CN108531454A - The purposes of Cord blood regenerated particle and composition in treating brain degenerative disease - Google Patents
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Abstract
The invention discloses a kind of purposes of the Cord blood regenerated particle and combinations thereof in treating brain degenerative disease, the Cord blood regenerated particle has following feature：Ranging from 15 μm of the diameter of the Cord blood regenerated particle；The Cord blood regenerated particle contains minim DNA ingredient；The Cord blood regenerated particle expresses Oct3/4, Nanog, Sox2 albumen.The study found that therapeutic agent prepared by Cord blood regenerated particle of the present invention and combinations thereof can effectively treat brain degenerative disease, the treatment for especially being used to regenerate cranial nerve cell and cerebral senility is delayed to move back.
Description
Technical field
The present invention relates to the fields such as biomedical sector, especially histocytology, regenerative medicine, disease treatment, especially
It is related to purposes of the Cord blood regenerated particle and combinations thereof in treating brain degenerative disease.
Background technology
Cerebral retrogressive disease is a kind of lesion for seriously endangering human health, mainly causes the specific god of mesencephalic centre nervous system
Through the abnormal dead atrophy of member, lead to cranial nerve function major defect, cognition or dyskinesia, this damage is irreversible
, it can deteriorate over time, eventually lead to brain disorder.
Cerebral retrogressive disease includes Alzheimer disease (AD), Parkinson's disease (PD), Huntington disease (HD), multiple sclerosis
Disease (MS), senile melancholia, is trembled and other encephalatrophy diseases (BA) etc. at Pick diseases at hand.Wherein Alzheimer disease and pa
The gloomy disease of gold is the highest two kinds of diseases of illness rate, and two diseases add up to 8,000,000 patients or so in China.
With the aggravation of China human mortality aging trend, the nervous systems such as cerebral ischemia, apoplexy and senile dementia, Parkinson
The prevalence proportions of damage and degenerative disease constantly increase.
Treatment for senile cerebral retrogressive disease, traditional drug therapy, operative treatment be all difficult play a role, and
Regenerative medicine has apparent advantage.Drug therapy plays a role often through certain signal paths are intervened, and regenerative medicine
It is then the tissue that regenerative cell substitutes damage, fundamentally cures disease.
Regenerative medicine refers to the tissue and device that loss or functional lesion are created using biology and the theoretical method of engineering science
Official makes it have normal structure and the mechanism and function of organ.
In recent years, exogenous cells therapy has been increasingly becoming the new strategy of cerebral retrogressive disease treatment.Exogenous cells are controlled
It treats and mainly restores the neuroid and cognitive function degenerated by introducing stem cell etc..These stem cells can be used as thin
Born of the same parents' transport system achievees the purpose that reparation using paracrine mechanism.Another thinking is the differentiation and promotion using stem cell
Neural circuit regenerated function achievees the purpose that treatment.This is fine, complicated, multi-step a process.
Seek a kind of safety and effective regenerative medicine therapy either cell preparation, is current treatment cerebral retrogressive
Property disease a hot spot and future drugs research and development an important directions.
Invention content
Based on hot technology problem existing for background, the present invention provides a kind of safe therapeutic agents or composition, this is controlled
Brain degenerative disease can effectively be delayed and treat by treating agent or composition.
Technical scheme is as follows：
The stem cell precursor active material that Cord blood regenerated particle is found as current research, can be followed by blood of human body
Loop system reaches the different parts of brain, by place position microenvironment stimulation and influence, can by self constantly aggregation and
Fusion and group core, develop for brain ancestral cells, and be further divided into cerebral granule cells, neuronal cell etc., Jin Eryou
Cerebral senility caused by the alleviation with age of effect is moved back, and a completely new method road is provided for further treatment brain degenerative disease
Road.
The Cord blood regenerated particle and combinations thereof of the present invention can regenerate brain granular cell, neuronal cell etc..
