CN1187058C - Stem cell medicine for repairing damage of central nerve and its preparing process - Google Patents
Stem cell medicine for repairing damage of central nerve and its preparing process Download PDFInfo
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Abstract
The present invention relates to a stem cell preparation for treating central nervous system diseases, and a related transplant technique thereof. Stem cells come from CD34 positive cells of hematopoietic tissues (cord blood, peripheral blood and marrow), and mesenchymal stem cells. The preparation technique relates to the separation, the purification and the cultivation of the stem cells, the preparation of cell suspension, etc. The transplant technique relates to the direct infusion of the CD34 positive cells and the mesenchymal stem cells through a spinal cord chamber. Because the brain and the spinal cord central nervous system are tissue positions which are exempted with immune functions and do not generate variant immunological rejection in a human body, the preparation has especially important purposes. A suitable stem cell preparation prepared by the technique can treat occupancy neurological function obstacles, such as the injury to the brain and the spinal cord central nervous system, implementation pathological changes, cerebral embolism, tumours, etc., via direct transplant. In addition, the stem cells prepared according to the technique can be used as gene therapy carriers for the transfection of relevant genes, and then the stem cells are directly transplanted to the brain and the spinal cord central nervous system to carry out the corresponding gene treatment so as to treat various tumours, thromboembolism and other gene defective diseases of the brain and the spinal cord central nervous system.
Description
Technical field:
The present invention relates to a kind of stem cell medicine for the treatment of nervous system injury, more particularly, relate to the CD34+ stem cell in hemopoietic tissue (Cord blood, peripheral blood and bone marrow) source and interstital stem cell preparation and preparation method thereof and treat the new technique of brain and spinal center nervous system injury and other disease with transplanting the CD34 positive (+) cell or interstital stem cell preparation.
Technical background:
For a long time, owing to usually seem at a loss what to do for the paralysis that causes by nervous system injury and other nervous system dysfunctions clinically, therefore it is believed that axoneure is non-renewable.Many research worker explore nervous system cell and transplant the functional rehabilitation that promotes nerve injury, but the donor nervous system cell is mainly derived from tire brain or other unexpected death person, and the source is not enough and have the ethics obstacle in clinical practice.Also have research to explore the probability that embryo stem cell transplantation promotes the nerve injury functional rehabilitation, can be divided into different tissue phenotype at the different parts of marrowbrain tissue after observing embryo stem cell transplantation, this shows that the host source intrinsic signal of property plays an important effect in the inducing cell atomization.Though fetal tissue transplantation may have therapeutical effect to neurodegeneration, cerebral spinal cord injury and cerebral ischemia infraction, because problems such as source deficiency, allosome immunologic rejection, ethics disputes, its clinical practice is very limited.Therefore, select another stem cell source transplantation treatment central nervous system disease to become more and more urgent.
Stem cell is the cells of origin that forms the various histoorgans of viable organism, has the function of self replication and multidirectional differentiation.In normal reproductive physiology process, stem cell is differentiated by germ cell.Germ cell is grown at first and is formed original embryonic stem cell, and the latter has the whole potential that form complete individuality.The further differentiation and proliferation of original embryonic stem cell forms the blastaea spline structure, and the cell of its internal layer cell mass also claims embryonic stem cell.The single embryonic stem cell of blastaea internal layer can not form a complete individuality, but has the totipotency that forms the various tissues of human body.The blastaea embryonic stem cell continues differentiation and development, progressively form the fetus of different gestational age, contain more rich stem cell in the Placenta Hominis Cord blood of connection fetus and parent during birth, the many tissues of birth back human body, particularly hemopoietic tissue comprises and contains multiple stem cell in bone marrow, peripheral blood, liver and the spleen.In these very long years of growing up, stem cell is lost its totipotency step by step at whole fetal development, and forming inferior all-round, pluripotent stem cell and tissue specially can stem cell, and the latter press differentiation function and divides have another name called hematopoietic stem cell, neural stem cell, skin progenitor cell, or the like.
