CN102533653A - 5-HT nerve cells induced by MSCs and ASCs and microencapsulation preparation method and application thereof - Google Patents
5-HT nerve cells induced by MSCs and ASCs and microencapsulation preparation method and application thereof Download PDFInfo
- Publication number
- CN102533653A CN102533653A CN2011104211099A CN201110421109A CN102533653A CN 102533653 A CN102533653 A CN 102533653A CN 2011104211099 A CN2011104211099 A CN 2011104211099A CN 201110421109 A CN201110421109 A CN 201110421109A CN 102533653 A CN102533653 A CN 102533653A
- Authority
- CN
- China
- Prior art keywords
- cell
- mscs
- neurocyte
- ascs
- microencapsulation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention relates to 5-HT (5-hydroxytryptamine) nerve cells induced by MSCs (mesenchymal stem cells (MSCs) and ASCs (adipose derived stem cells), which are prepared by the following steps: (1) culture and passage of MSCs and ASCs: (1.1) primary culture; and (1.2) digestion passage; (2) precursor induction; (3) functional induction; and (4) stable nature culture. A microencapsulation preparation method of the MSCs/ASCs-induced 5-HT nerve cells adopts an ANano-P-ANano (sodium alginate + nanoparticles-poly(amino acid)-sodium alginate + nanoparticles) method, and microencapsulates the MSCs/ASCs-induced 5-HT nerve cells using an electrostatic microcapsule generator;and each microcapsule contains 20 or 30 5-HT nerve cells, so that the cells in the microcapsule sufficiently contact with the microcapsule wall and are easy for adhesion and nutrient exchange, and microencapsulation induced 5-HT nerve cells are prepared. The microencapsulation induced 5-HT nerve cells are used for preparing medicines for treating depression or anticancer medicines. The central neural transplantation therapy using the microencapsulation induced 5-HT nerve cells will provide a new route for treating depression and cancer.
Description
Technical field
The invention belongs to biomedical sector, relate to stem cell and induce 5-HT neurocyte and cell to isolate (microencapsulation) technology and method, specifically, is MSCs, ASCs inductive 5-HT neurocyte and microencapsulation preparation method and application.
Background technology
(1) 5-HT neurocyte centering pivotly joins and is and regulates extensively
Serotonin (5-hydroxytryptamine; 5-HT) neurocyte or neurone mainly are positioned at brain stem nuclei of median raphe, black substance compact part, locus coeruleus afterbody and hypothalamus; It sends fiber and almost is projected to whole cns, goes upward to the extensive region of limbic system (hippocampus, separated nuclear, hypothalamus) and brain neocortex (volume, top, pillow, temporo, island leaf); Come downwards to anterior angle somatic movement and side angle internal organ motion (sympathetic and parasympathetic) neurone of spinal cord.Its physiological function shows that 5-HT neurone and fiber that nuclei of median raphe, nucleus tractus solitaril, hypothalamus, amygdala, hippocampus etc. are located are relevant with the sleep adjusting, and the neurone that hippocampus, amygdala and frontal lobe etc. are located is coordinated mood and behavior.We utilize the logical model experiment of animal 2.5% Paraformaldehyde 96 vola injection, and grey matter (PAG) and posterior horn of spinal cord 5-HT increasing expression around dorsal part hippocampus stimulation produced analgesia shows the midbrain central canal show 5-HT and neurone thereof participation analgesia.There is experiment to go back tamper indicating nuclei of median raphe head end; No rapid eye movement sleep completely dissolve; Can cause insomnia fully with the 5-HT synthetic inhibitor, and the induced brain wave paradoxical discharge, intracerebroventricular injection 5-HT acid can be eliminated this phenomenon; These experimental results confirm that 5-HT neurone and mediator 5-HT thereof participate in multi-functional adjustings such as body sleep, mood, behavior and analgesia, and promptly the 5-HT neurone has important regulatory role to the normal function of cns.
(2) compensatory-performance is depressed stress to cause 5-HT neurocyte function to be lost for a long time
Research data shows that 5-HT neurone release 5-HT participates in and stress regulate in the cental system, when cns 5-HT expression level reduces, is prone to take place the psychological stress damage.This psychological stress damage can cause two consequences: the severity increase that 1. makes the risk of dysthymia disorders generation and suffer from dysthymia disorders; Its mechanism of action mainly be maincenter 5-HT level low → the hypothalamic pituitary adrenal axis hyperfunction → when body receive traumatic incident stimulate back → vegetative nervous system be overexcited → make sympathin and the rising of suprarenin level through series reaction; Because the level of maincenter 5-HT neurone release 5-HT is low these two kinds of hormones are discharged the feedback adjusting of carrying out at this moment and can not reach normal, cause the superfunction of persistence hypothalamic pituitary adrenal axis; 2. maincenter 5-HT level low → not enough to the regulatory function of hippocampus; Because hippocampus belongs to the integral part of maincenter limbic system; 5-HT → hippocampus path stress have the prevention effect to the nocuity that unknown cause causes starting too early; Thereby the hippocampus imbalance joint due to the reduction of 5-HT level, can cause behavior " derepression " (to the environmental stimulus tetchiness), be easy to take place the injury of irritability body.Many research datas have proved that also the interior 5-HT concentration of blood and cerebrospinal fluid that mainly shows as unusually of neurotransmitter reduces in the patients with depression body, and postmortem finds that also 5-HT content reduces in the cerebral tissue.In addition, some have also pointed out the reduction of 5-HT content in the body about the research of the Vietnam War patients with depression, confidential relation are arranged with losing normal control wartime even suffer from after the war " syndromes after the war ".Also have research to show that 5-HT is inhibited to the high-order brain district of NE neuronal excitation; And NE neurone fever also can suppress the 5-HT neuron activity of nuclei of median raphe.It is excited when 5-HT reduces, then to cause the NE path to continue, and causes insomnia, anxiety, depression etc., and then causes 5-HT to reduce once again, more increases the weight of sleep and emotional handicap, and both reciprocal causations form vicious cycle thus.Therefore, according to the 5-HT minimizing of dysthymia disorders brain, perhaps 5-HT exhaustion or the low susceptible disease of its synthetic emission levels of script are the cause of disease, use 5-HT reuptake inhibitor treatment dysthymia disorders clinically, and part patient has obtained effect preferably.And the part patient is invalid, even severe side effect occurs, and this belongs to refractory type or severe dysthymia disorders most probably, and it is low excessively to come from its maincenter 5-HT level.We adopt internationally recognized chronic stress animal model; Show through subarachnoid space 5-HT neuron transplantation to stop the unusual generation with the multiple organ injury of animal behavior that showing that the 5-HT Transplanted cells can prevent and treat dysthymia disorders perhaps stress be to the injury due to the body.
