CN108379662A - A kind of application of stem cell in Heart Transplantation Model - Google Patents
A kind of application of stem cell in Heart Transplantation Model Download PDFInfo
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- CN108379662A CN108379662A CN201810130546.7A CN201810130546A CN108379662A CN 108379662 A CN108379662 A CN 108379662A CN 201810130546 A CN201810130546 A CN 201810130546A CN 108379662 A CN108379662 A CN 108379662A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/225—Fibrin; Fibrinogen
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N5/0602—Vertebrate cells
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- C12N5/0657—Cardiomyocytes; Heart cells
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
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- C12N2533/56—Fibrin; Thrombin
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Abstract
The invention particularly discloses a kind of stem cell Heart Transplantation Model application；Include the following steps：S1. the culture of heart flesh ball derived cell (CDCs)；S2. the preparation of blood platelet fibrin holder；S3. the observation of blood platelet fibrin holder；S4. heart flesh ball derived cell blood platelet fibrin holder combined transplantation.Show that cardiac function after myocardial infarction can preferably be corrected by combining CDCs in blood platelet fibrin holder；It can mutually promote between blood platelet fibrin holder and CDCs simultaneously, more vascular endothelial cells, cardiac muscle cell can be regenerated after combined transplantation, increase infarct location vessel density, it raises more stem cells and enters infarcted region performance regeneration function, to reverse negativity Myocardial Remodeling, improve cardiac function.
Description
Technical field
The present invention relates to Heart Transplantation Model technical fields, and in particular, to a kind of stem cell is in Heart Transplantation Model
Using.
Background technology
Myocardial infarction and heart failure are one of mankind's major causes of death of Western Countries, and seriously increase society
It can financial burden.Develop on a large scale very much although drug development and interventional treatment have, still can not reduce heart work(caused by myocardial infarction
Energy the failure total death rate and incidence are susceptible to the heart to find out its cause, being on the one hand a large amount of cardiomyocyte cell death after infarct
Malignant arrhythmia occurs for the serious failure of dirty function, eventually leads to death；And another aspect drug and interventional treatment
Though intervention makes quite a few patients of acute myocardial infarction survive, dead cell does not have neonatal cell to replace its work(
Can, when surviving cardiac muscle cell decompensation, left ventricle or even whole-heartedly function is badly damaged.Therefore it finds suitable approach and substitutes damage
The cardiac muscle of mistake promotes regeneration to have great significance in terms of changing prognosis.
The application of heart tissue engineering and biomaterial depends on synthetic material or natural material, by suitable
Approach recombinate the myocardium sample tissue to be formed with systolic and diastolic function, the heart of support function failure.In all material, injectable
Have the advantages that with biomaterial very prominent.Injectable combines stem cell with biomaterial, as bone marrow cell, fat source are dry thin
Born of the same parents and bone marrow derived inducing cardiac cell are proved that the function of heart can be improved.In recent years, continuous with recognizing CSCs
Deepen, many researchs think that it has the function of regeneration, can break up in vivo as a plurality of types of mature cells, such as
Cardiac muscle cell, vascular endothelial cell etc., therefore cause common concern.
Invention content
The present invention provides a kind of application of stem cell in Heart Transplantation Model to overcome the above-mentioned deficiency of the prior art.
To achieve the goals above, the present invention is achieved by the following technical programs：
Currently, being all based on the Clinical intervention of the cause of disease greatly for the treatment of ischemic heart disease, they are substituting death
The effect of cardiac muscle aspect is limited after the function and reparation infarct of cardiac muscle cell.Stem cell transplantation is the new for the treatment of ischemic heart disease
One of method, be used for Treating Ischemic Heart the best non-immunogenicity of ideal seed cell, have easily harvest, can be in vitro
Large amplification, the characteristic that can well grow up in heart microenvironment, and the reparation of damaged heart can be promoted.In recent years, greatly
The convictive evidence of measurer shows that there are stem cell pools in heart, if it is possible to be obtained from receptor itself heart dry thin
Born of the same parents, are not present immunogenicity and the case where be not suitable with heart microenvironment naturally, may have a treatment foreground well, and heart flesh
Glomus cell (CSp) and heart flesh ball derived cell (CDCs) (the cell mixing collection present in heart, containing cardiac stem cells
Group), the selective value as Treating Ischemic Heart must be inquired into.
A kind of application of stem cell in Heart Transplantation Model.
Preferably, the stem cell is heart flesh ball derived cell.
Application of the above-described heart flesh ball derived cell in Heart Transplantation Model, includes the following steps：
S1. the culture of heart flesh ball derived cell (CDCs)；
S2. the preparation of blood platelet fibrin holder；
S3. the observation of blood platelet fibrin holder；
S4. heart flesh ball derived cell-blood platelet fibrin holder combined transplantation.
Preferably, the culture of the heart flesh ball derived cell (CDCs) in the step S1 includes the following steps：Take health
The female 8 week old WKY rat heart muscle tissues of cleaning grade, harvest cell using tissue mass cell culture, then by suspend culture and
(or) adhere-wall culture mode obtains rat heart flesh ball derived cell, observes cellular morphology in vitro, lasting passage observation cell
Activity draws cell growth curve；Utilize the surface marker of Flow cytometry separate sources and different subculture
The expression of VEGF, IGF-1, SDF-1 and cell.
Preferably, the preparation of the blood platelet fibrin holder of the step S2, it is further comprising the steps of：
S1. Isoflurane deep anaesthesia rat is used；
S2. hara kiri skin finds out abdomen cardinal vein, adopts venous blood；
S3. by the venous blood being collected into immediately with 8% (v/v) sodium citrate mixing；
S4. it is put into centrifuge and gives 8000rpm centrifugation 5min, take supernatant, mix plastic success as follows, put
Enter -20 ° of 2ml centrifuge tubes to preserve for use；
S5.DMEM is put into 37 ° of water-baths and preheats；
S6.DMEM and platelet-poor plasma supernatant are according to 1-3：2-6 ratios mix, timing, gelation time are observed, in case in vivo
Experiment is used.
Preferably, the observation of the blood platelet fibrin holder in the step S3, it is further comprising the steps of：
S1. molding blood platelet fibrin holder is cleaned with PBS immediately；
S2. with 5% glutaraldehyde 30-60min is fixed in 0.2M sodium cacodylate buffers liquid (PH7.5)；
S3. it is cleaned 3 times with sodium cacodylate buffer liquid, each 5-15min；
S4. with 30%, 40%, 60%, 80%, 100% ethanol dehydration, each concentration is dehydrated 5-10min, then uses hexamethyl
Disilazane blots；
S5. it with ion sputtering film coating machine metalling is used, is taken pictures with scanning electron microscope.
Preferably, the heart flesh ball derived cell in the step S4-blood platelet fibrin holder combined transplantation, is also wrapped
Include following steps：
S1. 1 × 106CDCs and 50-100ul DMEM are taken to mix well, then mixed with 50-100ul Platelet-rich plasms；
S2. contain CDCs blood platelet fibrin holders before 100ul plastics are added in 24 orifice plates, be added in each hole
1ml is free of the IMDM of FBS；The blood platelet fibrin holder without CDCs, Mei Gekong is added in another 24 orifice plate simultaneously
The middle IMDM that 1ml is added and is free of FBS；
S3. the culture medium in collecting the 1st, 5,9,13,17 day respectively in each hole, while the fresh culture of equivalent is added
IMDM；
S4. according to the requirement of the ELISA kit of rat specificity, VEGF, IGF-1, SDF-1 in culture medium are detected.
Compared with prior art, the present invention has the advantages that：
CDCs can form blood vessel network structure in blood platelet fibrin holder, and can express cardiac muscle cell, interior
The marker of chrotoplast and vascular smooth muscle cells；It is co-cultured when with NRCMs, in the blood platelet fibrin holder containing CDCs
In NRCMs stretching, extensions, and cytochrome oxidase isozymes to be it play a role Walk are one of rapid, and more NRCMs have systolic and diastolic function, this
Illustrate blood platelet fibrin holder other than as carrier, additionally it is possible to provide good stem cell microenvironment, maintain stem cell
Activity promotes stem cells hyperplasia, promotes cardiac stem cells to other cell type differentiations, and advantage is provided for regeneration, and
And the growth of cell will not be not only fettered after the two mixing, additionally it is possible to promote the activity of recipient's heart cardiac muscle cell.
Specific implementation mode
The present invention is further elaborated with reference to specific embodiment, the embodiment is served only for explaining this hair
It is bright, it is not intended to limit the scope of the present invention.Test method used in following embodiments is routine unless otherwise specified
Method；Used material, reagent etc., unless otherwise specified, for the reagent and material commercially obtained.
A kind of application of stem cell in Heart Transplantation Model.
Preferably, the stem cell is heart flesh ball derived cell.
Application of the above-described heart flesh ball derived cell in Heart Transplantation Model, includes the following steps：
S1. the culture of heart flesh ball derived cell (CDCs)；
S2. the preparation of blood platelet fibrin holder；
S3. the observation of blood platelet fibrin holder；
S4. heart flesh ball derived cell-blood platelet fibrin holder combined transplantation.
Preferably, the culture of the heart flesh ball derived cell (CDCs) in the step S1 includes the following steps：Take health
The female 8 week old WKY rat heart muscle tissues of cleaning grade, harvest cell using tissue mass cell culture, then by suspend culture and
(or) adhere-wall culture mode obtains rat heart flesh ball derived cell, observes cellular morphology in vitro, lasting passage observation cell
Activity draws cell growth curve；Utilize the surface marker of Flow cytometry separate sources and different subculture
The expression of VEGF, IGF-1, SDF-1 and cell.
Preferably, the preparation of the blood platelet fibrin holder of the step S2, it is further comprising the steps of：
S1. Isoflurane deep anaesthesia rat is used；
S2. hara kiri skin finds out abdomen cardinal vein, adopts venous blood；
S3. by the venous blood being collected into immediately with 8% (v/v) sodium citrate mixing；
S4. it is put into centrifuge and gives 8000rpm centrifugation 5min, take supernatant, mix plastic success as follows, put
Enter -20 ° of 2ml centrifuge tubes to preserve for use；
S5.DMEM is put into 37 ° of water-baths and preheats；
S6.DMEM and platelet-poor plasma supernatant are according to 1-3：2-6 ratios mix, timing, gelation time are observed, in case in vivo
Experiment is used.
Preferably, the observation of the blood platelet fibrin holder in the step S3, it is further comprising the steps of：
S1. molding blood platelet fibrin holder is cleaned with PBS immediately；
S2. with 5% glutaraldehyde 30-60min is fixed in 0.2M sodium cacodylate buffers liquid (PH7.5)；
S3. it is cleaned 3 times with sodium cacodylate buffer liquid, each 5-15min；
S4. with 30%, 40%, 60%, 80%, 100% ethanol dehydration, each concentration is dehydrated 5-10min, then uses hexamethyl
Disilazane blots；
S5. it with ion sputtering film coating machine metalling is used, is taken pictures with scanning electron microscope.
Preferably, the heart flesh ball derived cell in the step S4-blood platelet fibrin holder combined transplantation, is also wrapped
Include following steps：
S1. 1 × 106CDCs and 50-100ul DMEM are taken to mix well, then mixed with 50-100ul Platelet-rich plasms；
S2. contain CDCs blood platelet fibrin holders before 100ul plastics are added in 24 orifice plates, be added in each hole
1ml is free of the IMDM of FBS；The blood platelet fibrin holder without CDCs, Mei Gekong is added in another 24 orifice plate simultaneously
The middle IMDM that 1ml is added and is free of FBS；
S3. the culture medium in collecting the 1st, 5,9,13,17 day respectively in each hole, while the fresh culture of equivalent is added
IMDM；
S4. according to the requirement of the ELISA kit of rat specificity, VEGF, IGF-1, SDF-1 in culture medium are detected.
1 experiment in vivo of experimental example
(1) experiment packet and sample time design
First group (DMEM rent, i.e. conventional medium group) 3 points of rats with acute myocardial infarction infarcted myocardium periphery, 6 points,
100ulDMEM is injected in 9 points and 12 positions, totally 15；
Second group (blood platelet fibrin holder group) 3 points of rats with acute myocardial infarction infarcted myocardium periphery, 6 points, 9
The point blood platelet fibrin holder dyed with 12 positions injection 100ul, totally 15；
Third group (blood platelet fibrin holder+CDCs groups) 3 points of rats with acute myocardial infarction infarcted myocardium periphery,
6 points, 9 points and 12 position injection no dyeings contain the blood platelet fibrin holder 100ul totally 15 of CDCs.
It is the 12nd hour, the 3rd day, the 7th day and the 14th day to test sampling time point.
(2) in blood platelet fibrin holder CDCs amplification
By CDCs cultures in blood platelet fibrin holder and in conventional organization culture plate, after 14 days, relative to culture
CDCs in Nostoc commune Vanch disk, the CDCs in blood platelet fibrin holder group extend and form blood vessel sample network structure.
CDCs quantity in blood platelet fibrin holder group and CDCs (cell number relative to 12h) under normal conditions are the 12nd
Hour, the 3rd day, the 7th day and the 14th day cell number is respectively 1.86 ± 0.42,3.17 ± 0.38,4.04 ± 0.46 and 1.96
± 0.54,2.37 ± 0.40,3.21 ± 0.65, two groups of each respective values (P ＜ 0.5), the former quantity increases more obvious.
(3) in blood platelet fibrin holder CDCs activity
When 14d analysis CDCs ratio of semi-minor axis length in blood platelet fibrin holder and under normal conditions be 1.57 ±
0.42 and 0.73 ± 0.97, the two difference is statistically significant (P ＜ 0.5).It is dyed with cell activity/toxic agent box when 14d
It is respectively (0.73 ± 0.97) % and (1.57 in blood platelet fibrin holder and in the Percent mortality of normal condition CDCs
± 0.42) %.
Show that cardiac function after myocardial infarction can preferably be corrected by combining CDCs in blood platelet fibrin holder；Simultaneously
It can mutually promote between blood platelet fibrin holder and CDCs, it is thin that more blood vessel endotheliums can be regenerated after combined transplantation
Born of the same parents, cardiac muscle cell increase infarct location vessel density, raise more stem cells and enter infarcted region performance regeneration function, from
And negativity Myocardial Remodeling is reversed, improve cardiac function.
Finally it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than the present invention is protected
The limitation of range is protected, although being explained in detail to the present invention with reference to preferred embodiment, those skilled in the art answer
Work as understanding, technical scheme of the present invention can be modified or replaced equivalently, without departing from the reality of technical solution of the present invention
Matter and range.
Claims (6)
1. a kind of stem cell is in the application of Heart Transplantation Model.
2. application of the stem cell according to claim 1 in Heart Transplantation Model, which is characterized in that the stem cell is
Heart flesh ball derived cell, includes the following steps：
S1. the culture of heart flesh ball derived cell (CDCs)；
S2. the preparation of blood platelet fibrin holder；
S3. the observation of blood platelet fibrin holder；
S4. heart flesh ball derived cell-blood platelet fibrin holder combined transplantation.
3. application of the heart flesh ball derived cell according to claim 2 in Heart Transplantation Model, which is characterized in that institute
The culture for stating the heart flesh ball derived cell (CDCs) in step S1 includes the following steps：Take female 8 week old of the cleaning grade of health
WKY rat heart muscle tissues harvest cell using tissue mass cell culture, then are obtained by suspension culture and (or) adhere-wall culture mode
Rat heart flesh ball derived cell, observes cellular morphology in vitro, and it is bent to draw cell growth for lasting passage observation cell activity
Line；Using Flow cytometry separate sources and different subculture surface marker VEGF, IGF-1, SDF-1 and
The expression of cell.
4. application of the heart flesh ball derived cell according to claim 2 in Heart Transplantation Model, which is characterized in that institute
The preparation of the blood platelet fibrin holder of step S2 is stated, it is further comprising the steps of：
S1. Isoflurane deep anaesthesia rat is used；
S2. hara kiri skin finds out abdomen cardinal vein, adopts venous blood；
S3. by the venous blood being collected into immediately with 8% (v/v) sodium citrate mixing；
S4. it is put into centrifuge and gives 8000rpm centrifugation 5min, take supernatant, mix plastic success as follows, be put into
- 20 ° of 2ml centrifuge tubes preserve for use；
S5.DMEM is put into 37 ° of water-baths and preheats；
S6.DMEM and platelet-poor plasma supernatant are according to 1-3：2-6 ratios mix, timing, gelation time are observed, in case in vivo studies
With.
5. application of the heart flesh ball derived cell according to claim 2 in Heart Transplantation Model, which is characterized in that institute
The observation of the blood platelet fibrin holder in step S3 is stated, it is further comprising the steps of：
S1. molding blood platelet fibrin holder is cleaned with PBS immediately；
S2. with 5% glutaraldehyde 30-60min is fixed in 0.2M sodium cacodylate buffers liquid (PH7.5)；
S3. it is cleaned 3 times with sodium cacodylate buffer liquid, each 5-15min；
S4. with 30%, 40%, 60%, 80%, 100% ethanol dehydration, each concentration is dehydrated 5-10min, then with two silicon of hexamethyl
Azane blots；
S5. it with ion sputtering film coating machine metalling is used, is taken pictures with scanning electron microscope.
6. application of the heart flesh ball derived cell according to claim 2 in Heart Transplantation Model, which is characterized in that institute
Heart flesh ball derived cell-blood platelet fibrin holder combined transplantation in step S4 is stated, it is further comprising the steps of：
S1. 1 × 106CDCs and 50-100ul DMEM are taken to mix well, then mixed with 50-100ul Platelet-rich plasms；
S2. contain CDCs blood platelet fibrin holders before 100ul plastics are added in 24 orifice plates, 1ml is added not in each hole
IMDM containing FBS；The blood platelet fibrin holder without CDCs is added in another 24 orifice plate simultaneously, is added in each hole
1ml is free of the IMDM of FBS；
S3. the culture medium in collecting the 1st, 5,9,13,17 day respectively in each hole, while the fresh culture IMDM of equivalent is added；
S4. according to the requirement of the ELISA kit of rat specificity, VEGF, IGF-1, SDF-1 in culture medium are detected.
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| CN201810130546.7A CN108379662A (en) | 2018-02-08 | 2018-02-08 | A kind of application of stem cell in Heart Transplantation Model |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110024742A (en) * | 2019-02-14 | 2019-07-19 | 广东省心血管病研究所 | A kind of animal model application method based on the stem cell of big data in heart transplant |
| EP4180050A1 (en) * | 2019-11-15 | 2023-05-17 | Secretome Therapeutics, Inc. | Immortalized cardiac stem cells for cardiac repair |
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| CN2824875Y (en) * | 2005-04-14 | 2006-10-11 | 南方医院 | An injection type tissue engineering bone engraft |
| CN101437938A (en) * | 2004-11-08 | 2009-05-20 | 约翰霍普金斯大学 | Cardiac stem cells |
| CN103860597A (en) * | 2014-03-03 | 2014-06-18 | 奥思达干细胞有限公司 | Stem cell preparation for treating ischemic cardiomyopathy and preparation method of stem cell preparation |
| JPWO2016043201A1 (en) * | 2014-09-16 | 2017-08-03 | 国立大学法人山口大学 | Method for producing cell sheet activated by low oxygen treatment |
| CN107028980A (en) * | 2009-05-20 | 2017-08-11 | 卡迪欧参生物科技有限公司 | Pharmaceutical composition for treating heart disease |
| CN108187141A (en) * | 2018-01-31 | 2018-06-22 | 广州沙艾生物科技有限公司 | A kind of application of heart flesh ball derived cell in biomaterial |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101437938A (en) * | 2004-11-08 | 2009-05-20 | 约翰霍普金斯大学 | Cardiac stem cells |
| CN2824875Y (en) * | 2005-04-14 | 2006-10-11 | 南方医院 | An injection type tissue engineering bone engraft |
| CN107028980A (en) * | 2009-05-20 | 2017-08-11 | 卡迪欧参生物科技有限公司 | Pharmaceutical composition for treating heart disease |
| CN103860597A (en) * | 2014-03-03 | 2014-06-18 | 奥思达干细胞有限公司 | Stem cell preparation for treating ischemic cardiomyopathy and preparation method of stem cell preparation |
| JPWO2016043201A1 (en) * | 2014-09-16 | 2017-08-03 | 国立大学法人山口大学 | Method for producing cell sheet activated by low oxygen treatment |
| CN108187141A (en) * | 2018-01-31 | 2018-06-22 | 广州沙艾生物科技有限公司 | A kind of application of heart flesh ball derived cell in biomaterial |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110024742A (en) * | 2019-02-14 | 2019-07-19 | 广东省心血管病研究所 | A kind of animal model application method based on the stem cell of big data in heart transplant |
| EP4180050A1 (en) * | 2019-11-15 | 2023-05-17 | Secretome Therapeutics, Inc. | Immortalized cardiac stem cells for cardiac repair |
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