CN104388384A - Application of oxidized low density lipoprotein for inducing mesenchymal stem cell to differentiate to myocardium-like cell - Google Patents
Application of oxidized low density lipoprotein for inducing mesenchymal stem cell to differentiate to myocardium-like cell Download PDFInfo
- Publication number
- CN104388384A CN104388384A CN201410598896.8A CN201410598896A CN104388384A CN 104388384 A CN104388384 A CN 104388384A CN 201410598896 A CN201410598896 A CN 201410598896A CN 104388384 A CN104388384 A CN 104388384A
- Authority
- CN
- China
- Prior art keywords
- cell
- bmmscs
- mesenchymal stem
- myocardium
- density lipoprotein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108010071584 oxidized low density lipoprotein Proteins 0.000 title claims abstract description 31
- 230000001939 inductive effect Effects 0.000 title claims abstract description 17
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 16
- 210000004027 cell Anatomy 0.000 title abstract description 42
- 230000004069 differentiation Effects 0.000 claims description 14
- 210000004271 bone marrow stromal cell Anatomy 0.000 claims description 7
- 210000000988 bone and bone Anatomy 0.000 claims description 6
- 230000004663 cell proliferation Effects 0.000 abstract description 7
- 108090000623 proteins and genes Proteins 0.000 abstract description 7
- 102000004169 proteins and genes Human genes 0.000 abstract description 7
- 238000006243 chemical reaction Methods 0.000 abstract description 6
- 238000002474 experimental method Methods 0.000 abstract description 3
- 210000004165 myocardium Anatomy 0.000 abstract description 3
- 239000001963 growth medium Substances 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 23
- 102100035071 Vimentin Human genes 0.000 description 16
- 108010065472 Vimentin Proteins 0.000 description 16
- 210000005048 vimentin Anatomy 0.000 description 13
- 210000000663 muscle cell Anatomy 0.000 description 9
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 7
- 210000004413 cardiac myocyte Anatomy 0.000 description 7
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 102100039819 Actin, alpha cardiac muscle 1 Human genes 0.000 description 6
- 101710170648 Actin, alpha cardiac muscle 1 Proteins 0.000 description 6
- 102000002723 Atrial Natriuretic Factor Human genes 0.000 description 6
- 101800001288 Atrial natriuretic factor Proteins 0.000 description 6
- 101800001890 Atrial natriuretic peptide Proteins 0.000 description 6
- 101800000407 Brain natriuretic peptide 32 Proteins 0.000 description 6
- 108050002772 E3 ubiquitin-protein ligase Mdm2 Proteins 0.000 description 6
- 230000006698 induction Effects 0.000 description 6
- 239000003550 marker Substances 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 208000010125 myocardial infarction Diseases 0.000 description 6
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 6
- 101800002247 Brain natriuretic peptide 45 Proteins 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 5
- 230000010307 cell transformation Effects 0.000 description 5
- 230000031700 light absorption Effects 0.000 description 5
- 210000000130 stem cell Anatomy 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 102100022900 Actin, cytoplasmic 1 Human genes 0.000 description 4
- 108010085238 Actins Proteins 0.000 description 4
- 101800000733 Angiotensin-2 Proteins 0.000 description 4
- 102400000345 Angiotensin-2 Human genes 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- CZGUSIXMZVURDU-JZXHSEFVSA-N Ile(5)-angiotensin II Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C([O-])=O)NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=[NH2+])NC(=O)[C@@H]([NH3+])CC([O-])=O)C(C)C)C1=CC=C(O)C=C1 CZGUSIXMZVURDU-JZXHSEFVSA-N 0.000 description 4
- 229920001213 Polysorbate 20 Polymers 0.000 description 4
- 229950006323 angiotensin ii Drugs 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 241000283707 Capra Species 0.000 description 3
- 108700039887 Essential Genes Proteins 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000000799 fluorescence microscopy Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 230000002107 myocardial effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- NMUSYJAQQFHJEW-UHFFFAOYSA-N 5-Azacytidine Natural products O=C1N=C(N)N=CN1C1C(O)C(O)C(CO)O1 NMUSYJAQQFHJEW-UHFFFAOYSA-N 0.000 description 2
