| There are pluripotent stem cells with specific myocardial differentiation potential in the adult heart,which are undifferentiated cells with high self-renewal and specific myocardial differentiation and proliferation potential existing in the mature heart tissue of the mature body.The damage cannot be repaired by self-regeneration,so saving the infarcted myocardial tissue and restoring its original function is the key to treating ischemic heart disease.Bone marrow mesenchymal stem cells(BMSCs)are common"seed cells"and have a multi-directional differentiation induction function.With this feature,under certain conditions,they can become fat,bone,muscle,differentiation of many types of cell tissues such as nerve,heart muscle,endothelium and so on.BMSCs can also be used as root cells to effectively delay aging and repair diseased tissues and organs.Implantation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells into damaged myocardial tissue has a certain effect on repairing myocardial injury.With the continuous development of transgenic technology,the transformation of stem cells through gene transfection has become a hot spot in the field of stem cell research.Lentiviral vectors are the most widely used gene vectors in transgenes and gene modification.Compared with vectors such as plasmids and adenoviruses,they have the advantages of high transfection efficiency,low cytotoxicity,and long expression time of target genes.Therefore,this paper mainly uses lentivirus as a carrier to mediate gene transformation of BMSCs and differentiation into cardiomyocytes.The research content includes the following three parts:Part ?:Isolation,purification and identification of bone marrow mesenchymal stem cells from SD ratsObjective:Bone marrow mesenchymal stem cells(BMSCs)are a kind of pluripotent stem cells,which are widely used in the fields of wound defect,tissue engineering research and tissue repair.BMSCs have the advantages of self-renewal and replication,multidifferentiation potential and no immune rejection in autotransplantation.At the same time,BMSCs are easy to be isolated,cultured and expanded in vitro,and are widely studied"seed cells".However,BMSCs can't propagate indefinitely as tumor cell lines.Therefore,in the research and application of BMSCs,it is necessary to extract and separate BMSCs from the donor,and then they can be used for subsequent experimental research after expanded culture,purification and identification in vitro.At present,there are four main separation methods for BMSCs:whole bone marrow adherence separation screening method,gradient density centrifugation method,flow cytometry and immunomagnetic bead method.The adherence separation screening method is easy to operate,does not require the assistance of precision equipment,and has relatively little damage to the cells,which is a classic separation method.Therefore,in this study,BMSCs were separated by adherence separation screening method.The isolated and purified BMSCs were first observed and identified,and the growth characteristics of the cultured cells were observed by inverted phase contrast microscopy;secondly,the specific molecular expression of cell surface molecules CD29,CD90,and CD45 were detected by flow cytometry;Identify its multi-directional differentiation ability.Method:In this study,BMSCs were isolated and screened with whole bone marrow adherent.Long bone marrow of 3 week-old clean SD rats was used as the test subjects.The adherent screen was used to isolate and extract BMSCs from rats.Observe whether the growth characteristics of cultured cells have the characteristics of BMSCs by phase contrast microscopy;analyze the expression of CD29,CD90 and CD45 molecules on the cell surface by flow cytometry.Results:1.Microscopic observation of the morphological changes of cells:the cells initially inoculated into the culture flasks were in a suspended state,and the cells were in a uniformly spherical shape with a large number.After 12hours of culture,some cells began to grow adherently,about 1-2 d All cells adhered to the wall and were round.After 4 d,the round cells gradually changed into irregular shaped cells,and some cells showed uneven protrusions.At this time,the nucleus was clearly visible.The full nucleus was mostly centered.The cells look like fibroblasts.After passage,the cells grow faster and grow well,and the shape and arrangement are regular and uniform long fusiform.2.Flow cytometry analysis of cell surface results:the positive rate of CD45 for hematopoietic stem cell markers CD45 detected by flow cytometry was 8.7%,the expression of cell fiber junction receptor CD29 was as high as96%,and the positive rate of cell adhesion molecule CD90 was as high as93.7%.This result indicates that the cells isolated and cultured in this study have high expression of surface antigen after passage purification,which is consistent with the characteristics of BMSCs,and proves that the cells we isolated and cultured are BMSCs.Conclusion:Through microscopic observation and flow cytometry detection,BMSCs were successfully isolated,cultured and expanded using whole bone marrow adherent separation and screening method.Part ?:Construction and titer detection of Wnt11 and BMP2overexpression lentiviral vectorsObjective:The continuous development of biotechnology and genetic engineering has made transgenic technology widely used in animal and plant research.Transformation of stem cells through genetic modification to differentiate them in specific directions has become a research hotspot in the field of stem cell transplantation.A large number of studies have proved that lentivirus can be used as a vector to integrate the