CN101812429A - Mouse cardiac muscle progenitor cell and preparation method thereof - Google Patents

Abstract

The invention discloses a mouse cardiac muscle progenitor cell and a preparation method thereof. The mouse cardiac muscle progenitor cell expresses a cardiac muscle progenitor cell mark ISL1 and a mouse cardiac muscle cell line early stage mark Tbx5. The preparation method comprises the following steps: firstly, separating mouse cardiac muscle cells of embryo culture for 15.5 days; infecting immortalizing plasmids SSR#69 for immortalizing the mouse cardiac muscle cells; obtaining immortalized monoclonal cardiac muscle cells by an antibiotic sieving method and an unlimited dilution method; then, sieving the cardiac muscle cells expressing ISL1 and Tbx5 from the immortalized monoclonal cardiac muscle cells; and obtaining the mouse cardiac muscle progenitor cells. The mouse cardiac muscle progenitor cells are reversible immortalized cells, can realize immortalization, have no risk for causing tumor and causing virus infection, belong to ideal tools for researching the influence on the multiplication and differentiation factors of the cardiac muscle progenitor cells under the condition of damaged adult muscle progenitor cells. The invention has good application prospects.

Description

Mouse cardiac muscle progenitor cell and preparation method thereof

Technical field

The present invention relates to a kind of progenitor cell, (cardiac progenitor cell CP), also relates to the preparation method of this cardiac muscle progenitor cell to particularly a kind of mouse cardiac muscle progenitor cell.

Background technology

Effectively treating to reduce mortality ratio to heart disease is one of great difficult problem of current medical science.The cardiac muscle regenerative power limited, in case sustain damage, functional myocardial cell's death or apoptosis will inevitably appear, influence the normal function of heart, finally cause chronic heart failure (chronic heart failure, CHF).Use the conventional medicament therapy to comprise receptor blocking agent, Zinc metallopeptidase Zace1 (ACE) inhibitor, aldosterone antagonist and diuretic(s) etc.; though can protect cardiac muscle to a certain extent; delay the process of heart disease; but can't make functional myocardial cell's regeneration; be difficult to fundamentally reverse the development process of CHF; still between 8～10%, admission rate is between 6～8% again for the mortality ratio of heart stalk back heart failure patient.And cardiac transplantation is also limited because of donor, and reason such as immunological rejection is difficult in large-scale promotion clinically in addition.

What allow now cardiologist and heart failure patient see dawn is the relevant research that utilizes Transplanted cells Regeneration and Repair damaged myocardium of rising in recent years, it has tempting prospect in theory, and its feasibility and effect are also confirmed by some experimentation on animals.In year surplus in the of nearly ten, Chinese scholars is being done number of research projects aspect the seed cell of screening Transplanted cells, filters out two class seed cells: become somatocyte and stem cell.But find simultaneously, cellular replacement therapy myocardial infarction with marrow and embryonic origin remains in deficiency, behind injection transplantation stem cell in the direct cardiac muscle, owing to transplant complicated component, stem cell still shows the polytropism to differentiation such as cardiac-like muscle cell and tumour cells under the inducing of myocardium microenvironment, and bring a series of complication of transplant thus, as little infarct, irregular pulse, calcification and tumour etc., its reason may be that the myocardium microenvironment after the myocardial damage is not quite similar with myocardium microenvironment and relevant regulatory gene in the embryo development procedure, and the stem cell of these virgin states can not be by normal regulation in the microenvironment of ripe myocardial damage; And behind the intravascular transplanting stem cell, find to have only the cell of only a few to rest on heart by the phenomenon of going back to the nest of cell, improve very little to heart function.Therefore, we need mobilize the cardiac muscle progenitor cell of heart existence itself to come compensatory damaged myocardium function.But how could effectively mobilize? what is the mechanism of its mobilization? in order to address this problem, and finally be implemented in laboratory animal and application clinically, we must at first obtain these cardiac muscle progenitor cells, study its characteristic, then the further factor of its propagation of analyzing influence and differentiation.

Summary of the invention

In view of this, one of purpose of the present invention is to provide a kind of mouse cardiac muscle progenitor cell that derives from heart tissue, and cardiac muscle progenitor cell is bred under adult myocardial cell damage situations and the factor of breaking up provides the ideal instrument for research influences; Two of purpose is to provide the preparation method of described mouse cardiac muscle progenitor cell.

For achieving the above object, the present invention adopts following technical scheme:

1, mouse cardiac muscle progenitor cell, it expresses cardiac muscle progenitor cell mark ISL1 and the myocardial cell is early sign thing Tbx5.

Further, also to express the myocardial cell be early sign thing Nkx2.5 to described cardiac muscle progenitor cell;

Further, described cardiac muscle progenitor cell is also expressed stem cell markers Oct3/4, Nanog and Sox2;

Further, described cardiac muscle progenitor cell is also expressed cardiac muscle progenitor cell mark Sca-1 and c-kit;

Further, described cardiac muscle progenitor cell express alpha-MyHC after inducing differentiation;

Further, described cardiac muscle progenitor cell is in inducing atomization, cardiac muscle progenitor cell mark ISL1 and myocardial cell are that early sign thing Nkx2.5 and Tbx5 expression are on a declining curve, myocardial cell's structural protein mark α-MyHC, α-actin and cTnT express in rising trend, and skeletal muscle mark MyoD and unstriated muscle mark SM-MHC do not express;

Further, described cardiac muscle progenitor cell is the reversibility immortalized cells;

Further, described cardiac muscle progenitor cell derives from the embryo heart tissue.

