CN108619169A - A kind of mesenchymal stem cell injection and preparation method for treating cerebral arterial thrombosis - Google Patents
A kind of mesenchymal stem cell injection and preparation method for treating cerebral arterial thrombosis Download PDFInfo
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Abstract
The invention discloses a kind of mesenchymal stem cell injection and preparation method for treating cerebral arterial thrombosis, the injection includes that quantity is 5 × 105‑2×106The physiological saline of 50% glucose injection and surplus that Compound vitamine injection that formula mannitol injection liquid that human serum albumin that mescenchymal stem cell, the mass volume ratio of the activation of a/mL are 1% 5%, mass volume ratio are 1% 8%, mass volume ratio are 1% 5%, mass volume ratio are 1% 5%.The mesenchymal stem cell injection for the treatment of cerebral arterial thrombosis disclosed by the invention can discharge the proinflammatory cytokines such as TNF α, IL 1, IL 6 after cerebral ischemia during inflammation damnification, be used for infusion of therapeutic cerebral arterial thrombosis.
Description
Technical field
The present invention relates to technical field of cell biology, it is more particularly related to which a kind for the treatment of ischemic cerebral apoplexy
In mesenchymal stem cell injection and preparation method.
Background technology
Ishemic stroke is most common type in apoplexy, it is since cerebral blood flow blocks, and intracerebral hypoxic-ischemic causes god
Through damage, long-term or permanent disability can be caused.The existing therapeutic choice of ishemic stroke is very limited, and only selection includes molten
Suppository such as tPA or surgical mechanical take bolt Reperfu- sion to perform the operation, and these means are all preferably several small after being limited to apoplexy generation
When (3-6h) in implement, be possible to obtain curative effect.Many patients because of various reasons, it is extremely of short duration to miss this often
Golden hour window, it is finally helpless to simply accept the nursing of supportive or Palliative.The long term medical cost of apoplexy
It is huge, many patients need to extend hospitalization, long-term physical therapy or rehabilitation, and may need long-term institutional care
Or residential care.
Mescenchymal stem cell (Mesenchymal Stem Cells, MSCs) is present in almost all of tissue, and
It can be isolated from marrow, adipose tissue, umbilical cord, placenta and amniotic fluid.MSCs not only has powerful self-renewal capacity
And multi-lineage potential, also there are the characteristics such as immunoregulation ability and low immunogenicity, be that fields of implantation has a extensive future
Renewable source cell, also therefore favored by more and more field of biology scientists and medical researchers；It is more next
More research confirms that mescenchymal stem cell (MSCs) can play immunological regulation by the cell factor of secretion and reduce inflammation
Effect.Cerebral arterial thrombosis is a complicated case process, and inflammatory reaction is in close relations with cerebral ischemia.All many cells because
The process and act on and differ that son generates generate protective effect to cerebral ischemia, pass through tune when pro-inflammatory cytokine is occupied an leading position
The expression for saving different cytokines, inhibits the treatment of inflammatory reaction that may have potential clinical value.The study found that
Mesenchymal stem cells can alleviate inflammatory reaction, the biological attribute that its this promotion inflammation changes to the multiplicative stage, serious
It is very important in inflammation suppression therapy.Therefore, it is related that mescenchymal stem cell was more and more extensive is applied to treatment inflammatory
Among disease.
However, since MSCs is influenced by vivo environment, therapeutic effect is uncertain, therefore stem cell repaiies inflammation at present
Recovering technology is still to be a problem to be solved.
Invention content
It is an object of the invention to solve at least the above, and provide the advantages of at least will be described later.
It is a still further object of the present invention to provide a kind of for alleviating acute inflammation and for treating ischemic cerebral apoplexy
Treat the mesenchymal stem cell injection and preparation method thereof of cerebral arterial thrombosis.
To achieve the goals above, the invention discloses a kind of mescenchymal stem cell injections for treating cerebral arterial thrombosis
Liquid, including following components：
Quantity is 5 × 105-2×106The mescenchymal stem cell of the activation of a/mL, people's blood that mass volume ratio is 1%-5%
The composite vitamin injection that formula mannitol injection liquid that albumin, mass volume ratio are 1%-8%, mass volume ratio are 1%-5%
The physiological saline of liquid, 50% glucose injection that mass volume ratio is 1%-5% and surplus.
Preferably, in the present invention, the mescenchymal stem cell of the activation is the mescenchymal stem cell of TNF-α activation, IL-1
Any one in the mescenchymal stem cell that the mescenchymal stem cell and TNF-α and IL-1 of activation activate simultaneously.
