CN111808797A - Application of Necrostatin-1 and preparation for promoting maturation of islet cells of newborn pigs - Google Patents
Application of Necrostatin-1 and preparation for promoting maturation of islet cells of newborn pigs Download PDFInfo
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- 230000035800 maturation Effects 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 241000282887 Suidae Species 0.000 title claims abstract description 20
- 230000001737 promoting effect Effects 0.000 title claims abstract description 18
- TXUWMXQFNYDOEZ-UHFFFAOYSA-N 5-(1H-indol-3-ylmethyl)-3-methyl-2-sulfanylidene-4-imidazolidinone Chemical compound O=C1N(C)C(=S)NC1CC1=CNC2=CC=CC=C12 TXUWMXQFNYDOEZ-UHFFFAOYSA-N 0.000 title claims description 18
- 238000000338 in vitro Methods 0.000 claims abstract description 7
- 239000001963 growth medium Substances 0.000 claims description 8
- 241000282898 Sus scrofa Species 0.000 claims 7
- 238000009472 formulation Methods 0.000 claims 2
- 239000000203 mixture Substances 0.000 claims 2
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- 238000011160 research Methods 0.000 abstract description 3
- 238000001727 in vivo Methods 0.000 abstract description 2
- 238000004113 cell culture Methods 0.000 abstract 1
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 20
- 102000004877 Insulin Human genes 0.000 description 10
- 108090001061 Insulin Proteins 0.000 description 10
- 229940125396 insulin Drugs 0.000 description 10
- 239000002609 medium Substances 0.000 description 8
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 6
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 5
- 230000000638 stimulation Effects 0.000 description 5
- UOFGSWVZMUXXIY-UHFFFAOYSA-N 1,5-Diphenyl-3-thiocarbazone Chemical compound C=1C=CC=CC=1N=NC(=S)NNC1=CC=CC=C1 UOFGSWVZMUXXIY-UHFFFAOYSA-N 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
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- 229940088872 Apoptosis inhibitor Drugs 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 208000013016 Hypoglycemia Diseases 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 206010022489 Insulin Resistance Diseases 0.000 description 1
- MIFGOLAMNLSLGH-QOKNQOGYSA-N Z-Val-Ala-Asp(OMe)-CH2F Chemical compound COC(=O)C[C@@H](C(=O)CF)NC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)OCC1=CC=CC=C1 MIFGOLAMNLSLGH-QOKNQOGYSA-N 0.000 description 1
- 239000000158 apoptosis inhibitor Substances 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
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- 238000011065 in-situ storage Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- RCTGMCJBQGBLKT-PAMTUDGESA-N scarlet red Chemical compound CC1=CC=CC=C1\N=N\C(C=C1C)=CC=C1\N=N\C1=C(O)C=CC2=CC=CC=C12 RCTGMCJBQGBLKT-PAMTUDGESA-N 0.000 description 1
- 229960005369 scarlet red Drugs 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
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- 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
- C12N5/0676—Pancreatic cells
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- 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
- C12N2501/70—Enzymes
- C12N2501/72—Transferases [EC 2.]
- C12N2501/727—Kinases (EC 2.7.)
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Abstract
The invention relates to the field of cell culture, and discloses application of Neocystatin-1 and a preparation for promoting maturation of islet cells of newborn pigs. The invention can promote the maturity of the islet of the newborn pig in the process of culturing the islet cells of the newborn pig in vitro in a short period, improve the function of the islet of the newborn pig, reduce the cost of the islet of the newborn pig, lay a research foundation for the maturity promotion of the islet of the newborn pig transplanted in vivo, and have great medical significance in the aspect of improving clinical islet transplantation.
Description
Technical Field
The invention relates to the field of cell biology, in particular to application of Necrostatin-1 and a preparation for promoting maturation of islet cells of newborn pigs.
Background
Adult pig islet cells can directly exert islet functions, but an anoxic process exists in the transplanting process, so that the adult pig islet cells are killed greatly after transplantation, and the transplantation failure is caused. Researches find that the islet cells of the newborn pigs have strong hypoxia tolerance and can survive in a large amount after transplantation, but the islet cells of the newborn pigs are still in an immature state, need to pass through a long maturation stage, cannot immediately regulate blood sugar after transplantation, and are easily induced to die by the hyperglycemia environment of a receptor. Therefore, the accelerated maturation of the islet cells of the newborn pigs is promoted, and the islet cells of the newborn pigs are transplanted with great significance.
Disclosure of Invention
The invention aims to provide application of Necrostatin-1, which is mainly used for preparing a preparation for promoting the maturation of islet cells of newborn pigs.
The concentration of the Necrostatin-1 in the preparation for promoting the maturation of the islet cells of the newborn pig is 20-500 mu M, preferably 20-200 mu M, further preferably 50-150 mu M, and most preferably 50-100 mu M.
The preparation for promoting the maturation of the islet cells of the newborn pig is prepared by adding Neocystatin-1 into a culture medium of the islet cells of the newborn pig.
Further, the preparation for promoting the maturation of the islet cells of the newborn pig is used during in vitro culture.
The second purpose of the invention is to provide another application of Necrostatin-1, mainly used for promoting the maturation of islet cells of newborn pigs.
When the maturation of islet cells of a newborn pig is promoted, a preparation with the concentration of Necrostatin-1 of 20-500 mu M is prepared; preferably 20 to 200. mu.M, more preferably 50 to 150. mu.M, most preferably 50 to 100. mu.M.
The preparation is prepared by adding Neocystatin-1 into a culture medium of the pancreatic islets of the newborn pigs.
Further, the preparation is used in vitro culture; the treatment time for promoting the maturation of the islet cells of the newborn pig is 3-5 days.
The third purpose of the invention is to provide a preparation for promoting the maturation of islet cells of newborn pigs, which comprises neocystatin-1.
The concentration of the Necrostatin-1 in the preparation is 20-500 mu M, preferably 20-200 mu M, further preferably 50-150 mu M, and most preferably 50-100 mu M.
The invention discovers for the first time that Neocystatin-1 can remarkably promote the maturation of islet cells in the in vitro culture process of a newborn pig; experiments show that after the Necrostatin-1 is added into the culture medium, the number of islet beta cells is obviously increased, the amount of insulin released by sugar stimulation and the content of insulin in a unit islet mass are obviously increased, and therefore, the Necrostatin-1 can improve the maturity of the neonatal pig islet.
The invention can promote the maturity of the islet of the newborn pig in the process of culturing the islet cells of the newborn pig in vitro in a short period, improve the function of the islet of the newborn pig, reduce the cost of the islet of the newborn pig, lay a research foundation for the maturity promotion of the islet of the newborn pig transplanted in vivo, and have great medical significance in the aspect of improving clinical islet transplantation.
Drawings
FIG. 1 shows DTZ staining of newly born pig islets after culturing Necrostatin-1 with different concentrations;
FIG. 2 is a graph showing the effect of different concentrations of Neostatin-1 on the number of islet beta cells in newborn pigs;
FIG. 3 shows glucose stimulation test of different concentrations of Neostatin-1 after culturing neonatal porcine islets;
FIG. 4 shows the effect of different concentrations of Neostatin-1 on the insulin content per islet mass.
Detailed Description
The following examples are intended to further illustrate the invention without limiting it.
Example 1
Neocystatin-1 promotes the number of beta cells of pancreatic islets of new-born pigs
Newborn pig islet culture medium (NICcMedium) containing 0. mu.M, 50. mu.M and 100. mu.M Neocystatin-1 was used at 37 ℃ with 5% CO2And in a 95% air incubator, 10000IEQ islet cells per dish are changed once every 2 days, the neonatal pig islet is cultured for 5 days, and then the neonatal pig islet is stained by Dithizone (DTZ) to observe the mature condition of the neonatal pig islet. A part of the newly born porcine islet cells cultured with NICC Medium containing 50 μ M echinostatin-1 stained scarlet red blood cellsThe color of the newborn pig islet cells cultured by the NICcMedium containing 100 mu M of Necrostatin-1 is mostly stained scarlet, which indicates that the maturity of the newborn pig islet cells cultured for 5 days is improved along with the increase of the concentration of the Necrostatin-1 in the culture medium. However, the newborn pig islet cells cultured by the NICC Medium without the addition of Neostatin-1 have no distinct scarlet color staining, and the result is shown in FIG. 1. Therefore, the Necrostatin-1 can increase the number of beta cells and promote the islet maturation when culturing the islet cells. The number of islet beta cells was analyzed using Image J software and the results are shown in figure 2.
Neocrostatin-1 was purchased from sigma reagent. The components of the NICC medium are as follows: 70% Ham's F-10 medium, 20% human serum albumin, 10% pig serum; and 50 mu mol of apoptosis inhibitor Z-VAD-FMK and 8mmol of pyruvic acid.
Example 2
Neocystatin-1 promotes functions of islet cells of newborn pigs
After 3 groups of islet cells were subjected to glucose stimulation (GSIS) test for 5 days by culturing neonatal pig islet culture Medium (NICCCMedium) containing 0. mu.M, 50. mu.M and 100. mu.M of Neostatin-1, the results in FIG. 3 show that neonatal pig islet cells cultured with NICC Medium containing 100. mu.M of Neostatin-1 release more insulin for sugar stimulation, while neonatal pig islet cells cultured with NICC Medium containing 50. mu.M of Neostatin-1 release more insulin for sugar stimulation, and neonatal pig islet cells cultured with NICC Medium without Neostatin-1 added release the least insulin, indicating that the islet cells cultured with Neostatin-1 added can promote the maturation of islet cells.
Example 3
Neocystatin-1 promotes insulin content of islet cells of newborn pigs
Neonatal pig islets were cultured for 5 days in neonatal pig islet medium (NICcMedium) containing 0. mu.M, 50. mu.M, 100. mu.M Necrostatin-1, respectively, under the same conditions as in example 1. Collecting 150IEQ islet cell mass, lysing islet cells with cell lysate for 30 minutes on ice, collecting lysate after the cells are completely lysed, quantifying insulin content and DNA content in the supernatant, and analyzing the ratio of insulin in terms of unit DNA amount, the results are shown in FIG. 4.
Claims (10)
- The application of Necrostatin-1, which is characterized in that the Necrostatin-1 is used for preparing a preparation for promoting the maturation of islet cells of newborn pigs.
- 2. The use according to claim 1, wherein the concentration of neostatin-1 in the preparation for promoting maturation of islet cells of a neonatal pig is 20-500 μ M, preferably 20-200 μ M, further preferably 50-150 μ M, most preferably 50-100 μ M.
- 3. The use of claim 1 or 2, wherein the preparation for promoting the maturation of islet cells of a newborn pig is prepared by adding Neocystatin-1 into a culture medium of islet cells of a newborn pig.
- 4. The use according to claim 1 or 2, wherein the preparation for promoting maturation of islet cells of neonatal swine is used in vitro culture.
- Use of Necrostatin-1 for promoting maturation of islet cells of neonatal pigs.
- 6. The use of claim 5, wherein the preparation of neostatin-1 is prepared at a concentration of 20-500 μ M in advance of the promotion of the maturation of islet cells of a neonatal pig; preferably 20 to 200. mu.M, more preferably 50 to 150. mu.M, most preferably 50 to 100. mu.M.
- 7. The use of claim 6, wherein the preparation is prepared by adding Neocystatin-1 to culture medium of islets of Langerhans of newborn pigs.
- 8. The use according to claim 6 or 7, wherein said preparation is for use in vitro culture; the treatment time for promoting the maturation of the islet cells of the newborn pig is 3-5 days.
- 9. A preparation for promoting the maturation of islet cells of a newborn pig, which comprises Neocystatin-1.
- 10. The formulation according to claim 9, characterized in that the concentration of nercrostatin-1 in the formulation is 20-500 μ M, preferably 20-200 μ M, further preferably 50-150 μ M, most preferably 50-100 μ M.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010740110.7A CN111808797A (en) | 2020-07-28 | 2020-07-28 | Application of Necrostatin-1 and preparation for promoting maturation of islet cells of newborn pigs |
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695367A (en) * | 2013-12-24 | 2014-04-02 | 湖南赛诺生物科技有限责任公司 | Improved culture medium for neonatal pig islet cells and use method of same |
| CN105078975A (en) * | 2015-07-22 | 2015-11-25 | 南方医科大学 | Application of Necrostatin-1 as neutrophile granulocyte apoptosis accelerant |
| CN105169367A (en) * | 2015-10-16 | 2015-12-23 | 中国人民解放军第三军医大学第一附属医院 | Application of TRPC6 in preparation of medicament for diagnosing and treating kidney ischemic reperfusion injury |
| CN106687575A (en) * | 2014-06-04 | 2017-05-17 | 美国安进公司 | Methods for harvesting mammalian cell cultures |
| CN107519165A (en) * | 2017-09-05 | 2017-12-29 | 中山大学中山眼科中心 | Applications of the Necrostatin 1 in nervus retrogression ocular disease drug is treated |
| WO2018132926A1 (en) * | 2017-01-23 | 2018-07-26 | Stemcell Technologies Canada Inc. | Media and methods for enhancing the survival and proliferation of stem cells |
| CN109880793B (en) * | 2019-03-14 | 2020-01-10 | 湖南赛诺生物科技股份有限公司 | Young pig islet cell culture medium and using method thereof |
-
2020
- 2020-07-28 CN CN202010740110.7A patent/CN111808797A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695367A (en) * | 2013-12-24 | 2014-04-02 | 湖南赛诺生物科技有限责任公司 | Improved culture medium for neonatal pig islet cells and use method of same |
| CN106687575A (en) * | 2014-06-04 | 2017-05-17 | 美国安进公司 | Methods for harvesting mammalian cell cultures |
| CN105078975A (en) * | 2015-07-22 | 2015-11-25 | 南方医科大学 | Application of Necrostatin-1 as neutrophile granulocyte apoptosis accelerant |
| CN105169367A (en) * | 2015-10-16 | 2015-12-23 | 中国人民解放军第三军医大学第一附属医院 | Application of TRPC6 in preparation of medicament for diagnosing and treating kidney ischemic reperfusion injury |
| WO2018132926A1 (en) * | 2017-01-23 | 2018-07-26 | Stemcell Technologies Canada Inc. | Media and methods for enhancing the survival and proliferation of stem cells |
| CN107519165A (en) * | 2017-09-05 | 2017-12-29 | 中山大学中山眼科中心 | Applications of the Necrostatin 1 in nervus retrogression ocular disease drug is treated |
| CN109880793B (en) * | 2019-03-14 | 2020-01-10 | 湖南赛诺生物科技股份有限公司 | Young pig islet cell culture medium and using method thereof |
Non-Patent Citations (3)
| Title |
|---|
| HIEN LAU ET AL.: "Dose-dependent effects of necrostatin-1 supplementation to tissue culture media of young porcine islets", 《PLOS ONE》 * |
| HIEN LAU ET AL.: "Necrostatin‐1 supplementation enhances young porcine islet maturation and in vitro function", 《XENOTRANSPLANTATION》 * |
| 叶斌 等: "Necrostatin-1对TNF-α诱导的胰岛细胞损伤的保护作用", 《中南大学学报（医学版）》 * |
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Application publication date: 20201023 |