CN101012452A - Formula constituted by cytokine and compound, its action of promoting nerve regeneration and action of researching and diagnosing nervous system disease - Google Patents
Formula constituted by cytokine and compound, its action of promoting nerve regeneration and action of researching and diagnosing nervous system disease Download PDFInfo
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Abstract
The invention discloses a formula of cell factor and compound and action to regenerate nerve and diagnose the nerve system, which is fit for insuline, bovine serum albumin, putrescine, hydrocortisone, luteohormone, transferrin, sodium selenite, alkaline cellulose cell growing factor and epiderm growing factor.
Description
Technical field:
The invention belongs to the nervous system disease field, specifically utilize between the cytokine, synergy between cytokine and compound, structure can promote the neurone division and have the cytokine of powerful neurotization effect and the prescription of compound, and the application of prescription in nervous system disease research, diagnosis and treatment.
Background technology
Neurotization difficulty, key are that neurone can not divide.Whether neurone can divide is significant problem in the life science.For a long time, neurone is considered to cell (Raedler, E.etal (1978) .Anat.Embryol.154, the 267-312. of end differentiation eventually; McConnell, S.K. (1988) .BrainRes.Rev.1: 1-23.; McKay, R.D.G. (1989) .Cell58,815-821.; Pincus, D.W.et al. (1988) .Neurosurgery, 42 (4), 858-868).General neurone has just broken away from the cell cycle after the animal birth soon, lost the ability (Jessel of division growth, T.M. (1991) .Reactions of neurons in jury.In Kandel E.R., Schwartz J., and JessellT.M. (eds) Principles of neural science (3
RdEdition), Appleton and Lange, pp.259-269).Therefore neurone is in case damage or pathology will cause permanent afunction.
Neural multiple disease, as degeneration and Spinal injuries such as senile dementia, Parkinson diseases, cerebral anoxia damage etc., all belong to more serious nervous system disease, be exactly because neuronal death or degeneration, can't repair or substitute and cause serious and the illness that can not treat.In recent years, a lot of scientists find to exist neurotization phenomenon (Altman, J. (1962) .Science 135,1127-1129. in animal brain; Kaplan, M.S.et al, (1977) .Science 197,1092-1094.; Bayer, S.A.et al, (1982) .Science 216,890-892.; Evans, J.et al, (2002) .JNeurophysiol.87,1076-1085; Kaplan, MS.et al (1981) .J.Comp.Neurol.195,323-338.; Huang, L.et al, (1990) .Dev.Brain Res.51,123-127.).Further research is also found, these neurotizations that occur in the brain are owing to be present in (Weiss, S. etc., (1996) .Trends Neurosci.387-393. due to the neural stem cell of brain; Stemple, D.L. etc., (1992) .Cell 71,1-20; Gritti, A. etc., (1996) .J.Neurosci.16 (3), 1091-1100; Magevi, S.S. etc., (2000) .Nature 405,951-955.).The discovery of neural stem cell is recognized the people and is had the possibility for the treatment of the central nervous system illness.Also having many difficulties but neural stem cell is used clinically, at first is neural stem cell source difficulty, and the neural stem cell source of self is limited; Next is that the allogeneic nerve stem cell involves ethics problem; The 3rd is nerve stem cell directional differentiation difficulty.Because the specificity of brain structure, according to its neurohumor, neurone have hundreds of more than.Want the directional induction cell differentiation of nerve cord to become hundreds of neurocyte, its difficulty is well imagined; The 4th is that the neural stem cell of implanting is difficult to form correct synaptic contact with host cell.Neurone also will be accepted the information that countless other neurones come, and is delivered at a distance by aixs cylinder and plays a role, even therefore neural stem cell can correctly be implanted to privileged site, still is difficult to reach repair.Therefore also can't realize in a short time by implantable neural stem-cell therapy nervous system disease.
Inventor herein's early-stage Study finds that the prescription that cytokine and compound constitute can clearly promote former generation neurone division, and multiple technologies proof splitted neurone has the 26S Proteasome Structure and Function feature of mature neuron.The discovery of neurone division phenomenon also inspires to the people, promptly might be by inducing neural division, thus reach the purpose of repairing nerve damage and neural system degeneration illness.
In other research, the contriver also finds can clearly promote neurotization based on what above-mentioned prescription constituted.Its effect is far longer than the effect of the single component promotion neurotization in any cytokine, compound or the prescription.So far the neurone of not finding relevant maincenter can be by the report of induced fission, and utilizes the synergy of cytokine to constitute the report of prescription therapeutic nerve injury.
Summary of the invention
The object of the invention provides and promotes neurone to divide and the cytokine of neurotization and the prescription that compound constitutes.
