CN104546916A - Novel stem cell preparation and application thereof in treating cerebral hemorrhage - Google Patents
Novel stem cell preparation and application thereof in treating cerebral hemorrhage Download PDFInfo
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- CN104546916A CN104546916A CN201510063669.XA CN201510063669A CN104546916A CN 104546916 A CN104546916 A CN 104546916A CN 201510063669 A CN201510063669 A CN 201510063669A CN 104546916 A CN104546916 A CN 104546916A
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- 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
- A61K35/48—Reproductive organs
- A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
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Abstract
The invention relates to a novel stem cell preparation and application thereof in treating cerebral hemorrhage, specifically the invention relates to the application of a novel stem cell preparation containing therapeutically effective amount of mesenchymal stem cells, a DMEM/F12 cell culture medium and bFGF in treating cerebral hemorrhage.
Description
Technical field
The present invention relates to a kind of novel stem cells preparation and it is used for the treatment of the purposes of cerebral hemorrhage, especially, the stem cell medicine that the present invention relates to from amniotic membrane is used for the treatment of cerebral hemorrhage.
Background technology
Cerebral hemorrhage (cerebral hemorrhage) is also known as cerebral hemorrhage, and refer to the intraparenchymatous hematostaxis of non-traumatic cerebral, the cause of disease is various, and the overwhelming majority is that the arteriolosclerotic angiorrhexis of hypertension causes.Cerebral hemorrhage is the common acute cerebrovascular disease of middle-aged and elderly people, and case fatality rate and disability rate are all very high, are the Clinical types that in China's cerebrovascular, mortality rate is the highest.The treatment of hypertensive cerebral hemorrhage can be divided into internal medicine conservative treatment and surgical operation therapy.Patient's bleeding amount is few, and nerve function lesion is comparatively light, or the poor inoperable patient-selectable of patient's ordinary circumstance selects internal medicine conservative treatment.Alleviate with medicine the secondary lesion that hematoma causes, promote neurological functional recovery; Prevent complication.Amount of bleeding is large, and the state of an illness is heavier needs surgical operation, removes hematoma, alleviates cerebral tissue pressurized, tries one's best and ensures function of nervous system.But function of nervous system all can be impaired to some extent.So the recovery of accelerator nerve function is problem demanding prompt solution.
In recent years, the upsurge of stem-cell therapy is started.Stem cell has multinomial differentiation potential and self-renewal capacity.At present, the kind of stem cell is more, as embryonic stem cell, bone marrow stem cell, umbilical cord stem cells, amniotic membrane stem cell, fat stem cell etc.
Embryonic stem cell remains at present in ethical issues, bone marrow and fat stem cell adult stem cell, relative maturity.Amniotic membrane, at the innermost layer of Placenta Hominis, is wrapped in fetus containing amniotic fluid.During fetal development 1 month, amniotic sac has been grown complete, amniotic membrane compared with umbilical cord grow more early.Amniotic membrane is the garbage of anemia of pregnant woman when producing, and there is not ethical issues and abundance, can be used as the splendid source of stem cell.
21 century, be the epoch of stem-cell therapy, stem cell can secrete various kinds of cell trophic factors, comprises EGF, bFGF, IGF, HGF, TNF-β etc., these factors can improve the microenvironment of body cell growth, promote propagation and the differentiation of body cell, promote the reparation of autologous tissue's damage, make it restore funcitons.Stem cell has multinomial differentiation potential in addition, can break up the histiocyte of alternative damage and play function.Therefore the aftertreatment for cerebral hemorrhage selects a kind of applicable stem cell and the feasible therapeutic scheme of science, is current problem demanding prompt solution.
The invention has the advantages that:
1 adopts amnion-derived mescenchymal stem cell, and abundance, does not exist ethical issues.Ability of cell proliferation is strong.
2 stem cell transplantation modes take different modes according to disease difference, and the present invention adopts Intraventricular damage local direct injection.And do drainage Post operation indwelling drainage tube at patients with cerebral hemorrhage, wait to select proper moment to be injected by stem cell by drainage tube, can avoid because cranium wound is opened in stem cell transplantation in brain again.
Summary of the invention
The invention provides a kind of stem cell medicine, wherein said stem cell medicine comprises the mescenchymal stem cell, DMEM/F12 cell culture medium and the bFGF that treat effective dose, and wherein said mescenchymal stem cell is from mammiferous amniotic membrane stem cell.
Preferably, according to stem cell medicine of the present invention, wherein treating effective dose is every kg patient body weight about 1 × 10
6individual mescenchymal stem cell.
Preferably, according to stem cell medicine of the present invention, wherein the concentration of interstital stem cell is 5 × 10
6-1.25 × 10
7/ ml, and containing 10ng/ml bFGF in 4ml DMEM/F12 cell culture fluid.
Further, according to stem cell medicine of the present invention, wherein said mammal is people.
Further, according to stem cell medicine of the present invention, it is formulated into stem cell suspension.
On the other hand, the invention provides a kind of method preparing stem cell medicine, said method comprising the steps of:
1) mescenchymal stem cell from mammalian amniotic membrane is cultivated;
2) go down to posterity amplification step 1) the middle mescenchymal stem cell cultivated;
3) with trypsinization go down to posterity amplification mescenchymal stem cell, with containing serum culture medium stop Digestion;
4) by step 3) mescenchymal stem cell with 5 × 10
6-1.25 × 10
7the concentration of/ml is suspended in the DMEM/F12 cell culture medium containing bFGF.
On the other hand, the invention provides according to stem cell medicine of the present invention or prepared according to the methods of the invention stem cell medicine the purposes in the medicine of the patient for the preparation for the treatment of mammal cerebral hemorrhage.
Preferably, according to purposes of the present invention, wherein said stem cell medicine is used by the ventricles of the brain.
Accompanying drawing explanation
Fig. 1 shows patient's boring and opens cranium, after drain, and indwelling drainage tube.
Stem cell suspension is injected into Intraventricular by Fig. 2 display.
Fig. 3 shows skin suture, is embedded in by drainage tube subcutaneous, and standby second time is transplanted.
Fig. 4 A-Fig. 4 H shows the cell surface marker of Flow cytometry amniotic membrane stem cell, wherein amnion mesenchymal stem cell number >=95% of CD105, CD73 and CD90 positive, amnion mesenchymal stem cell number≤2% of surface marker CD45, CD34, CD14, CD19, HLA-DR positive.
Should understand, appended accompanying drawing not shows the technique of painting slightly simplified of the exemplifying various feature of ultimate principle of the present invention pari passu.Specific design feature of the present invention disclosed herein comprises such as concrete size, direction, position and profile and will partly be determined by the environment specifically will applied and use.
In these figures, run through several figures of accompanying drawing, Reference numeral quotes equally or equivalent part of the present invention.
Detailed description of the invention
The object of the present invention is to provide a kind of stem cell medicine promoting neurological functional recovery for post operation of hypertensive cerebral hemorrhage, and clinical pathway.
First aspect content:
The preparation for the treatment of cerebral hemorrhage stem cell medicine
1 extracts cell:
A raw material: healthy pregnant women Cesarean esction fetus amniotic membrane, donor mark of hepatitis virus thing, syphilis, HIV detect all negative.Donor, to experiment informed consent, is ratified through Hospital Ethical Committee.
From placental separation amniotic membrane under B aseptic condition, with PBS washing several, remove the blood streak on amniotic membrane and dope.
Amniotic membrane, in Biohazard Safety Equipment, is cut into small pieces by C in culture dish, adds trypsinization, and serum neutralizes.
Membrane film shreds after removing and membrane film digesting the epithelial cell got off by D, and with PBS washing, the precipitation after centrifugal is inoculated in culture dish cultivates and amplification of going down to posterity.
2 prepare cerebral hemorrhage stem cell medicine
After A cell proliferation to some, cell is carried out trypsinization, do not stop Digestion containing the culture medium of serum with a large amount of.
The preparation of B stem cell medicine
The preparation of cell nutrient solution: composition: DMEM/F12 cell culture fluid, bFGF.
To suspend ready stem cell with the cell nutrient solution 4ml prepared, load in injection bottle after mixing, sterile sealing.
Second aspect
Stem-cell therapy scheme
1 patients with cerebral hemorrhage is preoperative, patient is handed over to the function and significance of stem-cell therapy
2 patients sign stem cell clinical trial treatment Informed Consent Form
3 row hematoma drainage.
At the end of 4 operations, the drainage tube placed at the ventricles of the brain or haematoma cavity is embedded in subcutaneous, sterile dressing.
5 postoperative 1 week, vital sign was steady, row stem-cell therapy.Open the drainage tube of wrapping, pull out syringe needle after extracting stem cell suspension out with syringe from bottle, syringe is received on drainage tube, slowly inject stem cell suspension.Inject residual cell in 2ml normal saline washing drainage tube again.
6 pull up syringe, are embedded in by drainage tube subcutaneous, sterile dressing, in order to second time stem cell transplantation.
7 these drainage tubes of common mistake transplant 3 times, after extract drainage tube, sew up scalp, bag gauze.
The third aspect
After stem-cell therapy, compared with not transplanting, limb function obviously improves.
Embodiment
1 extracts cell:
A raw material: healthy pregnant women Cesarean esction fetus amniotic membrane, donor mark of hepatitis virus thing, syphilis, HIV detect all negative.Donor, to experiment informed consent, is ratified through Hospital Ethical Committee.：
From placental separation amniotic membrane under B aseptic condition, with PBS washing several, remove the blood streak on amniotic membrane and dope.
Amniotic membrane, in Biohazard Safety Equipment, is cut into small pieces by C in culture dish, and adding concentration is after the trypsinization 30min of 0.125%, and serum neutralizes.
Membrane film shreds after removing and membrane film digesting the epithelial cell got off by D, and with PBS washing, the precipitation after centrifugal is inoculated in culture dish cultivates and amplification of going down to posterity.
Use Flow cytometry from the cell surface marker of the mescenchymal stem cell of amniotic membrane, wherein amnion mesenchymal stem cell number >=95% of CD105, CD73 and CD90 positive, amnion mesenchymal stem cell number≤2% of surface marker CD45, CD34, CD14, CD19, HLA-DR positive.
2 prepare stem cell medicine
The preparation of A cell nutrient solution: DMEM/F12 cell culture fluid 4ml is containing 10ng/mlbFGF.
B cell proliferation about reaches 5 × 10 to sum
7time individual, be the trypsinization of 0.25% by cell concentration, stop Digestion by the culture medium not containing serum being equivalent to 5 times of pancreatin amounts.1000RPM is centrifugal.With the stem cell of the cell nutrient solution suspended centrifugal precipitation prepared, load in injection bottle after mixing, sterile sealing, wherein, during the concentration of mescenchymal stem cell 1 × 10
7individual/ml.
The transplantation treatment of 3 patients with cerebral hemorrhage
Do the patients with cerebral hemorrhage of hematoma drainage
A is preoperative, patient is handed over to the function and significance of stem-cell therapy
B patient signs stem-cell therapy Informed Consent Form
At the end of C operation, at the ventricles of the brain or haematoma cavity placing drainage, sterile dressing.
Postoperative 1 week of D, vital sign is steady, and row stem-cell therapy, cell concentration is 1 × 10
7individual/ml, altogether 4ml.Open the drainage tube of wrapping, release the cerebrospinal fluid of 6ml, pull out syringe needle after extracting 4ml stem cell suspension out with syringe from bottle, syringe is received on drainage tube, slowly inject stem cell suspension.Inject 2ml normal saline again, residual cell in irrigation drainage tube.
E pulls up syringe, is embedded in by drainage tube subcutaneous, sterile dressing, in order to second time stem cell transplantation.
Second time and third time transplant step with first time.7 days, interval.1 course for the treatment of 3 times.
First time treats latter 5 days, and muscular tension also declines to some extent, and muscular strength increases.After treating a course for the treatment of, manifest curative effect successively, comprise patient's aphasis and alleviate, the dyskinesia that hemiplegia of limb causes all is improved.
Claims (8)
1. a stem cell medicine, wherein said stem cell medicine comprises the mescenchymal stem cell, DMEM/F12 cell culture medium and the bFGF that treat effective dose, and wherein said mescenchymal stem cell is from mammiferous amniotic membrane.
2. stem cell medicine according to claim 1, wherein treating effective dose is every kg patient body weight about 1 × 10
6individual mescenchymal stem cell.
3. stem cell medicine according to claim 1, wherein the concentration of interstital stem cell is 5 × 10
6-1.25 × 10
7/ ml, and containing 10ng/mlbFGF in 5ml DMEM/F12 cell culture fluid.
4. stem cell medicine according to claim 1, wherein said mammal is people.
5. stem cell medicine according to claim 1, it is formulated into stem cell suspension.
6. prepare a method for stem cell medicine, said method comprising the steps of:
1) mescenchymal stem cell from mammalian amniotic membrane is cultivated;
2) go down to posterity amplification step 1) the middle mescenchymal stem cell cultivated;
3) with trypsinization go down to posterity amplification mescenchymal stem cell, with containing serum culture medium stop Digestion;
4) by step 3) mescenchymal stem cell with 5 × 10
6-1.25 × 10
7the concentration of/ml is suspended in the DMEM/F12 cell culture medium containing bFGF.
7. the purposes of stem cell medicine in the medicine of the patient for the preparation for the treatment of mammal cerebral hemorrhage prepared by the stem cell medicine according to any one of claim 1-5 or method according to claim 6.
8. purposes according to claim 7, wherein said stem cell medicine is used by the ventricles of the brain.
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| CN201510063669.XA CN104546916A (en) | 2015-02-06 | 2015-02-06 | Novel stem cell preparation and application thereof in treating cerebral hemorrhage |
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| CN201510063669.XA CN104546916A (en) | 2015-02-06 | 2015-02-06 | Novel stem cell preparation and application thereof in treating cerebral hemorrhage |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114886921A (en) * | 2022-05-13 | 2022-08-12 | 深圳中检联新药检测有限责任公司 | Novel stem cell preparation and application thereof in treating cerebral hemorrhage |
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| CN102119936A (en) * | 2011-03-07 | 2011-07-13 | 李荣旗 | Method for preparing injection for treating ischemic brain damage by using human amniotic mesenchymal cells and injection |
| CN103642751A (en) * | 2013-12-06 | 2014-03-19 | 上海同泽和济生物科技有限公司 | Method for preparing stem cells from human amniotic membrane |
| CN103789258A (en) * | 2012-11-30 | 2014-05-14 | 陆华 | Separation method for human amniotic mesenchymal stem cells |
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2015
- 2015-02-06 CN CN201510063669.XA patent/CN104546916A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102119936A (en) * | 2011-03-07 | 2011-07-13 | 李荣旗 | Method for preparing injection for treating ischemic brain damage by using human amniotic mesenchymal cells and injection |
| CN103789258A (en) * | 2012-11-30 | 2014-05-14 | 陆华 | Separation method for human amniotic mesenchymal stem cells |
| CN103642751A (en) * | 2013-12-06 | 2014-03-19 | 上海同泽和济生物科技有限公司 | Method for preparing stem cells from human amniotic membrane |
Non-Patent Citations (4)
| Title |
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| 李国喜: "人羊膜间充质干细胞的培养、分化及移植治疗脑外伤的实验研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
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| 王兆福等: "不同来源人羊膜间充质干细胞的混合培养", 《中国组织工程研究与临床康复》 * |
| 罗焕: "《神经药理学基础》", 30 June 2011, 暨南大学出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114886921A (en) * | 2022-05-13 | 2022-08-12 | 深圳中检联新药检测有限责任公司 | Novel stem cell preparation and application thereof in treating cerebral hemorrhage |
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Application publication date: 20150429 |