CN105112368A - Stem cell preparation for treating retinosis and use method of stem cell preparation - Google Patents
Stem cell preparation for treating retinosis and use method of stem cell preparation Download PDFInfo
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Abstract
The invention provides a stem cell and a preparation for treating retinosis. Through optimum culture and induced differentiation on mesenchymal stem cells, the efficiency for differentiating the cell into the retina nerve precursor cell is improved; the similar effect to that of the retina precursor cell is achieved in the aspect of the transplantation effect, so that the retina function in the transplantation region is effectively improved; an ideal cell source is provided for the treatment of retinosis diseases through stem cell transplantation; high superiority is realized. A preparation method of the stem cell preparation provided by the invention has the advantages that the preparation process of the stem cell product obtained through separation, culture, posterity pass-down and induction is simple and reliable; the repeatability is good. A use method of the stem cell preparation for treating retinosis provided by the invention belongs to a cell replacing method with the advantages that the operation is simple, the popularization is convenient, and the injury is small. Wide application prospects are realized. The method belongs to the best path for treating the retinosis type degenerative diseases and rescuing the vision.
Description
Technical field
The present invention relates to regenerative medicine and stem cell biological technical field, relate to a kind of stem cell medicine and using method thereof for the treatment of retinal degeneration.
Background technology
About thick 0.1 ~ 0.5 millimeter of the retina of people, is histologically divided into 10 layers, and wherein mainly contain 4 confluent monolayer cells compositions, ecto-entad is pigment epithelial cell layer, visual cell's layer, bipolar cell layer and ganglion-cell layer successively.Layer of retina,pigment epithelium is closely connected with choroid, is made up of pigment epithelial cell (retinalpigmentepitheium, RPE), and normal RPE is the monolayer cell with polarity, is positioned at amphiblestroid outermost layer.It is the cellular layer of metabolism high activity, has support and nutrition photoreceptor cell, shading, heat radiation and the effect such as regeneration and reparation.Its major function comprises transhipment nutritive substance, and Vogan-Neu is recycled, chromogenesis and engulf the cone, rod photoreceptor cell acromere.
Retinal degenerative disease, comprises age-related macular degeneration, retinitis pigmentosa etc., and sickness rate raises gradually, is main blinding disease.These diseases are all with the afunction of Progressive symmetric erythrokeratodermia pigment epithelial cell, photoreceptor cell, and apoptosis or necrosis are feature, cause the irreversible infringement of eyesight.
Retinal degeneration main manifestations is that Bruch ' s film thickens, RPE hyperpigmentation and RPE cell depletion etc.Mainly be divided into atrophic type (dryness) and neovascular (moist or exudative type) two types clinically, the former mainly contains glassy membrane wart, RPE exception and geographic atrophy and changes; The latter mainly departs from choroidal neovascularization (choroidalneovascularization, CNV), RPE or plate-like fiber turns to pathological characteristic.
At present for retinal degeneration, particularly macular degeneration, modern medicine does not have specific methods for the treatment of.Clinical often according to the stage of the course of disease, use antioxidant, vitamins, hemostatic agent, and optic nerve nutritional drugs or cell-stimulating preparation, intravitreal injection anti-vascular endothelial growth factor (vascularendothelialgrowfactor, the therapeutic modality such as VEGF), but only can delay the progress of disease, fundamentally can not replace the retina cell of pathology, reverse the course of disease.The mode of past trial operation replaces the RPE of macula area, mainly contains macula lutea transposition operation and RPE autoplasty, and in long-term following up a case by regular visits to, confirmation can maintain or improve the eyesight of patient.This type of operation is transplant healthy RPE treatment retinal degenerative disease to provide foundation, but above-mentioned operation exists, and wound is large, complicated operation, and the shortcoming that complication is many, in addition, the case be badly damaged for sight sensor is then difficult to play a role.Therefore find a kind of simple to operate, be convenient to promote, traumatic little cell replacement method more and more comes into one's own.
Stem cell is the cell mass that a group has self and multidirectional potential.Utilize the plasticity-of stem cell, for the purpose of directed differentiation, cell carries out Transplanted cells or utilizes the secrete cytokines of stem cell to repair impaired retina and RPE, has become the study hotspot of retinal degenerative disease treatment.
The mechanism of cellular replacement therapy retinal degenerative disease: 1. cell replacement, is substituted the sick cell of loss by the stem cell of health; 2. nutritional support, the healthy stem cell of transplanting promotes the survival of peripheral cell; 3. the immunoregulation effect of graft area microenvironment, as promoted the inverse differentiation of graft area ortho states cell.In theory, setting up effectively new synaptic contact and the improvement of visual function on this basis, is the successful standard of stem cell transplantation.Research shows, only have and stem cell is induced to developmental condition and close maturation, competence exertion transplants effect.Desirable donorcells should have certain plasticity-, is namely in the critical period that photosensitive precursor cell is grown to mature cell.
