CN105925528B - Wang Shi progenitor cells and its application in promotion nerve regneration are applied derived from marrow neural crest cell - Google Patents

Wang Shi progenitor cells and its application in promotion nerve regneration are applied derived from marrow neural crest cell Download PDF

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CN105925528B
CN105925528B CN201610338493.9A CN201610338493A CN105925528B CN 105925528 B CN105925528 B CN 105925528B CN 201610338493 A CN201610338493 A CN 201610338493A CN 105925528 B CN105925528 B CN 105925528B
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施海燕
丁斐
强亮
余英奇
孙晓婷
胡静静
孙晓欢
何欢
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Nantong University
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Abstract

The present invention relates to tissue engineering technique fields, and in particular to a kind of to apply Wang Shi progenitor cells and its in the application promoted in nerve regneration derived from marrow neural crest cell.The present invention provides a kind of rat marrow source neural crest progenitor cells N1, deposit number is CCTCC NO:C201647, i.e. BM-NCC clones N1, has the performance for applying Wang Shi progenitor cells (SCP).The present invention also provides the secondary culture methods of BM-NCC clone N1, and the method to schwann's cell (SC) Induction of committed differentiation.BM-NCC clone N1 of the invention is expected to be applied to the rush treatment of nerve regeneration based on cell.

Description

Wang Shi progenitor cells are applied and its in promoting nerve regneration derived from marrow neural crest cell Application
Technical field
The present invention relates to tissue engineering technique fields, and in particular to it is a kind of derived from marrow neural crest cell to apply Wang Shi ancestral thin Born of the same parents and its application in promotion nerve regneration.
Background technique
In nervous system, schwann's cell (Schwann cell, SC) is the spongiocyte of peripheral nervous system, in axis Dash forward into myelin, the systematism of peripheral neurons, the maintenance of neuron normal function, the formation of cynapse and to neurotrosis and mind Reaction of dysmenorrhoea etc. plays a key role.Wallerian denaturation stage after peripheral nerve injury, SC dedifferente for Prematurity SC or precursor sample SC participates in the removing of myelin and aixs cylinder fragment, generates a variety of growth factors and cell factor, is proliferated And migrate and to form the Bungner band long conducive to axon regeneration, be finally divided into maturation again supports aixs cylinder weight at myelin SC It is new to form myelin.Currently, the neural tissue engineering based on SC has many application targets, including peripheral nerve disease or damage Treatment, the treatment potential and the treatment in SC related disease external model stage of central nervous system (CNS) disease.However SC Source and purity still limit its application in terms of cell therapy.
Applying Wang Shi progenitor cells (Schwann cell precursor, SCP) represents the early stage of schwann's cell.In In the growth course of vertebrate, SCP and immature SC originating from migration neural crest cell (neural crest cell, NCC), just disappeared to adulthood neural crest tissue.The SCP being enriched with from embryo's peripheral nerve and mature SC are transplanted to After CNS, the former has preferably existence and transfer ability than the latter.SCP can also be divided into the maincenter spongiocyte of myelin Repair brain damage.In addition, the neuronotropic differentiation of SCP is had also been observed that in nerve to occur after the birth of enteric nervous system.
Currently, be used to derivative SC there are many different exogenous stem cells types, including embryonic stem cell and lure Lead multipotential stem cell [Ma MS, Boddeke E, Copray S.Pluripotent stem cells for Schwann cell engineering.Stem Cell Rev.2015；11:205-18.][Li W,Huang L,Lin W,Ke Q,Chen R,Lai X,et al.Engraftable neural crest stem cells derived from cynomolgus monkey embryonic stem cells.Biomaterials.2015；39:75-84.][Liu Q,Spusta SC,Mi R, Lassiter RN,Stark MR,Hoke A,et al.Human neural crest stem cells derived from human ESCs and induced pluripotent stem cells:induction,maintenance,and differentiation into functional schwann cells.Stem Cells Transl Med.2012；1: 266-78.][Ren YJ,Zhang S,Mi R,Liu Q,Zeng X,Rao M,et al.Enhanced differentiation of human neural crest stem cells towards the Schwann cell lineage by aligned electrospun fiber matrix.Acta Biomater.2013；9:7727-36.] [Wang A,Tang Z,Park IH,Zhu Y,Patel S,Daley GQ,et al.Induced pluripotent stem cells for neural tissue engineering.Biomaterials.2011；32:5023-32.], all show it Repair to injured neurons.In fact, adult stem cell has a polyphyly differentiation potential in vitro, and they can be from It is self to obtain, can avoid the puzzlement of immunological rejection effect and ethics problem, therefore adult stem cell appear to be more prospect can NCC after the cell type in the substitution source SC, including mescenchymal stem cell (mesenchymal stem cell, MSC) and migration [Neirinckx V,Coste C,Rogister B,Wislet-Gendebien S.Concise review:adult mesenchymal stem cells,adult neural crest stem cells,and therapy of neurological pathologies:a state of play.Stem Cells Transl Med.2013；2:284- 96.]。
It has been reported that NCC and derivative SC can obtain [Nagoshi N, Shibata from dorsal root ganglion (DRG) after migration S,Kubota Y,Nakamura M,Nagai Y,Satoh E,et al.Ontogeny and multipotency of neural crest-derived stem cells in mouse bone marrow,dorsal root ganglia,and whisker pad.Cell Stem Cell.2008；2:392-403][Li HY,Say EH,Zhou XF.Isolation and characterization of neural crest progenitors from adult dorsal root ganglia.Stem Cells.2007；25:2053-65.][Vidal M,Maniglier M,Deboux C,Bachelin C, Zujovic V,Baron-Van Evercooren A.Adult DRG Stem/Progenitor Cells Generate Pericytes in the Presence of Central Nervous System(CNS)Developmental Cues, and Schwann Cells in Response to CNS Demyelination.Stem Cells.2015；33:2011- 24.][Zujovic V,Thibaud J,Bachelin C,Vidal M,Coulpier F,Charnay P,et al.Boundary cap cells are highly competitive for CNS remyelination:fast migration and efficient differentiation in PNS and CNS myelin-forming cells.Stem Cells.2010；28:470-9.], also it can go out pluripotency from impaired or intact peripheral nerve enrichment culture Schwann cell ball [Takagi T, Ishii K, Shibata