| Cardiovascular disease is the leading cause of death worldwide.The most common degenerative disease of the cardiovascular diseases is different kinds of slow arrhythmias and myocardial infarction.For various types of slow arrhythmia patients with clinical symptoms,traditional electronic pacemaker is still the first choice.However in the process of clinical use,there are still many unavoidable problems in electronic pacemakers,such as battery life problem,vein thrombosis,heart contractions not synchronized,lack of reactivity of neuro-transmitters.With the development of molecular biology and cell biology,especially in recent years,the dream of biological pacemaker is no longer distant.It is generally accepted that joint cell and gene pacing is most promising.A lot of researches have transfected different pacing genes to seed cells,and then transplanting it to the heart model,to make it show the ectopic pacemaker activities,which biological pacemaker has made the first step on the road.At present,the main understanding of cardiac pacing is concentrated on introverted hyperpolarization pacing current,and hyperpolarization-activated cyclonucleotide gate-controlled(HCN)gene family is the gene encoding the main pacing current,which is activated during membrane hyperpolarization and is most closely related to the production and regulation of cardiac pacemaker cells.The family of HCN and its encoded If are the key factors to generate pacing activity.HCN4 is the highest expression in sinus node.With the study of the sinoatrial node research over the past two years,it is found that the most abundant subtypes in the sinoatrial node and atrioventricular node,which are important not only for producing normal pacing potential and basic heart rate,but also involved in the regulation of the heart rate by the sympathetic nervous system.So far there is still no breakthrough in biological pacing research.Both vivo and vitro studies have proved that transfection gene is a feasible experimental method in animal models,but the experiments have not exceeded 6 weeks in vivo,which may be related to the gene carriers chosen by the researchers.Therefore,the choice of suitable vectors is still need to be studied.In recent years,with the deepening research of cardiovascular regenerative medicine based on stem cell,the era of clinical trial of stem cell repair for damaged myocardium has arrived.The application of stem cell types,which are recognized as great potential stem cells including induced pluripotent stem cells(iPSCs),mesenchymal stem cells(MSCs)and very small embryonic like stem cells(VSELs).Compared with MSCs and iPSCs,VSELs is smaller,more immature and more differentiated,and it is possible to solve the problem of coronary artery embolism.Analysis of these documents,although the biological pacemaker has made encouraging progress,but the duration of most of the biological pacemaker in vivo is not long,which not achieved the desired effect and there are several key issues need to be studied and solved which can be applied in clinical practice.Only by systematically studying these problems we can build a new generation of biological pacemaker with high efficiency,stability,long term and clinical application.Myocardial infarction(MI)is one of the most common cardiovascular diseases.There is no effective method in clinic.Nowadays,the research of stem cell mobilization for MI has become a hot issue.The number of VSELs is very small,limiting its clinical application.In normal condition,the VSELs are in static.Only when there is stress/injury,VSELs can mobilize the differentiation of other tissue specific stem cells into peripheral blood,but the number is still very small.Especially with the condition of the body’s stress,the number of VSELs mobilization into peripheral blood significantly.In recent years,many studies have found that the recombinant human granulocyte colony stimulating factor(G-CSF)can mobilize VSELs to peripheral blood,promote the effect of tissue repair and improve cardiac function after MI,and these VSELs has the ability of myocardial cells differentiation,which provide effective number of VSELs applied to clinic.Although the effect of western medicine treatment is limited,the curative effect is single,the effect time is not lasting and has the side effect.However,traditional Chinese medicine has received more and more attention due to its wide therapeutic effect.In Chinese medicine,the treatment of MI should be focused on activating blood circulation and removing blood stasis,invigorating qi and removing cold,nourishing blood and removing silt,and balancing Yin and Yang.Shexiang Baoxin Pills contain artificial musk,ginseng extract,artificial bezoar,cinnamon,styrax,toad venom,borneol,such as seven kinds of pharmaceutical ingredients,can remove cold evil,aeration machine,promoting blood circulation to remove blood stasis,blood vessels,enhance the curative effect of MI.This study through the establishment of rabbit model of MI,discussed on the basis of G-CSF mobilization combined application of Shexiang Baoxin Pills for the treatment of MI and the synergy of both,to provide a valid number of VSELs applied to clinic.This paper is divided into three parts,to discuss the following questions:1.VSELs is isolated and cultured in vitro,amplification and optimization of separation and identification;2.to establish appropriate VSELs cells into cardiac pacing cell differentiation system,explore the regulatory mechanism of HCN gene of cardiac pacemaker cells and key signaling pathways;3.The experimental study of rhG-CSF mobilization combined with Shexiangbaoxin pill for the treatment of myocardial infarction.Part 1:The isolation,amplification and identification of swine bone marrow very small embryonic stem cells.Objectives:In the present work,we established the method of isolating and culturing very small embryonic-like stem cells(VSELs)from swine in vitro,to set up the appropriate VSELs to directional differentiation system,observe the biological characteristics of VSELs.Methods:After obtaining the swine bone marrow,the mononuclear cells isolated from swine bone marrow by lysing buffer were stained by specific antibody CD45,Lin and CXCR4 designed for FACS of VSELs,after staining