CN106389470A - Stem cell preparation, method for preparing same and application of stem cell preparation - Google Patents
Stem cell preparation, method for preparing same and application of stem cell preparation Download PDFInfo
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Abstract
The invention relates to the field of biological treatment and discloses a stem cell preparation, a method for preparing same and application of the stem cell preparation. The stem cell preparation disclosed by the invention comprises the following components according to cell concentration: 5*10<4> to 1*10<6> umbilical cord mesenchymal stem cells per 20 Mu L; 1*10<5> to 5*10<6> retinal pigment epitheliums per 20 Mu L, wherein a ratio of the umbilical cord mesenchymal stem cells to the retinal pigment epitheliums is 1:2 to 1:8. The retinal pigment epitheliums are obtained through induction by the umbilical cord mesenchymal stem cells. The stem cell disclosed by the invention has a good curative efficacy to diabetic retinopathy.
Description
Technical field
The present invention relates to Biotherapeutics field, particularly to a kind of stem cell medicine and its preparation method and application.
Background technology
, as a kind of endocrine metabolism disease, its prevalence is just with the growth of population, aging, urbanization for diabetes
And the generalization of obesity and shortage motion is gradually increased.Diabetic retinopathy (Diabetic Retinopathy,
DR it is) one of its more serious microvascular complication, in China, the especially prevalence in the more flourishing big and medium-sized cities of economy just
Increasing year by year.Show through investigation, in ten thousand blind persons worldwide, account for 4.5% by DR blinding.Therefore, to cause
The treatment of blind property oculopathy DR is extremely urgent.
DR is a kind of multifactor retina PD leading to, and pathogeny is extremely complex, is related to a large amount of differences
Cell, the factor and molecule.Traditional theory thinks, DR is a kind of retinal microvascular disease, but in the last few years, gets more and more
Research confirm diabetes have impact on retina whole neural blood vessel unit, just cause neural blood vessel coupling numbers at the disease initial stage
The minimizing of amount, gradual neural degeneration, gliosiss and neuroinflamation sexually revise, and these pathological changes appear at and face
Before the visible retinal microvascular of bed changes.The neurocyte of retina, glial cell, microcirculqtory system and chronic inflammatory disease
Factor etc. interacts in DR pathogenic process and leads to the lasting development of the state of an illness, and then the microangiopathies of retina.
At present, the primary treatment measure of DR includes Drug therapy, laser therapy and 3 kinds of sides of Vitrectomy treatment
Formula.It is strict glycemic control and blood pressure, blood viscosity management, for middle severe non-proliferative type DR and the proliferous type of early stage at all
The feasible laser therapy of DR is to reduce blinding risk, and is only capable of going for the proliferous type DR of vitreous hemorrhage or detachment of retina
Vitrectomy.Operation can remove hematocele, anatomical reduction retina, but the function of retina suffers damage already, depending on
Power recovers unsatisfactory, and also operating difficulty is big, and operation itself is in the strike of retina and art and more than post-operative complication etc.
Unfavorable factor.
Ocular angiogenesis progress shows in recent years：VEGF (vascular endothelial
Growth factor, VEGF) it is to cause Diabetic Macular water (diabeticmacularedema, DME) and retina and arteries and veins
The critical stimuluses factor of network film new vesselses (choroidal neo vascularization, CNV).Targeting anti-vegf is biological
Although treatment can improve the blood retina barrier that DR causes damaging, however anti vegf agents treatment still fall within destruction new vesselses,
The field of angiogenesis inhibitor, can not fundamentally stop the development of DR, and this medicine of duplicate injection can cause retina god
Toxicity damage through cell.