S, Yasuda A, Sato M, Nagoshi N, et al.Schwann-spheres derived from injured peripheral nerves in adult mice-- their in vitro characterization and therapeutic potential.PLoS One.2011；6: e21497.][Martin I,Nguyen TD,Krell V,Greiner JF,Muller J,Hauser S,et al.Generation of Schwann cell-derived multipotent neurospheres isolated from intact sciatic nerve.Stem Cell Rev.2012；8:1178-87.], there is the identity of NCC and oriented to SC The potential of differentiation, these Schwann cell balls can provide autogenous cell source for nerve regneration, but corresponding biopsy means are to confession Damage caused by somatic nerves is in fact unpractical for translational medicine.So scholars, which are intended to search out, to be obtained Take the non-nervous tissue source of adult NCC and derivative SC.Have latest report [Mendez-Ferrer S, Michurina TV, Ferraro F,Mazloom AR,Macarthur BD,Lira SA,et al.Mesenchymal and haematopoietic stem cells form a unique bone marrow niche.Nature.2010；466: 829-34.][Isern J,Martin-Antonio B,Ghazanfari R,Martin AM,Lopez JA,del Toro R, et al.Self-renewing human bone marrow mesenspheres promote hematopoietic stem cell expansion.Cell Rep.2013；3:1714-24.][Isern J,Garcia-Garcia A,Martin AM, Arranz L,Martin-Perez D,Torroja C,et al.The neural crest is a source of mesenchymal stem cells with specialized hematopoietic stem cell niche function.Elife.2014；3:e03696.], find marrow stromal cell (the bone marrow of the Nestin positive Stromal cell, BMSC) with the SCP of neural crest origin there is common nerve-epithelial development to originate from, and both cells Long bone marrow may be resided in simultaneously, establish the microenvironment of candidate stem cell jointly；When in vitro culture, these Nestin sun Property, the BMSC of platelet-derived growth factor receptor (PDGFR) feminine gender can be formed with self-renewal capacity and more differentiation potentials Cell ball, and include stem cell of neural crest (neural crest stem in these mesenchyma balls (Mesensphere) Cell, NCSC) and neural crest progenitor cells (neural crest precursor, NCP), and thus further infer that in marrow have The residence of SCP cell mass.
Although BMSC is well-known high heterogeneous population, seminar's previous research work where the present inventor is Show [Shi H, Zhang T, Qiang L, Man L, Shen Y, Ding F.Mesenspheres of neural crest- derived cells enriched from bone marrow stromal cell subpopulation.Neurosci Lett.2013；532:70-5.], by CMC model, can from marrow response epidermal growth factor/fibroblastic growth BMSC (EGF/FGF2-responsive BMSC, the E/Fr-BMSC) subgroup of the factor raises the mesenchymal cell to suspension growth Ball (Mesensphere), i.e. residence NCC after the migration of marrow, it is critical that bone marrow neural crest cell (bone marrow Derived neural crest cell, BM-NCC) have self-renewal capacity and directed differentiation for neuron and spongiocyte Potential.
Summary of the invention
The purpose of the present invention is to provide a kind of BM-NCC clone N1 and BM-NCC clone N1 secondary culture method, Promote the application in nerve regneration drug in method from preparation to SC induction differentiation and BM-NCC clone N1.
The present invention is directed to how probe into from the derivative SCP of BM-NCC.Then, we isolate after obtaining rat BM-NCC Then multiple cell clones that can be frozen and recover are shown experimentally that BM-NCC clone can be induced to differentiate into S100 β sun Property SC, and SC purity derived from the cell of N1 therein clone is higher than other cell clones, without gene regulation or into one Step purifying, N1 cell-derived SC also have in vitro into myelin potential.
Testing result is also shown that, in protein level and mRNA level in-site, NCC mark is expressed in N1 cell height, and derivative SC expression is lowered；In derivative SC, SC mark related with SC differentiation state is then raised.We further use the condition of N1 cell Culture medium (N1-CM), i.e. cell culture supernatant processing oxygen sugar deprive the DRG neuron after (OGD) damage, the survival of neuron Growth with aixs cylinder is improved；In addition, N1 cell infusion is entered the rat sciatic nerve after crush injury by we, as a result show Show that the cell-derived SC (N1derived Schwann cell, N1-d-SC) of N1 cell ratio N1 has and preferably promotees nerve regneration With function repair, shows N1 cell in vivo and in vitro and can promote the reparation of injured neurons.Present invention demonstrates that N1 cell can It is identified as SCP identity (or undifferentiated SCP sample state), is expected to be applied to based on cell in the future derived from the SCP of BM-NCC Rush treatment of nerve regeneration.
The first aspect of the present invention provides a kind of rat marrow source neural crest progenitor cells N1, and deposit number is CCTCC NO: C201647。
Neural crest progenitor cells N1 in rat marrow source of the present invention, i.e. BM-NCC clone N1, have and apply Wang Shi progenitor cells (SCP) performance.
The present invention the results showed that N1 clone compared to simultaneously obtain other BM-NCC clone (such as N3, N4 and N13), there is the strongest potential broken up to SC, the SC ratio of the S100 β positive reaches 95% or more after Induction of committed differentiation, no Need subsequent cell sorting step；Meanwhile display have spongiocyte differentiation potential NCC mark CD44, HES1 and SOX10, mRNA expression are higher than others BM-NCC clone (such as N4) in N1 clone；And it shows latent with neuron differentiation The NCC of energy indicates that WNT1, ID2, p75NTR and CXCR4, mRNA expression are cloned then in N1 lower than BM-NCC grams other Grand (such as N4).
The second aspect of the present invention additionally provides the secondary culture method of BM-NCC clone N1.
The secondary culture method, be CCTCC NO:C201647 by deposit number cell recovery after, culture is to big Most cell balls increase and ball center's cell when starting obfuscation, are digested cell ball for single cell suspension with accutase enzyme, so Cell presses≤10 afterwards4The density of a/ml is inoculated into tissue culture plate, carries out balling-up secondary culture, and half amount is changed liquid 2 times weekly；Every 7 ~14d is passed on 1 time, or passage as needed.
The third aspect of the present invention additionally provides BM-NCC and clones N1 to the side of schwann's cell (SC) Induction of committed differentiation Method.
The Induction of committed differentiation method, be by deposit number be CCTCC NO:C201647 cell recovery after, culture To logarithmic growth phase, individual cells are separated into, and it is coated to be inoculated into Laminin lens/poly-D-lysine (laminin/PLL) It on culture plate, is incubated for and is cultivated with SC Induction of committed differentiation liquid, that is, contain 5% fetal calf serum (FBS), 35ng/ml all-trans retinoic acid (tRA), 5 μM of forskolin, 200ng/ml HRG-1 β, 10ng/ml FGF2,10ng/ml PDGF-AA and 1%N2 DMEM/F12 (v/v, 1:1) culture solution, every 2~3d are changed liquid 1 time.
The present invention provides a kind of method being simple and efficient, this induction differentiation scheme, which is cloned in 2d BM-NCC, to be started It works, no longer than 1 week.
The fourth aspect of the present invention additionally provides BM-NCC clone N1 in preparation and promotes the application in nerve regneration drug.
The application is specially the treatment based on cell, and BM-NCC clone N1 of the invention is to promote injured nerve again Raw research provides unlimited cell origin.
Treatment employing mode based on the cell can enter damaged tissues, be injected intravenously or be used for for locally injecting Prepare tissue engineering nerve graft row implant region etc..
The capsule that the drug can be secreted for cell suspension, the conditioned medium of cell, the factor of cell secretion, cell Steep vesica (excretion body etc.) extract etc. of (excretion body etc.) or cell secretion.
The specific technical solution of the present invention is as follows:
We have cultivated BM-NCC and separation obtains BM-NCC single cell clone, have detected the table of their NCC mark It reaches and self-renewal capacity.The efficiency broken up to SC is cloned according to each BM-NCC, we have selected N1 clone as further The cell subsets of research.N1 cell and embryo DRG neuron co-culture, with detect N1 cell at myelin potential, the results showed that N1 cell can derive SC and in vitro promote aixs cylinder at myelin.N1-CM is used to handle the DRG neuron of OGD damage, as a result table The bright survival for improving DRG neuron and axon growth situation.In addition, N1 cell or N1-d-SC are implanted into crush respectively Rat sciatic nerve, the results showed that can promote the regeneration of sciatic nerve and the recovery of function.
Existing research person analyzes the similar and difference of the MSC and NCC from adult mice marrow.It is logical that there are also researchers The effect for crossing Wnt1 and BMP2 is enriched to NCC single cell clone from adult BMSC, although experiment shows Nestin/SOX10/ The bone marrow cell of the p75NTR positive, it may be possible to there is the NCC to neuron and spongiocyte differentiation potential, but from different grams The percentage of GFAP or Tuj1 positive cell is different in the cell of grand differentiation, and some clones tend to derivative spongiocyte (GFAP positive cell), and other clones tend to derivative neuronal cell (Tuj1 positive cell).It is similar, the present invention In we separate and obtain multiple BM-NCC single cell clones, and it was found that the cell differentiations of 4 clones be the ratio of SC respectively not It is identical.The result shows that N1 clone has the strongest ability broken up to SC, the NCC of display SC differentiation potential indicates CD44, HES1 It is higher than others BM-NCC clone (such as N4) in N1 with the mRNA expression of SOX10, and shows the NCC of neuron differentiation potential Indicate that the mRNA expression of WNT1, ID2, p75NTR and CXCR4 are then lower than other BM-NCC clones (such as N4) in N1.
Break up for induction N1 cell to SC, has used laminin/PLL stromal surface and specific one group of cell factor.Point During change, we have the squamous epithelium like cell form [Lobsiger of of short duration presentation reported in the literature before not observing CS,Smith PM,Buchstaller J,Schweitzer B,Franklin RJ,Suter U,et al.SpL201:a conditionally immortalized Schwann cell precursor line that generates myelin.Glia.2001；36:31-47.][Xu Y,Xiong F,Liu L,Zhang C.Rat bone marrow stromal cells could be induced into Schwann cell precursor-like cells in vitro.Neurosci Lett.2011；488:229-33.].The difference of this form may be due to different inductive conditions With different cell origins.In contrast, the present invention in N1 cell to SC induction atomization in, 0h, 12h, for 24 hours and Gene expression pattern that 48h is detected respectively and existing document report be it is almost the same [Ziegler L, Grigoryan S, Yang IH,Thakor NV,Goldstein RS.Efficient generation of schwann cells from human embryonic stem cell-derived neurospheres.Stem Cell Rev.2011；7:394-403.] [Liu Z,Jin YQ,Chen L,Wang Y,Yang X,Cheng J,et al.Specific marker expression and cell state of Schwann cells during culture in vitro.PLoS One.2015；10: e0123278.][Finzsch M,Schreiner S,Kichko T,Reeh P,Tamm ER,Bosl MR,et al.Sox10is required for Schwann cell identity and progression beyond the immature Schwann cell stage.J Cell Biol.2010；189:701-12.].Absorbing to be, calcium is adhered Protein 19 (Cadherin 19) is used as the distinctive cell sign of SCP, as inverted U-shaped variation, In is presented to the differentiation of SC in N1 cell Induction 12h is apparently higher than 0h, is but significantly lower than 0h with 48h for 24 hours in induction.These time dependent variations, which can be compared to, simulates NCC Dynamic process [Jessen KR, Mirsky R, the Lloyd AC.Schwann Cells:Development broken up to mature SC and Role in Nerve Repair.Cold Spring Harb Perspect Biol.2015；7:a020487.] [Woodhoo A,Sommer L.Development of the Schwann cell lineage:from the neural crest to the myelinated nerve.Glia.2008；56:1481-90.][Takahashi M,Osumi N.Identification of a novel type II classical cadherin:rat cadherin19is expressed in the cranial ganglia and Schwann cell precursors during development.Dev Dyn.2005；232:200-8.], the results showed that the SCP feature of N1 cell may gradually weaken, and N1-d-SC may be as the extension of induction time be gradually from crudity to maturity state transition.