target gene into the host chromatin and can achieve a stable genetic effect.The use of lentiviruses to construct gene vectors and transfect bone marrow mesenchymal stem cells does not affect the phenotype of bone marrow mesenchymal stem cells,but it helps the differentiation of stem cells.Therefore,the construction of bone marrow mesenchymal stem cells with overexpression of the target gene is also a means to improve the effective differentiation of stem cells.Based on this feature,this study constructed BMP2 gene and Wnt11 gene overexpression plasmid lentiviral vector bone marrow mesenchymal stem cells to make a new type of stem cell"gene seed".Based on the research foundation and assumption of differentiation of BMSCs into cardiomyocytes by transfection of Wnt11 and BMP2 recombinant lentivirus,and following the progress of induction of BMSCs into cardiomyocytes at home and abroad,we constructed Wnt11 and BMP2 expression vectors mediated by lentivirus,single or double transfected BMSCs,and compared their differentiation efficiency.As a new way of stem cell induction,genetically modified bone marrow mesenchymal stem cells(BMSCs)are favored by researchers.Method:According to Wnt11 and BMP2 gene sequence,the target gene fragment was amplified and connected to the vector.After transformation,screening,identification and sequencing analysis,the target gene sequence was identified.WPRE sequence on lentivirus vector was detected by qRT-PCR.WPRE sequence can be integrated into cell genome along with the target gene.The titer of lentivirus can be obtained by sorting and converting the experimental data of qRT-PCR.Results:1.Wnt11 and BMP2 genes construct lentiviral pHS-AVC-LW-Wnt11 and pHS-AVC-LW-BMP2 vectors.The digested fragments are consistent with the expected molecular weight,that is,the vector construction is successful.2.Performed the Blast sequencing analysis on the vectors verified by the enzyme digestion,and the sequencing found that the Wnt11 and BMP2 genes were sequenced correctly.3.The amplification/detection object of the qRT-PCR experiment is the WPRE sequence on the lentiviral vector(the WPRE sequence can be integrated into the cell genome with the target gene),and it can be adjusted according to the qRT-PCR experimental data to obtain Titer.The virus titer of Wnt11 vector is 6.64×10~8 active virus particles per ml of virus supernatant,and the titer of BMP2 lentiviral vector is 2.71×10~8 active virus particles,and the virus titer is qualified.Conclusion:1.Successfully constructed the lentiviral vector pHS-AVC-LW-Wnt11and pHS-AVC-LW-BMP2 of Wnt11 and BMP2 genes.2.Qualified virus titer test can provide a basis for virus infection in subsequent experiments.Part ?:Lentivirus-mediated Wnt11 and BMP2 transfection of rat bone marrow mesenchymal stem cellsObjective:Based on Wnt11 and BMP2 recombinant lentivirus transfection of bone marrow mesenchymal stem cells to differentiate into cardiomyocyte-like cells in vitro induction of mesenchymal stem cells has attracted people's attention as a new way to induce stem cells.So far,there have been few studies on lentivirus-mediated Wnt11 and BMP2 double gene transfection to induce the differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells.Inducing differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells helps to increase the differentiation rate of cells.Therefore,in this study,lentiviral vectors were used to transfect Wnt11 and BMP2 into BMSCs respectively.In the dual transfection of Wnt11 and BMP2 into BMSCs,the efficiency of differentiation of Wnt11 and BMP2 single gene transfection and double gene transfection into cardiomyocytes was compared.Obtain long-lasting BMSCs overexpressing Wnt11 and BMP2,reveal the biological characteristics of lentivirus-mediated transgenic BMSCs,and explore whether gene transfection is superior to direct drug induction.It is expected to improve the cell differentiation efficiency and provide an excellent cell source for the clinical treatment of ischemic heart disease by cardiomyocyte transplantation.Method:1.Induction experiment grouping:Divided into 6 groups according to different inducers:group A(BMSCs blank control group),group B(Wnt11induction group),group C(Lenti-Wnt11-2-GFP lentivirus transfection group),group D(Lenti-BMP2-2-GFP lentivirus transfection group),group E(Lenti-control-GFPlentiviruscontrolgroup),groupF(Lenti-Wnt11-2-GFP/Lenti-BMP2-2-GFP double gene Lentiviral transfection group),the second-generation BMSCs in good culture condition were induced according to groups.Before induction,the optimal BMSCs induction density,drug induction optimal concentration,induction time and other parameters must be determined through preliminary experiments.Finally,each group was induced 72 h after the medium was changed,replaced with IMDM medium without inducer to continue the culture.2.Detection and identification of differentiated cardiomyocytes induced by BMSCs:5 aspects were used to detect and identify the differentiation effect and efficiency of each group of BMSCs to cardiomyocytes:(1)Immunocytochemical staining method was used to detect the expression of c Tn T,c Tn I and Cx43 protein in the myocardium of each group.(2)Immunocytofluorescence method was used to detect the expression of Desmin and Tm in each group of cardiomyocyte marker proteins.(3)qRT-PCR technology was used to detect the expression of GATA-4and Nkx2.5 genes in each group of cells.(4)Western blot technique was used to detect the expression of Desmin and Tm protein in cells induced to 28 d.