immune magnetic bead to enter Magnetic Activated Cell Sorting(MACS)for VSELs,which were cultured and amplified in vitro.The morphological characteristics,proliferation and ultrastructure of bone marrow VSELs were observed by using an optical microscope and transmission electron microscope with the muscle cells of swine cultured as the feeding layer.The biological characteristics of VSELs were studied in different aspects,such as the morphology,molecular,gene and protein levels to identify its stem cell characteristics respectively.Results:VSELs are major pluripotent stem cells as a rare population of very small primitive cells with pluripotent/embryonic characteristics,have a high nuclear-to-cytoplasmic ratio,and could express pluripotent markers SSEA-1,Oct-4,Nanog and Sox-2,alkaline phosphatase staining was positive.In VSELs,the organelle structure was observed in the nucleus,and the characteristics of the cytoplasm were immature.Quantitative PCR and gel electrophoresis showed that the VSELs cells were highly expressed CD133,SOX-2,OCT-4,NANOG and other stem cell specific genes.Conclusions:VSELs can be isolated from swine bone marrow by immunomagnetic beads and flow cytometry,which can express pluripotent stem cell markers.Part 2:Based on HCN transfection VSELs to construct biological pacing signal pathway screening inducer research.Objectives:In the present work,we set up the appropriate VSELs cells to directional differentiation system,explore the HCN gene regulate pacemaker cells mechanism and crucial signaling pathways.Methods:① Compared with bone marrow mesenchymal stem cells(MSCs)of isolation,culture,differentiation and identification for reference,groping and establishing VSELs differentiation system,appraising VSELs of differentiation ability system in vitro.Purification of P2 generation of VSELs cells inoculated in the 24 hole plate covered by matrix,induced into nerve cells(1640+10%fetal bovine serum+ RA/IBMX),induced into fat cells(including 10%fetal bovine serum 1640 + 10 mg/L insulin + 0.5μmol/L dexamethasone+ 0.5mmol/L IBMX + 50mol/L indomethacin),induced into myocardial cells(10μmol/L 5-Aza + serum-free culture medium)to replace the induction hole of VSELs cell cultures.After every 24h,the morphological changes of the cells were observed.Change fluid every 3d,the morphology of the cells was observed.②Making cell suspension in the logarithmic phase of P2 generation of VSELs with pancreatic enzyme,then inject 200μl cell suspension to the right side of the axillary subcutaneous and abdominal cavity in nude mice,observating growth,diet,mental activity of nude mice daily;③The signal pathway of key genes in MSCs and VSELs induced differentiation was established after transfection and transfection of HCN gene with lentiviral plasmid vectors.After successful building slow virus carrier,extracting plasmid standby,extend the MSCs and VSEL after vaccination in 24-well culture,cell proliferation to each hole area of 70%,transfection HCN 1,2,4 genes according to the Fugen transfection system respectively,and were cultured under standard cell culture conditions.Observing green fluorescent and counting efficiency of the transfection respectively in 12 h,24 h,and 48 h after transfection.Results:①The identification of VSELs differentiation in vitro was found that:VSELs induced neuronal cell toluidine blue staining was positive;VSELs induced fat cell oil red staining was positive;VSELs induced myocardial cell immunofluorescence showed that the myocardial cell specific protein was positive,which confirmed that VSELs had multiple potential differentiation ability.②It was found that the nude mice injected with VSELs had no tumor growth,with diet,mental activity and other conditions all favorable,with no obvious tumor mass in subcutaneous injection location and no mass formation in abdominal cavity.The nude mice were executed after 1 month of continuous observation,and the skin and abdominal tissues were removed,and the tumor tissues were not isolated at the injection site.③The HCN gene lentiviral plasmid was successfully transferred to MSCs and VSEL cells,and the fluorescence sign was strongest at 48h.Conclusions:We found these cells differentiate into cells belonging to all three germ layers in vitro,with no malignant proliferation of tumor.HCN gene lentivirus plasmid vector transfected to VSELs,established VSELs differentiation for cardiac pacemaker cells,directional differentiation system,explores the HCN gene regulation pacemaker cells and the mechanism of the key signaling pathways.Part 3:The Empirical Study on Shexiang Baoxin Pills based on rhG-CSF for Treating Myocardial InfarctionObjectives:To investigate the therapeutical effect of Shexiang Baoxin Pills for acute myocardial infarction(MI)based on recombinat human granulocyte colony-stimulating factor(rhG-CSF).Methods:MI was created by ligating left antierior descending(LAD)coronary artery in rabbits which were divided into control group(group I),rhG-CSF group(group II),Shexiang Baoxin Pills group(group Ⅲ),and the group of Shexiang Baoxin Pills based on rhG-CSF(group IV).General state of health and heart function in all groups were monitored at prior treatment and post-treatment.The hearts were harvested for pathologic detection after 6 weeks.Results:Compared to group Ⅰ,in other 3 groups,the general state of the animals took a favorable turn,left ventricular ejection fraction(LVEF)improved significantly(P<0.05),left ventricular end-diastolic dimension(LVEDd)was smaller(P<0.05)and the wall motion enhanced significantly.Infarct size was significantly smaller and the capillary density was significantly increased(P<0.05).Compared to group Ⅱ,LVEF improved further,LVEDd had no statistically significance(P>0.05),the wall motion enhanced further.The changes of infarct size was no statistically difference(P>0.05).The capillary density was significantly increased(P<0.05).Conclusions:Shexiang Baoxin Pills based on rhG-CSF or rhG-CSF alone can improve general state and heart function of rabbits with MI,decrease infarct size,increase capillary density,and a more significant improvement in heart function and capillary density of Shexiang Baoxin Pills based on rhG-CSF. |
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