2, the preparation method of described mouse cardiac muscle progenitor cell may further comprise the steps:

A, 15.5 days mouse cardiac muscle cell of separation and Culture embryo;

B, step a gained myocardial cell is infected immortalization plasmid SSR#69 and immortalization, by the mono-clonal myocardial cell of antibiotic-screening method and being immortalized of infinite dilution method;

C, from the mono-clonal myocardial cell of step b gained immortalization, filter out and express cardiac muscle progenitor cell mark ISL1 and the myocardial cell is the myocardial cell of early sign thing Nkx2.5 and Tbx5, promptly get mouse cardiac muscle progenitor cell.

Beneficial effect of the present invention is: mouse has up to 90% homology and both cardiovascular systemss very similar on gene to the people, the present invention selects for use mouse heart and myocardial cell to be research object, set up a kind of cardiac muscle progenitor cell that derives from heart tissue, this cardiac muscle progenitor cell is the reversibility immortalized cells, after knocking out immutalizing gene, can be returned to the primary cell state before the immortalization, thereby make the cell can infinite multiplication, there are not tumorigenesis and the danger that causes virus infection again, be that research influences cardiac muscle progenitor cell breeds and break up factor under adult myocardial cell damage situations ideal tools, good prospects for application is arranged.

Description of drawings

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is described in further detail below in conjunction with accompanying drawing, wherein:

Fig. 1 is vitro culture myocardial cell's a form (amplifying 200 times);

Fig. 2 is immortalization myocardial cell's a mono-clonal growing state (amplifying 300 times);

Fig. 3 detects the expression sequential (agarose gel electrophoresis figure) of specific heart gene in the heart development process for RT-PCR;

Fig. 4 detects the expression sequential (goal gene relative expression quantity statistical graph) of specific heart gene in the heart development process for RT-PCR;

Fig. 5 is RT-PCR screening cardiac muscle progenitor cell (agarose gel electrophoresis figure); 1～5,5A and 6～19 is respectively the immortalization myocardial cell and clones CP15-1～5, CP15-5A and CP15-6～19, and H9 is H9C2, and P19 is P19CL6;

Fig. 6 is RT-PCR screening cardiac muscle progenitor cell (goal gene relative expression quantity statistical graph); 1～5,5A and 6～19 is respectively the immortalization myocardial cell and clones CP15-1～5, CP15-5A and CP15-6～19;

Fig. 7 is that all-trans-retinoic acid (ATRA) is induced MyHC-GLuc screening active ingredients cardiac muscle progenitor cell; * represent P＜0.05;

Fig. 8 induces MyHC-GLuc screening active ingredients cardiac muscle progenitor cell (GTG figure) for RT-PCR and ATRA;

Fig. 9 detects ISL1 and the expression (amplify 200 times) of c-kit in the two positive colonies of ISL1/Tbx5 for immunofluorescence;

Figure 10 detects the expression of SV40T in the cardiac muscle progenitor cell for Western Blot;

Figure 11 is the cardiac muscle progenitor cell growth curve;

Figure 12 is the MyHC-GLuc activity that dexamethasone (Dex) is induced the differentiation cardiac muscle progenitor cell; * represent P＜0.05;

Figure 13 is the virus infection situation of cardiac muscle progenitor cell;

Figure 14 infects respectively behind AdRFP and the AdR-cre in the intravital luciferase imaging of nude mice for cardiac muscle progenitor cell;

Figure 15 infects respectively behind AdRFP and the AdR-cre in the intravital luciferase imaging of nude mice (average signal strength statistical graph) for cardiac muscle progenitor cell;

Figure 16 induces cardiac muscle progenitor cell differentiation (amplifying 200 times) for Dex;

Figure 17 detects the expression that Dex induces the myocardium development related gene of differentiation cardiac muscle progenitor cell for real-time quantitative PCR;

Figure 18 induces the cTnT of differentiation cardiac muscle progenitor cell to express for immunofluorescence detects Dex.

Embodiment

Hereinafter with reference to accompanying drawing, the preferred embodiments of the present invention are described in detail.Should be appreciated that preferred embodiment only for the present invention is described, rather than in order to limit protection scope of the present invention.

The main experiment material of using in the preferred embodiment:

(1) animal: the pregnant 15.5 days healthy CD1 mouse and the healthy immune deficiency BALB/c nude mice in 6～8 ages in week, the SPF level, provide by Univ Chicago USA's animal center, feed in the aseptic layer fluid room of strict sterilization, envrionment temperature maintains 25 ℃, atmospheric moisture is 60%～70%, feed and water ad lib after sterilizing.

(2) bacterium and cell strain: intestinal bacteria DH10B is available from U.S. invitrogen company.Mouse teratocarcinoma cell strain P19CL6 and mouse myocardial cell strain H9C2 are available from U.S. typical case DSMZ (ATCC), and mouse sarcoplast strain C2C12, human embryonic kidney cell's strain HEK293 and human colon cancer cell strain HCT116 are provided by Chicago University medical center molecular weight tumor research department.