Pluripotent stem cell in human cord blood and the people's bone marrow has advantages such as multiplication capacity is strong, the source is abundant, collection is convenient, and becoming another can be for the cell source of cell transplantation.Diseases such as these cellular replacement therapy leukemia, heritability hematopathy, serious immunodeficiency diseases, acute radiation injury are comparative maturity technically, has cured the hundreds of thousands patient.Yet it is not clear with differentiation and development whether hematopoietic stem cell transplantation can survive to the central nervous system, therefore.Prepare a kind of hemopoietic tissue stem cell medicine that can be widely used in the clinical treatment central nervous system disease and also set up the clinical meaning that relevant implantation technique then has particular importance.
The invention main points:
The object of the present invention is to provide a kind of stem cell product formulation and relevant implantation technique that can treat the central nervous system disease damage.The stem cell of this stem cell medicine is from the CD34+ stem cell and the interstital stem cell of hemopoietic tissue.Implantation technique relates to the CD34+ cell and interstital stem cell directly passes through the spinal cavity infusion.Because brain and spinal center nervous system are the tissue sites that the allosome immunologic rejection was exempted, do not produced to immunologic function in the human body, so the present invention has the purposes of particular importance:
1. according to technology of preparing provided by the invention, can be by present general stem cell separating and purifying method, from hemopoietic tissue separation of C D34 positive cell and interstital stem cells such as I or other people Placenta Hominis and Cord blood, bone marrow, peripheral blood, tire livers, the stem cell medicine that preparation is suitable;
2. can treat brain and spinal center nervous system injury by directly transplanting, carry out sexually transmitted disease (STD) change and cerebral embolism delayed ischemic neurological deficits according to the stem cell medicine of technology of preparing preparation of the present invention;
3. the stem cell according to technology of preparing preparation of the present invention can be used as gene therapy vector, the transfection related gene, be grafted directly to brain and spinal center nervous system then, the neural tumor of multiple brain and spinal center or other genetic flaw disease are treated in the gene therapy of being correlated with.
The example explanation:
Below, in conjunction with the embodiments, the present invention is described further, but the present invention is not limited to following embodiment.
The technology of preparing example of embodiment 1, navel blood stem cell preparation.Studying known CD34 molecule expresses in hematopoietic stem and vascular endothelial cell usually.At hemopoietic tissue, in Placenta Hominis and Cord blood, bone marrow, peripheral blood, vascular endothelial cell quantity is considerably less, therefore, is acknowledged as hematopoietic stem from the CD34 positive (+) cell of hemopoietic tissue.This example is got eutocous Cord blood, adopts magnetic bead affinity column (MACS) partition method separation of C D34+ cell, and flow cytometer (FACS) detects the CD34+ cell purity and reaches (Fig. 1) more than 97%.Isolating CD34+ cell counts, puts in the cell culture fluid standby at preceding 72 hours Brdu labellings (20 μ mol/L) of transplanting then.
The rat delayed ischemic neurological deficits example due to the artificial damage of human cord blood stem-cell therapy spinal cord is transplanted in embodiment 2, experiment.It is stand-by at first to isolate umbilical blood CD34+ cell by the method for example 1 description, prepares animal model by method described below then.Get 25 healthy Wistar rats, body weight 200-300g, male and female are not limit, random packet.Adopt abdominal cavity pentobarbital anesthesia, make spinal cord hemisection model by the Bregman legal system under the operating microscope.Then carry out umbilical blood CD34+ stem cell transplantation.Experimental design is as follows:
The PBS group: both sides are slowly injected 37 ℃ of PBS 3 μ l respectively end to end at the spinal cord injury position, are matched group; Umbilical blood CD34+ stem cell transplantation group: with umbilical blood CD34+ stem cell transplantation two zones of side end to end to spinal cord injury, each zone injection 3 μ l, every μ l contains cell number more than 25000, is experimental group.Transplant back 24h, 1 week, 2 weeks, 3 weeks, 4 weeks observed rat lower limb exercise situation, contrast improvement Tarlov standards of grading are carried out the motor function evaluation.