- NMUSYJAQQFHJEW-KVTDHHQDSA-N 5-azacytidine Chemical compound O=C1N=C(N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 NMUSYJAQQFHJEW-KVTDHHQDSA-N 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 239000002033 PVDF binder Substances 0.000 description 2
- 239000013614 RNA sample Substances 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 229960002756 azacitidine Drugs 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000013016 damping Methods 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000000763 evoking effect Effects 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000000751 protein extraction Methods 0.000 description 2
- 238000011476 stem cell transplantation Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- LYSDZXGDWHEQSY-KZXKDKCNSA-N 4-amino-1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1,3,5-triazin-2-one Chemical compound O=C1N=C(N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1.O=C1N=C(N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 LYSDZXGDWHEQSY-KZXKDKCNSA-N 0.000 description 1
- LHVIENWQGBWLJD-UHFFFAOYSA-N C(CCCCCCC)(=O)C1=C(C(=C2C(=C(NC2=C1)C)C)C)C Chemical compound C(CCCCCCC)(=O)C1=C(C(=C2C(=C(NC2=C1)C)C)C)C LHVIENWQGBWLJD-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 102000012199 E3 ubiquitin-protein ligase Mdm2 Human genes 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 101710165567 Extracellular signal-regulated kinase 1 Proteins 0.000 description 1
- 101710165576 Extracellular signal-regulated kinase 2 Proteins 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102000019149 MAP kinase activity proteins Human genes 0.000 description 1
- 108040008097 MAP kinase activity proteins Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102100024193 Mitogen-activated protein kinase 1 Human genes 0.000 description 1
- 102100024192 Mitogen-activated protein kinase 3 Human genes 0.000 description 1
- 206010028594 Myocardial fibrosis Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 210000001054 cardiac fibroblast Anatomy 0.000 description 1
- 208000015606 cardiovascular system disease Diseases 0.000 description 1
- NSQLIUXCMFBZME-MPVJKSABSA-N carperitide Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CSSC[C@@H](C(=O)N1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)=O)[C@@H](C)CC)C1=CC=CC=C1 NSQLIUXCMFBZME-MPVJKSABSA-N 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 210000003038 endothelium Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 230000004217 heart function Effects 0.000 description 1
- 231100000652 hormesis Toxicity 0.000 description 1
- 238000012151 immunohistochemical method Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 230000005976 liver dysfunction Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000010016 myocardial function Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- VLTRZXGMWDSKGL-UHFFFAOYSA-M perchlorate Inorganic materials [O-]Cl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-M 0.000 description 1
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 210000003954 umbilical cord Anatomy 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides application of oxidized low density lipoprotein for inducing mesenchymal stem cells to differentiate to myocardium-like cells. The experiment shows that when oxidized low density lipoprotein with concentration of 5 micrograms/mL is added into a culture medium of mesenchymal stem cells, BMMSCs have remarkable cell proliferation, the expression of myocardium-like cell specific protein and myocardium cell indicative molecules can be detected, which means that the mesenchymal stem cells are directionally differentiated to myocardium-like cells, and the conversion rate of the myocardium-like cells is as high as 30%.
Description
Technical field
The present invention relates to the method for a kind of inducing bone mesenchymal stem cell Cardiocytes differentiation, particularly relate to the application that oxidized low-density lipoprotein breaks up at inducing bone mesenchymal stem cell Cardiocytes.
