CN102091056A - Cell microcapsule for treating parkinsonism and composite cytoskeleton - Google Patents
Cell microcapsule for treating parkinsonism and composite cytoskeleton Download PDFInfo
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Abstract
The invention relates to a cell microcapsule for treating parkinsonism and a composite cytoskeleton, relating to the field of transplantation treatment of the parkinsonism. The cell microcapsule contains cells which are epithelial cells of retinochrome; and a microcapsule shell is internally mixed with neurotrophic factors. The invention provides a feasible method for treating the human parkinsonism by using the epithelial cells of heterogenous retinochrome, which treats the pathological brain tissues of a patient in a transplanting way together by adding the neurotrophic factors to the microcapsule shell. The invention not only directly plays the neuron repair function by solving the problems of clinical application, but also prolongs the survival time of transplanted cells with the epithelial cells of the retinochrome through synergistic action, thereby providing an ideal way for relieving the symptoms of the parkinsonism and repairing pathological cells.
Description
Technical field
The present invention relates to parkinson disease transplantation treatment field, Parkinsonian cell microcapsule of particularly a kind of treatment and compound cells support.
Background technology
Parkinson disease (parkinson ' s disease, be called for short PD) be central nervous system degenerative disease a kind of burst, that slowly make progress, its pathological characters mainly is substantia nigra of midbrain dopaminergic neuron selectivity degeneration death, Clinical symptoms mainly is slow in one's movements and disappearance, muscular rigidity, static tremor and posture shakiness, having another name called Parkinsonism, is modal neurodegenerative diseases.
Also do not have at present to prevent or thoroughly to cure Parkinsonian method, the Therapeutic Principle is that medicine and operative treatment combine.But advance in Drug Treatment of Parkinson and surgical method all can only improve partial symptoms, and long-term efficacy and pessimistic.Because parkinson disease mainly are to be caused due to dopamine level descends in the striatum by the degeneration of nigral dopaminergic neuron unit, therefore in the nigrostriatum system, transplant the cell that to secrete dopamine, recover the neural loop of dopamine, promptly become a kind of etiotropic ideal treatment direction.
From adrenal medulla tissue transplantation in 1985 first in clinical practice after the parkinson treatment and obtaining certain curative effect, experienced the development of two more than ten years, as a kind of reconstruction operation, become the worldwide focus of attention, Canadian D.Doudet has reported 9 with 1-methyl-4-phenyl-1,2,3, the Parkinsonian monkey that 6-tetrahydropyridine (MPTP) is made, in its pallidum, implant human retina pigment epithelium cell (RPE), eyeball from the donor, through In vitro culture and wrapped up the gel of the anti-rejection of one deck on its surface, the result shows that monkeys all in a year all improves significantly, and PET scanning shows that the picked-up of [18F]-L-dopa (dopamine) increases, ([11C]-raclopride) binding site reduces the tracer of d2 dopamine receptor, shows that the striatum dopamine level increases.Chinese patent application number is the injection of main component for the application for a patent for invention " a kind of treatment senile dementia, Parkinsonian neural stem cell injection " of " 200510123306.7 " discloses a kind of retinal pigment epithelium with the people source, directly inject patient's cerebral tissue diseased region, and have this injection of experimental example digital proof that the effective percentage that improves of parkinson disease symptom is reached 80%, further proving retinal pigment epithelium can play a role in the treatment parkinson disease.On the other hand, mention in the description of this patent application: brain and spinal cord are owing to the existence of blood brain barrier makes it can not produce immunological rejection (seeing description page 4 the 2nd row) in stem cell transplantation behind the central nervous system, traditional view thinks that also cerebral tissue has immunity and exempts characteristic simultaneously, main cause has: (1) cerebral tissue lacks lymphoid tissue and lymph pipeline, and exotic antigen can not cause immunne response; (2) cerebral tissue lacks antigen-presenting cell, can not bring out the allograft