| Myocardial infarction(MI)becomes one of the leading causes of death,and the incidence rate shows an upward trend,which deeply treathen healthy of mankind.But resently the standard treatment of MI usually depends on drug thrombolysis,intervention therapy and surgical ventricular reconstruction to restore blood supply,aiming for rescuing the dying cardiomyocyte.However,these conpensatory treatment hardly regenerate myocardium and also can not reverse the dead CMs foundamentally.Therefore,many patients died of heart failure caused by ventricular remodeling.In recent years,as the stem cell therapy study has advanced,there gradually comes a transformation and regeneration boom of stem cells to myocardial cells,many stem cells have been reported and confirmed to be potential of transferring into myocardial cells or vascular cells,including Mesenchymal Stem Cells(such as bone marrow mesenchymal stem cells,umbilical cord blood mesenchymal stem cells,etc.),Pluripotent Stem Cells(embryonic stem cells,induced pluripotent stem cells,etc.),Cardiac Progenitor Cells or Non-cardiac Progenitor Cells(source from other body tissues and organs),etc..However,with many pieces of good news about the basic research on transformation from stem cells to cardiomyocytes,the clinical application of stem cells has hit everywhere,for example,the survival rate of stem cell transplantation is less than 1%,the cardiac function of cell transplanted patients does not get obvious improvement,all these information makes the road of the cell transplantation a hard one to go.Reports on the past studies are mostly by applying the method of viruses or liposomes to carry genes to directly induce or re-programme so as to improve the formation rate,transplantation rate and survival rate of seed stem cells,but the immune rejection of viruses use and the risk of cancer caused by gene mutation have greatly limited the clinical application.Althought the popular reprogram method by small molecule chemical compound have eased the security concern,low indued efficiency and bad repetitiveness always accompanied with it cause over-dependence on signal transduction pathway and cells' states.Therefore,how can we get clinical stem cells suitable for myocardial repair and regeneration by a safe and efficient method have became an big challenge on stem cells' transition research.BMSCs are stem cells from mesoderm period with potential of self-renewing and multiple differentiation.They belong to the inhomogeneous cells with a comples transcriptome,and they can encode numerous protein for different development ways and physiological processes,in addition,they are popular in clinical application because of the low immunogenicity.However,most of recent study only focus on their paracrine function,the ability of directly differentiating into cardiomyocyte has always been controversial,hence there is no doubt that direct BMSCs transplantation will fail to develop its full potential.And cardiac stem cells is a kind of specific stem cells from heart tissue,besides the basic characteristics of normal stem cells,they can differentiate into three heart spectrum directly(including cardiomyocyte,endothelial cells and smooth muscle cells in the heart),acting as a good "sternness" source.As we all known,the development of heart is controlled by the network of myocardial transcription factor.In 2012,Nuture had reported that the key myocardial transcription factors Tbx5,Hand2,Mef2c and Gata4 were able to promote myocardial cell reprogram collaboratively,with the lack of these key transcription factors or abnormal expression,cells are destined to fail to differentiate into complete cardiomyocyte,from which shows that the regulation of transcription factor network has conbinded the induction signal in upstream with regulated genes in downstream on purpose of controllng the development and differentiation of myocardial line.Researchers have made several attempts to induce BMSCs converse to myocardial line by transfecting the four specific cardiac transcription factor genes,but eventually they cannot change the characteristics of BMSCs fundamentally because of its complex and powerful transcriptome.Based on above,originating from protein,we successfully desigh and build a nano carrier that is able to deliver protein---NG reagent,which can couple protein molecules quickly and gradually form nano/protein complex.These complexes possess highly efficiency to go through nuclear membrane and plasma membrane barrier,overcome the degradation of exogenous proteins caused by lysosome and protease in stem cells,and thus release proteins that will accomplish biological effect finally.This study takes bone marrow-derived mesenchymal stem cells as cell source,at first,we should master the best contructive conditions and influence factors of nano-protein complex in transfection stage;and analyze complex transfection efficiency and cell toxicity through testing the expression level of 4 intracellular myocardial transcription factors after transfection;finally,from the perspective of transformation,making a detection of cardiac progenitor stem cells' markers or related expression products after induction,this dissertation mainly studies the whole process and the differentiation efficiency of how protein induced BMSCs reprogramming to generate protein induced cardiac progenitors cells.Method:1.To build different mole numbers ratio of PEI(1200 kd)-DNase I(DNA enzyme I),NG5000-DNase I(5000 represents NG nanometer molecular