CN106474456A - A kind of cell preparation and its preparation method and application - Google Patents
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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Abstract
The present invention relates to cell preparation technical field, particularly to a kind of cell preparation and its preparation method and application.This cell preparation includes mescenchymal stem cell, bone morphogenetic protein 2 and normal saline.Present invention research finds, after mescenchymal stem cell and bone morphogenetic protein 2 are used in combination, it is remarkably improved LVEF level, and BNP level can be significantly reduced, effect is better than and is used alone mescenchymal stem cell or bone morphogenetic protein 2, and effective percentage is up to 80%～90%.It can be seen that, cell preparation of the present invention can effectively treatment heart failure.
Description
Technical field
The present invention relates to cell preparation technical field, particularly to a kind of cell preparation and its preparation method and application.
Background technology
Heart failure (heart failure) abbreviation heart failure, refers to the contractile function due to heart and (or) diastolic function
Occur obstacle it is impossible to fully venous return is discharged heart, lead to Venous system sludging, Arterial system hemoperfusion
Deficiency, thus causing cardiac cycle obstacle syndrome, shows as pulmonary venous pleonaemia, caval vein congestion in this kind of obstacle disease cluster.Root
The emergency occurring according to heart failure, clinic can be divided into acute heart failure and chronic heart failure.Acute heart failure can cause acute
Pulmonary edema, onset is anxious, and the state of an illness can develop rapidly to critical state.Burst severe dyspnea, orthopnea, gasp for breath,
Dysphoria simultaneously has fear, and respiratory frequency is up to 30～50 beats/min；Frequently cough simultaneously brings up a large amount of pink foam sample expectorant；
Heart rate is fast, apex Chang Kewen and gallop rhythm；The wet sound of two fullness of the lung cloth and wheezing sound.Also cardiogenic shock can be caused, show as
Hypotension, tissue low perfusion state, hemodynamics obstacle, metabolic acidosiss and hypoxemia.Acute heart failure often jeopardizes life
Life is it is necessary to urgent rescue.
Current clinical treatment mainly adopts the means such as Drug therapy, interventional therapy and surgical intervention：(1) initial therapy is
Through mask or nasal tube oxygen inhalation；Morphine, loop diuretic, cardiac tonic etc. give through vein.Patient is made to take seat or semireclining position, two lower limbs
Sagging, reduce veins of lower extremity backflow.(2) still not alleviation person should select application blood vessel to live according to systolic pressure and pulmonary venous pleonaemia situation to the state of an illness
Property medicine, such as inotropic agent, vasodilator and vasoconstrictor etc..(3) be in a bad way, blood pressure persistently reduce (<
90mmHg) or even cardiogenic shock person, monitor blood flow kinetics are answered, and supported using intra aortic balloon counterpulsation, mechanical ventilation,
The various non-drug therapy method such as blood purification, ventricle assist and surgical operation.(4) dynamic measurement BNP/NT-
ProBNP contributes to instructing the treatment of acute heart failure, and after treatment, its level still can be in any more person, points out poor prognosis, should strengthen controlling
Treat；After treatment, its level reduces and the range of decrease>30%, point out treatment effectively, good prognosis.But these Therapeutic Method can only be in certain journey
The generation of heart failure is alleviated on degree.
Stem cell is the pluripotent cell that a class has the of self-replication capacity and differentiation capability, has the various organizers of regeneration
Official and the potential function of human body, medical circle is referred to as " general-purpose cell ".Find, medulla mesenchyma is done through the research of forefathers
Cell directly or indirectly can be repaiied to impaired myocardial cell as a kind of medicine under suitable Cytokine
Multiple or supplementary, have become as one of study hotspot of heart failure treatment at present.Therefore, research and development a kind of can effectively treatment heart failure dry
Cell preparation becomes inexorable trend.
Content of the invention
In view of this, the invention provides a kind of cell preparation and its preparation method and application.This cell preparation can be effective
Treatment heart failure.