(3) plasmid: immortalization plasmid SSR#69 and plasmid pAmpho are provided by Chicago University medical center molecular weight tumor research department.The promoter plasmid MyHC-GLuc that carries promoter gene α-MyHC (myoglobulin heavy chain α) and reporter gene GLuc (secretor type luciferase) makes up (Gao-Hui Zhu.Differentiation.2009 Jun26.PMID:19560855) by the contriver in previous work, method is as follows: go up the fragment that α-MyHC gene order (accession number is NM_001091601) of logining designs following primer amplification α-the 4th exon upstream 5.5kb of MyHC gene according to geneseq database (GenBank), primer sequence is as follows: F:agg GgatccTgcaaggtcacacaagag (underscore partly is the BamHI restriction enzyme site), R:ccg CtcgagGtcactcaaactcttatgggggag (underscore partly is the XhoI restriction enzyme site); With the plasmid that contains α-MyHC gene is that template is carried out PCR, and the PCR reaction conditions is: 96 ℃ of pre-sex change 45 seconds, and 20 seconds, 55 ℃ annealing of 92 ℃ of sex change were extended 45 seconds for 30 seconds, 70 ℃ again, totally 28 circulations, last 70 ℃ were extended 5 minutes; The PCR product is after agarose gel electrophoresis is identified, cut glue and reclaim purifying, carry out double digestion with restriction enzyme BamHI and XhoI, plasmid pBGLuc (being provided by Chicago University medical center molecular weight tumor research department) with the same double digestion of warp is connected under the effect of T4 dna ligase again, connect the product electricity and be transformed into the big enterobacteria DH10B of competence, the converted product LB plate screening positive colony that contains kantlex, by bacterium colony PCR, digestion with restriction enzyme and determined dna sequence are identified positive colony, promptly get promoter plasmid MyHC-GLuc.This plasmid and myocyte are broken up irrelevant control plasmid TOP-Luc transfection C2C12 cell respectively, with to contain massfraction be the DMEM inducing culture of 2% horse serum or carry out inducing culture with Dex as inductor, by the GLuc reading, find that this plasmid has muscle specific, and its activity can effectively be activated in myocyte's atomization, express alpha-MyHC in cell differentiation procedure, it will start the expression of reporter gene GLuc, therefore can weigh the expression of α-MyHC in the cell by the expression amount of measuring GLuc in the cell culture supernatant, furthermore the dynamic process of clear-cells differentiation.

(4) adenovirus: the recombinant adenovirus AdRFP that carries red fluorescent protein (RFP) gene is provided by Chicago University medical center molecular weight tumor research department with the recombinant adenovirus AdR-cre that carries recombinase Cre gene.

(5) reagent: DMEM substratum, trypsinase, microbiotic (penicillin, penbritin, Streptomycin sulphate and Totomycin), RNA extracts reagent Trizol, six gather primer Hexamer at random, the Superscrip reversed transcriptive enzyme, RNA enzyme inhibitors RNasin, SDS-PAGE albumen sample-loading buffer, dna molecular amount standard, lipofectamine Lipofactamine and Western Blot related reagent are available from U.S. invitrogen company, foetal calf serum (FBS) and ultrapure water are available from U.S. Gibico company, the PCR primer is synthetic by American I DT company, dNTP, the Taq archaeal dna polymerase, dimethyl sulfoxide (DMSO) (DMSO), Realtime PCR test kit, the Gluc detection kit is available from U.S. NEB company, agarose, bromination second pyridine (EB) and polybrene (Polybrene) are available from U.S. Sigma company, glue reclaims purification column available from U.S. Millipore company, cell lysis buffer solution and plasmid a small amount of extraction agent box are available from U.S. Promega company, immunofluorescence antibody is available from Britain Abcam company, and avidin (Avdin)/vitamin H (Biotin) closed reagent box is available from U.S. VECTOR company.Concentration is that 0.1mol/L, pH are 7.2～7.4 phosphate buffered saline buffer (PBS) autogamy: take by weighing NaCl 9.0g, Na ₂HPO ₄12H ₂O 6.0g and NaH ₂PO ₄2H ₂O0.4g, make fully dissolving with distilled water after, regulate pH to 7.2～7.4, be settled to 1000ml with distilled water again, packing, 1.05kPa autoclaving 20min, 4 ℃ of preservations are standby.

(6) instrument: CO ₂Incubator (Thermo Forma 3110 types) and Bechtop are available from U.S. Thermo FisherScientific company, inverted microscope and gel imaging instrument are available from Japanese Olympus company, the fluorescence inverted microscope is available from Japanese Nikon company, PCR instrument (Gene Cycler ^TM) available from U.S. Bio-Rad company, spectrophotometer, chemoluminescence and fluor tester are available from U.S. Beckman company.

The experimental technique of unreceipted actual conditions in the preferred embodiment, usually according to normal condition, the molecular cloning experiment guide (third edition for example, J. work such as Sa nurse Brooker, Huang Peitang etc. translate, Science Press, 2002) described in condition, or the condition of advising according to manufacturer.

1, myocardial cell's separation and Culture

With pregnant 15.5 days healthy CD1 mouse CO ₂After sucking execution, take out the mice embryonic heart, put and fill in the Tissue Culture Dish of PBS that 25ml is chilled to 4 ℃ in advance, chopping and piping and druming repeatedly, adding 2ml massfraction is 0.25% trypsin solution, 37 ℃ digested 5 minutes, leaving standstill afterwards, transfer supernatant liquor to the collagen solution bag that through massfraction is 1% is spent the night and is filled in the Tissue Culture Dish of 15mlDMEM perfect medium, it is 0.25% trypsin solution that the residue heart tissue adds the 2ml massfraction again, 37 ℃ digested 5 minutes, leave standstill the back and shift supernatant liquor to same Tissue Culture Dish, at 37 ℃, volume fraction is 5% CO ₂With cultivated 1 hour under the condition of saturated humidity, cell culture fluid being transferred to another is to continue to cultivate in the Tissue Culture Dish that spent the night of 1% collagen solution bag through massfraction again, changes fresh DMEM perfect medium after 24 hours, removes the hemocyte that suspends.

Vitro culture myocardial cell's form as shown in Figure 1, the myocardial cell is into the fiber-like form, and cell is constantly bred single or agglomerating the beating of visible cell.