weight),NGlarge-DNase I complexes(large represents different molecular weight,average is about 30000),detect the particle size and zeta potential by using laser scattering method respectively,and screen for a suitable group,then observe the original appearance of the complexes under transmission electron microscope.At the same time,mix the three complexes above and the right amount of plasmid DNA for incubating,take an observation of the changes of DNA banding after the agarose gel electrophoresis,aim at verifying the protective effects of nanoparticles on protein;In addition,we take the larger molecular weight BSA(66 KD)as the research object,build the PEI-BSA,NG5000-BSA,NGlarge-BSA complex in the same condition,and also testify whether the nano-paiticles are successful building and its protective property by observing BSA banding change under the SDS-PAGE electrophoresis.At last,BMSCs are transfected by NG-GFP complexes with different mole number ratio we build,which shows unlike delivery efficiency by detection of immunofluorescence staining and Western Blot.2.By comparison,we make a deepen exploration on the transfection efficiency of NG-protein complexes,grope for the best conditions and influence factors for complexes building gradually,analyze fluorescent expression of intracellular proteins in different pH value,concentrations of serum and additives.When building conditions are mature enough,we begin to transfect different kinds of proteins(green fluorescent protein GFP,?-galactosidase,Tbx5,Hand2,Mef2c and Gata4 transcription factor)into the different types of cells(human bone marrow-mesenchymal stem cells and human dermal fibroblasts,human breast cancer cells and human umbilical endothelial cells).On the other side,we still use the three nanoparticles encapsulate GFP-N plasmid DNA for the sake of building NG-DNA complexes,by which transfect BMSCs,set lipo2000 as positive control,and then compare the transfection efficiency of gene carrier with protein carrier.3.BMSCs are transfected by NG5000-GFP complexes we build,lx ER-TRACKER is used for dyeing after 8 h,so as nuclear dyed by Hochest33324,which indicates fluorescence localization of intracellular GFP proteins and endoplasmic reticulum.This test lay a solid foundation on preliminary exploration of nano/protein complex' basic entrying mechanism,meanwhile,we can also validate the cytoplasm positioning of intracellular GFP.After the NG5000-Hand2 complexes delivery,we check the attenuation and enhancement degree of fluorescent expression in 1 h,4 h,8 h,16 h,24 h,48 h,72 h and 96 h,consequently,it reveals the short-time course of intracellular Hand2 and long time-metabolism.On one hand,we take an application of liposome lipo2000,PRO JECT commercial protein transfection reagent,NG conbind with Hand2 respectively to transfect BMSCs,so we can judge the three carriers' transfection efficiency through fluorescence intensity under infrared fluorescence detector.On the other hand,we keep further study on short-term and long-term toxicity of PEI,NG5000 and NGlarge to BMSCs,so as the apoptosis influence of three complexes(with Hand2).The former one would be determined by MTT in 1 d,3 d,5 d,7 d and 9 d respectively,the latter would be conduct AnnexinV-PI FC detection when transfected for 3 days.4.with the help of mall molecular compounds chir99021 and IWR-1,growth factors like bFGF,VEGF,IGF-1,relying on the successful building of modifeied Tbx5,Hand2,Mef2c,Gata4 complexes,We engage to induce hBMSCs differentiating into cardiac progenitor cells.After inducting for 1 d,3 d,8 d and 15 d,we observe the cells'morphological changes respectively,then extract total RNA and protein to detect the relative mRNA and protein's expression level as piCPCs marker.Results:1.Using the laser scatter instrument,we observed that when the mole number ratio of PEI or NG-DNase I was appropriate,the melting curve of the complexes they formed shows a single peak,and the particle diameters are able to keep between 200-500 nm(PEI)and 100-300 nm(NG).Consistently,transmission electron microscopy results revealed a compact and tight spherical structure of the complexes which evenly distributed.And the Zeta potential is positive with a relatively high absolute value.The solution of the complex is stable under this condition.But when the ratio was not appropriate(the number of carrier or protein relatively excess),there is an obstacle of forming a complex.The peak level of the melting curve is almost the same as it is in the protein control group,which is under 5 nm.The Zeta potential is strongly positive or moves in negative direction gradually and the absolute value becomes smaller tending to negative potential measured in the DNase I control group.By applying the principle of interaction between the enzymes and their substrates,we found that in the agarose gel electrophoresis,the enzyme DNase I can not act on plasmid DNA after modified and packed by the carrier.And in the SDS-PAGE electrophoresis,the substrate protein BSA can avoid being degraded by the proteinase K in a certain degree when the carrier coupled to BSA.Meanwhile,immunofluorescence assay indicates that the transfection effect is best when the mole number ratio of NG-GFP complex is 1:1.Western Blot consistently showed that when the ratio was 1:1,the expression of GFP in cell reaches the highest level.In addition,we also analyzed the effect of pH,serum concentration and additive PEG at the transfection.We found that pH 7.0 and the addition of 25 ug/mL PEG is in favor of the transfection while serum concentration seems not to affect the process.Based on these results,taking appropriate conditions,we successfully led GFP,?