CN105112368B - A kind of stem cell medicine and its application method for treating retinosis - Google Patents
A kind of stem cell medicine and its application method for treating retinosis Download PDFInfo
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Abstract
The present invention provides a kind of stem cell for treating retinosis and preparations, by the way that mesenchymal stem cell optimization culture and induction are broken up, it improves it and is divided into retina neural precursor like cell efficiency, its transplantation effect has similar effect to retinal precursor cells, graft area retinal function is set to be effectively improved, a kind of ideal cell origin is provided for cellular replacement therapy retinal degenerative disease, there is very strong superiority.The preparation method of stem cell medicine provided by the invention, the stem cell products obtained through separation, culture, passage and induction, preparation process is simple, reliable, favorable reproducibility.A kind of application method of stem cell medicine for treating retinosis of the present invention, it is a kind of easy to operate, it convenient for the cell replacement method promoted, wound is small, has broad application prospects, is the optimal path of current treatment retinosis class degenerative disease and redemption eyesight.
Description
Technical field
The present invention relates to regenerative medicines and stem cell biological technical field, are related to a kind of stem cell for treating retinosis
Preparation and its application method.
Background technology
The retina of people is about 0.1~0.5 millimeter thick, 10 layers is histologically classified as, wherein mainly there is 4 confluent monolayer cells groups
At ecto-entad is pigment epithelial cell layer, visual cell's layer, bipolar cell layer and ganglion-cell layer successively.Retinal pigment
Epithelial layer is closely coupled with choroid, is made of pigment epithelial cell (retinal pigment epitheium, RPE), normally
RPE be that there is polar cell monolayer, be located at retina outermost layer.It is the cellular layer for being metabolized high activity, has branch
The effects that holding with nutrition photoreceptor cell, shading, heat dissipation and regeneration and reparation.Its major function includes transhipment nutrients
Matter, retinol recycle, chromogenesis and the phagocytosis cone, rod cell acromere.
Retinal degenerative disease, including age-related macular degeneration, retinal pigment degeneration etc., incidence gradually rises
Height is main blinding disease.These diseases are lost with the function of progressive pigment epithelial cell, photoreceptor cell
It loses, with the characteristics of apoptosis or necrosis, causes the irreversible damage of eyesight.
Retinosis be mainly shown as Bruch ' s films thicken, RPE hyperpigmentations and RPE cell depletions etc..
Clinically it is broadly divided into atrophic type（Dryness）And neovascular（Moist or exudative type）Two types, the former mainly has glass-film
Wart, RPE exceptions and geographic atrophy change；The latter is mainly with choroidal neovascularization (choroidal
Neovascularization, CNV), RPE be detached from or plate-like fiber turn to pathological characteristic.
At present for retinosis, especially macular degeneration, modern medicine does not have specific therapy.Clinical normal root
According to the stage of the course of disease, antioxidant, vitamins, hemostat and optic nerve nutritional drugs or cell-stimulating preparation, glass are used
Internal injection anti-vascular endothelial growth factor（Vascular endothelialgrow factor, VEGF）Equal therapeutic modalities, but
It is only capable of delaying the progress of disease, cannot fundamentally replace the retina cell of lesion, reverse the course of disease.Past trial is performed the operation
Mode replace the RPE of macula area, mainly have macula lutea indexing operation and RPE autoplasties, being confirmed in long-term follow-up can
Maintain or improve the eyesight of patient.Such operation treats retinal degenerative disease for the RPE of transplanting health and provides foundation, however
Above-mentioned operation there are wounds big, complicated for operation, the disadvantage more than complication, in addition, the case being badly damaged for photoreceptor is then
It is difficult to play a role.Therefore it is easy to operate to find one kind, convenient for popularization, traumatic small cell replacement method increasingly by weight
Depending on.
Stem cell is the cell mass that a group has self-renewing and multidirectional potential.Utilize the plasticity of stem cell, orientation point
Retina and RPE that aim cell carries out cell transplantation or utilizes the secrete cytokines reparation of stem cell impaired are turned to,
As the research hotspot of retinal degenerative disease treatment.