In order to realize foregoing invention purpose, the present invention provides technical scheme below：
The invention provides a kind of cell preparation, including mescenchymal stem cell, bone morphogenesis protein-2 (BMP-2) and life
Reason saline.
Bone morphogenesis protein-2 (bone morphogenetic protein, BMP-2) as a member of BMP family,
Induced activity highest to heart development.Research shows, BMP can adjust various kinds of cell growth, propagation and differentiation it is possible to
Participate in heart development and morphogenetic regulation by adjusting the expression of some cardiac transcription factors.
The present invention, by after BMP-2 and mesenchyma stem cell combined use, can preferably play repairing of mescenchymal stem cell
Multiple effect, thus being remarkably improved left ventricular ejection fraction (LVEF) level, and can significantly reduce natriuretic peptide (BNP) level, effect
Really it is better than and is used alone mescenchymal stem cell or BMP-2, effective percentage is up to 80%～90%.It can be seen that, cell preparation of the present invention can
Effectively treatment heart failure.
Preferably, each amounts of components is in cell preparation：
Mescenchymal stem cell：(1～10) × 106Individual/mL；
Bone morphogenesis protein-2：0.05～0.1 μ g/mL；
Normal saline：Supply.
In the embodiment that the present invention provides, in cell preparation, each amounts of components is：
Mescenchymal stem cell：1×106Individual/mL；
Bone morphogenesis protein-2：0.05μg/mL；
Normal saline：Supply.
In the embodiment that the present invention provides, in cell preparation, each amounts of components is：
Mescenchymal stem cell：5×106Individual/mL；
Bone morphogenesis protein-2：0.1μg/mL；
Normal saline：Supply.
In the embodiment that the present invention provides, in cell preparation, each amounts of components is：
Mescenchymal stem cell：1×107Individual/mL；
Bone morphogenesis protein-2：0.1μg/mL；
Normal saline：Supply.
In the embodiment that the present invention provides, mescenchymal stem cell is mesenchymal stem cells MSCs.
Preferably, mesenchymal stem cells MSCs are P3 for mesenchymal stem cells MSCs.
Present invention also offers the preparation method of this cell preparation, comprise the steps：
Using bone morphogenesis protein-2 and mescenchymal stem cell mixing, resuspended using normal saline, obtain cell preparation.
In the present invention, the preparation method of mescenchymal stem cell is：Take the culture medium comprising mescenchymal stem cell, remove training
Foster base, is digested using Digestive system, obtains single cell suspension；Single cell suspension is centrifuged, washing, obtains mesenchyme and do
Cell.
Present invention also offers this cell preparation improves LVEF level in preparation or reduces the application in the horizontal medicine of BNP.
Present invention also offers application in preparation treatment heart failure medications for this cell preparation.
Present invention also offers a kind of medicine, the cell preparation providing including the present invention.This cell preparation includes mesenchyme
Stem cell, bone morphogenesis protein-2 (BMP-2) and normal saline.
In the embodiment that the present invention provides, medicine also includes pharmaceutically acceptable adjuvant.
The invention provides a kind of cell preparation and its preparation method and application.This cell preparation includes mesenchyme to be done carefully
Born of the same parents, bone morphogenesis protein-2 and normal saline.The present invention has the advantages that：
Present invention research finds, after mescenchymal stem cell and bone morphogenesis protein-2 are used in combination, is remarkably improved
LVEF level, and BNP level can be significantly reduced, effect is better than and is used alone mescenchymal stem cell or bone morphogenesis protein-2,
Effective percentage is up to 80%～90%.It can be seen that, cell preparation of the present invention can effectively treatment heart failure.
Specific embodiment
The invention discloses a kind of cell preparation and its preparation method and application, those skilled in the art can use for reference herein
Content, is suitably modified technological parameter and realizes.Specifically, all similar replacements and change are to people in the art
It is it will be apparent that they are considered as including in the present invention for member.The method of the present invention and application passed through preferably real
Apply example to be described, related personnel substantially can be to method described herein in without departing from present invention, spirit and scope
It is modified with application or suitably changes and combine, to realize and to apply the technology of the present invention.