immunorejection reaction in transplantation immunity; (3) there is blood brain barrier in cerebral tissue, and antibody in the blood and immunologically competent cell can not contact with exotic antigen, just can not produce immunoreation; (4) cerebral tissue lacks major histocompatibility complex (MHC) antigen, and it plays an important role in bringing out the allograft immunorejection reaction.But along with the further investigation of people to the cerebral tissue immunological characteristic, the immunity of cerebral tissue exemption property falls under suspicion, and this is because (1) cerebral blood vessel peripheral clearance plays the effect of lymph pipeline; (2) astrocyte, microglia and the vascular endothelial cell in the brain can be expressed MHC antigen under specific circumstances; (3) blood brain barrier exists the damage of local organization to cause opening, the lymphocyte activation of blood brain barrier in some zone, brings out immunological rejection.In addition, though in transplantation treatment, can adopt autogenous cell, the danger of the rarer immunological rejection of autogenous cell, but drawbacks limit such as source rareness and function difference its clinical practice, people source retinal pigment epithelium is mainly derived from the eyeball tissue in the body early embryo, it is equally very rare to obtain the source, therefore needs to seek more wide source of human stem cell.Fortunately, Affiliated Hospital of Fudan University finds in the research to the bovine retina pigment epithelium cell, and its growth curve shows the 1st～4 day and be its exponential phase of growth, and the dopamine secretion begins to detect at 2h, and 48h reaches the peak and slowly descends later on and become stable; In addition, they find that in the research to the pig retinal pigment epithelium pig RPE cell 2h after inoculation begins to secrete DA under the base state, and 4h reaches the peak, slowly descend afterwards and tend to be steady.This proof, animal retinal pigment epithelium are expected to play a role in the treatment parkinson disease, and this provides wide cell source for the cellular transplantation therapy parkinson; But these cells are used for the transplantation treatment parkinson disease, need overcome immunologic rejection on the one hand, and on the other hand, the transplanted cells time-to-live need reach several years to be enough to repair the cell of lesion locations.
There are some researches show in addition, the nutritious neuron of neurotrophic factor, stimulate axon regeneration, myelinization, the function of regulating microglia again, during the direct administration of nervus centralis, neurotrophic factor has positive effect to the timely parkinson disease symptom of improving, and effective percentage reaches about 80%.At present known four kinds of main neurotrophic factors of neuron Yellow Gentian Extract are separated from mammal, they are: nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT-3) and neurotrophic factor 4/5 (NT-4/5) (Guyton AC, Hall JE.Textbook of medicalphysiology, 11th Edition.Philadelphia:Elsevier Saunders.2006, medical physiology, English process plate, medical science publishing house of Peking University, 2007); Along with The Application of Technology such as serum-free culture neurons, in many tissue fluids and extracellular matrix, find the specific protein molecule that some are new successively, also can promote neuronic propagation, differentiation and survival.For example, ciliary neurotrophic factor (the ciliary neurotrophic factor that Scs and astrocyte produce, CNTF) can promote spinal neuron survival damaged and the embryo, and in treatment human motion neuronal degeneration disease, important value be arranged.And for example, glial cell derived neurotrophic factor (glial cell line-derived neurotrophic factor, GDNF) in isolated experiment, can support the existence of midbrain dopaminergic neuron, on various animal model for parkinsonism, can improve the density of the survival of dopaminergic neurons rate and the neural art tip and improve its symptom (Han Jisheng chief editor. Principles of Neural Science. the 2nd edition. Beijing: publishing house of Beijing Medical University, 1999).The effect that these neurotrophic factors are applied in clinically is dissatisfied.Main cause is (1): the neurotrophic factor molecular weight is big, is difficult to pass through blood brain barrier; (2): arrive nigrostriatum surely rapidly by differing behind the blood brain barrier and act on target spot.Therefore, employing is treated parkinson and must be solved above two problems clinically.