(3) the low excessively peripheral organs pathology-canceration susceptible that causes of maincenter 5-HT level
The research report of relevant tumour mechanism; Maincenter 5-HT level lowly possibly make some organ cell's canceration susceptible or cancer cells be easy to worsen (evolution); Its mechanism can be through maincenter → on every side (fan walk etc.) nerve → organ cell's the low and neuroendocrine hypothalamic pituitary adrenal axis hyperfunction of regulatory function; Through glucocorticosteroid, suprarenin and sympathin excessive secretion; Energy metabolism accumulation → organ cell low and toxicity molecule-excitatory amino acid and oxyradical (inducible nitric oxide iNO, mda MDA) of organ cell is resisted due to damage or the repairing effect obstacle; Like esophageal carcinoma Esophageal squamous cell carcinoma (ESCC); Though nosetiology points out factors such as oesophagus canceration and environment (food goes mouldy, NSC 223080), inheritance susceptible relevant already, research in recent years shows that the esophageal carcinoma belongs to chronic disease; Psychological stressful factors (mental wound or depression) is occupied critical role in the oesophagus canceration; And (cholinergic antiinflammatory pathway, CAP), vagus nerve also need be through the activation of brain stem nuclei of median raphe 5-HT function to the visceral injury provide protection that stress cause in the CAP path for nerve-cholinergic anti-inflammatory pathway.Because maincenter 5-HT participates in and stress regulate, chronic psychological stress or depression can cause the interior 5-HT of brain to be in low-level state for a long time, thereby psychological stress → maincenter 5-HT level lowly has substantial connection to oesophagus mucosal epithelium cell carcinogenesis.Other has Toh S etc. through to using the investigation of thymoleptic 5-HT reuptake inhibitor relatively, finds that medication crowd's lung cancer morbidity rate reduces.Pyroia S brings out liver tumor through hepatectomy+N-Nitrosodiethylamine (NDEA), finds that brain stem and pallium 5-HT2C acceptor binding force raise in the liver tumor forming process, promptly point out the 5-HT level low excessively.El-Salhy etc. have better antitumous effect with Sostatin (gi tract styptic), galanin (neurotransmitter is regulated peptide-adjustable appetite, cell death inducing etc.) and 5-HT etc. three to the gastrointestinal cancer cell.We adopt recently 5-HT cell subarachnoid space transplant control stress+experimentation on animals of the NSC 223080 esophageal carcinoma shows, chronic stress can cause the oesophagus canceration to quicken and worsen, and the 5-HT Transplanted cells can make the canceration of mouse oesophagus slow down or sickness rate reduces.
(4) one of not enough peripheral organs pathology mechanism of maincenter 5-HT nerve pathway malfunction
Research demonstration about maincenter 5-HT neurone → vagus nerve cholinergic path dysregulation and internal organ injury or its cell carcinogenesis relation; Path by maincenter 5-HT neurone → vagus nerve cholinergic → internal organ is clear and definite; Discharge the AChR (being embedded in the gp in the cytolemma) that ACh acts on cell through vagus nerve and postganglionic fibers thereof, to regulate internal organs or effector cell.Researchs such as Grozio report cholinocepter has the effect of regulating cell function, propagation, conversion and differentiation; The hypotype difference of acceptor plays an important role in cell proliferation-differentiation or tumour generation-vicious transformation, adopts nAChR-α 7 or mAChR-3 antagonist can suppress the lung carcinoma cell hyperplasia respectively.Have research to confirm that the cancer cells differentiation degree is lower, the AChRs hypotype reduces significantly, and binding site is lost the more.We cultivated respectively 21 days the high and low noble cells of the esophageal carcinoma (high differentiation EC9706, low differentiation EC109) through ACh; Find that ACh can make esophageal cancer cell AChR, gene damage reparative factor Prx1, cytodifferentiation calcium rely on and regulate albumin A nnexin2 and cytoskeletal protein Keratins up-regulated, the differentiation of form trend.
Our recent experimentation on animals result shows, cuts off vagus nerve and can quicken the canceration that NSC 223080 brings out epithelium of esophagus, and this maybe be relevant with the provide protection that has slackened the CAP path.In conjunction with the TPH2 gene polymorphic is redox; Property of participation susceptible and vagus nerve function weaken can make stress+NSC 223080 brings out the oesophagus canceration to quicken; This is soluble follows up a case by regular visits to the canceration of newly discovered oesophagus to esophageal carcinoma district occurred frequently in recent years and develops unsettled characteristics; Be the part patient with esophageal carcinoma do not treat and more or the lesion degree of surviving for many years remain unchanged; This prompter's quasi-cancer cell has the ability of the differentiation of inducing (can reverse), through the optimum adjusting of maincenter-vagus nerve-organ dysfunction, can play anticancer; Disease controlling or reach the purpose of healing, promptly neurotransmitter AChR function has positive impact to the conversion and the differentiation of relevant cancer cells after improving maincenter 5-HT system → vagus nerve path and saving.Show that thus maincenter 5-HT and vagus nerve have vital role to suppressing relevant internal organs cell carcinogenesis and anticancer.
(4) maincenter 5-HT neurocyte lose compensatory-come from acquired and congenital factor
Research prompting maincenter 5-HT level low reason has acquired and congenital two aspects: 1. acquired insufficient reason is because brain stem nuclei of median raphe 5-HT neurone sends body such as fiber to limbic system, brain neocortex and spinal cord and internal organ motor nuclei (sympathetic and parasympathetic) throws the basis that stress regulate path of genus 5-HT very widely.5-HT path adjustable NE and two systems of ACh in the maincenter, NE can cause 5-HT neurone generation mediator to reduce and exhaust.Chronic stress and patients with depression blood, cerebrospinal fluid (CSF), cerebral tissue 5-HT content reduce; The total content receptor of hippocampus 5-HT reduces; Rd then can recover after the treatment state of an illness is alleviated, and blocking-up 5-HT2A/2C acceptor all can cause anxiety and insomnia, and the 5-HT that aggravation NE excitement causes exhausts.This shows; The NE system of chronic stress startup continues the high state of opening and can cause maincenter 5-HT to exhaust, and maincenter 5-HT exhaustion can be aggravated excitement of NE system and emotional handicap, both reciprocal causations progressively; Form vicious cycle with this, cause carrying out property of maincenter 5-HT exhaustion thus.2. the low reason of congenital 5-HT level is the congenital little hippocampus of part patient; This type of congenital little hippocampus possibly come from the synthetic 5-HT of maincenter 5-HT neurone that current research finds rate-limiting enzyme--(Tryptophan hydroxylase, TPH) gene pleiomorphism is relevant for TPH.TPH1 mainly around system play a role, TPH2 is then at maincenter; Mouse C1473G sudden change causes 447 proline(Pro) to be replaced by l-arginine, and the level of its maincenter 5-HT can descend 30%～70%, and appearance and the disorderly relevant behavior change of maincenter 5-HT system function.When carrying the allelic TPH2 of G at the PC12 cell expressing, synthetic 5-HT is merely 45% of wild-type A.Prompting thus, low-level its former first gene function of going crazy of maincenter 5-HT is unusual with due to the risk factor associating, also is prone to suffer from the important foundation of dysthymia disorders or the easy generation of its organ cell canceration for the part crowd.