For various Derived Stem Cells is accurately induced to photosensitive precursor cell, the growth of the photosensitive precursor cell of Human embryo must be understood.Human embryo retinal development is by strict time variable control, and embryo 7 ~ 8 weeks, namely ganglion cell started appearance, subsequently, and cone cell, horizontal cell, amacrine cell directed differentiation gradually; Embryo about 12 weeks, amphiblestroid cardinal principle level start formed, and Beale's ganglion cells, rod photoreceptor cell, ortho states cell development a little later.The differentiation of the nuclear factor accuracy controlling photosensory cell of high degree of cooperation, these genes start according to time, spatial order or close, and regulate the growth and differ entiation of embryonic cell.There is the feature of the photosensitive precursor cell transplanting effect: be 1. in metamitosis, directed differentiation, no longer continue propagation; 2. the nuclear factors such as Otx2, PAX6, CRX, Nrl are expressed; 3. not yet grow for ripe photosensory cell, form various opsin in cell gradually, namely there is plasticity-.
Mesenchymal stem cells MSCs (bonemesenchymalstemcells, BMSCs) is a class versatile stem cell, and its source is relatively extensive, draws materials convenient and safe, there is not the restriction of ethics aspect; Have powerful paracrine action, the neurotrophic factor of its secretion and somatomedin can promote the reparation of damaged tissue; Have initiatively to the effect of damaged tissue zone migration; Without swollen neoplastic tendency after transplanting; Immunogenicity is low, can carry out autologous or allotransplantation, and the probability that rejection occurs is lower.BMSCs has multi-lineage potential equally, and except being divided into the osteocyte of mesoderm origin, chondrocyte, adipocyte, BMSCs can also be neurocyte, myocardial cell, spongiocyte etc. across differentiation of germinal layers under certain condition.Based on these characteristics above, make the clinical application potential of BMSCs in tissue repair and gene therapy receive increasing concern, in Transplanted Retina degenerative disease Therapy study, also enjoy high praise.
Current Transplanted cells approach mainly contains intravenous injection, intravitreal and subretinal injection.(1) intravenous injection: be by pallium cell injection in vein, cell arrives eye by body circulation, and under migrating to retina or in retina.Can the method be simple to operate, is convenient to repeatedly inject, and not by the restriction of volumetric injection, but there is inducing thrombosis is formed and the risk of allergy, and there is cell after systemic circulation, arrive dispute near retina.(2) through intravitreal: be by pallium cell injection in vitreous space, moved in retina by vitreous space.Due to the obstruction of internal limiting membrane, only can arrive in retina less than the cell of 1%, and easy infection in ball.(3) subretinal injection: directly cell infusion is transplanted to target site and between RPE layer and photoreceptor layer.Although be subject to volume restriction, the cell quantity of single injection is on the low side, but under the cell injected can enter retina, as long as have enough viable cell quantity and can break up to RPE and retinal light injury photoreceptor under the state of local microenvironment, just likely playing cell replacement effect, therefore should be optimal implantation method.
Although the research of cellular replacement therapy retinal degenerative disease achieves the progress of advancing by leaps and bounds in recent years, but still be faced with many obstacles.Distinct issues are transplanted cells in the survival rate only about 0.01% of subretinal space.Therefore, the survival and the transfer ability that improve transplanted cells are the keys improving transplantation effect.At present, although implantation method cell suspension directly being injected subretinal space is simple and easy to do, stem cell loses the microenvironment of maintaining its existence simultaneously.Along with the development and progress of science and technology, people constantly to find new medicine that treatment retinal degeneration is correlated with and one simple to operate, the cell replacement method being convenient to promote, wound is little becomes the focus of attention in the last few years gradually.
Summary of the invention
The object of this invention is to provide a kind of stem cell and preparation for the treatment of retinal degeneration, by by mesenchymal stem cells MSCs optimization culture and differentiation-inducing, improve it and be divided into retina neural precursor like cell efficiency, its transplantation effect and retinal precursor cells have similar effect, graft area retinal function is effectively improved, for cellular replacement therapy retinal degenerative disease provides a kind of desirable cell derived, there is very strong superiority.Another object of the present invention is to the preparation method providing above-mentioned stem cell medicine, the method is through being separated, cultivating, go down to posterity and induce the stem cell products obtained, and preparation process is simple, reliable, favorable reproducibility.
Another object of the present invention is to provide a kind of using method for the treatment of the stem cell medicine of retinal degeneration, is a kind of simple to operate, is convenient to the cell replacement method promoted, wound is little.
