CN101721430A - Application of amniotic epithelial cells in pharmacy - Google Patents
Application of amniotic epithelial cells in pharmacy Download PDFInfo
- Publication number
- CN101721430A CN101721430A CN200810043858A CN200810043858A CN101721430A CN 101721430 A CN101721430 A CN 101721430A CN 200810043858 A CN200810043858 A CN 200810043858A CN 200810043858 A CN200810043858 A CN 200810043858A CN 101721430 A CN101721430 A CN 101721430A
- Authority
- CN
- China
- Prior art keywords
- epithelial cells
- cell
- amniotic epithelial
- vascular diseases
- cerebral vascular
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention relates to the technical field of biomedicine, in particular to application of amniotic epithelial cells in a medicament for treating hemorrhagic cerebral vascular diseases. The invention discloses the application of the amniotic epithelial cells in treating or improving the hemorrhagic cerebral vascular diseases. The amniotic epithelial cells can effectively treat or improve the hemorrhagic cerebral vascular diseases, obviously improve the action function of patients with the hemorrhagic cerebral vascular diseases, and obviously reduce encephaledema symptoms caused by the hemorrhagic cerebral vascular diseases; the treatment do not need match because the amniotic epithelial cells do not express or weakly express MHC; the amniotic epithelial cells also do not express telomerase, and cell proliferation is controlled, so that the transplantation treatment is safe; meanwhile, the amniotic epithelial cells have wide sources, so that the phenomenon of ethic or morality is avoided. Therefore, the amniotic epithelial cells provide a new effective choice for clinically treating or improving the hemorrhagic cerebral vascular diseases in the future.
Description
Technical field
The present invention relates to the biological medicine technology field, be specifically related to the purposes of amniotic epithelial cells in the medicine of treatment hemorrhagic apoplexy.
Background technology
Cerebrovascular is meant the hemorrhage or thrombosis of rupture of blood vessel in brain, and what cause is one group of disease of main clinical manifestation with brain hemorrhagic or ischemia injury symptom, claims cerebrovascular accident or apoplexy again, is commonly called as to be apoplexy.This disease is common in crowd's above middle age acute attack, and disturbance of consciousness and quadriplegia can take place severe patient, be to cause human dead and disabled principal disease at present, and also be hyperpietic's the main cause of death.Cerebrovascular can be divided into two big classes by its character, one class is an ischemic cerebrovascular, clinically morely see, account for whole cerebrovascular patients' 70%～80%, be owing to reasons such as cerebral arteriosclerosis, make the cerebral arteries luminal stenosis, blood flow reduces or total blockage, the brain blood circulation obstacle, the impaired and a series of symptoms that take place of cerebral tissue.Another kind of is hemorrhagic apoplexy, how by due to the factors such as long-term hypertension, congenital cerebrovascular malformation.Because angiorrhexis, blood overflows, the compressing cerebral tissue, and blood circulation is obstructed, and patient often shows symptoms such as cerebral increases, obnubilation.This class patient accounts for about 20%～30% of cerebrovascular.Hemorrhagic apoplexy comprises: (1) cerebral hemorrhage means that brain essence angiorrhexis is hemorrhage.How by hypertension, cerebral arteriosclerosis, tumor or traumatic etc. causing.(2) subarachnoid hemorrhage, because the angiorrhexis of brain surface and brain bottom is hemorrhage, blood flows directly into due to the subarachnoid space.Common cause has aneurysm rupture, vascular malformation, hypertension, arteriosclerosis, hematopathy or cerebral trauma etc.
Epidemiological study shows that the case fatality rate of hemorrhagic apoplexy is 40-60%, accounts for the major part in the cerebrovascular death toll, and is all right both at home and abroad; 70-80% leaves invalidly among the survivor, and it is residual to attach most importance to about 40%, causes huge life to threaten to the patient, and quality of life is had a strong impact on simultaneously, causes white elephant also for patient family and even entire society.Doctor trained in Western medicine to the treatment of hemorrhagic apoplexy at present still with controlling blood pressure, dewater, fall cranium and press, symptomatic treatments such as hemorrhage application are main, to the big patient of part amount of bleeding or have the brain stem compressing, cerebral hernia, hydrocephalus sign person also needs operation in time to remove hematoma.Though the traditional Chinese medical science has cow-bezoar bolus for resurrection to Acute Cerebral Hemorrhage, Zhibao Dan, Styrax Pilulae, Therapeutic Method such as shenfu decoction, clinical application is extremely inconvenient.In recent years, the FUFANG DANSHEN ZHUSHEYE that has the scholar will be used for coronary heart disease and cerebral infarction originally is used for the treatment of acute cerebral hemorrhage, obviously being better than singly using the western medicine group aspect promotion patient consciousness recovery, absorption of hematoma and the reduction mortality rate, illustrate that the activating blood circulation to dissipate blood stasis method has definite therapeutical effect to acute cerebral hemorrhage.In addition, QINKAILING ZHUSHEYE that develops on the cow-bezoar bolus for resurrection basis and XINGNAOJING ZHUSHEYE have all adopted the intravenously administrable method.Since easy to use, the needs of clinical emergency treatment adapted to, so obtained extensive use clinically.But owing to these medicines are all only treated at a certain link of the complicated pathology of cerebral hemorrhage, so clinical effectiveness can not be fully up to expectations.
