CN1803193A - Use of stroma stem cell derived from bone marrow in preparing formulation for treating hypoxic ischemic cerebral palsy - Google Patents
Use of stroma stem cell derived from bone marrow in preparing formulation for treating hypoxic ischemic cerebral palsy Download PDFInfo
- Publication number
- CN1803193A CN1803193A CN 200510032737 CN200510032737A CN1803193A CN 1803193 A CN1803193 A CN 1803193A CN 200510032737 CN200510032737 CN 200510032737 CN 200510032737 A CN200510032737 A CN 200510032737A CN 1803193 A CN1803193 A CN 1803193A
- Authority
- CN
- China
- Prior art keywords
- cell
- bone marrow
- stem cell
- interstital stem
- preparation
- 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.)
- Pending
Links
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 43
- 206010021143 Hypoxia Diseases 0.000 title claims abstract description 15
- 210000001185 bone marrow Anatomy 0.000 title claims description 38
- 206010008129 cerebral palsy Diseases 0.000 title claims description 25
- 230000000302 ischemic effect Effects 0.000 title claims description 13
- 230000001146 hypoxic effect Effects 0.000 title claims description 12
- 238000009472 formulation Methods 0.000 title 1
- 239000000203 mixture Substances 0.000 title 1
- 210000004027 cell Anatomy 0.000 claims abstract description 40
- 238000002360 preparation method Methods 0.000 claims abstract description 24
- 210000004556 brain Anatomy 0.000 claims abstract description 10
- 206010070511 Hypoxic-ischaemic encephalopathy Diseases 0.000 claims abstract description 8
- 238000011552 rat model Methods 0.000 claims abstract description 6
- 208000037212 Neonatal hypoxic and ischemic brain injury Diseases 0.000 claims description 25
- 208000033300 perinatal asphyxia Diseases 0.000 claims description 25
- 241000700159 Rattus Species 0.000 claims description 23
- 210000001161 mammalian embryo Anatomy 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 13
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 239000001963 growth medium Substances 0.000 claims description 9
- 238000002347 injection Methods 0.000 claims description 8
- 239000007924 injection Substances 0.000 claims description 8
- 230000003321 amplification Effects 0.000 claims description 7
- 238000004500 asepsis Methods 0.000 claims description 7
- 208000009973 brain hypoxia - ischemia Diseases 0.000 claims description 7
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 7
- 238000000746 purification Methods 0.000 claims description 7
- 238000011084 recovery Methods 0.000 claims description 7
- 230000004927 fusion Effects 0.000 claims description 4
- 206010000210 abortion Diseases 0.000 claims description 2
- 231100000176 abortion Toxicity 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 230000002490 cerebral effect Effects 0.000 abstract description 15
- 208000002381 Brain Hypoxia Diseases 0.000 abstract description 2
- 206010033799 Paralysis Diseases 0.000 abstract 1
- 239000002504 physiological saline solution Substances 0.000 abstract 1
- 210000001519 tissue Anatomy 0.000 description 14
- 230000004083 survival effect Effects 0.000 description 12
- 238000002054 transplantation Methods 0.000 description 10
- 238000012360 testing method Methods 0.000 description 7
- 102100039289 Glial fibrillary acidic protein Human genes 0.000 description 6
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 description 6
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 description 6
- 238000011476 stem cell transplantation Methods 0.000 description 6
- 230000037396 body weight Effects 0.000 description 5
- 238000002560 therapeutic procedure Methods 0.000 description 5
- 241000972773 Aulopiformes Species 0.000 description 4
- 230000001054 cortical effect Effects 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 210000001320 hippocampus Anatomy 0.000 description 4
- 230000007087 memory ability Effects 0.000 description 4
- 230000001575 pathological effect Effects 0.000 description 4
- 235000019515 salmon Nutrition 0.000 description 4
- 238000010171 animal model Methods 0.000 description 3
- 238000002659 cell therapy Methods 0.000 description 3
- 210000001638 cerebellum Anatomy 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000010166 immunofluorescence Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000017074 necrotic cell death Effects 0.000 description 3
- 210000004498 neuroglial cell Anatomy 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 2
- 210000003710 cerebral cortex Anatomy 0.000 description 2
- 239000013256 coordination polymer Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 description 2
- 230000007773 growth pattern Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000011532 immunohistochemical staining Methods 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 210000001178 neural stem cell Anatomy 0.000 description 2
- 230000004770 neurodegeneration Effects 0.000 description 2
- 210000000449 purkinje cell Anatomy 0.000 description 2