To achieve these goals, the technical solution used in the present invention is: a kind of stem cell medicine for the treatment of retinal degeneration, is prepared by following steps:
(1) heparinization marrow blood is obtained;
(2) process of marrow blood and inoculation: with the volume ratio 1:2 LG-DMEM nutrient solution that to add containing volume fraction be 10%FBS, after mixing, be directly inoculated in culturing bottle;
(3) cultivation of primary cell: above-mentioned culturing bottle is placed in 5%CO
2, 37 DEG C of incubators cultivate, 48h does not clean first and directly changes liquid, and later every 2 ~ 3d changes liquid 1 time, and with buffered soln PBS cleaning before changing liquid, is full dose and changes liquid; Cell gathers in the crops primary cell with 0.25% trysinization after growing to 80% ~ 90% fusion, is designated as P0 for cell;
(4) cultivation of P1 passage cell: by above-mentioned P0 for cell with 5 × 10
4individual/ml density is inoculated in culturing bottle, and substratum is contain the LG-DMEM nutrient solution that volume fraction is 10%FBS, is placed in 5%CO
2, 37 DEG C of incubators cultivate, 24h does not clean first and directly changes liquid, and every 2d changes liquid 1 time later, and with buffered soln PBS cleaning before changing liquid, is full dose and changes liquid; Cell gathers in the crops primary cell with 0.025% trysinization after growing to 80% ~ 90% fusion, is designated as P1 for cell;
(5) cultivation of P2 ~ P4 passage cell: by above-mentioned P1 for cell with 5 × 10
4individual/ml density is inoculated in culturing bottle, repeats above-mentioned steps (4), can obtain P2 for cell; The like carry out Secondary Culture, P3 ~ P4 can be obtained for cell;
(6) qualification of mesenchymal stem cells MSCs: get P4 and carry out cell-surface antigens qualification for cell, wherein the expression of CD29, CD73, CD90 and CD105 is all more than 99%, and the expression of CD34, CD45 and HLA-DR is less than 3%;
(7) inducing culture of P5 passage cell: by above-mentioned P4 after qualified for cell with 5 × 10
4individual/ml density is inoculated in culturing bottle, substratum is the LG-DMEM nutrient solution of serum-free, wherein add 10ng/ml vitamin A, 10ng/ml transforminggrowthfactor-β1 (TGF β 1), the platelet-derived growth factor (PDGF) of 50 μ g/L and the dimethyl sulfoxide (DMSO) (DMSO) of 1%, be placed in 5%CO
2, 37 DEG C of incubators cultivate, 24h does not clean first and directly changes liquid, and every 2d changes liquid 1 time later, and with buffered soln PBS cleaning before changing liquid, is full dose and changes liquid; Cell gathers in the crops primary cell with 0.025% trysinization after growing to 80% ~ 90% fusion, obtains P5 for inducing cell, i.e. retina neural sample precursor cell;
(8) preparation of stem cell medicine: the P5 obtained in above-mentioned steps (7) is mixed with physiological saline for cell, and add 10ng/ml vitamin A, 10ng/ml transforminggrowthfactor-β1 (TGF β 1), 50 μ g/L platelet-derived growth factor (PDGF) and obtain stem cell medicine;
Wherein, P4 is also comprised for cell with (2 ~ 5) × 10 in step (5)
6individual/ml is frozen for subsequent use under lower than subzero 80 DEG C of environment;
Also comprise the recovery of P4 cell in step (5), be inoculated in 9ml nutrient solution after every 1ml freeze-stored cell recovery.
Further, in described step (8), the concentration of stem cell medicine is (1 ~ 10) × 10
9individual/ml.
A kind of preparation process for the treatment of stem cell in the stem cell medicine of retinal degeneration provided by the invention has above-mentioned steps (1) ~ (7) to form.
By the following technical solutions, concrete steps are a kind of using method for the treatment of the stem cell medicine of retinal degeneration:
The first step, the acquisition of stem cell medicine: the stem cell medicine obtaining treatment retinal degeneration with above-mentioned a kind of step (1) ~ (8) for the treatment of the stem cell medicine of retinal degeneration;
Second step, the storage of stem cell medicine and transport: stem cell medicine store with 2 ~ 15 DEG C of environment in and transport;
3rd step, the transplanting of stem cell medicine: adopt retinal pigment epithelium hemostasis after ball, injects the above-mentioned stem cell medicine of 3 μ l.
Further, in the described the first step, the concentration of stem cell medicine is (1 ~ 10) × 10
9individual/ml, preferably 5 × 10
9individual/ml.
Technical solution of the present invention also possesses following advantage:
1. the present invention adopts preferred method to prepare stem cell, inoculation and the marrow pluripotent stem cell cultivated grow fast, and the cycle is short, and cell quantity is many, and the abundant somatomedin saved in bone marrow microenvironment and short coherent substance, minimizing parting liquid and long normal-temperature operation are to the damage of cell.
2. in the stem cell medicine prepared by the present invention, add vitamin A, transforminggrowthfactor-β1 (TGF β 1), platelet-derived growth factor (PDGF), be of value to and safeguard normal vision function, promote the health of Epithelial cell and the synthesis of immunoglobulin (Ig), accelerate the differentiation promoting marrow pluripotent stem cell to retina cell.