2, myocardial cell's immortalization

With the blank substratum mixing of 2.0 μ g SSR#69,1.0 μ g pAmpho, 15 μ l Lipofectamine and 250 μ l DMEM, left standstill under the room temperature 30 minutes, make transfection mixture; HEK293 is inoculated in the Tissue Culture Flask with 60% density, is 5% CO at 37 ℃, volume fraction with the DMEM perfect medium ₂With cultivate under the condition of saturated humidity, after treating cell attachment, absorb substratum, with the blank substratum washing of 3ml DMEM 1 time, add the blank substratum of 2.5ml DMEM, hatched 5～10 minutes, add above-mentioned transfection mixture again, hatch the fresh DMEM perfect medium of replacing after 4 hours, the 36th hour sucking-off substratum is the filter filtration of 0.2 μ m with the aperture, filtrate transfers to transfection myocardial cell in myocardial cell's culturing bottle, change fresh DMEM perfect medium after 12 hours, add the Totomycin solution screening immortalization myocardial cell of 0.1mg/ml after 24 hours, screening time was 2 weeks.

3, immortalization myocardial cell mono-clonal

Using the infinite dilution method, the immortalization myocardial cell who filters out is inoculated into 96 orifice plates, stop dilution when every hole, 1/3 hole only contains 1 cell to 96 orifice plates, is 5% CO at 37 ℃, volume fraction with the DMEM perfect medium of the Totomycin that contains 0.1mg/ml ₂With continue screening and culturing under the condition of saturated humidity, when treating that the cell growth reaches 1/3 hole area substantially, use tryptic digestion, the Tissue Culture Flask of 24 orifice plates, 6 orifice plates, 25ml and the Tissue Culture Dish of 100mm successively go down to posterity, last frozen standby, before frozen, all use the DMEM perfect medium of the Totomycin that contains 0.1mg/ml to carry out screening and culturing, obtain 20 immortalization myocardial cell clones, called after CP15-1～5, CP15-5A and CP15-6～19 respectively altogether.

Immortalization myocardial cell's mono-clonal growing state as shown in Figure 2, the visible individual cells growth in the 3rd day of inoculation back, visible cell division in the 5th day forms 2～3 cells, occurs 1 little cell clone group in the time of the 10th day, and cell clone group reaches 1/3 hole area substantially in the time of the 15th day.

4, RT-PCR screening cardiac muscle progenitor cell

Because the isolation and identification method and the mark of the cardiac muscle progenitor cell of bibliographical information are had nothing in common with each other, therefore, extract embryo 13.5 days (E13.5) and 18.5 days (E18.5) at first respectively, back 1 day (D1), 3 days (D3), 7 days (D7) and 14 days (D14) are born, and the healthy CD1 mouse heart total tissue RNA of birth back 8 weeks (adult), adopt RT-PCR to detect the expression sequential of specific heart gene in the heart development process, with identify the early stage of myocardial cell and late period mark.

Concrete grammar is as follows: get the frozen heart tissue of 50mg, add 1ml Trizol, fully after the homogenate, add 360 μ l chloroforms, fully after the concussion, 14000rpm low-temperature centrifugation 20 minutes, get supernatant liquor, add 670 μ l Virahols and 5 μ l glycogens (Gly), fully after the concussion, 14000rpm low-temperature centrifugation 15 minutes, abandon supernatant liquor, add 600 μ l massfractions and be 70% ethanolic soln, fully after the concussion, 14000rpm low-temperature centrifugation 5 minutes, abandon supernatant liquor, dry, add 50 μ l and go endotoxic distilled water to make dissolving, get the heart tissue total rna solution ,-80 ℃ of preservations are standby.Go up the sequences Design PCR primer (primer sequence sees Table 1) of the mouse heart specific gene of login according to GenBank.The RT reaction method is: with Hexamer solution 2 μ l and the heart tissue total rna solution 10 μ l mixings of 0.5 μ g/ μ l, 70 ℃ were reacted 5 minutes, and made the Hexamer-RNA mixture; DNTPs solution 1 μ l, RNasin 0.2 μ l and the distilled water 1.6 μ l of dithiothreitol (DTT) solution 2 μ l, the 10mmol/L of 5 * damping fluid, 2 μ l, 0.1mol/L being mixed, make the RT reaction mixture on ice; Hexamer-RNA mixture, RT reaction mixture and reversed transcriptive enzyme 0.25 μ l are mixed, and 37 ℃ were reacted 60 minutes, and 95 ℃ were reacted 1 minute, were cDNA with the RNA reverse transcription, and being diluted to cumulative volume with distilled water again is 100 μ l, and-20 ℃ of preservations are standby.The PCR reaction system is: with dNTPs solution 2.4 μ l, the DMSO 1.2 μ l of 10 * damping fluid, 2.0 μ l, 10mmol/L, each 0.4 μ l of the primers F of 330ng/ μ l/R solution, cDNA template (RT reaction product) 10 μ l, Taq archaeal dna polymerase 0.2 μ l and distilled water 3.5 μ l mix, and cumulative volume is 20 μ l; Reaction conditions is: 95 ℃ of pre-sex change 2 minutes; 20 seconds, 68 ℃ annealing of 92 ℃ of sex change were extended 30 seconds for 40 seconds, 72 ℃ again, totally 12 circulations, and 1 annealing temperature of every circulation reduces by 1 ℃; 20 seconds, 56 ℃ annealing of 92 ℃ of sex change were extended totally 21～32 circulations 30 seconds for 40 seconds, 72 ℃ again; Last 72 ℃ were extended 5 minutes.It is 1% agarose gel electrophoresis that the PCR product carries out massfraction, the imaging of gel imaging instrument is preserved, the Bio-Rad image analysis system is measured optical density(OD) (OD) value of each DNA band, and be that confidential reference items are proofreaied and correct with the result with the GAPDH gene, goal gene relative expression quantity=goal gene OD value/GAPDH gene OD value.