The mechanism of cellular replacement therapy retinal degenerative disease：1. cell replacement is substituted by the stem cell of health and is lost
Sick cell；2. nutritional support, the healthy stem cell of transplanting promotes the survival of peripheral cell；3. graft area microenvironment is immune
Regulating and controlling effect such as promotes the inverse differentiation of graft area ortho states cell.Theoretically, effective new synaptic contact and herein basis are established
On visual function improve, be the successful standard of stem cell transplantation.Studies have shown that only stem cell is induced to developmental condition and is connect
Close ripe, competence exertion transplants effect.Ideal donorcells should have certain plasticity, that is, be in photosensitive precursor
The critical period developed to mature cell.
Accurately to induce various Derived Stem Cells to photosensitive precursor, it is necessary to understand the photosensitive precursor of Human embryo
Development.Human embryo retinal development is controlled by stringent program, and in embryo 7~8 weeks, gangliocyte was to start to occur, then,
The gradual directed differentiation of cone cell, horizontal cell, amakrine；Embryo 12 weeks or so, the substantially level of retina starts shape
At, and Beale's ganglion cells, rod cell, ortho states cell development are a little later.The nuclear factor accuracy controlling photosensory cell of high degree of cooperation
Differentiation, these genes according to the time, spatial order start or close, the growth and differentiation of embryonic cell are adjusted.Tool
There is the feature of the photosensitive precursor of transplanting effect：1. being in mitosis telophase, directed differentiation, does not continue to be proliferated；
2. expressing the nuclear factors such as Otx2, PAX6, CRX, Nrl；3. not yet development is ripe photosensory cell, intracellular gradual shape
At various opsins, that is, there is plasticity.
Mesenchymal stem cell（Bone mesenchymal stem cells, BMSCs）It is that a kind of multifunctional dry is thin
Born of the same parents, source is relatively extensive, and convenient material drawing safety, there is no the limitations in terms of ethics；With powerful paracrine action, point
The neurotrophic factor and growth factor secreted can promote the reparation of injury tissue；With active to the work of injury tissue zone migration
With；The tendency formed without tumour after transplanting；Immunogenicity is low, can carry out self or allograft, rejection occurs
Probability is relatively low.BMSCs equally has multi-lineage potential, osteocyte, cartilage cell, fat in addition to that can be divided into mesoderm origin
Fat cell, BMSCs can also be nerve cell, cardiac muscle cell, spongiocyte etc. across differentiation of germinal layers under certain condition.Based on
These upper characteristics so that clinical application potentials of the BMSCs in terms of tissue repair and gene therapy receives more and more attention,
High praise is also received in Transplanted Retina degenerative disease Therapy study.
Cell transplantation approach mainly has intravenous injection, intravitreal and subretinal injection at present.（1）Vein is noted
It penetrates：It is by pallium cell injection to vein, cell reaches eye by body circulation, and under migrating to retina or retina
It is interior.This method is easy to operate, convenient for injecting repeatedly, is not limited by volumetric injection, but there are inducing thrombosis formation and allergy
Risk, and can there are cells dispute near retina is reached after systemic circulation.（2）Through intravitreal：
It is to be moved in pallium cell injection to vitreous chamber in retina by vitreous chamber.Due to the obstruction of internal limiting membrane, only
Cell less than 1% can reach in retina, and easy infection in ball.（3）Subretinal injection：Directly cell infusion is transplanted
To target area, that is, RPE layers between photoreceptor layer.Although by volumetric constraint, the cell quantity of single injection is on the low side,
The cell injected can enter under retina, as long as having enough living cells quantities and can be in the state of local microenvironment
RPE and retinal light injury photoreceptor differentiation, it is possible to play cell replacement effect, therefore should be optimal transplanting side
Method.
Although the research of cellular replacement therapy retinal degenerative disease achieves the progress advanced by leaps and bounds in recent years,
So it is faced with many obstacles.Distinct issues are survival rate only about 0.01% of the transplanted cells in subretinal space.Therefore, it carries
The survival of high transplanted cells and transfer ability are the key that improve transplantation effect.Currently, cell suspension is directly injected into retina
The transplantation method of cavity of resorption is although simple and easy to do, but stem cell loses the microenvironment for maintaining its existence simultaneously.With the hair of science and technology
Exhibition and progress, people constantly search for treatment retinosis relevant new drug and one kind it is easy to operate, convenient for promote,
The small cell replacement method of wound is increasingly becoming the focus of attention in recent years.