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 206010002660 Anoxia Diseases 0.000 description 1
- 241000976983 Anoxia Species 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 206010051290 Central nervous system lesion Diseases 0.000 description 1
- 206010008190 Cerebrovascular accident Diseases 0.000 description 1
- 208000012661 Dyskinesia Diseases 0.000 description 1
- 102100028652 Gamma-enolase Human genes 0.000 description 1
- 101710191797 Gamma-enolase Proteins 0.000 description 1
- 101100091360 Homo sapiens RNPC3 gene Proteins 0.000 description 1
- 206010020880 Hypertrophy Diseases 0.000 description 1
- 208000036626 Mental retardation Diseases 0.000 description 1
- 238000012347 Morris Water Maze Methods 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 102100026085 RNA-binding region-containing protein 3 Human genes 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 108010017842 Telomerase Proteins 0.000 description 1
- 210000002718 aborted fetus Anatomy 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000001467 acupuncture Methods 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000007953 anoxia Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000000386 athletic effect Effects 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000004958 brain cell Anatomy 0.000 description 1
- 208000029028 brain injury Diseases 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 244000144987 brood Species 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 229960003638 dopamine Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 238000010185 immunofluorescence analysis Methods 0.000 description 1
- 238000002991 immunohistochemical analysis Methods 0.000 description 1
- 238000012309 immunohistochemistry technique Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000009533 lab test Methods 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 230000006386 memory function Effects 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- 239000003595 mist Substances 0.000 description 1
- 230000007659 motor function Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000003887 myelocyte Anatomy 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 230000000474 nursing effect Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 238000001050 pharmacotherapy Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000009256 replacement therapy Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 230000001228 trophic effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to the mesenchyme stem cell of marrow source in preparing preparation for treating anoxemia brain paralysis, wherein the preparation is prepared from mesenchyme stem cells of marrow source and physiological saline solution, the effective dosage for neonate rat models with hypoxic encephalopathy is 500000 cells/time, and the effective dosage for neonate cerebral pasly patients is 1000000-5000000 cells/time.
Description
Technical field
The invention belongs to stem cell and field of tissue engineering technology, relate to the new purposes of interstital stem cell, the interstital stem cell that especially relates to derived from bone marrow is used to prepare the purposes of the preparation for the treatment of hypoxic ischemic cerebral palsy.
Background technology
Enclosing the ischemic hypoxia brain injury (HIE) of newborn baby due to suffocating is the main cause that causes neonate cerebral palsy (cerebral palsy).Over nearly 20 years, although obstetrics and the development of neonate health care are fast, but cerebral palsy is fallen ill still not have obviously in neonate and is reduced, find according to unit samplings such as Beijing Medical University investigation in 1998, among China 0-6 year child CP children 310,000 is arranged approximately, and the U.S. enclosed a prospective study of living collaboration items and found that the cerebral palsy of children sickness rate reaches 4 ‰ with 4.6 ten thousand speed increase every year.Infant mainly shows as the non-sexual centre dyskinesia that carries out, or with mental retardation etc.Present Therapeutic Method mainly contains: (1) is based on the combined rehabilitation medical treatment of athletic rehabilitation; (2) based on trophic nerve, relaxed muscle, the pharmacotherapy of invigorating blood circulation etc.; (3) selective posterior rhizotomy therapy; (4) based on the tcm therapy of massage, acupuncture etc.But because cerebral palsy is caused that by fixed brain lesions the cause of disease can not be removed or correct to above Therapeutic Method, curative effect is not certainly.Yet for patients with cerebral palsy especially serious patient, infant not only bears huge human body misery, also can be because of suffocating that cerebral palsy can not be taken care of oneself and be caused, and make cerebral anoxia and make its intelligence be the decline of carrying out property.Therefore, if can study the effective cerebral palsy Therapeutic Method that makes new advances, will bring happiness to countless families.
Past adopts the cellular replacement therapy patients with cerebral palsy not appear in the newspapers as yet in decades, but has obtained certain progress in other disease of treatment central nervous system.
Source of human stem cell mainly contains: 1, embryo neural stem cells: the research report is arranged, adopt 6-9W aborted fetus brain cell treatment Parkinson`s condition of disease obviously to improve partly patient's function, even can also synthesize dopamine in some patient's body.But, because all multifactor clinical practice of embryo neural stem cells of having limited to such as cell source, ethics at the transplantation treatment nervous system disease.2, the interstital stem cell of derived from bone marrow: the seventies, separation and Culture from bone marrow such as Friedenstein goes out a kind of interstital stem cell, the deep interstital stem cell of discovering not only can survive, move at cerebral tissue, and express multiple neurocyte specificity markers such as neuron, neurogliocyte, obviously improve rat model functions such as apoplexy.At present, interstital stem cell is mainly derived from adult's bone marrow, but adult bone myelocyte source is limited, pathogen contamination is inevitable.Still do not have and adopt the report of embryo's bone marrow as source of human stem cell.
Obviously, under existence conditions, the relevant technologies and the method for hypoxic ischemic cerebral palsy therapeutical effect all existed many disadvantages and deficiency.
Summary of the invention
The object of the present invention is to provide a kind of interstital stem cell of derived from bone marrow to be used to prepare the purposes of the preparation for the treatment of hypoxic ischemic cerebral palsy.
Described preparation is formulated by the interstital stem cell and the normal saline of derived from bone marrow, and the effective dose that is used for the neonate rat model of hypoxic ischemic encephalopathy is 5 * 10
5Cell/time.This effective dose obtains therapeutic effect by animal model experiment and is definite.
The interstital stem cell of described derived from bone marrow prepares by the following method: utilize asepsis to separate pregnant rats embryo in mid-term bone marrow nucleated cell, be seeded in the culture bottle of the low sugar DMEM culture medium that contains hyclone, when cell attachment is bred near fusion, digest, go down to posterity, method goes down to posterity repeatedly according to this, reach the purpose of purification and amplification interstital stem cell, collecting cell is frozen, recovery is standby.
The preparation of the interstital stem cell preparation of described derived from bone marrow is used for the neonate rat is carried out locating injection in the brain, and dosage is 5.0 * 10
5Cell/time, in back 24 hours of the making of hypoxic ischemic encephalopathy model, transplant.
Described preparation is formulated by the interstital stem cell and the normal saline of derived from bone marrow, and the effective dose that is used for neonate cerebral palsy patient patient is 1.0～5.0 * 10
6Cell/time.This effective dose obtains therapeutic effect and human body general knowledge of using medicines by animal model experiment and is definite.
The interstital stem cell of described derived from bone marrow prepares by the following method: utilize asepsis to separate embryo's bone marrow nucleated cell from people's embryonic abortion material, be seeded in the culture bottle of the low sugar DMEM culture medium that contains hyclone, when cell attachment is bred near fusion, digest, go down to posterity, method goes down to posterity repeatedly according to this, reach the purpose of purification and amplification interstital stem cell, collecting cell is frozen, recovery is standby.
The preparation of the interstital stem cell preparation of described derived from bone marrow is used for neonate cerebral palsy patient is carried out locating injection in the brain, and dosage is 1.0～5.0 * 10
6/ time, after taking place, transplants in 24-48 hour hypoxic ischemic encephalopathy.
The present invention utilizes the interstital stem cell of embryo's derived from bone marrow to transplant to the scarce property CP children of anoxia by brain location infusion methods, promote its cerebral tissue neurocyte reparation, improve its balance coordination, motor function, improve study, memory ability, reach the purpose that improves infant quality of life, alleviates family and burden on society, the new way of opening up cell therapy for the clinical treatment cerebral palsy.
The preparation of the interstital stem cell preparation of described derived from bone marrow is used for the treatment of the effect assessment of hypoxic ischemic cerebral palsy:
1, survival ability: comprise
A) 1 all inner model survival of rats rates;
B) January inner model rat body weight growth pattern;
2, transplant back the 42nd day study, memory function;
3, the 10th week was dissected the cerebral tissue pathological characters;
4, SABC and the survival in cerebral tissue of indirect immunofluorescence analysis foreign cell, migration, neurocyte specific antigen are expressed.
The invention has the advantages that:
(1) mesenchymal stem cell transplantation with embryo's derived from bone marrow arrives in the newborn hypoxic ischemic rat model brain, can obviously improve the rat model survival ability; Improve its motion, study, memory ability; Nurse one's health the reparation of impaired cerebral tissue microenvironment, promotion nervous cell regenerating and/or function.
(2) open up the new way of cell therapy for the clinical treatment hypoxic ischemic encephalopathy.The present invention utilizes that the mescenchymal stem cell cell of embryo's derived from bone marrow is original, proliferation potential is big, preparation is easy, still has multidirectional differentiation potential behind continuous culture and the cryopreservation resuscitation, and cell homogeneity height has normal caryogram and telomerase activation.
(3) the present invention utilizes the interior location of the interstital stem cell brain injection for curing rat hypoxic ischemic cerebral palsy model of derived from bone marrow to prove, the interstital stem cell of derived from bone marrow can block and reverse the development of hypoxic ischemic cerebral palsy, the new way of opening up cell therapy for the clinical treatment hypoxic ischemic cerebral palsy.
Description of drawings
Fig. 1 is the check pathological section result (H﹠amp of SD rat cerebral tissue; E dyeing, * 200) figure.Wherein, A figure shows that normal control group spindle cell distributes; B figure shows neuronal degeneration necrosis (↑) in the HIE model; C figure shows cerebellum purkinje cell karyopycnosis (↑) in the HIE model; D figure shows that HIE/hFBMMSCs transplantation group cortical layer neurocyte extensively increases.
Fig. 2 is hFBMMSCs transplantation group cerebral tissue immunohistochemical staining testing result (DAB, * 100) figure, and A figure shows the anti-people NSE positive (↑); B figure shows the anti-people GFAP positive (↑).
Fig. 3 is HIE/hFBMMSCs indirect immunofluorescence testing result (* 100) figure.Wherein, 1A and 1B show: same the same position of section, and the green fluorescence person is the anti-people RNP positive among the A; Salmon pink fluorescence person is the anti-GFAP positive among the B; 2A and 2B show: same the same position of section, and the green fluorescence person is the anti-people RNP positive among the A; Salmon pink fluorescence person is the anti-NSE positive among the B.
The specific embodiment
Embodiment one: the preparation of the interstital stem cell of derived from bone marrow (MSC):
Utilize asepsis to separate second trimester embryo bone marrow nucleated cell, be seeded in the culture bottle of the low sugar DMEM culture medium that contains 8% hyclone, look growing state and change liquid every other day 1 time, cell attachment propagation is near merging when cell becomes 90% to merge attitude, digest, go down to posterity, method goes down to posterity repeatedly according to this, reaches the purpose of purification and amplification interstital stem cell, and collecting cell is frozen, recovery is standby.
Described low sugar DMEM increases the CAT NO.SH30002.01 product that foster base is selected HYCLONE company for use, the perhaps like product of GIBCO company.