Preferably, the present invention in, the mescenchymal stem cell be umbilical cord mesenchymal stem cells, mesenchymal stem cell,
Any one in fat mesenchymal stem cell.
The invention discloses a kind of preparation methods of mesenchymal stem cell injection that treating cerebral arterial thrombosis, including such as
Lower step：
Step 1: preparing mescenchymal stem cell, and cultivate forth generation mescenchymal stem cell；
Step 2: forth generation mescenchymal stem cell culture to 70%-80% is merged, the mesenchyma for preparing activation is dry thin
Born of the same parents；
Step 3: formula described in accordance with the claim 1, prepares and to weigh people's blood that mass volume ratio is 1%-5% white
Compound vitamine injection that formula mannitol injection liquid that albumen, mass volume ratio are 1%-8%, mass volume ratio are 1%-5%,
Mass volume ratio is 50% glucose injection of 1%-5%, and mixing；
Step 4: being 5 × 10 by quantity5-2×106The mescenchymal stem cell of the activation of a/mL is resuspended in step 3 mixing
In liquid afterwards, and the physiological saline of surplus is added, the mesenchymal stem cell injection for the treatment of cerebral arterial thrombosis is prepared.
Preferably, in the present invention, in the step 2, the preparation process of the mescenchymal stem cell of the activation is：
By the fusion of forth generation mescenchymal stem cell culture to 70-80%, when taking out forth generation mescenchymal stem cell culture
Culture medium in the mescenchymal stem cell culture dish used rinses forth generation mescenchymal stem cell with phosphate buffer；
With specific medium culture forth generation mescenchymal stem cell；
When the concentration of forth generation mescenchymal stem cell reaches 5 × 105-2×106When a/mL, the mesenchyma activated is dry
Cell.
Preferably, in the present invention, when the mescenchymal stem cell of the activation is the mescenchymal stem cell of TNF-α activation,
The specific culture medium is the culture medium of the α-MEM of the TNF-α containing a concentration of 20ng/mL；
When the mescenchymal stem cell of the activation is the mescenchymal stem cell of IL-1 activation, the specific culture medium is
The culture medium of the α-MEM of IL-1 containing a concentration of 50ng/mL；
When the mescenchymal stem cell of the activation is TNF-α and the IL-1 mescenchymal stem cells of activation simultaneously, the spy
Fixed culture medium is the culture of the TNF-α culture medium containing a concentration of 20ng/mL and the α-MEM of the IL-1 of a concentration of 50ng/mL
Base.
Preferably, in the present invention, in the step 1, the mescenchymal stem cell is umbilical cord mesenchymal stem cells, and the
The incubation of four generation umbilical cord mesenchymal stem cells is：
A) fresh mature healthy fetus umbilical cord, is taken, with the normal saline flushing containing 2 times of 100U/mL penicillin and streptomysin
Bloodstain is removed, Wal Tong Shi glue is removed, the Wal Tong Shi glue after stripping is cut into less than 1mm3Fritter；
B), the Wal Tong Shi glue shredded is spread evenly across to the bottom of cell culture apparatus, delayed into the cell culture apparatus
It is slow that 10mL serum free mediums are added, it is placed in 37 DEG C, 5v/v%CO2, cultivate in saturated humidity environment, by primitive cell culture 7
After it, serum-free medium is replaced, culture reaches 70% fusion to cell, removes serum-free medium, addition concentration difference
1min is digested for 0.25% and 0.1% trypsin-EDTA sodium, umbilical cord mesenchymal stem cells, which are shunk, is detached from bottle
The culture supernatant previously removed is added in wall at this time, neutralizes trypsin-EDTA solutions, and gently blow and beat using pipette, will
Umbilical cord mesenchymal stem cells suspension moves into 50mL centrifuge tubes, removes supernatant after centrifugation, rejoins Serum-free complete medium
Umbilical cord mesenchymal stem cells after centrifugation are resuspended；
C), according to 1 pass 1.5 inoculation 10cm culture dishes, umbilical cord mesenchymal stem cells culture fusion 70% when according to it is described b)
Operating process secondary culture, be trained forth generation umbilical cord mesenchymal stem cells.