Existing research report, using Laminin/PLL matrix and the beta induced skin progenitor cell (skin of HRG1 Precursor, SKP) to after SC differentiation, SC then is purified with different means again, a kind of cylindrical drum can be used (cylinder) select SC colony, digested and collect secondary culture after cell [Biernaskie JA, McKenzie IA, Toma JG,Miller FD.Isolation of skin-derived precursors(SKPs)and differentiation and enrichment of their Schwann cell progeny.Nat Protoc.2006； 1:2803-12.], can also carry out based on p75NTR immunofluorescence dyeing the sorting of streaming living cells [Khuong HT, Kumar R,Senjaya F,Grochmal J,Ivanovic A,Shakhbazau A,et al.Skin derived precursor Schwann cells improve behavioral recovery for acute and delayed nerve repair.Exp Neurol.2014；254:168-79.].In contrast, it is simple and efficient the present invention provides a kind of Method, with the method, 4 clone's subgroups in the present invention can have 40% to 95% cell to be induced to differentiate into SC, wherein N1 clone has as many as 95%, and induction N1 cell differentiation does not need subsequent cell sorting step after being SC.Many existing researchs Report adult stem cell can be induced to differentiate into SC, for example, from marrow [Caddick J, Kingham PJ, Gardiner NJ, Wiberg M,Terenghi G.Phenotypic and functional characteristics of mesenchymal stem cells differentiated along a Schwann cell lineage.Glia.2006；54:840-9.] [Park HW,Lim MJ,Jung H,Lee SP,Paik KS,Chang MS.Human mesenchymal stem cell- derived Schwann cell-like cells exhibit neurotrophic effects,via distinct growth factor production,in a model of spinal cord injury.Glia.2010；58:1118- 32.] or Cord blood [Peng J, Wang Y, Zhang L, Zhao B, Zhao Z, Chen J, et al.Human umbilical cord Wharton's jelly-derived mesenchymal stem cells differentiate into a Schwann-cell phenotype and promote neurite outgrowth in vitro.Brain Res Bull.2011；84:235-43.][Lee JH,Chung WH,Kang EH,Chung DJ,Choi CB,Chang HS,et al.Schwann cell-like remyelination following transplantation of human umbilical cord blood(hUCB)-derived mesenchymal stem cells in dogs with acute spinal cord injury.J Neurol Sci.2011；300:86-96.] source MSC [Pan Y, Cai S.Current state of the development of mesenchymal stem cells into clinically applicable Schwann cell transplants.Mol Cell Biochem.2012；368:127-35.], adipose-derived stem cell [Kingham PJ,Kalbermatten DF,Mahay D,Armstrong SJ,Wiberg M,Terenghi G.Adipose- derived stem cells differentiate into a Schwann cell phenotype and promote neurite outgrowth in vitro.Exp Neurol.2007；207:267-74.][Kaewkhaw R,Scutt AM, Haycock JW.Anatomical site influences the differentiation of adipose-derived stem cells for Schwann-cell phenotype and function.Glia.2011；59:734-49.], epidermis NCSC [Sieber-Blum M, Grim M, Hu YF, Szeder V.Pluripotent neural crest stem cells in the adult hair follicle.Dev Dyn.2004；231:258-69.] and dental pulp NCSC [Al-Zer H,Apel C,Heiland M,Friedrich RE,Jung O,Kroeger N,et al.Enrichment and Schwann Cell Differentiation of Neural Crest-derived Dental Pulp Stem Cells.In Vivo.2015；29:319-26.] etc., however the ratio that SC is divided into these researchs is only below 40% or does not report differentiation Rate.This programme can not only obtain more SC, and differentiation speed also than having (>=1 week) reported in the literature fastly, obtains so efficient Differentiation may be attributed to derived from SC be derived from NCP clone rather than NCSC or MSC.Special one is mentioned that, the spy with SCP Sign N1 clone is beneficial to establish an external SC differentiated system, also, this system includes prematurity SC at myelin SC Differentiation.
It is the mark of SC function maturation at myelin, is different from other SC hypotypes, such as prematurity SC and non-at the SC of myelin At myelin SC.In the present invention, myelin SC, also, MBP and MAG sun are formd into N1 cell and DRG neuron co-culture system The sheath structure of property confirms that it has the function of typical SC.The result shows that SCP directed differentiation be at myelin SC, in addition to needing Also need soluble factor other than paracrine signal, and with the intimate contact of aixs cylinder be also helpful [Joseph NM, Mukouyama YS,Mosher JT,Jaegle M,Crone SA,Dormand EL,et al.Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells.Development.2004；131:5599-612.].
The BM-NCC of immortality selected in the present invention clones N1, and the identity characteristic with SCP can freeze and answer repeatedly Soviet Union, therefore a unlimited cell origin can be provided for cell therapy, it can especially promote the regeneration of nerve.In addition, these are cloned Cell also can avoid the adverse effect of Cell differentials between different batches in primary cell preparation.Existing scholar reports from rat embryonic Tire sciatic nerve takes out SC, then establishes what a condition relied on the EGFR/neu fusion protein that retrovirus encodes Immortality SCP cell line (SpL201) [Lobsiger CS, Smith PM, Buchstaller J, Schweitzer B, Franklin RJ,Suter U,et al.SpL201:a conditionally immortalized Schwann cell precursor line that generates myelin.Glia.2001；36:31-47.].The present invention is moved from adult rat BM-NCC separation obtains BM-NCC cloned cell line after shifting, overcomes ethics problem, and avoid neurotrosis and gene changes It makes, being also not required to rely on the EGF in environment as SpL201 can just be survived.In addition, there are also scholars to apply different regulations The factor, from NCSC cell line [Sviderskaya EV, Easty DJ, Lawrence after the birth that mouse skin establishes immortality MA,Sanchez DP,Negulyaev YA,Patel RH,et al.Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells.FASEB J.2009；23:3179-92.], but need Wnt, shh and TGF signal beta access, epidermis NCSC can be used as one kind of SC Cell origin [Sakaue M, Sieber-Blum M.Human epidermal neural crest stem cells as a source of Schwann cells.Development.2015；142:3188-97.].In contrast, present invention first time Report can obtain from adult rat marrow and establish the NCC system of immortality, and this NCC system can be used as the relevant cell of biology Source provides SCP and further derivative SC.