(5) maincenter 5-HT Transplanted cells improve maincenter 5-HT lose compensatory-have a extensive future
So far, clinically be used for depression, anticancer medicine brings bigger toxic side effect.To sum up can know,,, and then can play adjusting maincenter or anticancer effect through the 5-HT of its release compensation if bestow the transplanting of 5-HT cell maincenter to being in long-term chronic stress state, dysthymia disorders and relevant cancer patients.And can be through this method prevention some special incident or Special Circumstances such as special violent earthquake, war etc., because psychology stimulates or pressure causes self-runaway condition or emotion abnormal syndrome also can play control effect.Association nerve system-mediator is also more and more paid attention in the control of dysthymia disorders or cancer just abroad at present and regulated the comprehensive therapy experiment that comprises its agonist and antagonist, demonstrated good prospect.
(6) clinical application 5-HT neurocyte source-stem cell inducing function cell and microencapsulation
The adult 5-HT neurocyte source that can be used for treating transplanting is very deficient, and there is rejection in heteroplastic transplantation, is difficult to apply as clinical transplantation.At present, stem cell has become the ideal scheme of cell or organ replacement therapy, and the 5-HT neurocyte that the stem cell inductive method acquisition that application autologous stem cells or allohistocompatibility are good can be used for the human implantation then becomes inexorable trend.Existing research shows, human bone marrow and fat is the stem cell bank of horn of plenty all, is prone to get safety, and its mesenchymal stem cells MSCs MSCs and ASCs can be to multi-functional differentiation.Yet ordering about stem cell must have corresponding method and condition towards the differentiation of the functioning cell of needs, finds to promote stem cell to report with method to the mature technology of 5-HT neurocyte differentiation both at home and abroad as yet.Owing to autologous stem cells induces the mature cell that comes to transplant also slight rejection can to take place sometimes, be with cell of transplanting and immunity system isolation--microencapsulation and avoid the effective ways of rejection.
Summary of the invention
The objective of the invention is to, a kind of MSCs, ASCs inductive 5-HT neurocyte are provided.
The objective of the invention is to, the microencapsulation preparation method of a kind of MSCs, ASCs inductive 5-HT neurocyte is provided.
The objective of the invention is to, a kind of application of microencapsulation induction type 5-HT neurocyte is provided, to overcome the above-mentioned shortcoming and defect of existing in prior technology.
The invention belongs to biomedical sector is microencapsulation self MSCs, ASCs inductive 5-HT neurocyte and preparation method thereof.
Thus, nutrient solution is at first adopted in contriver's design, and separation and Culture MSCs or ASCs are divided into the 5-HT neurocyte with it is induced, and then eliminate antigen, its long-term surviving of protection/nutrition/support and excretory effect through microencapsulation with isolation again.Use this method and induce the 5-HT neurocyte of differentiation, can be adapted to dysthymia disorders through microencapsulation, particularly refractory type or severe dysthymia disorders, stress nocuity disease and relevant treatment for cancer.This type microencapsulation self stem cell inductive 5-HT neurocyte can discharge the 5-HT quantum unit according to cell and give relatively individual cell microcapsule quantity and transplant; Can play physiological regulation and rehabilitative action to the patient; Have no side effect, have obvious superiority than other anti-depression drugs.Contriver's design and this method of founding are used for clinical 5-HT cell maturation to preparation, and reliable new technology and method can be provided.
The present invention includes: mesenchymal stem cells MSCs (bone mesenchymal stem cells, MSCs) or adipose stromal cells (adipose stromal stem cells ASCs) induces method and the induction type 5-HT cell micro-encapsulation technology of 5-HT neurocyte.
1. will separate the MSCs that obtains or ASCs induces through sets of conditions and becomes the 5-HT neurocyte;
2. (alginate-Nano--Poly lysine--alginate-Nano ANano-P-ANano) makes it microencapsulation to adopt sodium-alginate+nanometer-polylysine-sodium-alginate+nanometer again.
The technical problem that will solve required for the present invention, can realize through following technical scheme:
As first aspect of the present invention, MSCs, ASCs inductive 5-HT neurocyte is characterized in that, adopt nutrient solution that MSCs or ASSCs are become the 5-HT neurocyte through cultivating to induce step by step: may further comprise the steps:
(1) MSCs or ASSCs cultivate and go down to posterity:
1. former be commissioned to train foster;
2. had digestive transfer culture;
(2) precursor is induced and is adopted the precursor induced liquid to cultivate and induce, and this precursor induced liquid adds said precursor induced liquid with 3 cells of supporting 2 days of being commissioned to train, the next day change liquid, cultivated 4 days;
With MSCs induce into that projection attenuates, cell space and karyon are tending towards the precursor cell that becomes round, cell identifies that Nestin is positive;
(3) function is induced and is adopted the cultivation of function induced liquid and induce; This function induced liquid; Add the ripe 5-HT neurocyte of sealing coat, the next day change liquid, cultivated 6 days; With precursor cell continue to induce into that projection is many, cell space and karyon become circle or polygonal 5-HT neurocyte, cell identification of M AP2 (MAP 2), NeuN (specificity neuronal kernel albumen), 5-HT, TPH are positive, the nutrient solution ria-determination its discharge 5-HT concentration＞15ng/ml;
(4) stable performance is cultivated and is adopted neuronal cell cultures liquid to cultivate 1 day, and cell evaluation performance can be stablized, and the function of release 5-HT is vigorous, promptly becomes MSCs, ASCs inductive 5-HT neurocyte.