Another object of the present invention provides and promotes neurone to divide and the cytokine of neurotization and the application of prescription on the medicine of preparation treatment nervous system disease that compound constitutes.
The 3rd purpose of the present invention is to promote the cytokine of neurone division and neurotization and the application of prescription on research of preparation nervous system disease and diagnostic reagent that compound constitutes.
The present invention is achieved through the following technical solutions:
Promote the cytokine of neurone division and neurotization and the prescription that compound constitutes, contain Regular Insulin, bovine serum albumin, putrescine, hydrocortisone, progesterone, Transferrins,iron complexes, trilute, Sodium Selenite, Prostatropin and Urogastron.
The prescription that above-mentioned cytokine and compound constitute, its moiety and weight percent thereof (%) are:
Regular Insulin 0.04～0.4
Transferrins,iron complexes 0.8～8
Bovine serum albumin 0.04～0.4
Putrescine 0.12～1.2
Hydrocortisone 3.5 * 10
-5～3.5 * 10
-4
Sodium selenite 4 * 10
-5～4 * 10
-4
Trilute 8 * 10
-3～8 * 10
-2
Urogastron 8 * 10
-5～8 * 10
-4
All the other are water
The prescription that above-mentioned cytokine and compound constitute also contains Brain Derived Neurotrophic Factor and nerve growth factor.
The prescription that above-mentioned cytokine and compound constitute, its moiety and weight percent thereof (%) are:
Regular Insulin 0.04～0.4
Transferrins,iron complexes 0.8～8
Bovine serum albumin 0.04～0.4
Putrescine 0.12～1.2
Hydrocortisone 3.5 * 10
-5～3.5 * 10
-4
Sodium Selenite 4 * 10
-5～4 * 10
-4
Trilute 8 * 10
-3～8 * 10
-2
Urogastron 8 * 10
-5～8 * 10
-4
Brain Derived Neurotrophic Factor 1 * 10
-4～1 * 10
-3
All the other are water.
Water described in the prescription that above-mentioned cytokine and compound constitute is generally distilled water.
The application of the prescription that above-mentioned cytokine and compound constitute in the medicine of preparation treatment nervous system disease and research, diagnostic reagent.
The application of the prescription that above-mentioned cytokine and compound constitute in the medicine of preparation treatment nerve injury and neural system regression and pathology and research, diagnostic reagent.
The prescription that above-mentioned cytokine and compound constitute promotes neurone splitted medicine and the application of studying in the reagent in preparation.
The prescription that above-mentioned cytokine and compound constitute is at preparation treatment Spinal injury, the medicine of peripheral nerve injury equivalent damage illness and the application of studying in diagnosis, the reagent.
The application of the prescription that above-mentioned cytokine and compound constitute in the medicine of the various brain injurys due to preparation treatment cerebral anoxia, the ischemic and research, diagnostic reagent.
The prescription that above-mentioned cytokine and compound constitute is at preparation treatment senile dementia, Parkinson disease, and lateral spinal sclerosis waits the application in the medicine of illness such as nervus centralis regression change and research, the diagnostic reagent.
The preparation method of the prescription that above-mentioned cytokine and compound constitute: at first take by weighing hydrocortisone and progesterone, and be dissolved in respectively in the dehydrated alcohol, standby; Take by weighing Prostatropin be dissolved in Tris damping fluid or the water in (pH7.6 contains 0.1%BSA), standby; Proportionally take by weighing Regular Insulin, bovine serum albumin, putrescine, Transferrins,iron complexes, trilute, Sodium Selenite and Urogastron, they are joined in the distilled water dissolving, and the ethanol solution of hydrocortisone and progesterone and the Tris solution or the aqueous solution of Prostatropin joined in the distilled water, mixed evenly, and add distilled water and be settled to the volume required medicinal mixture of the present invention that promptly gets.
The preparation method of the prescription that above-mentioned cytokine and compound constitute can also be: at first proportionally takes by weighing hydrocortisone and progesterone, and is dissolved in respectively in the dehydrated alcohol, and standby; Take by weighing Prostatropin and be dissolved in (pH7.6 contains 0.1%BSA) in the Tris damping fluid, standby; Proportionally take by weighing Regular Insulin, bovine serum albumin, putrescine, Transferrins,iron complexes, trilute, Sodium Selenite, Urogastron, Brain Derived Neurotrophic Factor and nerve growth factor, they are joined in the distilled water dissolve, and the ethanol solution of hydrocortisone and progesterone and the Tris solution or the aqueous solution of Prostatropin joined in the distilled water, mixed evenly, and add distilled water and be settled to the volume required medicinal mixture of the present invention that promptly gets.