Invention content
The object of the present invention is to provide a kind of stem cell for treating retinosis and preparations, by doing medulla mesenchyma
Cell optimization culture and induction are broken up, and improve it and are divided into retina neural precursor like cell efficiency, transplantation effect with regard
Nethike embrane precursor has similar effect, and graft area retinal function is made to be effectively improved, and is cellular replacement therapy retina
Degenerative disease provides a kind of ideal cell origin, has very strong superiority.Another object of the present invention is to provide upper
State the preparation method of stem cell medicine, the stem cell products that this method is obtained through separation, culture, passage and induction, preparation process
Simply, reliably, favorable reproducibility.
Another object of the present invention is to provide a kind of application method of stem cell medicine that treating retinosis, is a kind of
It is easy to operate, convenient for the cell replacement method promoted, wound is small.
To achieve the goals above, the technical solution adopted by the present invention is：A kind of stem cell system for treating retinosis
Agent, through the following steps that prepare：
（1）Obtain test tube of hepari marrow blood；
（2）The processing and inoculation of marrow blood：With volume ratio 1：2 are added containing the LG-DMEM cultures that volume fraction is 10%FBS
Liquid is directly inoculated in culture bottle after mixing；
（3）The culture of primary cell：Above-mentioned culture bottle is placed in 5%CO2, 37 DEG C of incubators cultivated, 48h is for the first time not
Liquid is directly changed in cleaning, and later every 2~3d changes liquid 1 time, and is cleaned with buffer solution PBS before changing liquid, is that full dose changes liquid；Cell is long
Harvest primary cell is digested with 0.25% pancreatin after to 80%~90% fusion, is denoted as P0 for cell；
（4）The culture of P1 passage cells：By above-mentioned P0 for cell with 5 × 104A/ml density is inoculated in culture bottle, training
It supports base to be the LG-DMEM culture solutions for being 10%FBS containing volume fraction, is placed in 5%CO2, 37 DEG C of incubators cultivated, for the first time for 24 hours
It does not clean and directly changes liquid, change liquid 1 time per 2d later, and cleaned with buffer solution PBS before changing liquid, be that full dose changes liquid；Cell is long
Harvest primary cell is digested with 0.025% pancreatin after to 80%~90% fusion, is denoted as P1 for cell；
（5）The culture of P2~P4 passage cells：By above-mentioned P1 for cell with 5 × 104A/ml density is inoculated in culture bottle
In, it repeats the above steps（4）, P2 is can get for cell；And so on carry out secondary culture, P3~P4 can be obtained for cell；
（6）The identification of mesenchymal stem cell：Take P4 for cell carry out cell surface antigen identification, wherein CD29,
99% or more, the expression of CD34, CD45 and HLA-DR are less than 3% for the expression of CD73, CD90 and CD105；
（7）The Fiber differentiation of P5 passage cells：By the P4 after above-mentioned identified qualification for cell with 5 × 104A/ml density
It is inoculated in culture bottle, culture medium is the LG-DMEM culture solutions of serum-free, wherein addition 10ng/ml vitamin As, 10ng/ml turn
Change grouth factor beta 1（TGFβ1）, 50 μ g/L platelet derived growth factor（PDGF）With 1% dimethyl sulfoxide (DMSO)（DMSO）, set
In 5%CO2, 37 DEG C of incubators cultivated, do not clean for the first time directly change liquid for 24 hours, change liquid 1 time per 2d later, and change before liquid with slow
Solution PBS cleanings are rushed, are that full dose changes liquid；Cell is primary thin with 0.025% pancreatin digestion harvest after growing to 80%~90% fusion
Born of the same parents obtain P5 for inducing cell, i.e. retina neural sample precursor；
（8）The preparation of stem cell medicine：By above-mentioned steps（7）The P5 of middle acquisition, with physiological saline mixing, and adds for cell
Add 10ng/ml vitamin As, 10ng/ml transforminggrowthfactor-β1s（TGFβ1）, 50 μ g/L platelet derived growth factor
（PDGF）And obtain stem cell medicine；
Wherein, step（5）In further include P4 for cell with（2~5）×106A/ml freezes in less than subzero 80 DEG C of environment
Under it is spare；
Step（5）In further include P4 cells recovery, be inoculated in 9ml culture solutions after recovery per 1ml freeze-stored cells.
Further, the step（8）Middle stem cell medicine it is a concentration of（1~10）×109A/ml.
The preparation process of stem cell has above-mentioned step in a kind of stem cell medicine for treating retinosis provided by the invention
Suddenly（1）~（7）Composition.
