CN105797138A - Stem cell preparation for cornea repair and preparation method and application thereof - Google Patents
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Abstract
The invention relates to a stem cell preparation for cornea repair.The stem cell preparation comprises polysaccharide, KGF-2 and mesenchymal stem cells.By combining the polysaccharide, the KGF-2 and the mesenchymal stem cells, the anti-inflammatory effect is achieved, cornea surface cell proliferation can be promoted, cornea epithelial cell proliferation is stimulated, cornea epithelium repair ability and cornea transparency are improved, cornea excessive fibrosis can be prevented, and therefore healing of a damaged cornea is promoted.
Description
Technical field
The present invention relates to biological technical field, particularly to stem cell medicine and the system thereof of a kind of repairing corneal
Preparation Method and application.
Background technology
Corneal chemical burn is one of modal disease in Ophthalmic Emergency, is also a kind of more typically blinding
Oculopathy.External corneal chemical burn accounts for the 8% of whole ocular injury, and its morbidity has then accounted for outside eye at home
The 15% of wound, incidence rate is far above developed countries such as America and Europes.The at present various treatment angles of many research reports
The method of film alkali burn, including Drug therapy such as antibiotic, steroid hormone, ascorbic acid, collagenase
Inhibitor etc., and operative treatment such as corneal transplantation, amnion transplantation etc. achieve tradition the most to a certain extent
The therapeutic purposes of therapy.While it is true, these treatments still can not solve to involve cornea deep layer substrate very well and help
Damage and burnt degree corneal scarring and the problem of vascularization.Simultaneously as corneal transplantation appropriate donor lacks,
Autologous corneal limbus is drawn materials limited, and these problems also limit the clinical practice of corneal transplantation.Therefore, cornea
The treatment of alkali burn has been still filled with challenge, and exploring brand-new Therapeutic mode becomes the new direction of research.
Summary of the invention
The technical problem to be solved is, there is remediation efficiency for corneal restoration in prior art
The defects such as difference, poor operability, it is provided that one can improve corneal restoration efficiency, and operability is higher
The stem cell medicine of repairing corneal and its preparation method and application.
The technical solution adopted for the present invention to solve the technical problems is: provide the dry thin of a kind of repairing corneal
Born of the same parents' preparation, including polysaccharide, KGF-2 and mescenchymal stem cell.
In the stem cell medicine of the repairing corneal of present invention offer, also include heparin sodium, penicillin and chain
Mycin.
In the stem cell medicine of the repairing corneal of present invention offer, the mass fraction of described polysaccharide is
0.5～5%.
In the stem cell medicine of the repairing corneal of present invention offer, the concentration of described KGF-2 is
15～50 μ g/ml.
In the stem cell medicine of the repairing corneal of present invention offer, the concentration of described mescenchymal stem cell is
(2～8) × 105Individual/ml.
In the stem cell medicine of the repairing corneal of present invention offer, in described hepatocyte preparation, heparin sodium
Concentration be 15～20ng/ml, penicillin concn be the concentration of 200000 units/ml and streptomycin be 200000
Unit/ml.
The present invention provides the preparation method of the stem cell medicine of a kind of repairing corneal, comprises the following steps: will
After polysaccharide, KGF-2, heparin sodium, penicillin and streptomycin mixing, it is added dropwise in mescenchymal stem cell,
Interpolation limit, limit mixes.
In the preparation method of the stem cell medicine of the repairing corneal of present invention offer, described mesenchyme is dry thin
The acquisition process of born of the same parents, comprises the following steps:
Obtain Primary bone marrow mescenchymal stem cell, described Primary bone marrow mescenchymal stem cell is passed on training
Support to gathering in the crops the 3rd～5 mesenchymal stem cells MSCs acting as a generation.
In the preparation method of the stem cell medicine of the repairing corneal of present invention offer, the acquisition of described polysaccharide
Process, comprises the following steps:
Through distillation water extract-alcohol precipitation fall after being pulverized by the Pseudobulbus Bletillae (Rhizoma Bletillae), precipitate distilled water is resuspended, with chloroform and butanol
Mixture extracting after, dialysis, crosslinked glucosan G-100 is further purified, sulphation, the most thoroughly
Analysis, i.e. obtains polysaccharide.