-galactosidase,Hand2,Gata4 into human bone mesenchymal stem cells(hBMSCs),human dermal fibroblast,Michigan Cancer Foundation-7(MCF-7),human umbilical venous endothelial cells(hUVEC)respectively,while the proteins led in still had activity.Finally,compared to gene vector,we found that PEI(1200KD)and NG particle basically didn't have the ability of carrying DNA.2.We used NG to deliver GFP into hBMSCs and found that green fluorescence inside the cell almost remained in cytoplasm and nearly had a same distribution as the endoplasmic reticulum lay.In the case of NG transfecting Hand2,we observed that the protein had entered into the cell only 1 hour after transfection.As the time went,expression level of Hand2 increased,reaching the peak at 24 hours.And Hand2 got the ability of translocating into nucleus at this moment.48 hours after transfection,metabolism of the protein intensified.96 hours after transfection,transfected Hand2 protein almost disappeared.By comparing the abilities of Liposome 2000,Pro-Ject and NG reagent of carrying Hand2,we found that the efficiency of Pro-Ject is better than NG while NG better than Liposome 2000.But under the light microscope,we found that Lipsome 2000 and Pro-Ject caused an obvious harmfulness to the cell.So we used MTT assay and AnnexinV-PI apoptosis test to do a full toxic detection for the carriers and the protein complexes.The result indicated that no matter on a long time-course or a short time-course,PEI had an obvious toxicity to the cell than the NG particle had.Besides,the toxicity of NG became less after long time acting instead of being more toxic.And apoptosis test indicated that the complexes formed by NG particle with small molecular weight and protein hurt the cell least,so NG particle with small molecular weight was better than NG with larger molecular weight in protein transfection.3.Four transcription factors,Gata4,Hand2,Mef2c and Tbx5 all can entirely be targeted and led into the hBMSCs nucleus when modified by NG reagent.Fluorescence microscope observation revealed a near 100%efficiency of transfection.The whole process of transfection took about more than 15 d to differentiate into myocardial line.And at the 5d,the appearance of cell already had a preliminary change.At the 8 d,cells began to form an aggregate clone state-like cell cluster.At the 15 d,the clone state became more obvious.The cells elongated and crevices gradually formed,dividing cells into different blocks.Result of Q-PCR showed the mRNA level of specific markers of myocardial l line Tbx5 and Nkx2.5,marker of multipotential stem cell line Oct4 and marker of smooth muscle cell ?-SMA all had a certain degree of increase.But formation time of t each gene's peak differs.With regard to the expression of protein,we found the expression of makers of all three cell lines of cardiac progenitor cell:endothelial cell line(such as CD31),cardiomyocyte line(such as Nkx2.5)and smooth muscle cell line(such as sm_MHC and ?-SMA)all had expressions at the 15d after tansfection,with their own expression timings and variations deffers.Conclusions:1.We successfully coupled deoxybibonuclease I(DNase I),bovine serum albumin(BSA),green fluorescent protein(GFP),?-galactosidase and four cardial-specific transcription factors GHMT to NG forming a nano protein complex,which is characterized by diameter and Zeta potential analysis.We found NG particle had a good ability of protecting protein,and the best mole number ratio between NG and protein(we mentioned above)is about 1:1,while it is 4:1 between PEI and protein.2.Four proteins,GFP,?-galactosidase,Hand2,Gata4(including plasmosin and nucleoprotein)were led into four kinds of cell,hBMSCs,hFF,MCF-7 and hUVEC(including somatic cell,stem cell and tumor cell).Except serum concentration had no obvious effect on the transfection,a pH value of 7.0 really did have a help to the transportion of protein.3.The speed of NG-protein complexes going across the cell membrane was very fast.Such as Hand2 protein,1 hour after the transfection,signs of protein going across membrane can be detected,ang had a total time spanning of metabolism of about 48 hours that means this was a transient transfection.The efficiency of Pro-ject(commercial kits of protein transfection)is higher than Liposome2000 significantly,and slightly higher than NG without significant differences.But except NG,the other two had obvious toxicity to the cell.Further more,NG had a relatively weak toxicity to the cell after long time acting.Especially complexes formed by small molecular NG particle and protein,which almost had never caused any apoptosis in the process of the complex entering the cell with an appropriate concentration.4.Induced by the GHMT modified by the NG,some other helper cytokines and small molecule compound,hBMSCs represented as an initial state of cells'clone.After 15 d or more,the cells became cardiac progenitor cells that include three myocardial cell lines.The expression level of mRNA and protein specific in the each cell line all increased remarkably.And the cells became more multipotential.Therefore,NG is a excellent cell-reprogramming nano reagent,which can transduct protein into nuleus specifically,showing an uncomparable advantage when compares with virus or plasmid transfection. |
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