Can survive, grow and be converted into neurocyte for determining the CD34+ cell of being transplanted to spinal cavity, the injured nerve tissue is repaired in migration, experimental design is in 1 week of postoperative, 2 weeks, 3 weeks, 4 weeks, select 2 animal applications 4% paraformaldehydes carefully to pour into execution at random for every group, take out the damaged part spinal cord, fixing, paraffin embedding, continuous 6 μ m section.Adopt the SABC method to detect Brdu labelling umbilical hemopoietic stem cell, and use immunoenzyme mark and the neuron specific protein (NeuN) of immunofluorescence dual staining mensuration stem cell and the expression of glial fibrillary acidic protein (GFAP), judge the ability of its neurad cell differentiation.Table 1 and Fig. 2 are experimental results.The postoperative Tarlov scoring of enumerating from table 1 is a kind of method of general evaluation neurologic defict paralysis animal limb motor function, be divided into 0 to 5 integration, wherein 5 grades is that function is normal, 4 grades is that function is normal substantially, 3 grades for there being slight dysfunction, 2 grades for having than severe dysfunction, and 1 grade for there being severe dysfunction, and 0 grade then completely loses for function.Experimental result finds that its paralyzed limbs of rat of transplanting navel blood stem cell begins to improve after two weeks, 3 Zhou Houjun reach more than 3 grades, almost completely recover normal function after 5 weeks.In contrast to this, do not transplant its paralyzed limbs motor function of most rats of navel blood stem cell and can not recover all the time, have only a rat to return to 4 grades.Statistical analysis adopts Wiloxon two samples of ranked data to compare (rank test), the result shows that transplanting two groups of function of nervous system's scorings in back 24 hours compares P＞0.10, there was no significant difference, P＜0.05 is compared in postoperative one thoughtful two groups of function of nervous system's scorings all around, and significant difference is arranged on the statistics.Proof is transplanted the recovery that umbilical blood CD34+ cell can impel its extremity motor function of rat of paralysing due to the spinal cord injury.
Immunohistochemical staining is found the BrdU labelling human navel blood stem cell (Fig. 3) of survival in spinal cord, illustrate and use the technology of the present invention, can make the human umbilical blood stem cell of being transplanted in the spinal cord survive, organically combine, and further possess the possibility that is divided into nervous tissue's cell with spinal nervous tissue.
The technology of preparing example of embodiment 3, bone marrow interstital stem cell preparation.Get normal induced labor 20 age in week fetus, flushing, collect bones of limbs bone marrow, separate mononuclearcell, be inoculated in the 10%IMDM plastic culture bottle, attached cell passes the three generations.Transplant preceding 72 hours Brdu by 20 μ mol/L concentration labellings, detecting Brdu labelling positive rate is 85%, counts, puts in the cell culture fluid standby then.
Rat delayed ischemic neurological deficits example due to example 4, the artificial damage of experiment bone marrow interstital stem cell treatment spinal cord.It is stand-by to isolate umbilical blood CD34+ cell by the method for embodiment 1 description, prepares animal model then.Get 25 healthy Wistar rats, body weight 200-300g, male and female are not limit, random packet.Adopt abdominal cavity pentobarbital anesthesia, make spinal cord hemisection model by the Bregman legal system under the operating microscope.Experimental design is as follows:
The PBS group: both sides are slowly injected 37 ℃ of PBS of 3 μ l respectively end to end at the spinal cord injury position, are matched group; The bone marrow interstital stem cell transplantation group: bone marrow interstital stem cell is transplanted to two zones of side end to end of spinal cord injury, each zone injection 3 μ l, every μ l contains 25000 of cell number, is experimental group.Transplant back 24h, 1 week, 2 weeks, 3 weeks, 4 weeks observed rat lower limb exercise situation, carry out the motor function evaluation according to improvement Tarlov standards of grading.
Transplant human world matter stem cell at the survival of rat spinal cord intracavity, differentiation and vegetative state for understanding, the present invention is in 1 week of post-transplantation, 2 weeks, 3 weeks, 4 weeks, select 2 animal applications 4% paraformaldehydes carefully to pour into execution at random for every group, take out the damaged part spinal cord, fixing, paraffin embedding, continuous 6 μ m section.Adopt the SABC method to detect survival, growth, the migration and variation of Brdu labeled stem cells; Use immunoenzyme mark and immunofluorescence dual staining and measure the neuron specific protein (NeuN) of stem cell and the expression of glial fibrillary acidic protein (GFAP), judge the ability of its neurad cell differentiation.