The Cord blood regenerated particle and combinations thereof of the present invention can update big abr cell.
The Cord blood regenerated particle and combinations thereof of the present invention can improve cognition and the behavioral function of brain.
The Cord blood regenerated particle and combinations thereof of the present invention is specifically for use among the treatment of Alzheimer disease.
The Cord blood regenerated particle and combinations thereof of the present invention is also particularly applied among the treatment of Parkinson's disease.
The Cord blood regenerated particle and combinations thereof of the present invention is also particularly applied among the treatment of senile melancholia.
The Cord blood regenerated particle and combinations thereof of the present invention is also particularly applied among the treatment of alzheimer disease.
For this purpose, first aspect present invention provides Cord blood regenerated particle as therapeutic agent for treating brain degeneration disease
Disease for regenerating brain cells and delays the therapeutical uses that cerebral senility moves back.
Therapeutic agent according to a first aspect of the present invention, wherein the Cord blood regenerated particle, there is following feature：
The Cord blood regenerated particle is derived from the regenerated particle of Cord blood.
Ranging from 1-5 μm of the diameter of the Cord blood regenerated particle.
The Cord blood regenerated particle contains minim DNA ingredient.
The Cord blood regenerated particle expresses Oct3/4, the albumen such as Nanog, Sox2.
The Cord blood regenerated particle is for mammal, such as people, the dosage of the Cord blood regenerated particle
Be per kilogram patient's weight dosage be (1-10) x109A Cord blood regenerated particle.Such as per kilogram patient's weight dosage is 1x109
A Cord blood regenerated particle, per kilogram patient's weight dosage are 5x109A Cord blood regenerated particle, per kilogram patient's weight dosage
For 10x109A Cord blood regenerated particle.
Further, second aspect of the present invention provides a kind of for treating brain degenerative disease or for regenerating brain
Cell and delay the therapeutic agent that cerebral senility is moved back, in composition in the form of, the composition include mainly Cord blood regenerated particle liquid with
Injection buffer solution.
Composition according to a second aspect of the present invention, wherein the Cord blood regenerated particle liquid, there is following feature：
The Cord blood regenerated particle liquid comes from Cord blood.
Ranging from 1-5 μm of the diameter of Cord blood regenerated particle in the Cord blood regenerated particle liquid.
Cord blood regenerated particle in the Cord blood regenerated particle liquid contains minim DNA ingredient.
Cord blood regenerated particle in the Cord blood regenerated particle liquid expresses Oct3/4, the albumen such as Nanog, Sox2.
The Cord blood regenerated particle liquid is for mammal, such as people, the use of the Cord blood regenerated particle liquid
Dosage is that per kilogram patient's weight dosage is (1-10) x109A Cord blood regenerated particle.Such as per kilogram patient's weight dosage is
1x109A Cord blood regenerated particle, per kilogram patient's weight dosage are 5x109A Cord blood regenerated particle, per kilogram patient's weight
Dosage is 10x109A Cord blood regenerated particle.
Therapeutic agent according to a second aspect of the present invention, wherein the injection buffer solution is when for mammal, example
Such as people, the injection buffer solution in the therapeutic agent is 0.9% sodium chloride injection.
According to a first aspect of the present invention and the purposes of second aspect, ability may be used in the Cord blood regenerated particle used in
It is prepared by method known to domain.Such as the Cord blood regenerated particle obtained by Cord blood, it is referred to Chinese Patent Application No.
Method in CN201610871433.3 obtains.
The invention has the beneficial effects that：
The present invention provides a kind of Cord blood regenerated particles and combinations thereof to prepare for treating brain degenerative disease
Or for regenerating brain cells and delay the purposes in the therapeutic agent that cerebral senility moves back.The study found that therapeutic agent of the present invention can be effective
Treatment brain degenerative disease, be especially used for the treatment of regenerating brain cells and improve brain cognition and behavioral function is controlled
It treats.