Background technology
Mesenchymal stem cells MSCs (Bone Marrow Mesenchymal Stem Cells, BMMSCs) be the class adult stem cell be present in marrow, there is very strong self-renewal capacity and multi-lineage potential, under specific physiological environment or under external evoked culture condition, can to the multiple adult cell differentiation such as skeletonization, cartilage, fat, one-tenth flesh, nerve and endothelium.BMMSCs is current one of most widely used seed cell in organizational project and regenerative medicine, is applied to the treatment of neural system and cardiovascular system diseases clinically.Animal experiment study shows, is transplanted to by BMMSCs in particular organization and organ, and it can break up to the functioning cell of this tissue and organ.
Myocardial cell is non-renewable cell, once dead, can only lean on the propagation of the Cardiac Fibroblasts of adjacent tissue to supplement.But inoblast can secrete a large amount of collagen proteins, this will cause myocardial fibrosis, and myocardial function can be caused to decline, and severe patient can cause heart failure.And utilize the Myocardium Differentiation characteristic of stem cell and stem cell transplantation to treat myocardial infarction, become cardiovascular and a hot issue that is stem cell field.In recent years, about the research of BMMSCs myocardiac differentiation and Cells Transplantation in Treatment of Myocardial Infarction has become one of emphasis problem of research both at home and abroad.Correlative study shows, and the BMMSCs being transplanted to site of myocardial infarction supplements the intrinsic myocardial cell of heart of partial loss by myocardiac differentiation, improve heart function to some extent, reducing myocardial infarction area.But still there are problems, the problem that wherein most importantly cells into cardiomyocytes transformation efficiency is lower in relevant Effects of Stem Cell Transplantation on Myocardial Infarction Patients at present.Correlative study shows, and is transplanted to the stem cell of site of myocardial infarction, comprises the stem cell in umbilical cord source, the stem cell of Cord Blood-Derived and mesenchymal stem cells MSCs, all can not produce the myocardial cell of effectively treatment quantity.
Research display, Angiotensin II (Angiotensin II, Ang II) can promote that BMMSCs Cardiocytes transforms, by active cells extracellular signal-regulated kinase 1/2 (Extracellular Signal-Regulated Protein 1/2, ERK1/2), 5-aza-cytidine (5-Azacytidine) also promotes that BMMSCs Cardiocytes transforms.But, adopt Angiotensin II external evoked BMMSCs Cardiocytes differentiation phase, there is the problems such as consumption is large, cost is high, Induction Transformation efficiency is low, and Angiotensin II easily causes the side effects such as hypertension in vivo.5-aza-cytidine then belongs to chemical inducer, can produce pessimal stimulation effect, such as liver dysfunction, oligoleukocythemia etc. to body, and in addition, 5-aza-cytidine is also used as chemotherapeutics clinically, and it easily causes apoptosis.
Summary of the invention
Based on this, be necessary for the problem existing for prior art, provide that a kind of Induction Transformation efficiency is high, the inductor of few side effects, to induce BMMSCs Cardiocytes directed differentiation efficiently.
The technical solution adopted in the present invention is as follows:
The application of oxidized low-density lipoprotein in inducing bone mesenchymal stem cell (BMMSCs) Cardiocytes directed differentiation.
Wherein in an embodiment, in the substratum of mesenchymal stem cells MSCs, add the oxidized low-density lipoprotein that concentration is 5 μ g/mL.
Experiment proves, oxidized low-density lipoprotein (Oxidized Low Density Lipoprotein, ox-LDL) can inducing bone mesenchymal stem cell (BMMSCs) Cardiocytes directed differentiation, cardiac-like muscle cell transformation efficiency is up to 30%.BMMSCs is after ox-LDL inducing culture, short cell proliferation genes Mdm2 expresses and increases, remarkable promotion BMMSCs cell proliferation, and cardiac-like muscle cell specific proteins can be detected---the expression of vimentin (Vimentin), simultaneously, cardiomyocyte marker molecule---atrial natriuretic peptide (AtrialNatriuretic Peptide, ANP), Type B sodium element (B-type Natriuretic Peptide, BNP), α-myoglobulin heavy chain (α-MHC), the mrna expression amount of β-myoglobulin heavy chain (β-MHC) and α-cardiac actin (α-CardiacActin) also significantly increases.