A kind of application method of stem cell medicine that treating retinosis uses following technical scheme, the specific steps are：
The first step, the acquisition of stem cell medicine：The step of with a kind of above-mentioned stem cell medicine for treating retinosis（1）
~（8）Obtain the stem cell medicine for the treatment of retinosis；
Second step, the storage and transport of stem cell medicine：Stem cell medicine is stored and in 2~15 DEG C of environment and is transported；
Third walks, the transplanting of stem cell medicine：Using being injected under the retinal pigment epithelium after ball, 3 μ l of injection are above-mentioned dry
Cell preparation.
Further, in the first step stem cell medicine it is a concentration of（1~10）×109A/ml, preferably 5 × 109
A/ml.
Technical solution of the present invention is also equipped with following advantages：
1. the present invention prepares stem cell using preferred method, inoculation and the marrow pluripotent stem cell growth cultivated are fast,
Period is short, and cell quantity is more, and saves the abundant growth factor in bone marrow microenvironment and promote coherent substance, reduces separation
The damage of liquid and prolonged normal-temperature operation to cell.
2. adding vitamin A, transforminggrowthfactor-β1 in the stem cell medicine prepared by the present invention（TGFβ1）, blood platelet
Source property growth factor（PDGF）, it is beneficial to safeguard normal vision function, promotes the health and immunoglobulin of Epithelial cell
Synthesis, accelerate promote differentiation of the marrow pluripotent stem cell to retina cell.
3. the present invention makes stem cell directly reach on retina using the method injected under the retinal pigment epithelium after ball
Cortex, and it is closely coupled with choroid, have the advantages that site of action is direct, curative effect is fast, administration is accurate, is a kind of operation letter
It is single, convenient for popularization, traumatic small cell replacement method.
4. the present invention uses marrow derived stem cell, compared to other stem cells, marrow derived stem cell has the following advantages:1)
With the ability broken up to the histocyte of three germinal layers, such as osteocyte, cartilage cell, cardiac muscle cell, nerve cell；2) it takes
The problem of material is easy, and is easy to expand, no ethics；3) there is powerful paracrine action, secretory nerve trophic factors and life
The long factor promotes the reparation of injury tissue；4) have the function of immunological regulation and inhibit inflammatory factor release, be conducive to of the same race different
The transplanting of body reduces the use of immunological rejection drug；5) actively to the effect of injury tissue zone migration；6) without tumour after transplanting
The tendency of formation.
Description of the drawings
Attached drawing described herein is used to provide further understanding of the present invention, and is constituted part of this application, this hair
Bright illustrative embodiments and their description do not constitute improper limitations of the present invention, in the accompanying drawings for explaining the present invention：
Fig. 1 is stem cell medicine, stem cell preparation and its application method flow chart of the present invention.
Specific implementation mode
It is below with reference to the accompanying drawings and in conjunction with the embodiments, next that the present invention will be described in detail.
Referring to Fig.1 shown in stem cell medicine of the present invention, stem cell preparation and its application method flow chart, the present invention is specifically real
It is as follows to apply mode.