The PCR primer of table 1 mouse heart specific gene

RT-PCR detect the specific heart gene in the expression sequential in the heart development process shown in Fig. 3～4, visible ISL1 is expressed in the commitment of myocardial cell E13.5 specifically, almost detected during E18.5 less than; In the growth course that becomes systemic heart, expression amount reduces c-kit gradually at E13.5, and the expression trend of Sca-1 is opposite with c-kit, is trend of rising gradually; Cardiac muscle is early expression gene Nkx2.5 with the reaching maturity and reduce gradually of heart, and gap junction protein Cx43 does not have considerable change trend, and late structural proteins α-MyHC, α-actin and cTnT then reach maturity with heart and increase gradually.

According to above-mentioned detected result and in conjunction with bibliographical information, choose specificity higher progenitor cell marker thing ISL1, myocardial cell and be mark Nkx2.5 and Tbx5 leading indicator as the screening cardiac muscle progenitor cell; Stem cell markers Oct3/4, Nanog and Sox2 are used to detect institute's isolated cells and whether have the marker gene of stem cell self; Other progenitor cell marker thing and myocardial cell are that mark is used for the auxiliary residing myocardial cell's etap of cell of weighing.According to These parameters, adopt RT-PCR from 20 immortalization myocardial cells clones, to screen cardiac muscle progenitor cell, and be usually used in studying P19CL6 that the myocardial cell grows and H9C2 in contrast with bibliographical information.The concrete grammar of RT-PCR is the same, and the PCR primer sequence sees Table 2.

The PCR primer of myocardial cell's marker gene of table 2 heart development different steps

RT-PCR screening cardiac muscle progenitor cell such as Fig. 5～6 and shown in Figure 8, stem cell markers Oct3/4, Nanog and Sox2 express in 20 immortalization myocardial cell clones and P19CL6 basically, but do not express (Fig. 5 A) in H9C2; Specificity cardiac muscle progenitor cell mark ISL1 expresses in CP15-5A, CP15-9, CP15-10, CP15-13, CP15-18 and P19CL6, high expression level in CP15-9 particularly, non-specific cardiac muscle progenitor cell mark Sca1 and c-kit also express in most of immortalization myocardial cell clones, but ISL1, Sca1 and c-kit still do not express (Fig. 5 B) in H9C2; The myocardial cell is that early sign thing Nkx2.5, GATA4, Tbx5, Hand2 and Flk-1 all have expression in various degree in CP15-5A, CP15-9, CP15-10, CP15-13, CP15-18 and P19CL6, wherein the Tbx5 expression amount is higher and difference is more obvious, but the relatively low Hand2 of expression specificity only among the H9C2, other marks almost do not have expression (Fig. 5 C); Myocardial cell's structural protein mark α-MyHC, Actc1, cTnI and ANF be the relatively low and no significant difference of expression amount in 20 immortalization myocardial cell clones and P19CL6, and cTnI high expression level (Fig. 5 D) in H9C2.

5, ATRA induces MyHC-GLuc screening active ingredients cardiac muscle progenitor cell

Myosin (MyHC) is the important structure integral part of Muscle contraction unit, the wherein existence of α-MyHC and express specificity in a organized way, and therefore, the existence of α-MyHC has the important hints meaning to the muscle cell atomization.

With 2 μ g MyHC-GLuc, 15 μ l Lipofactamine and the blank substratum mixing of 500 μ l DMEM, placed under the room temperature 10～30 minutes, make transfection mixture; 20 immortalization myocardial cells clone that will be in exponential phase of growth respectively is inoculated in the Tissue Culture Flask with 60～80% density, is 5% CO at 37 ℃, volume fraction with the DMEM perfect medium ₂With cultivate under the condition of saturated humidity, after treating cell attachment, absorb substratum, with the blank substratum washing of 3.0ml DMEM 1 time, add 2.5ml DMEM perfect medium, hatched 10 minutes, add above-mentioned transfection mixture again, hatch after 4 hours and be replaced by the DMEM perfect medium, the ATRA solution that adds final concentration after 12 hours and be 0.5 μ mol/L is induced differentiation, activates the MyHC-GLuc activity, respectively at inducing the back to draw 20 μ l cell culture supernatants on the the 3rd, 6,12 and 18 day, with the expression amount of GLuc detection kit detection GLuc, operate according to the test kit specification sheets.

ATRA induces MyHC-GLuc screening active ingredients cardiac muscle progenitor cell (Fig. 7 has only listed through RT-PCR and detected data for ISL1/Tbx5 two positive colony CP15-5A, CP15-9, CP15-10, CP15-13 and CP15-18 and 3 ISL1/Tbx5 jack to jack adapter sex clone CP15-2, CP15-3 and CP15-19) shown in Fig. 7～8, as seen ATRA can induce the MyHC-GLuc activity of the two positive colonies of ISL1/Tbx5 effectively, particularly in the time of the 18th day (p＜0.05).

6, immunofluorescence detects ISL1 and the expression of c-kit in the two positive colonies of ISL1/Tbx5

In order further to verify RT-PCR result, adopt immunofluorescence technique to detect ISL1 and the expression of c-kit in ISL1/Tbx5 two positive colony CP15-5A, CP15-9, CP15-10, CP15-13 and CP15-18, simultaneously in contrast with 2 ISL1/Tbx5 jack to jack adapter sex clone CP15-12 and CP15-17.