The appearance that " artificial cell " is accompanied by technique for packing and microencapsulation technology begins to have obtained extensive use at biomedical sector.The notion of artificial cell is by Chang, T.M.S is set forth in nineteen sixty-five, has the research history of four more than ten years so far, and its notion is that the living material that will comprise cell, enzyme and adsorbent wraps up with macromolecule hyper-film, thereby formation microcapsule, i.e. cell micro-encapsulation.Permoselective membrane by microcapsule carries out immunoprotection to tissue or cell; can need not to carry out allogeneic or xenotransplantation under the condition of immunosuppressant; replenish the biological active substances that lacks in the disease body; simultaneously, heteroplastic realization can overcome the limitation of human transplant organ donor source.The material that microencapsulation is commonly used mainly contains natural, semi-synthetic and tens of kinds of synthetic high polymer three major types, and method commonly used has sodium alginate, poly-D-lysine (APA) microcapsule preparation method; Sodium alginate, chitosan (ACA) microcapsule preparation method, agar capsule heart preparation method and synthetic polyelectrolyte multiplayer microcapsule preparation method etc.; The cell microcapsule preparation process has one-step method and two-step method.One-step method is that the mixed solution with chitosan and calcium chloride splashes into directly that reaction obtains microcapsule in the sodium alginate soln, cyst wall contains totally 3 layers of chitosan sedimentary deposit, sodium alginate-chitosan complexation layer and calcium alginate coacervates, and traditional microcapsule preparation method is a two-step method.The first step is the mixed liquor of cell and the sodium alginate soln micropore by syringe pump to be splashed into prepare calcium alginate microsphere in the calcium chloride solution, second the step calcium alginate microsphere that obtains is placed the chitosan solution film forming, again with in the sodium alginate soln and film forming after calcium alginate microsphere promptly obtain sodium alginate-chitosan microcapsules.Another better microcapsule system is with sodium alginate, poly-D-lysine is a microencapsulated material, the steps include: that the first step splashes into the mixed liquor of cell and the sodium alginate soln micropore by syringe pump prepares calcium alginate microsphere in the calcium chloride solution, the second step calcium alginate microsphere is immersed in the surface with the polylysine aqueous solution and forms Na-alginate-polylysine polyelectrolyte coordination compound layer, then this microsphere is dipped in the sodium citrate aqueous solution, sodium alginate stripping from microcapsule makes microsphere be transformed into the semipermeable membrane film layer structure of sodium alginate-polylysine-sodium alginate (APA), cell is easily free movable in this microcapsule than in calcium alginate gel, and film-strength height, cell can not leak, and is suitable for life-time service.
At present, do not see the cytochrome epithelial cell and make artificial cell overcoming the report of immunological rejection, also not seeing simultaneously combines with neurotrophic factor is used to prolong the report of transplanted cells survival period.
Summary of the invention
The present invention provides Parkinsonian cell microcapsule of a kind of treatment and compound cells support according to defective and blank that above-mentioned field exists, and cell microcapsule provides feasible way for allogenic retinal pigment epithelium is used for the treatment of the human Parkinson's disease; Transplanted cells survival rate height among the present invention, survival period are long, provide a kind of ideal approach for alleviating the parkinson disease symptom and repairing sick cell.
The Parkinsonian cell microcapsule of a kind of treatment is characterized in that: including cell is retinal pigment epithelium, is mixed with neurotrophic factor in the capsule casing material of microcapsule.
Described softgel shell is followed successively by sodium alginate layer, chitosan layer, sodium alginate layer from the inside to surface; Or sodium alginate layer, poly-D-lysine layer, sodium alginate layer.
Described neurotrophic factor is the neurotrophic factor microsphere that gelatin coatings forms.
Described neurotrophic factor microsphere is 20% gelatin solution with neurotrophic factor with volume mass than 5～10ml: 50～200 μ g make by coacervation.
Described softgel shell is that 3% sodium alginate soln, 1.5% chitosan solution and neurotrophic factor microsphere make; The volume ratio of described 3% sodium alginate soln, 1.5% chitosan solution and neurotrophic factor microsphere is 0.5～1.5: 0.5～1.5: 0.5～1.5, and described chitosan solution participates in reaction after mixing with described neurotrophic factor microsphere is first again.
Described softgel shell is that 1.5% sodium alginate soln, 0.05% Poly-L-Lysine Solution and neurotrophic factor microsphere make; The volume ratio of described 1.5% sodium alginate soln, 0.05% Poly-L-Lysine Solution and neurotrophic factor microsphere is 0.5～1.5: 0.5～1.5: 0.5～1.5, and described chitosan solution participates in reaction after mixing with described neurotrophic factor microsphere is first again.
In the described cell microcapsule, retinal pigment epithelium is 1 * 10
8～1 * 10
9Cells/L.
Described neurotrophic factor is nerve growth factor, brain derived neurotrophic factor, neurotrophic factor-3 and/or neurotrophic factor-4/5.
Described retinal pigment epithelium be the 3rd～5 generation passage cell.
The source of described retinal pigment epithelium is people, cattle, pig, rabbit.
The Parkinsonian compound cells support of a kind of treatment makes by following steps:
(1) retinal pigment epithelium is mixed with sodium alginate soln, add chitosan or Poly-L-Lysine Solution mixing again, add the neurotrophic factor mixing at last, place 0～7 ℃ of refrigerator freezing;
(2) by default three dimensional structure, obtain assembling product by three-dimensional controlled assemble method;
(3) on the assembling product, drip calcium chloride and make it that cross-linking reaction take place,
Described neurotrophic factor is the neurotrophic factor microsphere that gelatin coatings.
The cell microcapsule of treatment parkinson provided by the invention, retinal pigment epithelium is wrapped in the microencapsulated material, the softgel shell of microcapsule plays a part semipermeable membrane, allows secretory substance, the nutrient substance turnover of small-molecular weight, and the immunologic active material of macromolecule is had shielding action.After this microcapsule is transplanted to the cerebral diseased position, make the capsule inner cell avoid human immune system's attack, the success rate and the cell survival rate of cell transplantation have been improved, simultaneously, the cell source that also is used in transplanting no longer is confined to autogenous cell or human archeocyte, also can be the retinal pigment epithelium of animals such as cattle, pig, rabbit; And the excretory Dopaminergics material of capsule inner cell is diffused into outside the capsule, the required nutrient substance of cell can be spread in the capsule, therefore synthetic dopamine can be secreted into diseased region in the capsule, and the nutrient molecule of implanting in the environment can be spread to the vigor that the interior trophocyte of capsule makes it to continue to keep the secretion dopamine; In the softgel shell of cell microcapsule of the present invention, added neurotrophic factor, neural battalion trophic factors directly contacts with diseased region by transplanting, and makes the function of its performance repair of neuron, and sick cell is initiatively repaired; On the other hand, neurotrophic factor can stimulate induces capsule inner cell growth, thereby prolongs the survival period of capsule inner cell, reaches the continuous release dopamine, prolongs effect duration of transplantation treatment; Simultaneously neurotrophic factor is joined the capsule casing material that is used for microcapsule, solved it and used the difficult problem of the existence of treatment parkinson clinically.
The material that the present invention makes microcapsule adopts sodium alginate and chitosan microcapsules system commonly used at present; perhaps sodium alginate and poly-D-lysine system; these two kinds of system biocompatibility are better, and the semi permeability of rete is stable, and film strength is enough to provide protection for wherein cells physiological metabolic activity.Among the present invention, blended neurotrophic factor can work for a long time in the capsule casing material in order to make, the present invention preferably is wrapped in neurotrophic factor and forms gelatine microsphere in the gelatin, gelatine microsphere and the capsule casing material making cell microcapsule that is mixed, gelatin plays a part slow release, neurotrophic factor is slowly released, prolong, reach gradually and make the purpose of repairing at the bottom of the part pathological changes neuron side neuronic repair time; Among the present invention, preferred gelatine microsphere is 20% gelatin solution and neurotrophic factor with 5～10ml: the ratio of 50～200 μ g is made, manufacture method adopts coacervation commonly used, sustainable 2～3 years of the slow releasing function of the gelatine microsphere of making, be enough to cooperate retinal pigment epithelium thoroughly to treat the pathological changes cerebral tissue of disturbances in patients with Parkinson disease together, but the invention is not restricted to this ratio and method.
In a preferred version of the present invention, capsule casing material is 1～3% sodium alginate soln and 1.5% chitosan solution, the present invention preferred 3% sodium alginate soln, 1.5% chitosan solution, neurotrophic factor microsphere mix by volume at 0.5～1.5: 0.5～1.5: 0.5～1.5, this proportion make and microcapsule have the semipermeable membrane hole of suitable film-strength and suitable dopamine secretion and micromolecule nutrient substance turnover exchange.
In cell microcapsule; cell and capsule casing material should have a suitable ratio; should and implant between the environment for cell big as far as possible contact area is provided; guarantee the mechanism that signal and the unimpeded formation of mass transfer approach well give mutual protection between the cell simultaneously; if hold too many cell in the microcapsule; then the excretory inadequately dopamine material of contact area between cell and the implantation environment can not be diffused into target spot efficiently, and cell is then dead easily very little, influences the transplanted cells survival period.Therefore, the present invention gropes experiment by a large amount of contrasts, determines that the volume ratio of retinal pigment epithelium in microcapsule is 1 * 10 among the present invention
8～1 * 10
9Cells/L can achieve the above object.
The present invention is directed to the cell microcapsule that treatment parkinson this purpose provides, neurotrophic factor wherein can be in nerve growth factor, brain derived neurotrophic factor, neurotrophic factor-3, neurotrophic factor-4/5 one or more, and above-mentioned four kinds is the neurotrophic factor that the proof that has been separated in mammalian body at present has the neuron repair.In one embodiment of the present of invention, preferred brain derived neurotrophic factor.
There is data to prove, after going down to posterity, retinal pigment epithelium can keep secreting the DA characteristic, the secretion of obviously visible dopamine in 4 generation cells, the retinal pigment epithelium that the present invention adopts is preferably the go down to posterity cell in 3～5 generations of stem cell, make the cell microcapsule of making like this after being transplanted to diseased region, can secrete dopamine immediately, improve patient's symptom immediately.
Retinal pigment epithelium among the present invention preferably studies have shown that cattle, pig, the rabbit of dopamine secretory function certainly at present, also can adopt the retinal pigment epithelium in the people source of cultivating in the biotech firm.
The present invention also provides a kind of compound cells support for the treatment of parkinson, retinal pigment epithelium is mixed into the cytoskeleton mixture with capsule casing material earlier, adopt then and Li Man, (" the direct three-dimensional controlled package technique external structure organization engineered cartilages of cell " such as Zhao's iridium people, practical stomatology magazine, Sep in 2008,24 (5)) Bao Dao three-dimensional controlled assemble method and equipment thereof, two-dimentional circuit by the three dimensional structure of default compound cells support is successively piled up the compound cells support that molding makes blank, carry out crosslinkedization or fixing step at last again, have certain macrostructure compound cells support thereby make.The effect of compound rest is and can provides a kind of macroscopical supporting construction that is beneficial to cultivation for the microcapsule cell, can directly structural transplantation be arrived lesion locations, for cell provides good living environment, the material of support just is has the little capsule casing material in semi-permeable porous crack, it is long-pending that the three dimensional structure of support helps improving the contact surface of implant and internal milieu, be convenient to implant the nutrient substance exchange and the therapeutant release of environment and microcapsule inside, improve therapeutic effect and prolong effective treatment cycle.
Description of drawings
The effect curve of Fig. 1 cell microcapsule treatment parkinson mice
Abscissa is represented transplant time, and vertical coordinate is represented the ratio that number of revolutions reduces
Fig. 2. the structure of compound cells support
Upper left: in the compound cells groups of holders process of assembling
Upper right: the compound cells support that assembling is finished
The lower-left: microscopically is observed the situation of hole
The bottom right: ultramicroscope is observed the situation of cell attachment on the compound cells support down
The specific embodiment
The cultivation of going down to posterity of embodiment 1. retinal pigment epitheliums.
The retinal pigment epithelium primary cell is available from U.S. ScienCell company
Used culture fluid is the EpiCM culture medium (EpiCM of U.S. ScienCell company, Catalog Number is 4101), this nutrient media components is that every 500ml culture medium contains 10ml hyclone FBS (Catalog No.0010), 5ml epithelial cell nutrient (Catalog No.4152) and 5ml penicillin streptomycin antibody (P/S, Catalog No.0503).
Passage is cultivated the trypsin Trypsine of use 0.25% as Digestive system.The primary cell of purchasing is that the 75cm2 cultivation is bottled, places 37 ℃, 5%CO
2Cultivate in the incubator, every 3d changes a culture fluid, goes down to posterity until cell fusion.
Cell division propagation should in time go down to posterity when merging, the culture fluid suction is gone, PBS buffer flushing 3 times, 0.25% trypsinization 3～5 minutes, when the observation of cell edge begins to shrink under the inverted microscope, inhale and remove Digestive system, add culture fluid and end digestion, the piping and druming mixing divides to be filled to the cultivation of going down to posterity in the new culture bottle.
Embodiment 2: single coacervation preparation comprises the gelatine microsphere of neurotrophic factor
The preparation quality mark is 20% gelatin solution 5ml, 80 ℃ of high temperature sterilizes of baking oven 2～3 times, each 4～5 hours.Aseptic condition adds 100ug neurotrophic factor (ScienCell, NGS, Catalog No.1562) down, drips acetone under the pH3.8 condition gelatin solution is condensed, and adds glutaraldehyde cross-linking again and obtains diameter 200-400nm, ganoid gelatine microsphere.
Embodiment 3. makes sodium alginate-poly-D-lysine-sodium alginate cell microcapsule
Step 3, it is 1 * 10 that the sodium alginate of RPE and 1.5% is mixed and made into density
6The cell suspension of/ml.Cell suspension is splashed into the 1.1%CaCl of mild agitation with No. 4 syringe needles
2In, make it form gel beads.
Step 4, the neurotrophic factor microsphere that embodiment 2 makes mixes with equal-volume with 0.05% poly-D-lysine,
Step 5. gel beads successively with mixed liquor, the sodium alginate combination reaction of step 4, with the core of the sodium citrate liquefaction microsphere of concentration about 5.5%, promptly obtain cell microcapsule at last.
Embodiment 4. makes the sodium alginate-chitose-sodium alginate cell microcapsule
It is 1 * 10 that step 3. is mixed and made into density with the sodium alginate of RPE and 3%
6The cell suspension of/ml.Cell suspension is splashed into the 1.1%CaCl of mild agitation with No. 4 syringe needles
2In, make it form gel beads.
The neurotrophic factor microsphere that step 4. embodiment 2 makes mixes with equal-volume with 1.5% chitosan solution,
Step 5. gel beads successively with mixed liquor, the sodium alginate combination reaction of step 4.
Embodiment
The compound rest of preparation retinal pigment epithelium microencapsulation
Step 3. is 1 * 10 with being mixed and made into density under the RPE cell that extracts and the 1mL 3% sodium alginate soln aseptic condition
6The cell suspension of/ml adds the chitosan solution mixing of 1mL 1.5% again, adds the neurotrophic factor microsphere of the 2 1mL gelatin coatings that make in implementing again, and mix homogeneously was placed on 0～7 ℃ of refrigerator freezing 20 minutes.
Step 4. with the gel state mixture that freezes pack into the three-dimensional controlled kludge I generation 5mL syringe that matches in, install syringe needle, be installed in cell three-dimensional controlled group installation go up (be Li Man, Zhao's iridium people etc., " the direct three-dimensional controlled package technique external structure organization engineered cartilage of cell ", practical stomatology magazine, Sep in 2008, the equipment of the employing in 24 (5)), the start-up control program, according to default alveolate texture, assemble.
The cellular compound cells support that step 5. assembles is seen Fig. 2, is positioned on the wave carrier piece, and wave carrier piece is taken out, and aseptic condition adds the calcium chloride (CaCl of 100mmol/L down
2) solution 500ul carries out cross-linking reaction, solidify promptly to obtain the compound cells support after 5～30 minutes.
Step 6. is rinsed well with cell culture fluid (with embodiment 1), and the compound rest on the wave carrier piece is scraped into the porous culture plate, adds 3～5ml culture fluid (with embodiment 1) and cultivates observation, after cultivation 3～5 cell division cycle (D), takes a morsel and detects.Adopt the cytoactive on the trypan blue staining checking bracket, only the painted explanation cytoactive of small amounts of cells is good, can be used for transplantation treatment.
Experimental example 1. mouse tests
The cell microcapsule that adopts embodiment 3 to make adopts the parkinson rat model, 6-OHDA rat model postoperative 3 all lumbar injection apomorphines (0.5mg/kgBW), with rotating cycle greater than 210 times/30min for becoming the mould standard.
Set normal saline (NS) matched group, the cell microcapsule group (RPE-APA) that null cell group (RPE) and the present invention make.
Wherein the NS group is 12, each 15 of RPE group and RPE-APA groups.During transplanting RPE cell suspension is adjusted to 1 * 10
6Cells/ml, RPE-APA group concentration is adjusted to 2 * 10
6Individual capsule/L is used for transplanting, and adopts 2 transplanting of right side striatum.The NS group is by same procedure injection 20ul normal saline, and let the acupuncture needle remain at a certain point 15 minutes; RPE group and RPE-APA group adopt injection.
Behind cell transplantation, observed in the 1st, 2,4,8,14 weeks respectively and respectively organize the inductive circling behavior variation of rat apomorphine, measure the rotating cycle of 30min.Contrast before and after the circling behavior evaluation employing self, promptly the number of revolutions of bringing out with transplanting back apomorphine is represented circling behavior intensity of variation (see figure 1) than the percentage ratio (%) of minimizing before transplanting.Observed result shows that beginning in number of revolutions the 2nd week after transplanting that the rat apomorphine brings out in the RPE-APA group reduces, and the 4th all minimizing amplitudes are more obvious, are 54.79%, and the minimizing of number of revolutions lasted till for the 14th week always, and the minimizing amplitude is 60.25%.Since the rat number of revolutions of RPE group reduced to some extent in the 2nd week, but the amplitude that reduces subsequently fluctuates back and forth, contrasts no significant difference with the normal saline group.The experiment structure shows that adopting cell microcapsule to transplant has obvious curative effects, and compares with directly transplanting RPE cell, and it is longer that the former continues curative effect time, more remarkable effect.The cell of directly transplanting only can produce therapeutic effect at transplant early.
Experimental example 2. clinical trials
Adopt cell microcapsule transplantation treatment 2 example parkinson disease patients in late period of the present invention, three-dimensional location is adopted in operation, by CT or MRI scanning location, determines the operation target spot, again by area of computer aided navigation system software, determines the three-dimensional coordinate of target spot.With cell microcapsule, be adjusted to 2 * 10
6Individual capsule/L injects striatum district target spot in Parkinsonian's brain (shell nuclear, nucleus ventralis posterolateralis thalami).All patients all do not use immunosuppressive drug, and (Unified Parkinson ' sDisease Rating Scale UPDRS) assesses patient to use unified parkinson disease grade scoring before and after the treatment.The result shows, adopts the transplanting curative effect of cell microcapsule more lasting, can significantly improve patient's motion function, and its function was improved sustainable 3 years.Patient's pair cell migration process well-tolerated does not have apparent side effect.
In sum, the present invention provides feasible method for allogenic retinal pigment epithelium is used for the treatment of the human Parkinson's disease, neurotrophic factor has joined in the softgel shell transplantation treatment patient pathological changes cerebral tissue together, both overcome the problem that exists in the clinical practice, directly brought into play the function of its repair of neuron; By synergism, prolong the survival period of transplanted cells again and between the retinal pigment epithelium, provide a kind of ideal approach for alleviating the parkinson disease symptom and repairing sick cell.