In cell preparation that the present invention provides and its preparation method and application, biomaterial used all can be buied by market.
With reference to embodiment, the present invention is expanded on further：
Embodiment 1：The preparation of human marrow mesenchymal stem cell
Obtain bone marrow 10mL from regular purchasing channel, Percoll (1.073 × 10 after anticoagulant heparin-3G/L) density gradient from
The heart, is taken middle mononuclear cell layer to be washed 2 times with low sugar DMEM, inoculates into 75cm2Culture bottle, with low sugar DMEM containing 10%FBS
Culture.
Change liquid after 24h, abandon not adherent cell, remaining attached cell is MSCs.
Change liquid within every 2～3 days, 0.25% trypsinization passes on after cell growth is converged to 90%.
Embodiment 2：The preparation of cell preparation
The P3 that Example 1 obtains, for mesenchymal stem cells MSCs, sucks culture medium, adds Digestive system to be digested, it
Terminate digestion with appropriate serum afterwards, gently blow and beat into single cell suspension.1500rpm, is centrifuged 5min, abandons supernatant, washed with 4 DEG C of PBS
Wash resuspended mixing after cell 3 times, adjustment cell concentration is about 107Individual/mL.
Formula ratio P3 is mixed for mesenchymal stem cells MSCs and BMP-2, uses normal saline re-suspended cell.
Table 1 embodiment 2 cell preparation forms
| Component | Content |
| P3 is for mesenchymal stem cells MSCs | 1×106Individual/mL |
| BMP-2 | 0.05μg/mL |
| Normal saline | Supply |
Embodiment 3：The preparation of cell preparation
The P3 that Example 1 obtains, for mesenchymal stem cells MSCs, sucks culture medium, adds Digestive system to be digested, it
Terminate digestion with appropriate serum afterwards, gently blow and beat into single cell suspension.1500rpm, is centrifuged 5min, abandons supernatant, washed with 4 DEG C of PBS
Wash resuspended mixing after cell 3 times, adjustment cell concentration is about 107Individual/mL.
Formula ratio P3 is mixed for mesenchymal stem cells MSCs and BMP-2, uses normal saline re-suspended cell.
Table 2 embodiment 3 cell preparation forms
| Component | Content |
| P3 is for mesenchymal stem cells MSCs | 5×106Individual/mL |
| BMP-2 | 0.1μg/mL |
| Normal saline | Supply |
Embodiment 4：The preparation of cell preparation
The P3 that Example 1 obtains, for mesenchymal stem cells MSCs, sucks culture medium, adds Digestive system to be digested, it
Terminate digestion with appropriate serum afterwards, gently blow and beat into single cell suspension.1500rpm, is centrifuged 5min, abandons supernatant, washed with 4 DEG C of PBS
Wash resuspended mixing after cell 3 times, adjustment cell concentration is about 107Individual/mL.
Formula ratio P3 is mixed for mesenchymal stem cells MSCs and BMP-2, uses normal saline re-suspended cell.
Table 3 embodiment 4 cell preparation forms
| Component | Content |
| P3 is for mesenchymal stem cells MSCs | 1×107Individual/mL |
| BMP-2 | 0.1μg/mL |
| Normal saline | Supply |
Embodiment 5：Preparation evaluation
The cell preparation that embodiment 2-4 is obtained carries out microorganism detection and endotoxin detection.Wherein, microorganism detection
(antibacterial, funguses) are detected according to the 4th general rule 1100 of version Pharmacopoeia of People's Republic of China in 2015；Endotoxin detects basis
The 4th general rule 1143 of version Pharmacopoeia of People's Republic of China in 2015 is detected.
Testing result：The biological preparation that embodiment 2-4 is obtained, in antibacterial, funguses and endotoxic detection, is all negative.
Illustrate the up-to-standard of this stem cell medicine, can be used for treating heart failure.
Embodiment 6：The therapeutic effect detection of preparation
Test method：Using intramyocardial injection mode, to heart failure modeling, successful mice carries out preparation injection.