Table 2 and Fig. 4 are experimental results.The postoperative Tarlov scoring of enumerating from table 1 is a kind of method of general evaluation neurologic defict paralysis animal limb motor function, be divided into 0 to 5 integration, wherein 5 grades is that function is normal, 4 grades is that function is normal substantially, 3 grades for there being slight dysfunction, 2 grades for having than severe dysfunction, and 1 grade for there being severe dysfunction, and 0 grade then completely loses for function.Experimental result finds that its paralyzed limbs of the rat of bone marrow interstital stem cell begins to improve after two weeks, 3 Zhou Houjun reach more than 3 grades, almost completely recover normal function after 5 weeks.In contrast to this, its paralyzed limbs motor function of most rats of bone marrow interstital stem cell can not recovered all the time, has only a rat to return to 4 grades.Statistical analysis adopts Wiloxon two samples of ranked data to compare (rank test), the result shows that transplanting two groups of function of nervous system's scorings in back 24 hours compares P＞0.10, there was no significant difference is transplanted back one thoughtful two groups of function of nervous system's scorings all around and is compared P＜0.05, and significant difference is arranged on the statistics.
Immunohistochemical staining is found the BrdU labelling human bone marrow interstital stem cell (Fig. 5) of survival in spinal cord, illustrate and use the technology of the present invention, can make the human marrow-interstitial stem cell of being transplanted in the spinal cord survive, organically combine, and further possess the possibility that is divided into nervous tissue's cell with spinal nervous tissue.This example proof is transplanted the recovery that human marrow-interstitial stem cell can impel its extremity motor function of rat of paralysing due to the spinal cord injury.The result of above-mentioned example shows that transplanting human cord blood stem cell or bone marrow interstital stem cell can change significantly to the artificial rat spinal cord that damages
For with the defective of function, but its mechanism of action it be unclear that.The major function effect that central nervous system cell is transplanted has following several respects: 1) Nutrition, the cell that is implanted into the host can produce or secrete trophic factors and directly or indirectly support survival and function thereof impaired or dead host neuron soon, or the stimulation of host neuron produces blastogenesis; 2) biological function effect, the cell that is implanted into the host can be divided into neuron, neurogliocyte, and differentiated neuron can form functional synapse with host neuron; 3) transplanted cells and central nervous system's integration, transplanted cells and axoneure form import into widely and spread out of get in touch, nervous pathway rebuilds.
In this research, can in spinal cord, find the BrdU labelling human navel blood stem cell and the bone marrow interstital stem cell (Fig. 3 and Fig. 5) of survival by the SABC method.These stem cell transplantations can provide the cell source of a trophic factors to damage location, produce and secretion central nervous system's somatomedin, play an important effect in the propagation of nervous tissue and atomization.Experiment finds that also the stem cell of transplanting is incorporated in the myeloid tissue well.Though this part cell quantity seldom, may be not enough to repair whole nerve injury, because they provide the cell source of a trophic factors, by producing and secrete cytokines can the neural reparation of stimulation of endogenous, the recovery of promotion function of nervous system.
2. in a word, the present invention adopts transplanting human cord blood CD 34+stem cell and bone marrow interstital stem cell effective to the delayed ischemic neurological deficits that spinal cord injury position treatment spinal cord injury causes, for brain and spinal center nervous system injury and relevant disease neurological functional recovery provide new treatment technology and products thereof.The stem cell of hemopoietic tissue such as umbilical blood and bone marrow can obtain from patient self, also can provide by other people, brain and spinal center nervous system are again to have the tissue that immunity is exempted in the human body, therefore navel blood stem cell or other hemopoietic tissue stem cell transplantation are the available strategies of treatment brain and spinal center nervous system injury and relevant disease, have broad clinical application prospect and cell therapy formulation development and are worth.
Description of drawings:
Fig. 1: reach 97.88% through FACS from the isolating CD34+ cell purity of cord blood cells.