Preferably, in the present invention, the mescenchymal stem cell is mesenchymal stem cell, and forth generation medulla mesenchyma is dry
The incubation of cell is：
1), in anterior superior spine point of puncture, 50mL bone marrow fluids is extracted with medullo-puncture needle, are injected in the sterile centrifugation tube of 50mL；With
PBS liquid of the equivalent containing 20 μ/mL heparin dilutes, and it is 1.073g/mL's that isometric proportion, which is added, along the tube wall of sterile centrifugation tube
Lymphocyte separation medium is centrifuged, centrifugal rotational speed 2000rpm, and centrifugation time 30min draws mononuclearcell after centrifugation
Layer；Then sterile saline centrifuge washing 2 times is used, the rotating speed of each centrifugal process is 1000rpm, centrifugation time 5min,
Liquid is discarded supernatant, precipitation is taken；
2) precipitation that centrifugation obtains is suspended with serum free medium, cell is counted, by 5 × 106A/mL is inoculated in
In culture bottle, 37 DEG C are set, cultivated in the incubator of the carbon dioxide that volume fraction is 5%, saturated humidity；After culture 3 days, replace
Serum-free medium discards non-attached cell, later every 3 days 1 time replacement culture solution；After 7-9 days, mesenchymal stem cell melts
When closing up to 50%-60%, first time passage is carried out；Serum-free medium is removed, is respectively 0.25% and 0.1% using concentration
37 DEG C of digestion 1min of trypsase-EDTA, marrow MSC, which is shunk, is detached from bottle wall, and the culture supernatant previously removed is added, neutralizes
Trypsase-EDTA, and gently blown and beaten using pipette, medulla mesenchyma cell suspension is moved into 50mL sterile centrifugation tubes；
1000rpm centrifuges 8min, discards supernatant liquid；Serum-free complete medium resuspension is rejoined, is counted；
3), 1-1.5 is passed according to 1 be inoculated with 10cm culture dishes；When 60%-80% is merged in mesenchymal stem cell culture, press
Secondary culture is carried out according to the operating process 2).
Preferably, in the present invention, the mescenchymal stem cell is fat mesenchymal stem cell, and forth generation fat mesenchymal is dry
The incubation of cell is：
S1, separation abdomen white adipose tissue, reject visible capilary and musculature, and sterile PBS washs three times, then
Adipose tissue is shredded to 1mm with sterile scissors3Paste below adds sterile digestive juice, 45- is at the uniform velocity stirred in 37 DEG C of water-baths
50min, until tissue block digestion is clean；Filtrate is collected by filtration in 200 mesh screens, then uses the low sugar containing 10% fetal calf serum
DMEM culture medium equivalent neutralizes, and 1500rpm centrifuges 10min, discards supernatant liquid, obtains fat mesenchymal stem cell；
S2, with 1 × 106The cell density of a/mL is resuspended fat mesenchymal stem cell with serum free medium, and is inoculated with
In culture bottle, 37 DEG C, volume fraction be 5% carbon dioxide, cultivate in saturated humidity incubator, work as fat mesenchymal stem cell
When growing to 70%-80% fusions, fat mesenchymal stem cell is digested using 0.25% trypsin solution, with 1：3 inoculation
Ratio, which is inoculated into new culture bottle, carries out secondary culture；Fat mesenchymal stem cell be fused to no more than 80%, 1000rpm from
Heart 8min, discards supernatant liquid, rejoins Serum-free complete medium and fat mesenchymal stem cell is resuspended, count；
S3, according to 1 pass 1-1.5 be inoculated with 10cm culture dishes, fat mesenchymal stem cell culture merge 60%-80% when according to
S2 secondary cultures.
The mesenchymal stem cell injection for the treatment of cerebral arterial thrombosis provided by the invention is for treating ischemic cerebral apoplexy.
The present invention includes at least following advantageous effect：
1, it is dry to be conducive to the mesenchyma activated for the mesenchymal stem cell injection for the treatment of cerebral arterial thrombosis provided by the invention
Cell in vitro under suspension microenvironment stabilization, the mescenchymal stem cell of activation can keep higher work in injection
Power, the mescenchymal stem cell for being conducive to activation maintains activity, and ensure that the safety of clinical infusion.
2, the mesenchymal stem cell injection for the treatment of cerebral arterial thrombosis provided by the invention can significantly improve acute inflammation,
For treating ischemic cerebral apoplexy.
Part is illustrated to embody by further advantage, target and the feature of the present invention by following, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Description of the drawings
Fig. 1 be it is of the present invention treatment cerebral arterial thrombosis mesenchymal stem cell injection to intravenous injection after
RT-PCR testing results.
Fig. 2 is the nervous function after the mesenchymal stem cell injection injection for the treatment of cerebral arterial thrombosis of the present invention
Testing result.
Specific implementation mode
The present invention is described in further detail with reference to the accompanying drawings and embodiments, to enable those skilled in the art's reference
Specification word can be implemented according to this.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein are not precluded one or more
The presence or addition of a other elements or combinations thereof.