Take the successful mice of 60 heart failure modelings, be divided into 6 groups, including negative control group, 3 groups of experimental grouies, stem cell group and
BMP-2 group：
Negative control group：Using beta-blocker conventional therapy means；
Experimental group 1：Treated with the preparation that embodiment 2 is obtained respectively；
Experimental group 2：Treated with the preparation that embodiment 3 is obtained respectively；
Experimental group 3：Treated with the preparation that embodiment 4 is obtained respectively；
Stem cell group：Treated using the stem cell that embodiment 1 obtains；
BMP-2 group：Treated using BMP-2.
Each group is as follows to the injection dosage of mice：
The injection dosage to mice for table 4 each group
| Group | Mice quantity (only) | Dosage |
| Negative control group | 26 | —— |
| Experimental group 1 | 26 | 1mL |
| Experimental group 2 | 26 | 1mL |
| Experimental group 3 | 26 | 1mL |
| Stem cell group | 26 | 2×107Individual |
| BMP-2 group | 26 | 0.2μg |
Observe the indexs such as the LVEF and BNP level of mice after 8 weeks, pass judgment on the effective percentage for the treatment of.
Effective percentage calculation is：Mice quantity/15 that LVEF and BNP index significantly improves.
Result is as shown in the table：
The indexs such as the LVEF and BNP level of table 5 mice
Note：* represent that there is compared with matched group significant difference, p<0.05.
From above-mentioned result of the test, after mescenchymal stem cell and BMP-2 are used in combination, it is remarkably improved LVEF water
Flat, and can significantly reduce BNP level, effect is better than and is used alone mescenchymal stem cell or BMP-2, effective percentage up to 80%～
90%.It can be seen that, cell preparation of the present invention can effectively treatment heart failure.
The above is only the preferred embodiment of the present invention it is noted that ordinary skill people for the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of cell preparation is it is characterised in that include mescenchymal stem cell, bone morphogenesis protein-2 and normal saline.
2. cell preparation according to claim 1 it is characterised in that in described cell preparation each amounts of components be：
Mescenchymal stem cell：(1～10) × 106Individual/mL；
Bone morphogenesis protein-2：0.05～0.1 μ g/mL；
Normal saline：Supply.
3. cell preparation according to claim 1 and 2 is it is characterised in that described mescenchymal stem cell is medulla mesenchyma
Stem cell.
4. cell preparation according to any one of claim 1 to 3 is it is characterised in that described mesenchymal stem cells MSCs
For P3 for mesenchymal stem cells MSCs.
5. as any one of Claims 1-4 the preparation method of cell preparation it is characterised in that comprising the steps：
Using bone morphogenesis protein-2 and mescenchymal stem cell mixing, resuspended using normal saline, obtain described cell preparation.
6. as any one of Claims 1-4, cell preparation improves LVEF level in preparation or reduces in the horizontal medicine of BNP
Application.
7. application in preparation treatment heart failure medications for the cell preparation as any one of Claims 1-4.
8. a kind of medicine is it is characterised in that include cell preparation as any one of Claims 1-4.
9. medicine according to claim 8 is it is characterised in that described medicine also includes pharmaceutically acceptable adjuvant.
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| CN103221058A (en) * | 2010-08-27 | 2013-07-24 | 迈阿密大学 | Bone marrow derived CD271 precursor cells for cardiac repair |
| CN105238746A (en) * | 2015-09-21 | 2016-01-13 | 深圳华毓造血干细胞研究有限公司 | Inducing method and inducing liquid of mesenchymal stem cells |
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- 2016-10-18 CN CN201610906954.8A patent/CN106474456A/en active Pending
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|---|---|---|---|---|
| CN100475956C (en) * | 2003-12-09 | 2009-04-08 | 安特罗根有限公司 | Method of preparing cell for transplantation |
| CN102438636A (en) * | 2009-05-20 | 2012-05-02 | 卡迪欧参生物科技有限公司 | Pharmaceutical composition for treating heart diseases |
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