CN107012124A - 钠钙交换体1启动子联合丹参酚酸B诱导iPSCs定向心肌分化的方法 - Google Patents
钠钙交换体1启动子联合丹参酚酸B诱导iPSCs定向心肌分化的方法 Download PDFInfo
- Publication number
- CN107012124A CN107012124A CN201710272058.5A CN201710272058A CN107012124A CN 107012124 A CN107012124 A CN 107012124A CN 201710272058 A CN201710272058 A CN 201710272058A CN 107012124 A CN107012124 A CN 107012124A
- Authority
- CN
- China
- Prior art keywords
- red
- phenolic acid
- rooted salvia
- ipscs
- root
- 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
- 150000007965 phenolic acids Chemical class 0.000 title claims abstract description 49
- 235000017276 Salvia Nutrition 0.000 title claims abstract description 48
- 229910001424 calcium ion Inorganic materials 0.000 title claims abstract description 28
- 230000004069 differentiation Effects 0.000 title claims abstract description 25
- 230000006698 induction Effects 0.000 title claims abstract description 22
- 210000004165 myocardium Anatomy 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims abstract description 17
- 240000007164 Salvia officinalis Species 0.000 title description 38
- 102100035088 Sodium/calcium exchanger 1 Human genes 0.000 claims abstract description 26
- 108010067207 sodium-calcium exchanger 1 Proteins 0.000 claims abstract description 26
- 239000002245 particle Substances 0.000 claims abstract description 3
- 241001072909 Salvia Species 0.000 claims abstract 12
- 230000008859 change Effects 0.000 claims description 5
- 230000014509 gene expression Effects 0.000 abstract description 12
- 230000000747 cardiac effect Effects 0.000 abstract description 10
- 102000007469 Actins Human genes 0.000 abstract description 5
- 108010085238 Actins Proteins 0.000 abstract description 5
- 238000005516 engineering process Methods 0.000 abstract description 5
- 102000004987 Troponin T Human genes 0.000 abstract description 4
- 108090001108 Troponin T Proteins 0.000 abstract description 4
- 102000010825 Actinin Human genes 0.000 abstract description 3
- 108010063503 Actinin Proteins 0.000 abstract description 3
- 206010000891 acute myocardial infarction Diseases 0.000 abstract description 3
- 230000001737 promoting effect Effects 0.000 abstract description 3
- 210000002966 serum Anatomy 0.000 abstract description 2
- 239000011734 sodium Substances 0.000 description 20
- 210000004027 cell Anatomy 0.000 description 18
- 239000000243 solution Substances 0.000 description 10
- 241000700605 Viruses Species 0.000 description 9
- 239000005089 Luciferase Substances 0.000 description 7
- 235000015097 nutrients Nutrition 0.000 description 7
- 108060001084 Luciferase Proteins 0.000 description 6