Fig. 2: umbilical blood CD34+ cell transplantation is to the effect of neurologic defict rat neurological functional recovery.
Fig. 3: transplant Brdu labelling positive human navel blood stem cell (arrow shows the dark brown cell) in the section of back myeloid tissue, amplification 100 *
Fig. 4: people's bone interstital stem cell is transplanted the effect to neurologic defict rat neurological functional recovery.
Fig. 5: Brdu labelling positive human bone interstital stem cell (arrow shows the dark brown cell) amplification 200 in myeloid tissue's section after transplanting *
Rat extremity motor function Tarlov scoring after table 1, the positive transplanting of bleeding of the umbilicus CD34
| The experimental group numbering | Postoperative 24h | One week of postoperative | Two weeks of postoperative | Three weeks of postoperative | Around the postoperative |
| A3-2 | 2 | 4 | 5 | 5 | 5 |
| A4-2 | 0 | 1 | 4 | 4 | 5 |
| A5-2 | 2 | 4 | 4 | 4 (execution) | |
| B1-2 | 0 | 0 | 4 | 4 | 4 |
| B3-2 | 0 | 0 | 4 | 5 | 5 |
| B5-2 | 0 | 2 | 4 | 4 | 4 |
| C2-2 | 0 | 2 | 4 | 4 | 4 |
| C3-2 | 0 | 2 | 4 | 4 | 4 |
| C4-2 | 1 | 2 | 3 | 3 (execution) | |
| C5-2 | 2 | 2 | 4 | 4 | 4 |
| D1-2 | 0 | 0 | 5 | 5 | 5 |
| D2-2 | 0 | 1 | 4 | 4 | 4 |
| D4-2 | 1 | 2 | 4 | 4 | 4 |
| Mean value | 0.615 | 1.692* | 4.077* | 4.154* | 4.364* |
| The control group numbering | Postoperative 24h | One week of postoperative | Two weeks of postoperative | Three weeks of postoperative | Around the postoperative |
| D3-1 | 0 | 2 | 2 | Dead | |
| D5-1 | 0 | 1 | 1 | 1 | 1 |
| D3-2 | 1 | 0 | 4 | 4 | 4 |
| D5-2 | 0 | 1 | 2 | 3 | 3 |
| E1-2 | 0 | 0 | 0 | 0 | 0 |
| E2-2 | 0 | 0 | 0 | 0 | 0 |
| E3-2 | 0 | 0 | Dead | ||
| E4-2 | 0 | 0 | 0 | Dead | |
| Mean value | 0.125 | 0.555 | 1.286 | 1.600 | 1.600 |
Indicate putting to death in the table is artificially this rat to be put to death, and gathers its spinal cord injury position and prepares histotomy and dye do immunity Look. Indicating death in the table refers to because of spinal cord injury and fails the natural death that recovers to cause.
* symbol refers to compare P＜0.05 with control group.
Rat extremity motor function Tarlov scoring behind table 2. transplantation of mesenchymal stem cells
| The experimental group numbering | Postoperative 24h | One week of postoperative | Two weeks of postoperative | Three weeks of postoperative | Around the postoperative |
| A1-1 | 0 | 1 | 4 | 5 | 5 |
| B1-1 | 0 | 1 | 3 | 4 | 5 |
| B2-1 | 0 | 1 | 3 | 5 (execution) | |
| B3-1 | 0 | 1 | 2 | 3 | 4 |
| B4-1 | 0 | 2 | 4 | 4 | 4 |
| B5-1 | 0 | 0 | 2 | 2 | 3 |
| C2-1 | 0 | 0 | 2 (execution) | ||
| C3-1 | 0 | 2 | 4 | 4 | 5 |
| C5-1 | 0 | 1 | 4 | 4 | 5 |
| D1-1 | 0 | 2 | 3 (execution) | ||
| D2-1 | 0 | 2 | 2 | 3 (execution) | |
| D4-1 | 3 | 2 | 4 | 4 | 4 |
| E5-1 | 0 | 3 | 4 | 5 | 5 |
| The control group numbering | Postoperative 24h | One week of postoperative | Two weeks of postoperative | Three weeks of postoperative | Around the postoperative |
| D3-1 | 0 | 2 | 2 | Dead | |
| D5-1 | 0 | 1 | 1 | 1 | 1 |
| D3-2 | 1 | 0 | 4 | 4 | 4 |
| D5-2 | 0 | 1 | 2 | 3 | 3 |
| E1-2 | 0 | 0 | 0 | 0 | 0 |
| E2-2 | 0 | 0 | 0 | 0 | 0 |
| E3-2 | 0 | 0 | Dead | ||
| E4-2 | 0 | 0 | 0 | Dead | |
| Mean value | 0.125 | 0.500 | 1.286 | 1.600 | 1.600 |
Indicate putting to death in the table is artificially this rat to be put to death, and gathers its spinal cord injury position and prepares histotomy and exempt from Epidemic disease dyeing. Indicating death in the table refers to because of spinal cord injury and fails the natural death that recovers to cause.
* symbol refers to compare P＜0.05 with control group.
1. the preparation of bone marrow interstital stem cell:
Get normal induced labor 20 age in week fetus, flushing, collect four limbs bone marrow, separate mononuclearcell, inoculation In 10%IMDM plastic culture bottle, attached cell passes the three generations. Transplant front 72 hours Brdu by 20 μ mol/L The concentration mark, detecting Brdu mark positive rate is 85%, numeration, for subsequent use.
2. animal model preparation:
Get 25 healthy Wistar rats, body weight 200-300g, male and female are not limit, random packet. Adopt the abdominal cavity Amobarbital anesthesia is made spinal cord hemisection model by the Bregman legal system under the surgical operation microscope.
3. transplantation of mesenchymal stem cells:
(1) PBS group: 370 ℃ of PBS 3 μ l are slowly injected respectively in both sides end to end at the spinal cord injury position, are control group;
(2) transplantation of mesenchymal stem cells group: with transplantation of mesenchymal stem cells to two of the end to end sides of spinal cord injury The zone, each zone injection 3 μ l, every μ l contains 25000 of cell number, is experimental group. 4. postoperative 24h, 1 Week, 2 weeks, 3 weeks, 4 weeks are observed rat lower limb exercise situation, move according to improvement Tarlov standards of grading Functional evaluation.
5. in 1 week of postoperative, 2 weeks, 3 weeks, 4 weeks, select 2 animal applications 4% paraformaldehydes carefully to pour into execution at random for every group, take out the damaged part spinal cord, fixing, paraffin embedding, continuous 6 μ m section.
6. adopt the SABC method to detect survival, growth, the migration and variation of Brdu labelling bone marrow interstital stem cell; Use immunoenzyme mark and immunofluorescence dual staining and measure the neuron specific protein (NeuN) of bone marrow interstital stem cell and the expression of glial fibrillary acidic protein (GFAP), judge the ability of its neurad cell differentiation.
Statistical analysis adopts Wiloxon two samples of ranked data to compare (rank test).
Postoperative two groups of function of nervous system's scorings in 24 hours are P＞0.10 relatively, there was no significant difference.P＜0.05 is compared in postoperative one thoughtful two groups of function of nervous system's scorings all around, has statistical significance.
Claims (1)
1, comes from the CD34 of human cord blood
+The application of stem cell medicine in preparation reparation neurologic defict medicine.
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| CA2564679C (en) | 2004-03-22 | 2015-06-23 | Osiris Therapeutics, Inc. | Mesenchymal stem cells and uses therefor |
| WO2008055224A2 (en) * | 2006-11-01 | 2008-05-08 | Rutgers, The State University Of New Jersey | Lithium stimulation of cord blood stem cell proliferation and growth factor production |
| EP2624846A2 (en) | 2010-10-08 | 2013-08-14 | Osiris Therapeutics, Inc. | Nanoparticle-loaded cells |
| KR20200088445A (en) | 2017-11-22 | 2020-07-22 | 메조블라스트 인터내셔널 에스에이알엘 | Cell composition and treatment method I |
| CN110314172A (en) * | 2019-07-17 | 2019-10-11 | 中国医科大学附属第一医院 | A kind of cell preparation and preparation method thereof for treating spinal cord injury |
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