Concrete grammar is as follows: the immortalization myocardial cell clone that will be in exponential phase of growth is inoculated into 60～80% density and cultivates in the chamber slide system, is 5% CO at 37 ℃, volume fraction with the DMEM perfect medium ₂With cultivate under the condition of saturated humidity, after treating cell attachment, absorb substratum, get the chamber slide, with PBS washing 1 time, cold acetone is fixed 10 minutes, drying is 10 minutes under the room temperature, and PBS washing 1 time is that the 5% and two anti-serum that belong to the source together sealed 20 minutes with volume fraction, PBS washing 1 time, add anti-ISL1 monoclonal antibody or anti-c-kit monoclonal antibody (extent of dilution is 1: 200), establish an anti-control group (monoclonal antibody that adds IgG hypotype of the same race) simultaneously, hatched under the room temperature 60 minutes or 4 ℃ of overnight incubation, PBS washing 3 times, add corresponding two anti-(extent of dilution is 1: 200) of DyLight 594 (green fluorescence) mark again, hatched under the room temperature 30 minutes, PBS washing 3 times, mounting is put under the fluorescent microscope and is observed.

Immunofluorescence detects ISL1 and the expression of c-kit in the two positive colonies of ISL1/Tbx5 as shown in Figure 9, and ISL1 expresses the strongest in CP15-9 and a little less than expressing in CP15-13, an anti-control group is not almost expressed, a little less than control group is expressed (Fig. 9 A); C-kit all has expression in all the other each groups except almost not expressing in an anti-control group, especially express in CP15-9 and CP15-13 strong (Fig. 9 B); Consistent with RT-PCR result.

Comprehensive above-mentioned RT-PCR result, ATRA induce active result of MyHC-GLuc and immunofluorescence result, filter out 4 clone CP15-5A, CP15-9, CP15-10 and CP15-18 with cardiac muscle progenitor cell characteristic.

7, Western Blot detects the recoverability of cardiac muscle progenitor cell

Contain the SV40T immutalizing gene in the SSR#69 structure, and the action target spot LoxP that before and after the SV40T gene, has added recombinase Cre respectively, when Cre existed, the SV40T gene can be acted on 2 LoxP sites by Cre and excise, thereby makes immortalized cells be returned to the normal growth state.

CP15-9 and CP15-10 are infected AdR-Cre respectively, and collecting cell cracking total protein after 48 hours with the expression of Western Blot detection SV40T, is observed the recoverability of cardiac muscle progenitor cell.Concrete grammar is as follows: total protein is mixed with SDS-PAGE albumen sample-loading buffer, water-bath was boiled 10 minutes, last sample carries out SDS-PAGE, after electrophoresis finishes, with the protein electrotransfer to pvdf membrane, with massfraction is that 1% skim-milk solution room temperature lower seal was closed 1～2 hour, (with containing massfraction is that the TBST of 1% skim-milk dilutes to add anti-SV40T monoclonal antibody again, extent of dilution is 1: 500), 4 ℃ of overnight incubation, TBST washing 3 times, colour developing, the imaging of gel imaging instrument is preserved, and is that confidential reference items are proofreaied and correct with the result with β-actin.

The expression that Western Blot detects SV40T in the cardiac muscle progenitor cell as shown in figure 10, visible Cre can effectively remove the SV40T gene, reduces its expression in cardiac muscle progenitor cell, thereby makes the state before cardiac muscle progenitor cell returns to immortalization.

8, immutalizing gene is to the influence of cardiac muscle progenitor cell propagation and differentiation

CP15-5A, CP15-9, CP15-10 and CP15-18 are infected AdRFP and AdR-Cre respectively, carried out cell counting on the the 1st, 2,3,4,5 and 6 day, observe the influence of the existence of SV40T gene cardiac muscle progenitor cell propagation in infecting the back.The cardiac muscle progenitor cell growth curve as shown in figure 11, the growth tendency of visible Ad-RFP group and AdR-Cre group is consistent, but the cell proliferation that Ad-Cre organizes is slower, shows that the existence of SV40T gene can improve the multiplication capacity of cardiac muscle progenitor cell.

Behind CP15-9 transfection MyHC-GLuc, infect Ad-RFP and AdR-Cre more respectively, Dex solution with 0.5 μ mol/L is induced differentiation, activate the MyHC-GLuc activity, drew 20 μ l cell culture supernatants on the the 3rd, 6,9 and 12 day in inducing the back, with the expression amount of GLuc detection kit detection GLuc,, observe the influence of the existence of SV40T gene to the cardiac muscle progenitor cell differentiation according to the operation of test kit specification sheets.When the active detection of MyHC-GLuc, for the cell concentration of balanced Ad-RFP group with the AdR-Cre group, application cell cracking total protein concentration comes stdn GLuc reading.Dex induce the differentiation cardiac muscle progenitor cell the MyHC-GLuc activity as shown in figure 12, with respect to the blank group, Dex can effectively induce the MyHC-GLuc activity (p＜0.05) of differentiation cardiac muscle progenitor cell, and blank group and Dex induce in the group AdR-Cre little to the active influence of MyHC-GLuc, show that the existence of SV40T gene is less to the influence of cardiac muscle progenitor cell differentiation.

9, the tumorigenicity of cardiac muscle progenitor cell detects

With plasmid pSEB-Luc and pAmpho cotransfection HEK293, at 37 ℃, volume fraction 5% CO ₂With cultivate under the condition of saturated humidity, begin to collect viral liquid after 36 hours, collected 1 time in per 12 hours, and collected altogether 3 times, after every ml virus liquid adds the polybrene of 6 μ l2mg/ml, divide 4 subinfection CP15-9 or CP15-10, each effect 4～8 hours was cultivated 8～12 hours with fresh DMEM perfect medium between each, and the blasticidin of adding 2.0 μ g/ml screened for 1 week behind last 1 subinfection, form the cell strain CP15-9* or the CP15-10* of stably express luciferase, it is frozen standby to go down to posterity again; Cultivate CP15-9* or CP15-10* with the DMEM perfect medium, treat that cell attachment infects Ad-RFP and AdR-Cre respectively when growing to 80% fusion, change fresh DMEM perfect medium after 12 hours, put in the 48th hour under the fluorescent microscope and observe, the cell infection rate is about 60～80% (as shown in figure 13) substantially, use tryptic digestion, collecting cell, centrifugal 5 minutes of 1000rpm, abandon supernatant liquor, cell precipitation with the PBS washing that contains phosphatidylserine after, be resuspended among the 50 μ l PBS, put preserve on ice standby; With 1 * 10 ⁶It is subcutaneous that individual cell through above-mentioned processing is transplanted to nude mice, carried out the luciferase imaging on the 1st, 3 and 7 day in transplanting the back, and carry out signal strength analysis.