3. the present invention adopts the method for retinal pigment epithelium hemostasis after ball, stem cell is made directly to arrive retinal epithelium layer, and be closely connected with choroid, have that site of action is direct, curative effect is fast, administration advantage accurately, be a kind of simple to operate, be convenient to popularization, traumatic little cell replacement method.
4. the present invention adopts marrow derived stem cell, and compared to other stem cells, marrow derived stem cell has the following advantages: 1) have the ability that the histocyte to three germinal layers breaks up, as osteocyte, chondrocyte, myocardial cell, neurocyte etc.; 2) draw materials easily, be easy to amplification, without the problem of ethics; 3) have powerful paracrine action, secretory nerve nutritional factor and somatomedin promote the reparation of damaged tissue; 4) there is the effect of immunomodulatory and the release of the inflammation-inhibiting factor, be conducive to allochthonous transplanting, reduce the use of immunological rejection medicine; 5) initiatively to the effect of damaged tissue zone migration; 6) without swollen neoplastic tendency after transplanting.
Accompanying drawing explanation
Accompanying drawing described herein is used to provide a further understanding of the present invention, and form a application's part, schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention, in the accompanying drawings:
Fig. 1 is stem cell medicine of the present invention, stem cell preparation and using method schema thereof.
Embodiment
Below with reference to the accompanying drawings and in conjunction with the embodiments, describe the present invention in detail.
Shown in Fig. 1 stem cell medicine of the present invention, stem cell preparation and using method schema thereof, the specific embodiment of the invention is as follows.
Embodiment 1 treats the preparation of the stem cell medicine of retinal degeneration
Treat a stem cell medicine for retinal degeneration, specifically comprise following preparation process:
(1) heparinization marrow blood is obtained;
(2) process of marrow blood and inoculation: with the volume ratio 1:2 LG-DMEM nutrient solution that to add containing volume fraction be 10%FBS, after mixing, be directly inoculated in culturing bottle;
(3) cultivation of primary cell: above-mentioned culturing bottle is placed in 5%CO
2, 37 DEG C of incubators cultivate, 48h does not clean first and directly changes liquid, and later every 2 ~ 3d changes liquid 1 time, and with buffered soln PBS cleaning before changing liquid, is full dose and changes liquid; Cell gathers in the crops primary cell with 0.25% trysinization after growing to 80% ~ 90% fusion, is designated as P0 for cell;
(4) cultivation of P1 passage cell: by above-mentioned P0 for cell with 5 × 10
4individual/ml density is inoculated in culturing bottle, and substratum is contain the LG-DMEM nutrient solution that volume fraction is 10%FBS, is placed in 5%CO
2, 37 DEG C of incubators cultivate, 24h does not clean first and directly changes liquid, and every 2d changes liquid 1 time later, and with buffered soln PBS cleaning before changing liquid, is full dose and changes liquid; Cell gathers in the crops primary cell with 0.025% trysinization after growing to 80% ~ 90% fusion, is designated as P1 for cell;
(5) cultivation of P2 ~ P4 passage cell: by above-mentioned P1 for cell with 5 × 10
4individual/ml density is inoculated in culturing bottle, repeats above-mentioned steps (4), can obtain P2 for cell; The like, P3 ~ P4 can be obtained for cell;
(6) qualification of mesenchymal stem cells MSCs: get P4 and carry out cell-surface antigens qualification for cell, wherein the expression of CD29, CD73, CD90 and CD105 is all more than 99%, and the expression of CD34, CD45 and HLA-DR is less than 3%;
(7) inducing culture of P5 passage cell: by above-mentioned P4 after qualified for cell with 5 × 10
4individual/ml density is inoculated in culturing bottle, substratum is the LG-DMEM nutrient solution of serum-free, wherein add 10ng/ml vitamin A, 10ng/ml transforminggrowthfactor-β1 (TGF β 1), the platelet-derived growth factor (PDGF) of 50 μ g/L and the dimethyl sulfoxide (DMSO) (DMSO) of 1%, be placed in 5%CO
2, 37 DEG C of incubators cultivate, 24h does not clean first and directly changes liquid, and every 2d changes liquid 1 time later, and with buffered soln PBS cleaning before changing liquid, is full dose and changes liquid; Cell gathers in the crops primary cell with 0.025% trysinization after growing to 80% ~ 90% fusion, obtains P5 for inducing cell, i.e. retina neural sample precursor cell;
(8) preparation of stem cell medicine: the P5 obtained in above-mentioned steps (7) is mixed with physiological saline for cell, and add 10ng/ml vitamin A, 10ng/ml transforminggrowthfactor-β1 (TGF β 1), 50 μ g/L platelet-derived growth factor (PDGF) and obtain stem cell medicine;
Wherein, P4 is also comprised for cell with (2 ~ 5) × 10 in step (5)
6individual/ml is frozen for subsequent use under lower than subzero 80 DEG C of environment;
Also comprise the recovery of P4 cell in step (5), be inoculated in 9ml nutrient solution after every 1ml freeze-stored cell recovery;
In step (8), the concentration of stem cell medicine is (1 ~ 10) × 10
9individual/ml.
The flow cytometry of embodiment 2 retina neural sample precursor cell
1. authentication step is as follows:
A) collect respectively differentiation-inducing in step 4 in above-described embodiment 1 before P4 for the P5 after induction in cell and step 7 for cell, be placed in centrifuge tube, the centrifugal 5min of 1000rpm, abandons supernatant liquor;
B) add PBS6ml Eddy diffusion cell, divide and be filled in 6 streaming pipes, the centrifugal 5min of 1000rpm, abandons supernatant liquor;
C) fix 30min under adding 4% paraformaldehyde room temperature, the centrifugal 5min of 1000rpm, abandons supernatant liquor;
D) add PBS Eddy diffusion cell, fully after concussion, the centrifugal 5min of 1000rpm, abandons supernatant liquor;
E) drip 10% lowlenthal serum confining liquid, close the centrifugal 5min of 30min, 1000rpm under room temperature, abandon supernatant liquor;
F) primary antibodie of antibody diluent dilution is dripped: mouse-anti people Nestin monoclonal antibody (1:100), mouse-anti people MAP2 monoclonal antibody (l:100), mouse-anti people Rhodopsin monoclonal antibody (l:100), mouse-anti people NSE monoclonal antibody (1:100), mouse-anti people GFAP monoclonal antibody (1:100), negative control is set, replaces primary antibodie with PBS, place 4 DEG C of overnight incubation in wet box;
G) repeating step 4) twice;
H) add antibody diluent dilution two resist: the goat anti-mouse IgG antibodies (1:100) of FITC mark, and 37 DEG C of lucifuges hatch lh;
I) repeating step 4) twice;
J) 1% paraformaldehyde 500 μ l Eddy diffusion cell is added, fixing to be measured;
K) 200 eye mesh screens filter, flow cytomery fluorescent value, and often pipe counts 10000 cells;
L) flow cytometer and software kit thereof is adopted to carry out peeking and analyzing.
2. qualification result:
A) flow cytometry situation: before and after induction, 3 detections have been carried out in the expression of nidogen (Nestin), microtubule-associated protein 2 (MAP2), neuronspecific enolase (NSE), glial fibrillary acidic protein (GFAP) and Visual purple albumen (Rhodopsin), represent with means standard deviation.
B) result display: after induction, Nestin positive cell drops to 79.3% from 29.8%, and MAP2 positive cell rises to 92.5% from 16.1%, Rhodopsin positive cell rises to 98.2% from 9.8%, NSE positive cell rises to 95.1% from 18.8%, GEAP positive cell rises to 21.3% from 1.2%, has significance (P<0.05) through statistical analysis difference.
C) show after inducing culture, major part BMSCs cell can express the labelled protein Rhodopsin (>90%) of ripe neuronic labelled protein MAP2, NSE and photoreceptor cell, and the gene of the mark Nestin of neural ancestral cells and protein expression increase (>75%) in remarkable afterwards in induction; The mark one of small part cell expressing neurogliocyte remembers Protein G FAP (<20%); Show after the induction of different inducing cultures, the differentiation due of BMSCs cell strengthens further, occur that the expression of certain organ-specific genes significantly raises, with regard to providing for the cellular replacement therapy of field of ophthalmology, one new, source is abundanter and seed cell source more easily of drawing materials for this.
Embodiment 3 animal model experiment
1. set up choroidal neovascularization (CNV) animal model
Application Krypton-red laser carries out light to mouse experiment retina and coagulates, and the power of laser, light coagulate spot diameter and the time shutter is respectively 300mW, 50 μm and 0.060s induction CNV animal model.
2. cell preparation transplant experiment
1) stem cell medicine for preparing of Example 1, cell concn is 5 × 10
9individual/ml, is loaded on the stem cell medicine prepared in EP pipe, and is placed in 2 ~ 15 DEG C of cold-storage insulation boxes stand-by, and in experimentation, cell can precipitate after leaving standstill, and notes using front mixing;
2) get experimental rat 1, weigh;
3) with 3% vetanarcol 30mg/kg intraperitoneal injection of anesthesia;
4) 0.5% dicaine is as anterior corneal surface narcotic eye drip, with compound tropine phthalein amine for mydriatic eye drip once loose large pupil;
5) under operating microscope, observe eyeground, after confirming that eyeground is without exception, prepare to start injection;
6) hold with left hand and fear son, clamp conjunctiva gently, fixing eyes, the right hand holds sharp syringe needle, under operating microscope direct-view, and 1mm after nasal side corneal limbus, inclined-plane thrusts upward, too dark without inserting needle, can puncture sclera, cause a tunnel;
7) handheld holder subsequently, syringe needle is entered intraocular along former tunnel, avoid crystal, under operating microscope direct-view, syringe needle arrives temporo side-looking nethike embrane from nasal side, choose territory, avascular area, when a needle tip touches retina, finger force forward impelling slightly, the resistance that front is slight can be felt, under heading into retinal pigment epithelium, assistant is now allowed to push away microsyringe, inject gap under 3 μ L cell suspensions to retinal pigment epithelium, keep the several seconds, for ensureing that the complete suspension of 3 μ L cell is pushed into after under retinal pigment epithelium, extract syringe needle, inject complete.Visible injection position retina protuberance, becomes disengaged position, without retina profuse bleeding, is considered as injecting successfully;
8) postoperative to rat painting ofloxacin eye ointment, protect from infection.Note in whole process, drip hyaluronic acid sodium, keep cornea moistening, in order to avoid bitot's patches, mist are only, affect the observation on eyeground;
9) animal sends animal center continuation raising back to after reviving.
3. transplant after experimental observation and sample disposal
In postoperative 3d, 1w, 21d, 10% hydration chloric acid anesthetized rat, after mydriasis, under operating microscope, observe eye ground with plane mirror, then rat is put to death in excessive anesthesia, unties manadesma along corneal limbus, from disconnected optic nerve, extract eyeball, pare off and remove excess tissue around, drop in 30% sucrose solution, 4 DEG C of preservations, subsequently capable freezing serial section.Under fluorescence microscopy, pick out the section of green fluorescence, carry out HE dyeing and immunofluorescence dyeing.
4. optical check
The inspections of retina optical coherence tomography (OCT), FFA (FFA) and Indocyanine-Green (ICGA) have been carried out respectively with postoperative 21d before Transplanted cells.
5. experimental result
1) fundoscopy of 15 experimental rats: postoperative 0d, all visible local retinal protuberance, departs from, without retinal hemorrhage, vitreous hemorrhage etc.Postoperative 1d, 30 eye conjunctivas are slightly congested, and cornea is all transparent, pupil circle, and 1 left eye occurs that crystal is slightly muddy, and eyeground is peeped unclear, and all the other crystal are all transparent, mixes only, hemorrhage and retina is obviously hemorrhage without vitreum, and the visible local retinal of transplanting place departs from, swells.Postoperative 7d, 30 conjunctival congestions disappear, corneal transparency, and 1 left eye crystal is obviously muddy, and eyeground is peeped unclear, and all the other crystal are transparent, and all without vitreous opacity, hemorrhage and retinal hemorrhage, original retina raised position is calm.Postoperative 21d, 30 equal corneal transparencies of rathole, pupil circle, 1 left eye crystal is obviously solely mixed, and eyeground is peeped unclear, and all the other crystal are transparent, and eyeground is normal.
2) situation that after transplanting, stem cell survives within the eye:
The frozen section of a, stem cell injection eye, during postoperative 3d, can see ceasma, and postoperative 7d, 21d eyeball serial section, is showed no ceasma, and ceasma can self-heal, and retina structure is complete, little to retina injury.Each time point, the cell of all visible EGFP mark, so gap under cells survive retinal pigment epithelium, cellular form is good, As time goes on cell, and EGFP (+) cell moves, mainly be colonizated in injection position, also do not observe and be integrated into neural retina layer.
B, get the frozen section with EGFP (+) cell further, after carrying out HE dyeing, the amphiblestroid form of finding more clearly, on partially sliced during 3d after surgery, can see that cell enters gap under retinal pigment epithelium, the position of pin hole, cell is gap survival under retinal pigment epithelium, section during postoperative 7d, do not observe ceasma, when 7d is described, ceasma can self-heal, little to retinal tissue damage, cell is thrown away and is gathered in injection site, cell arrangement is more tight, it is calm that the section of postoperative 21d can be observed the upper retina of most section, cell survival is gap under retinal pigment epithelium, between cell, arrangement closely, form laminate structure, have no heterocyst and become knurl tendency.
3.OCT check result
OCT can be observed the change of the optical characteristics caused due to the retinal tissue even change of cellular form, comprises the inflammatory exudation of any level of retina, fibrosis, hard exudate, hemorrhage and new vessel and film hyperplasia etc.OCT experimental result display in the present embodiment: after treatment, central fovea of macula retinal thickness significantly reduces, and macular area textural anomaly is restored.
