CN1330142A - Method for exosomatic separation and amplification of filled stem cells between human marrow and funic blood and directional induction toward nerve cells - Google Patents

Abstract

The percoll with 1.073 of specific weight and the lymphocyte separating liquid with 1.077 of specific weight are respectively used to separate single nuclear cell from marrow and funicle blood. The Mesencult (Stem Cell Co) culture medium is used to purify and amplify the mesenchyme stem cells. The strong antioxidizing agent with proper concentration is used for exosomatic induction of said mesenchyme stem cells for directional differentiation toward nerve cells.

Description

People's marrow and amplification of Cord blood mescenchymal stem cell in-vitro separation and neuralward cell directional inductive method

The present invention relates to people's marrow and Cord blood mescenchymal stem cell (mesenchymal stem cells, in-vitro separation amplification MSCs) and the directional induction differentiation of neuralward cell.

Along with organizational project and engineered rise and development, the research of stem cell becomes one of focus of 21 century.In the individual genesis and development process of humans and animals, in embryo and adult tissue, all exist stem cell with height updating ability and multidirectional differentiation potential.But stem cell in-vitro separation, amplification and freezing preservation, and can be induced to differentiate into various kinds of cell and tissue under suitable condition, this provides the ideal model system for inquiring into developmental biology problems such as humans and animals embryo generation, histocyte differentiation, gene expression regulation, has also opened up new way for the cell replacement treatment and the gene therapy (goal gene imports stem cell) of clinical tissue defective disease and heredopathia simultaneously.At present, stem cell tentatively is divided into embryonic stem cell and tissue stem cell two big classes.Though embryonic stem cell has more totipotency, people's embryonic stem cell is studied owing to be subjected to ethics and the restriction of the factors such as difficulty of drawing materials, study less.And tissue stem cell is present in fetus and the various histoorgans of adult, it is extensive relatively to originate, and the further investigation of tissue stem cell do not relate to ethics problem, and infusion is given patient after taking from patient's tissue stem cell directional induction differentiation, does not have immunological rejection.Therefore, tissue stem cell is the main research object of a class of Stem Cell Engineering.

MSCs is the tissue stem cell that the class that receives much attention at present has multidirectional differentiation potential.The bone marrow MSCs of vitro culture has been widely used in research field.Also there is MSCs in report in the Cord blood recently.Cord blood MSCs and bone marrow MSCs are similar, express kinds of surface sign such as SH3, SH4, CD29, CD90 etc.; But do not express the surface marker of hemopoietic stem cell system, as lipopolysaccharides acceptor CD14, CD34 and leukocyte surface antigens c D45 etc.This group cell characteristics is stable, still has multidirectional differentiation potential after continuous passage cultivation and the freezing preservation.Experiment shows that under external specific inductive condition, bone marrow MSCs can be divided into various kinds of cell such as bone, cartilage, fat, tendon, muscle; Cord blood MSCs also has the ability to bone and adipocyte differentiation.This carries out cell therapy for organizational project provides new seed cell, and very big potential applicability in clinical practice is arranged.

The in-vitro separation, purifying, amplification and the directed differentiation that the objective of the invention is to explore marrow and Cord blood MSCs are the condition of neurocyte, provide more competent theoretical foundation and technological method for marrow and Cord blood MSCs are used for the damaging and heredity sacred disease of clinical treatment.

With embodiment the present invention is further elaborated down.

Separation, purifying and the amplification cultivation of embodiment 1. bone marrow MSCs

One, material and method

Aseptic condition is gathered people's marrow of non-disease in the blood system down, with the α that contains 15% foetal calf serum (Hyclone)-5 times of dilutions of MEM nutrient solution, the centrifugal 5min of 400g abandons supernatant and lipid layer, adds α-MEM nutrient solution that 5mL contains 15% foetal calf serum, make cell suspension, the proportion that is added to gently is that the centrifugal 20min of 400g gets interfacial layer on 1.073 the Percoll parting liquid, add 5ml PBS and make single cell suspension, centrifuge washing. with 2.0 * 10 ⁵/ cm ²The density of (T-25 culturing bottle) is inoculated in the Mesencult nutrient solution (StemCell Co.), places 37 ℃, 5%CO ₂With cultivate in the incubator of saturated humidity. after cultivating 3d, change substratum, discard not attached cell.When cell of the liquid length to 80% of changing later every 3d merges with 1: 1 0.25% trypsinase and 0.02%EDTA mixed solution had digestive transfer culture (controlling digestion time), with 8.0 * 10 at microscopically ³/ cm ²Density be inoculated in the culturing bottle that goes down to posterity (T-25) and carry out amplification cultivation.