Oxidized low-density lipoprotein (ox-LDL) is as inductor, BMMSCs Cardiocytes not only effectively can be induced to break up, and can cell proliferation be promoted, simultaneously, oxidized low-density lipoprotein belongs to endogenous material, can not produce bad hormesis to body.
Accompanying drawing explanation
Fig. 1 is the cellular form figure of BMMSCs;
Fig. 2 is the absorption detection figure of BMMSCs to Dil-ox-LDL;
Fig. 3 is the MTT detected result of BMMSCs after ox-LDL induction;
Fig. 4 is the expression that Western-blotting detects that BMMSCs urgees cell proliferation genes Mdm2;
Fig. 5 is the expression of Vimentin in Immunofluorescence test BMMSCs, and wherein, A is Heochest3342 staining cell, and B is Vimentin positive cell, and C is Heochest3342 staining cell and the superposing of Vimentin positive cell;
Fig. 6 is the transformation efficiency that ox-LDL induces the conversion of BMMSCs Cardiocytes;
Fig. 7 is the expression amount that Western-blotting detects Vimentin;
Fig. 8 A to Fig. 8 E is the mrna expression amount that RT-PCR detects cardiomyocyte marker molecule.
Embodiment
Embodiment one: the separation of Marrow Mesenchymal Stem Cells (BMMSCs) and cultivation
Aseptically, from mouse hind leg femur bone marrow, isolate mesenchymal stem cells MSCs, add in the DMEM substratum containing 20% foetal calf serum, with 5 × 10
5the concentration of/mL is inoculated in Tissue Culture Flask, is placed in 37 DEG C, 5%CO
2incubator in cultivate, cultivate after 3 weeks and obtain highly purified BMMSCs (as shown in Figure 1).
Embodiment two: Marrow Mesenchymal Stem Cells (BMMSCs) differentiation-inducing
Get the BMMSCs of cultivation after 3 weeks, use 0.25% tryptic digestion, after PBS buffer solution for cleaning, add in the DMEM substratum containing 20% foetal calf serum and fully mix; With 5 × 10
6bMMSCs is inoculated in culturing bottle by the cell concn of/mL, and in substratum, adds the oxidized low-density lipoprotein (ox-LDL) of 5 μ g/mL, then at 37 DEG C, 5%CO
2condition under carry out inducing culture, within every 3 days, change liquid once and add the ox-LDL of 5 μ g/mL, differentiation-inducing 4 weeks.
The enchylema of get induction respectively 1 week, 2 weeks, 3 weeks and 4 weeks, cellular form after the induction of fluorescence microscopy Microscopic observation, and measure cell proliferation quantity by mtt assay, detected quantity and the cardiac-like muscle cell transformation efficiency of Vimentin positive cell by Immunohistochemical method, and detected the mrna expression situation (detailed in Example three to embodiment eight) of cardiomyocyte marker molecule by RT-PCR method.
Embodiment three: two capryloyl tetramethyl-indole cyanine perchlorate (Dil) fluorescently-labeled ox-LDL (Dil-ox-LDL) is taken in and is detected
By BMMSCs with 1 × 10
5/ cm
2density be inoculated in 12 well culture plates, cultivate the Dil-ox-LDL adding 5 μ g/mL for 4 days in backward substratum, be placed in 37 DEG C, 5%CO
2incubator in hatch 30 minutes, then use the DMEM substratum washed cell 2 times of serum-free, be placed in fluorescence microscopy Microscopic observation and take pictures, detected result is as shown in Figure 2.
Detected result shows, BMMSCs has the ability of very strong picked-up ox-LDL.