Behind the hemorrhagic apoplexy, often cause a series of pathophysiology reactions of the local generation of body and cerebral tissue, the blood of obstruction cerebral tissue supplies and causes and the disorder of environment in the neurocyte causes neuronal apoptosis or necrosis.Therefore, the crucial cranial nerve cell that should repairing damage of the convalescent treatment of hemorrhagic apoplexy.Adopt stem cell reparation or regeneration of damaged cranial nerve cell to become the focus in current neuroscience field, neural stem cell and embryo stem cell transplantation once once opened up a new way for the treatment of brain injury, but because both come source problem, clinical practice is limited.
Summary of the invention
Technical problem to be solved by this invention is at an existing hemorrhagic apoplexy treatment difficult problem, and new medicine or method is provided.
For this reason, on the one hand, the invention discloses the purposes of amniotic epithelial cells in preparation treatment hemorrhagic apoplexy.
In some embodiments, amniotic epithelial cells of the present invention derives from the mankind.
In some embodiments, hemorrhagic apoplexy of the present invention for by organic diseases because of or wound the accident cerebral hemorrhage or the subarachnoid hemorrhage that cause, wherein preferred cerebral hemorrhage.
On the other hand, the cell preparation that the invention also discloses the described amniotic epithelial cells that uses effective dose or contain described amniotic epithelial cells is united the method for the treatment of and/or improve hemorrhagic apoplexy of using separately or with other medicines.
Hemorrhagic apoplexy effectively be treated and/or be improved to amniotic epithelial cells of the present invention can, improves the action function of hemorrhage patients with cerebrovascular disease significantly, reduced the cerebral edema symptom that hemorrhagic apoplexy caused significantly; Amniotic epithelial cells is not because of or weak expression MHC, and treatment need not to join type; Amniotic epithelial cells is not expressed telomerase yet, and cell proliferation is controlled, and transplantation treatment is safe; Simultaneously, the amniotic epithelial cells source is very extensive, can not produce ethics or moral issue.Therefore, the present invention is for clinical treatment from now on or improve hemorrhagic apoplexy new effective selection is provided.
Description of drawings
Fig. 1. the balance beam appraisal result.
Fig. 2. the asymmetric application appraisal result of forelimb.
The histology location of the hAEC of Fig. 3 .Hoechst33258 labelling in tricorn.
Fig. 4. the water content of different time points cerebral tissue.
Fig. 5. the content of monoamine neurotransmitter in the cerebrospinal fluid.
The specific embodiment
The amnion tissue sample treatment.
Separate amniotic membrane from the mammal Placenta Hominis that exsomatizes, adopt the physiological buffer flushing to remove hemocyte, machinery is rejected residual chorion and blood vessel.
As used herein, term " mammal " is the most high in a vertebrates monoid, is evolved by reptile, and principal character is: body surface hairiness, general parted hair, neck, trunk, extremity and five parts of tail; Use pulmonary respiration; Thermostasis is a Homoiotherm; Brain is big and flourishing; Suckling; Viviparous.Suckling and viviparity are the notable attribute of mammal.
As used herein, term " amniotic membrane " (Amnion) also can be called fetal membrane, is the vital tissue that carries out mass exchange between parent and fetus.Amniotic membrane mainly can be divided into epithelial layer, basal layer, compacted zone, fibroblast layer and spongy layer five parts.Main amniotic epithelial cells (the human amniotic epithelial cells of human amniotic cell (Amniotic cells) by epithelial layer, hAEC) and the mesenchymal cell of basal layer constitute, be the early stage product of fetal development closely of getting in touch with developmental fetus.Amniotic epithelial cells can express early stage stem cell some not isolabeling as the gtelatinous fibre associated protein that neurocyte is special, neuronal specificity labelling (MAP2) neural stem cell specific marker (Nestin), liver parenchyma cell albumen, alpha-fetoprotein etc.Since this explanation amniotic membrane keeps the PD cell characteristic of immaturity, has the multipotency differentiation potential.[KnezevicV，Anat?1996；189(Pt?1)：1-7；Yugel，Transplantation?2004；77(9)：1452-4；Takashima?S，CellStruct?Funct?2004；29(3)：73-84；Sakuragawa?N，Neurosci?Lett?1996；209(1)：9-12；Wei?J.P，Cell?Transplant?2003；12(5)：545-52]。Simultaneously, amnion cell itself self lack I, II class antigen such as HLA-A ,-B ,-C and-R antigen and β 2 immunoglobulins [Akle CA, Lance 1981; 2 (8254): 1003-5; M.Adinolfi, nature vol 295:28] can not produce immunogenicity after transplanting, amnion cell exists HLA-E, G antigen simultaneously in addition, and they have immunosuppressive activity [Ueta M et al Clin Exp Imunol 2002; 129 (3): 464-70].