In the present invention, N1-CM can repair the DRG neuron of OGD damage in vitro, and sciatic nerve can be repaired by transplanting N1 in vivo It crushes, shows that N1 can play the therapeutic effect based on cell.Neurotrophic factor contained in N1-CM is in cell culture The survival of the DRG neuron of OGD damage and the growth of aixs cylinder are promoted, and N1 cell infusion enters the rat sciatic nerve crushed Afterwards, the repair risen is better than N1-d-SC.For preferably application cell transplantation treatment neurotrosis, the cell of injection exists Intracorporal survival condition is an importance in need of consideration, is in addition also required to consider to inject precellular differentiation state, from And it builds together good regeneration microenvironment [Walsh S, Midha R.Practical considerations with surrounding tissue concerning the use of stem cells for peripheral nerve repair.Neurosurg Focus.2009；26:E2.].
BM-NCC clone N1 of the invention is expected to be applied to the rush treatment of nerve regeneration based on cell.
Detailed description of the invention
Fig. 1 is the aspect graph (A) and immunocytochemical stain figure (B) of BMSC and derivative Mesensphere.
Fig. 2 is flow cytometer (FCM) analysis chart of E/Fr-BMSC and derivative Mesensphere.
Fig. 3 is BM-NCC single cell clone aspect graph (A) and growth curve (B) and growth curve chart (C).
Fig. 4 is mRNA expression of the MSC and NCC mark in N1, N4 and E/Fr-BMSC: expression is in N1 and N4 high Indicate (A) in the NCC of E/Fr-BMSC, expression indicates (B) in MSC of the N1 and N4 lower than E/Fr-BMSC, and expression exists The mark (C) of N1, N4 and E/Fr-BMSC no significant difference.
Fig. 5 is that BM-NCC clones differentiation to SC, and: BM-NCC clones N1 to the aspect graph (A and B) of SC differentiation 12h and 48h, The ratio of each BM-NCC clonal derivation SC (S100 β is positive) compares (C), and BM-NCC clones the immunocytochemistry of SC derived from N1 Colored graph (D).
Fig. 6 be SC and NCC mark in the derivative SC induction differentiation 0h, 12h of N1, for 24 hours with the mRNA expression of 48h: SC mark The expression of will changes (A), and the expression of NCC mark changes (B), and the expression of SCP mark changes (C), the table of SC and the shared mark of NCC Up to variation (D).
Fig. 7 is the detection figure of N1 cell and DRG neuron co culture system in vitro at myelin: co-culturing 0d, 1d, 14d and 21d Aspect graph and immunocytochemical stain figure (A) co-culture the myelin aspect graph (B) of 28d, and the myelin for co-culturing 28d is immune thin Born of the same parents' chemical staining figure (C), at myelin mark in the mRNA expression (D) for co-culturing 7d and 28d.
Fig. 8 is the DRG neuron β Tubulin III immunocytochemical stain figure that N1-CM repairs OGD damage in vitro.
Fig. 9 is the rat sciatic nerve form and function detection figure that cell infusion reparation crushes: after cell infusion in 3 weeks not Each experimental group sciatic nerve function index (SFI) with time point is compared (A), each experimental group composite muscle after cell infusion 3 weeks Action potential (CMAP) wave amplitude compares (B), the immunohistochemical staining figure of each experimental group regenerating nerve after cell infusion 3 weeks (C)。
Preservation information: cell line of the invention is named as rat marrow source neural crest progenitor cells N1, is preserved in Chinese Typical Representative Culture collection (abbreviation CCTCC), address: Wuhan, China university, preservation date on April 16th, 2016, deposit number CCTCC NO:C201647.
It is the 50th and 51 generation cells in the rat marrow source neural crest progenitor cells N1 of CCTCC preservation.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention will be described in detail.But the following example should not be regarded as to the present invention The limitation of range.
Experimental method in following embodiments is unless otherwise specified conventional method.
The culture and identification of 1. marrow neural crest cell (BM-NCC) of embodiment
The culture of BM-NCC cultivates using following duplicate two steps CMC model program and obtains BM-NCC:
The first step flushes out marrow from shin bone and femur immediately, by bone after adult Wistar rat (8 week old) is condemned to death Myelocyte inoculated and cultured is attached the characteristic of plastic culture dish growth using BMSC, changed repeatedly in the IMDM culture solution containing 10%FBS The candidate stem cell of liquid removal floating, is separately cultured out Primary rat BMSC；
Second step, with contain 20ng/ml EGF, 20ng/ml FGF2,1%N2 (v/v), 2%B27 (v/v)), 2mM paddy ammonia Amide, 100U/ml penicillin, DMEM/F12 (v/v, 1:1) balling-up culture solution of 0.1mg/ml streptomysin carry out primary BMSC Balling-up culture, inoculating cell density are 2~3 × 105A/ml is formed primary cell ball (Sphere).Balling-up culture solution is namely The culture solution of NCC.
Then, the first step is repeated, that is, collecting the primary cell ball of suspension, inoculated and cultured is trained in the IMDM containing 10%FBS again Nutrient solution attaches the characteristic of plastic culture dish growth using BMSC, obtains E/Fr-BMSC；
Repeat second step, with DMEM/F12 balling-up culture solution, balling-up culture carried out to E/Fr-BMSC subgroup, formation it is thin Born of the same parents' ball is also referred to as mesenchymal cell ball (Mesensphere).
Cell or cell ball phase contrast microscope in the above incubation are observed and acquire picture；Use immunocytochemistry (ICC) colouring method detects MSC and NCC marker protein；With the expression of flow cytometer (FCM) analysis cell sign albumen.
Primary BMSC and E/Fr-BMSC shows the fibroblast-like cells form of adherent growth；Sphere derived from BMSC and Mesensphere suspension balling-up derived from E/Fr-BMSC grows (Figure 1A).
Separation obtains the process of Mesensphere: (1) separating primary BMSC cell ball (a2) from primary BMSC (a1)；(2) It is derivative E/Fr-BMSC (a3) from primary BMSC cell ball (a2)；(3) Mesensphere (a4) is separated from E/Fr-BMSC (a3). ICC coloration result display E/Fr-BMSC expression MSC marker protein CD90, and Mesensphere expression NCC marker protein CD29, CD133 and p75NTR.E/Fr-BMSC and Mesensphere expresses stem-cell marker albumen Vimentin and Nestin (figure 1B)。
FCM analysis is the results show that E/Fr-BMSC high expresses MSC marker protein CD90 and CD73, and Mesensphere high Express NCC marker protein CD133.High expression CD44, CD29 and Nestin (Fig. 2) of E/Fr-BMSC and Mesensphere.