Embodiment four: mtt assay detects cell proliferative conditions
By BMMSCs with 1 × 10
3/ cm
2density be inoculated in 96 well culture plates, arrange control group and sample sets, control group adopts conventional DMEM substratum simultaneously, and sample sets adds the ox-LDL of 5 μ g/mL in DMEM substratum, in 37 DEG C, 5%CO
2incubator in hatch 24 hours after, add the MTT solution that 10 μ L concentration are 5mg/mL to every hole, continue to hatch 2 hours, then add 100 μ LDMSO solution to every hole, then after hatching 2 hours, adopt enzyme-linked immunosorbent assay instrument to detect the light absorption value at 570nm place.
Detected result shows, and the light absorption value of control group is 0.14OD, and the light absorption value of sample sets is 0.21OD, and by the Relative cell proliferation numerical value of the ratio calculation sample sets of sample sets light absorption value and control group light absorption value, result as shown in Figure 3.The results show, compared with control group, the BMMSCs of sample sets after 24 hours of incubation cell quantity significantly increases, and shows that ox-LDL can stimulate the propagation of BMMSCs.
Embodiment five: immunoblotting (Western-blotting) detects the expression of short amplified gene Mdm2
The BMMSCs that obtains is cultivated as a control group with embodiment one, cell in embodiment two after differentiation-inducing 4 weeks is as sample sets, the total protein of control group, sample sets cell is extracted respectively with total protein extraction test kit, with the polyacrylamide gel electrophoresis protein isolate of 12%, and forwarded to pvdf membrane (PVDF membrane) upper (electric current is 200mA, and the transferring film time is 2 hours); Close pvdf membrane 1 hour with the BSA of 5% under room temperature, then add rabbit anti-mouse Mdm2 antibody (1: 1000 dilution), to be placed on shaking table night incubation at 4 DEG C; Wash film 3 times with the Tris alkali damping fluid (TBS) containing 0.1%Tween20, then add the goat antirabbit two anti-igg-HR (1: 1000 dilution) of horseradish peroxidase mark, be placed on shaking table and at room temperature hatch 1 hour; Film is washed 3 times with the TBS containing 0.1%Tween20, luminous and expose substrate with the super quick luminescent solution of ECL (purchased from American Thermo Fisher Scientific Inc.).Contrast as internal reference using housekeeping gene β-actin, compare respectively after gray scale scanning with the gray-scale value of β-actin, the cardiac-like muscle cell of calculating control group, sample sets urgees the expression amount of amplified gene Mdm2, and result as shown in Figure 4.
Detected result shows, and the BMMSCs of sample sets is after inducing culture, and short amplified gene Mdm2 expression amount significantly raises, and shows that ox-LDL can stimulate the propagation of BMMSCs.
Embodiment six: the expression of Immunofluorescence test cardiac-like muscle cell specific proteins Vimentin
By BMMSCs with 1 × 10
5/ cm
2density be inoculated in 12 well culture plates, arrange control group and sample sets, control group adopts conventional DMEM substratum simultaneously, and sample sets adds the ox-LDL of 5 μ g/mL in DMEM substratum after 4 days in cultivation, in 37 DEG C, 5%CO
2condition under inducing culture 3 weeks; With the neutral formalin fixed cell of 4%, PBS buffer solution cell 2 times, at room temperature use the bovine serum albumin (BSA) of 1% as confining liquid closing cell 30 minutes, then rabbit anti-mouse Vimentin antibody (1: 200 dilution) is dripped to cell, incubated at room is after 1 hour, with PBS buffer solution cell 3 times; Then the anti-IgM of goat antirabbit two (1: 200 dilution) of fluorescein Cy3 mark is added, after room temperature lucifuge hatches 30 minutes, with PBS buffer solution cell 2 times; Then Heochest3342 dyestuff (nucleus specific dye is dripped, purchased from American ImmunoChemistry Technologies, LLC) solution (1: 500 dilution), incubated at room temperature is after 5 minutes, with PBS buffer solution cell 3 times; Be placed in fluorescence microscopy Microscopic observation, take pictures, and add up total cellular score (i.e. Heochest3342 staining cell quantity), Vimentin positive cell quantity respectively, and calculating myocardium like cell transformation efficiency, result is as shown in Figure 5, Figure 6.
