CN106177918A - A kind of mesenchymal stem cell injection and its preparation method and application - Google Patents
A kind of mesenchymal stem cell injection and its preparation method and application Download PDFInfo
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- A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
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Abstract
The present invention relates to stem cells technology field, particularly to a kind of mesenchymal stem cell injection and its preparation method and application.This mesenchymal stem cell injection includes mescenchymal stem cell, human albumin, low molecular heparin calcium, vitamin C, lentinan, dimethyl sulfoxide and solvent.The mesenchymal stem cell injection that the present invention provides can effectively maintain the vigor of stem cell, it is possible to increase Striatal Dopamine Content, can effectively treat parkinson disease.
Description
Technical field
The present invention relates to stem cells technology field, particularly to a kind of mesenchymal stem cell injection and preparation method thereof and
Application.
Background technology
Parkinson disease (Parkinson ' s disease, PD), also referred to as parkinson, is a kind of common with striatum
Dopaminergic neuron function Progressive symmetric erythrokeratodermia loses the nervous system degeneration disease being characterized.British physician James in 1817
First this disease is described in detail by Parkinson, and its clinical manifestation mainly includes static tremor, bradykinesia, flesh
Tetanic and posture gait disorder, patient can be with non-motor symptoms such as depressed, constipation and sleep disorder simultaneously.China's over-65s
The prevalence of crowd PD is about 1.7%.Major part Parkinsonian is Sporadic cases, and only having the patient less than 10% has house
Race's history.The definite cause of disease of parkinson disease is the clearest and the most definite, and inherited genetic factors, environmental factors, age ageing, oxidative stress etc. all may
Participate in the degeneration death process of PD dopaminergic neuron.Its prominent pathological change is substantia nigra of midbrain dopamine (DA) neuron
Degeneration, disappearance cause Dopamine In Striatum level to decline and eosinophilic inclusion occur in black substance remaining neuron kytoplasm,
I.e. Lewy body (Lewy body).Occur clinical symptoms time, substantia nigra dopaminergic neuron death at least more than 50%, stricture of vagina
Shape body DA content reduces more than 80%.In addition to dopaminergic system, the non-dopaminergic system of Parkinsonian also has bright
Aobvious is impaired, such as the cholinergic neuron of Meynert basal nuclei, the noradrenergic neuron of locus coeruleus, brain stem rapheal nuclei
Serotoninergic neuron, and the autonomic neuron in cerebral cortex, brain stem, spinal cord and periphery.Face in a large number
Bed and animal experiment study find, the abnormal β ripple occurred in ganglion basal is also closely related with the muscular tension of PD.
Treatment PD mainly uses Drug therapy at present, but chemicals has certain side effect, along with biotechnology
Development, stem cell is the most, owing to it has self renewal and can be divided in the research treated in various disease models
Polytype cell, has a wide range of applications, also the most right by the main research as treatment neurodegenerative diseases
As.The behavior observing animal model for parkinsonism becomes the important means of animal model for parkinsonism and drug verification.The most main
If using mesenchymal stem cells MSCs to carry out the research treated, but bone marrow collection can bringing the misery on health to patient.Tire
The stem cell such as dish mescenchymal stem cell have that differentiation potential is big, multiplication capacity is strong, immunogenicity is low, it is low to remain, draw materials conveniently, nothing
The restriction of problems of morals principles, being prone to the feature such as preparation of industrialization and low cost, above biological characteristics makes it possible to become
For tissue repair, the generation of suppression immunological rejection and the multipotency of metabolic disease cell therapy most potential applicability in clinical practice
Stem cell.Can effectively treat Parkinsonian stem cell medicine therefore it provides a kind of there is important medical value.
Summary of the invention
In view of this, the invention provides a kind of mesenchymal stem cell injection and its preparation method and application.Fill between Gai
Matter stem cell injection liquid can effectively maintain the vigor of stem cell, it is possible to increase Striatal Dopamine Content, can effectively treat handkerchief
The gloomy disease of gold.
In order to realize foregoing invention purpose, the present invention provides techniques below scheme:
The invention provides a kind of mesenchymal stem cell injection, including mescenchymal stem cell, human albumin, low molecule
Calciparine, vitamin C, lentinan, dimethyl sulfoxide and solvent.
In the present invention, human albumin is clinical injection level composition, for cells with nutrient；Vitamin C can maintain
The activity of oxide enzyme, thus keep the activity of cell；Low molecular heparin calcium can effectively reduce cell conglomeration, prevents cell infusion
Time blocking filter for transfusion reduce loss cell；DMSO is clinical rank；Solvent can keep the osmotic pressure of cell, beneficially cell
Keep activity.The present invention can preferably maintain the vigor of mescenchymal stem cell after several combinations of substances being used, thus has
Preferably curative effect.
As preferably, every 100mL mesenchymal stem cell injection includes:
Mescenchymal stem cell: (0.5～l) × 108Individual；
Human albumin: 1～5g；
Low molecular heparin calcium: 0.5g；
Vitamin C: 0.3～0.7g；
Lentinan: 0.2～0.6g；
Dimethyl sulfoxide: 5～9mL；
Solvent: supply.
Preferably, every 100mL mesenchymal stem cell injection includes:
Mescenchymal stem cell: 1 × 108Individual；
Human albumin: 1～5g；
Low molecular heparin calcium: 0.5g；
Vitamin C: 0.3～0.7g；
Lentinan: 0.2～0.6g；
Dimethyl sulfoxide: 5～9mL；
Solvent: supply.
In the embodiment that the present invention provides, every 100mL mesenchymal stem cell injection includes:
Mescenchymal stem cell: 1 × 108Individual；
Human albumin: 3g；
Low molecular heparin calcium: 0.5g；
Vitamin C: 0.5g；
Lentinan: 0.4g；
Dimethyl sulfoxide: 7mL；
Solvent: supply.
In another embodiment that the present invention provides, every 100mL mesenchymal stem cell injection includes:
Mescenchymal stem cell: 1 × 108Individual；
Human albumin: 1g；
Low molecular heparin calcium: 0.5g；
Vitamin C: 0.3g；
Lentinan: 0.2g；
Dimethyl sulfoxide: 5mL；
Solvent: supply.
In another embodiment that the present invention provides, every 100mL mesenchymal stem cell injection includes:
Mescenchymal stem cell: 1 × 108Individual；
Human albumin: 5g；
Low molecular heparin calcium: 0.5g；
Vitamin C: 0.7g；
Lentinan: 0.6g；
Dimethyl sulfoxide: 9mL；
Solvent: supply.
As preferably, mescenchymal stem cell is placenta mesenchyma stem cell.
As preferably, placenta mesenchyma stem cell is the 2nd～5 generation placenta mesenchyma stem cells.
In the embodiment that the present invention provides, placenta mesenchyma stem cell is the 3rd generation placenta mesenchyma stem cell.
As preferably, solvent is Multiple electrolytes injection, glucose injection or normal saline.Clinical infusion is with compound recipe
Electrolyte injection is optimal.
As preferably, Multiple electrolytes injection is Bomaili A Multiple electrolytes injection.
In the embodiment that the present invention provides, normal saline is 0.9% sodium chloride injection.
Present invention also offers this mesenchymal stem cell injection preparation improve Striatal Dopamine Content medicine or
Treat the application in Parkinsonian medicine.
Present invention also offers the preparation method of this mesenchymal stem cell injection, including: by human albumin, low molecule
Calciparine, vitamin C, lentinan and solvent mixing, filtration sterilization, add dimethyl sulfoxide, pre-cooling, obtain mixed liquor；
Use the resuspended mescenchymal stem cell of mixed liquor, obtain mesenchymal stem cell injection.
As preferably, filtration sterilization uses degerming level hydrophilic filters filtration sterilization.
As preferably, the aperture of degerming level hydrophilic filters is 0.1～1 μm.
As preferably, the temperature of pre-cooling is 4 DEG C.
The invention provides a kind of mesenchymal stem cell injection and its preparation method and application.This mescenchymal stem cell is noted
Penetrate liquid and include mescenchymal stem cell, human albumin, low molecular heparin calcium, vitamin C, lentinan, dimethyl sulfoxide and molten
Matchmaker.There is advantages that
1, the present invention uses placenta mesenchyma stem cell to prepare stem cell injection liquid, draws materials conveniently, does not violate ethical issues；
2, mesenchymal stem cell injection prepared by the present invention can effectively maintain the vigor of stem cell, under low temperature state
Frozen 48 months stem cell motility rates, easy to use；
3, the mesenchymal stem cell injection of the present invention can improve Striatal Dopamine Content, can effectively treat parkinson
Sick.
Accompanying drawing explanation
Fig. 1 shows the qualification result of placenta mesenchyma stem cell；Wherein 1-1 shows that P3 cultivates after 72h under the microscope for cell
Amplify the picture of 40 times；1-2 shows that P3 amplifies the picture of 100 times under the microscope after cultivating 72h for cell；
Fig. 2 shows the dyeing qualification result of placenta mesenchyma stem cell；2-1 shows that P3 is for placenta mesenchyma stem cell induction differentiation
For amplifying the picture of 40 times after osteoblast under the microscope；2-2 shows that P3 is induced to differentiate into skeletonization for placenta mesenchyma stem cell
The picture of 100 times is amplified under the microscope after cell；After 2-3 shows that P3 is induced to differentiate into lipoblast for placenta mesenchyma stem cell
Amplify the picture of 40 times under the microscope；2-4 shows that P3 is induced to differentiate into after lipoblast micro-for placenta mesenchyma stem cell
The picture of 100 times is amplified under mirror.
Detailed description of the invention
The invention discloses a kind of mesenchymal stem cell injection and its preparation method and application, those skilled in the art can
To use for reference present disclosure, it is suitably modified technological parameter and realizes.Special needs to be pointed out is, all similar replacements and change are to this
Being apparent from for skilled person, they are considered as being included in the present invention.The method of the present invention and application are
Being described by preferred embodiment, related personnel substantially can be to herein in without departing from present invention, spirit and scope
Described methods and applications are modified or suitably change and combine, and realize and apply the technology of the present invention.
Mescenchymal stem cell (mesenchymal stem cells, MSC) is the important member of stem cell line, derives from
Grow mesoderm in early days and ectoderm, belong to pluripotent stem cell, in vivo or under external specific inductive condition, can be divided into
The Various Tissues cells such as islets of langerhans, nerve, blood vessel endothelium, bone, cartilage, muscle, liver, cardiac muscle.
In mesenchymal stem cell injection that the present invention provides and its preparation method and application, raw materials used medicine or adjuvant are equal
Can be buied by market.
Below in conjunction with embodiment, the present invention it is expanded on further:
The preparation of embodiment 1 placenta mesenchyma stem cell
1, the primary separation of placenta mesenchyma stem cell
Aseptically, take the fresh human placenta thrown aside puerperal, peel off Placenta Hominis, rinse with PBS buffer solution, by Placenta Hominis group
Knit and be cut into 5 × 5cm2, add 0.25% trypsinization of 3-5 times of volume, be placed on 37 DEG C of constant-temperature tables of 200r/min, digestion
30min, every the violent shaken several times of 10min, to remove epithelial cell.
After having digested, transfer them in liquid containing bottle, add 150mL PBS buffer solution, be aggressively shaken, repeat this behaviour
Make 2 times.Placenta tissue is transferred in small beaker, shreds to 1mm3.Transfer in liquid containing bottle, add 20mL 0.5%I type
Collagenase and complete medium (DMEM in high glucose+10%FBS), make the final concentration of 0.1-0.2% of collagenase.It is placed in 37 DEG C,
In the constant-temperature table of 250r/min, until piece of tissue is melted substantially.Postdigestive tissue fluid is diluted with PBS buffer solution,
1500r/min, centrifugal 5min, discard supernatant, by the resuspended precipitation of PBS buffer solution, uses 100 μm filter screens to filter, filtrate
1500r/min, centrifugal 5min, it is thus achieved that placenta mesenchyma stem cell.
Resuspended with complete medium, by 0.3-1 × 106Cells/mL is inoculated in 10cm culture dish, and every ware adds
100 μ L 1 μ g/mL EGF and the SHH of final concentration of 100ng/mL.It is placed in CO2Incubator is cultivated, after 48h, changes liquid, follow-up every 2
～3d changes a subculture.
2, Human plactnta mescenchymal stem cell Secondary Culture
When primary Human plactnta growth of mesenchymal stem cells to 80% merges, i.e. disappear with the EDTA-Trypsin of 0.25%
Change, carry out Secondary Culture, after reaching 2-5 generation, observation of cell growth conditions, with using the washing of PBS buffer solution after trypsinization,
Centrifugal collection.
3, the 2-5 that system of identification is standby is for placenta mesenchyma stem cell, sees including basis of microscopic observation, flow cytometer detection and dyeing
Examining, qualification result is with reference to the standard customized in " international cell therapy association (ISCT) ", and result is as follows:
1) basis of microscopic observation
The 3rd generation placenta mesenchyma stem cell after cultivating 3d as seen from Figure 1 has been formed and has filled between more typical Placenta Hominis
Matter sample, and have obvious near-wall air curtain.
2) flow cytometer detection
The placenta mesenchyma stem cell taking for the 3rd generation carries out flow cytomery.As can be seen from Table 1, this cell is expressed
CD105, CD73, CD90 and HLA-ABC (expression be higher than 90%), do not express CD45, CD34 and HLA-DR (expression in
1%).
The flow cytometer detection result of table 1 placenta mesenchyma stem cell
| Traget antibody | Positive rate (%) | Traget antibody | Positive rate (%) |
| CD34 | 0.06 | CD105 | 99.15 |
| CD90 | 99.89 | HLA-ABC | 99.23 |
| CD45 | 0.13 | HLA-DR | 0.42 |
| CD73 | 97.8 | / | / |
3) dyeing is observed
Take the placenta mesenchyma stem cell in the 3rd generation carrying out Differentiation Induction in vitro is lipoblast and osteoblast, dyeing
Observe.The placenta mesenchyma stem cell that as seen from Figure 2 prepared by the present invention through suitable condition can to lipoblast with become
Bone cell differentiation, it was demonstrated that placenta mesenchyma stem cell prepared by the present invention has Multidirectional Differentiation ability.
Conclusion: above-mentioned result of the test may certify that the cell prepared by the present invention possesses the characteristic of mescenchymal stem cell.
The preparation of embodiment 2 placenta mesenchyma stem cell preparation
The method of preparation 100mL stem cell injection liquid: by human albumin's stock solution 30mL (matter that albumin concentration is 10%
Amount volume ratio final concentration of 3%), low molecular heparin calcium 0.5mL (mass volume ratio final concentration of 0.5%), vitamin C 0.5g
(mass volume ratio final concentration of 0.5%), lentinan 0.4g (mass volume ratio final concentration of 0.4%) and 0.9% sodium chloride
Injection 61.6mL mixes, and is slowly added to DMSO 7mL (volumn concentration is 7%), puts into pre-cooling in 4 DEG C of refrigerators, by Placenta Hominis
Mescenchymal stem cell (final concentration of cells l × 106Individual/mL) resuspended in mixed liquor, by the placenta mesenchyma stem cell note of preparation
Penetrate liquid to be sub-packed inIn cell cryopreservation bag, prepare complete.
The preparation of embodiment 3 placenta mesenchyma stem cell preparation
The method of preparation 100mL stem cell injection liquid: by human albumin's stock solution 5mL (matter that albumin concentration is 20%
Amount volume ratio final concentration of 1%), low molecular heparin calcium 0.5mL (mass volume ratio final concentration of 0.5%), vitamin C 0.3g
(mass volume ratio final concentration of 0.3%), lentinan 0.2g (mass volume ratio final concentration of 0.2%) and 0.9% sodium chloride
Injection 88.8mL mixes, and is slowly added to DMSO 5mL (volumn concentration is 5%), puts into pre-cooling in 4 DEG C of refrigerators, by Placenta Hominis
Mescenchymal stem cell (final concentration of cells l × 106Individual/mL) resuspended in mixed liquor, by the placenta mesenchyma stem cell note of preparation
Penetrate liquid to be sub-packed inIn cell cryopreservation bag, prepare complete.
The preparation of embodiment 4 placenta mesenchyma stem cell preparation
The method of preparation 100mL stem cell injection liquid: by human albumin's stock solution 25mL (matter that albumin concentration is 20%
Amount volume ratio final concentration of 5%), low molecular heparin calcium 0.5mL (mass volume ratio final concentration of 0.5%), vitamin C 0.7g
(mass volume ratio final concentration of 0.7%), lentinan 0.6g (mass volume ratio final concentration of 0.6%) and vigorous arteries and veins power 64.4mL
Mixing, is slowly added to DMSO 9mL (volumn concentration is 9%), puts into pre-cooling in 4 DEG C of refrigerators, by placenta mesenchyma stem cell
(final concentration of cells l × 106Individual/mL) resuspended in mixed liquor, the placenta mesenchyma stem cell injection of preparation is sub-packed inIn cell cryopreservation bag, prepare complete.
The conventional sense of embodiment 5 placenta mesenchyma stem cell injection
Placenta mesenchyma stem cell injection prepared by above-described embodiment 2-4 all should keep sample and carry out endotoxin detection, antibacterial
Fungal detection, detection of mycoplasma, testing result is negative for qualified.
The Function Identification of embodiment 6 placenta mesenchyma stem cell injection
(1) test packet:
Test group 1: the placenta mesenchyma stem cell injection of embodiment 2 preparation；
Test group 2: the placenta mesenchyma stem cell injection of embodiment 3 preparation；
Test group 3: the placenta mesenchyma stem cell injection of embodiment 4 preparation；
Matched group 1: matched group 1 is substantially the same manner as Example 2, simply without vitamin C, its preparation method is: will be white
Protein concentration is human albumin's stock solution 60mL (mass volume ratio final concentration of 6%), the low molecular heparin calcium 1mL (matter of 10%
Amount volume ratio final concentration of 1%), lentinan 0.8g (mass volume ratio final concentration of 0.8%) and 0.9% sodium chloride injection
25mL mixes, and is slowly added to DMSO 14mL (volumn concentration is 14%), puts into pre-cooling in 4 DEG C of refrigerators, by placenta mesenchyma
Stem cell (final concentration of cells l × 106Individual/mL) resuspended in mixed liquor, the placenta mesenchyma stem cell injection of preparation is divided
It is loaded onIn cell cryopreservation bag, prepare complete.
Matched group 2: matched group 2 is substantially the same manner as Example 2, simply without vitamin C and lentinan, its preparation side
Method is: by human albumin's stock solution 30mL (mass volume ratio final concentration of 3%) that albumin concentration is 10%, Low molecular heparin
Calcium 0.5mL (mass volume ratio final concentration of 0.5%) and 0.9% sodium chloride injection 662.5mL mixing, is slowly added to DMSO
7mL (volumn concentration is 7%), puts into pre-cooling in 4 DEG C of refrigerators, by placenta mesenchyma stem cell (final concentration of cells l × 106
Individual/mL) resuspended in mixed liquor, the placenta mesenchyma stem cell injection of preparation is sub-packed inCell freezes
Deposit in bag, prepare complete.
Matched group 3: only add vitamin C in matched group 3 injection, its preparation method is: by vitamin C 1g (mass body
Long-pending ratio final concentration of 1%) and 0.9% sodium chloride injection 93mL mixing, (volumn concentration is to be slowly added to DMSO7mL
7%), pre-cooling in 4 DEG C of refrigerators is put into, by placenta mesenchyma stem cell (final concentration of cells l × 106Individual/mL) weight in mixed liquor
Outstanding, the placenta mesenchyma stem cell injection of preparation is sub-packed inIn cell cryopreservation bag, prepare complete.
(2) test method and result
1) frozen test
Mescenchymal stem cell injection prepared by placenta mesenchyma stem cell injection and the matched group 1-4 of embodiment 2-4
Liquid is lowered the temperature through programmed cooling instrument, places in liquid nitrogen and preserves.The cell cryopreservation bag of several frozen formula more than taking out from liquid nitrogen,
Carry out 37 DEG C of water-baths are recovered, in 2min, complete resuscitation process, take the cell suspension after a small amount of recovery and carry out Trypan Blue
Counting, same method measures frozen 6 months, 12 months, 24 months and the Cell viability of 48 months respectively, and result is as shown in table 2:
Table 2 each test group Cell viability testing result
| The frozen time | Embodiment 2 | Embodiment 3 | Embodiment 4 |
| 6 months | 98.7% ± 0.4% | 98.7% ± 0.4% | 98.7% ± 0.4% |
| 12 months | 95.8% ± 0.2% | 95.5% ± 0.1% | 96.6% ± 0.2% |
| 24 months | 91.1% ± 0.3% | 90.1% ± 0.3% | 91.8% ± 0.4% |
| 48 months | 87.8% ± 0.2% | 87.2% ± 0.1% | 89.6% ± 0.3% |
Table 3 each cellular control unit survival rate test result
| The frozen time | Matched group 1 | Matched group 2 | Matched group 3 |
| 6 months | 98.7% ± 0.4% | 98.7% ± 0.4% | 98.7% ± 0.4% |
| 12 months | 90.1% ± 0.3% | 85.1% ± 0.1% | 80.1% ± 0.3% |
| 24 months | 86.4% ± 0.1% | 80.3% ± 0.2% | 68.3% ± 0.2% |
| 48 months | 70.6%+0.2% | 68.7% ± 0.4% | 55.7% ± 0.3% |
From table 2,3 it can be seen that the placenta mesenchyma stem cell injection of the present invention is recovered after long-time preservation, its
Vigor remains unchanged higher, and the general cell therapy motility rate reaching national requirements is more than or equal to 85%, and Cell viability passes through
After the cryopreservation resuscitation of 48 months, motility rate is above comparative example 1-4.
2) differentiation culture test
The cell of recovery after frozen for embodiment 2 injection 48 months is carried out the training of serum-free mescenchymal stem cell culture medium
Supporting, observation of cell form, to becoming fat, becoming cartilage and Osteoblast Differentiation, dyed examining under a microscope all becomes positive, it was demonstrated that it depends on
Old holding Multidirectional Differentiation ability.
3) flow cytometer detection
The cell of recovery after frozen for embodiment 2 injection 48 months is carried out flow cytometer detection, testing result such as following table:
Flow cytometer detection result after the recovery of table 4 placenta mesenchyma stem cell
| Traget antibody | Positive rate (%) | Traget antibody | Positive rate (%) |
| CD34 | 0.08 | CD105 | 98.87 |
| CD90 | 99.74 | HLA-ABC | 98.62 |
| CD45 | 0.31 | HLA-DR | 0.63 |
| CD73 | 96.8 | / | / |
From can be seen that with up flow type result through between the placenta mesenchyma stem cell of frozen 24 months and fresh preparation
Mesenchymal stem cells phenotype is consistent, it was demonstrated that placenta mesenchyma stem cell prepared by the present invention preserve for a long time to cell phenotype and dryness without
Impact.
Embodiment 7 placenta mesenchyma stem cell injection is to the treatment Parkinsonian experiment effect of mice
(1) qualification of PD model:
Buying 60 PD rat models, model starts after setting up for 1 week, induces it with 0.5mg/kg apomorphine lumbar injection
Rotating to strong side, record rat number of revolutions, measure every time and continue 40 minutes, 1 times a week, all numbers of revolutions are more than 7 times/min
Above, it is successful model.Buy PD model and be successful model.
(2) treatment of MSCs:
60 PD rat models are randomly divided into 3 groups, often group 20, respectively A, B, C tri-groups:
A group: for blank；
B group: for normal saline group；
C group: the embodiment of the present invention 2 placenta mesenchyma stem cell preparation transplantation group；
Use CM-Dil labeled cell: the stem cell medicine compositions of the present invention is taken 200ul cell suspension, add 1ul
CM-Dil fluorescent dye, 37 DEG C of lucifuges hatch 5min, and then 4 DEG C of lucifuges hatch 15min, with brine twice, are placed in
Fluorescence microscopy Microscopic observation, PD rat model 10% chloral hydrate (0.2mL/100g) intraperitoneal injection of anesthesia, routine disinfection, Gu
On brain solid positioner, keep anterior-posterior horizontal, transplant injection site consistent with modeling injection site.Transplanted cells quantity
For A group blank, i.e. it is left intact, B group injection 10ul equal-volume normal saline, C group orthotopic transplantation MSCs, 1 ×
106Individual/10ul.All sewing up the incision for 3 groups, every day normally feeds cleaning.
The detection method of therapeutic effect generally uses animal behavioral study, SABC to detect, and concrete test method is as follows:
(1) animal behavioral study
1) after transplanting MSCs, each group PD rat timing lumbar injection apomorphine (0.5mg/kg) induction PD sample disease weekly
Shape, about 10min are after rat spin stabilization, and shooting records rat number of revolutions in 5min, continuous induction, observes 4 weeks.
2) the 2nd week, C group (MSCs transplantation group) was after transplanting 2 weeks, and the number of revolutions of PD rat starts to reduce, and transplants continuously
Latter 4th week, number of revolutions was considerably less than A group (blank group) (P ＜ 0.05), and the circling behavior of PD rat obtains the most notable
Improvement.B group starts for the 2nd week after transplanting until the 8th week (observing the end of term) number of revolutions and matched group are without significant change.
3) after PD rat cell is transplanted, its rotating cycle is down to 3～6 circles/min by 12 before transplanting～15 circle/min.
(2) expression of SABC detection TH
1), after the fixing matched group of perfusion and cell transplantation group PD rat, take cerebral tissue and prepare frozen section and do TH immuning tissue
Chemical staining, observes the damage situation of rat brain nigral dopamine cell；And at fluorescence microscopy Microscopic observation CM-Dil labelling
Transplanted cells survival in brain and growing state.
2) TH immune histochemical evidence: the TH immuning positive cell of matched group PD rat damage side black substance is less,
Postoperative 4 weeks of cell transplantation, it can be seen that the TH immuning positive cell that damage side black substance is dispersed in, cell space presents ellipse or vertebral body
Shape.
3) ELISA detects PD rat brain striatum DOPAMINE CONTENT IN RABBIT, each group of rat of 4w, TLISA method detection after cell transplantation
Brain striatal DOPAMINE CONTENT IN RABBIT, each group content is the most as shown in table 5:
Rat brain Striatal Dopamine Content testing result respectively organized by table 5
As can be seen from Table 5, C group after cell transplantation 4 weeks the Striatal Dopamine Content of PD rat apparently higher than it
He is two groups.Visible, the placenta mesenchyma stem cell compositions of the present invention can effectively treat parkinson disease.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For Yuan, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a mesenchymal stem cell injection, it is characterised in that include mescenchymal stem cell, human albumin, low molecule liver
Element calcium, vitamin C, lentinan, dimethyl sulfoxide and solvent.
Mesenchymal stem cell injection the most according to claim 1, it is characterised in that every 100mL mescenchymal stem cell note
Penetrate liquid to include:
Mescenchymal stem cell: (0.5～l) × 108Individual；
Human albumin: 1～5g；
Low molecular heparin calcium: 0.5g；
Vitamin C: 0.3～0.7g；
Lentinan: 0.2～0.6g；
Dimethyl sulfoxide: 5～9mL；
Solvent: supply.
Mesenchymal stem cell injection the most according to claim 1 and 2, it is characterised in that every 100mL mescenchymal stem cell
Injection includes:
Mescenchymal stem cell: 1 × 108Individual；
Human albumin: 1～5g；
Low molecular heparin calcium: 0.5g；
Vitamin C: 0.3～0.7g；
Lentinan: 0.2～0.6g；
Dimethyl sulfoxide: 5～9mL；
Solvent: supply.
Mesenchymal stem cell injection the most according to any one of claim 1 to 3, it is characterised in that described mesenchyme
Stem cell is placenta mesenchyma stem cell.
Mesenchymal stem cell injection the most according to any one of claim 1 to 4, it is characterised in that described solvent is
Multiple electrolytes injection, glucose injection or normal saline.
Mesenchymal stem cell injection the most according to claim 5, it is characterised in that described Multiple electrolytes injection is
Bomaili A Multiple electrolytes injection.
7. as according to any one of claim 1 to 6, mesenchymal stem cell injection improves Striatal Dopamine Content in preparation
Medicine or treat the application in Parkinsonian medicine.
8. the preparation method of mesenchymal stem cell injection as according to any one of claim 1 to 6, it is characterised in that including:
Human albumin, low molecular heparin calcium, vitamin C, lentinan and solvent are mixed, filtration sterilization, add dimethyl sub-
Sulfone, pre-cooling, obtain mixed liquor；
Use the resuspended mescenchymal stem cell of described mixed liquor, obtain mesenchymal stem cell injection.
Preparation method the most according to claim 8, it is characterised in that described filtration sterilization uses degerming level hydrophilic filter
Device filtration sterilization, the aperture of described degerming level hydrophilic filters is 0.1～1 μm.
Preparation method the most according to claim 8 or claim 9, it is characterised in that the temperature of pre-cooling is 4 DEG C.
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