Amniotic epithelial cells separates and cultivates.
" separation " refers to from tissue sample emigrated cells and separates with other non-tissue stem cell.Using any routine techniques or method is unicellular with complete separate tissue, these technology or method comprise mechanical force (shred-ability or shearing force), with a kind of protease of or combination for example collagenase, trypsin, lipase, as US patent 5, disclosed release enzyme (liberase) H1 and pepsin carry out the combination of enzymic digestion or machinery and enzyme method in 952,215.For example, can be by the following method organize segmental digestion: the method for using the tissue digestion of collagenase mediation with complete; Or be disclosed in US patent 5,830,714 and 5,952 with reference to other method that is used for use collagenase of the present invention, in 215.Similarly, can use neutral protease to replace collagenase, as at Twentyman, P.R. and J.M.Yuhas (CancerLett 1980:9 (3): disclosed method 225-228).In addition, method can adopt the combination of enzyme, for example collagenase and tryptic combination, and as at Russell, people such as S.W.F.Doc (Int J Cancer 1976:18 (3): disclosed method 322-30); Or enzyme such as trypsin and mechanical dissociative combination, as at Engelholm.S.A, people such as M.Spang.Thomsen (Br J Cancer 1985:51 (1): disclosed method 93-98).
Other method well known by persons skilled in the art concentrates competent cell colony. and these processing after scouring/concentration steps can be implemented individually or simultaneously.In one embodiment, by allowing the cell colony Continuous Flow cross rotation film system or similar system for example in US patent 034,135 and 5,234, disclosed system is with cell concentration with remove enzyme in 608.
Except said method, also can be further purified or enrichment competent cell colony behind the cell washing or after cultivating, reduce heteroproteose cell and dead cell.Cell in the separate out suspended liquid can be realized by following technology: the adhesion of buoyant density sedimentation and centrifugation, differentiated and solid phase and eluting, immune magnetic pearl, fluorescence laser cell sorting (FACS) or other technology on the solid phase.These different technologies and carry out the example of the device of these technology can be referring to prior art and traded commodity.As immunomagnetic beads method, at disclosed amniotic epithelial cells antigen among the WO03042405, adopt specific antibodies such as SSEA-4, Nanog, CK-3, be further purified or the amniotic epithelial cells of enrichment SSEA-4 (+), Nanog (+), CK-3 (+) by immunomagnetic beads method.
The type of the basal medium that the present invention is used without limits, as long as can be used for the culture medium of cell culture.Preferred culture medium comprises DMEM culture medium and NPBM culture medium.To other component types that may contain in the basal medium above-mentioned without limits, preferred composition comprises F-12, FCS and the nerve survival factor.In this class culture medium, for example, the concentration of F-12 and FCS is respectively 50% and 10%.The concentration of CO2 preferably 5% in this class culture medium, but the invention is not restricted to this.
And, in another preferred embodiment of the present invention, add bFGF (basic fibroblast growth factor) or EGF (epidermal growth factor) in the basal medium of mentioning upward.In this case, can add a kind of or both's adding.The concentration for example of bFGF above-mentioned or EGF is 1ng/ml to 100ng/ml, and preferred concentration is 10ng/ml.To time of adding and method without limits.Preferably, add reagent every day when amniotic epithelial cells above-mentioned is cultivated in basal medium.
According to the present invention, the basal medium of mentioning upward or contain in the basal medium of other composition and also can add human whole serum, cord serum or artificial cerebrospinal fluid.
The amniotic epithelial cells preparation.
Amniotic epithelial cells preparation of the present invention comprises active component amniotic epithelial cells and pharmaceutically acceptable carrier.
Consider the type of the disease that will treat, those skilled in the art can suitably select the proper states of cell: without the cell (slightly putting forward part) of the collection of any processing; Partially purified cell; The cell of purification and then through cultivating amplification; Or the cell of neurad differentiation of stem cells.
At this paper, " pharmacy is acceptable " component is meant and is suitable for people and/or animal not have the material with suitable useful/relative risk of over-drastic adverse side effect (as toxicity, zest and anaphylaxis).