Claims (10)
1. treat Parkinsonian cell microcapsule for one kind, it is characterized in that: including cell is retinal pigment epithelium, is mixed with neurotrophic factor in the capsule casing material of microcapsule.
2. cell microcapsule according to claim 1, described softgel shell are followed successively by sodium alginate layer, chitosan layer, sodium alginate layer from the inside to surface; Or sodium alginate layer, poly-D-lysine layer, sodium alginate layer.
3. cell microcapsule according to claim 2, described neurotrophic factor are the neurotrophic factor microsphere that gelatin coatings forms.
4. cell microcapsule according to claim 3, described neurotrophic factor microsphere are 20% gelatin solution with neurotrophic factor with volume mass than 5～10ml: 50～200 μ g make by coacervation.
5. cell microcapsule according to claim 4, described softgel shell are that 3% sodium alginate soln, 1.5% chitosan solution and neurotrophic factor microsphere make; The volume ratio of described 3% sodium alginate soln, 1.5% chitosan solution and neurotrophic factor microsphere is 0.5～1.5: 0.5～1.5: 0.5～1.5, and described chitosan solution participates in reaction after mixing with described neurotrophic factor microsphere is first again; Perhaps described softgel shell is that 1.5% sodium alginate soln, 0.05% Poly-L-Lysine Solution and neurotrophic factor microsphere make; The volume ratio of described 1.5% sodium alginate soln, 0.05% Poly-L-Lysine Solution and neurotrophic factor microsphere is 0.5～1.5: 0.5～1.5: 0.5～1.5, and described chitosan solution participates in reaction after mixing with described neurotrophic factor microsphere is first again.
6. cell microcapsule according to claim 1, the volume ratio of retinal pigment epithelium number and described cell microcapsule are 1 * 10
8～1 * 10
9Cells/L.
7. according to the arbitrary described cell microcapsule of claim 1～6, described neurotrophic factor is nerve growth factor, brain derived neurotrophic factor, neurotrophic factor-3 and/or neurotrophic factor-4/5.
8. according to the arbitrary described cell microcapsule of claim 1～6, described retinal pigment epithelium be the 3rd～5 generation passage cell.
9. according to the arbitrary described cell microcapsule of claim 1～6, the source of described retinal pigment epithelium is people, cattle, pig, rabbit.
10. treat Parkinsonian compound cells support for one kind, make by following steps:
(1) retinal pigment epithelium is mixed with sodium alginate soln, add chitosan or Poly-L-Lysine Solution mixing again, add the neurotrophic factor mixing at last, place 0～7 ℃ of refrigerator freezing;
(2) by default three dimensional structure, obtain assembling product by three-dimensional controlled assemble method;
(3) on the assembling product, drip calcium chloride and make it that cross-linking reaction take place,
Described neurotrophic factor is the neurotrophic factor microsphere that gelatin coatings.
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| US11141510B2 (en) | 2015-04-07 | 2021-10-12 | Revotek Co., Ltd. | Compositions for cell-based three dimensional printing |
| CN113801776A (en) * | 2021-10-11 | 2021-12-17 | 浙江大学 | Five-channel cell micromanipulation paw based on microfluidics and control method |
| US11224680B2 (en) | 2015-04-07 | 2022-01-18 | Revotek Co., Ltd | Compositions for cell-based three dimensional printing |
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2009
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11141510B2 (en) | 2015-04-07 | 2021-10-12 | Revotek Co., Ltd. | Compositions for cell-based three dimensional printing |
| US11224680B2 (en) | 2015-04-07 | 2022-01-18 | Revotek Co., Ltd | Compositions for cell-based three dimensional printing |
| CN113801776A (en) * | 2021-10-11 | 2021-12-17 | 浙江大学 | Five-channel cell micromanipulation paw based on microfluidics and control method |
| CN113801776B (en) * | 2021-10-11 | 2024-02-09 | 浙江大学 | Five-channel cell microscopic operation paw based on microfluidics and control method |
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| CN102091056B (en) | 2012-10-24 |
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