradually.The shape of cultured cells was arranged consistent.2 The positive rate of CD29,CD45,and CD90 was 94.3%,1.3%,95.9%,respectively,by flow cytometry.It revealed that these cells expressed stromal cell antigen rather than hematopoietic stem cell antigen.These results conformed to the characteristics of mesenchymal stem cells.3 During osteogenic induction,BMSCs gradually transformed to polygonal or irregular-shaped cells and grew intensively.The induced cells were positive for alizarin red staining after 21 days.During adipogenic induction,small lipid droplets accumulated and became larger gradually in some cells.The induced cells were positive for oil red O staining after 14 days.During cartilage induction,the local part of BMSCs gradually changed into round or oval.Afte 14 days,the cells and its related structures were positive for alcian blue staining.Conclusions:SD rat BMSCs were successfully isolated,amplified and characterized.Part two Myocardial differentiation of BMSCs induced by expression of TGF-?1 gene in vitroObjective: Recent studies have shown that transforming growth factor beta 1(TGF-?1)can improve the cardiogenic differentiation of BMSCs in vitro.For an onangiogenesis effect,TGF-?1 displays a biphasic role.Low concentrations of TGF-?1 synergistically enhance,whereas high concentrations decrease the vascular invasion of cultured endothelial cells induced by angiogenic factors.Therefore,different concentrations of TGF-?1 may play different roles in cell proliferation and differentiation,bone formation,angiogenesis,cell cycle progression and cellular migration.In the present study,three different concentrations of TGF-?1 were tested to find the optimal concentration that would efficiently differentiate BMSCs into cardiomyocytes(CMs)or cardiomyocyte-like cells(CLCs)in vitro.Methods:1 Experimental groups: the 2nd-generation cells were co-incubated with 250 ng/mL 5-AZA(Group A),2 ng/mL TGF-?1(Group B),5 ng/mL TGF-?1(Group C),10 ng/mL TGF-?1(Group D),for 72 h.After which,the cells were washed three times with PBS,and the medium was replaced by complete medium without any inductor.The control group(Group E)was BMSCs which cultured without any inductive substance.The medium was changed every 3 days till 28 days,and then the cells were prepared for the succeeding experiments.2 To evaluate cardiomyogenic differentiation of BMSCs in each group,immunohistochemistry staining and immunofluorescence staining of the CMs proteins,Desmin,tropomyosin(Tm),Cx43 and Cardiac Troponin I(cTnI),was performed.The relative mRNA expression level of ?-MHC,GATA-4 and Nkx2.5 in different group was detected by RT-qPCR.The expression of GATA-4 and Nkx2.5 in different group at 14 d after induction was detected by Western blot.Ultrastructure of BMSCs in each treatment group was also observed.Results:1 After the 2nd-generation BMSCs were induced by 5-AZA and different concentrations of TGF-?1 for 72 h,the morphological differentiation from BMSCs to CLCs was initiated.The differentiated cells appeared to be the single or group distribution.Cells were spindle-shaped or branched,with one or two round nucleus located in the center.On day 28,BMSCs in the induction group showed fusiform shape,orientating with one accord.The morphous of BMSCs in all the induction groups was similar.2 The results of immunohistochemistry staining showesd that BMSCs in Group A,Group B,Group C and Group D could all be identified by the positive staining for Desmin,Tm,Cx43 and cTnI.The positive rates were all higher than that of the Group E(P<0.05).The expression of Desmin,Tm,Cx43 and cTnI in Group C was significantly higher than that in Group B(P<0.05).Furthermore,the expression of Desmin and cTnI in Group C was also higher than that in Group D(P<0.05).3 The results of RT-q PCR showed that cells in all group did not express ?-MHC mRNA,but expressed GATA-4 and Nkx2.5 at 7 days,14 days and 28 days after induction.The expression of GATA-4 mRNA and Nkx2.5 mRNA in Group A,Group B,Group C and Group D was significantly higher than those in Group E(P<0.05).For GATA-4 mRNA,the expression level in Group C was 3.3-fold higher than that in Group E.For Nkx2.5 mRNA,it was 2.7 times higher than that in Group E(P<0.05).4 The results of Western blot showed that after 28 days of induction,cells of Group A,Group C and Group E can express GATA-4 and Nkx2.5 protein.The expression of GATA-4 and Nkx2.5 in Group A and Group C was all significantly higher than those in Group E,respectively(P<0.05).5 Transmission electron microscope(TEM)revealed that,organelles such as a mass of smooth endoplasmic reticulum,mitochondria,meanwhile,some glycogen and ribosomes were visible in the cytoplasm of these cells in each induced group.In addition,myofilament arranged parallel in the cytoplasm.BMSCs in induction groups had an oval nucleus which located in the center.These ultrastructural characteristics of BMSCs in induction groups were similar to myocardial cells.6 The results of immunofluorescence staining showed that on day 28 after induction,most of the cells in induction groups were present both Desmin proteins(red)and cTnI proteins(green).Analysis of the fluorescence intensity revealed that in Group C,the expression of Desmin and cTnI was noticeably higher than that of Group D,respectively(P<0.05).Conclusions:TGF-?1 may induce BMSCs to acquire cardiogenic phenotype in vitro and 5ng/mL may be a suitable induced concentration.Part three Myocardial differentiation of rat BMSCs transfected with TGF-?1 gene through the inhibition of Wnt/?-catenin signaling pathwayObjective: In the research of gene therapy related to cardiovascular disease,gene modification of lentiviral vector has been widely used.TGF-?1 is a pleiotropic cytokine with many and complex effects in cell and tissue physiology.It is a multifunctional cytokine involved in the differentiation,growth,and survival of a variety of cells.Wnt signaling,including the canonical pathway(Wnt/?-catenin signaling pathway)and non-canonical pathway,plays a key role in cell differentiation,embryonic development,tumorigenesis and angiogenesis.Research has shown that Wnt/?-catenin signaling pathway and TGF-? signaling pathway interacted and involved in the process of Epithelial-Mesenchymal Transition(EMT)and fibrous disease induced by TGF-?.Therefore,TGF-?1 has been transfected by lentivirus vector into BMSCs to promote the cardiomyocyte differentiation.We also investigated the relationship between this process and Wnt/?-catenin signaling pathway.Methods:1 Experimental groups: Group A(transfected with Lenti-TGF-?1-GFP),Group B(transfected with Lenti-empty vector-GFP),Group C(blank group).Then the cultured cells were transfected and the best MOI value was determined.2 The expression of TGF-?1 in different group at 96 h after transfection was detected by Western blot.3 The concentration of TGF-?1 in cell culture medium of different group was detected by ELISA.4 The expression of Desmin and cTn I in each group at 28 d after transfection was detected by immunohistochemistry staining.5 The expression of ?-catenin,GSK3?,p-GSK3?,c-Myc,cyclinD1 and MMP-7 in different group was detected by Western blot.6 Intracellular distribution of ?-catenin in BMSCs was detected by immunofluorescence staining.7 The relative mRNA expression level of c-Myc,cyclin D1 and MMP-7 in different group was detected by RT-qPCR.8 Western blot was used to detect the effect of LiCl on the expression of ?-catenin,p-?-catenin in different group.Results:1 Preliminary experiment showed that the best MOI value was 150.After 96 hours of transfection,the morphology of the cells was arranged closely,some of them contracted and became round,and the nucleus was not clear.2 Western blot results showed that after 96 hours of transfection,cells in Group A can express TGF-?1 while there was no expression of TGF-?1 in Group B or Group C.3 The results of ELISA showed that the concentration of TGF-?1 in cells of blank group,cells transfected with Lenti-empty vector-GFP and cells treated with 5 ng/mL TGF-?1 were 1.245 ng/mL,1.395 ng/mL and 1.865 ng/mL,respectively.However,the concentration of TGF-?1 in cells transfected with Lenti-TGF-?1-GFP was 2.055 ng/mL(P<0.05).4 The results of immunohistochemistry staining showed that compared with Group B or Group C,BMSCs in Group A could be identified by the positive staining for Desmin and cTn I(P<0.05).5 Following transfection of TGF-?1,the levels of intranuclear ?-catenin and total ?-catenin were all significantly decreased in BMSCs(P<0.05).However,the levels of cytoplasmic ?-catenin were largely unchanged.In addition,the levels of GSK-3? were largely unchanged in BMSCs,whereas phosphorylated GSK-3? was significantly decreased in BMSCs(P<0.05).The expression of c-Myc,cyclinD1 and MMP-7 protein in Group A was significantly lower than those in the blank group,respectively.6 After transfection of TGF-?1,?-catenin in the nucleus was significantly weaker than that in the control group,indicating that ?-catenin which translocated from cytoplasm to nucleus was decreased after transfection of TGF-?1.7 The results of RT-qPCR showed that cells in all groups can express c-Myc,cyclinD1 and MMP-7 mRNA at 7 days,14 days and 28 days after transfection.The expression of c-Myc,cyclinD1 and MMP-7 mRNA in Group A was significantly lower than those in the blank group,respectively.For c-Myc mRNA,the expression levels in Group A was 0.61-fold lower than that in the blank group.For cyclinD1 mRNA,it was 0.68 times lower than that in the blank group(P<0.05).8 Compared with the control group,the expression of ?-catenin was decreased significantly in single transfection group while the expression of phosphorylated ?-catenin was increased(P<0.05).However,the expression of ?-catenin in the LiCl group and transfection+LiCl group was all increased,while the expression of phosphorylated ?-catenin was decreased(P<0.05).Conclusions:1 SD rat BMSCs were transfected with TGF-?1 by lentivirus transfection method and differentiated toward cardiomyocytes.2 In the process of promoting the differentiation of BMSCs into cardiomyocytes by transfecting with TGF-?1 gene,it may be achieved by promoting the phosphorylation of ?-catenin and interfering with ?-catenin shifts from cytoplasm to nucleus.3 In the process of promoting the differentiation of BMSCs into cardiomyocytes by transfecting with TGF-?1 gene,it may be achieved by inhibiting the expression of c-Myc,cyclinD1 and MMP-7 in Wnt/?-catenin signaling pathway. |
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