One, the preparation of umbilical cord mesenchymal stem cells
Operated in superclean bench, a), umbilical cord separation：Fresh mature healthy fetus umbilical cord is taken, with containing 2 times
The normal saline flushing of 100U/mL penicillin and streptomysin removes bloodstain, Wal Tong Shi glue is removed, by the Wall Tong Shi after stripping
Glue is cut into less than 1mm3Fritter；
B), mesenchymal cell original cuiture：The Wal Tong Shi glue shredded is spread evenly across to the bottom of cell culture apparatus, to
It is slowly added to 10mL serum free mediums in the cell culture apparatus, is placed in 37 DEG C, 5v/v%CO2, train in saturated humidity environment
It supports, after primitive cell culture 7 days, replaces serum-free medium, culture reaches 70% fusion to cell, removes serum-free training
Nutrient solution, addition concentration are respectively 0.25% and 0.1% trypsin-EDTA sodium digestion 1min, umbilical cord mesenchyma
Stem cell, which shrinks, is detached from bottle wall, and the culture supernatant previously removed is added at this time, neutralizes trypsin-EDTA solutions, and use
Pipette is gently blown and beaten, and umbilical cord mesenchymal stem cells suspension is moved into 50mL centrifuge tubes, 1000rpm centrifuges 8min, removed after centrifugation
Supernatant is removed, Serum-free complete medium is rejoined and the umbilical cord mesenchymal stem cells after centrifugation is resuspended；
C), secondary culture：1.5 inoculation 10cm culture dishes are passed according to 1, umbilical cord mesenchymal stem cells culture is pressed when merging 70%
According to operating process secondary culture b), it is trained forth generation umbilical cord mesenchymal stem cells.
Two, the preparation of mesenchymal stem cell
1), using conventional method in anterior superior spine point of puncture, 50mL bone marrow fluids is extracted with medullo-puncture needle, inject the sterile of 50mL
In centrifuge tube；It is diluted with PBS liquid of the equivalent containing 20 μ/mL heparin, and isometric proportion is added along the tube wall of sterile centrifugation tube and is
The lymphocyte separation medium of 1.073g/mL is centrifuged, centrifugal rotational speed 2000rpm, and centrifugation time 30min inhales after centrifugation
Take mononuclearcell layer；Then sterile saline centrifuge washing is used 2 times, the rotating speed of each centrifugal process is 1000rpm, centrifugation
Time is 5min, discards supernatant liquid, takes precipitation；
2) precipitation that centrifugation obtains is suspended with serum free medium, cell is counted, by 5 × 106A/mL is inoculated in
In culture bottle, 37 DEG C are set, cultivated in the incubator of the carbon dioxide that volume fraction is 5%, saturated humidity；After culture 3 days, replace
Serum-free medium discards non-attached cell, later every 3 days 1 time replacement culture solution；After 7-9 days, mesenchymal stem cell melts
When closing up to 50%-60%, first time passage is carried out；Serum-free medium is removed, is respectively 0.25% and 0.1% using concentration
37 DEG C of digestion 1min of trypsase-EDTA, mesenchymal stem cell, which is shunk, is detached from bottle wall, is added in the culture previously removed
Clear liquid is neutralized trypsase-EDTA, and is gently blown and beaten using pipette, and it is sterile that medulla mesenchyma cell suspension is moved into 50mL
In centrifuge tube；1000rpm centrifuges 8min, discards supernatant liquid；Serum-free complete medium resuspension is rejoined, is counted；
3), 1-1.5 is passed according to 1 be inoculated with 10cm culture dishes；When 60%-80% is merged in mesenchymal stem cell culture, press
Secondary culture is carried out according to the operating process 2).
Three, the preparation of fat mesenchymal stem cell
S1, abdomen white adipose tissue is conventionally detached, rejects visible capilary and musculature, sterile PBS
Washing three times, is then shredded adipose tissue to 1mm with sterile scissors3Paste below adds sterile digestive juice, 37 DEG C of water-baths
In at the uniform velocity stir 45-50min, until tissue block digestion is clean；Filtrate is collected by filtration in 200 mesh screens, then uses and contains 10% tire
The low sugar DMEM culture medium equivalent of cow's serum neutralizes, and 1500rpm centrifuges 10min, discards supernatant liquid, and it is dry thin to obtain fat mesenchymal
Born of the same parents；
S2, with 1 × 106The cell density of a/mL is resuspended fat mesenchymal stem cell with serum free medium, and is inoculated with
In culture bottle, 37 DEG C, volume fraction be 5% carbon dioxide, cultivate in saturated humidity incubator, work as fat mesenchymal stem cell
When growing to 70%-80% fusions, fat mesenchymal stem cell is digested using 0.25% trypsin solution, with 1：3 inoculation
Ratio, which is inoculated into new culture bottle, carries out secondary culture；Fat mesenchymal stem cell be fused to no more than 80%, 1000rpm from
Heart 8min, discards supernatant liquid, rejoins Serum-free complete medium and fat mesenchymal stem cell is resuspended, count；
S3, according to 1 pass 1-1.5 be inoculated with 10cm culture dishes, fat mesenchymal stem cell culture merge 60%-80% when according to
S2 secondary cultures.
Four, prepared by the mesenchymal stem cell injection for the treatment of cerebral arterial thrombosis
The preparation method of the mescenchymal stem cell of the activation includes the steps that being：By forth generation mescenchymal stem cell culture
To the fusion of 70-80%, the culture medium in mescenchymal stem cell culture dish is taken out, it is dry thin to rinse mesenchyma with phosphate buffer
Born of the same parents, with the culture medium of the α-MEM containing TNF-α, α-MEM containing IL-1 culture medium or contain TNF-α and contain IL-1
Medium culture 12h.TNF-α and the concentration of IL-1 are respectively 20ng/ml, 50ng/ml.If the concentration of mescenchymal stem cell
5 × 10 can be reached5-2×106A/mL shows that mescenchymal stem cell has been successfully activated.
The mesenchymal stem cell injection for the treatment of cerebral arterial thrombosis provided by the invention comprising following components：Quantity
It is 5 × 105-2×106The mescenchymal stem cell of the activation of a/mL, mass volume ratio are the human serum albumin of 1%-5%, quality
Volume ratio is the formula mannitol injection liquid of 1%-8%, and mass volume ratio is the Compound vitamine injection of 1%-5%, quality volume
Than being physiological saline for 50% glucose injection and surplus of 1%-5%.
Stem cell injection liquid is filled between preparation 100mL activation of the present invention, including：The mass volume ratio of 5mL is dense eventually
50% grape of human serum albumin stoste, the formula mannitol injection liquid of 2mL, the Compound vitamine injection of 3mL, 5mL that degree is 1%
The mescenchymal stem cell of sugared injection, the physiological saline of 85mL and activation.In addition to the mescenchymal stem cell of activation, injection remaining
Ingredient need to be prepared in advance, and 4 DEG C of pre- cold standbies.MSC is finally resuspended in injection, and single cell suspension, every milliliter of injection is made
The quantity of the mescenchymal stem cell of middle activation is 5 × 105-2×106It is a.The mescenchymal stem cell of the activation prepared is in 2-10
In DEG C condition, 2 hours inner cells remain single cell suspension state, and cell viability (Trypan Blue meter vigor) is maintained at
95% or more.The ratio of the equal representation quality (g) of mass volume ratio and volume (ml) of the present invention.
Human serum albumin, composite vitamin, mannitol, glucose are clinical injection liquid ingredient, can provide battalion for cell
Support, conducive to cell metabolism, maintain good stem cell in vitro microenvironment.
The mesenchymal stem cell injection for the treatment of cerebral arterial thrombosis provided by the invention is especially advantageous for the mesenchyma of activation
Stem cell in vitro under suspension microenvironment stabilization, be conducive to stem cell and maintain activity, and clinical infusion safety.Between activation
Mesenchymal stem cells can keep higher vigor the short time in this suspension, utmostly maintain the characteristic of stem cell so that defeated
The treatment potential of bigger can be played by noting internal cell.
In order to verify activation provided by the invention mescenchymal stem cell the effect in alleviating acute inflammation, the present invention
Following experiment is carried out：
1, cerebral arterial thrombosis model foundation
The random male mice 30 for using C57BL/6J, 23-27 grams of weight.With penta bar of 10% chloraldurate or 50mg/kg
Than appropriate sodium, intraperitoneal injection of anesthesia is carried out to rat with 0.33-0.35ml/100g.Iodophor disinfection carries out blunt separation after cutting skin.
Along right side, nutator tendon finds downwards arteria carotis.Arterial sheath is carefully detached, vagus nerve is not injured.Neck is subsequently isolated
Always, neck is outer, internal carotid.Ligature that neck is total, external carotid artery.Internal carotid is closed using artery clamp folder, and uses eye at arteria carotis communis
An osculum is cut by section.Enter the fishing line for dipping in wax, head end rounding well prepared in advance, ligatures.It sews up a wound and is filled again after 2 hours
Note.After rat is awake, if there is astasia, left limb paralysis turn-takes to side when carrying tail, illustrates MCAO (in brain
Arterial occlusion) model foundation success.Postoperative 2h monitors the indices of rat, and by tail vein injection MSC, postoperative 8h is again
Secondary monitoring rat indices.It postoperative 12 hours, carries out Neuroscore and breaks end to take brain, the body of brain death is analyzed in TTC dyeing
Product.
2, the mesenchymal stem cell injection transplantation treatment cerebral arterial thrombosis of cerebral arterial thrombosis is treated
The rat for having become mould is divided into 4 groups using completely random method, every group of 6 rats：
(1) blank treatment group：Animal tail vein is injected using 1mL acellular injections, the acellular note
It includes the human serum albumin that mass volume ratio is 1-5% to penetrate liquid, and mass volume ratio is the formula mannitol injection liquid of 1-8%, mass body
For product than the Compound vitamine injection for being 1-5%, 50% glucose injection and surplus that mass volume ratio is 1-5% are physiology
Brine；
(2) the umbilical cord mesenchymal stem cells injection for treating group of TNF-α activation：Using 1mL injections to animal tail vein
It is injected, which contains acellular injection and a concentration of 5 × 105-2×106The mesenchyma of the TNF-α activation of a/mL
Stem cell.
(3) the umbilical cord mesenchymal stem cells injection for treating group of IL-1 activation：Using 1mL injections to animal tail vein into
Row injection, the injection include acellular injection and a concentration of 5 × 105-2×106The mesenchyma of the IL-1 activation of a/mL is dry thin
Born of the same parents.
(4) TNF-α+IL-1 activation umbilical cord mesenchymal stem cells treatment group, using 1mL injections to animal tail vein into
Row injection, the injection include acellular injection and a concentration of 5 × 105-2×106It is filled between the TNF-α+IL-1 activation of a/mL
Matter stem cell.This experiment is by the way of single injection.
As a result：
1, the mesenchyma of TNF-α+IL-1 activation significantly reduces containing for the inflammatory factor in cerebral arterial thrombosis rat body
Amount and expression
Postoperative 2h and postoperative 8h extracts rat blood, and inflammatory factor TNF-α in rat body, IL-1 are detected by RT-PCR
With IL-6 contents.Fig. 1 with control group, TNF-α activation group with IL-1 activation groups the results show that compare, TNF-α+IL-1 activation groups
Rat body in the activation of inflammatory factor obviously inhibited.
2, the MSC of TNF-α+IL-1 activation considerably reduces the cerebral infarct volume of rat：
It postoperative 24 hours, carries out Neuroscore and breaks end to take brain, the volume of brain death is analyzed in TTC dyeing.Such as Fig. 2 institutes
Show that the MSC groups with control group, the MSC groups of TNF-α activation, IL-1 activation are compared, the rat of the MSC groups of TNF-α and IL-1 activation
Cerebral infarct volume significantly reduces.
Result above confirms, TNF-α and IL-1 jointly pretreated mesenchymal stem cell injection can significantly improve it is acute
Inflammation and cerebral arterial thrombosis rat cerebral infarction volume.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily
Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and legend shown and described herein.
Claims (9)
1. a kind of mesenchymal stem cell injection for treating cerebral arterial thrombosis, which is characterized in that including following components：
Quantity is 5 × 105-2×106The mescenchymal stem cell of the activation of a/mL, the white egg of people's blood that mass volume ratio is 1%-5%
In vain, Compound vitamine injection that mass volume ratio is 1%-8% formula mannitol injection liquid, mass volume ratio are 1%-5%, matter
Measure the physiological saline of 50% glucose injection and surplus that volume ratio is 1%-5%.
2. a kind of mesenchymal stem cell injection for treating cerebral arterial thrombosis according to claim 1, which is characterized in that
The mescenchymal stem cell of the activation is the mescenchymal stem cell and TNF-α that mescenchymal stem cell, the IL-1 that TNF-α activates are activated
With any one in the IL-1 simultaneously mescenchymal stem cell of activation.
3. a kind of mesenchymal stem cell injection for treating cerebral arterial thrombosis according to claim 2, which is characterized in that
The mescenchymal stem cell is arbitrary in umbilical cord mesenchymal stem cells, mesenchymal stem cell, fat mesenchymal stem cell
It is a kind of.