Wherein, in the step (1), said former be commissioned to train to support get marrow 5～10ml or fatty tissue 3～5g, the direct cell counting of marrow is with 1.5 * 10
6The cell density of/ml; Fatty tissue through trace digestion, carries out cell counting, with 1.5 * 10 earlier again
7The cell density of/ml in 50ml plastic culture bottle, contains the DMEM nutrient solution of 10% serum with cell inoculation, places saturated humidity, 37 ℃, 5%CO
2Cultivate in the constant incubator;
With full marrow, or whole fat cell attachment method isolation and purification MSCs or ASSCs: primary cell culture 48h changes liquid, and carefulness discards not attached cell;
After this change liquid the next day, inverted microscope is observed down, cover with bottle sidewall to 7 days cells at the bottom of.
Wherein, in the step (1), said had digestive transfer culture MSCs or ASCs reach contact inhibition at the bottom of being cultured to and covering with bottle, and the sucking-off nutrient solution adds the CMF-PBS flushing; Add 0.25% pancreatin, place 37 ℃ of digestion 1min, add the nutrient solution that contains 10% serum and digest with termination, suction pipe piping and druming makes cell come off from the bottle wall;
The nutrient solution that every bottle of adding contains the DMEM nutrient solution 10ml of 10% serum goes down to posterity by 1: 3 bottle, continue to cultivate, the next day change liquid.
At the bottom of covering with in 5 days bottle, 3 generations of had digestive transfer culture to the once more;
This method cultured cells is fusiformis, the shaft-like or shape of dashing forward more, identifies CD44, the CD133 positive.
Wherein, in the step (2), said precursor induced liquid staple is according to the parts by weight meter: high sugared DMEM 1000ml, Sodium.alpha.-ketopropionate 80～120mg, Sodium Selenite 5～30 μ g, L-glutaminate 250～350mg, 2 mercapto ethanol 3～5 μ l, NaHCO
33～5g, HEPES5～7g, the helper-inducer factor are Prostatropin (bFGF) 100～200ng/ BDNF (BDNF) 100～200ng/ all-trans retinoic acid (ATRA) 5～10mg, 1～3 part of/tissue juice and cytokine.
Wherein, in the step (3), said function induced liquid staple (according to the parts by weight meter): high sugared DMEM 1000ml, Sodium.alpha.-ketopropionate 80～120mg, Sodium Selenite 5～30 μ g, L-glutaminate 250～350mg, 2 mercapto ethanol 3～5 μ l, NaHCO
33～5g, HEPES5～7g, B27 5～10ml, tryptophane 1～5mg, the helper-inducer factor are 1～3 part of Prostatropin (bFGF) 100～200ng/ BDNF (BDNF) 100～200ng/ATRA5～10mg/ tissue juice and cytokine.
Wherein, In the step (4), the staple of said neuronal cell cultures liquid (according to the parts by weight meter): Neuronal Medium 1000ml, B2710～20ml, tryptophane 5～10mg, the helper-inducer factor are BDNF (BDNF) 100～200ng.
Wherein, In said step (2) and the step (3), said tissue juice is Regular Insulin 4～8 μ, Transferrins,iron complexes 1～5～mg, progesterone 10～50ng, Kendall compound 1～5mg, hydrocortisone 1～5mg, the former propylhomoserin 1～5mg of 3 iodine, vitamin H 1～5mg, human serum albumin 1g, putrescine 1～3mg etc.
As second aspect of the present invention, the microencapsulation preparation method of MSCs, ASCs inductive 5-HT neurocyte is characterized in that, may further comprise the steps:
Adopt ANano-P-ANano (sodium-alginate+nanoparticle-gather propylhomoserin-sodium-alginate+nanoparticle) method, make MSCs, ASCs inductive 5-HT neurocyte microencapsulation microencapsulation through static microcapsule generator;
Every micro-capsule contains 20 or 30 5-HT neuron clusters (Fig. 4), makes the cell in the micro-capsule fully contact the micro-capsule wall, is easy to adhere to and the nutrition exchange, promptly processes microencapsulation induction type 5-HT neurocyte.
As the third aspect of the invention, a kind of application of microencapsulation induction type 5-HT neurocyte is characterized in that, is used to prepare the medicine of treating dysthymia disorders.
As fourth aspect of the present invention, a kind of application of microencapsulation induction type 5-HT neurocyte is characterized in that, is used to prepare cancer therapy drug.
Beneficial effect of the present invention:
Microencapsulation induction type 5-HT cell of the present invention; This microencapsulation people 5-HT neurocyte maincenter transplantation therapy; To new approach be provided for clinical treatment dysthymia disorders and anticancer therapy; And, wide application prospect is arranged, thereby can produce huge economic and social benefit for other correlative studys provide experimental tool cell etc.The microencapsulation induction type 5-HT cell characteristics and the advantage of this method preparation are following:
(1) microencapsulation induction type 5-HT Transplanted cells belongs to physiological regulatory action.
(2) this induction type 5-HT neurocyte does not have the foreign gene influence.
(3) this stem cell revulsion high-efficient simple adopts this method that MSCs and MSCs, ASCs are induced to become 5-HT neurocyte positive rate and reaches more than 95%, and the 5-HT release function is strong.This method only adopts cell to change the liquid culture method, is easy to hold and operation.
(4) micro-capsule of preparation has good physicals, can play support/nutrition/provide protection.
(5) micro-capsule of preparation has good biologically stable.
(6) micro-capsule of preparation has suitable little perviousness.
(7) no immunogenicity.
(8) micro-capsule of favorable tissue consistency 5-HT neurocyte.
(9) have no side effect.
Description of drawings
Fig. 1 is that 3 generation MSCs cultivated 5 days
Fig. 2 is MSCs precursor inducing culture 4 days
Fig. 3 A is that MSCs induces 5-HT to cultivate 6 days (Toluidine blue staining)
Fig. 3 B is that MSCs induces 5-HT to cultivate 6 days (5-HT immunohistochemical staining)
Fig. 4 is the ANano-P-Anano micro-capsule
Fig. 5 is a microencapsulation induction type 5-HT neurocyte action principle synoptic diagram
Embodiment
Below in conjunction with specific embodiment, the present invention is described further.Should be understood that following examples only are used to the present invention is described but not are used to limit scope of the present invention.
The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition that manufacturer provides is carried out.
The present invention includes: mesenchymal stem cells MSCs (bone mesenchymal stem cells, MSCs) or adipose stromal cells (adipose stromal stem cells ASCs) induces method and the induction type 5-HT cell micro-encapsulation technology of 5-HT neurocyte.
