CN108187141A - A kind of application of heart flesh ball derived cell in biomaterial - Google Patents
A kind of application of heart flesh ball derived cell in biomaterial Download PDFInfo
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- CN108187141A CN108187141A CN201810098368.4A CN201810098368A CN108187141A CN 108187141 A CN108187141 A CN 108187141A CN 201810098368 A CN201810098368 A CN 201810098368A CN 108187141 A CN108187141 A CN 108187141A
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- heart
- blood platelet
- derived cell
- ball derived
- platelet fibrin
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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
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/225—Fibrin; Fibrinogen
-
- 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
- A61L27/3826—Muscle cells, e.g. smooth muscle cells
-
- 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
- A61L27/3886—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 comprising two or more cell types
-
- 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
- A61L27/3895—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 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
Abstract
The invention particularly discloses a kind of application of heart flesh ball derived cell in biomaterial；Include the following steps：S1. the culture of heart flesh ball derived cell (CDCs)；S2. the preparation of blood platelet fibrin stent；S3. the observation of blood platelet fibrin stent；S4. heart flesh ball derived cell blood platelet fibrin stent combined transplantation.Show that cardiac function after myocardial infarction can preferably be corrected by combining CDCs in blood platelet fibrin stent；It can mutually promote between blood platelet fibrin stent 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, so as 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 heart flesh ball derived cell is in biology
Application in material.
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, heart work(caused by still can not reducing myocardial infarction
The total death rate of energy failure and incidence to find out its cause, being on the one hand a large amount of cardiomyocyte cell death after infarct, are susceptible to the heart
Malignant arrhythmia occurs for the serious failure of dirty function, eventually leads to death；And another aspect drug and interventional treatment
Though intervention survives quite a few patients of acute myocardial infarction, 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 prognosis is changed.
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 done carefully
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, it is 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 heart flesh ball derived cell in biomaterial to overcome the above-mentioned deficiency of the prior art
Application.
To achieve these goals, the present invention is achieved by the following technical programs：
At present, it is 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 of cardiac muscle cell and reparation infarct.Stem cell transplantation is the new for the treatment of ischemic heart disease
One of method, for the best non-immunogenicity of preferable seed cell of Treating Ischemic Heart, have easily harvest, can be in vitro
A large amount of characteristics that expands, 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 naturally there is no immunogenicity and are not suitable with the situation of heart microenvironment, may have a treatment prospect 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 heart flesh ball derived cell in biomaterial, the biomaterial are a kind of heart transplant mould
Type.
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 stent；
S3. the observation of blood platelet fibrin stent；
S4. heart flesh ball derived cell-blood platelet fibrin stent 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 Flow cytometry separate sources and the surface marker of different subculture
The expression of VEGF, IGF-1, SDF-1 and cell.
Preferably, the preparation of the blood platelet fibrin stent 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 stent in the step S3, it is further comprising the steps of：
S1. molding blood platelet fibrin stent is cleaned immediately with PBS；
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 using ion sputtering film coating machine metalling, is taken pictures with scanning electron microscope.
Preferably, the heart flesh ball derived cell in the step S4-blood platelet fibrin stent combined transplantation, is also wrapped
Include following steps：
S1. 1 × 106CDCs and the abundant mixings of 50-100ul DMEM are taken, then is mixed with 50-100ul Platelet-rich plasms；
S2. contain CDCs blood platelet fibrin stents before 100ul plastics are added in 24 orifice plates, added in each hole
1ml is free of the IMDM of FBS；The blood platelet fibrin stent without CDCs, Mei Gekong are added in another 24 orifice plate simultaneously
The middle IMDM for adding in 1ml and being free of FBS；
S3. the culture medium in collecting the 1st, 5,9,13,17 day respectively in each hole, while add in the fresh culture of equivalent
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 stent, 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 stent 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 stent 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 embodiment
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 heart flesh ball derived cell in biomaterial, the biomaterial are a kind of heart transplant mould
Type.
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 stent；
S3. the observation of blood platelet fibrin stent；
S4. heart flesh ball derived cell-blood platelet fibrin stent 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 Flow cytometry separate sources and the surface marker of different subculture
The expression of VEGF, IGF-1, SDF-1 and cell.