4. a kind of mesenchymal stem cell injection for treating cerebral arterial thrombosis according to claims 1 to 3 any one of them
Preparation method, which is characterized in that include the following steps：
Step 1: preparing mescenchymal stem cell, and cultivate forth generation mescenchymal stem cell；
Step 2: forth generation mescenchymal stem cell culture to 70%-80% is merged, the mescenchymal stem cell of activation is prepared；
Step 3: formula described in accordance with the claim 1, prepare and weigh human serum albumin that mass volume ratio is 1%-5%,
Compound vitamine injection that formula mannitol injection liquid that mass volume ratio is 1%-8%, mass volume ratio are 1%-5%, quality
Volume ratio is 50% glucose injection of 1%-5%, and mixing；
Step 4: being 5 × 10 by quantity5-2×106After the mescenchymal stem cell of the activation of a/mL is resuspended in step 3 mixing
In liquid, and the physiological saline of surplus is added, the mesenchymal stem cell injection for the treatment of cerebral arterial thrombosis is prepared.
5. a kind of preparation method of mesenchymal stem cell injection for treating cerebral arterial thrombosis according to claim 4,
It is characterized in that, in the step 2, the preparation process of the mescenchymal stem cell of the activation is：
By the fusion of forth generation mescenchymal stem cell culture to 70-80%, used when taking out forth generation mescenchymal stem cell culture
Mescenchymal stem cell culture dish in culture medium, with phosphate buffer rinse forth generation mescenchymal stem cell；
With specific medium culture forth generation mescenchymal stem cell；
When the concentration of forth generation mescenchymal stem cell reaches 5 × 105-2×106When a/mL, the mescenchymal stem cell that is activated.
6. a kind of preparation method of mesenchymal stem cell injection for treating cerebral arterial thrombosis according to claim 5,
It is characterized in that, when the mescenchymal stem cell of the activation is the mescenchymal stem cell of TNF-α activation, the specific culture
Base is the culture medium of the α-MEM of the TNF-α containing a concentration of 20ng/mL；
When the mescenchymal stem cell of the activation be IL-1 activation mescenchymal stem cell when, the specific culture medium be containing
The culture medium of the α-MEM of the IL-1 of a concentration of 50ng/mL；
It is described specific when the mescenchymal stem cell of the activation is TNF-α and the IL-1 mescenchymal stem cells of activation simultaneously
Culture medium is the culture medium of the TNF-α culture medium containing a concentration of 20ng/mL and the α-MEM of the IL-1 of a concentration of 50ng/mL.
7. a kind of preparation method of mesenchymal stem cell injection for treating cerebral arterial thrombosis according to claim 4,
It is characterized in that, in the step 1, the mescenchymal stem cell is umbilical cord mesenchymal stem cells, and forth generation umbilical cord mesenchyma
The incubation of stem cell is：
A) fresh mature healthy fetus umbilical cord, is taken, is removed with the normal saline flushing containing 2 times of 100U/mL penicillin and streptomysin
Bloodstain removes Wal Tong Shi glue, the Wal Tong Shi glue after stripping is cut into less than 1mm3Fritter；
B), the Wal Tong Shi glue shredded is spread evenly across to the bottom of cell culture apparatus, slowly added into the cell culture apparatus
Enter 10mL serum free mediums, is placed in 37 DEG C, 5v/v%CO2, cultivate in saturated humidity environment, after primitive cell culture 7 days,
Serum-free medium is replaced, culture reaches 70% fusion to cell, removes serum-free medium, and addition concentration is respectively
0.25% and 0.1% trypsin-EDTA sodium digests 1min, and umbilical cord mesenchymal stem cells, which are shunk, is detached from bottle wall,
The culture supernatant previously removed is added at this time, trypsin-EDTA solutions are neutralized, and gently blow and beat using pipette, by navel
Band mesenchyma stem cell suspension moves into 50mL centrifuge tubes, and supernatant is removed after centrifugation, and rejoining Serum-free complete medium will
Umbilical cord mesenchymal stem cells after centrifugation are resuspended；
C) 1.5 inoculation 10cm culture dishes, are passed according to 1, according to behaviour b) when umbilical cord mesenchymal stem cells culture merges 70%
Make process secondary culture, is trained forth generation umbilical cord mesenchymal stem cells.
8. a kind of preparation method of mesenchymal stem cell injection for treating cerebral arterial thrombosis according to claim 4,
It is characterized in that, the mescenchymal stem cell is mesenchymal stem cell, the culture of forth generation mesenchymal stem cell
Cheng Wei：
1), in anterior superior spine point of puncture, 50mL bone marrow fluids is extracted with medullo-puncture needle, are injected in the sterile centrifugation tube of 50mL；Use equivalent
PBS liquid dilution containing 20 μ/mL heparin, and the lymph that isometric proportion is 1.073g/mL is added along the tube wall of sterile centrifugation tube
Cell separating liquid is centrifuged, centrifugal rotational speed 2000rpm, centrifugation time 30min, and mononuclearcell layer is drawn after centrifugation；
Then sterile saline centrifuge washing is used 2 times, the rotating speed of each centrifugal process is 1000rpm, and centrifugation time 5min is discarded
Supernatant takes precipitation；
2) precipitation that centrifugation obtains is suspended with serum free medium, cell is counted, by 5 × 106The culture bottle that a/mL is inoculated in
In, it sets 37 DEG C, cultivated in the incubator of the carbon dioxide that volume fraction is 5%, saturated humidity；After culture 3 days, serum-free is replaced
Culture solution discards non-attached cell, later every 3 days 1 time replacement culture solution；After 7-9 days, mesenchymal stem cell fusion reaches
When 50%-60%, first time passage is carried out；Serum-free medium is removed, is respectively 0.25% and 0.1% pancreas egg using concentration
37 DEG C of digestion 1min of white enzyme-EDTA, mesenchymal stem cell, which is shunk, is detached from bottle wall, and the culture supernatant previously removed is added,
Trypsase-EDTA is neutralized, and is gently blown and beaten using pipette, medulla mesenchyma cell suspension is moved into 50mL sterile centrifugation tubes
In；1000rpm centrifuges 8min, discards supernatant liquid；Serum-free complete medium resuspension is rejoined, is counted；
3), 1-1.5 is passed according to 1 be inoculated with 10cm culture dishes；When 60%-80% is merged in mesenchymal stem cell culture, according to institute
The operating process stated 2) carries out secondary culture.
9. a kind of preparation method of mesenchymal stem cell injection for treating cerebral arterial thrombosis according to claim 4,
It is characterized in that, the mescenchymal stem cell is fat mesenchymal stem cell, the culture of forth generation fat mesenchymal stem cell
Cheng Wei：
S1, separation abdomen white adipose tissue, reject visible capilary and musculature, and sterile PBS is washed three times, then uses nothing
Bacterium scissors shreds adipose tissue to 1mm3Paste below adds sterile digestive juice, 45- is at the uniform velocity stirred in 37 DEG C of water-baths
50min, until tissue block digestion is clean；Filtrate is collected by filtration in 200 mesh screens, then uses the low sugar containing 10% fetal calf serum
DMEM culture medium equivalent neutralizes, and 1500rpm centrifuges 10min, discards supernatant liquid, obtains fat mesenchymal stem cell；
S2, with 1 × 106The cell density of a/mL is resuspended fat mesenchymal stem cell with serum free medium, and is inoculated in culture
In bottle, 37 DEG C, volume fraction be 5% carbon dioxide, cultivate in saturated humidity incubator, when fat mesenchymal stem cell is grown to
When 70%-80% is merged, fat mesenchymal stem cell is digested using 0.25% trypsin solution, with 1：3 inoculative proportion connects
Secondary culture is carried out in kind to new culture bottle；Fat mesenchymal stem cell is fused to be centrifuged no more than 80%, 1000rpm
8min discards supernatant liquid, rejoins Serum-free complete medium and fat mesenchymal stem cell is resuspended, count；
S3,1-1.5 inoculation 10cm culture dishes are passed according to 1, fat mesenchymal stem cell culture passes when merging 60%-80% according to S2
It is commissioned to train foster.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110055216A (en) * | 2019-05-09 | 2019-07-26 | 张秀明 | A method of improving interstital stem cell biological function |
| CN111346051A (en) * | 2020-03-19 | 2020-06-30 | 瑞太干细胞中心(沈阳)有限公司 | Preparation method of umbilical cord mesenchymal stem cell injection for treating cerebral infarction |
| CN113057937A (en) * | 2021-03-23 | 2021-07-02 | 辽宁何氏医学院 | Stem cell intravenous injection and preparation method and application thereof |
-
2018
- 2018-06-27 CN CN201810676861.XA patent/CN108619169A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110055216A (en) * | 2019-05-09 | 2019-07-26 | 张秀明 | A method of improving interstital stem cell biological function |
| CN111346051A (en) * | 2020-03-19 | 2020-06-30 | 瑞太干细胞中心(沈阳)有限公司 | Preparation method of umbilical cord mesenchymal stem cell injection for treating cerebral infarction |
| CN113057937A (en) * | 2021-03-23 | 2021-07-02 | 辽宁何氏医学院 | Stem cell intravenous injection and preparation method and application thereof |
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