Cardiac muscle progenitor cell infects respectively behind Ad-RFP and the AdR-Cre in the intravital luciferase imaging of nude mice shown in Figure 14～15, as seen prolong in time after transplanting, the luciferase signal of Ad-RFP group and AdR-Cre group weakens gradually and finally disappears, the blackout of AdR-Cre group is morning, and the cardiac muscle progenitor cell of transplanting does not form tumour in nude mouse.

10, Dex induces the differentiation of cardiac muscle progenitor cell

CP15-5A, CP15-9, CP15-10 and CP15-18 are induced differentiation with the Dex solution of 0.5 μ mol/L respectively, put observation of cell form change under the bright field microscope in the time of the 14th day, as shown in figure 16, CP15-5A, CP15-9 and CP15-10 induce differentiation after 14 days at Dex, it is roomy that cellular form becomes, queueing discipline is tight between the cell, and the CP15-18 cellular form is elongated, and fasciculation is arranged.

11, real-time quantitative PCR detects the expression that Dex induces the myocardium development related gene of differentiation cardiac muscle progenitor cell

CP15-5A, CP15-9, CP15-10 and CP15-18 are induced differentiation with the Dex solution of 0.5 μ mol/L respectively, extract cell total rna when the 7th day and the 11st day, obtain cDNA by RT, the real-time quantitative PCR that carries out myocardium development related gene again detects (primer sequence sees Table 3), and is that confidential reference items are proofreaied and correct with GAPDH with the result.

The PCR primer of the myocardium development related gene of table 3

Real-time quantitative PCR detect Dex induce the differentiation cardiac muscle progenitor cell myocardium development related gene expression as shown in figure 16, totally it seems, compare with blank group (not adding Dex), CP15-9 and CP15-10 induce in the atomization at Dex, early gene ISL1, Nkx2.5 and Tbx5 express on a declining curve, late gene α-MyHC, α-actin and cTnT express in rising trend, and skeletal muscle marker gene MyoD and unstriated muscle marker gene SM-MHC express hardly; And in CP15-5A and CP15-18, do not have fixedly variation tendency.

12, immunofluorescence detects the cTnT expression that Dex induces the differentiation cardiac muscle progenitor cell

CP15-5A, CP15-9, CP15-10 and CP15-18 are induced differentiation with the Dex solution of 0.5 μ mol/L respectively, and fixed cell in the time of the 14th day adopts immunofluorescence technique to detect the expression of myocardial cell's structural protein cTnT.As seen the result compares with blank group (not adding Dex) as shown in figure 18, and cTnT induces the expression in the differentiation cardiac muscle progenitor cell stronger, wherein relative stronger with the expression of CP15-9 and CP15-10 again at Dex, and has form to change.

Generally speaking, preferred embodiment is 15.5 days a mouse cardiac muscle cell of first separation and Culture embryo, make it infect immortalization plasmid SSR#69 and immortalization, mono-clonal myocardial cell by antibiotic-screening and being immortalized of infinite dilution method, induce the MyHC-GLuc activity according to genetic expression sequential and ATRA in myocardial cell's growth course again, and immunofluorescence technique checking, filter out 4 clone CP15-5A with cardiac muscle progenitor cell characteristic, CP15-9, CP15-10 and CP15-18, in further real-time quantitative PCR and immunofluorescence screening process, find that CP15-9 and CP15-10 induce in the atomization at Dex, early gene ISL1, Nkx2.5 and Tbx5 express gradually and reduce, late gene α-MyHC, α-actin and cTnT express obviously and raise, and skeletal muscle and unstriated muscle gene are expressed hardly, are more excellent clone.Result of study also shows, 4 cardiac muscle progenitor cell mono-clonals that preferred embodiment filters out are the reversibility immortalized cells, after knocking out immutalizing gene, can be returned to the primary cell state before the immortalization, thereby make the cell can infinite multiplication, there are not tumorigenesis and the danger that causes virus infection again, be that research influences cardiac muscle progenitor cell breeds and break up factor under adult myocardial cell damage situations ideal tools, good prospects for application is arranged.

Explanation is at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although by invention has been described with reference to the preferred embodiments of the present invention, but those of ordinary skill in the art is to be understood that, can make various changes to it in the form and details, and the spirit and scope of the present invention that do not depart from appended claims and limited.

Sequence table

＜110〉Children's Hospital Attached to Chongqing Medical Univ.