Description of the drawings
Fig. 1：The form that Cord blood regenerated particle is presented when cultivating in the medium；
Fig. 2：It is sliced colored graph；The slice colored graph that Fig. 2A -2B are 7 days, wherein 2A are nonspecific antibody protein staining, figure
2B dyes for GFP antibody proteins；The slice colored graph that Fig. 2 C-2D are 4 weeks, wherein 2C are nonspecific antibody protein staining, Fig. 2 D
It is dyed for GFP antibody proteins；Fig. 2A -2D are shot under 2.5 times of lens；Band-like original mold formula is distributed in Fig. 2 B and 2D by arrow
It points out.
Fig. 3：Brain part co-expresses GFP, nestin and DAPI.
Fig. 4：The particle spline structure that the regenerated particle newly formed is fused into.
Fig. 5：Mouse brain regenerated particle situation schematic diagram, wherein Fig. 5 A are the brain part that regenerated particle is dispersed in 5 days
A border circular areas；Fig. 5 B are 8 days brain parts, and most of regenerated particles are connected to each other to form dendron in border circular areas
Shape spline structure.
Fig. 6：Big eucaryotic cell structure or preceding neuronal cell co-express GFP and nestin.
Fig. 7：The original cilium spline structure of the length of GFP and nestin coexpressions.
Fig. 8：The nestin and Oct4 of 4 weeks brain part coexpressions.
Fig. 9：Mouse finds the probability scenarios of underwater platform.
Figure 10：Mouse finds the incubation period situation of underwater platform.
Specific implementation mode
The present invention can be further described by the following examples, and under the scope of the present invention is not limited to
State embodiment.
The present invention carries out general and/or specific description to material and experimental method used in experiment.Although being
Realize that many materials and operating method used in the object of the invention are it is known in the art that still the present invention still makees to the greatest extent may be used herein
It can detailed description.
Embodiment 1
The preparation of Cord blood regenerated particle
In the present embodiment, the preparation of Cord blood regenerated particle is illustrated.
Blood source：Umbilical cord blood collection to the blood bag containing anti-coagulants in, anti-coagulants is original sodium citrate in blood bag, is passed through
Cord blood is transported to and prepares laboratory by cold chain transportation mode (temperature is maintained at 2 to 8 DEG C).
Condition：The processing procedure for obtaining Cord blood regenerated particle needs whole sterile working.
It is as follows：
(1) Cord blood is taken to carry out 200g centrifugations, centrifugation time is 10 minutes, is divided into two parts after centrifugation, upper solution A and
Lower sediment A.
(2) a layer precipitate A is removed, is by volume 1:2-5 is added PBS and slightly washs, and carries out 200g centrifugations, and centrifugation time is
10 minutes, two parts, upper solution B and lower sediment B are divided into after centrifugation.
(3) a layer precipitate B is removed, is by volume 1:5-10 is added erythrocyte cracked liquid and is placed in horizontal shaker, shakes 20 points
Clock carries out 2000g centrifugations later, and centrifugation time is 10 minutes, and two parts, upper solution C and lower sediment C are divided into after centrifugation,
Upper solution C is discarded.
(4) a layer precipitate C is removed, is by volume 1:5-10 be added PBS acutely shake, jitter time be 2 minutes, it is laggard
Row 200g centrifugations, centrifugation time is 10 minutes, two parts, upper solution D and lower sediment D is divided into after centrifugation, lower sediment D is abandoned
Fall.
(5) upper solution A, upper solution B, upper solution D are taken respectively, carries out 5000g centrifugations, and centrifugation time is 10 points
Clock after centrifugation, collects all precipitations.
(6) culture medium (the mixing serum of umbilical cord blood for containing 20%) that the precipitation being collected into is placed in MEM is cultivated, and is cultivated
Condition is 37 DEG C, and in 5%CO2 incubators, it is primary that culture medium was replaced per 2-3 days.
(7) culture medium by culture 10 days is collected, and 5000g centrifugations is carried out, to be enriched with Cord blood regenerated particle (regenerated particle
Form is shown in Fig. 1).
Embodiment 2
The cerebral hemisphere that regenerated particle moves into damage is in belt pattern
The regenerated particle (GFP labels) that experimental group is obtained by tail vein injection embodiment 1 is small to ischemic brain damage
Mouse, control group injecting normal saline.
When 7 days, the sliced section of control group brain is all GFP negative (Fig. 2A), but in GFP antibody responses portion
Divide positive (Fig. 2 B) in GFP.When 4 weeks, the continuous part of control group part brain is all GFP negative (Fig. 2 C), still
It is positive (Fig. 2 D) in GFP in GFP antibody responses part.When 7 days, entire brain part shows almost all of left hemisphere group
Knit be ischemic and damage, and be enhance and it is widened.The substance of the GFP positives is in belt pattern (shown in Fig. 2 B arrows)
Arrangement is centered around half brain ball of damage.
Embodiment 3
Regenerated particle forms particulate cellular
The regenerated particle (GFP labels) that experimental group is obtained by tail vein injection embodiment 1 is small to ischemic brain damage
Mouse, control group injecting normal saline.
Regenerated particle migrates into after brain tissue, they are to be fused into a seedless cell spline structure first, later
Further form karyocyte.High power microexamination further confirms the regenerated particle for coming from capillary (see the width of Fig. 4
Shown in arrow) it is first merged before forming granular cell, and further development forms new particulate cellular.
Embodiment 4
Regenerated particle forms dendron shape neuron
The regenerated particle (GFP labels) that experimental group is obtained by tail vein injection embodiment 1 is small to ischemic brain damage
Mouse, control group injecting normal saline.
In the brain of ischemic injuries, the regenerated particle of dispersion is connected with each other fusion and forms fiber-like structures, and further shape
The neuron expressed at dendron shape.5 days brain parts, one group of regenerated particle was dispersed in a circular region (see figure
5A).A other regenerated particle may be determined clearly.However, 8 days brain parts, most regenerated particles linked each other
In dendron shape spline structure in a circular region, at the same time sub-fraction is still in the pattern of dispersion (see Fig. 5 B).2 weeks it
Afterwards, we have confirmed that there is the big eucaryotic cell structure or preceding neuronal cell coexpression GFP and nestin of core (see Fig. 6).
Embodiment 5
Regenerated particle participates in updating big abr cell
Regenerated particle can be connected to each other to form long fiber-like structures to generate new cell.It is observed that one
The original cilium spline structure of the length of GFP and nestin coexpressions is discharging weak DAPI (see Fig. 7) and is dyeing small cell two
It holds (in DAPI shown in arrow), this shows that long cilium spline structure may update new cell.
In order to further confirm that the cell of these nestin expression is stem cell, we are had checked using Laser Scanning Confocal Microscope
In the nestin and Oct4 (see Fig. 8) of 4 weeks brain parts coexpression, those nestin common location Zonal expressions are found
Oct4, therefore they are stem cells.
Embodiment 6
Regenerated particle is obviously improved cognition and the behavioral function of brain dementia mouse
In the present embodiment, experiment is divided into 3 groups：SAMR1 groups, SAMP8- brine groups, SAMP8- regenerated particle groups.
SAMR1 groups do not make any processing, and SAMP8- brine groups give 0.15ml physiological saline tail vein injection processing/moon, and SAMP8- is again
Raw particle group gives 0.15ml regenerated particles tail vein injection processing/moon.
Mouse is found in the probability scenarios of underwater platform, at 0 month, SAMP8- brine group and SAMP8- regenerated particle groups
Low, indifference between SAMP8- brine group and SAMP8- regenerated particle groups all more notable than SAMR1 groups.At 6 months, SAMP8- salt
Water group and SAMP8- regenerated particles group are all significantly lower than SAMR1 groups, and SAMP8- brine group ratio SAMP8- regenerated particle groups are significantly low
(see Fig. 9).
Mouse was found in the incubation period of underwater platform, and at 0 month, SAMP8- brine group and SAMP8- regenerated particles group were all
It is more significantly high than SAMR1 groups, indifference between SAMP8- brine group and SAMP8- regenerated particle groups.At 6 months, SAMP8- brine
Group and SAMP8- regenerated particles group are all more significantly high than SAMR1 groups, SAMP8- brine group ratio SAMP8- regenerated particle groups it is significantly high (see
Figure 10).
Two above experiment shows that regenerated particle can obviously improve cognition and the behavioral function of brain dementia mouse.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Any one skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (10)
1. a kind of Cord blood regenerated particle for treating brain degenerative disease, which is characterized in that the Cord blood regenerated particle
Ranging from 1-5 μm of diameter；The Cord blood regenerated particle contains minim DNA ingredient；The Cord blood regenerated particle expression
Oct3/4, Nanog, Sox2 albumen.
2. Cord blood regenerated particle as described in claim 1, which is characterized in that the Cord blood regenerated particle can regenerate brain
Granular cell, neuronal cell.
3. Cord blood regenerated particle as described in claim 1, which is characterized in that the Cord blood regenerated particle can update greatly
Abr cell.
4. Cord blood regenerated particle as described in claim 1, which is characterized in that the Cord blood regenerated particle can improve greatly
The cognition of brain and behavioral function.
5. Cord blood regenerated particle as described in claim 1, which is characterized in that the Cord blood regenerated particle can be applied to
Among the treatment of Alzheimer disease.
6. Cord blood regenerated particle as described in claim 1, which is characterized in that the Cord blood regenerated particle can be applied to
Among the treatment of Parkinson's disease.
7. Cord blood regenerated particle as described in claim 1, which is characterized in that the Cord blood regenerated particle can be applied to
Among the treatment of senile melancholia.
8. Cord blood regenerated particle as described in claim 1, which is characterized in that the Cord blood regenerated particle can be applied to
Among the treatment of alzheimer disease.
9. Cord blood regenerated particle as described in claim 1, which is characterized in that it can be used for preparing treatment brain degeneration
Disease for regenerating brain cells and delays therapeutic agent or composition that cerebral senility moves back.
10. Cord blood regenerated particle as claimed in claim 9, which is characterized in that the therapeutic agent includes mainly Cord blood
Regenerated particle liquid and injection buffer solution；The injection buffer solution is 0.9% sodium chloride injection.
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Cited By (4)
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| CN109771443A (en) * | 2018-09-29 | 2019-05-21 | 孔五一 | A kind of application of Cord blood regenerated particle in preparation treatment acute kidney injury drug |
| CN114159472A (en) * | 2021-12-16 | 2022-03-11 | 上海华颜医药科技有限公司 | A method for delaying brain atrophy by using cord blood |
| US20220088086A1 (en) * | 2020-09-24 | 2022-03-24 | Therapeutic Solutions International, Inc. | Personalized Immunotherapies for Reduction of Brain Inflammation and Suicide Prevention |
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| CN109777772A (en) * | 2018-09-29 | 2019-05-21 | 孔五一 | A kind of Cord blood regenerated particle and the preparation method and application thereof |
| CN109771443A (en) * | 2018-09-29 | 2019-05-21 | 孔五一 | A kind of application of Cord blood regenerated particle in preparation treatment acute kidney injury drug |
| US20220088086A1 (en) * | 2020-09-24 | 2022-03-24 | Therapeutic Solutions International, Inc. | Personalized Immunotherapies for Reduction of Brain Inflammation and Suicide Prevention |
| CN114159472A (en) * | 2021-12-16 | 2022-03-11 | 上海华颜医药科技有限公司 | A method for delaying brain atrophy by using cord blood |
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