Embodiment two: the preparation of the interstital stem cell of derived from bone marrow (MSC):
Utilize asepsis to separate second trimester embryo bone marrow nucleated cell, be seeded in the culture bottle of the low sugar DMEM culture medium that contains 10% hyclone, look growing state and changed liquid in 3 days 1 time, cell attachment propagation is near merging when cell becomes 80% to merge attitude, digest, go down to posterity, method goes down to posterity repeatedly according to this, reaches the purpose of purification and amplification interstital stem cell, and collecting cell is frozen, recovery is standby.
Described low sugar DMEM culture medium is selected the CAT NO.SH30002.01 product of HYCLONE company, the perhaps like product of GIBCO company for use.
Embodiment three: the preparation of the interstital stem cell of derived from bone marrow (MSC):
Utilize asepsis to separate second trimester embryo bone marrow nucleated cell, be seeded in the culture bottle of the low sugar DMEM culture medium that contains 15% hyclone, look growing state and changed liquid in 4 days 1 time, cell attachment propagation becomes 70% to merge attitude near merging to cell, digest, go down to posterity, method goes down to posterity repeatedly according to this, reaches the purpose of purification and amplification interstital stem cell, and collecting cell is frozen, recovery is standby.
Described low sugar DMEM culture medium is selected the CAT NO.SH30002.01 product of HYCLONE company, the perhaps like product of GIBCO company for use.
Embodiment four: the modeling of hypoxic ischemic cerebral palsy neonate rat:
1 day age newborn F344 rat (body weight 5.85 ± 0.39g), dorsal position is fixed after the ether inhalation anesthesia, neck medisection skin, free left carotid, sew up the incision after No. 5 toe-in is pricked, insert behind the 2h in 37 ℃ of constant-temperature enclosed containers, import the mist of 8%O2,92%N2 with the speed of 1L/min and keep 3h, take out to be placed on and accept female Mus nursing in the former feeding environment.
Embodiment five: the interstital stem cell infusion experiment of embryo's derived from bone marrow:
1, treatment opportunity: after the modelling in 24 hours.
2, dosage: 5.0 * 105/g.
3, approach: brain locating injection.
4, test control group: adopt design in groups, brood rat is divided into 4 groups of model/stem cell transplantation group, model/normal saline group, model/treatment, normal controls.
Effect assessment:
1, survival ability
Survival of rats situation in (1) 1 week: the stem cell transplantation group, more there are not significant difference in survival rate and normal control group; Be higher than normal saline group and not treatment group.See table 1 for details
(2) neonate rat body weight gain situation in January: transplant the back and rose in the 5th day, increased by the transplantation group rat body weight and accelerate, do not have significant difference with the normal control group on the 30th day, overweight normal saline group and not treatment group.See table 2 for details
2, changing function: behind the cell transplantation the 42nd day, adopt Morris water maze laboratory test transplantation group rat learning and memory ability, possess the study close, memory ability with normal rats, be better than normal saline injection and not treatment group.See table 3 for details
3, anatomical results: 10W carries out the cerebral tissue structural analysis to experimental rat.Stem cell transplantation group cerebral tissue atrophy occurs and the edema ratio is starkly lower than normal saline injection group and not treatment group, does not have significant difference with the normal control group.
4, cerebral tissue check pathological section result: H﹠amp; E dyeing, under the optical microscope, the visible brain nervous cell hypertrophy of stem cell transplantation group is especially at positions such as cerebral cortex layer, Hippocampus; Cerebral blood vessel wall partly, visible a large amount of cell migration around; The visible a large amount of cells in stem cell transplantation inserting needle position are agglomerating or be dispersed in distribution, are directivity and move towards periphery.And regional neurocytes such as not treatment group and the visible cerebellum of normal saline group, cortical layer, Hippocampus obviously reduce, and neuronal kernel pyknosis, necrosis, glial cell are engulfed and the glial cell reactive hyperplasia forms pathological changes phenomenons such as glial cell tuberosity.See Fig. 1 for details.Fig. 1 is the check pathological section result (H﹠amp of SD rat cerebral tissue; E dyeing, * 200) figure.Wherein, A figure shows that normal control group spindle cell distributes; B figure shows neuronal degeneration necrosis (↑) in the HIE model; C figure shows cerebellum purkinje cell karyopycnosis (↑) in the HIE model; D figure shows that HIE/hFBMMSCs transplantation group cortical layer neurocyte extensively increases.
5, immunohistochemical analysis result: adopt the immunohistochemistry technique analysis, have anti-people NSE, GFAP reacting cells in the transplantation group rat cerebral tissue, more obvious with zones such as cortical layer and Hippocampus; Other 3 groups of laboratory animals there is no NSE or GFAP positive cell.See Fig. 2 for details.Fig. 2 is hFBMMSCs transplantation group cerebral tissue immunohistochemical staining testing result (DAB, * 100) figure, and A figure shows the anti-people NSE positive (↑), and B figure shows the anti-people GFAP positive (↑).
6, indirect immunofluorescence testing result: location detection such as transplantation group both sides cerebral cortex layer, Hippocampus are to anti-people RNP/CD45, anti-people RNP/NSE or the two positive intact cells of anti-people RNP/GFAP, especially at the inserting needle position, a large amount of two positive cells are agglomerating or be dispersed in existence, migration and density descend in gradient towards periphery, and anti-people RNP, CD45, NSE, GFAP positive cell all do not appear in other 3 treated animal brain tissue slice.See Fig. 3 for details.Fig. 3 is HIE/hFBMMSCs indirect immunofluorescence testing result (* 100) figure.Wherein, 1A and 1B show: same the same position of section, and the green fluorescence person is the anti-people RNP positive among the A; Salmon pink fluorescence person is the anti-GFAP positive among the B; 2A and 2B show: same the same position of section, and the green fluorescence person is the anti-people RNP positive among the A; Salmon pink fluorescence person is the anti-NSE positive among the B.
Table 1 neonate rat survival rate in 1 week
| Nest is other | Newborn rat sum (N) | HIE/hFBMMSC survival rate (%) | HIE/NS survival rate (%) | HIE/ does not treat survival rate (%) | Normal control survival rate (%) |
| 1 | 10 | 100.0％(3/3) | 66.7％(2/3) | 50.0％(1/2) | 100.0％(2/2) |
| 2 | 14 * | 75.0％(3/4) | 33.3％(1/3) | 66.7％(2/3) | 100.0％(3/3) |
| 3 | 13 ** | 66.7％(2/3) | 0.0％(0/2) | 50.0％(1/2) | 100.0％(3/3) |
| 4 | 8 * | 100.0％(2/2) | 50.0％(1/2) | 50.0％(0/1) | 100.0％(2/2) |
| 5 | 16# | 66.7％(2/3) | 33.3％(1/3) | 66.7％(2/3) | 33.3％(1/3) |
| 6 | 9## | 100.0％(2/2) | 0.0％(0/2) | 100.0％(1/1) | 100.0％(2/2) |
| 7 | 11## | 33.3％(1/3) | 50.0％(1/2) | 0.0％(0/2) | 50.0％(1/2) |
| Sum (N) | 81 | 75.0％(15/20) | 35.3％(6/17) | 50.0％(7/14) | 82.4％(14/17) |
Body weight growth pattern in 4 groups of neonate rat 1M of table 2 (x ± SD)
| Natural law (d) | Normal control (g) | HIE/ hFBMMSCs(g) | HIE/NS (g) | HIE/ does not treat (g) | |
| 5 | 8.89±0.82 | 5.48±0.91 | 4.28±0.39 | 4.82±0.61 | |
| 10 | 15.87±1.34 | 13.99±0.93 | 12.44±0.93 | 12.59±0.93 | |
| 15 | 22.80±1.84 | 20.96±1.85 | 17.93±1.68 | 17.98±1.38 | |
| 20 | 33.94±1.62 | 33.61±2.63 | 27.39±1.84 | 27.82±1.01 | |
| 25 | 52.54±1.67 | 51.80±3.12 | 45.79±1.53 | 46.79±1.14 | |
| 30 | 73.92±6.27 | 71.01±2.66 | 63.53±5.35 | 64.93±4.25 | |