Two, result

MSCs is the spindle shape than homogeneous, and three all left and right sides cell growths reach 80%～90% merges, and each clone is hundreds of approximately to thousands of cells.These passages are cultivated, and the MSCs after going down to posterity reaches fusion about one week, every bottle of average (5.5 ± 0.17) * 10 that obtain, MSCs digestion back that individual layer merges ⁵Individual cell.5.0 * 10 ⁵Individual former generation adult bone marrow MSCs obtains 7.5 * 10 at amplification in vitro after 15 generations ¹²Individual cell increases about 1.36 * 10 ⁷Doubly.

Separation, purifying and the amplification cultivation of embodiment 2. Cord blood MSCs

One, material and method

Aseptic condition is gathered down the human cord blood 50-100mL of the non-disease in the blood system of anticoagulant heparin, with the PBS of 0.01M PH7.4 by 1: 1 mixing, by 4: 1 and 0.5% methylcellulose gum mixing, leave standstill the 30min sedimented red cell again.Suct and clear the heart, abandon supernatant, make single cell suspension with PBS, on the lymphocyte separation medium of the proportion 1.077 that is added to, the centrifugal 20min of 400g gets interfacial layer, adds PBS and makes single cell suspension, and centrifuge washing is with 1.0 * 10 ⁶The density of/cm2 (T-25 culturing bottle) is inoculated in the Mesencult nutrient solution (Stem Cell Co.), places 37 ℃, 5%CO ₂With cultivate in the incubator of saturated humidity.After one week, changing substratum, discard not attached cell. later every 3-4d changes liquid once.When cell length to 80% merges with 1: 1 0.25% trypsinase and 0.02%EDTA mixed solution had digestive transfer culture (controlling digestion time), with 8.0 * 10 at microscopically ³/ cm ²Density be inoculated in the culturing bottle that goes down to posterity (T-25) and carry out amplification cultivation.

Two, result

The cultured umbilical blood mononuclear cell, full dose is changed liquid after the week, removes not attached cell, and the umbilical cord blood sample mononuclearcell that has is cultivated the back and broken bone like cell occurred, mescenchymal stem cell occurs after the umbilical cord blood sample mononuclearcell that the has cultivation.Broken bone like cell cell space is bigger, rounded, a plurality of nuclears.The mescenchymal stem cell cell space is fusiformis, be dispersed in existence at first, the back formation tens of two weeks is to the clones of a hundreds of cell, rapid propagation along with cell, the back cell growth of three weeks reaches 80%～90% merges, and the about hundreds of of each clone is to several thousand cells, and the MSCs of this moment is the spindle shape than homogeneous, form is similar to bone marrow MSCs, but slightly little than bone marrow MSCs.Every bottle of average (8.25 ± 0.49) * 10 that obtain, Cord blood MSCs digestion back that individual layer merges ⁵Individual MSCs cultivates these passages 6.6 * 10 ⁵The former generation MSCs of individual Cord blood obtains 9.9 * 10 at amplification in vitro after 10 generations ⁸Individual cell increases about 1.5 * 10 ³Doubly.

The surface antigen characteristic of embodiment 3. marrow and Cord blood MSCs

One, method

Get amplification monobasic marrow and Cord blood MSCs respectively, remove nutrient solution, PBS washes twice, and with 1: 1 0.25% trypsinase and the digestion of 0.02%EDTA mixed solution, making concentration respectively after washing with the PBS that contains 2%BSA was 1.0 * 10 ⁶The single cell suspension of/mL.The bone marrow MSCs single cell suspension adds 3 Eppendof pipe respectively, and each Eppendof pipe adds 500 μ l, manages negative contrast, adds 5 μ lIgG1-FITC and the 5 μ l IgG1-PE monoclonal antibodies of anti-mouse for No. 1; No. 2 pipes add anti-people's 5 μ lCDlla-FITC and 5 μ l CD29-PE monoclonal antibodies; No. 3 pipes add anti-people's 5 μ l CD71-FITC and 5 μ l CD34-PE monoclonal antibodies.Cord blood MSCs single cell suspension adds 6 Eppendof pipe respectively, manages negative contrast, adds 5 μ lIgG1-FITC and the 5 μ l IgG1-PE monoclonal antibodies of anti-mouse respectively for No. 1 and No. 2; All the other four pipes add anti-people's 5 μ l CD71-FITC, 5 μ l CD34-PE, 5 μ lCD11a-FITC, 5 μ l CD29-PE monoclonal antibodies respectively.Incubated at room 20min, flow cytometer detects.

Two, result

Detect discovery through flow cytometer, amplification monobasic marrow and Cord blood MSCs do not express CD34, CD11a, strongly expressed CD29, weak expression CD71.CD34 is the specific surfaces sign of hemopoietic stem cell, and CD11a (LFA-1 α chain) is the lymphocyte sign, and CD29 is the member of integrin family, and CD71 is a TfR.This shows that MSCs is the non-directional ancestral cells that a group that is different from hematopoietic cell in marrow and the Cord blood is in undifferentiated state.

Inducing of the neuralward cell of embodiment 4. marrow and Cord blood MSCs broken up and evaluation

One, method

Get the bone marrow MSCs and 2,5 of amplification in vitro after 2,6,10 generations, the Cord blood MSCs after 8 generations respectively, with 8.0 * 10 ³/ cm ²Concentration is inoculated in six orifice plates that are placed with the disinfection cap slide, the preparation cell climbing sheet, every hole adds 2ml Mesencult nutrient solution, when reaching the 60-70% fusion, change the DMEM nutrient solution that contains 20% foetal calf serum (Hyclone) and 3 μ mol/L beta-mercaptoethanols into and induce 24h in advance, PBS (PH7.4) washing then changes the DMEM nutrient solution that contains 2%DMSO and 200 μ mol/L fourth hydroxyanisols (BHA) into and induces.Begin after half an hour to examine under a microscope, observe once every half an hour later on, do not have considerable change up to cellular form.

For marrow and Cord blood MSCs, get the cell climbing sheet of inducing behind 2h, 4h, 6h, the 12h respectively, with reference to Hi stostain ^TM-SP (chain enzyme avidin-peroxidase) test kit (Beijing Zhong Shan biotech company) working method is advanced immunohistochemical assay, detect neurofilament protein (neurofilament, NF) and neuronspecific enolase (neuron-specific enolase, NSE).Get the cell climbing sheet of inducing back 6h, 8h simultaneously respectively, use Toluidine blue staining, observe Nissl body.

Two, result

Inducing back 12h to find that original big and flat MSCs cell space shrinks in advance, the irregularity that cell edges becomes has many thin digitations.Induce in advance when finishing, a part of cell cell space has become sub-circular.Tangible morphological change takes place in the 3h inner cell after formally inducing, and the cell cell space further shrinks, and forms circle, the taper of irregularity, trilateral, and the cell that has has a plurality of projections, and sends branch, and whole end has the termination of similar neuronal cell; The projection that has is extended gradually, forms coniform end eventually, and it is prominent to be similar to the neuronic major axis of Golgi I type.Considerable change substantially no longer takes place cellular form behind the 5h, observe to find: bone marrow MSCs has and presents typical neuron phenotype more than 80%, and Cord blood MSCs has about 70% to present typical neuron phenotype.

Immunohistochemical methods is the result show, marrow and Cord blood MSCs induce the back different time all to have NSE and NF to express, and see Table 1.Positive cell is brown.It is painted that the NSE positive cell shows as the diffusivity endochylema, and NF all has expression in nuclear week and projection.

Table 1 marrow and Cord blood MSCs induce the NSE of back different time and NF to express

2h 4h 6h 12hNSE ++ ++ ++ ++ NF ++ ++++++++ +++be dyeing strong positive (dark brown brown), ++ be stained positive (brown)

Toluidine blue staining finds, induces in the kytoplasm of the neuron cell behind 6h and the 8h and exists navy blue bulk or granular Nissl body.

This shows the potential that marrow and Cord blood MSCs all have the neuralward cytodifferentiation.

Claims (2)

1. the method for in-vitro separation, amplification and purifying marrow and Cord blood mescenchymal stem cell, it is characterized in that utilizing the lymphocyte separation medium of the percoll of proportion 1.073 and proportion 1.077 to separate mononuclearcell in marrow and the Cord blood respectively, use Mesencult then ^TM(Stem Cell Co.) substratum purifying amplification of mesenchymal stem cells.

2. the external neuralward cell directional of mescenchymal stem cell that separation amplification method as claimed in claim 1 obtains is induced, it is characterized in that adding in the pre-inducing culture 3 μ mol/L beta-mercaptoethanols, add 2%DMS0 and 200 μ mol/L fourth hydroxyanisols (BHA) in the formal inducing culture.