Embodiment 1 treats the preparation of the stem cell medicine of retinosis
A kind of stem cell medicine for treating retinosis, specifically includes following preparation process：
（1）Obtain test tube of hepari marrow blood；
（2）The processing and inoculation of marrow blood：With volume ratio 1：2 are added containing the LG-DMEM cultures that volume fraction is 10%FBS
Liquid is directly inoculated in culture bottle after mixing；
（3）The culture of primary cell：Above-mentioned culture bottle is placed in 5%CO2, 37 DEG C of incubators cultivated, 48h is for the first time not
Liquid is directly changed in cleaning, and later every 2~3d changes liquid 1 time, and is cleaned with buffer solution PBS before changing liquid, is that full dose changes liquid；Cell is long
Harvest primary cell is digested with 0.25% pancreatin after to 80%~90% fusion, is denoted as P0 for cell；
（4）The culture of P1 passage cells：By above-mentioned P0 for cell with 5 × 104A/ml density is inoculated in culture bottle, training
It supports base to be the LG-DMEM culture solutions for being 10%FBS containing volume fraction, is placed in 5%CO2, 37 DEG C of incubators cultivated, for the first time for 24 hours
It does not clean and directly changes liquid, change liquid 1 time per 2d later, and cleaned with buffer solution PBS before changing liquid, be that full dose changes liquid；Cell is long
Harvest primary cell is digested with 0.025% pancreatin after to 80%~90% fusion, is denoted as P1 for cell；
（5）The culture of P2~P4 passage cells：By above-mentioned P1 for cell with 5 × 104A/ml density is inoculated in culture bottle
In, it repeats the above steps（4）, P2 is can get for cell；And so on, P3~P4 can be obtained for cell；
（6）The identification of mesenchymal stem cell：Take P4 for cell carry out cell surface antigen identification, wherein CD29,
99% or more, the expression of CD34, CD45 and HLA-DR are less than 3% for the expression of CD73, CD90 and CD105；
（7）The Fiber differentiation of P5 passage cells：By the P4 after above-mentioned identified qualification for cell with 5 × 104A/ml density
It is inoculated in culture bottle, culture medium is the LG-DMEM culture solutions of serum-free, wherein addition 10ng/ml vitamin As, 10ng/ml turn
Change grouth factor beta 1（TGFβ1）, 50 μ g/L platelet derived growth factor（PDGF）With 1% dimethyl sulfoxide (DMSO)（DMSO）, set
In 5%CO2, 37 DEG C of incubators cultivated, do not clean for the first time directly change liquid for 24 hours, change liquid 1 time per 2d later, and change before liquid with slow
Solution PBS cleanings are rushed, are that full dose changes liquid；Cell is primary thin with 0.025% pancreatin digestion harvest after growing to 80%~90% fusion
Born of the same parents obtain P5 for inducing cell, i.e. retina neural sample precursor；
（8）The preparation of stem cell medicine：By above-mentioned steps（7）The P5 of middle acquisition, with physiological saline mixing, and adds for cell
Add 10ng/ml vitamin As, 10ng/ml transforminggrowthfactor-β1s（TGFβ1）, 50 μ g/L platelet derived growth factor
（PDGF）And obtain stem cell medicine；
Wherein, step（5）In further include P4 for cell with（2~5）×106A/ml freezes in less than subzero 80 DEG C of environment
Under it is spare；
Step（5）In further include P4 cells recovery, be inoculated in 9ml culture solutions after recovery per 1ml freeze-stored cells；
Step（8）Middle stem cell medicine it is a concentration of（1~10）×109A/ml.
The flow cytometry of 2 retina neural sample precursor of embodiment
1. authentication step is as follows：
a）The P4 in above-described embodiment 1 in step 4 before induction differentiation is collected respectively for the P5 after being induced in cell and step 7
It for cell, is placed in centrifuge tube, 1000rpm centrifuges 5min, abandons supernatant；
b）PBS 6ml suspension cells again are added, in packing to 6 streaming pipes, 1000rpm centrifuges 5min, abandons supernatant；
c）4% paraformaldehyde is added and fixes 30min at room temperature, 1000rpm centrifuges 5min, abandons supernatant；
d）It is added PBS suspension cells again, after fully shaking, 1000rpm centrifuges 5min, abandons supernatant；
e）10% lowlenthal serum confining liquid is added dropwise, closes 30min at room temperature, 1000rpm centrifuges 5min, abandons supernatant；
f）The diluted primary antibody of antibody diluent is added dropwise:The anti-human Nestin monoclonal antibodies of mouse (1：100), the anti-human MAP2 of mouse is mono-
Clonal antibody (l:100), the anti-human Rhodopsin monoclonal antibodies (l of mouse:100), the anti-human NSE monoclonal antibodies of mouse (1:100),
The anti-human GFAP monoclonal antibodies of mouse (1:100), negative control is set, primary antibody is replaced with PBS, places 4 DEG C of overnight incubations in wet box；
g）Repeat step 4）Twice；
h）The diluted secondary antibody of antibody diluent is added：The goat anti-mouse IgG antibodies (1 of FITC labels:100) it, is protected from light for 37 DEG C
It is incubated lh；
i）Repeat step 4）Twice；
j）1% paraformaldehyde, 500 μ l suspension cells again are added, fixation is to be measured；
k）200 mesh screens filter, flow cytomery fluorescent value, and often pipe counts 10000 cells；
l）It is fetched and is analyzed using flow cytometer and its software kit.
2. qualification result：
a）Flow cytometry situation：The front and back nestin (Nestin) of induction, microtubule associated protein 2 (MAP2), nerve
The expression of first specificity olefinic alcohol enzyme (NSE), glial fibrillary acid protein (GFAP) and rhodopsin albumen (Rhodopsin)
3 detections have been carried out, have been indicated with means standard deviation.
b）As a result it shows：Nestin positive cells drop to 79.3% from 29.8% after induction, and MAP2 positive cells from
16.1%, which rises to 92.5%, Rhodopsin positive cells, rises to 98.2%, NSE positive cells from 9.8% and is risen to from 18.8%
95.1%, GEAP positive cell rise to 21.3% from 1.2%, through the significant (P of statistical analysis difference<0.05).
c）Show after Fiber differentiation, most of BMSCs cells can express mature neuron labelled protein MAP2,
NSE and photoreceptor cell labelled protein Rhodopsin (>90%), the gene of the marker Nestin of neural ancestral cells
With protein expression in dramatically increasing after induction（>75%）；The mark one that small part cell expresses Deiter's cells remembers Protein G FAP
(<20%)；Show after different inducing culture inductions, the differentiation due of BMSCs cells further enhances, and certain device occurs
The expression of official's specific gene significantly increases, this just provides a new, source for the cellular replacement therapy of field of ophthalmology
More rich and materials more easily seed cell source.
3 animal model experiment of embodiment
1. establishing choroidal neovascularization (CNV) animal model
Light is carried out using Krypton-red laser to mouse experiment retina to coagulate, when power, the light of laser coagulate spot diameter and exposure
Between be respectively that 300mW, 50 μm and 0.060s induce CNV animal models.
2. cell preparation transplant experiment
1）The stem cell medicine that Example 1 prepares, cell concentration are 5 × 109A/ml, by prepared stem cell
Preparation is loaded in EP pipes, is placed in 2~15 DEG C of cold-storage insulation boxes for use, and in experimentation, cell can precipitate after standing, and pay attention to making
With preceding mixing；
2）Experimental rat 1 is taken, is weighed；
3）With 3% yellow Jackets 30mg/kg intraperitoneal injection of anesthesia；
4）0.5% dicaine once dissipates big as anterior corneal surface anesthetic eye drip using compound tropine phthalein amine as mydriatic eye drip
Pupil；
5）Under surgical operation microscope, observing eye bottom, after confirming that eyeground is without exception, preparation starts to inject；
6）It is held with left hand and fears son, gently clamp conjunctiva, fixed eyes, the right hand holds sharp syringe needle, under surgical operation microscope direct-view,
The 1mm after nasal side corneal limbus, inclined-plane are pierced into upward, too deep without inserting needle, can be punctured sclera, be caused a tunnel；
7）Syringe needle is entered intraocular along former tunnel, avoids crystal by subsequent handheld holder, is looked at straight in surgical operation microscope
Under, syringe needle reaches temporo side view nethike embrane from nasal side, chooses avascular area domain, when a needle tip touches retina, finger slightly
Firmly forward impelling can feel the slight resistance in front, head under retinal pigment epithelium, allow assistant to push away micro-injection at this time
Device injects gap under 3 μ L cell suspensions to retinal pigment epithelium, is kept for the several seconds, to ensure that the 3 complete suspensions of μ L cells are pushed into
After under retinal pigment epithelium, syringe needle is extracted, injection finishes.It can be seen that injection position retina swells, and at disengaged position, no view
Film massive haemorrhage, is considered as and injects successfully；
8）It is postoperative to apply ofloxacin eye ointment to rat, it prevents from infecting.Pay attention in the whole process, sodium hyaluronate being added dropwise, protect
Cornea moistening is held, in order to avoid bitot's patches, mist are only, influences the observation on eyeground；
9）Animal center is sent back to after animal revival to continue to raise.
3. the Germicidal efficacy after transplanting and sample disposal
In postoperative 3d, 1w, 21d, 10% hydration chloric acid anesthetized rat after mydriasis, under surgical operation microscope, is seen with plane mirror
Eye ground is examined, rat is put to death in then excessive anesthesia, unlocks fascia along corneal limbus, detachment optic nerve extracts eyeball, pares off and remove
Surrounding excess tissue is put into 30% sucrose solution, and 4 DEG C of preservations, subsequent row freezes serial section.Under fluorescence microscopy, pick out
There is the slice of green fluorescence, carries out HE dyeing and immunofluorescence dyeing.