4.FFA and ICGA check result
Experimental result shows: before treatment, the performances such as macular region hemorrhage, eyeground height fluorescence and exudative process can appear in optical fundus blood vessel associating radiography, and CNV focus is in active period.After treatment, macular area fluorescence leakage stops, and absorption of hemorrhage, loses high fluorescence, and CNV focus is in stationary phase.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (6)
1. treat a stem cell medicine for retinal degeneration, it is characterized in that, prepared by following steps:
(1) heparinization marrow blood is obtained;
(2) process of marrow blood and inoculation: with the volume ratio 1:2 LG-DMEM nutrient solution that to add containing volume fraction be 10%FBS, after mixing, be directly inoculated in culturing bottle;
(3) cultivation of primary cell: above-mentioned culturing bottle is placed in 5%CO
2, 37 DEG C of incubators cultivate, 48h does not clean first and directly changes liquid, and later every 2 ~ 3d changes liquid 1 time, and with buffered soln PBS cleaning before changing liquid, is full dose and changes liquid; Cell gathers in the crops primary cell with 0.25% trysinization after growing to 80% ~ 90% fusion, is designated as P0 for cell;
(4) cultivation of P1 passage cell: by above-mentioned P0 for cell with 5 × 10
4individual/ml density is inoculated in culturing bottle, and substratum is contain the LG-DMEM nutrient solution that volume fraction is 10%FBS, is placed in 5%CO
2, 37 DEG C of incubators cultivate, 24h does not clean first and directly changes liquid, and every 2d changes liquid 1 time later, and with buffered soln PBS cleaning before changing liquid, is full dose and changes liquid; Cell gathers in the crops primary cell with 0.025% trysinization after growing to 80% ~ 90% fusion, is designated as P1 for cell;
(5) cultivation of P2 ~ P4 passage cell: by above-mentioned P1 for cell with 5 × 10
4individual/ml density is inoculated in culturing bottle, repeats above-mentioned steps (4), can obtain P2 for cell; The like carry out Secondary Culture, P3 ~ P4 can be obtained for cell;
(6) qualification of mesenchymal stem cells MSCs: get P4 and carry out cell-surface antigens qualification for cell, wherein the expression of CD29, CD73, CD90 and CD105 is all more than 99%, and the expression of CD34, CD45 and HLA-DR is less than 3%;
(7) inducing culture of P5 passage cell: by above-mentioned P4 after qualified for cell with 5 × 10
4individual/ml density is inoculated in culturing bottle, substratum is the LG-DMEM nutrient solution of serum-free, wherein add 10ng/ml vitamin A, 10ng/ml transforminggrowthfactor-β1 (TGF β 1), the platelet-derived growth factor (PDGF) of 50 μ g/L and the dimethyl sulfoxide (DMSO) (DMSO) of 1%, be placed in 5%CO
2, 37 DEG C of incubators cultivate, 24h does not clean first and directly changes liquid, and every 2d changes liquid 1 time later, and with buffered soln PBS cleaning before changing liquid, is full dose and changes liquid; Cell gathers in the crops primary cell with 0.025% trysinization after growing to 80% ~ 90% fusion, obtains P5 for inducing cell, i.e. retina neural sample precursor cell;
(8) preparation of stem cell medicine: the P5 obtained in above-mentioned steps (7) is mixed with physiological saline for cell, and add 10ng/ml vitamin A, 10ng/ml transforminggrowthfactor-β1 (TGF β 1), 50 μ g/L platelet-derived growth factor (PDGF) and obtain stem cell medicine;
Wherein, P4 is also comprised for cell with (2 ~ 5) × 10 in described step (5)
6individual/ml is frozen for subsequent use under lower than subzero 80 DEG C of environment;
Also comprise the recovery of P4 cell in described step (5), be inoculated in 9ml nutrient solution after every 1ml freeze-stored cell recovery.
2. a kind of stem cell medicine for the treatment of retinal degeneration according to claim 1, is characterized in that, in described step (8), the concentration of stem cell medicine is (1 ~ 10) × 10
9individual/ml.
3. a kind of stem cell treated in the stem cell medicine of retinal degeneration according to claim 1, is characterized in that, be prepared from by following steps:
(1) heparinization marrow blood is obtained;
(2) process of marrow blood and inoculation: with the volume ratio 1:2 LG-DMEM nutrient solution that to add containing volume fraction be 10%FBS, after mixing, be directly inoculated in culturing bottle;
(3) cultivation of primary cell: above-mentioned culturing bottle is placed in 5%CO
2, 37 DEG C of incubators cultivate, 48h does not clean first and directly changes liquid, and later every 2 ~ 3d changes liquid 1 time, and with buffered soln PBS cleaning before changing liquid, is full dose and changes liquid; Cell gathers in the crops primary cell with 0.25% trysinization after growing to 80% ~ 90% fusion, is designated as P0 for cell;
(4) cultivation of P1 passage cell: by above-mentioned P0 for cell with 5 × 10
4individual/ml density is inoculated in culturing bottle, and substratum is contain the LG-DMEM nutrient solution that volume fraction is 10%FBS, is placed in 5%CO
2, 37 DEG C of incubators cultivate, 24h does not clean first and directly changes liquid, and every 2d changes liquid 1 time later, and with buffered soln PBS cleaning before changing liquid, is full dose and changes liquid; Cell gathers in the crops primary cell with 0.025% trysinization after growing to 80% ~ 90% fusion, is designated as P1 for cell;
(5) cultivation of P2 ~ P4 passage cell: by above-mentioned P1 for cell with 5 × 10
4individual/ml density is inoculated in culturing bottle, repeats above-mentioned steps (4), can obtain P2 for cell; The like carry out Secondary Culture, P3 ~ P4 can be obtained for cell;
(6) qualification of mesenchymal stem cells MSCs: get P4 and carry out cell-surface antigens qualification for cell, wherein the expression of CD29, CD73, CD90 and CD105 is all more than 99%, and the expression of CD34, CD45 and HLA-DR is less than 3%;
(7) inducing culture of P5 passage cell: by above-mentioned P4 after qualified for cell with 5 × 10
4individual/ml density is inoculated in culturing bottle, substratum is the LG-DMEM nutrient solution of serum-free, wherein add 10ng/ml vitamin A, 10ng/ml transforminggrowthfactor-β1 (TGF β 1), the platelet-derived growth factor (PDGF) of 50 μ g/L and the dimethyl sulfoxide (DMSO) (DMSO) of 1%, be placed in 5%CO
2, 37 DEG C of incubators cultivate, 24h does not clean first and directly changes liquid, and every 2d changes liquid 1 time later, and with buffered soln PBS cleaning before changing liquid, is full dose and changes liquid; Cell gathers in the crops primary cell with 0.025% trysinization after growing to 80% ~ 90% fusion, obtains P5 for inducing cell, i.e. retina neural sample precursor cell.
4. a kind of using method for the treatment of the stem cell medicine of retinal degeneration according to claim 1, is characterized in that, be prepared from by following steps:
(1) heparinization marrow blood is obtained;
(2) process of marrow blood and inoculation: with the volume ratio 1:2 LG-DMEM nutrient solution that to add containing volume fraction be 10%FBS, after mixing, be directly inoculated in culturing bottle;
(3) cultivation of primary cell: above-mentioned culturing bottle is placed in 5%CO
2, 37 DEG C of incubators cultivate, 48h does not clean first and directly changes liquid, and later every 2 ~ 3d changes liquid 1 time, and with buffered soln PBS cleaning before changing liquid, is full dose and changes liquid; Cell gathers in the crops primary cell with 0.25% trysinization after growing to 80% ~ 90% fusion, is designated as P0 for cell;
(4) cultivation of P1 passage cell: by above-mentioned P0 for cell with 5 × 10
4individual/ml density is inoculated in culturing bottle, and substratum is contain the LG-DMEM nutrient solution that volume fraction is 10%FBS, is placed in 5%CO
2, 37 DEG C of incubators cultivate, 24h does not clean first and directly changes liquid, and every 2d changes liquid 1 time later, and with buffered soln PBS cleaning before changing liquid, is full dose and changes liquid; Cell gathers in the crops primary cell with 0.025% trysinization after growing to 80% ~ 90% fusion, is designated as P1 for cell;
(5) cultivation of P2 ~ P4 passage cell: by above-mentioned P1 for cell with 5 × 10
4individual/ml density is inoculated in culturing bottle, repeats above-mentioned steps (4), can obtain P2 for cell; The like carry out Secondary Culture, P3 ~ P4 can be obtained for cell;
(6) qualification of mesenchymal stem cells MSCs: get P4 and carry out cell-surface antigens qualification for cell, wherein the expression of CD29, CD73, CD90 and CD105 is all more than 99%, and the expression of CD34, CD45 and HLA-DR is less than 3%;
(7) inducing culture of P5 passage cell: by above-mentioned P4 after qualified for cell with 5 × 10
4individual/ml density is inoculated in culturing bottle, substratum is the LG-DMEM nutrient solution of serum-free, wherein add 10ng/ml vitamin A, 10ng/ml transforminggrowthfactor-β1 (TGF β 1), the platelet-derived growth factor (PDGF) of 50 μ g/L and the dimethyl sulfoxide (DMSO) (DMSO) of 1%, be placed in 5%CO
2, 37 DEG C of incubators cultivate, 24h does not clean first and directly changes liquid, and every 2d changes liquid 1 time later, and with buffered soln PBS cleaning before changing liquid, is full dose and changes liquid; Cell gathers in the crops primary cell with 0.025% trysinization after growing to 80% ~ 90% fusion, obtains P5 for inducing cell, i.e. retina neural sample precursor cell;
(8) preparation of stem cell medicine: the P5 obtained in above-mentioned steps (7) is mixed with physiological saline for cell, and add 10ng/ml vitamin A, 10ng/ml transforminggrowthfactor-β1 (TGF β 1), 50 μ g/L platelet-derived growth factor (PDGF) and obtain stem cell medicine;
(9) storage of stem cell medicine and transport: stem cell medicine store with 2 ~ 15 DEG C of environment in and transport;
(10) transplanting of stem cell medicine: adopt retinal pigment epithelium hemostasis after ball, injects the above-mentioned stem cell medicine of 3 μ l.
5. a kind of using method for the treatment of in the stem cell medicine of retinal degeneration according to claim 4, is characterized in that, in described step (8), the concentration of stem cell medicine is (1 ~ 10) × 10
9individual/ml, preferably 5 × 10
9individual/ml.
6. the stem cell medicine described in Claims 1 to 5 and the application in retinal degenerative disease thereof.
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