Transferrins,iron complexes (Transferrin) is that cell fission propagation is necessary as a kind of somatomedin, plays a role by combining with the TfR of cell surface.The short cell fission effect of Transferrins,iron complexes depend on its transportation iron ion to cell (Jennifer L. etc., Experimental cell research, 17:687-695).Iron ion is the important composition composition of ribonucleotide reductase, and it is active closely related with DNA synthetic speed, in the active obviously enhancing of S phase.
Regular Insulin (Insulin) promotes the cell growth, obtains basic nutritive substance by helping cell, and its acceptor has activity (Crouch, 1991 of Tyrosylprotein kinase; Csaba, 1991)
Urogastron (Epidermal Growth Factor, be called for short EGF) and Prostatropin (basic Fibroblast Growth Factor, be called for short bFGF) all be the mitogen of using always, can promote the division growth of broad variety cell, play a role by combining respectively with its Receptor EGFR, bFGFR.Wherein bFGF has neurotrophic effect, can promote speed (J.Kungnickel, the K.Haase of extension, germinating growth and the raising neurotization of aixs cylinder after the nerve injury, J.Konitzer, M.Timmer, C.Grothe, J.Neurobiol 66:940-948,2006).EGF and FGF can promote regeneration (MC.Jimenez Hamann, CH.Tator, MS.Shoichet, Exp Neurol.194:106-19,2005) after the Spinal injury.
Neurotrophic factor is the special protein of a class, and it has special neurotrophic activity effect.Experiment confirm, neurotrophic factor can be blocked the expression of the upright early gene that caused by nerve injury (IEG ' s).The strong growth promoting function of neurotrophic factor is pointed out and it can be used for preventing in the nerve injury around or reduce neurone loss of function or death, promotes axon regeneration.All neurotrophic factors all are to combine by the tyrosine kinase receptor with target cell surface particular category to play a role, every kind of acceptor is in case activated by the ligand receptor binding substances, to produce protein phosphorylation effect, activated gene subsequently by signalling channel in the cell.
Nerve growth factor (Nerve Growth Factor, be called for short NGF) not only can promote the survival of adult animals cholinergic neuron, and can induce the injured nerve fiber of central nervous system in suitable matrix, to regenerate, also can promote perineural regeneration (WJ.Freed, Brain Res Bull.1:393-412 (1976); FH.Gage, G.Buzsaki, DM.Armstrong, Prog Brain Res 83:357-70 (1990); H.Horie, Y.Bando, H.Chi, T.Takenaka, Neurosci Lett 121:125-8 (1991); LF.Kromer, CJ.Cornbrooks, Ann N Y Acad Sci 495:207-24 (1987) .) NGF is one of most important bioactive molecules in the neural system, around the influence and some neuronic survival and differentiation in the nervus centralis.After the nerve injury, the effect of neuroprotective unit, promotion axon regeneration is arranged around.NGF is that to keep the survival of specific Sensory neurone and sympathetic neuron necessary.Behind the axonal injury, due to the changing unit of neurone genetic expression ground is reduced by the neuronic NGF of absorption.After the peripheral nerve injury, can promote Sensory nerve fibre regeneration, promote to set up functional the contact between sympathetic nerve rudiment and the Sensory neurone if give exogenous NGF.
Brain Derived Neurotrophic Factor (Brain-derived Neurotrophic Factor, be called for short BDNF) can promote the shoot regeneration that of central nervous system neurons, increase connection (LA.Mamounas etc., J Neurosci.20:771-82 (2000) between the injury rats spinal neuron; R.Vavrek etc., Brain129:1534-45 (2006) .).BDNF is a kind of important motor neuron and Sensory neurone nutritional factor, with the NGF sequence 50% homology is arranged.BDNF can promote the regeneration and the myelinization of rodent sciatic nerve injury.In situ hybridization analyze to show, BDNF expresses in the schwann cell of injured nerve far-end and Denervated myofiber.Use BDNF antibody and can obviously suppress rat sciatic nerve regeneration.Exogenous BDNF alleviates the function of the degeneration that peripheral nerve injury causes in addition.
Prostatropin (basic Fibroblast Growth Factor is called for short bFGF) has short widely neurotization effect as a kind of peptide growth factor.All there is bFGF to exist at normal neurosome, aixs cylinder, dendron near-end, comprises waist section spinal neuron, sciatic schwann cell, ranvier's constrictions.Discover that Exogenous bFGF can promote to damage back schwann cell hyperplasia, keeps the neurone normal function, reduces neuronal death, promote axon regeneration.After peripheral nerve was cross-section, endogenous bFGF also can promote neural vasculogenesis except enlarging axon diameter as neurotrophic factor, increasing the relevant major diameter aixs cylinder quantity.
Other component such as bovine serum albumin is mainly the necessary base substance of growth metabolism of cell, keeps the neuronic growth in the serum free medium.