(5)Transmission electron microscope was used to observe the ultrastructure of BMSCs after induction culture to 28 d.Result:1.Observed the morphological changes of BMSCs transfected with lentivirus under microscope:after cultured for 1 week,BMSCs were further expanded and diversified in size,and blood cells and hematopoietic stem cells were gradually eliminated in the process of fluid change and passage;no matter in the drug-induced group or the lentivirus transfected group,after induced for 96 h,BMSCs were gradually eliminated.The proliferation of BMSCs was significant,and the morphology of BMSCs was significantly changed,most of them had changed into long fusiform or polygonal shape.After 4 weeks,the arrangement of BMSCs had obvious directionality,the adjacent cells were closely connected,some cells formed vortex like structure.Compared with the control group,the number and morphology of BMSCs changed after induction.However,it was obviously observed that there were a few suspended dead cells after transfected with lentivirus.The higher the concentration of virus,the more dead cells.With the increase of culture time,the number of dead cells decreased,and the cells gradually recovered their vitality and began to proliferate.After 96 hours of transfection,the fluorescent transfection efficiency of the cells was higher,among which the transfection efficiency of MOI=150 was the highest.Due to the experimental reasons,there are many dead cells and almost no living cells in Wnt11 and BMP2double transfected cells and no relevant data were obtained.Therefore,the double transfection of lentivirus was not successful.There was no cell death and fluorescence in the blank control group.The morphological changes of the cells after induction and transfection were in accordance with the expectation,and the later experiment could be carried out.2.Immunocytochemical staining to observe the expression of c Tn I,c Tn T,and Cx43 in each group of cells:after induction for 4W,c Tn I,c Tn T,and Cx43were positively expressed in the cytoplasm of cells.The object has the strongest integrated optical density(IOD)value.BMSCs blank control group and Lenti-control-GFP lentivirus transfection group)showed weak positive or negative expression of Cx43 and c Tn I.There was a significant difference in the positive expression of each marker between each induction group(P<0.05).3.Immunofluorescence detection of Desmin and Tm protein expression:Immunofluorescence cytochemistry detection results showed that after 4W of each experimental group,the BMSCs cell nuclei were mostly empty stained,and the cytoplasm showed positive expression of Desmin and Tm protein.Green or red fluorescence,green fluorescence in the direct induction group,red fluorescence in the transfection group,and weakly positive fluorescence expression in the BMSCs empty plasmid transfection group.The lentivirus transfection group had the highest positive rate of MOI=150 Desmin and Tm protein.After statistical analysis,the positive expression differences of Desmin and Tm in each experimental group were statistically significant(P<0.05).4.Results of relative fluorescence quantitative PCR:the results of qRT-PCR showed that GATA-4 and Nkx2.5 cells were expressed in each group.GAPDH was used as the internal reference control to determine the expression of GATA-4 and Nkx2.5 genes by relative quantitative analysis.The results showed that the expression level of BMSCs blank control group was similar to that of lentivirus blank plasmid transfection group,and the expression level of Wnt11 and BMP2 lentivirus transfection group was relatively high,and GATA-4 and Nkx2.5 were induced 4 weeks later in Wnt11and BMP2 lentivirus transfection group.The relative expression of GATA-4and Nkx2.5 genes was higher than that of other experimental groups at all time points,the difference was statistically significant(P<0.05).5.Western blot test results:the deproteinization was extracted from BMSCs blank control group,Wnt11 direct induction group,Wnt11 lentivirus transfection group,BMP2 lentivirus transfection group and lentivirus transfection control group after 4 weeks induction.The expression level of desmin and Tm protein was higher in the induction group and the transfection group than in the control group.The expression level of desmin protein was the highest in the transfection group,followed by the induction group.The expression of Tm protein was the lowest in the transfection control group,and higher in the induction group than in the transfection group.Statistical analysis showed that the positive expression of desmin in each experimental group was statistically significant(P<0.05).6.TEM results:The inner structure of BMSCs in each group was observed by transmission electron microscope,and it was found that the nucleus of BMSCs induced by lentivirus was located in the center of the cell,which was oval;myofilaments,rough endoplasmic reticulum,mitochondria,ribosomes and other organelles were obviously seen in the cytoplasm;the parallel arranged myofilaments were obviously more than other induction groups,which accorded with the structural characteristics in the development of cardiomyocytes.Conclusion:1.Wnt11 lentivirus vector and BMP2 lentivirus vector were successfully constructed to induce BMSCs cells.2.The Wnt11 or BMP2 direct drug group can induce rat BMSCs to differentiate into cardiomyocytes.3.Wnt11 lentiviral vector transfection can induce rat BMSCs todifferentiate into cardiomyocytes,and BMP2 lentiviral vector transfection can induce rat BMSCs to differentiate into cardiomyocytes.4.The efficiency of lentivirus-mediated transfection to induce the differentiation of rat BMSCs into cardiomyocyte-like cells is better than that of direct induction of differentiation. |
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