＜120〉mouse cardiac muscle progenitor cell and preparation method thereof

<160>40

<210>1

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primers F of ISL1 gene

<400>1

aaggacaaga?aacgcagcat 20

<210>2

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primer R of ISL1 gene

<400>2

ccatcatgtc?tctccggact 20

<210>3

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primers F of Sca-1 gene

<400>3

cctggagccc?tctagtgatg 20

<210>4

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primer R of Sca-1 gene

<400>4

gagcagcaat?ccacaacaaa 20

<210>5

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primers F of c-kit gene

<400>5

gggctagcca?gagacatcag 20

<210>6

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primer R of c-kit gene

<400>6

aggagaagag?ctcccagagg 20

<210>7

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primers F of Nkx2.5 gene

<400>7

gagcctggta?gggaaagagc 20

<210>8

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primer R of Nkx2.5 gene

<400>8

ggtgggtgtg?aaatctgagg 20

<210>9

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primers F of Cx43 gene

<400>9

cctcaacatg?gcatttcctt 20

<210>10

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primer R of Cx43 gene

<400>10

tccacaccta?gagggtcagg 20

<210>11

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primers F of α-MyHC gene

<400>11

gaggaccagg?ccaatgagta 20

<210>12

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primer R of α-MyHC gene

<400>12

gctgggtgta?ggagagcttg 20

<210>13

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primers F of α-actin gene

<400>13

ctgacagagg?caccactgaa 20

<210>14

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primer R of α-actin gene

<400>14

catctccaga?gtccagcaca 20

<210>15

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primers F of cTnT gene

<400>15

ctgagacaga?ggaggccaac 20

<210>16

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primer R of cTnT gene

<400>16

accaagttgg?gcatgaagag 20

<210>17

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primers F of Oct3/4 gene

<400>17

ctgggcgttc?tctttgga 18

<210>18

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primer R of Oct3/4 gene

<400>18

ggcttcctcc?acccactt 18

<210>19

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primers F of Nanog gene

<400>19

aagtacctca?gcctccagca 20

<210>20

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primer R of Nanog gene

<400>20

gtgctgagcc?cttctgaatc 20

<210>21

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primers F of Sox2 gene

<400>21

ccggggagat?acatgctg 18

<210>22

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primer R of Sox2 gene

<400>22

gaacccgact?gggaacct 18

<210>23

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primers F of GATA4 gene

<400>23

tctcccagga?acatcaaacc 20

<210>24

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primer R of GATA4 gene

<400>24

gtgtgaaggg?gtgaaaagga 20

<210>25

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primers F of Tbx5 gene

<400>25

cgctgtgact?tcgtaccaga 20

<210>26

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primer R of Tbx5 gene

<400>26

actttgcatc?cgagacatcc 20

<210>27

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primers F of Hand2 gene

<400>27

ctacttccac?ggctggctta 20

<210>28

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primer R of Hand2 gene

<400>28

ccataatggg?agtggtccag 20

<210>29

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primers F of Flk-1 gene

<400>29

ggcggtggtg?acagtatctt 20

<210>30

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primer R of Flk-1 gene

<400>30

gtcactgaca?gaggcgatga 20

<210>31

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primers F of Actc1 gene

<400>31

ccctggattt?tgagaacgag 20

<210>32

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primer R of Actc1 gene

<400>32

gagtctctgg?acagcggaag 20

<210>33

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primers F of cTnI gene

<400>33

ctgcaggaga?tgattgacga 20

<210>34

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primer R of cTnI gene

<400>34

tgtagccatc?agcgtttttg 20

<210>35

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primers F of ANF gene

<400>35

gggggtagga?ttgacaggat 20

<210>36

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primer R of ANF gene

<400>36

ggatcttttg?cgatctgctc 20

<210>37

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primers F of MyoD gene

<400>37

tggcagcgag?cactacag 18

<210>38

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primer R of MyoD gene

<400>38

gcggtgtcgt?agccattc 18

<210>39

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primers F of SM-MHC gene

<400>39

gcagaaggct?cagaccaaag 20

<210>40

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PCR primer R of SM-MHC gene

<400>40

tatccagaat?gcccaggaag 20

Claims (9)

1. mouse cardiac muscle progenitor cell is characterized in that: it expresses cardiac muscle progenitor cell mark ISL1 and the myocardial cell is early sign thing Tbx5.

2. mouse cardiac muscle progenitor cell according to claim 1 is characterized in that: it is early sign thing Nkx2.5 that described cardiac muscle progenitor cell is also expressed the myocardial cell.

3. mouse cardiac muscle progenitor cell according to claim 1 is characterized in that: described cardiac muscle progenitor cell is also expressed stem cell markers Oct3/4, Nanog and Sox2.

4. mouse cardiac muscle progenitor cell according to claim 1 is characterized in that: described cardiac muscle progenitor cell is also expressed cardiac muscle progenitor cell mark Sca-1 and c-kit.

5. mouse cardiac muscle progenitor cell according to claim 1 is characterized in that: described cardiac muscle progenitor cell is express alpha-MyHC after inducing differentiation.

6. mouse cardiac muscle progenitor cell according to claim 1, it is characterized in that: described cardiac muscle progenitor cell is in inducing atomization, cardiac muscle progenitor cell mark ISL1 and myocardial cell are that early sign thing Nkx2.5 and Tbx5 expression are on a declining curve, myocardial cell's structural protein mark α-MyHC, α-actin and cTnT express in rising trend, and skeletal muscle mark MyoD and unstriated muscle mark SM-MHC do not express.

7. according to the described mouse cardiac muscle progenitor cell of the arbitrary claim of claim 1 to 6, it is characterized in that: described cardiac muscle progenitor cell is the reversibility immortalized cells.

8. mouse cardiac muscle progenitor cell according to claim 7 is characterized in that: described cardiac muscle progenitor cell derives from the embryo heart tissue.

9. the preparation method of the described mouse cardiac muscle progenitor cell of claim 1 is characterized in that: may further comprise the steps:

A, 15.5 days mouse cardiac muscle cell of separation and Culture embryo;

B, step a gained myocardial cell is infected immortalization plasmid SSR#69 and immortalization, by the mono-clonal myocardial cell of antibiotic-screening method and being immortalized of infinite dilution method;

C, from the mono-clonal myocardial cell of step b gained immortalization, filter out and express cardiac muscle progenitor cell mark ISL1 and the myocardial cell is the myocardial cell of early sign thing Tbx5, promptly get mouse cardiac muscle progenitor cell.

Images