The present invention protects the application in the medicine preparing repairing corneal of the above-mentioned stem cell medicine further.
Stem cell medicine of repairing corneal of present invention offer and its preparation method and application, Ke Yida are provided
To following beneficial effect: polysaccharide and KGF-2 combine mescenchymal stem cell, not only have antiinflammatory action, and
And anterior corneal surface cell proliferation can be promoted, stimulate corneal epithelial cell propagation, improve repairing of corneal epithelium
Reactivation power and the transparency of cornea, and it is prevented from cornea excess fibrosis, thus promote corneal injury
Healing.
Detailed description of the invention
The stem cell medicine of the repairing corneal that the present invention provides, including polysaccharide, KGF-2 (Keratinocyte
Growth Factor-2, body keratinized cell growth factor-2) and mescenchymal stem cell；Further, also include
Heparin sodium, penicillin and streptomycin.
Wherein, polysaccharide is to be connected, by long-chain monosaccharide, the polymeric carbohydrate being combined into through glycosidic bond, its
There is various biological activity, including antiinflammatory, promote cell proliferation, immunosuppressant etc.；Polysaccharide is composition
Corneal stroma and the important component of extracellular matrix；Endogenous polysaccharide in corneal penetrating injury agglutination by
The fewest, polysaccharide participates in corneal restoration process, and after corneal injury, polysaccharide can be by promoting anterior corneal surface cell
Propagation and suppression inflammatory reaction and healing acceleration.It addition, the hyaluronate sodium in polysaccharide to irritate epithelium thin
Born of the same parents' propagation, strengthen epithelial cell to the adhesive capacity of substrate the infiltration that suppresses inflammatory cell, thus promote
The healing of corneal injury；Additionally, new vessels and inflammatory reaction are controlled in keratopathy is treated by polysaccharide
Treatment effect, it is possible to increase the repair ability of corneal epithelium and corneal transparence.
Owing to polysaccharide has good hygroscopicity and hydrophilic, it is possible to promote oxygen, nutrient substance and other
The permeation of water soluble metabolites, can also strengthen the adhesiveness of epithelium and stromal cell simultaneously；And
The paracrine of mesenchymal stem cells has the various factor, can not only promote propagation and the migration of corneal epithelium,
And corneal epithelium reparation and suppression inflammatory reaction and the function of new vessels can be promoted.Therefore, polysaccharide
Propagation and the migration of corneal epithelium all can be promoted with mescenchymal stem cell.Preferably, in stem cell medicine,
The concentration of mescenchymal stem cell is (2～8) × 105Individual/ml.
Preferably, polysaccharide origin of the present invention is in the Pseudobulbus Bletillae (Rhizoma Bletillae), and the Pseudobulbus Bletillae (Rhizoma Bletillae) has hemostasis, subsides a swelling, accelerates
The function of injury recovery, it is possible to for hemostasia and detumescence and burn etc..The polysaccharide Main Ingredients and Appearance in Pseudobulbus Bletillae (Rhizoma Bletillae) source is
Mannan, β-D-pyranglucoside, β-D-diglucoside, have higher osmotic pressure, with cornea
Epithelium has good biocompatibility.Preferably, in stem cell medicine, the mass fraction of polysaccharide is
0.5～5%.
KGF-2 is a member of keratinocyte growth factor family in FGFs superfamily, mainly thin by mesenchyme
Born of the same parents synthesize, and on the one hand can promote the propagation of corneal epithelial cell, break up and migrate；On the other hand, KGF-2
Fibroblast transition hypertrophy can be suppressed, prevent cornea excess fibrosis, maintain the normal structure of cornea
Structure；In addition, KGF-2 is conducive to recovery and the contracting of Corneal Burn muddiness speckle of corneal transparence
Little；Preferably, in stem cell medicine, the concentration of KGF-2 is 15～50 μ g/ml.
Heparin sodium has the effect of anticoagulant, antisolvent precipitation, to prevent mescenchymal stem cell to be packed together, and shadow
Ring its cytoactive etc.；The present invention preferentially use low molecular sodium heparin, i.e. mean molecule quantity less than 8000
Daltonian heparin sodium, to improve its biocompatibility.Preferably, in stem cell medicine, heparin sodium
Concentration is 15～20ng/ml.
Streptomycin and penicillin are used in combination the antibacterial etc. that can suppress almost all kind, it is to avoid antibacterial pair
The harmful effect that mescenchymal stem cell causes, thus improve the life-span of stem cell medicine, improve stem cell system
The therapeutic effect of agent.Preferably, in stem cell medicine, the concentration of penicillin is 200000 units/ml, chain
The concentration of mycin is 200000 units/ml.
To sum up, the stem cell medicine that the present invention provides not only has antiinflammatory action, and can promote cornea
Superficial cell is bred, and stimulates corneal epithelial cell propagation, improves the repair ability of corneal epithelium and cornea
Transparency, and it is prevented from cornea excess fibrosis, thus promote the healing of corneal injury.
Further, the preparation method of stem cell medicine of the present invention, comprise the following steps:
S1, acquisition mescenchymal stem cell；
S2, extraction polysaccharide；
S3, it is mixed with stem cell medicine.
In step S1, the present invention preferentially uses the 3rd～5 mesenchymal stem cells MSCs acting as a generation, specifically
Acquisition process is:
S11, density－gradient centrifuga－tion method combine stationary culture bolting house mesenchymal stem cells；
Bone marrow extraction, is sufficiently mixed with basal medium, filters, and adds Percoll liquid, centrifugal, collects
Tunica albuginea layer, is mononuclearcell, and washing, then with containing hyclone, penicillin and streptomycin
Basal medium is cultivated, and changes culture medium, remove the most adherent cell, every 2～4 after 24～72 hours
It changes liquid once, reaches Fusion Strain to cell, i.e. results Primary bone marrow mescenchymal stem cell.
S12, the Secondary Culture of Primary bone marrow mescenchymal stem cell；
After Primary bone marrow mescenchymal stem cell reaches 80% fusion, trypsinization 3～5 minutes, see cell
Gap increases, and cellular morphology retraction, for time circular, stops digestion, then in 1:(2～4) ratio inoculate
Cultivate in the basal medium containing Ox blood serum, change liquid every other day, merge to cell, be repeatedly conveyed to obtain
Obtain arbitrary generation mesenchymal stem cells MSCs in the 3rd～5 generations.
S2, the present invention preferably, extract polysaccharide from the Pseudobulbus Bletillae (Rhizoma Bletillae), concretely comprise the following steps: warp after being pulverized by the Pseudobulbus Bletillae (Rhizoma Bletillae)
Distillation water extract-alcohol precipitation fall, precipitate distilled water is resuspended, after extracting with the mixture of chloroform and butanol, thoroughly
Analysis, crosslinked glucosan G-100 is further purified, and sulphation is again dialysed, i.e. obtained polysaccharide.
S21, take the Pseudobulbus Bletillae (Rhizoma Bletillae) and pulverize, with dispersing and dissolving 2 in 50～100 DEG C of distilled water～after 6 hours, filter,
Collect filtrate；
S22, will filtrate add 3 times of volumes ethanol in soak 12～24 hours, precipitate distilled water weight
Outstanding；
The mixed liquor mixing of S23, addition chloroform and butanol is centrifugal, extract proteins composition and the liquid of aqueous layer；
S24, dialyse through dialysis cartridge, and make lyophilized powder, obtain the concise thing of polysaccharide；
The concise thing of S25, polysaccharide is further purified through cross-linking dextran G-100 after dissolving；
S26, modify after purification polysaccharide sulfated with pyridine chlorosulfonic acid method；
S27, Sulfated polysaccharide are dissolved in after purification through DE-52 and cross-linking dextran G-100 dialysis cartridge
In PBS, make the polysaccharide solution that mass fraction is 0.5～5%.
S3, by step S2 obtain polysaccharide solution, KGF-2, heparin sodium, penicillin and streptomycin mix
Closing uniformly, be added dropwise in step S12 by mixture in the mesenchymal stem cells MSCs obtained, limit is added
Limit mixes, after mix homogeneously, frozen, stand-by.
The present invention protects the application in preparing repairing corneal medicine of this stem cell medicine further.
In order to make the purpose of the present invention, technical scheme and advantage clearer, below in conjunction with embodiment,
The present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to
Explain the present invention, be not intended to limit the present invention.
Embodiment 1
S1, acquisition mescenchymal stem cell；
S11, density－gradient centrifuga－tion method combine stationary culture bolting house mesenchymal stem cells；
By the bone marrow of extraction, inject in the centrifuge tube containing 5ml low sugar DMEM immediately and be sufficiently mixed, warp
After 200 mesh stainless steel filtering nets filter, join preset equal-volume Percoll liquid (density gently by adherent for suspension
1.082kg/L, is purchased from GE company, the U.S., and Percoll is through polyvinylpyrrolidone (polyvinyl
Pyrolidone, PVP) the silica gel particle suspension that processes, to cytotoxic and zest) centrifuge tube
In, 2400r/min is centrifuged 20min, collects the mononuclearcell of cloud tunica albuginea layer, and PBS washes twice
(1200r/min, 10min), with containing 100mL/L hyclone, 100U/mL penicillin, 100u/mL chain
The low sugar DMEM culture medium Eddy diffusion cell of mycin, adjusts cell density with 5 × 105/ mL is inoculated in
In 50mL culture bottle, it is positioned over CO2Incubator is hatched, changes culture medium after 72h, remove the most adherent
Cell, later every 3d changes liquid 1 time, until propagation reaches to merge, it is thus achieved that Primary bone marrow mesenchyme is dry thin
Born of the same parents, observation of cell growth and morphological characteristic under inverted microscope, select one bottle of best cell of growth to carry out
Secondary Culture.
S12, Secondary Culture
After Primary bone marrow mescenchymal stem cell reaches 80% fusion, 2.5mL/L trypsin is i.e. can use to paste
Parietal cell digestion separates (37 DEG C, 3-5min), sees that intercellular substance increases cellular morphology retraction for being finished time circular
Full culture medium stops digestion.Make cell suspension with suction pipe piping and druming, then carry out passing on connecing in the ratio of 1:2
Cultivate kind in the DMEM culture bottle containing 100mL/L new-born calf serum, the next day change liquid, until adherent carefully
Born of the same parents' fusion closer to each other, is paved with the bottom surface of whole culture bottle, repeats above operation, repeatedly passes on, and passes
To the 4th generation, standby.
S2, polysaccharide purify；
Through distillation water extract-alcohol precipitation fall after being pulverized by the Pseudobulbus Bletillae (Rhizoma Bletillae), precipitate distilled water is resuspended, with chloroform and butanol
Mixture extracting after, dialysis, crosslinked glucosan G-100 is further purified, sulphation, the most thoroughly
Analysis, i.e. obtains polysaccharide.
S21, take the Pseudobulbus Bletillae (Rhizoma Bletillae) and pulverize, after dispersing and dissolving in 80 DEG C of distilled water 4 hours, filter, collect
Filtrate；
S22, will filtrate add 3 times of volumes ethanol in soak 12 hours, precipitate distilled water is resuspended；
The mixed liquor mixing of S23, addition chloroform and butanol is centrifugal, extract proteins composition and the liquid of aqueous layer；
S24, dialyse through dialysis cartridge, and make lyophilized powder, obtain the concise thing of polysaccharide；
The concise thing of S25, polysaccharide is further purified through cross-linking dextran G-100 after dissolving；
S26, modify after purification polysaccharide sulfated with pyridine chlorosulfonic acid method；
S27, Sulfated polysaccharide are dissolved in after purification through DE-52 and cross-linking dextran G-100 dialysis cartridge
In PBS, make the polysaccharide solution that mass fraction is 0.5～5%.
S3, the preparation of stem cell medicine；
Polysaccharide, KGF-2, heparin sodium, penicillin and the streptomycin obtained in S31, mixing step S2；
The 3rd generation mesenchymal stem cells MSCs obtained in S32, use resuspended step S12 of normal saline so that
Cell concentration is 5 × 105Individual/ml.
S33, the medulla mesenchyma being added dropwise in step S32 by the mixture obtained in step S31 are dry thin
In cytosol, interpolation limit, limit mixes, and to make the mass fraction of polysaccharide be 2%, and KGF-2's is final concentration of
25 μ g/ml, the final concentration of 20ng/ml of heparin sodium, the final concentration of 200000 units/ml of penicillin and
The final concentration of 200000 units/ml of streptomycin；
After mix homogeneously, it is dispensed in pre-charge injector, is put in-80 DEG C of refrigerators frozen, stand-by.
Microencapsulation immunocyte for checking present invention offer further is frozen and method for resuscitation has significantly
Beneficial effect, arranges following experiment and verifies.
Test experience one, by the embodiment of the present invention prepare stem cell medicine train altogether with Human glioma
Support, the impact of detection corneal epithelial cell propagation；
The non-matched group co-cultured of Setup Experiments: co-culture as compareing with the polysaccharide solution that mass fraction is 2%
Group 1, co-cultures as matched group 2 with mesenchymal stem cells MSCs.
Mtt assay is each group cell proliferation change, testing result such as table after detecting respectively 24,48 and 72 hours
Shown in 1,
Table 4 is the optical density of detection group and experimental group corneal epithelial cell
Testing result: detection group and matched group 1-2, after acting on corneal epithelial cell 72h, and compare
Group 1-2 data are compared and be there is significant difference；Thus illustrating, polysaccharide, KGF-2 associating mesenchyme is dry thin
Born of the same parents, the propagation of energy couple Human glioma plays obvious facilitation.
Above embodiments of the invention are described, but the invention is not limited in above-mentioned concrete
Embodiment, above-mentioned detailed description of the invention is only schematic rather than restrictive, this area
Those of ordinary skill under the enlightenment of the present invention, without departing from present inventive concept and claimed
Ambit under, it may also be made that a lot of form, within these belong to the protection of the present invention.
Claims (10)
1. the stem cell medicine of a repairing corneal, it is characterised in that include polysaccharide, KGF-2 and fill
Matter stem cell.
The stem cell medicine of repairing corneal the most according to claim 1, it is characterised in that also include
Heparin sodium, penicillin and streptomycin.
The stem cell medicine of repairing corneal the most according to claim 2, it is characterised in that described many
The mass fraction of sugar is 0.5～5%.
The stem cell medicine of repairing corneal the most according to claim 3, it is characterised in that described
The concentration of KGF-2 is 15～50 μ g/ml.
The stem cell medicine of repairing corneal the most according to claim 4, it is characterised in that between described
The concentration of mesenchymal stem cells is (2～8) × 105Individual/ml.
The stem cell medicine of repairing corneal the most according to claim 5, it is characterised in that described liver
In cell preparation, the concentration of heparin sodium is 15～20ng/ml, penicillin concn be 200000 units/ml and
The concentration of streptomycin is 200000 units/ml.
7. a preparation method for the stem cell medicine of the repairing corneal as described in any one of claim 2～6,
It is characterized in that, comprise the following steps:
After polysaccharide, KGF-2, heparin sodium, penicillin and streptomycin being mixed, it is added dropwise over mesenchyme dry thin
In born of the same parents, interpolation limit, limit mixes.
The preparation method of the stem cell medicine of repairing corneal the most according to claim 7, its feature exists
In, the acquisition process of described mescenchymal stem cell, comprise the following steps:
Obtain Primary bone marrow mescenchymal stem cell, described Primary bone marrow mescenchymal stem cell is passed on training
Support to gathering in the crops the 3rd～5 mesenchymal stem cells MSCs acting as a generation.
The preparation method of the stem cell medicine of repairing corneal the most according to claim 7, its feature exists
In, the acquisition process of described polysaccharide, comprise the following steps:
Through distillation water extract-alcohol precipitation fall after being pulverized by the Pseudobulbus Bletillae (Rhizoma Bletillae), precipitate distilled water is resuspended, with chloroform and butanol
Mixture extracting after, dialysis, crosslinked glucosan G-100 is further purified, sulphation, the most thoroughly
Analysis, i.e. obtains polysaccharide.
10. preparing the medicine of repairing corneal according to the stem cell medicine described in any one of claim 1～6
In application.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105396125A (en) * | 2015-11-26 | 2016-03-16 | 山东省眼科研究所 | Application of IL-6 to repairing of corneal epithelial injuries |
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