Cardiac-like muscle cell transformation efficiency=Vimentin positive cell quantity/Heochest3342 staining cell quantity
The results show, Vimentin positive cell quantity and the cardiac-like muscle cell transformation efficiency of sample sets are all significantly higher than control group, show that ox-LDL can induce BMMSCs Cardiocytes to break up.
Embodiment seven: immunoblotting (Western-blotting) detects the expression amount of cardiac-like muscle cell specific proteins Vimentin
The BMMSCs that obtains is cultivated as a control group with embodiment one, cell in embodiment two after differentiation-inducing 4 weeks is as sample sets, the total protein of control group, sample sets cell is extracted respectively with total protein extraction test kit, with the polyacrylamide gel electrophoresis protein isolate of 12%, and forwarded to pvdf membrane (PVDF membrane) upper (electric current is 200mA, and the transferring film time is 2 hours); Close pvdf membrane 1 hour with the BSA of 5% under room temperature, then add rabbit anti-mouse Vimentin antibody (1: 1000 dilution), to be placed on shaking table night incubation at 4 DEG C; Wash film 3 times with the Tris alkali damping fluid (TBS) containing 0.1%Tween20, then add the goat antirabbit two anti-igg-HR (1: 1000 dilution) of horseradish peroxidase mark, be placed on shaking table and at room temperature hatch 1 hour; Film is washed 3 times with the TBS containing 0.1%Tween20, luminous and expose substrate with the super quick luminescent solution of ECL (purchased from American Thermo Fisher Scientific Inc.).Contrast as internal reference using housekeeping gene β-actin, compare respectively after gray scale scanning with the gray-scale value of β-actin, the expression amount of the cardiac-like muscle cell specific proteins Vimentin of calculating control group, sample sets, result as shown in Figure 7.
The results show, the Vimentin expression amount of sample sets is significantly higher than control group, shows that ox-LDL can induce BMMSCs Cardiocytes to break up.
Embodiment eight: inverse transcription polymerase chain reaction (RT-PCR) detects the mrna expression amount of cardiomyocyte marker molecule ANP, BNP, α-MHC, β-MHC and α-Cardiac Actin
The BMMSCs that obtains is cultivated as a control group with embodiment one, cell in embodiment two after differentiation-inducing 4 weeks is as sample sets, the cell extraction total serum IgE of test kit from control group, sample sets is extracted respectively with RNA, and process RNA sample, to remove DNA pollution with DnaseI (deoxyribonuclease).The RNA sample of control group, sample sets being diluted respectively is 100 μ g/mL, gets 10 μ L samples respectively, synthesizes the first chain cDNA with Reverse Transcription box.Respectively using the first chain cDNA of control group, sample sets as template, primer shown in employing table 1 carries out PCR reaction, with measure BMMSCs through ox-LDL induction after cardiomyocyte marker molecule ANP, BNP, α-MHC, β-MHC and α-Cardiac Actin expression amount.
PCR reaction system: cDNA template 100ng, primer 0.3 μM, 10 × PCR reaction solution 2 μ L, add deionized water to 20 μ L.
PCR reaction conditions: 94 DEG C of denaturations 5 minutes, 94 DEG C of sex change 30 seconds, 55 DEG C of annealing 30 seconds, 72 DEG C extend 2 minutes, and after 30 circulations, 72 DEG C extend 10 minutes eventually.
Table 1 PCR reacts primer
Contrast using housekeeping gene GAPDH as internal reference, compare with the gray-scale value of GAPDH respectively after gray scale scanning, calculate control group, cardiomyocyte marker molecule ANP, BNP, α-MHC, the β-MHC of sample sets and the expression amount of α-Cardiac Actin respectively, result is as shown in Fig. 8 A to Fig. 8 E.
The results show, the expression amount of ANP (Fig. 8 A), the BNP (Fig. 8 B) of sample sets, α-MHC (Fig. 8 C), β-MHC (Fig. 8 D) and α-Cardiac Actin (Fig. 8 E) is all significantly higher than control group, shows that ox-LDL can induce BMMSCs Cardiocytes to break up.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (2)
1. the application of oxidized low-density lipoprotein in inducing bone mesenchymal stem cell Cardiocytes directed differentiation.
2. application according to claim 1, is characterized in that: in the substratum of mesenchymal stem cells MSCs, add the oxidized low-density lipoprotein that concentration is 5 μ g/mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410598896.8A CN104388384B (en) | 2014-10-29 | 2014-10-29 | The application that oxidized low-density lipoprotein breaks up in inducing bone mesenchymal stem cell Cardiocytes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410598896.8A CN104388384B (en) | 2014-10-29 | 2014-10-29 | The application that oxidized low-density lipoprotein breaks up in inducing bone mesenchymal stem cell Cardiocytes |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104388384A true CN104388384A (en) | 2015-03-04 |
| CN104388384B CN104388384B (en) | 2018-07-20 |
Family
ID=52606345
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410598896.8A Expired - Fee Related CN104388384B (en) | 2014-10-29 | 2014-10-29 | The application that oxidized low-density lipoprotein breaks up in inducing bone mesenchymal stem cell Cardiocytes |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104388384B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105238746A (en) * | 2015-09-21 | 2016-01-13 | 深圳华毓造血干细胞研究有限公司 | Inducing method and inducing liquid of mesenchymal stem cells |
| CN106350481A (en) * | 2016-09-12 | 2017-01-25 | 青海七彩花生物科技有限公司 | Method for induced differentiation of mesenchymal stem cells to cardiomyocyte-like cells |
| CN114622019A (en) * | 2022-03-23 | 2022-06-14 | 新乡医学院 | Application of BIRC5 gene as osteogenic differentiation inhibitor of human bone marrow mesenchymal stem cells |
| CN114657121A (en) * | 2022-03-23 | 2022-06-24 | 新乡医学院 | Application of LOX1 gene as BMSCs osteogenic differentiation promoter under action of fluid shear force |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101914493A (en) * | 2010-07-16 | 2010-12-15 | 山东省齐鲁干细胞工程有限公司 | Inducing method for differentiating umbilical cord mesenchymal stem cells into nerve cells |
-
2014
- 2014-10-29 CN CN201410598896.8A patent/CN104388384B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101914493A (en) * | 2010-07-16 | 2010-12-15 | 山东省齐鲁干细胞工程有限公司 | Inducing method for differentiating umbilical cord mesenchymal stem cells into nerve cells |
Non-Patent Citations (2)
| Title |
|---|
| FENXI ZHANG ET AL: "Lectin-like oxidized LDL receptor-1 expresses in mouse bone marrow-derived mesenchymal stem cells and stimulates their proliferation", 《EXPERIMENTAL CELL RESEARCH》 * |
| 武俊芳等: "氧化型低密度脂蛋白对小鼠骨髓间充质干细胞迁移的影响", 《郑州大学学报（医学版）》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105238746A (en) * | 2015-09-21 | 2016-01-13 | 深圳华毓造血干细胞研究有限公司 | Inducing method and inducing liquid of mesenchymal stem cells |
| CN106350481A (en) * | 2016-09-12 | 2017-01-25 | 青海七彩花生物科技有限公司 | Method for induced differentiation of mesenchymal stem cells to cardiomyocyte-like cells |
| CN114622019A (en) * | 2022-03-23 | 2022-06-14 | 新乡医学院 | Application of BIRC5 gene as osteogenic differentiation inhibitor of human bone marrow mesenchymal stem cells |
| CN114657121A (en) * | 2022-03-23 | 2022-06-24 | 新乡医学院 | Application of LOX1 gene as BMSCs osteogenic differentiation promoter under action of fluid shear force |
| CN114657121B (en) * | 2022-03-23 | 2023-07-25 | 新乡医学院 | Application of LOX1 gene as BMSCs osteogenic differentiation promoter under action of fluid shear force |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104388384B (en) | 2018-07-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Pietronave et al. | Monophasic and biphasic electrical stimulation induces a precardiac differentiation in progenitor cells isolated from human heart | |
| Thorpe et al. | European Society of Biomechanics SM Perren Award 2012: the external mechanical environment can override the influence of local substrate in determining stem cell fate | |
| Perán et al. | Human cardiac tissue induces transdifferentiation of adult stem cells towards cardiomyocytes | |
| WO2009027563A1 (en) | Population of adult stem cells derived from cardiac adipose tissue and use thereof in cardiac regeneration | |
| US20100098739A1 (en) | Compositions and methods for modular soft tissue repair | |
| Chang et al. | Multiple directional differentiation difference of neonatal rat fibroblasts from six organs | |
| Xiao et al. | Extracellular matrix from human umbilical cord-derived mesenchymal stem cells as a scaffold for peripheral nerve regeneration | |
| CN105518125A (en) | Methods and compositions for generating epicardium cells | |
| WO2012096461A2 (en) | Composition for suspension culturing of stem cells | |
| CN104388384A (en) | Application of oxidized low density lipoprotein for inducing mesenchymal stem cell to differentiate to myocardium-like cell | |
| Jiang et al. | Differentiation of cardiomyocytes from amniotic fluid‑derived mesenchymal stem cells by combined induction with transforming growth factor β1 and 5‑azacytidine | |
| Xiang et al. | Lentivirus-mediated Wnt11 gene transfer enhances Cardiomyogenic differentiation of skeletal muscle-derived stem cells | |
| Meyer et al. | Epimuscular fat in the human rotator cuff is a novel beige depot | |
| Bylund et al. | Coordinated proliferation and differentiation of human-induced pluripotent stem cell-derived cardiac progenitor cells depend on bone morphogenetic protein signaling regulation by GREMLIN 2 | |
| Cho et al. | Regeneration of infarcted mouse hearts by cardiovascular tissue formed via the direct reprogramming of mouse fibroblasts | |
| KR20170023188A (en) | Cardiac cell culture material | |
| Yang et al. | Effect of neuron‐derived neurotrophic factor on rejuvenation of human adipose‐derived stem cells for cardiac repair after myocardial infarction | |
| Davis et al. | Induction of myofibrillogenesis in cardiac lethal mutant axolotl hearts rescued by RNA derived from normal endoderm | |
| Gomes-Alves et al. | In vitro expansion of human cardiac progenitor cells: exploring'omics tools for characterization of cell-based allogeneic products | |
| Deng et al. | Metformin pre-treatment of stem cells from human exfoliated deciduous teeth promotes migration and angiogenesis of human umbilical vein endothelial cells for tissue engineering | |
| Wu et al. | Angiotensin II promotes cardiac differentiation of embryonic stem cells via angiotensin type 1 receptor | |
| CN109554351A (en) | The application of Rspo1 inducing bone mesenchymal stem cell Cardiocytes differentiation | |
| Ghosh et al. | Keratin mediated attachment of stem cells to augment cardiomyogenic lineage commitment | |
| US7534607B1 (en) | Method of producing cardiomyocytes from mesenchymal stem cells | |
| Xu et al. | Using bovine pituitary extract to increase proliferation of keratocytes and maintain their phenotype in vitro |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20180613 Address after: 453003 Xin Yan Road, Hongqi District, Xinxiang, Henan Applicant after: XINXIANG MEDICAL University Address before: 453003 floor 22, block B, chamber building, 688 Jinsui Avenue, Xinxiang, Henan. Applicant before: HENAN HUALONG BIOTECHNOLOGY Co.,Ltd. Applicant before: XINXIANG MEDICAL University |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180720 |