1. will separate the MSCs that obtains or ASCs induces through sets of conditions and becomes the 5-HT neurocyte;
2. (alginate-Nano--Poly lysine--alginate-Nano ANano-P-ANano) makes it microencapsulation to adopt sodium-alginate+nanometer-polylysine-sodium-alginate+nanometer again.
Marrow, promptly (bone mesenchymal stem cells MSCs) derives from Shanghai, Shanghai City, China biomedical tissue ERC of The 2nd Army Medical College to mesenchymal stem cells MSCs, and mesenchymal stem cells MSCs can be from body or allogeneic.
Fatty tissue, promptly (adipose stromal stem cells ASCs) derives from Shanghai, Shanghai City, China biomedical tissue ERC of The 2nd Army Medical College to adipose stromal cells, and adipose stromal cells can be from body or allogeneic.
Microencapsulation induction type 5-HT neurocyte is used for processing promptly.
Embodiment 1
One, MSCs or ASSCs two-step approach are induced the 5-HT neurocyte
Adopt the sets of conditions nutrient solution of design and preparation, MSCs or ASSCs are become the 5-HT neurocyte through cultivating to induce step by step.
(1) MSCs or ASSCs cultivate and go down to posterity:
1. former being commissioned to train supported the state of an illness and the body weight according to the patient, gets himself marrow 5～10ml or fatty tissue 3～5g g, and the direct cell counting of marrow is with 1.5 * 10
6The cell density of/ml; Fatty tissue through trace digestion, carries out cell counting, with 1.5 * 10 earlier again
7～1.5 * 10
9The cell density of/ml in 5～10 100ml plastic culture bottles, contains the DMEM nutrient solution of 10% serum with cell inoculation, places saturated humidity, 37 ℃, 5%CO
2Cultivate in the constant incubator.
With full marrow, or whole fat cell attachment method isolation and purification MSCs or ASSCs: primary cell culture 48h changes liquid, and carefulness discards not attached cell.
After this change liquid the next day, inverted microscope is observed down, cover with bottle sidewall to 7 days cells at the bottom of.
2. at the bottom of had digestive transfer culture MSCs or ASCs are cultured to and cover with bottle, reach contact inhibition, the sucking-off nutrient solution adds the CMF-PBS flushing; Add 0.25% pancreatin, place 37 ℃ of digestion 1min, add the nutrient solution that contains 10% serum and digest with termination, suction pipe piping and druming makes cell come off from the bottle wall.
The DMEM nutrient solution 10ml that every bottle of adding contains 10% serum goes down to posterity by 1: 3 bottle, continue to cultivate, the next day change liquid.
At the bottom of covering with in 5 days bottle, 3 generations of had digestive transfer culture to the once more.
This method cultured cells is fusiformis, the shaft-like or shape (Fig. 1) of dashing forward more, identifies CD44, the CD133 positive.
(2) precursor is induced and is adopted the cultivation of precursor induced liquid and induce this precursor induced liquid staple: high sugared DMEM 1000ml, Sodium.alpha.-ketopropionate 80～120mg, Sodium Selenite 5～30 μ g, L-glutaminate 250～350mg, 2 mercapto ethanol 3～5 μ l, NaHCO
33～5g, HEPES5～7g, the helper-inducer factor are Prostatropin (bFGF) 100～200ng/ BDNF (BDNF) 100～200ng/ all-trans retinoic acid (ATRA) 5～10mg; / tissue juice (said tissue juice is Regular Insulin 4～8 μ, Transferrins,iron complexes 1～5～mg, progesterone 10～50ng, Kendall compound 1～5mg, hydrocortisone 1～5mg, the former propylhomoserin 1～5mg of 3 iodine, vitamin H 1～5mg, human serum albumin 1g, putrescine 1～3mg etc.) and 1～3 part of cytokine; 3 cells of supporting 2 days of being commissioned to train are added the precursor induced liquid; The next day change liquid, cultivated 4 days.
With MSCs induce into that projection attenuates, cell space and karyon are tending towards the precursor cell (Fig. 2) that becomes round, cell identifies that Nestin is positive.
(3) function is induced and is adopted the cultivation of function induced liquid and induce this function induced liquid staple (according to the parts by weight meter): high sugared DMEM, pyruvic acid, L-glutaminate, 2 mercapto ethanol, NaHCO
3, HEPES, B27, tryptophane, helper-inducer factor bFGF/BDNF/ATRA/ tissue juice (said tissue juice is Regular Insulin 4～8 μ, Transferrins,iron complexes 1～5～mg, progesterone 10～50ng, Kendall compound 1～5mg, hydrocortisone 1～5mg, the former propylhomoserin 1～5mg of 3 iodine, vitamin H 1～5mg, human serum albumin 1g, putrescine 1～3mg etc.) and cytokine etc.; Add the ripe 5-HT neurocyte of sealing coat; The next day change liquid; Cultivated 6 days; With precursor cell continue to induce into that projection is many, cell space and karyon become circle or polygonal 5-HT neurocyte, cell identifies that MAP 2 (MAP2), specificity neuronal kernel albumen (NeuN), 5-HT, TPH are positive, the nutrient solution ria-determination its discharge 5-HT concentration＞15ng/ml.
(4) stable performance is cultivated and is adopted neuronal cell cultures liquid to cultivate; The staple of this neuronal cell cultures liquid; According to the parts by weight meter: Neuronal Medium 1000ml, B2710～20ml, tryptophane 5～10mg, helper-inducer factor B DNF100～200ng; Cultivated 1 day, cell evaluation performance can be stablized, and discharges the function vigorous (Fig. 3 A and Fig. 3 B) of 5-HT.
Two, inductive 5-HT neurocyte microencapsulation
1, adopts ANano-P-ANano (sodium-alginate+nanoparticle-gather propylhomoserin-sodium-alginate+nanoparticle) method, make MSCs, ASCs inductive 5-HT neurocyte microencapsulation microencapsulation through static microcapsule generator;
Every micro-capsule contains 20 or 30 5-HT neuron clusters (Fig. 4), makes the cell in the micro-capsule fully contact the micro-capsule wall, is easy to adhere to and the nutrition exchange, promptly processes microencapsulation induction type 5-HT neurocyte.
2, microencapsulated material performance and evaluation:
Experimental observation: 1. the micro-capsule cell was cultivated 2,4,6 days through adopting petridish, and inverted microscope is observed micro-capsule wall periphery form and kept (no distortion, depression or shrinkage), shows that the micro-capsule physicals is good, and biological property is stable; 2. cultivated 2,4,6 days, and detected culture supernatant liquid 5-HT content and can reach 8～12ng/ml, reflection cell survival and function are good, show that little perviousness is suitable; 3. animal (rat) subarachnoid space is transplanted the no fervescence in back, show that this micro-capsule histocompatibility is strong, and buffer action is good, does not have the pyrogen that causes exothermic reaction; 4. the no leukocyte infiltration of check is learned by myeloid tissue; The centrifugal no white corpuscle of cerebrospinal fluid increases, and shows that this type of micro-capsule has no side effect to the part.