Take various modes to reduce the incidence rate of diabetes or to stop disease before visible microangiopathies occur
The progress of disease is only effective.It is true that treatment diabetic retinopathy the most effectively method be to reduce
The progress that disease occurs or stoped before visible microangiopathies occur of diabetes.There is the forward sight of pathological changes in retina
Just apoptosis in nethike embrane neuronal cell and M ü ller cell, can be simultaneously right in DR early stage if there are a kind of Therapeutic Method
These aspects are intervened, then the treatment of DR may be made more comprehensively more meaningful.Retinal epithelial cells (retinal
Pigmented epithelium, RPE), important function is had on the maintenance of retinal neuronal cell and self renewal, such as：
The photoreceptor cell outer segment membranous disc that phagocytosis digestion comes off；Promote the regeneration of 11 1 cis retinenes；Adjust the immunoreation of ophthalmic；
Participate in forming retinal vessel barrier etc..There are some researches show, RPE cell is transplanted to pathological changes subretinal space patient can be delayed to regard
Feel that the process lost or part recover its visual performance.Subretinal space autologous RPE cell transplantation is more stable, and has long-term
Curative effect.But its source and quantity limit and hinder the method extensive application clinically.
At present, with DR pathogenesis progressively understand and its novelty therapeutic modality appearance successively, stem cell injection
Therapy increasingly causes everybody concern.Stem cell is the pluripotent cell that a class has the of self-replication capacity.With stem cell it is
The therapeutic modality on basis is central authorities and peripheral nervous system, and the damage of especially neural gaps creates good for neuranagenesis
Good environment.Wherein, mescenchymal stem cell (MSC), gradually enters into the visual field of researcher.It has very high plasticity with self more
New ability, immunogenicity weak moreover it is possible to secrete multiple factors, become organizational project preferable seed cell source.
Treat the Drug therapy medicine of diabetic retinopathy at present, although as anti vegf agents can improve retina
Damage, but to blood vessel, there is toxicity damage；There is the stem cell medicine of efficient curative effect special due to its source of human stem cell
Property and quantity finiteness, limit clinic application.Therefore, research and develop one kind to be used for treating diabetic by mescenchymal stem cell
The preparation of retinopathy is those skilled in the art's technical issues that need to address.
Content of the invention
The invention discloses a kind of stem cell medicine and its preparation method and application, described stem cell medicine treatment diabetes
Property retinopathy is evident in efficacy, toxic and side effects are little.
The present invention is achieved by the following technical solutions：
The invention discloses a kind of stem cell medicine, by cell gauge, including following component：
Umbilical cord mesenchymal stem cells 5 × 104～1 × 106Individual/20 μ L；
Retinal pigment epithelium 1 × 105～5 × 106Individual/20 μ L；
The ratio of described umbilical cord mesenchymal stem cells and described retinal pigment epithelium is 1:4.
Preferably, described stem cell medicine presses cell gauge, including following component：
Umbilical cord mesenchymal stem cells 1 × 105Individual/20 μ L；
Retinal pigment epithelium 4 × 105Individual/20 μ L；
The ratio of described umbilical cord mesenchymal stem cells and described retinal pigment epithelium is 1:4.
Preferably, the described retinal pigment epithelium in described stem cell medicine is to be done carefully by described umbilical cord mesenchyma
Born of the same parents' induction differentiation is prepared from.
The separation and Culture of umbilical cord mesenchymal stem cells and authentication method list of references《Mesenchymal stem cell transplantation glycosuria
Sick rat early stage retinopathy experimentation》.
The preparation of retinal pigment epithelium of umbilical cord mesenchymal stem cells induction and identification list of references《Transcription factor
Induction human umbilical cord mesenchymal stem cells transdifferentiation is the research of retinal pigment epithelium like cell》, Tongji University's journal, 2015,
36(5).
The invention discloses the preparation method of described stem cell medicine, comprise the following steps：
Described umbilical cord mesenchymal stem cells are mixed with described retinal pigment epithelium, described stem cell system is obtained
Agent.
The invention also discloses the stem cell medicine that described stem cell medicine or described preparation method are obtained is in preparation treatment
Application in the pharmaceutical preparation of diabetic retinopathy.
In clinical studies, the application of the mescenchymal stem cell (MSCs) of embryonic stem cell and derived from bone marrow is relatively broad.
However, from the point of view of Point of View of Clinical, the acquisition of bone marrow is a kind of invasive mode, increase patient suffering, limited source, and with
Its propagation/inducibility of the increase at age progressively declines；The limited source of embryonic stem cell and its safety and moral check are asked
There is dispute always in topic.
The present invention adopts the mescenchymal stem cell in umbilical cord source and its group of the retinal pigment epithelium of induction differentiation
Close the treatment that preparation carries out DR.Umbilical cord mesenchymal stem cells have wide material sources, convenience of drawing materials, proliferative ability is strong, possess multidirectional point
Change potential and the feature possessing immunoregulation effect.It is thin that human umbilical cord mesenchymal stem cells can be induced to differentiate into islets of langerhans sample in vitro
Born of the same parents, this noble cellss cluster transplantation is entered in liver, and internal peptide, insulin secretion increase, and blood sugar level is controlled.And umbilicuss
Band mesenchymal cell can be substituted or repaired the retinal pigment damaging by drug-induced differentiation chemical conversion retinal epithelial cells
Epithelial cell.Neural stem cell in retina can be promoted in secretory nerve trophic factors under suitable microenvironment after local injection
And the proliferation and differentiation of precursor, carry out immunomodulating, the local inflammation reaction on suppression optical fundus, nutrition and protection fundus tissue.
The umbilical cord mesenchymal stem cells in source and its retinal pigment epithelium inducing differentiation into can reduce to body
Immunogenicity, and the administering mode of stem cell medicine of the present invention is to be injected into eye vitreous chamber, is injected into vitreous chamber and draws
Play immunological rejection probability little.
Animal experiment, experiment results proved embodiment of the present invention system are carried out to the stem cell medicine of embodiment of the present invention preparation
Standby stem cell medicine has good curative effect to diabetic retinopathy.
Prior art is often alone mescenchymal stem cell, is not used in combination using two kinds of cell types.With existing
Technology is compared, and stem cell medicine disclosed by the invention has good curative effect to diabetic retinopathy, and described stem cell
Cellular component in preparation is drawn materials easily, and wide material sources are it is easy to popularization and application.
Specific embodiment
The invention discloses a kind of stem cell medicine and its application, described stem cell medicine is to diabetic retinopathy
Evident in efficacy, and toxic and side effects are little.Those skilled in the art can use for reference present disclosure, be suitably modified technological parameter and realize.Special
Not it is pointed out that all similar replacements and change apparent to those skilled in the art, they all by
It is considered as including in the present invention.The method of the present invention and application are described by preferred embodiment, and related personnel is obvious
In without departing from present invention, spirit and scope, method described herein and application can be modified or suitably change and group
Close, to realize and to apply the technology of the present invention.
With reference to embodiment, the present invention is expanded on further.
Using rat as subjects, umbilical cord mesenchymal stem cells used are the umbilicuss drawn materials in rat body to the present embodiment
Band mescenchymal stem cell, retinal epithelial cells used are to form from the induction of rat umbilical cord mesenchymal stem cells.Should be noted
, method disclosed in the present embodiment has universality however it is not limited to apply on rat, applies also for other species,
Can be applicable to human body.
The separation and Culture of umbilical cord mesenchymal stem cells and authentication method list of references《Mesenchymal stem cell transplantation glycosuria
Sick rat early stage retinopathy experimentation》.
The preparation of retinal pigment epithelium of umbilical cord mesenchymal stem cells induction and identification list of references《Transcription factor
Induction human umbilical cord mesenchymal stem cells transdifferentiation is the research of retinal pigment epithelium like cell》, Tongji University's journal, 2015,
36(5).
Embodiment 1 prepares stem cell medicine
By umbilical cord mesenchymal stem cells 0.2 × 105Individual with retinal pigment epithelium 0.8 × 105Individual mixing, makes 20 μ
L mixing stem cell medicine, the solvent of use is normal saline.Wherein, umbilical cord mesenchymal stem cells are thin with retinal pigment epithelium
The ratio of born of the same parents is 1:4.
Embodiment 2 prepares stem cell medicine
By umbilical cord mesenchymal stem cells 0.33 × 105Individual with retinal pigment epithelium 0.67 × 105Individual mixing, makes
20 μ L mixing stem cell medicines, the solvent of use is normal saline.Wherein, umbilical cord mesenchymal stem cells and retinal pigment epithelium
The ratio of cell is 1:2.
Embodiment 3 prepares stem cell medicine
By umbilical cord mesenchymal stem cells 0.1 × 105Each and every one and retinal pigment epithelium 0.8 × 105Individual mixing, makes
20 μ L mixing stem cell medicines, the solvent of use is normal saline.Wherein, umbilical cord mesenchymal stem cells and retinal pigment epithelium
The ratio of cell is 1:8.
Embodiment 4 prepares stem cell medicine
By umbilical cord mesenchymal stem cells 5 × 104Individual with retinal pigment epithelium 1 × 105Individual mixing, makes 20 μ L and mixes
Close stem cell medicine, the solvent of use is normal saline.Wherein, umbilical cord mesenchymal stem cells and retinal pigment epithelium
Ratio is 1:2.
Embodiment 5 prepares stem cell medicine
By umbilical cord mesenchymal stem cells 1 × 106Individual with retinal pigment epithelium 5 × 106Individual mixing, makes 20 μ L and mixes
Close stem cell medicine, the solvent of use is normal saline.Wherein, umbilical cord mesenchymal stem cells and retinal pigment epithelium
Ratio is 1:5.
The foundation of embodiment 6DR animal model and identification
1st, the foundation of diabetes model
Take healthy female male SD rat totally 38,8～10 week old, body weight 200～220g.It is randomly divided into Normal group (n
=5) and model group (n=33).All row slit lamp, after direct ophthalmoscopy, exclude ocular disease.Rat adaptability word supports 3
After it, every rat carries out measured body weight, calculates streptozotocin (STZ) consumption with 45mg/kg.STZ is dissolved in fresh joining
System Chinese catalpa lemon acid-lemon acid sodium buffer in, after being made into 2%STZ solution, carry out tail vein injection at once.After injection when 1 week
Tail point blood surveys blood glucose, blood glucose>16.7mmol/L is successfully established for diabetes model.After Cheng Mo, Rat Standard is raised and diet, sees
Examine the ordinary circumstances such as diet drinking-water, defecation, hair color, mobility, the mental status, weekly timing detection blood glucose and body weight, periodically
Observe anterior ocular segment and optical fundus, monthly row fundus photography, fundus fluorescein angiography.Period finds that dead and blood sugar recovery rat is timely
Remove.
2. the identification of diabetic retinopathy model
Raise after diabetes rats modeling success 10 weeks, randomly choose 2 from Normal group and model group respectively
Carry out FITC-dextran fluoroscopic visualization inner nuclear layer retina, to confirm whether DR model is successful.FITC-dextran burns light radiography
The method of inner nuclear layer retina is as follows：
(1) join liquid：50mgFITC-dextran fluorescein is dissolved in 1mL distilled water, standby after being extracted with syringe.
(2) anaesthetize：Using 10% hydration chloric acid to experimental rat row intraperitoneal injection of anesthesia.
(3) irrigate：Fixing rat head, extremity, open rapidly breast and expose heart, irrigated by apex inserting needle row left ventricle,
It is that perfusion sufficiently indicates that yellow in the positions such as the ear of rat, nose.
(4) collection of specimens：Extract eyeball at once, be fixed on 4% paraformaldehyde 30 minutes.
(5) inner nuclear layer retina：Under ophthalmic operating microscope, cut eyeball along corneoscleral junction, remove prosthomere tissue and glass
Glass body, careful separation goes out retina as complete as possible, and radial centered on optic disc cut off, be laid on microscope slide, drip
2% neutral gum 1, covered.
(6) observe：Observe retinal vascular morphologies using prominent light microscope and take pictures, take excitation wavelength 490nm, filtration
Optical wavelength 520nm.
Result shows the equal modeling success of 38 rats.
The clinical trial of different component proportioning in embodiment 7 stem cell medicine
Successful diabetic retinopathy rat model is modeled as subjects using embodiment 6, chooses 16 rats
It is divided into 4 groups, every group is 4 rats (8 eyes).
3 test group of setting, the respectively stem cell medicine of embodiment 1 to 3 preparation.Separately set a blank control group, empty
The normal saline of white matched group local injection equivalent.
The method of intravitreal stem cell medicine is as follows：
(1) by operating theater instruments high pressure steam sterilizations such as microsyringe (volume 20 μ L) and the micro- tweezers of ophthalmology before injecting,
Cool drying is standby.
(2) anaesthetize and fix：Rat weight, is anaesthetized by dosage lumbar injection 10% chloral hydrate of 3mL/kg.Greatly
Mus conjunctiva is intracapsular to instill the abundant mydriasis of Tropicamide Phenylephrine Eye Drops, instills Proparacaine Hydrochloride Eye Drops topical anesthesia, treats four
Limb is weak and limp, absent corneal reflex, is fixed on homemade foam operating-table.
(3) sterilize：Iodophor disinfection conjunctival sac is clean with normal saline flushing after 1 minute.
(4) extract each test group cell suspension or normal saline 20 μ L with microsyringe, exclude air, in ophthalmologic operation
Under microscope, after limbus of corneae, about 2mm sentences 45° angle inserting needle and is injected in rat vitreous chamber, and avoids damage to crystalline lenses.
Through the visible syringe needle point of crystalline lenses, slowly inject, after stopping 10 seconds, pull out pin, pressed 1 minute with cotton swab is light at puncture, prevent
Injecting fluid backflows.
(5) finally in intracapsular Levofloxacin Eye Drops of conjunctiva and erythromycin eye ointment prevention infection.
(6) rat is placed in warm environment, observes to revival.
Retinal EB seepage quantitative analysis method is as follows：
Azovan blue (evans blue, EB) is a kind of four sodium diazo colours, and molecular weight is 980Da, energy after intravenous injection
With plasma albumin with 10:1 ratio is combined closely, and when vascular permeability increases, EB protein complex passes through blood vessel wall
Leak into surrounding tissue, be a kind of comparatively ideal tracer.
(1) Normal group takes 4 rats with bilateral eyeballs (8 eyes), and DR model experiment group takes 4 rat right eyes (8 respectively
Eye), carry out EB seepage quantitative analyses.
(2) anaesthetize：Rat weight, is anaesthetized by dosage lumbar injection 10% chloral hydrate of 3ml/kg.
(3) irrigate EB：Cut off left side pars inguinalis skin with shear and expose left femoral vein, pressed with 1ml syringe
45mg/kg
Extract the EB solution that concentration is 30g/L, inject femoral vein in 1 minute, rat body at once becomes indigo plant and shows to fill
Form work(.
(4) circulate 2 hours in warm environment, the revival of period rat need to add to be supplemented with chloral hydrate anaesthetizes.
(5) irrigate PBS：Fixing rat extremity and head, open thoracic cavity, expose heart, insert perfusion syringe needle by left ventricle,
Cut off right auricle, perfusion PBS (37 DEG C of preheatings), injection pressure is 120mm Hg (irrigation flow is equivalent to 66mL/ minute), the time
2 minutes.
(6) draw materials：Perfusion is rapid after terminating to extract eyeball, cuts off in operating microscope lower edge corneoscleral junction, removes prosthomere
And the tissue such as crystalline lenses, vitreous body, carefully separately retinal tissue.
(7) drying is weighed：Retinal tissue lucifuge, be placed in Constant Temp. Oven be dried 45 minutes, claim its dry weight.
(8) every retina and 70 DEG C of 120 μ L first phthalein amine are incubated 18 hours, 4 DEG C of 15000rpm high speed centrifugations of extracting solution
30 minutes, take supernatant to survey the absorbance of 620nm and 740nm respectively, seek its net absorbance.
(9) ultraviolet spectrophotometer record concentration from the standard EB solution of 75ng/ μ L～1000ng/ μ L 620nm,
Absorbance at 740nm, both differences are net absorbance, set up EB standard curve.Inhaled with net according to EB solution concentration
The relation of shading value, obtains EB concentration in sample real solution, is multiplied by 120 μ L with this concentration and draws retinal EB leakage
(ng).Finally use retina dry weight (mg) markization EB leakage (ng), result is expressed as ng/mg.
Result of the test is as follows
| Group | Matched group | Embodiment 1 | Embodiment 2 | Embodiment 3 |
| EB infiltration capacity | 24.67±2.26 | 10.25±2.56* | 14.58±3.26* | 15.38±1.26* |
* represent P compared with matched group<0.05, there is significant difference.
Statistical procedures are carried out using SPSS 20.0 software (IBM SPSS Statistics), through comparing, matched group
There were significant differences and between each test group (P<0.05), and the difference between embodiment 1, embodiment 2 and embodiment 3 also have statistics anticipate
Adopted (P<0.05).Preferably, the ratio that is, two kinds of cells in stem cell medicine are worked as in explanation is 1 to the therapeutic effect of embodiment 1:When 4
Therapeutic effect is best.
Repeat above-mentioned test with the stem cell medicine of embodiment 4 and embodiment 5 preparation, obtain similar conclusion.
Embodiment 8
The present embodiment has carried out the curative effect contrast test of stem cell medicine and single cell component
Test is grouped as follows：
1st, Normal group：Take healthy SD rat 4 (8 eyes).
Below the rat of 2 to 5 groups be taken from embodiment 6 preparation DR rat model, every group 4.
2nd, blank control group：Normal saline solution
3rd, test group A：The stem cell medicine of embodiment 1 preparation.
4th, experiment group B：The retinal pigment epithelium 1 × 10 of mesenchymal stem cells MSCs induction5Individual.
5th, test group C：The retinal pigment epithelium 1 × 10 of induced differentiation of embryonic stem cells5Individual.
Two eyes of rats in normal control group do not give any intervention, in addition to Normal group, blank control group eyes glass
Intracoelomic injection 20 μ L normal saline, other each group rat eyes intravitreal 20 μ L cell suspension (totally 1 × 105Individual).
Inject cell suspension after diabetes Cheng Mo, after 4 months, put to death rat, collect Eyeball exemplar.Before setting up model, after diabetes Cheng Mo
And detect rat blood sugar when making a collection of specimens and record body weight.Retina is observed using EB Perfused vessel inner nuclear layer retina detection by quantitative
Vascular leakage situation.
Result of the test is as follows：
| Group | Normal group | Blank control group | Test group A | Experiment group B | Test group C |
| EB infiltration capacity | 3.92±0.48 | 26.84±4.57 | 11.75±2.55* | 18.63±4.69* | 19.08±5.12* |
* represent P compared with blank control group<0.05, there is significant difference.
Statistical procedures are carried out using SPSS 20.0 software (IBM SPSS Statistics), through comparing, matched group
There were significant differences and between each test group (P<, and the also statistically significant (P of the difference between test group A and experimental group B and C 0.05)<
0.05).The therapeutic effect of test group A preferably, that is, illustrates when in stem cell medicine, the therapeutic effect containing two kinds of cells is better than list
Plant the effect of cell re-injection.
Repeat above-mentioned test with stem cell medicine prepared by embodiment 2 to embodiment 5, obtain same conclusion.
The above is only the preferred embodiment of the present invention it is noted that ordinary skill people for the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (7)
1. a kind of stem cell medicine is it is characterised in that include following component：
Umbilical cord mesenchymal stem cells 5 × 104～1 × 106Individual/20 μ L；
Retinal pigment epithelium 1 × 105～5 × 106Individual/20 μ L；
The ratio of described umbilical cord mesenchymal stem cells and described retinal pigment epithelium is 1:2～1:8.
2. stem cell medicine according to claim 1 is it is characterised in that the cell concentration of described umbilical cord mesenchymal stem cells is
1×105Individual/20 μ L.
3. stem cell medicine according to claim 1 is it is characterised in that the cell concentration of described retinal pigment epithelium
For 4 × 105Individual/20 μ L.
4. stem cell medicine according to claim 1 is it is characterised in that described umbilical cord mesenchymal stem cells and described view
The ratio of membranochromic pigments epithelial cell is 1:4.
5. the stem cell medicine according to Claims 1-4 any one is it is characterised in that described retinal pigment epithelium
Cell is to be induced by described umbilical cord mesenchymal stem cells.
6. the preparation method of the stem cell medicine described in claim 1 to 5 any one is it is characterised in that comprise the following steps：
Described umbilical cord mesenchymal stem cells are mixed with described retinal pigment epithelium, described stem cell medicine is obtained.
7. the stem cell medicine described in claim 1 to 5 any one or prepared the doing carefully of the preparation method described in claim 6
Application in the pharmaceutical preparation of preparation treatment diabetic retinopathy for born of the same parents' preparation.
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| CN111658609A (en) * | 2020-07-24 | 2020-09-15 | 北京诺赛启研再生医学研究院有限公司 | Eye drops and preparation method thereof |
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Application publication date: 20170215 |