The separation of embodiment 2.BM-NCC clone and proliferation and cell sign detect
BM-NCC, which is obtained, using limiting dilution assay culture clones [Glejzer A, Laudet E, Leprince P, Hennuy B,Poulet C,Shakhova O,et al.Wnt1and BMP2:two factors recruiting multipotent neural crest progenitors isolated from adult bone marrow.Cell Mol Life Sci.2011；68:2101-14.].Cell ball secondary culture to the 4th it is more than generation after, with accutase enzyme by cell ball digestion be Cell is inoculated into 96 porocyte culture plates by the density in 0.7/hole and carries out unicellular balling-up culture (often by individual cells suspension Hole cell number is 0,1 or 2 etc. at random), half amount is changed liquid 2 times weekly.The micro- every hole cell of sem observation, when individual cells increase When growing the cell number contained by the individual cells ball to be formed and reaching 100 or more, just this cell ball is disappeared with accutase enzyme Single cell suspension is turned to, the balling-up culture passed on is further continued for.Single cell suspension is with≤5 × 104The cell density of a/ml connects Kind, every 7~14d is passed on 1 time, or passage as needed.
Obtained cell clone subgroup routine passage, cell clone N1, N3, N4 and N13 Long-term Proliferation therein, to draw Make the Long-term Proliferation curve of above-mentioned 4 cell clones, each cell clone every generation (P) accumulative total number of cells with following Formula, which calculates, to be obtained: Cn=Cn-1×Tn/Sn, CnIn=the n-th generation, added up total number of cells, Cn-1In=the (n-1)th generation, added up total number of cells, Tn= In n-th generation, added up total number of cells, Sn=the n-th pickup kind cell number.
To detect vitro growth rates, N1 the and N4 clone cell for being passaged to P31, P32 and P33 is seeded in 6 holes respectively In the 2ml culture solution of culture plate, inoculum density is 1 × 104A/ml.Incubation time is collected cell 1 time and is counted thin every for 24 hours Born of the same parents' sum, co-cultures 10d, draws out growth curve with the average cell number that 10 time points count.
The results show that BM-NCC single cell clone subgroup N1, N3, N4 and N13 (Fig. 3 A) of 4 Long-term Proliferations are obtained, and Continuous passage at least 8 generations in vitro, N1 and N4 passage are more than 37 generations up to 16 months or more (Fig. 3 B).In addition, P31, P32 and P33 The 10d growth curve (Fig. 3 C) of the N1 and N4 cell in generation shows that single cell clone N1 and N4 more than generation is also still protected in passage to 30 Hold good self-renewal capacity.
Quantitative real-time PCR (qRT-PCR) has statistical difference as the result is shown: NCC, which indicates, expresses water in the mRNA of N1 and N4 Averagely be apparently higher than the mRNA expression in E/Fr-BMSCs, including Nestin, Musashi1, CD133, TFAP2 α, HNK1, CD49d, WNT1, ID2, p75NTR, CD44, HES1 and SOX10 (Fig. 4 A)；However, MSC mark expresses water in the mRNA of N1 and N4 The average mRNA expression significantly lower than in E/Fr-BMSC, including CD90, CD73, Vimentin, N-Cadherin and CXCR4 (Fig. 4 B).In addition, the mRNA expression of CD29, SOX2 and Snail in N1 or N4 is similar to E/Fr-BMSCs's MRNA expression (Fig. 4 C).The experimental results showed that N1 and N4 cell clone maintains BM-NCC identity.
Induction of committed differentiation of the embodiment 3.BM-NCC to schwann's cell (SC)
Break up using specific differentiation liquid induction BM-NCC monoclonal cell (N1, N3, N4 and N13) to SC.
Separate each cell clone for obtaining P5~P7 for when be separated into individual cells, and be inoculated into PLL/laminin On coated roundlet slide, with containing 5%FBS, 35ng/ml tRA, 5 μM of forskolin, 200ng/ml HRG-1 β, 10ng/ml DMEM/F12 (v/v, 1:1) culture solution (i.e. SC Induction of committed differentiation liquid) of FGF2,10ng/ml PDGF-AA and 1%N2 are incubated for Culture, every 3d are changed liquid 1 time.
After 12h is broken up in induction, adherent cell starts to extend N1 cell, and cellular morphology becomes spindle (figure from circle 5A)；After 48h, sample arrangement (Fig. 5 B) shoulder to shoulder is presented in fusiform cell；After 60h, cell gradually aging and death.Selection induction The ICC that the cell of 48h carries out S100 β is dyed to confirm differentiation of the BM-NCC monoclonal cell to SC, and counts S100 β positive table The cell percentages reached compare the differentiation efficiency of this 4 BM-NCC monoclonals of N1, N3, N4 and N13, there is statistics as the result is shown Difference, N1 clone's directed differentiation are that the ratio of the SC of the S100 β positive is apparently higher than other 3 BM-NCC clones (Fig. 5 C).In addition, ICC coloration result also shows N1-d-SC coexpression GFAP, CNPase and p75NTR (Fig. 5 D) of the S100 β positive.
QRT-PCR analyzes the not isogeneous induction divergaence time point (0h, 12h, for 24 hours and 48h) in 48h, and SC and NCC mark exist The mRNA expression of N1-d-SC changes.Have statistical difference as the result is shown: SC mark, including S100 β, GFAP, CNPase, O4 and ErbB3 gradually increases (Fig. 6 A) with the extension expression of induction time；And NCC indicates, including CD44, CD29, CD49d, CXCR4, WNT1, Nestin, Musashi1, CD133, HNK1, SOX2, ID2 and HES1, then prolonging with induction time Long mRNA expression gradually decreases (Fig. 6 B)；In addition, SCP indicates Cadherin19 in the mRNA expression ratio of induction 12h Significantly increase in 0h, but be substantially reduced for 24 hours with 48h ratio in 0h in induction, it is notable that is significantly higher than in induction 12h Induction for 24 hours with 48h (Fig. 6 C)；In addition, shared mark of the SOX10 and p75NTR as SC and NCC, p75NTR is with induction time Extension mRNA expression gradually increase, SOX10 is then that mRNA expression significantly increases and protects always after inducing 12h High level is held to 48h (Fig. 6 D).
The culture of embodiment 4.DRG neuron and DRG neuron and N1 cell co-culture to form myelin
(1) originally culture of DRG neuron [Shea GK, Tsui AY, Chan YS, Shum as described in existing document DK.Bone marrow-derived Schwann cells achieve fate commitment--a prerequisite for remyelination therapy.Exp Neurol.2010；224:448-58.][Gu Y,Wang J,Ding F,Hu N,Wang Y,Gu X.Neurotrophic actions of bone marrow stromal cells on primary culture of dorsal root ganglion tissues and neurons.J Mol Neurosci.2010；40: 332-41.]。
DRG is got from fetal rat (embryo 14.5~15.5) or neonate rat backbone back two sides vertebral foramen, is successively answered It after digesting 30 minutes respectively with 3mg/ml clostridiopetidase A and 0.25% pancreatin, inhales and abandons enzyme solution, the DMEM culture solution containing 5%FBS is added, Gently mechanical piping and druming to be to obtain single cell suspension, then using differential attachment method remove adherent faster Deiter's cells and at Fibrocyte reapplies the neuronal cultured solution processing containing 10 μM of fluorodeoxyuridines of anti-mitosis medicine and 10 μM of uridines, Inhibit the growth of non-neuronal cell neuron is further purified.Cell is with 5 × 104A/cm2Density be inoculated in PLL coating Culture dish, the every hole of 24 orifice plates about 1 × 105A neuron, neuronal cultured solution be containing 1%B27 (v/v), 50ng/ml NGF and The Neurobasal culture solution of the glutamine of 2mM, every 2~3d change liquid 1 time, at least culture 7d.
When cell forms fine and close neural process network, fetal rat DRG neuron at myelin for testing.It is big after birth Mouse DRG neuron is used for neure damage model experiment, sees embodiment 5.
(2) DRG neuron and N1 cell co-culture to form myelin
[Shea GK, Tsui AY, Chan YS, Shum are carried out referring to having document report at myelin experiment in vitro DK.Bone marrow-derived Schwann cells achieve fate commitment--a prerequisite for remyelination therapy.Exp Neurol.2010；224:448-58.].
After N1 cell inoculation is cultivated 6h in the coated culture dish of PLL/laminin with NCC culture solution, it is changed to SC orientation Break up liquid and be incubated for 12h, the N1 cell after induction is divided into the short spindle cell of stretching, extension by round cell, and the collection of N1 cell is followed by In the network that the DRG neuron neural process in kind to the above-mentioned every hole of 24 orifice plate is formed, every hole inoculation about 5 × 104A N1 cell.Training altogether The condition of supporting are as follows: DMEM/F12 the and Neurobasal culture medium (1:1, v/v) without glutamine adds whole SC inductions point Change the factor and whole DRG Neuronal Growth Factors (seeing above), every 2~3d half, which is measured, to be changed liquid 1 time.
After co-culturing 1 week, it is spindle shape SC that it is cell-derived that N1, which can be observed, and is arranged along the aixs cylinder of neuron, then Start to add L-AA (50 μ g/ml)) and glucose (4mg/ml), tRA, PDGF-AA, FGF2 and HRG-1 β are removed, with Stimulate the formation of aixs cylinder myelin.Co-cultivation continues to 3 weeks, and every 2~3d is changed liquid 1 time.
DRG neuron with N1 cell as described above the step of co-culture.N1-d-SC is kept in contact with DRG neuron, The bipolar morphology that elongation is gradually presented, after co-culturing 14d and 21d, many cells initially form the myelin of DRG neuron, 21d Shi Gengjia is significant (Fig. 7 A)；After co-culturing 28d, the morphosis (figure of ring sample is presented in the myelin that DRG neuron axon is formed 7B)；ICC dyeing display myelin ring spline structure coexpression is at myelin marker protein MBP and MAG (Fig. 7 C)；QRT-PCR is further Analysis shows that there is statistical difference, MBP, MAG, P0, PMP22, PLP and Krox20 are bright in the mRNA expression for co-culturing 28d It is aobvious to be higher than when co-culturing 7d (Fig. 7 D).
Embodiment 5.N1-CM repairs the DRG neuron of OGD damage in vitro
With the N1 cell of Neurobasal culture medium culture logarithmic growth phase, growth factor is not added, cell density is 2 × 105A/ml collects culture medium, i.e. N1-CM afterwards for 24 hours, is stored in -20 DEG C.
With the neonate rat DRG neuron of sugar-free Neurobasal culture medium culture purified, condition of culture is 37 DEG C, 5% CO2Concentration and 0.1%O2The damage of concentration, i.e. OGD.Liquid processing is changed with N1-CM after 8h, condition of culture is 37 DEG C, 5%CO2Concentration and 21%O2Concentration is cultivated for 24 hours, i.e. N1-CM repair process after OGD damage.
ICC dyeing detection N1-CM repairs the expression of the OGD damage DRG neuron β Tubulin III of front and back.As a result it shows Show, with conventional medium processing DRG neuron compared with, N1-CM processing DRG neuron survival and Neurite Outgrowth this Two aspects make moderate progress (Fig. 8).
N1 cell is transplanted after 6. sciatic nerve crush of embodiment or N1-d-SC carries out cell therapy
(1) cell transplantation is performed the operation after sciatic nerve crush
Be injected intraperitoneally combined anesthesia medicine (10mg/kg xylazine, 95mg/kg ketamine, 0.7mg/kg acepromazine) with Anesthetized rat；Side is performed an operation notch in the middle part of hind leg thigh, exposure left sciatic；Sciatic nerve separate nervus tibialis and The position proximal end of nervus peroneus communis, to stop blooding clamp injury sciatic nerve 3 times, for 10 seconds every time, every minor tick 10 seconds is careful not to Block the blood circulation of surrounding tissue；It is through with No. 9/0 silk thread of 1 nonabsorable horizontal in being carried out on the muscle for crush position Label；It is given immediately after in the far-end for crushing position along the parallel inserting needle of nerve and crushes nerve injection 1 × 106A N1 cell or N1-d-SC (induction N1 breaks up 48h)；Operative incision is finally sutured.
Rat is divided into saline injection (NS) group, N1 cell transplantation group, N1-d-SC cell transplantation group, and every group 10.
(2) the functional rehabilitation situation of foot printing test test rat
Carry out rat foot printing test with 8d, 12d, 16d and 20d after operation before surgery respectively, method is with reference to existing document [Yuan Y,Shen H,Yao J,Hu N,Ding F,Gu X.The protective effects of Achyranthes bidentata polypeptides in an experimental model of mouse sciatic nerve crush injury.Brain Res Bull.2010；81:25-32.].Be summarized as follows text: the rat biped rear solid end palm dips in red stamp-pad ink, walks The gallery (wide 15cm, high 15cm, long 80cm) for being covered with blank sheet of paper rice paper is crossed, red footprint is left.It checks and chooses clearly Footprint, and measure normal and art parapodum toe width (normal toe spread, NTS；experimental toe Spread, ETS), footmark length (normal print length, NPL；Experimental print length, EPL), Intermediate toes width (normal intermediary toe spread, NIT；experimental intermediary toe Spread, EIT), sciatic nerve function index (the sciatic function of every rat is finally calculated according to formula Index, SFI), SFI indicates that nervous function is normal close to 0, and SFI is damaged completely close to -100 expression nervous functions.Bain etc. gives Formula out is as follows:
SFI=-38.3 [(EPL-NPL)/NPL]+109.5 [(ETS-NTS)/NTS]+13.3 [(EIT-NIT)/NIT]- 8.8
In foot printing test, SFI value represents walk activity, is used to refer to rat hindlimb after crushing sciatic nerve and feels and transport Dynamic recovery situation.Operation consent SFI value is normal value 0, and SFI value sharply falls to -100 after operation, then as sciatic nerve Regeneration gradually rise.That compares NS processing group, N1-d-SC processing group and N1 cell processing group has statistics poor as the result is shown Different: 20d is apparently higher than NS processing group to the SFI value of N1-d-SC processing group after surgery；The SFI value of N1 cell processing group is being performed the operation 12d, 16d and 20d are apparently higher than NS processing group afterwards；In addition, 20d is apparently higher than N1- to the SFI value of N1 cell processing group after surgery D-SC processing group (Fig. 9 A).
(3) electro physiology detection compound muscle action potential (CMAP)
The electro physiology detection of CMAP is carried out within 3 weeks after surgery.Left sciatic is exposed after anesthetized rat again, in ischium After nerve cord proximal part gives electro photoluminescence, ipsilateral gastrocnemius abdomen record CMAP, using the CMAP of the non-operated hind limb in opposite side as Normal control, to inject NS group as negative control.Testing result shows statistical difference, the CMAP width of N1 cell processing group Degree peak value is apparently higher than N1-d-SC processing group；Meanwhile the recovery of two kinds of cell processing group electrophysiological functions is substantially better than NS processing Group (Fig. 9 B).
(4) immunohistochemistry (IHC) of sciatic nerve dyes detection
The regenerated sciatic nerve of each group is taken out after performing the operation 3 weeks, the non-surgical denervation in opposite side is as normal control.Neural sample into It is fixed after row, nerves transected face frozen section is prepared, IHC detects the expression of S100 β and NF200.The results show that rat ischium mind After crushing through cell infusion handle and regenerated nervous fibers, the aixs cylinder of positive expression NF200 are surrounded by positive expression The myelin of S100 β；Also, the axon diameter and Myelin thickness of N1 cell processing composition myelin are all larger than N1-d-SC processing group；Together When, the regenerating nerve axon diameter and Myelin thickness of two kinds of cell processing groups are all larger than NS processing group (Fig. 9 C).
Conclusion:
BM-NCC is a kind of cell origin for being easy to obtain from myeloid tissue after migration.It, can since the height of BMSC is heterogeneous Separation obtains multiple BM-NCC monoclonal cell subgroups, and wherein N1 cell clone can most effectively derive a large amount of high-purity SC, And it does not need genetic modification or is further purified.The major progress of this research is after can deriving the high SC of homogenieity from BM-NCC Generation, and being expressed according to Cadherin19 in the height of N1 cell, differentiation capability from N1 cell to SC, N1 cell at myelin ability, And repair of the N1 cell to injured nerve, it was demonstrated that N1 cell clone has SCP (or undifferentiated SCP sample state) identity Inference.In conclusion being expected in the future derived from the SCP (BM-NCC clones N1) of BM-NCC applied to the rush nerve based on cell Regenerative therapy.
" BM-NCC " comes from mammal in the present invention, including but is not limited to rat, mouse, rabbit, cat, dog, monkey, people.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this The principle of invention, various changes and improvements may be made to the invention without departing from the spirit and scope of the present invention, these changes Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its Equivalent defines.

Claims (4)

1. a kind of rat marrow source neural crest progenitor cells N1, deposit number is CCTCC NO:C201647.
2. neural crest progenitor cells N1 in rat marrow source according to claim 1, which is characterized in that it has, and to apply Wang Shi ancestral thin The performance of born of the same parents.
3. the secondary culture method of neural crest progenitor cells N1 in rat marrow source as described in claim 1, which is characterized in that described Secondary culture method it is as follows:
After the cell recovery for being CCTCC NO:C201647 by deposit number, culture to most cells ball increases and ball center's cell When starting obfuscation, cell ball digest for single cell suspension with accutase enzyme, then cell is by≤104The density of a/ml connects Kind arrives tissue culture plate, carries out balling-up secondary culture, and half amount is changed liquid 2 times weekly；Passage as needed.
4. rat marrow source neural crest progenitor cells N1 as described in claim 1 is to the side of schwann's cell Induction of committed differentiation Method, which is characterized in that the Induction of committed differentiation method is as follows:
After the cell recovery for being CCTCC NO:C201647 by deposit number, culture to logarithmic growth phase is separated into individual cells, and It is inoculated on the coated culture plate of Laminin lens/poly-D-lysine, is incubated for and is cultivated with schwann's cell Induction of committed differentiation liquid, Every 2~3d is changed liquid 1 time；
The schwann's cell Induction of committed differentiation liquid are as follows: contain 5% fetal calf serum, 35ng/ml all-trans retinoic acid, 5 μM Forskolin, 200ng/ml HRG-1 β, 10ng/ml FGF2, the 10ng/ml PDGF-AA and 1%N2 that volume ratio is 1:1 DMEM/F12 culture solution.
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