Preferably, the preparation of the blood platelet fibrin stent 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 stent in the step S3, it is further comprising the steps of：
S1. molding blood platelet fibrin stent is cleaned immediately with PBS；
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 using ion sputtering film coating machine metalling, is taken pictures with scanning electron microscope.
Preferably, the heart flesh ball derived cell in the step S4-blood platelet fibrin stent combined transplantation, is also wrapped
Include following steps：
S1. 1 × 106CDCs and the abundant mixings of 50-100ul DMEM are taken, then is mixed with 50-100ul Platelet-rich plasms；
S2. contain CDCs blood platelet fibrin stents before 100ul plastics are added in 24 orifice plates, added in each hole
1ml is free of the IMDM of FBS；The blood platelet fibrin stent without CDCs, Mei Gekong are added in another 24 orifice plate simultaneously
The middle IMDM for adding in 1ml and being free of FBS；
S3. the culture medium in collecting the 1st, 5,9,13,17 day respectively in each hole, while add in the fresh culture of equivalent
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,
9 points and 12 positions injection 100ulDMEM, totally 15；
Second group (blood platelet fibrin stent group) 3 points of rats with acute myocardial infarction infarcted myocardium periphery, 6 points, 9
The point blood platelet fibrin stent dyed with 12 positions injection 100ul, totally 15；
Third group (blood platelet fibrin stent+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 stent 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 stent CDCs amplification
By CDCs cultures in blood platelet fibrin stent and in conventional organization culture plate, after 14 days, relative to culture
CDCs in Nostoc commune Vanch disk, the CDCs in blood platelet fibrin stent group extend and form blood vessel sample network structure.
CDCs quantity in blood platelet fibrin stent group and CDCs (relative to the cell number of 12h) under normal conditions are the 12nd
Hour, the cell number of the 3rd day, the 7th day and the 14th day 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 stent CDCs activity
During 14d analysis CDCs ratio of semi-minor axis length in blood platelet fibrin stent and under normal conditions for 1.57 ±
0.42 and 0.73 ± 0.97, the two difference is statistically significant (P ＜ 0.5).It is dyed during 14d with cell activity/toxic agent box
It is respectively (0.73 ± 0.97) % and (1.57 in blood platelet fibrin stent 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 stent；Simultaneously
It can mutually promote between blood platelet fibrin stent 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 with reference to preferred embodiment to the present invention, those of ordinary skill in the art should
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. application of a kind of heart flesh ball derived cell in biomaterial, which is characterized in that the biomaterial is a kind of heart
Dirty transplantation model.
2. application of the heart flesh ball derived cell according to claim 1 in Heart Transplantation Model, which is characterized in that packet
Include following steps：
S1. the culture of heart flesh ball derived cell (CDCs)；
S2. the preparation of blood platelet fibrin stent；
S3. the observation of blood platelet fibrin stent；
S4. heart flesh ball derived cell-blood platelet fibrin stent 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 cleaning grade of health
WKY rat heart muscle tissues harvest cell, then obtain by suspension culture and (or) adhere-wall culture mode using tissue mass cell culture
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 stent 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 stent in step S3 is stated, it is further comprising the steps of：
S1. molding blood platelet fibrin stent is cleaned immediately with PBS；
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 using ion sputtering film coating machine metalling, 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 stent combined transplantation in step S4 is stated, it is further comprising the steps of：
S1. 1 × 106CDCs and the abundant mixings of 50-100ulDMEM are taken, then is mixed with 50-100ul Platelet-rich plasms；
S2. contain CDCs blood platelet fibrin stents before 100ul plastics are added in 24 orifice plates, 1ml is added in not in each hole
IMDM containing FBS；The blood platelet fibrin stent 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 add in the fresh culture IMDM of equivalent；
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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| CN201810098368.4A CN108187141A (en) | 2018-01-31 | 2018-01-31 | A kind of application of heart flesh ball derived cell in biomaterial |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108379662A (en) * | 2018-02-08 | 2018-08-10 | 深圳大图科创技术开发有限公司 | A kind of application of stem cell in Heart Transplantation Model |
| 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 |
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