- 230000001939 inductive effect Effects 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 210000001778 pluripotent stem cell Anatomy 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 210000002242 embryoid body Anatomy 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 239000006180 TBST buffer Substances 0.000 description 3
- 210000004413 cardiac myocyte Anatomy 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000005138 cryopreservation Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 210000001671 embryonic stem cell Anatomy 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000004806 packaging method and process Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 210000000130 stem cell Anatomy 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 229930193140 Neomycin Natural products 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000006160 differential media Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- MURGITYSBWUQTI-UHFFFAOYSA-N fluorescin Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC=C(O)C=C2OC2=CC(O)=CC=C21 MURGITYSBWUQTI-UHFFFAOYSA-N 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 230000004660 morphological change Effects 0.000 description 2
- 230000002107 myocardial effect Effects 0.000 description 2
- 229960004927 neomycin Drugs 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 235000005412 red sage Nutrition 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- SNKFFCBZYFGCQN-UHFFFAOYSA-N 2-[3-[3-[1-carboxy-2-(3,4-dihydroxyphenyl)ethoxy]carbonyl-2-(3,4-dihydroxyphenyl)-7-hydroxy-2,3-dihydro-1-benzofuran-4-yl]prop-2-enoyloxy]-3-(3,4-dihydroxyphenyl)propanoic acid Chemical compound C=1C=C(O)C=2OC(C=3C=C(O)C(O)=CC=3)C(C(=O)OC(CC=3C=C(O)C(O)=CC=3)C(O)=O)C=2C=1C=CC(=O)OC(C(=O)O)CC1=CC=C(O)C(O)=C1 SNKFFCBZYFGCQN-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 201000000628 Gas Gangrene Diseases 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 206010061216 Infarction Diseases 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- SNKFFCBZYFGCQN-VWUOOIFGSA-N Lithospermic acid B Natural products C([C@H](C(=O)O)OC(=O)\C=C\C=1C=2[C@H](C(=O)O[C@H](CC=3C=C(O)C(O)=CC=3)C(O)=O)[C@H](OC=2C(O)=CC=1)C=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 SNKFFCBZYFGCQN-VWUOOIFGSA-N 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 235000011135 Salvia miltiorrhiza Nutrition 0.000 description 1
- 102000001794 Sodium-Calcium Exchanger Human genes 0.000 description 1
- 108010040240 Sodium-Calcium Exchanger Proteins 0.000 description 1
- VEUACKUBDLVUAC-UHFFFAOYSA-N [Na].[Ca] Chemical compound [Na].[Ca] VEUACKUBDLVUAC-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 102000041060 alpha-actinin family Human genes 0.000 description 1
- 108091060828 alpha-actinin family Proteins 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 230000004094 calcium homeostasis Effects 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000001537 electron coincidence spectroscopy Methods 0.000 description 1
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 1
- 239000000686 essence Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000007574 infarction Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 206010028320 muscle necrosis Diseases 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 244000132619 red sage Species 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- STCJJTBMWHMRCD-UHFFFAOYSA-N salvianolic acid B Natural products OC(=O)C(Cc1ccc(O)c(O)c1)OC(=O)C=Cc2cc(O)c(O)c3OC(C(C(=O)OC(Cc4ccc(O)c(O)c4)C(=O)O)c23)c5ccc(O)c(O)c5 STCJJTBMWHMRCD-UHFFFAOYSA-N 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 1
- 238000009168 stem cell therapy Methods 0.000 description 1
- 238000009580 stem-cell therapy Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 230000002537 thrombolytic effect Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0657—Cardiomyocytes; Heart cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Rheumatology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cardiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明公开一种钠钙交换体1启动子联合丹参酚酸B诱导iPSCs定向心肌分化的方法，属于医药技术领域。该方法是通过构建NCX1慢病毒颗粒并转染至iPSCs，辅以丹参酚酸B联合诱导。所述的丹参酚酸B的浓度为1～100μM。本发明证实钠钙交换体1启动子联合丹参酚酸B可有效诱导iPSCs心肌定向分化。丹参酚酸B可有效促进NCX1表达，同时促进心肌标志物α肌动蛋白(α‑actin)、心肌肌钙蛋白T(cTnT)表达显著提高。且iPSCs形态可见心肌细胞样梭形改变。iPSCs作为修复急性心肌梗死的种子细胞，具有广阔的应用前景。
Description
技术领域
本发明属于医药技术领域，具体涉及一种钠钙交换体1启动子联合丹参酚酸B诱导iPSCs定向心肌分化的方法。
背景技术
急性心肌梗死的干细胞治疗仍存在诸多方面的困难，如种子细胞的选择问题、心肌坏死范围广泛、细胞移植归巢效率低下及局部心肌微环境缺血缺氧等，因此，其临床应用仍然未能得以广泛的开展。诱导性多能干细胞(induced pluripotent stem cells，iPSCs)是通过重编程体细胞获得的具有与胚胎干细胞(embryonic stem cells，ECSs)相似的高度增殖和多向分化潜能的一种多能干细胞，又因其缺少胚胎干细胞的免疫原性及道德伦理限制，诱导性多能干细胞有望成为修复急性心肌梗死后心肌的具有巨大临床应用前景的种子细胞。
丹参为唇形科植物丹参Salvia miltiorrhiza Bge.的干燥根及根茎，除了传统的活血化瘀、养心安神的功效外，还有抗氧化，抑制血小板聚集、促进血栓溶解，抗肿瘤，调节免疫功能的作用，其在心血管疾病方面的新药理作用、作用机制和药代动力学的研究日趋深入，使其拥有非常广阔的临床应用前景。丹参酚酸B是其中一种单体成份。
作为心肌分化最早的标志物钠钙交换体钠钙交换体启动子广泛分布于心肌细胞膜上，双向转运胞膜内外Na+和Ca2+，参与心肌兴奋-收缩耦联及胞内钙稳态的维持，钠钙交换体1是一个糖基化的具有12个跨膜部分的跨膜蛋白，对维持心肌钙平衡有重要作用。
目前利用iPSCs定向心肌分化的方法很多，本研究旨在提供一种新型的诱导方法，进一步推动iPSCs的临床应用。
发明内容
为了克服现有技术的缺点与不足，本发明的目的在于提供一种钠钙交换体1启动子联合丹参酚酸B诱导iPSCs定向心肌分化的方法。
目前尚无有效诱导iPSCs心肌定向分化的方法，本研究为了推进iPSCs的临床应用，旨在提供一种有效安全的诱导心肌分化方法。
本发明的目的通过下述技术方案实现：
一种钠钙交换体1启动子联合丹参酚酸B诱导iPSCs定向心肌分化的方法，包括如下步骤：
通过构建NCX1慢病毒颗粒并转染至iPSCs，辅以丹参酚酸B联合诱导，倒置显微镜、细胞免疫技术以及Western Blots观察其转染情况、形态学以及心肌标志物表达情况。实验证实钠钙交换体1启动子联合丹参酚酸B可有效诱导iPSCs定向心肌分化。
优选的，所述的丹参酚酸B的浓度为1～100μM；更优选为10～100μM。
优选的，所述的培养的条件为5％CO2、37℃培养15～20d。
本发明的机理是：拟通过钠钙交换体1启动子(sodium-calcium exchangerpromoter，NCX1)联合丹参酚酸B(Salvianolic acid B，Sal B)诱导诱导性多能干细胞(induced pluripotent stem cells，iPSCs)定向心肌细胞分化，iPSCs作为修复急性心肌梗死的种子细胞，具有广阔的应用前景。
本发明相对于现有技术，具有如下的优点及效果：
本发明证实钠钙交换体1启动子联合丹参酚酸B可有效诱导iPSCs心肌定向分化。丹参酚酸B可有效促进NCX1表达，同时促进心肌标志物α肌动蛋白(α-actin)、心肌肌钙蛋白T(cTnT)表达显著提高。且iPSCs形态可见心肌细胞样梭形改变。
附图说明
图1是NCX1慢病毒转染诱导性多能干细胞后NCX1表达情况；其中，A：EGFP；B：Oct4；C：NCX1；D：Merged。
图2是钠钙交换体1启动子联合丹参酚酸B诱导iPSCs分化后形态改变；其中，A：对照组；B：丹参酚酸B(1μM)；C：丹参酚酸B(10μM)；D：丹参酚酸B(100μM)；E：NCX1组；F：NCX1+丹参酚酸B(1μM)；G：NCX1+丹参酚酸B(10μM)；H：NCX1+丹参酚酸B(100μM)。
图3是钠钙交换体1启动子联合丹参酚酸B诱导iPSCs分化后心肌特异性标志物表达情况。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
下列实施例中未注明具体实验条件的实验方法，通常按照常规实验条件或按照制造厂商所建议的实验条件。未注明具体来源的试剂及生物材料，均为市售产品。
实施例1制备iPSCs
1)滋养层(MEF)的制备：0.1％(体积分数)的明胶加入T25培养瓶，于37℃细胞培养箱静置20min后将其吸除，加入5～6mL预热37℃的MEF培养液，与此同时将小鼠胚胎成纤维细胞(MEF)(购自上海中科院细胞库)从液氮中快速取出，置于37℃水浴中快速融解并立即用体积分数为75％的酒精擦拭冻存管后拿到超净台内，将冻存管内的细胞悬液转移至含MEF培养液的15mL离心管内，以1000rpm离心5min，弃上清液重悬后加入到T25培养瓶中，放置到CO2恒温培养箱中，培养24h后得到滋养层即可加入iPSCs；
2)iPSCs的培养以及传代：步骤(1)中培养24h后得到的滋养层，4倍显微镜下可见已经均匀铺满T25培养瓶，此时可迅速从液氮中取出iPSCs(中科院上海生科院生化细胞所和中科院上海生科院生化细胞所干细胞平台提供)冻存管，复苏过程类似小鼠胚胎成纤维细胞，将复苏的iPSCs直接传入MEF铺板的T25培养瓶，置于37℃细胞培养箱，每日换液并且观察细胞集落形态的变化。待细胞集落长到合适大小及时传代，PBS(不含钙镁离子)轻轻冲洗一遍后加入0.25％(体积分数)胰酶(含EDTA)消化，小鼠iPSCs培养液终止消化，以1000rpm离心5min，弃上清加培养液吹打制备成单细胞悬液。
实施例2钠钙交换体1启动子制备
1)慢病毒Neo-Promoter_419bp>Luciferase:IRES:DsRed_Express2构建和鉴定：
引物设计：根据Genebank中钠钙交换体1启动子基因的cDNA序列，设计钠钙交换体1启动子引物序列为：
上游引物：5′-GCAACAGACATACAAACTAAAGAAT-3′；
下游引物：5′-GGAGCAACATAGTTAAGAATACC-3′。
PCR扩增：在上述引物的作用下，以钠钙交换体1启动子的cDNA为模板进行扩增，电泳检测PCR产物，切胶回收纯化目的条带。将载体Neo-Promoter_419bp>Luciferase:IRES:DsRed_Express2与扩增钠钙交换体1启动子在内切酶作用下重组，取2μL重组反应液转化50μL感受态细胞，新霉素筛选重组阳性克隆并扩增，菌液行PCR扩增验证，挑取构建正确的菌落培养过夜，提取质粒DNA，构建正确标记。
2)慢病毒的包装和浓缩：
将慢病毒包装质粒Vira Power TM Lentiviral Packaging Mix和载体质粒pLV[Exp]-Neo-Promoter_419bp>Luciferase:IRES:DsRed_Express2以1：1的摩尔比例混合，在Lipofectamine2000的作用下共转染293T细胞，置于37℃、体积分数5％CO2的培养箱中培养。12h后换液，加入新鲜293T细胞培养液。72h后观察有强红色色荧光及细胞融合现象时收集培养上清，4℃、4500r/min离心15min，将病毒上清液用0.45μm滤膜过滤以去除细胞碎片，4℃、50 000×g高速离心90min。用RNase-free L-DMEM悬浮病毒颗粒沉淀，-80℃冻存备用。同法构建只含红色色荧光蛋白的空病毒，标记pLV[Exp]-Neo-Luciferase:IRES:DsRed_Express2。
3)慢病毒的滴度测定：接种293T细胞于6孔板，每孔接种2×105个细胞，37℃培养过夜；第2天将慢病毒溶液用无血清DMEM(不含抗生素)按1×10-2，1×10-3，1×10-4，1×10-5，1×10-6，1×10-7进行系列稀释，将其分别加入到相应的孔中，加入聚酰胺(Polybrene)终浓度至8mg/L，置37℃、体积分数5％CO2培养箱培养。12h后全量换液，加入新鲜的293T细胞培养液。第4天观察荧光表达情况，荧光细胞数随稀释倍数增加而减少，计数出表达荧光的细胞个数，将得到的数值乘以相应的稀释倍数就得到病毒原液的滴度数。
4)pLV[Exp]-Neo-Promoter_419bp>Luciferase:IRES:DsRed_Express2包装iPSCs：将对数生长期诱导性多能干细胞(iPSCs)消化离心后，以分化培养基重悬成2×107L-1细胞悬液，分化培养基为含体积分数1％非必需氨基酸、1％谷氨酰胺、0.25％β-巯基乙醇，体积为15％胎牛血清的DMEM。细胞悬液以20μL/滴接种到直径10cm的培养皿上，培养皿内加少许PBS。悬滴48h后将形成的拟胚体悬浮到含新鲜分化培养液的培养皿中，将病毒pLV[Exp]-Neo-Promoter_419bp>Luciferase:IRES:DsRed_Express2和对照病毒转染诱导性多能干细胞的拟胚体。
处理分组如下：A：对照组；B：丹参酚酸B(1μM)；C：丹参酚酸B(10μM)D：丹参酚酸B(100μM)；E：NCX1组；F：NCX1+丹参酚酸B(1μM)；G：NCX1+丹参酚酸B(10μM)；H：NCX1+丹参酚酸B(100μM)。比较各组之间心肌细胞的诱导效率，每天更换一次培养基。病毒感染24～48h后，使用新霉素进行筛选。第10天后将悬浮的拟胚体接种到0.1％明胶铺板的培养瓶中，用上述分化培养液继续培养，在倒置显微镜下每日观察其生长情况，且收集经诱导分化处理的细胞后续实验使用。
实施例3细胞免疫荧光以及形态学鉴定
诱导培养20d后，PBS洗涤3次，以4％多聚甲醛固定，PBS冲洗，DAPI复染细胞核10min，树脂胶封片固定。荧光显微镜以及倒置显微镜下下观察，每孔观察10个视野共30个视野。
NCX1慢病毒转染诱导性多能干细胞后NCX1表达情况，如图1所示。结果表明：荧光显微镜定性分析可见NCX1有效表达于iPSCs。
钠钙交换体1启动子联合丹参酚酸B诱导iPSCs分化后形态改变，如图2所示；其中，A：对照组；B：丹参酚酸B(1μM)；C：丹参酚酸B(10μM)；D：丹参酚酸B(100μM)；E：NCX1组；F：NCX1+丹参酚酸B(1μM)；G：NCX1+丹参酚酸B(10μM)；H：NCX1+丹参酚酸B(100μM)。结果表明：相比对照组而言，B～H各组拟胚体均可见边缘不规则，iPSCs如心肌细胞样呈梭形生长。
实施例4心肌特异性标志物(α肌动蛋白(α-actin)、心肌肌钙蛋白T(cTnT))表达情况分析
Western-blot技术：将细胞接种至6孔板，37℃，5％CO2常规条件下培养24h。待细胞稳定后按实施例2中的各分组处理方法处理。提取各组蛋白并进行SDS-PAGE电泳，电泳后转膜用含5％脱脂奶粉的TBST 37摄氏度封闭60min，用相应抗体4℃孵育过夜。TBST漂洗3次后加入相应二抗后37℃孵育60min。然后用TBST漂洗5次后检测。采用图像分析软件进行图像分析，计算条带与β-actin的相对积分光密度值。
钠钙交换体1启动子联合丹参酚酸B诱导iPSCs分化后，心肌特异性标志物表达情况，如图3所示。结果表明：相比对照组而言，其他各组α肌动蛋白(α-actin)和心肌肌钙蛋白T(cTnT)的表达量均有统计学意义(p<0.05)；相比NCX1组而言，不同浓度的丹参酚酸B可有效提高NCX1的心肌分化诱导效率，特别是10～100μM的丹参酚酸B。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 暨南大学
<120> 钠钙交换体1启动子联合丹参酚酸B诱导iPSCs定向心肌分化的方法
<130> 1
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> 上游引物
<400> 1
gcaacagaca tacaaactaa agaat 25
<210> 2
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> 下游引物
<400> 2
ggagcaacat agttaagaat acc 23
Claims (4)
1.一种钠钙交换体1启动子联合丹参酚酸B诱导iPSCs定向心肌分化的方法，其特征在于包括如下步骤：
通过构建NCX1慢病毒颗粒并转染至iPSCs，辅以丹参酚酸B联合诱导。
2.根据权利要求1所述的钠钙交换体1启动子联合丹参酚酸B诱导iPSCs定向心肌分化的方法，其特征在于：
所述的丹参酚酸B的浓度为1～100μM。
3.根据权利要求1或2所述的钠钙交换体1启动子联合丹参酚酸B诱导iPSCs定向心肌分化的方法，其特征在于：
所述的丹参酚酸B的浓度为10～100μM。
4.根据权利要求1所述的钠钙交换体1启动子联合丹参酚酸B诱导iPSCs定向心肌分化的方法，其特征在于：
所述的培养的条件为5％CO2、37℃培养15～20d。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710272058.5A CN107012124B (zh) | 2017-04-24 | 2017-04-24 | 钠钙交换体1启动子联合丹参酚酸B诱导iPSCs定向心肌分化的方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710272058.5A CN107012124B (zh) | 2017-04-24 | 2017-04-24 | 钠钙交换体1启动子联合丹参酚酸B诱导iPSCs定向心肌分化的方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107012124A true CN107012124A (zh) | 2017-08-04 |
| CN107012124B CN107012124B (zh) | 2019-10-18 |
Family
ID=59446799
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710272058.5A Expired - Fee Related CN107012124B (zh) | 2017-04-24 | 2017-04-24 | 钠钙交换体1启动子联合丹参酚酸B诱导iPSCs定向心肌分化的方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107012124B (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108939075A (zh) * | 2018-07-26 | 2018-12-07 | 陕西师范大学 | Ncx1基因作为治疗呼吸暂停综合症的药物干预靶点的应用 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050255590A1 (en) * | 2004-04-09 | 2005-11-17 | Goldstein Ronald S | Methods for generating neuronal cells from human embryonic stem cells and uses thereof |