Pharmaceutically acceptable carrier is meant pharmacy acceptable solvent, suspending agent or excipient, helps cell survival, and the cell that transmissibility is prepared is to the human or animal.Carrier is selected according to the administering mode of plan suitably.Carrier of the present invention includes but not limited to various physiological buffers, as normal saline, phosphate buffer, artificial cerebrospinal fluid; Or whole serum, cord serum; Also various man-made supports be can comprise, gelfoam, decalcification bone, polyglycolic acid (PGA), polylactic acid (PLA) and their copolymer included but not limited to.
Treatment is used
Adopt the method that is fit to arbitrarily all can give the patient with this cell.A kind of suitable method is the spinal cord intracavitary administration, can directly inject in the cerebrospinal fluid.Usually these cells are included in the pharmaceutically acceptable fluid medium.Cell gives can repeat or carry out continuously (for example, being infused in the cerebrospinal fluid by continuous irrigation).Generally speaking, multiple administering mode takes usually at interval and used respectively in 7-10 days at least.Another method be with cell seeding in bioabsorbable material such as gelfoam, adopt operation to have the bioabsorbable material of cell to implant required position kind.Above-mentioned two kinds of methods can obtain better therapeutic in conjunction with application.
The suitable consumption of cell will change according to spinal cord lesion that the patient suffered or destructive total amount, patient's body weight and other factors.Usually, the dosage range that at every turn gives is about 10
3-10
9Cell is typically about 10
6-10
7Cell.
Cell of the present invention or cell preparation can be treated from body patient and/or allosome patient, preferably join the identical or close allosome patient of type from the body patient or with cell source body MHC, most preferably from the body patient.
Particularly, when cell and/or the preparation that contains cell are administered to the allosome patient, use one or more immunosuppressant can for the patient who accepts this cell and/or tissue, to alleviate, preferably prevent the transplant rejection effect. the example that is applicable to the immunosuppressant of the inventive method comprises can stimulate the activating agent of approach by suppressor T cell/B cell jointly, for example in United States Patent (USP) 20020182211 disclosed interference T cell and B cell via the link coupled activating agent of CTLA4 and B7 approach.Other example comprises Cyclosporin A, myophenylate mofetil, rapamycin and antithymocyte globulin.
As used herein, term " from the body patient " is the patient that the cell used or cell preparation derive from self.
As used herein, cell or cell preparation that term " allosome patient " is used derive from allosome or xenogeneic patient.
In some embodiments of the present invention, cell is administered to the patient with one or more medicines.
Described medicine has been meant the neuroprotective cell, reduces the medicine of the effect of broken ends of fractured bone necrosis and promotion axon growth, includes but not limited to NGF (nerve growth factor), Brain Derived Neurotrophic Factor, neurenergen 3 and neurenergen-4/5.
Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment 1.hAEC separates and cultivates
Get healthy cesarean pregnant women placental, from the chorion denuded amniotic membrane.Hemocyte is removed in the PBS flushing, 37 ℃ of digestion of 0.25% trypsin 30min, and piping and druming adds the RPMI1640 that contains 10%FBS and stops digestion, and 100 mesh sieves filters, and 1,500rpm is centrifugal, abandons supernatant, with 1 * 10
4/ cm
2Be seeded in the 10cm plate.HAECs places 37 ℃, 5%CO in RPMI-1640 (10%FBS, 100 μ g/ml streptomycins, 100U/ml penicillin, 10ng/ml EGF and 0.3mg/ml glutaminase)
2Cultivate in the cell culture incubator.
The preparation of embodiment 2.hAEC cell preparation
Embodiment 3.hAECs treatment rat cerebral hemorrhage mold
1 animal
The female adult Sprague-Dawley rat of SPF level is adopted in experiment, available from Shanghai Si Laike Experimental Animal Center, and body weight 250 ± 10g, single cage is raised, and can freely absorb water and food, and illumination formation daytime/night, 12h-12h circulated.Laboratory animal is divided into matched group, operation (cerebral hemorrhage) group, treatment (cell transplantation) group, and wherein operation group and treatment group are divided into operation back and treatment 2d, 3d, 7d, 14d and 28d again.