The composition of the prescription that above-mentioned cytokine and compound constitute can be bought from the market.
The contriver induces the neurone that comprises that cell differentiation of nerve cord is come to the prescription and the composition thereof of cytokine of the present invention and compound formation, the mature neuron that the PC12 differentiation comes, former generation cortical neuron and cerebellum Purkinje neurone splitted ability etc. studies show that the prescription of cytokine of the present invention and compound has induces neurone splitted effect preferably.Show as: the sub-neurone that (1) its induced fission produces can keep original neuronic chemical property preferably; (2) the sub-neurone that produces of induced fission can keep the structural nexus with target cell; (3) target tissue of domination can easily be sought and reach to sub neuronic nerve fiber; These factors all are beneficial to the reconstruction and the central nervous system illness functional rehabilitation of newborn neuron greatly.
The most important condition of neurotization is to make the neurone of damage be in good existing state, and cytokine prescription of the present invention can promote the neurone division, and the splitted neuronal survival is in good condition.Secondly neurotization is that the neurite of damage is sprouted fast, and promotes its growth.The contriver studies confirm that the cytokine synergy is powerful in the cytokine prescription of the present invention, can obviously promote neurotization.Wherein the prescription in conjunction with cytokine of the present invention and compound has the cell survival of maintenance state and promotes the neurone splitting action, BDNF, NGF have the effect that clear and definite promotion nerve sprouts fast and promotes its growth, therefore the prescription of cytokine of the present invention and compound not only can promote neurotization, also has clear and definite treatment nerve injury effect.
Advantage of the present invention and beneficial effect: (1), the present invention can keep the neuron survival state, and can promote neuronic division, for the treatment nervous system disease provides possibility; (2), the present invention can also promote neurotization, and the sub-neurone of induced fission generation can keep the structural nexus of original neuronic chemical property and maintenance and target cell preferably, the target tissue of domination can easily be sought and reach to the neuronic nerve fiber of son, thereby can reach the purpose of treatment nerve injury; (3), the present invention utilizes the neurocyte of cerebral tissue original position, implantation that needn't foreign cell, thereby technical simple possible; (4), component of the present invention all is to utilize existing composition, production method is simple, cost is low.
Description of drawings
Fig. 1. be former generation neurone division phase phase microscope photo.
Phase microscope photo mitosis prophase that a representing;
B represents the phase microscope photo of mitosis metaphase;
C represents the phase microscope photo of mitosis anaphase;
D represents the mitotic division phase microscope photo in late period.
Fig. 2. former generation Purkinje neurone splitted phase microscope photo.
Fig. 3. the regression curve of vitro culture each day purkinje cell counting.
Fig. 4. the shared histogram of different incubation time three type purkinje cells.。
The sub-neurone that the division of Fig. 5 .Purkinje neurone forms continues the division photo.
Fig. 6. be in the Purkinje neurone laser co-focusing photo of metaphase.
Fig. 7. the Purkinje neurone laser co-focusing photo the anaphase of being in.
Fig. 8. former generation cortical neuron laser co-focusing photo.
A and b demonstration are in the early stage neurone of mitotic division;
C, d and e show the neurone that is in mitosis metaphase, and chromatin is by PI mark (redness).
Fig. 9. the cortical neuron photo the anaphase of being in.
A shows that neurone is saturated by the PI mark, and kytoplasm is by the NSE immunocytochemical stain.
It is saturated by the PCNA mark that b-d shows, kytoplasm is respectively by NF-160, SOM and GAD mark.
Figure 10. the mitosis prophase the neurone electromicroscopic photograph.
Figure 11. the neurone electromicroscopic photograph of mitosis metaphase.
Figure 12. the neurone electromicroscopic photograph of mitosis anaphase.
Figure 13. the neurone electromicroscopic photograph in mitotic division late period.
Figure 14. the new neurone that forms and other neurone form the electromicroscopic photograph of functional cohesion.
Figure 15. shown the sodium, the potassium-channel current potential that divide neurone and have typical mature neuron.
Figure 16. after the cytokine prescription was expelled to cortex, injection zone peripheral nerve unit was by BrdU mark situation photo.
Figure 17. the cytokine prescription can pass through two nerve node chalaza (A-D), enters the photo of L1 spinal cord.
Figure 18. the T7 intercostal nerve that showed cell factor prescription promotes T to originate from the 6-8 spinal cord enters PHA-L direct motion mark (A-C) photo of T12-L2 spinal cord.
Embodiment
Further the present invention is described in detail with specific embodiment below, but the present invention is not constituted any restriction.
Embodiment 1
The preparation of the prescription that cytokine of the present invention and compound constitute: take by weighing hydrocortisone 1mg, be dissolved in the 10ml dehydrated alcohol, be mixed with 0.01% mother liquor; Take by weighing progesterone 1mg, be dissolved in the 10ml dehydrated alcohol, be mixed with 0.01% mother liquor; Take by weighing Regular Insulin 100mg, be dissolved in the 10ml distilled water, be mixed with 1% mother liquor; Take by weighing bovine serum albumin 100mg, be dissolved in the 10ml distilled water, be mixed with 1% mother liquor; Take by weighing Transferrins,iron complexes 2g, be dissolved in the 10ml distilled water, be mixed with 20% mother liquor; Take by weighing putrescine 300mg, be dissolved in the 10ml distilled water, be mixed with 3% mother liquor; Take by weighing trilute 20mg, be dissolved in the 10ml distilled water, be mixed with 0.2% mother liquor; Take by weighing Sodium Selenite 1mg, be dissolved in the 10ml distilled water, be mixed with 0.01% mother liquor; 100 μ gEGF are dissolved in the 1ml distilled water, are mixed with 0.01% mother liquor; 10 μ g bFGF are dissolved in the 100 μ 110mM Tris damping fluids (pH7.6 contains 0.1%BSA), are mixed with 0.01% mother liquor; Draw 0.01% hydrocortisone mother liquor, 9.1 μ l, 0.01% progesterone mother liquor, 126 μ l, 1% Regular Insulin mother liquor 10ml, 1% bovine serum albumin mother liquor 10ml, 20% Transferrins,iron complexes mother liquor 10ml, 3% putrescine mother liquor 10ml, 0.2% trilute mother liquor 10ml, 0.01% Sodium Selenite mother liquor 1ml respectively, add distilled water and be settled to 100ml, evenly mixed.Be distributed into the 1ml/ pipe then, be stored in-70 ℃ of refrigerators.In experimentation, add EGF and bFGF again, it is 2 * 10 that its final concentration is respectively
-4%.Each concentration of component is Regular Insulin 0.1%, bovine serum albumin 0.1%, putrescine 0.32%, hydrocortisone 9.1 * 10 in the solution
-5%, progesterone 1.26 * 10
-4%, Transferrins,iron complexes 2%, trilute 2 * 10
-2%, Sodium Selenite 1.04 * 10
-4%, EGF2 * 10
-4% and bFGF2 * 10
-4%.
The preparation of the prescription that cytokine of the present invention and compound constitute: 10 μ g BDNF are dissolved in the 100 μ l distilled waters, are mixed with 0.01% mother liquor; 10 μ g NGF are dissolved in the 100 μ l distilled waters, are mixed with 0.01% mother liquor; On the basis of embodiment 1 made composition, add BDNF again, making its final concentration is 2.5 * 10
-4%; Add NGF then, making its final concentration is 5 * 10
-4%.Evenly mixed, the distilled water constant volume makes prescription of the present invention.
Embodiment 3 induces former generation cortex and spinal neuron division experiment
Rat spinal cord is separated with cortical neuron, add heavy dose of cytosine arabinoside then, but remove neural stem cell and other somatoblast.Use Nestin then, GFAP and NSE immunocytochemistry are checked, confirm no any Nestin immuning positive cell, former generation neuronal cell account for more than 95% of total cell proportion.The prescription that adds embodiment 1 gained cytokine and compound formation is then induced the neurone division, and (see figure 1) finds that considerable neurone all is in the M phase (m period) as a result.Picture shows is the former generation cortex and spinal neuron that is in the mitotic division different times.
The rat cerebellum cell is cultivated, added heavy dose of cytosine arabinoside then, but remove other somatoblast.The specific Calbindin D28k of utilization purkinje cell antibody carries out the immunocytochemistry of culturing cell to be identified.The prescription that adds embodiment 1 cytokine and compound formation is then induced the neurone division, observes under phase microscope, and (see figure 2) shows that part Purkinje neurone is in the M phase in the culturing cell as a result.
Carry out neuronic cultivation of cerebellum Purkinje and induced fission according to the method for embodiment 4, the cerebellar neuron of getting respectively with the equal densities inoculation in the 2nd, 4,6,8,10 day at induced fission, under opticmicroscope, observe behind the Calbindin D28k immunocytochemical stain, count calbindinD28k male purkinje cell quantity in 20 times of object lens visuals field.The neuronic quantity of former generation Purkinje obviously increased after the result showed adding embodiment 1 cytokine prescription.The result is carried out the regression curve analysis, find that the quantity of Calbindin D28k positive cell has also increased (see figure 3) along with the prolongation of cultivating fate, increasing the most tangible time is at 6-8 days that cultivate.From the composition (Fig. 4) of cell type, Fig. 4 shows, in the early stage still late period of no matter cultivating, I type (Fig. 4 left side post) purkinje cell (morphological development is inmature, and cell space is less, and projection is few, mostly is bipolar neuron, and branch seldom) all occupies the majority; The more sophisticated III type of form (Fig. 4 right side post) cell (cell space is obviously big than peripheral cell, and projection is more, and the one-level projection also has a lot of branches); (the cell cell space increases II type (Fig. 4 intermediolateral column) cell, projection increases, branch is also more, but mostly be the one-level projection, the dendron ridge is also less) belong to from the transition cell of I type to III type cell development process, therefore, it changes than the increase and decrease of regular meeting with these two kinds of cell quantities, when I type cell quantity increased, its shared ratio obviously reduced.Demonstration is with the increase of cultivating fate, and cell quantity also increases.
Under the phase microscope that can take continuously, observe the former foster neurone of being commissioned to train of embodiment 1 cytokine prescription induced fission, seek the neurone that is in the mitotic division different times, to take continuously then, frequency is 1 time/30min.Found that induce down at the prescription of embodiment 1 cytokine and compound formation, the new filial generation neurone that forms can divide (see figure 5) continuously.Fig. 5 is presented at the prescription of embodiment 1 cytokine and compound formation and induces down, and the sub-neurone that the division of Purkinje neurone forms still can continue division.
Former being commissioned to train of embodiment 7 induced fissions supported the mark that neurone has mature neuron
For the neurone that proves induced fission is a mature neuron, the contriver checks with the Nestin immunocytochemistry earlier before the prescription that adds embodiment 1 cytokine and compound is induced the neurone division, does not find any Nestin immunity positive mark cell.Get rid of neural stem cell and precursor cell and be divided into neuronic possibility.And then further use immunofluorescence Double Labelling Technique (Hoechst/PI shows somatoblast), immunocytochemistry Double Labelling Technique (PCNA shows somatoblast), having proved the marker (Calbindin D28k/NSE/NF-160/SOM/GAD) of expressing ripe neuronal specificity in the neurone of induced fission, is that the neurone of differentiation and maturation (is seen Fig. 6～Fig. 9).
Fig. 6 is in the Purkinje neurone laser co-focusing photo of metaphase, pericaryon Purkinje neuronal specificity mark Calbindin D28k mark (redness).Chromatin is positioned at the equator, by Hoechst mark (blueness).
Purkinje neurone laser co-focusing photo anaphase that Fig. 7 being in, karyon Hoechst mark (blueness).Kytoplasm is by Calbindin D28k mark (redness).
Fig. 8 cortical neuron laser co-focusing of former generation photo, show be in mitotic division early stage (a, b) and mid-term (c, d, neurone e), chromatin is by PI mark (redness).Its tenuigenin is by neuronal specificity mark NSE dyeing (green);
Cortical neuron anaphase that Fig. 9 being in, picture a shows that the neurone karyon is by PI mark (redness), kytoplasm is by NSE immunocytochemical stain (green), picture b-d shows that karyon is by PCNA mark (hyacinthine), kytoplasm is respectively by NF-160, SOM and GAD mark (brown), these all are the marks of mature neuron.
Adopt the method for embodiment 3 to carry out rat cerebral cortex neuronic former be commissioned to train foster and induced fission, after carrying out the anchored in place, embedding of electron microscopy, under phase microscope, seek the neuron cell that is in the mitotic division different times, behind the mark, original position cuts, and sticks in the resin holder finishing embedded block, carry out ultrathin section(ing), carry out immuno-electron microscope dyeing then.The result shows that former generation neurone of phase when these are being in different division has specific neuronal structure, and just can be by the NSE colloid gold label at the splitted neurone.The mark (Figure 10-13) that just has mature neuron at the splitted neurone is described.Even just capture and to form typical cynapse (seeing Figure 14) with other neurone at the splitted neurone.Having pointed out the splitted neurone is mature neuron, also points out this a fewly just can bring into play the function of neurons effect at the splitted neurone.
Neurone electromicroscopic photograph mitosis prophase that Figure 10 showing does not have complete nucleus (A and B) in the neuronic perikaryon, have many chromatinal masses that come in every shape (Cr) in cytoplasm.Can also observe a large amount of plastosome (mi), microtubule, lysosome and a spot of rough surfaced endoplasmic reticulum (RER) in the cytoplasm.Shown in the arrow (arrow) is the colloid gold particle of NSE mark.
Figure 11 shows the neurone electromicroscopic photograph of mitosis metaphase, and spindle fibre is present between two sister chromatids (Cr).Divide existence is dispersed in a large number in the neuronic cytoplasm polyribosome (stars) and the neurotubule (mt) of plastosome (mi) with marshalling.
Figure 12 shows the neurone electromicroscopic photograph of mitosis anaphase, and neuronic central authorities have formed a dark depression in division.Plastosome (mi) structure and colloid gold particle (arrow) can clearly be observed in the two poles of the earth of two close cells of sister chromatid.
Figure 13 shows two nucleus of the interior existence of neurone electromicroscopic photograph division neurone of metamitosis, lay respectively at the two poles of the earth (A) of cell, divide and have a large amount of polyribosome (stars), rough surfaced endoplasmic reticulum (RER) and plastosome (mi) in the neuronic cytoplasm.In the neuronic cytoplasm of division, there are many colloid gold particles (arrow).
Figure 14 show metamitosis neurone and other neurone form close functional cohesion, even typical synaptic structure; Three terminal oval-shaped varicosities of big formation that become of nervous process have formed typical asymmetry synaptic structure with the neuronic projection of daughter cell; The local cells film obviously thickens, and electron density increases; The presynaptic structure contains a large amount of limpid vesicles.
The neurone that embodiment 9 is in telophase shows mature neuron electricity physiological characteristic
Adopt the method for embodiment 3 to carry out rat cerebral cortex neuronic former be commissioned to train foster and induced fission, the neurone of selecting to be in mitotic division period at microscopically carries out single celled patch clamp and detects then, observes the electric physiological characteristic whether it has mature neuron.The result shows, their resting potential is all between-60-70mV.After accepting a series of depolarize electricity irritation, can see the outward current of inward electric current and time-delay fast.Data processing and analysis revealed, these division neurones have sophisticated Na
+Electric current and K
+Electric current, Na
+The threshold value of electric current is about-50mV.These division neurones have the excitability of height, have the typical electric physiological characteristic (seeing Figure 15) of mature neuron.Figure 15. show the sodium, the potassium-channel current potential that just have typical mature neuron at the splitted neurone.Figure shows the excitability current potential that just has mature neuron at the splitted neurone.
The prescription 5 μ l of embodiment 1 are expelled to the adult rat cortex, and consumption is 5 μ l/, and after 5 days, injection 120mg BrdU is in rat abdominal cavity.After 5 days, the utilization Paraformaldehyde 96 pours into fixing to animal.After taking out brain, carry out immuno-chemical marker.Result's (seeing Figure 16) shows that the part cortical neuron is by the BrdU mark, even the cortex giant pyramidal cells illustrates that also by BrdU and NSE immunity double-tagging local neuron is stimulated by the cytokine prescription of embodiment 1, has the splitted ability.
After Figure 16 showed that the cytokine prescription of embodiment 1 is expelled to cortex, injection zone peripheral nerve unit by BrdU mark situation (among Figure 16 a); Figure B is toluylene red and BrdU double labeling cells.B-e is NSE and BrdU immunity double-tagging neurone among Figure 16.
Embodiment 11 treatment Spinal injury experiments
The cytokine that embodiment 2 is obtained and the prescription part of compound apply to the broken ends of fractured bone of spinal cord, can make the nerve fiber of the broken ends of fractured bone be easy to grow into the spinal cord in distally, are beneficial to the reparation of Spinal injury.As after scheming to show that spinal cord is fully cross-section,, make nerve fiber pass through the broken ends of fractured bone by N4 effect, enter distally myeloid tissue, and get in touch by varicosity and the foundation of the next spinal cord and (to see Figure 17, Figure 18).Figure 17. after the spinal cord transection, behind the cross-section fully rat spinal cord of T7 intercostal nerve-L1 Dorsal root bridge joint, the cytokine of embodiment 2 and the prescription of compound can pass through two nerve node chalaza (A-D among Figure 17), enter L1 spinal cord (E, material is from the little frame e of figure B).And, the neural juncture of T7 and L1, L1 enters the spinal cord place does not have clear and definite neuroma and abnormal pigmentary deposit on the skin trace; Figure 18, the T7 intercostal nerve that the cytokine of demonstration embodiment 2 and the prescription of compound promote T to originate from the 6-8 spinal cord enters the PHA-L direct motion mark (A-C among Figure 18) of T12-L2 spinal cord.High power lens shows that down these markers are nerve fiber and varicosity.
Claims (10)
1, promotes the cytokine of neurone division and neurotization and the prescription that compound constitutes, contain Regular Insulin, bovine serum albumin, putrescine, hydrocortisone, progesterone, Transferrins,iron complexes, trilute, Sodium Selenite, Prostatropin and Urogastron.