| WO2011125055A2 (en) * | 2010-04-09 | 2011-10-13 | Consorcio De Tecnologia E Innovacion En Salud S.A. | Synthetic constructs for polynucleotide and protein expression |
| CN103173451A (zh) * | 2013-04-15 | 2013-06-26 | 江苏省人民医院 | 一种心肌特异启动子 |
| CN105687209A (zh) * | 2016-02-25 | 2016-06-22 | 暨南大学 | 丹参单体化合物在制备增强iPS细胞生物学特性产品中的应用 |
-
2017
- 2017-04-24 CN CN201710272058.5A patent/CN107012124B/zh not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050255590A1 (en) * | 2004-04-09 | 2005-11-17 | Goldstein Ronald S | Methods for generating neuronal cells from human embryonic stem cells and uses thereof |
| WO2011125055A2 (en) * | 2010-04-09 | 2011-10-13 | Consorcio De Tecnologia E Innovacion En Salud S.A. | Synthetic constructs for polynucleotide and protein expression |
| CN103173451A (zh) * | 2013-04-15 | 2013-06-26 | 江苏省人民医院 | 一种心肌特异启动子 |
| CN105687209A (zh) * | 2016-02-25 | 2016-06-22 | 暨南大学 | 丹参单体化合物在制备增强iPS细胞生物学特性产品中的应用 |
Non-Patent Citations (4)
| Title |
|---|
| 戴波 等: "钠钙交换体启动子促进诱导多能干细胞向心肌细胞转化", 《中国组织工程研究》 * |
| 李伟望 等: "丹酚酸B体外诱导大鼠骨髓间充质干细胞分化为心肌样细胞", 《中华老年心脑血管病杂志》 * |
| 江灿 等: "丹参各单体成份对诱导性多能干细胞生物学特性的影响", 《暨南大学学报(自然科学与医学版)》 * |
| 魏文娟: "丹参及其三种单体成分体外诱导胚胎干细胞向心肌细胞分化的实验研究", 《中国学位论文全文数据库》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108939075A (zh) * | 2018-07-26 | 2018-12-07 | 陕西师范大学 | Ncx1基因作为治疗呼吸暂停综合症的药物干预靶点的应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107012124B (zh) | 2019-10-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106636210B (zh) | 转录因子组合诱导成纤维细胞转分化为类睾丸间质细胞的方法 | |
| CN104195103B (zh) | 一种诱导多能干细胞的培养基及其用途 | |
| CN109385404B (zh) | 一种诱导干细胞分化为神经元的方法及神经元与应用 | |
| CN105255826A (zh) | 人诱导多功能干细胞向睾丸间质细胞的诱导分化方法及其用途 | |
| CN107034238A (zh) | 一种永生化特络细胞系及其构建方法 | |
| CN102286532B (zh) | 一种获得诱导性多能干细胞的方法 | |
| CN101497872A (zh) | 诱导人胚胎干细胞定向分化为肝细胞的方法及专用培养基 | |
| CN109897815B (zh) | 一种无需包被的脂肪内皮祖细胞的高效分离和培养方法 | |
| CN107794244A (zh) | Vero‑pAPN细胞系及其制备方法 | |
| CN107012124B (zh) | 钠钙交换体1启动子联合丹参酚酸B诱导iPSCs定向心肌分化的方法 | |
| CN110229790A (zh) | 永生化人源神经干细胞系、制备方法、重组病毒载体及应用 | |
| CN104805122A (zh) | Runx2重组慢病毒感染骨髓间充质干细胞表达成骨基因的方法 | |
| CN104830907A (zh) | 骨髓间充质干细胞表达成骨基因的构建方法 | |
| CN108486156A (zh) | 一种永生化树鼩小肠上皮细胞系及其构建方法与应用 | |
| CN109833328A (zh) | miR-27及其类似物在体外抑制骨髓间充质干细胞向肥大软骨细胞分化中的应用 | |
| CN105233303B (zh) | 一种通过基因改造血管内皮细胞促进创面愈合的方法及其应用 | |
| CN113151153A (zh) | 一种小鼠腹膜血管周细胞的纯化方法和鉴定方法 | |
| CN109680000B (zh) | 利用树鼩骨髓间充干细胞建立hcv细胞模型的方法 | |
| CN104450622B (zh) | 一种重组细胞系、其制备方法和应用 | |
| CN107177555B (zh) | 一种H9C2心肌细胞培养液诱导iPSCs定向心肌分化的方法 | |
| CN108893492B (zh) | 一种重组慢病毒生产试剂盒及其应用 | |
| CN105018527A (zh) | 一种慢病毒转染鱼类或两栖类细胞系的方法 | |
| CN106497883A (zh) | 一种稳定表达抑癌基因REIC/Dkk3的人脐带间充质干细胞及其构建方法和应用 | |
| CN105936888A (zh) | 人扁桃体上皮细胞的分离培养与鉴定方法 | |
| CN106011065B (zh) | 人眼球结膜胬肉干细胞细胞株 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20191018 |