4. optical check
Before cell transplantation and postoperative 21d has carried out retina optical coherence tomography (OCT), fluorescein eye respectively
Bottom angiography (FFA) and Indocyanine-Green（ICGA）It checks.
5. experimental result
1）The funduscopy of 15 experimental rats：Postoperative 0d, visible local retinal protuberance, is detached from, no retina goes out
Blood, vitreous hemorrhage etc..Postoperative 1d, 30 eye conjunctivas are slightly congested, and cornea is transparent, and pupil circle, 1 left eye crystal occurs slightly
Muddiness, eyeground are peeped unclear, remaining crystal is transparent, and no vitreum is mixed solely, bleeding and the apparent bleeding of retina, visible office at transplanting
Portion's detachment of retina, protuberance.Postoperative 7d, 30 conjunctival congestions subside, and corneal transparency, 1 left eye crystal is obviously muddy, eyeground
Peep unclear, remaining crystal is transparent, and without vitreous opacity, bleeding and retinal hemorrhage, original retina raised position is calm.Art
21d afterwards, 30 equal corneal transparencies of rathole, pupil circle, 1 left eye crystal is obviously mixed only, and eyeground is peeped unclear, remaining crystal is transparent,
Eyeground is normal.
2）The case where stem cell survives within the eye after transplanting：
A, the frozen section of stem cell injection eye, when postoperative 3d, it can be seen that ceasma, postoperative 7d, 21d eyeball serial section,
Be showed no ceasma, ceasma can self-heal, retina structure is complete, small to retinal damage.Each point in time, it is visible
The cell of EGFP labels, it is gap under retinal pigment epithelium that cell, which can survive in, and cellular morphology is good, over time
Cell, EGFP (+) cell do not migrate, are mainly colonized in injection position, also do not observe and are integrated into neural retina layer.
B, the frozen section with EGFP (+) cell is further taken, after carrying out HE dyeing, clearer finding retina
Form, it is partially sliced when 3d after surgery on, it can be seen that cell enters gap under retinal pigment epithelium, the position of pin hole,
Cell gap under retinal pigment epithelium is survived, and slice when postoperative 7d does not observe ceasma, when illustrating 7d, ceasma can from
Row healing, to retinal tissue damage it is small, cell, which is thrown away, is gathered in injection site, cell arrangement more closely, the slice of postoperative 21d
Most slices can be observed, and above retina is calm, cell survival gap under retinal pigment epithelium, is arranged closely between cell,
Layer structure is formed, has no heterocyst and tumor formation tendency.
3.OCT inspection results
The change of the optical characteristics caused by the retinal tissue even variation of cellular morphology, packet can be observed in OCT
Include inflammatory exudation, fibrosis, hard exudate, bleeding and the new vessels and film hyperplasia etc. of any level of retina.The present embodiment
Middle OCT experimental results are shown：Central fovea of macula retinal thickness significantly reduces after treatment, and macular area textural anomaly is restored.
4. FFA and ICGA inspection results
Experimental result is shown：Optical fundus blood vessel joint radiography may occur in which the high fluorescence of macular region hemorrhage, eyeground and exudation before treatment
Performances, the CNV lesions such as venereal disease change are in active stage.After treatment, macular area fluorescence leakage stops, and absorption of hemorrhage loses high fluorescence,
CNV lesions are in resting stage.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, any made by repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (2)
1. a kind of stem cell medicine for treating retinosis, which is characterized in that through the following steps that prepare：
（1）The culture of P1 passage cells：By P0 for mesenchymal stem cell with 5 × 104A/mL density is inoculated in culture bottle,
Culture medium is the LG-DMEM culture solutions for being 10%FBS containing volume fraction, is placed in 5%CO2, 37 DEG C of incubators cultivated, it is first for 24 hours
Secondary do not clean directly changes liquid, changes liquid 1 time per 2d later, and cleaned with buffer solution PBS before changing liquid, is that full dose changes liquid；Cell
Harvest primary cell is digested with 0.025% pancreatin after length to 80%~90% fusion, is denoted as P1 for cell；
（2）The culture of P2~P4 passage cells：By above-mentioned P1 for cell with 5 × 104A/mL density is inoculated in culture bottle, is repeated
Above-mentioned steps（1）, P2 is can get for cell；And so on carry out secondary culture, P3~P4 can be obtained for cell；
（3）The identification of mesenchymal stem cell：Take P4 for cell carry out cell surface antigen identification, wherein CD29, CD73,
99% or more, the expression of CD34, CD45 and HLA-DR are less than 3% for the expression of CD90 and CD105；
（4）The Fiber differentiation of P5 passage cells：By the P4 after above-mentioned identified qualification for cell with 5 × 104A/mL density inoculation
In culture bottle, culture medium is the LG-DMEM culture solutions of serum-free, wherein addition 10ng/mL vitamin As, 10ng/mL conversion lifes
Long factor-beta 1（TGFβ1）, 50 μ g/L platelet derived growth factor（PDGF）With 1% dimethyl sulfoxide (DMSO)（DMSO）, it is placed in 5%
CO2, 37 DEG C of incubators cultivated, do not clean for the first time directly change liquid for 24 hours, change liquid 1 time per 2d later, and change molten with buffering before liquid
Liquid PBS cleanings, are that full dose changes liquid；Cell digests harvest cell with 0.025% pancreatin after growing to 80%~90% fusion and obtains P5 generations
Inducing cell, i.e. retina neural sample precursor；
（5）The preparation of stem cell medicine：By above-mentioned steps（4）The P5 of middle acquisition, with physiological saline mixing, and is added for cell
10ng/mL vitamin As, 10ng/mL transforminggrowthfactor-β1s（TGFβ1）, 50 μ g/L platelet derived growth factor（PDGF）
And obtain stem cell medicine；
Wherein, the step（2）In further include P4 for cell with（2~5）×106A/mL freezes in less than subzero 80 DEG C of environment
Under it is spare；
The step（2）In further include P4 cells recovery, be inoculated in 9mL culture solutions after recovery per 1mL freeze-stored cells.
2. a kind of stem cell medicine for treating retinosis according to claim 1, which is characterized in that the step
（5）Middle stem cell medicine it is a concentration of（1~10）×109A/mL.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101407787A (en) * | 2008-11-21 | 2009-04-15 | 中山大学中山眼科中心 | Method for preparing retina neural ganglia progenitor cells |
| CN101688178A (en) * | 2007-04-18 | 2010-03-31 | 哈达锡特医学研究服务及发展有限公司 | Stem cell-derived retinal pigment epithelium |
-
2015
- 2015-09-30 CN CN201510636044.8A patent/CN105112368B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101688178A (en) * | 2007-04-18 | 2010-03-31 | 哈达锡特医学研究服务及发展有限公司 | Stem cell-derived retinal pigment epithelium |
| CN101407787A (en) * | 2008-11-21 | 2009-04-15 | 中山大学中山眼科中心 | Method for preparing retina neural ganglia progenitor cells |
Non-Patent Citations (8)
| Title |
|---|
| "Identification of retinal ganglion cell neuroprotection conferred by platelet-derived growth factor through analysis of the mesenchymal stem cell secretome";Thomas V.Johnson et al.;《Brain》;20131030;第137卷;第503-519页 * |
| "PDGF受体、TGF-β1及其受体在视网膜下液中的表达";许讯等;《眼科新进展》;20020131;第22卷(第1期);第9-11页 * |
| "Stem/progenitor cells:A potential source of retina-specific cells for retinal repair";YongYan Bi et al.;《Neuroscience Research》;20090730;第65卷;第215-211页 * |
| "人骨髓间充质干细胞向光感受器样细胞诱导分化的研究";徐国兴等;《国际眼科杂志》;20110131;第11卷(第1期);第14-18页 * |
| "成人骨髓间充质干细胞体外向视网膜细胞的诱导分化";俞海燕等;《中国药理学通报》;20140521;第30卷(第6期);第787-788页材料与方法和结果部分 * |
| "维生素A酸、锌等诱导大鼠骨髓间质干细胞分化为神经元样细胞的研究";唐云安等;《四川大学学报（医学版）》;20031231;第34卷(第3期);第377-380页 * |
| "视网膜色素上皮细胞培养上清液联合视黄酸对骨髓间充质干细胞的诱导分化作用";冯月兰;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20091015(第10期);第3页摘要、第7-8页和第31-33页讨论部分 * |
| "骨髓间充质干细胞诱导分化为视网膜细胞的研究";冯月兰等;《海峡科学》;20071231(第12期);第22-23页 * |
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