2 research methoies
(1) making of rat cerebral hemorrhage mold
With reference to CrystalL[Crystal L MacLellan, Gergely Silasi, Candice C Poon, et al.Journal ofCerebral Blood Flow ﹠amp; Metabolism, 2007:1-10] etc. method and improve.To be fixed on the stereotaxic instrument after the rat usefulness chloral hydrate anesthesia (10% chloral hydrate is by 0.3ml/100g body weight lumbar injection), be equipped with hair, the local wiping field of operation of povidone iodine cuts rat head skin, and (bregma is the center to be positioned to basal nuclei with position finder, 3.5mm is opened on the side, dark 5.5mm).Get 0.375U/ μ l Collagenase IV (Sigma product) the 1 μ l of the preparation of PBS under the aseptic condition, inject the rat basal nuclei with the speed of 2 μ l/min, let the acupuncture needle remain at a certain point 5min pulls out behind the pin no liquid and flows from needle track is counter.Dental base acrylic resin powder sealing boring, aseptic operation needlework skin suture, povidone iodine wiping; The intramuscular injection mycillin is with infection.Monitor the heart rate of animal in the operation process in real time, blood pressure and body temperature keep the anus temperature at 37.5 ℃.After laboratory animal is revived fully, press Bederson[Bederson JB, Pitts LH, Tsujim, et al.Stroke, 1986,17:472-477] scoring: it is unsettled that rat is carried tail, and 0 is divided into and stretches two forelimbs earthward, does not see dystropy; The I level is mentioned the Mus tail, and brain injury offside forelimb reclaims post-buckling under abdomen; The II level, except I level symptom, during to the thruster rat, resistance obviously reduces than offside from brain injury; The III level, except I level and II level symptom, the sign that the oriented brain injury offside of rat side is outstanding; The IV level, the animal confusion.Except IV level animal model, other all includes research range in.Postoperative 2d, 3d, 7d, 14d and 28d carry out the model scoring to rat to be observed:
Balance beam walking test [Altumbabic M, Peeling J, Bigio MRD, et al.Stroke, 1998,29:1917-1923.] is measured motion and is integrated and the coordination ability.The long 80cm of balance beam, wide 2.5cm lies in the place apart from the high 10cm in ground.The standard of keeping the score: 0 minute, pass balance beam, can not fall; 1 minute, pass balance beam, the chance of falling is less than 50%; 2 minutes, pass balance beam, the chance of falling is greater than 50%; 3 minutes, can pass balance beam, but the paralysis rear flank limb of getting involved can not help to move forward; 4 minutes, can not pass balance beam, but above can being sitting in: 5 minutes, rat is placed on the balance beam and can falls down.
The asymmetric experiment scoring of forelimb [Ya Hua, Timothy Schallert, Richard F, et al.] rat is put into high 30cm, in the clean glass of bore 20cm, observe its number of times that finds new outlets with left and right sides upper limb contact glass wall, according to the motion frequency record of animal 3-10 minute.
Formula is score value=[R/ (R+L+B)]-[L/ (R+L+B)]
Wherein R is for using the number of times of right upper extremity; L is for using the number of times of left upper extremity; B is the number of times that uses two upper limb simultaneously.
(2) labelling HAEC
Adopt Hoechst33258 (Sigma product) labelling HAEC: add fluorescent marker Hochest33258 in adherent HAEC culture dish, the working concentration that makes Hochest33258 in the culture medium is 1 μ g/ml.HAEC is placed 37 ℃, 5%CO
2Labelling 12h in the incubator removes culture medium then, and the HAEC adherent with 0.25% trypsinization is centrifugal, then uses recentrifuge after the PBS liquid 2ml rinsing, removes the Hochest33258 fluorescent marker on unmarked repeatedly for 3 times.Resuspended adjustment cell concentration is 10
5/ μ l is standby.The expression of observing Hoechst33258 fluorescence on the HAEC be implanted into tricorn under fluorescence microscope is followed the trail of in the brain of HAEC and is located.
(3) transplant hAEC
24h transplants behind the cerebral hemorrhage.Be fixed on the stereotaxic instrument after rat anaesthetized once more, the local wiping field of operation of povidone iodine is cut off surgical thread, exposes skull, is positioned to left side tricorn (0.9mm behind the bregma, 1.4mm is opened on a left side, dark 4.9mm) with position finder.Get 10 μ lhAECs suspensions of embodiment 2 preparation, inject the rat basal nuclei with the speed of 5 μ l/min, let the acupuncture needle remain at a certain point 5min pulls out behind the pin no liquid and flows from needle track is counter.Dental base acrylic resin powder sealing boring, aseptic operation needlework skin suture, povidone iodine wiping; The intramuscular injection mycillin is with infection.
(4) brain water content
Broken end behind each group rat deep anaesthesia is got brain, along midline incision, distinguish the both sides cerebral hemisphere, the precise electronic balance claims weight in wet base (wwt); Put in 80 ℃ of baking boxs, toast continuously 48h to constant weight (last twice of poor quality≤0.2mg) back claims dry weight (dw),
Adopt the Elliott formula to calculate brain water content (%).
Brain water content %=(wwt-dw)/wwt * 100%
(5) liquid chromatograph-electrochemical detector detects the kind and the changes of contents of neurotransmitter in the rat brain spinal fluid.
During collect cerebrospinal fluid,, cut off dorsal body setae and expose skin with wet gauze wiping rat nape portion skin.Two ear lines are cut a transverse incision (about 1.5cm), put caudal ward therein along the subcutaneous 2cm that cuts off, and skin is separated both sides, broaden one's vision.Be close to the rat skull and successively shear each flesh layer successively, and the broken ends of fractured bone draws caudal ward to broaden one's vision successively.Near behind the neck during ligamenta flava, separate the muscle that covers with No. 7 injection needles carefully, expose occipito-axial ligaments.(the pin inclined-plane upwards with the 1ml insulin syringe, the nearly level of needle tip is thrust subarachnoid space, fixing needle body, slowly extract cerebrospinal fluid, general collection capacity is 50-120 μ l, drips 10% cold concentration perchloric acid mixing in 10: 1 ratios, in 4 ℃, the centrifugal 10min of 10000r/min, get-80 ℃ of preservations after the supernatant packing, to be checked.
3 interpretations of result
Adopt the spss13.0 statistical analysis software, image acquisition and analytical system, and the processing method of corresponding reagent box description.Data all use mean ± standard deviation to represent, the data between experimental group relatively adopt variance analysis to carry out statistical procedures, and test level is 0.05.
Result and discussion:
1. behavior scoring
The balance beam of cerebral hemorrhage and each time point of treatment group rat and the asymmetric application appraisal result of forelimb are seen Fig. 1 and Fig. 2.Balance beam walking test is used to measure the motion integration and the coordination ability of rat.The balance beam walking test score shows: 24h behind the injection collagenase, the unusual of sports coordination ability promptly appears in animal, the balance beam scoring of operation group 2d, 3d, 7d, 14d, 28d animal is compared with the matched group of corresponding time period, all have notable difference (* among Fig. 1: with compare P＜0.05 with time point operation group; *: with compare P＜0.01 with time point operation group).
Collagenase injection causes rat brain can cause the asymmetric application phenomenon of significant forelimb after hemorrhage, and from appraisal result: the scoring of operation treated animal is significantly higher than matched group; Compare with operation group rat, treatment group 2d, 3d, 7d, 14d, 28d all have significant difference, the tool statistical significance (* among Fig. 2: with compare P＜0.05 with time point operation group; *: with compare P＜0.01 with time point operation group; #: compare P＜0.05 with matched group; ##: compare P＜0.01 with matched group).
Ethological improvement has promptly appearred in cerebral hemorrhage animal 24h after having transplanted hAEC, and the balance beam scoring of treatment group 2d, 3d, 7d, 14d, 28d animal is used asymmetric scoring with forelimb and compared with the operation group of corresponding time period, and notable difference is all arranged.
2.hAEC the location
The hAEC of the Hoechst33258 labelling of transplanting through tricorn observes discovery under fluorescence microscope: hAEC is distributed in tricorn, ventriculus tertius, aqueduct of midbrain, and cell attachment is also arranged on the choroid plexus; Cell is light blue fluorescence, and nucleus subcircular and nuclear are bigger, the kytoplasm non-coloring, and cell state is good.
Conventional cresyl viollet dyeing finds that tricorn has agglomerate near circular cell, even dyeing, and cell boundaries is clear, structural integrity, cellular morphology is normal; Ventriculus tertius, aqueduct of midbrain also have a small amount of subcircular cell, and be obvious with the choroid plexus cell architectural difference.(see that Fig. 3 AD is a same brain section; BE is a same brain section; CF is a same brain section; A, B, C are the photo under the fluorescence microscope; D, E, F are the painted photo of conventional cresyl viollet)
3. water content
Each is organized the water content of rat the two cerebral hemispheres different time and sees Fig. 4.From the result, rat brain hemorrhage back 2d and 3d are the peak periods of cerebral edema, compare with control rats all to have significant difference; The rat brain water content increase that cerebral hemorrhage causes can continue until 7d behind the cerebral hemorrhage, and involves left hemisphere.
The hemorrhage big brain water content increase that causes of rat brain can obviously improve after tricorn is transplanted hAEC, (*: compare P＜0.05 with the side of performing the operation with time point from Fig. 4; #: compare P＜0.05 with matched group; ##: compare P＜0.01 with matched group.) as can be seen: the right brain water content of treatment group 2d, 3d is starkly lower than the water content of operation group 2d, 3d, and the P value is all＜0.05.
Each is organized rat brain left hemisphere water content and changes little.
4 monoamine neurotransmitters change
Each changes of contents of organizing monoamine neurotransmitter in the rat brain spinal fluid see Fig. 5 (*: with compare P＜0.05 with time point operation group; *: with compare P＜0.01 with time point operation group; #: compare P＜0.05 with matched group; ##: compare P＜0.01 with matched group.)。
The content difference in time that causes the monoamine neurotransmitter of cerebrospinal fluid after rat brain is hemorrhage changes, and during 2d-7d, the content of monoamine transmitters dopamine is to be tangible rising trend behind cerebral hemorrhage, and in the cerebral hemorrhage later stage, 14-8d, the content of dopamine reduces gradually, and is lower than normal value.
From the result of this research, during 7d, the content of dopamine is significantly higher than the matched group level behind the cerebral hemorrhage, P＜the 0.01 and content of transplanting the treatment treated animal of hAEC significantly is lower than the operation group, and, compare no significant difference near the matched group level with matched group.
Scope of the present invention is not subjected to the restriction of described specific embodiments, and described embodiment is only desired also to comprise the method and the component of functional equivalent in the scope of the invention as the single example of illustrating various aspects of the present invention.In fact, except content as herein described, those skilled in the art can easily grasp multiple improvement of the present invention with reference to above description and accompanying drawing.Described improvement also falls within the scope of appended claims.Every piece of list of references mentioned above is listed this paper in as a reference all in full.
Claims (4)
1. amniotic epithelial cells is in preparation treatment and/or improve purposes in the hemorrhagic apoplexy medicine.
2. purposes as claimed in claim 1 is characterized in that described amniotic epithelial cells derives from the mankind.
3. purposes as claimed in claim 1, it is characterized in that described hemorrhagic apoplexy for by organic diseases because of or wound the accident cerebral hemorrhage or the subarachnoid hemorrhage that cause.
4. purposes as claimed in claim 3 is characterized in that described hemorrhagic apoplexy is a cerebral hemorrhage.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008100438580A CN101721430B (en) | 2008-10-22 | 2008-10-22 | Application of amniotic epithelial cells in pharmacy |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008100438580A CN101721430B (en) | 2008-10-22 | 2008-10-22 | Application of amniotic epithelial cells in pharmacy |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101721430A true CN101721430A (en) | 2010-06-09 |
| CN101721430B CN101721430B (en) | 2013-01-23 |
Family
ID=42443381
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008100438580A Active CN101721430B (en) | 2008-10-22 | 2008-10-22 | Application of amniotic epithelial cells in pharmacy |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101721430B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102229528A (en) * | 2011-04-15 | 2011-11-02 | 广西南宁灵康赛诺科生物科技有限公司 | Method for extracting unsaturated fatty acid composition from humen amniotic epithelial cells, and application thereof |
| CN110090227A (en) * | 2018-04-18 | 2019-08-06 | 浙江大学 | Purposes of the human amnion membrane in treatment graft versus host disease(GVH disease) |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1884495A (en) * | 2005-06-24 | 2006-12-27 | 和泓生物技术（上海）有限公司 | Human amnion cell capable of expressing extraneous gene and its preparation method and uses |
| CN101199550A (en) * | 2007-11-16 | 2008-06-18 | 中国医学科学院血液学研究所 | Method of preparing containing endothelium ancestor cell preparation utilizing umbilical or placenta and uses thereof |
| CN101249101A (en) * | 2008-03-27 | 2008-08-27 | 孔祥飞 | Applications of human placenta interstitial fluid for curing brain and cerebrovascular disease medicament |
-
2008
- 2008-10-22 CN CN2008100438580A patent/CN101721430B/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102229528A (en) * | 2011-04-15 | 2011-11-02 | 广西南宁灵康赛诺科生物科技有限公司 | Method for extracting unsaturated fatty acid composition from humen amniotic epithelial cells, and application thereof |
| CN102229528B (en) * | 2011-04-15 | 2014-04-16 | 广西南宁灵康赛诺科生物科技有限公司 | Method for extracting unsaturated fatty acid composition from humen amniotic epithelial cells, and application thereof |
| CN110090227A (en) * | 2018-04-18 | 2019-08-06 | 浙江大学 | Purposes of the human amnion membrane in treatment graft versus host disease(GVH disease) |
| CN110090227B (en) * | 2018-04-18 | 2021-03-26 | 浙江大学 | Use of human amniotic epithelial cells in treatment of graft-versus-host disease |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101721430B (en) | 2013-01-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Deng et al. | Collagen scaffold combined with human umbilical cord-mesenchymal stem cells transplantation for acute complete spinal cord injury | |
| JP5871865B2 (en) | Method for inducing differentiation and proliferation of neural progenitor cells or neural stem cells into neurons, composition for inducing differentiation and proliferation, and pharmaceutical preparation | |
| WO2018103406A1 (en) | Neural stem cell injection for treating brain damage diseases and preparation method and use method thereof | |
| US8613906B2 (en) | Treatment of ischemia using stem cells | |
| JPS58501817A (en) | Fiber lattice seeded with cells | |
| CN103040860B (en) | A kind of method starting mammalian stem cell and chlorine dioxide are in the application for preparing drug for starting mammalian stem cell | |
| Khalili et al. | Therapeutic benefit of intravenous transplantation of mesenchymal stem cells after experimental subarachnoid hemorrhage in rats | |