1. physicals is good; 2. biological stable; 3. little perviousness is suitable; 4. histocompatibility is strong, and buffer action is good; 5. have no side effect.
3, the 5-HT neurocyte Function Identification of microencapsulation:
Neurocyte the survival synthetic and release good, 5-HT that shows clear and definite microencapsulation is vigorous, confirms that the individual quantum of micro-capsule discharges titre and individual patients micro-capsule Transplanted cells quantity.
The neuron cluster Function detection of microencapsulation, every 50ml culturing bottle inoculation 1 * 10
3～5 * 10
3Individual micro-capsule groups of cells, the next day get that please liquid to detect 5-HT content be 5～18ng.Need according to maincenter adjusting, homeostasis balance and compensate function, individual transplanting amount is 3 * 10
4～10 * 10
4Micro-capsule.
Three, microencapsulation induction type 5-HT neurocyte action principle
(1) micro-capsule is kept survival of capsule inner cell and function, and exempts immunoreation
1. the material sodium-alginate+nanoparticle of cell microcapsule-gather propylhomoserin-sodium-alginate+3 layers of mocromembrane of nanoparticle; The cell that can be in the micro-capsule provides suitable adhering to and nutrition intake; Keep its stability; Improve the synthetic and release function of its 5-HT, can be the outer slow liquid flow zone spinal cord mantle of micro-capsule micro-capsule is adapted to affine, more be prone to build the environment of survival for it;
2. this micro-capsule is that inductive 5-HT cell provides isolation barrier, makes it avoid contacting with immunocyte, avoids safeguarding its good survival by its immunoreation that causes or immunity injury;
3. this micro-capsule has suitable perviousness, can make the outer nutritive substance of capsule be easy to infiltrate in the capsule used with enough cells, keeps the homeostasis in the capsule, can guarantee that again the 5-HT of its release is easy to ooze out.
(2) 5-HT that the 5-HT compensation cerebrospinal fluid that the 5-HT cell discharges in the micro-capsule reduces and the 5-HT of brain cell exhaust, recover or improve the anti-stress damage or the tolerance of maincenter.
Dysthymia disorders or exhaust with patient that stress be relevant or part cancer patients maincenter 5-HT or level low excessively; And maincenter 5-HT exhaust can come from stress due to 5-HT cell function depleted (acquired weakening); Or inherent 5-HT cell TPH polymorphum and irritability is injured high susceptible (congenital deficiency); Cause the 5-HT cell of himself progressively to develop into the stress tolerance incapability, lose compensatory pathological phenomenon and appear, show as worry, irritability ... etc.; Cause thus that neuronal function that maincenter 5-HT regulates is unusual and cause its descending conduction path organ cell's illness susceptible of being arranged, or pathology malignant development.
As shown in Figure 5; Microencapsulation induction type 5-HT nerve cells transplantation is in subarachnoid space 1; The 5-HT that the 5-HT cell discharges in the capsule through osmosis to subarachnoid space; The 5-HT neurocyte release 5-HT that transplants oozes out micro-capsule and goes into cerebrospinal fluid 2, diffuses to corresponding neurone with the cerebrospinal fluid circulation and brings into play its mediator effect, and 5-HT diffuses to hippocampus, hypothalamus, dorsal nucleus of vagus nerve cell 3 in the cerebrospinal fluid.5-HT after the effect is through the neurone re-uptake and inactivation or play Nutrition, and hippocampus, hypothalamus, dorsal nucleus of vagus nerve improve malfunction 4 through physiological regulation.
Microencapsulation induction type 5-HT cell of the present invention; This microencapsulation people 5-HT neurocyte maincenter transplantation therapy; To new approach be provided for clinical treatment dysthymia disorders and anticancer therapy; And, wide application prospect is arranged, thereby can produce huge economic and social benefit for other correlative studys provide experimental tool cell etc.The microencapsulation induction type 5-HT cell characteristics and the advantage of this method preparation are following:
(1) microencapsulation induction type 5-HT Transplanted cells belongs to the microencapsulation induction type cell maincenter transplanting that this preparation method of physiological regulatory action obtains, and the 5-HT that exhausts through the release compensation belongs to normal neurotransmitter.This type of microencapsulation inductive 5-HT cell is compared with existing depression, stress damage or anticarcinogen, has better physiological regulation curative effect, and can not bring undesirable action.
(2) this induction type 5-HT neurocyte does not have foreign gene influences this revulsion and belongs to chemically induced; There is not the importing of exogenous carrier (virus or liposome etc.) and gene; Thereby resulting Transplanted cells can not bring due to illness poison infection or foreign gene to integrate producer group abnormal change to body and cause other cell generation pathological changes in transplanted cells or the body to body.
(3) this stem cell revulsion high-efficient simple adopts this method that MSCs and MSCs, ASCs are induced to become 5-HT neurocyte positive rate and reaches more than 95%, and it is strong to have a vigorous 5-HT release function.This method only adopts cell to change the liquid culture method, need not complicated technology such as vector construction and gene transfection or gene importing, thereby is easy to hold and operation.
(4) micro-capsule of preparation has good physicals provides the suitable space of survival to the capsule inner cell, can play supports/nutrition/provide protection, and through being implanted into human body, the gap of blister cavities and cyst wall does not have constriction or expansion variation.
(5) micro-capsule of preparation have transplant in the good Biostatic gonosome after, drive or mantle adheres to and all keeps its stable morphology through the cerebrospinal fluid circulation.
(6) micro-capsule of preparation has suitable little perviousness micro-capsule wall rich suitable porosity and wetting ability; Can allow the cerebrospinal fluid nutritive ingredient that suits or adhere to the 5-HT that nutritive ingredient is infiltrated and the 5-HT neurocyte discharges in the pia mater spinalis capillary vessel of back in capsule outside capsule, to ooze out, promptly be easy to the inside and outside exchange of substance of micro-capsule.Simultaneously, capsule inner cell and vivo immuning system are had the good isolation effect, avoid transplanted cells to cause any rejection.
(7) no immunogenicity microencapsulation inductive 5-HT neurocyte is to derive from autogenous cell; Thereby the 5-HT cell in the capsule itself is same somatocyte; Say that from theory the inductive cell does not have immunogenicity or only atomic weak immunogenicity thus, promptly can not cause the body graft-rejection, after promptly transplanting in the body; Do not cause subarachnoid space and systemic inflammatory response, phenomenons such as cerebrospinal fluid or blood leukocytes rising do not take place.
(8) micro-capsule of favorable tissue consistency 5-HT neurocyte; Adopt the sodium-alginate histocompatibility of ANano-P-ANano good; The nanoparticle tissue is abundant; Further strengthen histocompatibility, make the cell microcapsule of transplanting suitable each other, exempted the incidental foreign body reaction of existing medicament with the tissue that contacts.
(9) the have no side effect 5-HT neurocyte that uses microencapsulation is that 5-HT through its release plays a role; 5-HT is that essential neurotransmitter is kept and regulated to the maincenter function; Also can promote cell proliferation of nerve cord and grow,, can play good physiological action for maincenter increases or replenish the 5-HT of requirement; And without any side effects, promptly there are not toxic reactions such as the heating of the back of transplanting, liver, kidney, neural system.
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; The present invention is not restricted to the described embodiments; That describes in the foregoing description and the specification sheets just explains principle of the present invention; The present invention also has various changes and modifications under the prerequisite that does not break away from spirit and scope of the invention, and these variations and improvement all fall in the scope of the invention that requires protection.The present invention requires protection domain to be defined by appending claims and equivalent thereof.
Claims (10)
1.MSCs, ASCs inductive 5-HT neurocyte, it is characterized in that adopting nutrient solution that MSCs or ASSCs are cultivated to induce through substep becomes the 5-HT neurocyte: may further comprise the steps:
(1) MSCs or ASSCs cultivate and go down to posterity:
1. former be commissioned to train foster;
2. had digestive transfer culture;
(2) precursor is induced and is adopted the precursor induced liquid to cultivate and induce, and this precursor induced liquid adds said precursor induced liquid with 3 cells of supporting 2 days of being commissioned to train, the next day change liquid, cultivated 4 days;
With MSCs induce into that projection attenuates, cell space and karyon are tending towards the precursor cell that becomes round, cell identifies that Nestin is positive;
(3) function is induced and is adopted the cultivation of function induced liquid and induce; This function induced liquid; Add the ripe 5-HT neurocyte of sealing coat, the next day change liquid, cultivated 6 days; With precursor cell continue to induce into that projection is many, cell space and karyon become circle or polygonal 5-HT neurocyte, cell identification of M AP2 (MAP 2), NeuN (specificity neuronal kernel albumen), 5-HT, TPH are positive, the nutrient solution ria-determination its discharge 5-HT concentration＞15ng/ml;
(4) stable performance is cultivated and is adopted neuronal cell cultures liquid to cultivate 1 day, and cell evaluation performance can be stablized, and the function of release 5-HT is vigorous, promptly becomes MSCs, ASCs inductive 5-HT neurocyte.
2. MSCs according to claim 1, ASCs inductive 5-HT neurocyte is characterized in that, in the step (1), said former be commissioned to train to support get marrow 5～10ml or fatty tissue 3～5g, the direct cell counting of marrow is with 1.5 * 10
6The cell density of/ml; Fatty tissue through trace digestion, carries out cell counting, with 1.5 * 10 earlier again
7～1.5 * 10
9The cell density of ml in 100ml plastic culture bottle, contains the DMEM nutrient solution of 10% serum with cell inoculation, places saturated humidity, 37 ℃, 5%CO
2Cultivate in the constant incubator;
With full marrow, or whole fat cell attachment method isolation and purification MSCs or ASSCs: primary cell culture 48h changes liquid, and carefulness discards not attached cell;
After this change liquid the next day, inverted microscope is observed down, cover with bottle sidewall to 7 days cells at the bottom of.
3. MSCs according to claim 1, ASCs inductive 5-HT neurocyte is characterized in that, in the step (1), said had digestive transfer culture MSCs or ASCs reach contact inhibition at the bottom of being cultured to and covering with bottle, and the sucking-off nutrient solution adds the CMF-PBS flushing; Add 0.25% pancreatin, place 37 ℃ of digestion 1min, add the nutrient solution that contains 10% serum and digest with termination, suction pipe piping and druming makes cell come off from the bottle wall;
The DMEM nutrient solution 10ml that every bottle of adding contains 10% serum goes down to posterity by 1: 3 bottle of branch bottle, continue to cultivate, the next day change liquid;
At the bottom of covering with in 5 days bottle, 3 generations of had digestive transfer culture to the once more;
This method cultured cells is fusiformis, the shaft-like or shape of dashing forward more, identifies CD44, the CD133 positive.
4. MSCs according to claim 1, ASCs inductive 5-HT neurocyte; It is characterized in that; In the step (2), said precursor induced liquid staple is according to the parts by weight meter: high sugared DMEM 1000ml, Sodium.alpha.-ketopropionate 80～120mg, Sodium Selenite 5～30 μ g, L-glutaminate 250～350mg, 2 mercapto ethanol 3～5 μ l, NaHCO
33～5g, HEPES5～7g, the helper-inducer factor are Prostatropin (bFGF) 100～200ng/ BDNF (BDNF) 100～200ng/ all-trans retinoic acid (ATRA) 5～10mg, 1～3 part of/tissue juice and cytokine.
5. MSCs according to claim 1, ASCs inductive 5-HT neurocyte; It is characterized in that; In the step (3), said function induced liquid staple (according to the parts by weight meter): high sugared DMEM 1000ml, Sodium.alpha.-ketopropionate 80～120mg, Sodium Selenite 5～30 μ g, L-glutaminate 250～350mg, 2 mercapto ethanol 3～5 μ l, NaHCO
33～5g, HEPES5～7g, B27 5～10ml, tryptophane 1～5mg, the helper-inducer factor are 1～3 part of Prostatropin (bFGF) 100～200ng/ BDNF (BDNF) 100～200ng/ATRA5～10mg/ tissue juice and cytokine.
6. MSCs according to claim 1, ASCs inductive 5-HT neurocyte; It is characterized in that; In the step (4), the staple of said neuronal cell cultures liquid (according to the parts by weight meter): Neuronal Medium 1000ml, B2710～20ml, tryptophane 5～10mg, the helper-inducer factor are BDNF (BDNF) 100～200ng.
7. MSCs according to claim 1, ASCs inductive 5-HT neurocyte; It is characterized in that; In said step (2) and the step (3), said tissue juice is Regular Insulin 4～8 μ, Transferrins,iron complexes 1～5～mg, progesterone 10～50ng, Kendall compound 1～5mg, hydrocortisone 1～5mg, the former propylhomoserin 1～5mg of 3 iodine, vitamin H 1～5mg, human serum albumin 1g, putrescine 1～3mg.
8. the microencapsulation preparation method of a MSCs as claimed in claim 1, ASCs inductive 5-HT neurocyte is characterized in that, may further comprise the steps:
Adopt ANano-P-ANano (sodium-alginate+nanoparticle-gather propylhomoserin-sodium-alginate+nanoparticle) method, make MSCs, ASCs inductive 5-HT neurocyte microencapsulation microencapsulation through static microcapsule generator;
Every micro-capsule contains 20 or 30 5-HT neuron clusters, makes the cell in the micro-capsule fully contact the micro-capsule wall, is easy to adhere to and the nutrition exchange, promptly processes microencapsulation induction type 5-HT neurocyte.
9. the application of a microencapsulation induction type 5-HT neurocyte as claimed in claim 8 is characterized in that, is used to prepare the medicine of treating dysthymia disorders.
10. the application of a microencapsulation induction type 5-HT neurocyte as claimed in claim 8 is characterized in that, is used to prepare cancer therapy drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110421109.9A CN102533653B (en) | 2011-12-15 | 2011-12-15 | 5-HT nerve cells induced by MSCs and ASCs and microencapsulation preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110421109.9A CN102533653B (en) | 2011-12-15 | 2011-12-15 | 5-HT nerve cells induced by MSCs and ASCs and microencapsulation preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102533653A true CN102533653A (en) | 2012-07-04 |
| CN102533653B CN102533653B (en) | 2014-12-10 |
Family
ID=46341689
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110421109.9A Expired - Fee Related CN102533653B (en) | 2011-12-15 | 2011-12-15 | 5-HT nerve cells induced by MSCs and ASCs and microencapsulation preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102533653B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106350490A (en) * | 2016-11-02 | 2017-01-25 | 中国科学院广州生物医药与健康研究院 | Method for acquiring nerve cells from fibroblasts by utilizing serum-free medium |
| CN111073855A (en) * | 2018-10-19 | 2020-04-28 | 中国科学院上海生命科学研究院 | Method for inducing astrocyte to transdifferentiate into serotonin neurons and application |
| CN113730372A (en) * | 2021-10-15 | 2021-12-03 | 中国人民解放军陆军军医大学第二附属医院 | Phospholipid membrane microcapsule of neural stem cell and preparation method thereof |
-
2011
- 2011-12-15 CN CN201110421109.9A patent/CN102533653B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| 廖华等: "微囊化大鼠松果体细胞的体外培养研究", 《解剖学报》 * |
| 李积胜等: "缺锌大鼠脑内5-HT 神经细胞及其受体的变化", 《解剖科学进展》 * |
| 潘国强等: "骨髓间充质干细胞分化为神经元样细胞的研究进展", 《中国煤炭工业医学杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106350490A (en) * | 2016-11-02 | 2017-01-25 | 中国科学院广州生物医药与健康研究院 | Method for acquiring nerve cells from fibroblasts by utilizing serum-free medium |
| CN106350490B (en) * | 2016-11-02 | 2019-10-11 | 中国科学院广州生物医药与健康研究院 | The method of nerve cell is obtained from fibroblast using serum free medium |
| CN111073855A (en) * | 2018-10-19 | 2020-04-28 | 中国科学院上海生命科学研究院 | Method for inducing astrocyte to transdifferentiate into serotonin neurons and application |
| CN113730372A (en) * | 2021-10-15 | 2021-12-03 | 中国人民解放军陆军军医大学第二附属医院 | Phospholipid membrane microcapsule of neural stem cell and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102533653B (en) | 2014-12-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7235509B2 (en) | Implantable living electrode and method of making same | |
| CN103340890B (en) | NADPH is as the preparation of the application preventing and treating cerebral infarction medicine aspect | |
| AU2013299377A1 (en) | Injectable brown adipose microtissues for treatment and prevention of obesity and diabetes | |
| CN109589337B (en) | Myocardial cell preparation and preparation method and application thereof | |
| JP2015508654A (en) | Methods and compositions for brown adipose-like cells | |
| US20190314419A1 (en) | Composition containing adipose stem cell-derived exosomes as active ingredient for preventing or treating liver fibrosis | |
| Fukuda et al. | The intraperitoneal space is more favorable than the subcutaneous one for transplanting alginate fiber containing iPS-derived islet-like cells | |
| Chan et al. | Therapeutic potential of human stem cell implantation in alzheimer’s disease | |
| CN110317788A (en) | A kind of preparation method and application of NPC culture medium, excretion body | |
| CN102533653B (en) | 5-HT nerve cells induced by MSCs and ASCs and microencapsulation preparation method and application thereof | |
| Zhao et al. | Sigma 1 receptor contributes to astrocyte-mediated retinal ganglion cell protection | |
| CN102625707A (en) | Novel applications of HIP/PAP or derivatives thereof | |
| US10017740B2 (en) | Deriving brown adipose tissue cells | |
| TWI590830B (en) | Active ingredients for enhancing stem cell differentiation and used in health care after recovery from myocardial infarction | |
| Mishra et al. | Mitochondria transfer as a potential therapeutic mechanism in Alzheimer’s disease-like pathology | |
| Darwish et al. | Intranigral injection of endotoxin suppresses proliferation of hippocampal progenitor cells | |
| US11746329B2 (en) | Systems and methods for producing injectable enhanced stem cell exosomes, improved exosomes and methods of use | |
| CN113388580B (en) | Method for inducing adipose-derived stem cells to differentiate into functional dopaminergic neurons and application | |
| Sun et al. | Protective effect of bone morphogenetic protein-7 induced differentiation of bone marrow mesenchymal stem cells in rat with acute spinal cord injury | |
| Xu et al. | Protective effect of mesenchymal stromal cell-derived exosomes on traumatic brain injury via miR-216a-5p | |
| CN101496815B (en) | Medicament for treating hepatic failure and preparation method thereof | |
| CN106139241B (en) | Application of the curcumin under diabetic conditions in medical titanium alloy implants | |
| Gokuladhas et al. | Ocular progenitor cells and current applications in regenerative medicines–review | |
| CN110025767A (en) | A kind of osteocalcin is preparing the application in Alzheimer disease drugs | |
| Zhang et al. | Rps6ka2 enhances iMSC chondrogenic differentiation to attenuate knee osteoarthritis through articular cartilage regeneration in mice |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20141210 Termination date: 20161215 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |