| At present the heart failure is the primary cause which causes to die and to cripple. The mortality rate is high. The pathogenesis is myocardial cell diminution in number, But the intersititial cell is mainly becomes fibroblast to increase obviously. Because the myocardial is finally end the differentiation cell, Multiplication ability is limited, the method of treatment cannot reverse already necrosis's cardiac muscle and is unable progress which interrupts the heart to fade now. cell transplant has become the treatment heart failure the new strategy. Formerly studied have made certain progress, But because the variant body cell transplant existence immunity repel, cannot form the effective excited contraction coupling with the host cell, And has donor questions and so on cell origin and ethics morals, therefore has the good clinical prospect from oneself-cell transplant. Specially inducts through the exogenous gene from the somatic cell improves its living thing character, The induction differentiate"the cardiac muscle type"the cell, substitutes necrosis's myocardial cell, thus improvement heart function.The fibroblast comes from the embryo time's mesenchymic cell differentiation, being the knot contracts in the organization the most active cell. The participation constitution organism wound repairs an important cell group, in organism organization repairs in the reconstruction play the strong character, in each kind of wound repair influential role, fibroblast including complete set genome team DNA accepts the material base which the gene extension. The fibroblast gained and grown easily in vitro, also had the comprehensive understanding to its ex vitro growth rule, can become seed cell through induction ex vitro. The myogenic determination gene is one which of leading regulation genes in the embryonic development process the muscle occurs, and differentiate the decision function to myoblast's growth, Changes over to the non-muscle cell the MyoD gene, can start the myo- process, At the same time, the external MyoD gene's instantaneous expression, can activate the endocardial gene, promotes the myo- production. how does cell transplant technical requirement solution's another crucial question is realize between the transplant cell and the original myocardial cell's function conformity, Cx43 is main gap junction protein in the ventricle, through Cx43 gene expression cell gap junction protein, Improving the intercellular communication, causes the transplant cell to form the effective cell gap junction with own myocardial cell, maintains the synchronized contraction.In recent years once had the research to report the fibroblast induces into through the gene may the excitatory cells, based on above reason this experimental design MyoD and the Cx43 gene to cause it to differentiate into"the cardiac muscle"the cell , transplants to dead cardiac muscle, thus improvement heart function. Therefore, organize the engineering technology and the transgene technology has developed the oneself muscle cell transplant origin.Objective:Discussion exogenetic MyoD and Cx43gene induces rat fibroblast to differentiate into"the cardiac muscle"the cell, carries on from the autologous cell transplant treatment chronic heart failure experiment feasibility.Methods:(1) Construct efficient eukaryotic expression plasmid lentiviral vector pLenti6/v5-DEST-MyoD and pLenti6/v5-DEST-Cx43 encoding exogenetic rat MyoD cDNA or Cx43 cDNA respectively, which were identified with the agarose electrophoresis and gene sequent examination;(2) Gains rat fibroblast in vitro separation, the cultivating , the reproduction; After medicine screening, Carries on the Vimentin immunocytochemistry to stain to the DFs appraisal, The DFs applies 5- bromine deaeration uracil nuclear glucoside (bromodeoxyuridine, BrdU) carries on the DFs tracing mark.(3) The cultured rat DFs were transfected with the recombinant lentiviral vector method, the mophological and ultrastructure changes of the transfected cells were observed by microscopy, and the expression of MyoD and Cx43 genes in the transfected cells was detected by immunocyto-chemical methods;(4) Rat chronic heart failure models were established by ligating the middle-proximal 1/3 segment of left anterior descending coronary artery (LAD) after operation for 4 weeks.(5) The converted cells were autologous transplanted into the infarcted myocardium four weeks after coronary artery occlusion;(6) The changes of electrocardiogram (ECG) was observed by multiple peripheral adapter (MPA) physiologic recorder system, Four weeks later after cell transplantation cardiac function of left ventricular were evaluated by Langendorff perfusion of the isolated heart instrument with MPA physiologic recorder system, and myocardial infarction size (MIS) were be detected by 2, 3, 5-triphenyltetrazolium chloride (TTC) method;,Capillary vessel density,Ultrastructure and typical cross gap junction of the grafted cells in infarcted myocardium were characterized by transmission electron microscopy.(7) BrdU-labeled immunohistochemistry was used to observe grafted cells differentiation and propagation ex vivo, andconnexin43 immuno- histochemistry studies were performed to assess the function of these implantated cells;(8) Microelectrode studies with measurements changes of membrane currents .Results:(1) The MyoD cDNA or Cx43 cDNA were cloned into Lentiviral vector respectively, and the cDNA sequencing results of pLenti6/v5-DEST- MyoD and pLenti6/v5-DEST-Cx43 were identified with sequent examination;(2) The success separation and propagation high-purity rat DFs, the anti-prof, and immunostaining of vimentin was positive in cultured DFs;(3) These transfected cells were detected out the expression of MyoD and Cx43 genes by RT-PCR and Western blot;(4) The efficiented transduced DFs with the MyoD and Cx43-encoding vector underwent myogenic conversion, as evidenced by the positive immunostaining of immunocytochemical methods for desmin and alpha-actin, and detection of typical cross gap junction coupling in these adjacent converted DFs by electron microscopy; The membrane currents increases through microelectrode measurements(5) Operating the chest to tie up LAD, creates the medium degree cardiac muscle ischemia. after 4w, examines the heart function, LVEDP≥15mmHg for the chronic heart failure model success symbol;(6) After cell transplant for 4w, MyoD/Cx43 gene transplant group, demonstrated that may reduce the heart failure symptom, to improve the heart the function,that is higher than other transplant group obviously, but has the difference with the normal group;(7) By four weeks after the converted DFs by MyoD and Cx43 genes myocardial transplantation, the MIS in accepted myocardial transplantation group were lower than that in other control groups, and the dysfunctional ECG and cardial function also improved in the same myocardial transplantation group;(8) Histological study showed that transplantation cells in infarcted myocardium were detected by BrdU-labeled immunocytochemistry staining in host heart, and the gap junctions protein was postive by connexin43 immunocytochemistry, But the MyoD/Cx43groups cell BrdU-labeled are many, cell arrangement order;(9) DFs transfected by both MyoD and Cx43 genes could connect with original myocardium tissue through gap junctional coupling observed by electron microscopy.Conclusions:(1) DFs can be genetically transfected by both MyoD and Cx43 genes to differentiate into myocytes ex vivo;(2) The DFs converted by both MyoD and Cx43 genes can survive and proliferation in the hosts'myocardial scar tissue, forming gap junctional coupling, it may be attenuating the ventricular remodeling process to improve heart function after transplantation in CHF rats,... |
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