| RU2394593C2 (en) | Implanted neuroendoprosthetic system, method of obtaining it and method of carrying out reconstructive neurosurgical operation | |
| ES2550456T3 (en) | Use of a composition containing mesenchymal stem cells derived from human umbilical cord blood to induce differentiation and proliferation of neural precursor cells or neural stem cells to neural cells | |
| CN104212762B (en) | Method for culture of urine-derived pluripotent stem cells by virtue of in vitro small molecule induction | |
| US20230201423A1 (en) | Cell sheet construct for neurovascular reconstruction and manufacture thereof | |
| Mao et al. | Skin-derived precursor cells promote angiogenesis and stimulate proliferation of endogenous neural stem cells after cerebral infarction | |
| KR20090055691A (en) | Composition for inducing differentiation and proliferation of neural precursor cells or neural stem cells to neural cells, comprising a human umbilical cord blood-derived mesenchymal stem cell as an active ingredient | |
| CN101721430B (en) | Application of amniotic epithelial cells in pharmacy | |
| CN109055300A (en) | The isolated culture method of people's endometrial tissue derived endothelial progenitor cells | |
| JP2013517854A (en) | Sewable tissue graft structure for reconstructing human or animal organs | |
| PL212052B1 (en) | Method for the acquirement of gley smell cells and their applications | |
| RU2303462C1 (en) | Biotransplant and method for treating mental development delay cases | |
| TW201444973A (en) | Cell therapy for the treatment of neurodegeneration | |
| CN101721429A (en) | Application of amniotic epithelial cells and cell preparations thereof | |
| RU2299073C1 (en) | Biotransplant and method for treating chronic cardiac failure (variants) | |
| Dezawa | Mesenchymal Stem/Stromal Cells: ENDOGENOUS NON-TUMORIGENIC PLURIPOTENT MUSE CELLS; BASIC RESEARCH AND CLINICAL TRIALS | |
| Alzhanuly et al. | Perspectives of using stem cell technologies for the treatment of diabetes mellitus | |
| US9750848B2 (en) | Method of preparing an implantable neuroendoprosthetic system | |
| Li et al. | Transplantation of a new composite of neural cells and marrow stromal cells into rat brain after stroke |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: SINO-AMERICAN GUOLIAN (SHANGHAI) BIOTECHNOLOGY RES Effective date: 20120220 Owner name: SINO-AMERICAN ICELL (SHANGHAI) BIOTECHNOLOGY CO., Free format text: FORMER OWNER: HEHONG BIO-TECH (SHANGHAI) CO., LTD. Effective date: 20120220 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 201203 PUDONG NEW AREA, SHANGHAI TO: 200333 PUTUO, SHANGHAI |
|
| TA01 | Transfer of patent application right |
Effective date of registration: 20120220 Address after: 200333 building 4, building 879, Lane 15, Zhongjiang Road, Shanghai, Putuo District Applicant after: Cell-star Biological Technology (Shanghai) Co., Ltd. Co-applicant after: Guolian (Shanghai) Biotechnology Research Co., Ltd. Address before: 201203 Shanghai Guo Shou Jing Road, Zhangjiang High Tech Park of Pudong New Area No. 351 Building No. 2 room 694-11 Applicant before: Hehong Biotechnology (Shanghai) Co., Ltd. |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C56 | Change in the name or address of the patentee |
Owner name: SHANGHAI CICELL BIOTECHNOLOGY CO., LTD. Free format text: FORMER NAME: ZHONGMEI SAIAO (SHANGHAI) BIOTECHNOLOGY CO., LTD. |
|
| CP01 | Change in the name or title of a patent holder |
Address after: 200333 building 4, building 879, Lane 15, Zhongjiang Road, Shanghai, Putuo District Patentee after: SINO-AMERICAN ICELL (SHANGHAI) BIOTECHNOLOGY CO., LTD. Patentee after: Shanghai United Stem Cell Technology Co., Ltd. Address before: 200333 building 4, building 879, Lane 15, Zhongjiang Road, Shanghai, Putuo District Patentee before: Cell-star Biological Technology (Shanghai) Co., Ltd. Patentee before: Guolian (Shanghai) Biotechnology Research Co., Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20171017 Address after: 200333 A block, 4 floor, building 15, 879 lane, Zhongjiang Road, Shanghai, Putuo District Co-patentee after: Beijing victory Biotechnology Co., Ltd. Patentee after: SINO-AMERICAN ICELL (SHANGHAI) BIOTECHNOLOGY CO., LTD. Co-patentee after: Guangzhou victory Biotechnology Co., Ltd. Co-patentee after: Chongqing United Stem Cell Technology Co., Ltd. Co-patentee after: Xuzhou cell Medical Co., Ltd. Co-patentee after: Shanghai United Stem Cell Technology Co., Ltd. Address before: 200333 building 4, building 879, Lane 15, Zhongjiang Road, Shanghai, Putuo District Co-patentee before: Shanghai United Stem Cell Technology Co., Ltd. Patentee before: SINO-AMERICAN ICELL (SHANGHAI) BIOTECHNOLOGY CO., LTD. |