2, according to the described prescription of claim 1, its moiety and weight percent thereof (%) are:
Regular Insulin 0.04～0.4
Transferrins,iron complexes 0.8～8
Bovine serum albumin 0.04～0.4
Putrescine 0.12～1.2
Hydrocortisone 3.5 * 10
-5～3.5 * 10
-4
Progesterone 5 * 10
-5～5 * 10
-4
Sodium selenite 4 * 10
-5～4 * 10
-4
Trilute 8 * 10
-3～8 * 10
-2
Urogastron 8 * 10
-5～8 * 10
-4
Prostatropin 8 * 10
-5～8 * 10
-4
All the other are water
3, according to the described prescription of claim 1, also contain Brain Derived Neurotrophic Factor and nerve growth factor.
4, according to the described prescription of claim 3, its moiety and weight percent thereof (%) are:
Regular Insulin 0.04～0.4
Transferrins,iron complexes 0.8～8
Bovine serum albumin 0.04～0.4
Putrescine 0.12～1.2
Hydrocortisone 3.5 * 10
-5～3.5 * 10
-4
Progesterone 5 * 10
-5～5 * 10
-4
Sodium Selenite 4 * 10
-5～4 * 10
-4
Trilute 8 * 10
-3～8 * 10
-2
Urogastron 8 * 10
-5～8 * 10
-4
Prostatropin 8 * 10
-5～8 * 10
-4
Brain Derived Neurotrophic Factor 1 * 10
-4～1 * 10
-3
Nerve growth factor 2 * 10
-4～2 * 10
-3
All the other are water.
5, claim 1,2,3 or 4 described prescriptions are in the medicine of preparation treatment nervous system disease and the application in nervous system disease research and the diagnostic reagent.
6, claim 1,2,3 or 4 described prescriptions are in preparation treatment nerve injury and the medicine of neural system degeneration and the application in nerve injury and neural system degeneration research and the diagnostic reagent.
7, claim 1,2,3 or 4 described prescriptions promote neurone splitted medicine and neurone to divide the application in research and the diagnostic reagent in preparation.
8, claim 1,2,3 or 4 described prescriptions are in preparation treatment Spinal injury, the medicine of peripheral nerve injury equivalent damage illness and the application in damaging illness research and the diagnostic reagent.
9, claim 1,2, the medicine of the various brain injurys of 3 or 4 described prescriptions due to preparation treatment cerebral anoxia, ischemic and the application in brain injury research and the diagnostic reagent.
10, claim 1,2,3 or 4 described prescriptions are in the medicine of illness such as nervus centralis degeneration such as preparation treatment senile dementia, Parkinson disease or lateral spinal sclerosis and the application in nervus centralis degeneration illness research and the diagnostic reagent.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI476010B (en) * | 2013-01-25 | 2015-03-11 | Agricultural Technology Res Inst | A composition for promoting hair growth |
| CN104560876A (en) * | 2014-12-31 | 2015-04-29 | 广州赛吉生物科技有限公司 | Clinical-grade serum-free medium for adherent culture of human neural stem cells |
| CN107603951A (en) * | 2016-07-12 | 2018-01-19 | 中国人民解放军军事医学科学院基础医学研究所 | It is a kind of to be used in new growth/neurotrophic factor composition of body induced maturation neuron division and application thereof |
| WO2022008609A1 (en) * | 2020-07-08 | 2022-01-13 | Grifols Worldwide Operations Limited | Compositions having neuroregenerative applications |
| WO2022008586A1 (en) * | 2020-07-08 | 2022-01-13 | Grifols Worldwide Operations Limited | Compositions having neuroregenerative applications |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2006269884A1 (en) * | 2005-07-15 | 2007-01-25 | Primegen Biotech, Llc | Therapeutic reprogramming of germ line stem cells |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI476010B (en) * | 2013-01-25 | 2015-03-11 | Agricultural Technology Res Inst | A composition for promoting hair growth |
| CN104560876A (en) * | 2014-12-31 | 2015-04-29 | 广州赛吉生物科技有限公司 | Clinical-grade serum-free medium for adherent culture of human neural stem cells |
| CN104560876B (en) * | 2014-12-31 | 2018-03-16 | 广州吉帝生物科技有限公司 | A kind of clinical grade serum free medium for adhere-wall culture human nerve stem cell |
| CN107603951A (en) * | 2016-07-12 | 2018-01-19 | 中国人民解放军军事医学科学院基础医学研究所 | It is a kind of to be used in new growth/neurotrophic factor composition of body induced maturation neuron division and application thereof |
| CN107603951B (en) * | 2016-07-12 | 2020-08-14 | 中国人民解放军军事医学科学院基础医学研究所 | Novel growth/neurotrophic factor composition for in vivo induction of mature neuronal division and use thereof |
| WO2022008609A1 (en) * | 2020-07-08 | 2022-01-13 | Grifols Worldwide Operations Limited | Compositions having neuroregenerative applications |
| WO2022008586A1 (en) * | 2020-07-08 | 2022-01-13 | Grifols Worldwide Operations Limited | Compositions having neuroregenerative applications |
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