CN106729642A - A kind of stem cell medicine and its preparation method and application - Google Patents
A kind of stem cell medicine and its preparation method and application Download PDFInfo
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- CN106729642A CN106729642A CN201710161077.0A CN201710161077A CN106729642A CN 106729642 A CN106729642 A CN 106729642A CN 201710161077 A CN201710161077 A CN 201710161077A CN 106729642 A CN106729642 A CN 106729642A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/38—Albumins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The present invention relates to biological therapy field, a kind of stem cell medicine and its preparation method and application is disclosed, the stem cell medicine has good curative effect to Macular lesion.Stem cell medicine disclosed by the invention, including：Mesenchymal stem cells MSCs, neural-like cells, albumin and solvent.The invention discloses the preparation method of the stem cell medicine, comprise the following steps：(1) albumin and solvent are mixed, prepares albumin suspension；(2) the resuspended mesenchymal stem cells MSCs of albumin solution prepared with step (1), obtains the mesenchymal stem cells MSCs preparation；(3) the resuspended neural-like cells of mesenchymal stem cells MSCs preparation are prepared with step (2), product is obtained.The invention also discloses the stem cell medicine or application of the stem cell medicine obtained in the preparation method in the Macular lesion biological agent for the treatment of is prepared.
Description
Technical field
The present invention relates to biological therapy field, more particularly to a kind of stem cell medicine and its preparation method and application.
Background technology
Macular area is an important area of retina, positioned at eye Posterior pole, mainly regards work(with epicritic vision and colour vision etc.
Can be relevant.Once lesion occurs in macular area, usually there is visual impairment, muscae genetic vision or metamorphopsia.ARM generally has
Two types.One kind is Dry macular lesion, and one kind is wet macular lesion.Wet MD accounts for the 10% of total incidence,
It is, due to having abnormal angiogenic growth, new vessels Rupture haemorrhag under retina, and to cause scar tissue growth, makes eyesight unexpected
Decline, can rapidly severely impact the central vision of patient, even result in central light loss.The main disease of wet macular lesion
Shape shows as：Central vision decrease fast, metamorphopsia, words and expressions bending on paper, there is shadow or fuzzy region in vision center.Mesh
The preceding treatment for ARM is usually using methods such as the traditional Chinese medical science or clinical operations, but such method is only capable of playing and delays disease
Feelings, and radical cure can not be played a part of.Additionally, existing treatment method high recurrence rate, long-term blind rate is high.
At present, mesenchymal stem cells MSCs (BMSCs) is applied to PVR (ROP) by increasing researcher
Treatment.The mechanism performance of BMSCs treatments ROP is as follows：1. cell migration gathers damage field, after retinal vessel injury,
BMSCS can produce substantial amounts of growth factor, such as VEGF, platelet derived growth factor and ICAM
Deng, improve vascularization microenvironment in terms of play an important role；2. cell transplantation participates in repairing：1. the stem cell being transplanted to
Pathological tissues are migrated, breed, merge and supplement the gangliocyte being damaged, and are formed closely with the nerve cell of host
Synaptic contact, rebuild nerve pathway, improve corresponding function；2. by autocrine and the function of paracrine, after playing its transplanting
Neuroprotection and trophism；3. immunoregulation effect and downward inflammatory reaction, suppress the related inflammatory factor to a certain extent
Damage to retinal vascular cell and nerve fiber, and reduce the probability of happening of related complication after clinical transplantation.Therefore,
A kind of stem cell medicine to Macular lesion with good efficacy of research and development is that those skilled in the art's technology urgently to be resolved hurrily is asked
Topic.
The content of the invention
In view of this, the invention discloses a kind of stem cell medicine and its preparation method and application, the stem cell medicine
There is good curative effect to Macular lesion.
The invention discloses a kind of stem cell medicine, including：Mescenchymal stem cell, neural-like cells and albumin.
Preferably, the neural-like cells are differentiated by stem cell induction.
Preferably, the mescenchymal stem cell is mescenchymal stem cell of the second generation to the 5th generation.
Preferably, the content of the mescenchymal stem cell is 1 × 106~5 × 106Individual/mL.
Preferably, the content of the neural-like cells is 1 × 106~5 × 106Individual/mL.
Preferably, the content of the albumin is 2~15%.
The invention also discloses the preparation method of the stem cell medicine, comprise the following steps：
A1) albumin and physiological saline are mixed, albumin suspension is prepared；
A2) using step a1) the resuspended mesenchymal stem cells MSCs of albumin preparation for preparing, between obtaining marrow
Mesenchymal stem cells preparation；
A3) using step a2) the resuspended neural-like cells of mesenchymal stem cells MSCs preparation that prepare, obtain institute
State stem cell medicine.
Preferably, the concentration of the albumin preparation is 2~15%.
Preferably, the mescenchymal stem cell is obtained according to following steps：
(1) marrow is centrifuged, obtains primary mescenchymal stem cell；
(2) the resuspended primary mescenchymal stem cell, is cultivated；
(3) change liquid and remove non-attached cell, add culture medium to continue to cultivate the primary mescenchymal stem cell；
(4) cell cultivated is digested, obtains mescenchymal stem cell.
Preferably, the neural-like cells are obtained by the mescenchymal stem cell by inducing differentiation.
Preferably, doing thin the invention also discloses described stem cell medicine or according to obtained in described preparation method
Application of born of the same parents' preparation in Macular lesion is treated.
In sum, the invention discloses a kind of stem cell medicine and its preparation method and application, the stem cell medicine bag
Include mesenchymal stem cells MSCs, neural-like cells and albumin.Combine by medulla mesenchyma by by mesenchymal stem cells MSCs
The neural-like cells of stem cell induction differentiation carry out subretinal space transplanting, can avoid blood flow and cause excretion body
Loss.Additionally, mesenchymal stem cells MSCs can secrete the reparation that cytokine profiles directly participate in injury tissue, enhancing is not
The resistivity of injury tissue, shortens the tissue repair time.At the same time, neural-like cells can directly participate in neurotrosis tissue
Replacement reparation, shorten the time of differentiation, greatly reduce healing the time limit.Therefore, therapeutic effect is obvious, and occurs without irreversible
Blind.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
The accompanying drawing to be used needed for having technology description is briefly described, it should be apparent that, drawings in the following description are only this
Inventive embodiment, for those of ordinary skill in the art, on the premise of not paying creative work, can also basis
The accompanying drawing of offer obtains other accompanying drawings.
Fig. 1 is the optical microscope of the mesenchymal stem cells MSCs of original cuiture in the embodiment of the present invention；
Fig. 2A is the control tube flow cytometer detection result figure of mesenchymal stem cells MSCs in the embodiment of the present invention；
Fig. 2 B are the sample cell flow cytometer detection result figure of mesenchymal stem cells MSCs in the embodiment of the present invention；
Fig. 3 is the shows fluorescent microscopy images of neural-like cells in the embodiment of the present invention.
Specific embodiment
The invention discloses a kind of stem cell medicine and its preparation method and application, the stem cell medicine can avoid blood
Flow and cause the loss of excretion body, strengthen the resistivity of non-injury tissue, shorten repair time, have to Macular lesion
Good curative effect.Those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realization.In particular
It is that all similar replacements and change are apparent to those skilled in the art, they are considered as being included in this
Invention.The method of the present invention and application are described by preferred embodiment, and related personnel can substantially not depart from this
Method described herein and application be modified in the content of the invention, spirit and scope or suitably change with combining realizing and
Using the technology of the present invention.
With reference to embodiment, the present invention is expanded on further.
Embodiment 1 prepares the induction differentiation of mesenchymal stem cells MSCs and neural-like cells
1st, the original cuiture of mesenchymal stem cells MSCs
The marrow for gathering is taken, Ficoll density gradient centrifugation methods are used in super-clean bench, isolate medulla mesenchyma
Stem cell；With DMEM+10%FBS complete mediums it is resuspended after be seeded in the plate of 15cm specifications, be transferred to 5%CO2、37℃
Environment in cultivate.
Liquid is changed after culture 48h, new complete medium is supplemented and is continued to cultivate, the primary cell that acquisition can be passed on；
2nd, the Secondary Culture of mesenchymal stem cells MSCs
1) P0 that can be passed on is taken for cell, original fluid in ware is discarded, and adds 10mL PBSs gently to rinse cell
Aufwuchsplate, discards washing lotion；
2) adding 2mL0.25% trypsase, the plate that rolls makes pancreatin uniform fold in ware bottom, sees under the microscope
The increase of visible cell gap is examined, kytoplasm retraction, when spindle shape cell rounding brightens, adds 10 times of cultures completely of volume immediately
Base terminates digestion；
3) blown and beaten repeatedly with liquid-transfering gun, until cell all comes off into single cell suspension, suspension be transferred in centrifuge tube,
1500rpm room temperatures are centrifuged 5min；
4) supernatant is abandoned, adds DMEM+10%FBS complete medium re-suspended cells, cell density to be adjusted to 1 × 105Cells,
It is placed in 37 DEG C, cultivates in the cell culture incubator of 5% carbon dioxide and saturated humidity；
5) whne cell growth to merge when, repeat step 2), obtain the second generation used by the present embodiment to the 5th generation marrow
Mescenchymal stem cell.Figure 1A is the microscope figure that second generation mesenchymal stem cells MSCs amplifies 40 times, and Figure 1B is second generation marrow
Mescenchymal stem cell amplifies 100 times of microscope figure.
3rd, the identification of mesenchymal stem cells MSCs
Collect the mesenchymal stem cells MSCs P of Secondary Culture3For cell 3 × 106Cell, respectively in three EP pipes, adds
After 10%FBS+PBS piping and druming is uniform, 1500rpm centrifugation 5min abandon supernatant.After cleaning 2 times, 10%FBS+ is added per solencyte
Each 200uL of PBS, are respectively labeled as control tube (C), sample cell 1 (S1), sample cell 2 (S2).Wherein C pipes without anything,
S1 pipes add CD45, CD73, CD90, HLA-DR；S2 pipes add CD105, CD11b, CD19, CD34；After piping and druming is uniform, lucifuge 4
DEG C be incubated 20min, has been incubated addition 10%FBS+PBS piping and druming it is uniform after, after 1500rpm is centrifuged 5min, abandon supernatant.Cleaning 2 times
Afterwards, with the strainer filtering of 70um, up flow type instrument detection surface antigen.Streaming result is as shown in Fig. 2 Fig. 2A is control tube (C)
Flow cytometer detection result, Fig. 2 B are the flow cytometer detection result of sample cell 1 (S1) and sample cell 2 (S2).
According to ISCT promulgations in 2006《The minimum standard of mescenchymal stem cell identification》, tri- kinds of CD73, CD90, CD105
The expression of surface antigen is not less than 95.0%；The expression of CD34, CD45, CD11b, CD19, HLA-DR is not higher than 2.0%.Fig. 2A
It is 8 kinds of corresponding Isotype controls of antigen of mesenchymal stem cells MSCs, Fig. 2 B are 8 kinds of expressions of antigen：CD73、CD90、
CD105 expression is higher than 95.0%；HLA-DR, CD45, CD19, CD34, CD11b expression are less than 2.0%, meet the surface of stem cell
Antigen property, illustrates that stem cell does not occur the phenomenon of differentiation, the characteristics of still maintain stem cell.
4th, mesenchymal stem cells MSCs induction is differentiated to form neural-like cells
Observation mesenchymal stem cells MSCs, collects cell in two 15mL centrifuge tubes when cell density is up to 70% or so,
Centrifugation, PBS is washed one time, the appropriate resuspended precipitation of pre-induced nutrient solution containing Basic Fibroblast Growth Factor is added, by cell
Being inoculated in carries out cell climbing sheet in the Tissue Culture Dish that placed aseptic slide, in 5%CO2, cultivate in 37 DEG C of environment；
Pre-induced culture medium is sucked after 24h, after rinsing 2 times with PBS, addition is containing dimethyl sulfoxide (DMSO) and anethole htpb
Induction broth, is transferred to 5%CO2, cultivated in 37 DEG C of environment；
Inducing culture is sucked after 24h, after washing twice with PBS, the complete of nerve growth factor containing 25ng/mL (NGF) is added
Full culture medium；The mescenchymal stem cell culture medium that control group more renews, continuous culture 12 days, and every other day to adding in culture medium
Plus appropriate nerve growth factor (NGF), it is maintained the final concentration of 25ng/mL.
Authentication step to neural-like cells is as follows：
Treat that about 80 μm of neural bulb diameter removes complete medium, use the culture without basic fibroblast growth factor instead
Base, and the hyclone that volume fraction is 10% is added, adjustment cell concentration is with 5 × 108×L-1It is inoculated in and relies ammonia containing poly
In 24 orifice plates of the coated aseptic slide of acid, per hole inoculating cell suspension 2mL, then to adding 200 μ L β-Tubulin in every hole
III monoclonal antibody, sends culture plate back to incubator and continues to cultivate 4d, and midway does not carry out changing liquid, and close observation is unicellular and nerve
The morphological change and differentiation situation of ball.Anti- β-Tubulin III dyeing is carried out in induction differentiation within the 1st day.After induction differentiation 4d
Immunofluorescence dyeing carries out the identification of phenotypic differentiation, and sets control group, adds the goat anti-rabbit igg secondary antibody of TRITC marks, in
Property gummy sealing, fluorescence microscopy Microscopic observation simultaneously takes pictures.
Neural-like cells are carried out with anti-β-Tubulin III dyeing, it is found that coloration result is positive, as shown in Figure 3.Control group
Do not develop the color.Treatment is further analysed to image with NIH ImageJ softwares, the fluorescent stainings of β-Tubulin III are positive
Picture fusion, superposition, are shown in that the composite multiple fluorescent stainings of β-Tubulin III are positive.Cell body is in fusiformis or ellipse, cell
Cell space reduces to be drawn close to core, and projection is in bipolar or multipole shape, and elongated projection is woven into net, and stereoscopic ring is formed in space
Formula is contacted.
β-Tubulin III are one of expressed earliest neuronal markers in original neural epithelium, are neurons
Structural proteins, it is widely used in neurobiological study as the peculiar mark of neuron.Knowable to picture, culture
Cell expresses the albumen of β-Tubulin III, and the cell for illustrating culture is neural-like cells.
The preparation of the stem cell medicine of embodiment 2
Albumin is mixed with physiological saline, preparation obtains the albumin suspension that concentration is 5%；
The second generation of the preparation of embodiment 1 is collected to the 5th mesenchymal stem cells MSCs for acting as a meaning generation, physiological saline is used
Cleaning 2 times, with the mesenchymal stem cells MSCs of the resuspended collection of albumin suspension, by the content of mesenchymal stem cells MSCs adjust to
3×106Cells, obtains mesenchymal stem cells MSCs preparation, is placed in 4 DEG C of environment and saves backup；
With the resuspended neural-like cells of mesenchymal stem cells MSCs, and the content of neural-like cells is adjusted to 3 ×
106Cells, is obtained stem cell medicine.
The preparation of the stem cell medicine of embodiment 3
Albumin is mixed with physiological saline, preparation obtains the albumin suspension that concentration is 2%；
The second generation of the preparation of embodiment 1 is collected to the mesenchymal stem cells MSCs in the 5th generation, is cleaned with physiological saline 2 times,
With the mesenchymal stem cells MSCs of the resuspended collection of albumin suspension, the density of mesenchymal stem cells MSCs is adjusted to 1 ×
106Cells, obtains mesenchymal stem cells MSCs preparation, is placed in 4 DEG C of environment and saves backup；
With the resuspended neural-like cells of mesenchymal stem cells MSCs, and the density of neural-like cells is adjusted to 1 ×
106Cells, is obtained stem cell medicine.
The preparation of the stem cell medicine of embodiment 4
Albumin is mixed with physiological saline, preparation obtains the albumin suspension that concentration is 15%；
The second generation of the preparation of embodiment 1 is collected to the mesenchymal stem cells MSCs in the 5th generation, is cleaned with physiological saline 2 times,
With the mesenchymal stem cells MSCs of the resuspended collection of albumin suspension, the density of mesenchymal stem cells MSCs is adjusted to 5 ×
106Cells, obtains mesenchymal stem cells MSCs preparation, is placed in 4 DEG C of environment and saves backup；
With the resuspended neural-like cells of mesenchymal stem cells MSCs, and the density of neural-like cells is adjusted to 5 ×
106Cells, is obtained stem cell medicine.
The animal experiment of embodiment 5
1st, animal：The rat model of wet macular lesion is chosen, body weight is close, regardless of male and female, totally 40.Modeling method is
After rat anesthesia matrigel will be injected toward subretinal space.
2nd, experiment packet：The rat of wet macular lesion is divided into 4 groups, every group each 10, packet is as shown in table 1；Often
The transplanting volume of group preparation is 50uL, is detected after 2 weeks.
3rd, checked that inspection parameter is using 3D Topcon OCT-1000：Scan depths 6.0mm × 6.0mm, scanning
Length is adjusted with extent of disease, and scan mode is horizontal or vertical linear scan.Result of the test is shown in Table 1.
The experiment of table 1 packet and result of the test
As can be known from the results, the stem cell medicine for being prepared using embodiment 2~5 carries out re-injection, can be to wet macular lesion
The more obvious improvement result of acquirement, cure rate be 75.0%~95.0%.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of stem cell medicine, it is characterised in that including：Mesenchymal stem cells MSCs, neural-like cells and albumin.
2. stem cell medicine according to claim 1, it is characterised in that the neural-like cells are by the stem cell
Induction is differentiated.
3. stem cell medicine according to claim 1, it is characterised in that the content of the mescenchymal stem cell is 1 × 106
~5 × 106Individual/mL.
4. stem cell medicine according to claim 1, it is characterised in that the content of the neural-like cells is 1 × 106~5
×106Individual/mL.
5. stem cell medicine according to claim 1, it is characterised in that the content ratio of the albumin is 2~15%.
6. stem cell medicine according to claim 1, it is characterised in that the stem cell medicine also includes solvent, described
Solvent is physiological saline.
7. the preparation method of the stem cell medicine described in a kind of claim 1 to 6 any one, it is characterised in that including following
Step：
A1) albumin and physiological saline are mixed, albumin suspension is prepared；
A2) using step a1) the resuspended mesenchymal stem cells MSCs of albumin suspension for preparing, obtain medulla mesenchyma
Stem cell medicine；
A3) using step a2) the resuspended neural-like cells of the mesenchymal stem cells MSCs preparation that prepare, it is obtained and produces
Product.
8. preparation method according to claim 7, it is characterised in that the mesenchymal stem cells MSCs is according to following steps
Obtain：
B1) marrow is centrifuged, primary mescenchymal stem cell is obtained；
B2) the resuspended primary mescenchymal stem cell, is cultivated；
B3) change liquid and remove non-attached cell, add culture medium to continue to cultivate the primary mescenchymal stem cell；
B4) cell cultivated is digested, mescenchymal stem cell is obtained.
9. preparation method described in the stem cell medicine or claim 7 or 8 described in claim 1 to 6 any one is obtained
Application of the stem cell medicine in the Macular lesion biological agent for the treatment of is prepared.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104771414A (en) * | 2015-01-30 | 2015-07-15 | 广州赛莱拉干细胞科技股份有限公司 | Adipose-derived stem cell preparation and preparation method thereof |
| CN105112368A (en) * | 2015-09-30 | 2015-12-02 | 奥思达干细胞有限公司 | Stem cell preparation for treating retinosis and use method of stem cell preparation |
| CN106492197A (en) * | 2016-11-30 | 2017-03-15 | 广州赛莱拉干细胞科技股份有限公司 | A kind of stem cell medicine and its preparation method and application |
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2017
- 2017-03-17 CN CN201710161077.0A patent/CN106729642A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104771414A (en) * | 2015-01-30 | 2015-07-15 | 广州赛莱拉干细胞科技股份有限公司 | Adipose-derived stem cell preparation and preparation method thereof |
| CN105112368A (en) * | 2015-09-30 | 2015-12-02 | 奥思达干细胞有限公司 | Stem cell preparation for treating retinosis and use method of stem cell preparation |
| CN106492197A (en) * | 2016-11-30 | 2017-03-15 | 广州赛莱拉干细胞科技股份有限公司 | A kind of stem cell medicine and its preparation method and application |
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Application publication date: 20170531 |