CN101306194A - Medicine for treating nerves damage and preparation method thereof - Google Patents
Medicine for treating nerves damage and preparation method thereof Download PDFInfo
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- CN101306194A CN101306194A CNA2008100585698A CN200810058569A CN101306194A CN 101306194 A CN101306194 A CN 101306194A CN A2008100585698 A CNA2008100585698 A CN A2008100585698A CN 200810058569 A CN200810058569 A CN 200810058569A CN 101306194 A CN101306194 A CN 101306194A
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Abstract
The invention provides a medicine used for curing nerve damage and a method used for preparing the medicine. The invention belongs to the technical field of a biologic medicine. The medicine is prepared with snake venom nerve growth factors NGF, neurotrophy factors NTF, nerve stem cells and Schwann cells according to the following method: the entire spinal cord of a newborn animal or a fetus which can not be conceived because of natural and man-made calamities is implanted in aseptic PBS liquid or culture fluid; required ganglia are cleaned and shorn again and again. After the required ganglia are cultured in a thermostat of 37 DEG C for 48 hours, little culture fluid is added; the required ganglia are cultured for eight days before the required ganglia are frozen and deposited for passage. Before operation, the frozen and deposited cells are revived, and are implanted in the pia mater spinalis of the damaged spinal cord. The medicine can provide the cultured cells and the damaged neurocyte with new nutriments and NGF required for promoting the growth. The implanted cells can be separated and added to the damaged neural parts to repair the damaged, dead or feeble cells with safe and remarkable cure effect.
Description
Technical field
The present invention relates to a kind of nerve injury medicine and preparation method thereof, belong to the biological medicine technology field.
Background technology
At present both at home and abroad aspect the treatment nerve injury, the report that adopts single nerve growth factor NGF or neurotrophic factor NTF class medicine is all arranged, have and adopt oral (Bears Yu Liang etc.: the preparation method of the medicine of treatment diseases in peripheral nerve system and male genetic defect, number of patent application 00116192.X, July 25 calendar year 2001 is open), also have and adopt intramuscular injection, though have certain effect, weak curative effect.Its reason is the pairing medicines of these conventional administering modes, and nerve growth factor NGF decline speed in vivo is very fast, and at the dosage at nerve injury position seldom, so curative effect is also just limited, particularly the curative effect of oral drugs is just poorer.In order to make damage location that higher NGF be arranged, have both at home and abroad and adopt single implantation NGF research and test, because damage location lacks living environment, lack and promote the nerve growth differentiation and the factors such as NGF, NTF that promote the nerve fiber growth, thereby cause implanting the too early apoptosis of cell, be difficult to make the neurocyte self-recovery Growth and Differentiation of damaged part.Evidence is arranged in addition, and excessive injected NGF may cause the nerve fiber growth overstocked, and arrangement disorder influences formation such as Lang Shi knot, can cause nerve block on the contrary, produces paralysis, so adopt safer intramuscular injection mostly.
Summary of the invention
Technical problem to be solved by this invention provides a kind of nerve injury medicine and preparation method thereof, described implanted medicine is beneficial to and makes cultured cell and damage location neurocyte both obtain new nutrition and the required NGF of growth promotion, implant cell can separate as early as possible again, rise in value repair to the injured nerve position formerly be damaged, downright bad or decline cell, therapeutic effect is safer, more remarkable.
Solving the technical scheme that technical problem of the present invention adopts is:
Medicine of the present invention is for being formed by the treatment neural stem cell of animal and nerve growth factor NGF or neurotrophic factor NTF co-cultivation, wherein living cells content is not less than 85%, NGF or NTF are no less than 2500 units～5000 units, and cell content is no less than 400 * 10
4Individual/ml～600 * 10
4Individual/ml.Described nerve growth factor NGF or neurotrophic factor NTF are the materials of separation and Extraction from cobra-venom, also can adopt the nerve growth factor or the neurotrophic factor that obtain from people, Mus or other animal species.And the neural stem cell in the effective ingredient of described medicine can comprise from Cord blood and nervi olfactory cell extraction.
Preparation technology's method of the present invention is: the neuroganglion of the neural stem cell that will obtain from people embryo or tissue is placed on the culture fluid and cleans, constant temperature is cultivated after 48 hours for 37 ℃, add nerve growth factor NGF or neurotrophic factor NTF and culture fluid co-cultivation after 8 days, make cell content be no less than 400 * 10
4Individual/ml～600 * 10
4Individual/ml, living cells content is not less than 85%, and NGF or NTF are no less than 2500 units～5000 units, is prepared into the special-purpose medicaments of implant damage position or spinal cord spinal pia mater.
Preparation method also comprises: nerve growth factor NGF or neurotrophic factor NTF are separation and Extraction from cobra-venom; At first neuroganglion is cleaned used culture fluid and adopt 1640 or 199, to NGF or the used mixed-culture medium of NTF co-cultivation be then, account for 1640 or 199 basic culture solutions of weight 85% and 15% newborn calf serum, and also having added penicillin 100 units/ml, streptomycin 100 μ g/ml in addition, culture fluid remains on pH7.0; Neural stem cell adopts and can not continue the spinal cord of conceived fetus, and fresh spinal cord takes off in 30 minutes, after cleaning repeatedly, shreds into the neuroganglion of 1mm.
This project is separation and purification of nerve growth factor NGF from homemade cobra-venom, and the structure determination result shows with people β chain, mouse submandibular gland NGF homology and reach more than 83% that but stable, content is 2/10000, is higher than other animals and tissue content.Experiment at first utilizes gossypol to cause male rat genetic defect model, prove that it can significantly increase the sperm count that causes after the damage and descend, improve motility of sperm, hormone FSH, LH significantly reduce, mate the back give birth to plant number, pregnancy rate significantly increases and near the normal control group, compare P＜0.1 with normal saline treatment group, this shows that this medicine has therapeutical effect preferably to male genetic defect.Pathologic finding proof Interstitial cell, sustenticular cell obviously recover, and seminal vesicle is full, proves that the reproductive system that is damaged has obvious recovery.
Experiment also proves has remarkable facilitation, activity to be higher than people β chain nerve growth factor to chick embryonic dorsal root ganglion, the growth of PC12 cellular neural.At home and abroad proving first has remarkable promotion division, cell proliferation and function in delaying senility to Tamarin and the growth of people's schwann's cell.The normal cultivation after 8 days reduced, and adds this factor and cultivates well-grown after 16 days.Research proves that also rat, cat injury of sciatic nerve are caused the paralysis animal obvious restitution, bring out current potential proof sensation and nervus motorius current potential and obviously recover fast P＜0.01 compared with the control, pathological section proof nerve fiber rises appreciably, and receptor increases, and is dose-effect relationship.We at first prove at the excessive application nerve growth factor of damage location will cause the nerve fiber excess growth, and conduction Lang Shi knot is disorderly, and the otherwise impact curative effect for medicinal application provides important evidence, may be to solve the key of such clinical drug weak curative effect both at home and abroad.We prove that also the GAP-43 positive particle obviously increases this medicine in the part Dorsal root cat cornu dorsale medullae spinalis to going, and are dose-effect relationship.Prove the spinal cord half cut-off simultaneously, cut off the rat palsy model entirely remarkable dose-effect effect is arranged, quadriplegia is treated through March and can be creeped substantially, swimming has significant curative effect compared with the control, finish the research of 3 monkey palsy model simultaneously, cooperate multiple that health, schwann's cell transplantation treatment can creep or walk, jump substantially after March, the growth of pathological section proof damage location nerve fiber, hypertrophy, injured nerve cells has remarkable recovery.At home and abroad at first nerve growth factor NGF, NTF, neural stem cell etc. are united and be used for the treatment of 50 many cases paralytics, all significantly reactivate function and standing of patient, part patient can walk.
Beneficial effect of the present invention also comprises: with NGF and neural stem cell co-cultivation, do and help making cultured cell and damage location neurocyte both to obtain new nutrition and the required NGF of growth promotion, implant simultaneously cell can separate as early as possible, rise in value repair to the injured nerve position formerly be damaged, downright bad or decline cell.Breeding newborn neural stem cell in a large number for damage location neurocyte hypertrophy provides, making damaged part obtain repairing.This method is except that culture period adds NGF, also add a large amount of NGF during transplanting, can make the neural stem cell of rising in value in the culture fluid that sufficient nutritional labeling is arranged like this, also remedy the NGF secretory volume minimizing of impaired back simultaneously and caused damaged cell to stop to grow phenomenons such as neuroganglion, necrosis, decline.Therefore, the two mixes to use and is better than prior art and implants NGF or neural stem cell separately with NGF and neural stem cell.Existing both at home and abroad research only limits to animal experiment, and this project has been finished rat, monkey, animal experiment, and carries out 50 many cases people clinical researches simultaneously, and total effective rate is more than 80%.Part patient can stand or walk by instrument.
Description of drawings
Fig. 1 cultivates chick embryonic dorsal root ganglion figure after 30 hours for the present invention;
Fig. 2 cultivates PC12 cell after 10 days for the present invention;
Fig. 3 is people's schwann's cell of the present invention and nerve growth factor co-cultivation experimental result;
Fig. 4 is a Tamarin schwann's cell and NGF co-cultivation experimental result after 8 days;
Fig. 5 is the experimental result of schwann's cell and NGF treatment paralysis monkey;
Fig. 6 is the experimental result of schwann's cell and NGF treatment paralysis monkey;
Fig. 7 is the therapeutic outcome of schwann's cell and NGF treatment paralysed patient;
Fig. 8 is the therapeutic outcome of schwann's cell and NGF treatment paralysed patient;
Fig. 9 is the therapeutic outcome of schwann's cell and NGF treatment paralysed patient.
The specific embodiment
Embodiment 1: will clean repeatedly with 1640 or 199 culture fluid from the fresh spinal cord that the people embryo took off in 30 minutes, shred into the neuroganglion about 1mm, in 37 ℃ calorstat, cultivated 48 hours, add cobra-venom nerve growth factor NGF and culture fluid co-cultivation after 8 days, this culture fluid is 1640 or 199 and 15% the newborn calf serum mixed-culture medium that accounts for weight 85%, and added penicillin 100 units/ml, streptomycin 100 μ g/ml in addition, culture fluid remains on pH7.0.Finally make co-cultivation become the cell content in the composition of medicine to be no less than 400 * 10
4Individual/ml～600 * 10
4Individual/ml, living cells content is not less than 85%, and NGF is no less than 2500 units～5000 units.Frozen, go down to posterity.Recovery freeze-stored cell before the operation is implanted in the damage location spinal cord spinal pia mater.
Embodiment 2: take off fresh spinal cord from the Tamarin body, in 30 minutes, clean repeatedly with 1640 or 199 culture fluid, shred into the neuroganglion about 1mm, in 37 ℃ calorstat, cultivated 48 hours, add mouse submandibular gland nerve growth factor NGF and culture fluid co-cultivation after 8 days, this culture fluid is 1640 or 199 and 15% the newborn calf serum mixed-culture medium that accounts for weight 85%, and has added penicillin 100 units/ml, streptomycin 100 μ g/ml in addition, and culture fluid remains on pH7.0.Finally make co-cultivation become the cell content in the composition of medicine to be no less than 400 * 10
4Individual/ml～600 * 10
4Individual/ml, living cells content is not less than 85%, and NGF is no less than 2500 units～5000 units.Frozen, go down to posterity.Recovery freeze-stored cell before the operation is implanted in the Tamarin damage location spinal cord spinal pia mater, and the measurement result of the angular movement N cell speed of growth before the Tamarin is seen for details table 1.
Embodiment 3: take off fresh spinal cord from the mice body, in 30 minutes, clean repeatedly with 1640 or 199 culture fluid, shred into the neuroganglion about 1mm, in 37 ℃ calorstat, cultivated 48 hours, add cobra-venom nerve growth factor NGF and culture fluid co-cultivation after 8 days, this culture fluid is 1640 or 199 and 15% the newborn calf serum mixed-culture medium that accounts for weight 85%, and has added penicillin 100 units/ml, streptomycin 100 μ g/ml in addition, and culture fluid remains on pH7.0.Finally make co-cultivation become the cell content in the composition of medicine to be no less than 400 * 10
4Individual/ml～600 * 10
4Individual/ml, living cells content is not less than 85%, and NGF is no less than 2500 units～5000 units.Frozen, go down to posterity.Recovery freeze-stored cell before the operation is implanted around the sciatic nerve that mice hindered by clamp, the pain sensation of mice is recovered measurement result see table 2 for details.
Below be the experiment case
Experiment in vitro:
Case 1: adopt Rhinopithecus bieti schwann's cell (SC) and snake venom NGF co-cultivation, and under condition of equivalent, add equivalent mannitol and be contrast, the result proves that the cell culture propagation phase, the peak period was at the 8th day at the 4th day, add the nerve growth factor experimental group and be significantly higher than matched group in culture period division growth, matched group begins apoptosis after cultivating 8 days, the experimental group apoptosis significantly delays, and sees Table 1.
Table 1: the preceding angular movement N cell speed of growth is measured
Case 2: (10～100 units/ml) add the chick embryonic dorsal root ganglion co-cultivation can promote the nerve fiber phenomenal growth, and matched group does not add the NGF (see figure 1) and cultivates chick embryonic dorsal root ganglion after 30 hours with nerve growth factor or NTF
A: matched group, a small amount of short and thick nerve fiber grows (* 40) from neuroganglion;
B:NGF organizes (30ng/ml NGF), and a large amount of nerve fibers grow (* 40) from neuroganglion is radial.
Case 3:PC12 cell experiment adds NGF or NTF, and (5～30ng/ml) nerve fibers of significantly growing, matched group does not add NGF and then almost maintains the original state, and seldom grows the neuroganglion (see figure 2).
C: matched group, cell are spheroidal (* 400);
D:NGF organizes (20ng/ml NGF), and a large amount of PC12 cell differentiations become neuron cell (* 400).
Research in the body:
Case 4:NGF, NGF+ Culture of neural stem cells are carried out the pain sensation and are recovered experiment after hindering the mice sciatic nerve with clamp, and the result shows that NGF+ schwann's cell group is recovered better, and the pain sensation is recovered obviously to shift to an earlier date average time, is better than the NGF high dose group.See Table 2
Table 2:NGF recovers influence (n=2) to the mice pain sensation that the sciatic nerve clamp is hindered
Experimental group is compared P＜0.01 with matched group
Case 5: adopt cat clamp sciatic nerve injury to cause palsy model mensuration NGF paralysis treatment of animals exercising result is shown that two groups all there were significant differences.Check pathological section is the result show, the nerve fiber growth obviously recovers, and Lang Shi joint appears, SABC shows that the neurocyte receptor obviously increases, and mainly is distributed in around the nuclear, receptor necessarily increases, but there is not significance, whether distributed more widely relevant in animal, human body with NGF, wait further discussion.See Table 3～table 4.
Table 3:NGF observes cat injury of sciatic nerve behavioristics
Case 6: rat test: rat spinal cord cuts off the animal model measurement result entirely and shows, every group of 10 adult rats body weight, 200 ± 22 grams inject and cultivate back rat neural stem cell 400 * 10
4Individual/ml～600 * 10
4Individual/ml, NGF 2500～5000 units/ml.Transplant that the animal swimming test proves after one month, significantly be better than transplanting separately the neural stem cell group and inject 2500～5000 units/ml group and normal saline group (seeing Table 4) separately weekly.
Table 4: experimental group matched group swimming time statistics of different treatment time (n=6)
| Group | Swimming time (minute, second) before the treatment | Treat swimming time (minute, second) after 1 month | Treat swimming time (minute, second) after 3 months |
| Normal mice | 1′30″±21″ | 1′41″±33″ | 1′29″±30″ |
| Operation back normal saline group | 1′38″±33″ | 21″±7″ | 33″±12″ |
| The NGF group | 1′29″±24″ | 43″±12″ | 1′12″±22″ |
| NGF+ schwann's cell group | 1′36″±28″ | 53″±21″ | 1′33″±34″ |
| NTF+ schwann's cell group | 1′32″±22″ | 1′01″±8″ | 1′38″±1.8″ |
| The schwann's cell group | 1′33″±31″ | 37″±13″ | 1′05″±22″ |
The result shows: the treatment group is compared with matched group, there were significant differences P＜0.01, and wherein " NTF+ schwann's cell group " and " NGF+ schwann's cell group " recovers fine, and single also have certain curative effect with NGF and schwann's cell, but the recovery of paralysing is not share.
Check pathological section nerve fiber growth, Nissl body, nerve synapse, neural myelin all have obvious recovery, compare with matched group that there were significant differences, and obviously pathological changes such as necrosis, cavity, hemorrhage, demyelination appears in contrast, cut into slices and during SABC just carrying out.
We and domestic correlational study result prove that also NGF is hindered by the rat sciatic nerve clamp and alloxan causes diabetes nerve tip damage model sensory evoked potential that remarkable recovery is arranged, reach about 60%, and the nervus motorius damage have certain recovery, but not remarkable.
Case 7: monkey test: the half cut-off of three monkey spinal cords implanted 400 * 10 after 30 days
4Individual/ml～600 * 10
4Individual/the ml neural stem cell (with the NGF co-cultivation), after raising in 2 months, the monkey walking of standing.Wherein two are recovered normal substantially after treatment half a year.See Fig. 5,6.The matched group surgery recovery can not be stood.The animal pathological section proves that nerve injury significantly recovers
Case 8: human therapy result: ratify routine patient treatment test surplus cooperating to carry out 30 with the hospital through relevant department.The result proves, the patient paralyses and significantly improves, and wherein example can be by the mechanical assistant walking of standing surplus in the of 20.See accompanying drawing 7～9.
Claims (6)
1, a kind of nerve injury medicine, it is characterized in that: medicine is for being formed by the treatment neural stem cell of animal and nerve growth factor NGF or neurotrophic factor NTF co-cultivation, wherein living cells content is not less than 85%, NGF or NTF are no less than 2500 units～5000 units, and cell content is no less than 400 * 10
4Individual/ml～600 * 10
4Individual/ml.
2, by the described nerve injury medicine of claim 1, it is characterized in that: nerve growth factor NGF or neurotrophic factor NTF are the materials of separation and Extraction from cobra-venom.
3, by the described nerve injury medicine of claim 1, it is characterized in that: nerve growth factor NGF or neurotrophic factor NTF are the materials that obtains from people, Mus or other animal species.
4, by claim 2 or 3 described nerve injury medicines, it is characterized in that: neural stem cell comprises Cord blood and nervi olfactory cell extraction in the effective ingredient.
5, a kind of preparation method of nerve injury medicine, it is characterized in that: the neuroganglion of the neural stem cell that will obtain from people embryo or tissue is placed on the culture fluid and cleans, constant temperature is cultivated after 48 hours for 37 ℃, add nerve growth factor NGF or neurotrophic factor NTF and culture fluid co-cultivation after 8 days, make cell content be no less than 400 * 10
4Individual/ml～600 * 10
4Individual/ml, living cells content is not less than 85%, and NGF or NTF are no less than 2500 units～5000 units, is prepared into the special-purpose medicaments of implant damage position or spinal cord spinal pia mater.
6, the preparation method of nerve injury medicine according to claim 5 is characterized in that: nerve growth factor NGF or neurotrophic factor NTF are separation and Extraction from cobra-venom; At first neuroganglion is cleaned used culture fluid and adopt 1640 or 199, to NGF or the used mixed-culture medium of NTF co-cultivation be then, account for 1640 or 199 basic culture solutions of weight 85% and 15% newborn calf serum, and also having added penicillin 100 units/ml, streptomycin 100 μ g/ml in addition, culture fluid remains on pH7.0; Neural stem cell adopts and can not continue the spinal cord of conceived fetus, and fresh spinal cord takes off in 30 minutes, after cleaning repeatedly, shreds into the neuroganglion of 1mm.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107148278A (en) * | 2014-08-15 | 2017-09-08 | 财团法人卫生研究院 | Strengthen the method for nerve regneration using NSC and IL12p40 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107148278A (en) * | 2014-08-15 | 2017-09-08 | 财团法人卫生研究院 | Strengthen the method for nerve regneration using NSC and IL12p40 |
| CN107148278B (en) * | 2014-08-15 | 2021-06-29 | 财团法人卫生研究院 | Method for enhancing nerve regeneration by using neural stem cells and IL12p40 |
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Open date: 20081119 |