| Sum (N) | 30 | 9 | 10 | 4 | 4 |
4 groups of experimental rat Morris of table 3 water maze training and testing result (sec of X ± SD)
| Time (d) | Normal control (n=4) | HIE/hFBMMSCs (n＝4) | HIE/NS (n＝4) | HIE/ does not treat (n=4) |
| 1 | 105.0±1.5 | 110.0±7.1 | 111.0±1.4 * | 118.0± 1.5# |
| 2 | 50.0±2.7 | 90.0±2.5 | 96.0±22.6 * | 100.0±2.5 |
| 3 | 30.0±9.8 | 47.5±12.5 | 71.7±24.7 | 80.0±15.3 |
| 4 | 13.0±3.2 | 14.0±4.3 | 33.0±20.9 | 29.0±11.0 |
| 5 | 7.0±2.1 | 7.5±2.1 | 23.7±14.9 | 24.0±9.0 |
*: the experimental result of 2 Mus; The experimental result of #:3 Mus
Claims (7)
1, the interstital stem cell of derived from bone marrow is used to prepare the purposes of the preparation for the treatment of hypoxic ischemic cerebral palsy.
2, purposes according to claim 1 is characterized in that, described preparation is formulated by the interstital stem cell and the normal saline of derived from bone marrow, and the effective dose that is used for the neonate rat model of hypoxic ischemic encephalopathy is 5 * 10
5Cell/time.
3, purposes according to claim 2, it is characterized in that, the interstital stem cell of described derived from bone marrow prepares by the following method: utilize asepsis to separate pregnant rats embryo in mid-term bone marrow nucleated cell, be seeded in the culture bottle of the low sugar DMEM culture medium that contains hyclone, when cell attachment is bred near fusion, digest, go down to posterity, method goes down to posterity repeatedly according to this, reach the purpose of purification and amplification interstital stem cell, collecting cell is frozen, recovery is standby.
4, purposes according to claim 2 is characterized in that: the preparation of the interstital stem cell preparation of described derived from bone marrow, be used for the neonate rat is carried out locating injection in the brain, and dosage is 5 * 10
5/ time, in back 24 hours of the making of hypoxic ischemic encephalopathy model, transplant.
5, purposes according to claim 1 is characterized in that, described preparation is formulated by the interstital stem cell and the normal saline of derived from bone marrow, and the effective dose that is used for neonate cerebral palsy patient is 1.0～5.0 * 10
6Cell/time.
6, purposes according to claim 5, it is characterized in that, the interstital stem cell of described derived from bone marrow prepares by the following method: utilize asepsis to separate embryo's bone marrow nucleated cell from people's embryonic abortion material, be seeded in the culture bottle of the low sugar DMEM culture medium that contains hyclone, when cell attachment is bred near fusion, digest, go down to posterity, method goes down to posterity repeatedly according to this, reach the purpose of purification and amplification interstital stem cell, collecting cell is frozen, recovery is standby.
7, purposes according to claim 5 is characterized in that: the preparation of the interstital stem cell preparation of described derived from bone marrow, be used for neonate cerebral palsy patient is carried out locating injection in the brain, and dosage is 1.0～5.0 * 10
6/ time, after taking place, transplants in 24-48 hour hypoxic ischemic encephalopathy.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510032737 CN1803193A (en) | 2005-01-13 | 2005-01-13 | Use of stroma stem cell derived from bone marrow in preparing formulation for treating hypoxic ischemic cerebral palsy |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510032737 CN1803193A (en) | 2005-01-13 | 2005-01-13 | Use of stroma stem cell derived from bone marrow in preparing formulation for treating hypoxic ischemic cerebral palsy |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1803193A true CN1803193A (en) | 2006-07-19 |
Family
ID=36865424
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200510032737 Pending CN1803193A (en) | 2005-01-13 | 2005-01-13 | Use of stroma stem cell derived from bone marrow in preparing formulation for treating hypoxic ischemic cerebral palsy |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1803193A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110461344A (en) * | 2017-01-16 | 2019-11-15 | 美迪因诺医疗创新科技有限公司 | For treating the composition of newborn HIE |
| CN112236152A (en) * | 2018-04-04 | 2021-01-15 | 杜克大学 | Method for treating cerebral palsy and hypoxic-ischemic encephalopathy using human umbilical cord tissue-derived mesenchymal stromal cells |
| 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 |
-
2005
- 2005-01-13 CN CN 200510032737 patent/CN1803193A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110461344A (en) * | 2017-01-16 | 2019-11-15 | 美迪因诺医疗创新科技有限公司 | For treating the composition of newborn HIE |
| CN110461344B (en) * | 2017-01-16 | 2024-01-09 | 美迪因诺医疗创新科技有限公司 | Composition for treating neonatal HIE |
| CN112236152A (en) * | 2018-04-04 | 2021-01-15 | 杜克大学 | Method for treating cerebral palsy and hypoxic-ischemic encephalopathy using human umbilical cord tissue-derived mesenchymal stromal cells |
| 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 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Deckel et al. | Anatomical predictors of behavioral recovery following fetal striatal transplants | |
| CN1171991C (en) | Culture process of human nerve stem cell | |
| Sensenbrenner et al. | Cultivation and growth of dissociated neurons from chick embryo cerebral cortex in the presence of different substrates | |
| CN105039245A (en) | Method for promoting in-vitro maturing of human immature oocyte by utilizing 3D printing technology | |
| Sakamoto et al. | Widespread neuronal degeneration in rats following oral administration of methylmercury during the postnatal developing phase: a model of fetal-type Minamata disease | |
| CN106722934B (en) | Anti-aging health food containing earthworm and clam worm peptide-containing extract | |
| CN1820069A (en) | Selection and propagation of progenitor cells | |
| CN1803193A (en) | Use of stroma stem cell derived from bone marrow in preparing formulation for treating hypoxic ischemic cerebral palsy | |
| Okamoto | Observations on neurons and neuroglia from the area of the reticular formation in tissue culture | |
| CN109152801A (en) | Using the improvement and treatment of the brain injury in perinatal period of multipotential stem cell | |
| EP1073420A2 (en) | Specially devised neuronal implants for reconstruction of damaged central nervous system | |
| CN1839922A (en) | Traditional Chinese medicine preparation for treating leukoderma, psoriasis and its preparation method | |
| CN115927201A (en) | Preparation method and application of transplantable cell strain for treating spinal cord injury | |
| Sun et al. | Electroacupuncture-enhanced differentiation of bone marrow stromal cells into neuronal cells | |
| RU2798554C2 (en) | Biomedical cell product for the treatment of oncological, neurodegenerative, autoimmune diseases and injuries of the brain and spinal cord | |
| Aguillon-Gutierrez et al. | Effects of endogenous bioregulators from mammalian blood serum and bone tissue on amphibian limb regeneration | |
| CN1270727C (en) | Method of curing injured spinal cord and therapeutic agents for that | |
| CN1752208A (en) | A kind of novel method that improves human marrow mesenchymal stem cell to the dopaminergic neuron-like cell differentiation efficiency | |
| CN1824310A (en) | Preparation method of medicine for treating pelada | |
| CN106138098A (en) | Natural killer cell associating antidepressants application in improving depressive symptom | |
| RU2646792C1 (en) | Horse remedy with regenerative activity | |
| Ren et al. | Effect of Confucius’ Pillow Elixir on Cognitive Ability of Agrypnia Mice and Its Mechanism | |
| LIN | Bushen Tiaogan prescription reverses prednisone-induced osteoporosis in zebrafish | |
| Kempka et al. | Application of biotechnological methods in stem cell transplantation | |
| Nikitin | Russian Studies on Age-associated Physiology, Biochemistry, and Morphology: Historic Description with Extensive Bibliography |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |