CN104419660A - Method for preparing autologous hematopoietic stem cells, kit, the stem cells and application - Google Patents
Method for preparing autologous hematopoietic stem cells, kit, the stem cells and application Download PDFInfo
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Abstract
The invention provides a method for preparing autologous hematopoietic stem cells. The method includes following steps of cultivating somatic cells in a cell culture fluid for 3-6 days and collecting the generated autologous hematopoietic stem cells. The cell culture fluid is an RPMI1640 culture fluid containing: 1-100 [mu]M of Y-27632 (C14H21N3O.2HCl), 1-100 ng/ml of stem cell factors, 1-100 ng/ml of interleukin 3, 1-100 ng/ml of interleukin 6, 1-100 ng/ml of interleukin 11, 1-100 ng/ml of a macrophage colony stimulating factor (M-CSF), 1-100 ng/ml of a granulocyte colony-stimulating factor (G-CSF), 1-100 [mu]g/ml of fucoidin, 1-100 [mu]g/ml of dextran sulfate, 1-100 nM of AMD3100 (1,1'-[1,4-phenylenebis(methylene)]-bis-1,4,8,11-tetrazacyclotetradecane), 1-100 [mu]M of M-alendronate sodium (Malendronate sodium trihydrate), and 1-100 [mu]M of disodium pamidronate. The invention provides a method for preparing the autologous hematopoietic stem cells, a kit, the stem cells and an application. In addition, the method is advantaged in yield, purity, and producing speed of the production of the protein-induced autologous hematopoietic stem cells.
Description
Technical field
The present invention relates to biomedical sector, specifically, relate to prepare autologous stem cell method, test kit, stem cell and application.
Background technology
Stem cell is the multipotential cell that a class has the of self-replication capacity, and under certain condition, it can be divided into several functions cell.Etap residing for stem cell is divided into embryonic stem cell and adult stem cell.Embryonic stem cell is the body early embryo cell that a class possesses unlimited self and differentiation potential, and be the cell of the internal layer cell pattern when zygote division develops into blastaea, it has can to the ability of the cytodifferentiation of each germinal layer category.Adult cell refers to the stem cell with reparation and regenerative power being present in (comprising marrow, nerve, muscle, epidermis, testis or kidney etc.) in people and Mammals particular organization or organ, under given conditions, adult stem cell produces new stem cell or is differentiated to form new functioning cell by certain program, thus make tissue and organ keep the running balance of growth and decline, with embryonic stem cell unlike, adult stem cell breaks up with the organization type of settling down to it only.Stem cell is that one is not fully broken up, still jejune cell, and have the potential function of the various histoorgan of regeneration and human body, medical circle is called " general-purpose cell ".
In decades, hemopoietic stem cell is applied to the treatment of disease in the blood system clinically, is proved to be the effective ways for the treatment of malignant hematologic disease.Hemopoietic stem cell is also proved to be has transdifferentiationof ability, has the potential being differentiated to form the various kinds of cell such as osteocyte, neurocyte and myocardial cell.So autologous stem cell is also widely used in the reproducibility repairing and treating of the various diseases in regenerative medicine and repair medical science field.
Because allosome or xenogenic stem cells transplanting inevitably can produce immunological rejection, autologous stem cells has become one of most important field of modern life science development.After humans and animals birth, the hemopoietic stem cell quantity retained in body is extremely rare.Therefore, the source of autologous stem cells difficulty and production quantity deficiency are annoying further developing of modern regenerative medicine and autologous tissue's organ engineering always.The autologous stem cell how obtaining huge quantity is an important technical bottleneck of current international artery cell disease treatment.
In recent years, applying gene recombinant technology is verified, somatocyte can be re-coded and reverse differentiation, generation is called as the autologous multipotential stem cell of inducibility (induced pluripotent stem cell, iPS stem cell), the production technology for autologous stem cells provide new by way of.Therefore, the reverse differentiation of somatocyte is utilized to produce the focus that autologous multipotential stem cell has become stem cells technology research and development.
The source of current hemopoietic stem cell can be divided three classes: marrow hemopoietic stem cells, mobilizes Peripheral blood stem cell and cord blood stem cell.Marrow hemopoietic stem cells and the general number of nucleated cells of mobilization Peripheral blood stem cell only reach 5-10 × 10
8/ Kg, CD34+ cell only reaches 1-5 × 10
6/ Kg; 80ml cord blood stem cell quantity is less, and CD34+ cell only reaches 1-5 × 10
6.Research display, in the sick disease in field of non-blood and the repairing and treating of wound, the consumption of hemopoietic stem cell becomes positive correlation with the effect size of histoorgan disease and injury repairing.The quantity of above three kinds of hemopoietic stem cells, also can not meet great substantive tissue organ disease far away, and as heart trouble, cerebral nerve disease and the regeneration of damage, hepatopathy, renal failure etc. disease and the needs of repairing and treating, its curative effect is obviously not enough.
Therefore, be necessary to research and develop the derived from hematopoietic precursor cells made new advances.In addition, how to obtain the autologous stem cell of sufficient amount, high security, being one has a Medical Biology difficult problem yet-to-be developed.
Summary of the invention
For the problem that in prior art, autologous stem cell source is not enough, retrievable quantity is few and security is low, according to an aspect of the present invention, provide a kind of method preparing autologous stem cell, the method comprises:
Gather the somatocyte of human or animal.The somatocyte obtained is with 5 × 10
6density be inoculated in Tissue Culture Dish, change cell culture fluid.
Preferably, gather the peripheral blood of human or animal and/or bone marrow fluid, apply conventional Ficoll centrifugal separation technique, centrifugal, be separated and obtain blood mononuclear cells.The mononuclearcell obtained is with 5 × 10
6density be inoculated in Tissue Culture Dish, change cell culture fluid.
In the method preparing autologous stem cell, somatic sources in the in vitro tissue of human or animal, comprise in blood, bone marrow fluid, skin and fat one or more.
In the method preparing autologous stem cell, somatocyte comprises cord blood cell, placenta cells, skin cells, blood karyocyte and/or adipocyte.
37 DEG C and 5% CO
2environment in, somatocyte is cultivated 3-6 days in cell culture fluid, and described nutrient solution is RPMI1640 nutrient solution.Wherein, contain in nutrient solution:
The Y-27632(C of 1-100 μM
14h
21n
3o2HCl), preferred value is 10 μMs;
The STEM CELL FACTOR of 1-100ng/mL, preferred value is 10ng/mL;
The interleukin Ⅲ of 1-100ng/mL, preferred value is 10ng/mL;
The interleukin 6 of 1-100ng/mL, preferred value is 10ng/mL;
The interleukin-11 of 1-100ng/mL, preferred value is 10ng/mL;
The macrophage colony stimulating factor (M-CSF) of 1-100ng/mL, preferred value is 10ng/mL;
The granulocyte colony-stimulating factor (G-CSF) of 1-100ng/mL, preferred value is 10ng/mL;
The fucoidin of 1-100 μ g/mL, preferred value is 10 μ g/mL;
The T 500 of 1-100 μ g/mL, preferred value is 10 μ g/mL;
The AMD3100(1 of 1-100nM, 1 '-[Isosorbide-5-Nitrae-phenylene two (methylene radical)]-two-Isosorbide-5-Nitrae, 8,11-tetraazacyclododecane tetradecane), preferred value is 10ng/mL;
The M-alendronate sodium (Malendronate sodium trihydrate) of 1-100 μM, preferred value is 10 μMs;
The Pamidronate Disodium of 1-100 μM, preferred value is 10 μMs.
Blow and beat cultured cells gently, after cell is suspended completely, collecting cell is in centrifuge tube, carry out the cell cleaning operation that centrifugal-physiological saline suspends, after repeating 3 times, to suspend protein induced property autologous stem cell with physiological saline, obtain autologous stem cell.
By the autologous stem cell freezen protective prepared, the method for freezen protective is: by the autologous stem cell that obtains with 1 × 10
5individual/ml to 1 × 10
6the cell density of individual/ml and isopyknic dimethyl sulfoxide (DMSO) and 10% the mixed solution of low molecular dextran mix, mixture is cooled to-80 DEG C, then transfers to deep-bed drying in the liquid nitrogen of-186 DEG C.
In the above-mentioned methods, preferably, contain in the RPMI164 nutrient solution used: the Y-27632(C of 10 μMs
14h
21n
3o2HCl); The STEM CELL FACTOR of 10ng/mL; The interleukin Ⅲ of 10ng/mL; The interleukin 6 of 10ng/mL; The interleukin-11 of 10ng/mL; The macrophage colony stimulating factor (M-CSF) of 10ng/mL; The granulocyte colony-stimulating factor (G-CSF) of 10ng/mL; The fucoidin of 10 μ g/mL; The T 500 of 10 μ g/mL; The AMD3100(1 of 10nM, 1 '-[Isosorbide-5-Nitrae-phenylene two (methylene radical)]-two-Isosorbide-5-Nitrae, 8,11-tetraazacyclododecane tetradecane); (M-alendronate sodium) Malendronate sodium trihydrate of 10 μMs; The Pamidronate Disodium of 10 μMs.
According to another aspect of the present invention, a kind of test kit for the preparation of autologous stem cell is additionally provided.Test kit comprises a kind of cell culture fluid (RPMI1640 nutrient solution), wherein contains:
The Y-27632(C of 1-100 μM
14h
21n
3o2HCl), preferred value is 10 μMs;
The STEM CELL FACTOR of 1-100ng/mL, preferred value is 10ng/mL;
The interleukin Ⅲ of 1-100ng/mL, preferred value is 10ng/mL;
The interleukin 6 of 1-100ng/mL, preferred value is 10ng/mL;
The interleukin-11 of 1-100ng/mL, preferred value is 10ng/mL;
The macrophage colony stimulating factor (M-CSF) of 1-100ng/mL, preferred value is 10ng/mL;
The granulocyte colony-stimulating factor (G-CSF) of 1-100ng/mL, preferred value is 10ng/mL;
The fucoidin of 1-100 μ g/mL, preferred value is 10 μ g/mL;
The T 500 of 1-100 μ g/mL, preferred value is 10 μ g/mL;
The AMD3100(1 of 1-100nM, 1 '-[Isosorbide-5-Nitrae-phenylene two (methylene radical)]-two-Isosorbide-5-Nitrae, 8,11-tetraazacyclododecane tetradecane), preferred value is 10ng/mL;
The M-alendronate sodium (Malendronate sodium trihydrate) of 1-100 μM, preferred value is 10 μMs;
The Pamidronate Disodium of 1-100 μM, preferred value is 10 μMs.
Preferably, the cell culture fluid (RPMI1640 nutrient solution) in mentioned reagent box contains: the Y-27632(C of 10 μMs
14h
21n
3o2HCl); The STEM CELL FACTOR of 10ng/mL; The interleukin Ⅲ of 10ng/mL; The interleukin 6 of 10ng/mL; The interleukin-11 of 10ng/mL; The macrophage colony stimulating factor (M-CSF) of 10ng/mL; The granulocyte colony-stimulating factor (G-CSF) of 10ng/mL; The fucoidin of 10 μ g/mL; The T 500 of 10 μ g/mL; The AMD3100(1 of 10nM, 1 '-[Isosorbide-5-Nitrae-phenylene two (methylene radical)]-two-Isosorbide-5-Nitrae, 8,11-tetraazacyclododecane tetradecane); The M-alendronate sodium (Malendronate sodium trihydrate) of 10 μMs; The Pamidronate Disodium of 10 μMs.
In the above-mentioned methods, the cell culture fluid of application multiple protein composition, make the reverse differentiation of autoblood mononuclearcell of people or mouse, produce protein induced property autologous stem cell, we are referred to as: protein induced property autologous stem cell (Protein induced Hematologic Stem Cell, PiHPS).
Autologous stem cell can be prepared according to method provided by the invention.Autologous stem cell may be used for setting up autologous stem cell storehouse, prepare the medicine that antiaging agent or healthcare products and preparation relate to the disease that neomorph is reproduced and repaired.Test kit provided by the invention can be used for setting up autologous stem cell storehouse.
The invention provides a kind of prepare autologous stem cell method, test kit, stem cell and application, and the production method of protein induced property autologous stem cell provided by the invention has significant advantage in output, purity, production rate and security.
Accompanying drawing explanation
Figure 1A to Fig. 1 D is from reverse form and CD34 and the CD133 specific phenotypes qualification figure breaking up the people's autologous stem cell produced of human blood mononuclearcell;
Fig. 1 E to Fig. 1 H is from reverse form and CD34 and the CD133 specific phenotypes qualification figure breaking up the mouse autologous stem cell produced of mouse blood mononuclearcell;
Fig. 1 I to Fig. 1 P is CD34 and the CD133 specific phenotypes qualification comparison diagram of mouse blood mononuclearcell, mouse autologous stem cell;
Fig. 2 A surveys view from the flow cytometer of people's autologous stem cell of the reverse differentiation production of human blood mononuclearcell (PMBC);
Fig. 2 B is the graphic representation of the transformation efficiency of the people's autologous stem cell produced from the reverse differentiation of human blood mononuclearcell (PMBC);
Fig. 2 C is the graphic representation of the transformation efficiency of the mouse autologous stem cell produced from the reverse differentiation of mouse blood mononuclearcell (PMBC);
Fig. 3 A is the photo of Balb/c nude mice vein transplantation inducibility autologous stem cell after 105 days;
Fig. 3 B is the photo of Balb/c nude mice vein transplantation B16 tumour cell after 105 days;
Fig. 3 C is the photo of after Balb/c nude mice by subcutaneous transplanting+vein transplantation inducibility autologous stem cell 105 days;
Fig. 3 D is that Balb/c nude mice by subcutaneous transplants the photo of B16 tumour cell after 105 days;
Fig. 3 E is the internal anatomy of the lung tumor that Balb/c nude mice vein transplantation B16 tumour cell produces for 21 days afterwards;
Fig. 3 F is the Tumor incidence histogram of each test group and control group.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, the every other embodiment that those of ordinary skill in the art obtain, all belongs to the scope of protection of the invention.
The preparation of embodiment 1 test kit
Operate in the safe operating table of 10-100 level cleanliness factor, prepare under the cold condition of 4-10 degree.In the RPMI640 nutrient solution of 500ml, add respectively: the Y-27632(C14H21N3O2HCl of 10 μMs); The STEM CELL FACTOR of 10ng/mL; The interleukin Ⅲ of 10ng/mL; The interleukin 6 of 10ng/mL; The interleukin-11 of 10ng/mL; The macrophage colony stimulating factor (M-CSF) of 10ng/mL; The granulocyte colony-stimulating factor (G-CSF) of 10ng/mL; The fucoidin of 10 μ g/mL; The T 500 of 10 μ g/mL; The AMD3100(1 of 10nM, 1 '-[Isosorbide-5-Nitrae-phenylene two (methylene radical)]-two-Isosorbide-5-Nitrae, 8,11-tetraazacyclododecane tetradecane); The M-alendronate sodium (Malendronate sodium trihydrate) of 10 μMs; The Pamidronate Disodium of 10 μMs.
Abundant mixing, dissolves, then through frit, the sterilization of 0.22 micron pore size, is mixed with cell culture fluid.
Cell culture fluid is placed in test kit, thus makes the test kit for calpastatin inducibility autologous stem cell.
The preparation of the protein induced property autologous stem cell (M-PiHPS) of embodiment 2 mouse
The Balb/C male mice at 3-5 monthly age is selected (to buy from radiological medicine institute of the Chinese Academy of Medical Sciences (Tianjin), animal conformity certification number: SCXK(Tianjin) 2010-0002), gather its whole blood, use conventional Ficoll centrifugal separation technique to blood carry out centrifugal, be separated thus obtain blood mononuclear cells.By the mononuclearcell of acquisition with 5 × 10
6the density of individual/ml, is inoculated in Tissue Culture Dish, and to add in embodiment 1 cell culture fluid of preparation wherein, 37 DEG C and 5% CO
2incubator in cultivate 5 days thus obtain protein induced property autologous stem cell.Blow and beat cultured cells gently, after cell is suspended completely, collecting cell is in centrifuge tube, carry out the cell cleaning operation that centrifugal-physiological saline suspends, after repeating 3 times, to suspend protein induced property autologous stem cell with physiological saline, obtain protein induced property autologous stem cell.By the autologous stem cell that obtains with 1 × 10
5individual/ml to 1 × 10
6the cell density of individual/ml and isopyknic dimethyl sulfoxide (DMSO) and 10% the mixed solution of low molecular dextran mix, mixture is cooled to-80 DEG C, then transfers to deep-bed drying in the liquid nitrogen of-186 DEG C.
The preparation of the protein induced property autologous stem cell (M-PiHPS) of embodiment 3 people
Gather the peripheral blood 50ml of human body, use conventional Ficoll centrifugal separation technique to blood carry out centrifugal, be separated thus obtain blood mononuclear cells.By the mononuclearcell of acquisition with 5 × 10
6the density of individual/ml, is inoculated in Tissue Culture Dish, and to add in embodiment 1 cell culture fluid of preparation wherein, 37 DEG C and 5% CO
2incubator in cultivate 5 days thus obtain protein induced property autologous stem cell.Blow and beat cultured cells gently, after cell is suspended completely, collecting cell is in centrifuge tube, carry out the cell cleaning operation that centrifugal-physiological saline suspends, after repeating 3 times, to suspend protein induced property autologous stem cell with physiological saline, obtain protein induced property autologous stem cell.By the protein induced property autologous stem cell that obtains with 1 × 10
5individual/ml to 1 × 10
6the cell density of individual/ml and isopyknic dimethyl sulfoxide (DMSO) and 10% the mixed solution of low molecular dextran mix, mixture is cooled to-80 DEG C, then transfers to deep-bed drying in the liquid nitrogen of-186 DEG C.
By following test, type qualification and secure authentication are carried out to the PiHPS that embodiment 2 and embodiment 3 obtain.
Test 1 cell-specific phenotypic evaluation
With the slide glass of poly-D-Lys obtained in advance, select the cell suspending liquid concentration of the best of the male self-generation stem cell of non-testis endogenous binding protein inducibility of preparation in embodiment 2 and embodiment 3, under 1800RCF, rotate throwing sheet with sectioning cells whizzer make slide and carry out mark for 2 minutes, throw cell density on sheet (cell is even, and density is suitable).Deposit in-20 degree refrigerators frozen after throwing sheet dries.From-20 degree refrigerators, take out Cell sheet glass dry in room temperature, use marking pen defined area at reverse side.Then protein induced property autologous stem cell physiological saline suspension embodiment 2 prepared uses 4% paraformaldehyde (to configure with PBS respectively, purchased from solarbio company, production code member is P1010) fixing, through 0.2%Triton X-100 punch 10 minutes and phosphate buffered saline buffer (PBS) cleaning after, use 10% Normal Goat Serum to close 1 hour under the condition of room temperature, obtain sample.Repeat sample making step, use the protein induced property autologous stem cell obtained in embodiment 2 to obtain sample A and sample B, use the protein induced property autologous stem cell obtained in embodiment 3 to obtain sample C and sample D.First antibody reagent rabbit against murine CD34 is added in sample A; First antibody reagent rabbit antihuman CD 34 is added in sample C; First antibody reagent rabbit against murine CD133 is added in sample B; The anti-human CD133 of first antibody reagent rabbit is added in sample D.Under the condition of 4 DEG C, make the antigen in four samples react with first antibody 12 hours (or spending the night) respectively.Then after sample is cleaned in use phosphate buffered saline buffer (PBS), second antibody joined in four samples respectively, second antibody is the goat anti-rabbit igg (1:200 using FITC mark; Buy from Chemicon company, production code member is AP132F).Under the condition of room temperature, make the first antibody in four samples react 1 hour with second antibody respectively.Then, with DAPI(1:1000) carry out dye core, carry out 1 minute under room temperature condition.After use phosphate buffered saline buffer PBS cleaning, carry out the operation of glycerine mounting to sample, institute uses edge sealing agent for glycerine/PBS mountant (glycerine: PBS=1:9, pH8.0-8.5, the manufacture of art designing biotech firm).Finally use Nikon fluorescence microscope to take pictures, in 6-8 hour, complete various taking pictures.
Test-results is shown in Figure 1A to Fig. 1 P, and 1A to Fig. 1 P shows cell-specific phenotypic evaluation figure, and Figure 1A to Fig. 1 D sequentially show form and CD34 and the CD133 specific phenotypes qualification figure of the people's autologous stem cell produced from the reverse differentiation of human blood mononuclearcell; Fig. 1 E to Fig. 1 H sequentially show form and CD34 and the CD133 specific phenotypes qualification figure of the mouse autologous stem cell produced from the reverse differentiation of mouse blood mononuclearcell; Fig. 1 I to Fig. 1 P shows CD34 and the CD133 specific phenotypes qualification comparison diagram of mouse blood mononuclearcell, mouse autologous stem cell.
Wherein, Figure 1A and Figure 1B shows unconverted human blood mononuclearcell.
Fig. 1 C shows the obtained cell of embodiment 3 after reacting with primary antibodie (rabbit antihuman CD 34) and two anti-(goat anti-rabbit iggs that FITC marks), at fluorescence microscope to fluorescing fractions (light tone region), thus cell behaviour autologous stem cell obtained in embodiment 3 can be described.
Fig. 1 D shows the obtained cell of embodiment 3 after reacting with primary antibodie (the anti-human CD133 of rabbit) and two anti-(goat anti-rabbit iggs that FITC marks), at fluorescence microscope to fluorescing fractions (light tone region), thus cell behaviour autologous stem cell obtained in embodiment 3 can be described.
Fig. 1 E and Fig. 1 shows unconverted mouse blood mononuclearcell.
Fig. 1 G shows the obtained cell of embodiment 2 after reacting with primary antibodie (rabbit antihuman CD 34) and two anti-(goat anti-rabbit iggs that FITC marks), at fluorescence microscope to fluorescing fractions (light tone region), thus can illustrate that cell obtained in embodiment 2 is mouse autologous stem cell.
Fig. 1 H shows the obtained cell of embodiment 2 after reacting with primary antibodie (the anti-human CD133 of rabbit) and two anti-(goat anti-rabbit iggs that FITC marks), at fluorescence microscope to fluorescing fractions (light tone region), thus can illustrate that cell obtained in embodiment 2 is mouse autologous stem cell.
Test the mensuration of 2 autologous stem cell transformation efficiencys
Method one: the transformation efficiency of cells were tested by flow cytometry autologous stem cell
Test principle: use immunofluorence technic to mark CD34, CD133 positive cell, determine autologous stem cell transformation efficiency (productivity) by CD34, CD133 positive cell of certification mark.
Testing sequence:
1) thaw cell: also the human blood mononuclearcell (PBMC) of cryopreservation and the 4 increment product of people's autologous stem cell and mouse blood mononuclearcell (PBMC) and mouse autologous stem cell will made in embodiment 2 and embodiment 3 are put into 37 degree of water-baths respectively and thawed.
2) PiHPS of the PBMC of human body and mouse, human body and mouse is transferred in EP pipe respectively, centrifugal 5min under normal temperature under 2500RCF.
3) remove supernatant liquor, often pipe adds confining liquid (2%FCS, 1%SA PBS dilutes) the resuspension cell precipitation of 400 μ L.
4) centrifugal 5min under normal temperature under 2500RCF.
5) remove supernatant liquor, often pipe adds the confining liquid of 50 μ L, resuspension cell precipitation.
6) the CD34 antibody reagent of 20 μ L is added in pipe.Be placed on dark place after mixing and hatch 30min, carry out flow cytometer (U.S. BD FACSCalibur) analysis afterwards, automatically calculate the CD34 positive cell quantity in sample and per-cent.
Test-results is shown in Fig. 2 A, and as shown in Figure 2 A, Fig. 2 A surveys view from the flow cytometer of people's autologous stem cell of the reverse differentiation production of human blood mononuclearcell (PMBC).Can 38% be reached by the transformation efficiency of the autologous stem cell of cells were tested by flow cytometry.
Method two: immunofluorescence technique detects CD34, CD133 positive cell and counting
Testing sequence: 1) prepare PiHPS cell according to the preparation method of embodiment 2, difference is, incubation time is respectively 1 day, 2 days, 3 days, 4 days, 5 days.Prepare the PiHPS sample of 5 groups of mouse.Prepare PiHPS cell according to the preparation method of embodiment 3, difference is, incubation time is respectively 1 day, 2 days, 3 days, 4 days, 5 days.Prepare the PiHPS sample of 5 groups of people.
2) then cell-specific phenotypic evaluation is carried out according to the grouping of test 1 and method.Under Nikon fluorescent microscope, FITC is labeled as green and DAPI is labeled as blueness, the mean number calculating CD34, CD133 specific staining cell of ten border downward views, except the mean number of nucleus (DAPI colouring) giving ten border downward views, equals positive cell percentage.
Test-results
As shown in fig. 2 b and fig. 2 c, Fig. 2 B is the graphic representation of the transformation efficiency of the people's autologous stem cell produced from the reverse differentiation of human blood mononuclearcell (PMBC); Fig. 2 C is the graphic representation of the transformation efficiency of the mouse autologous stem cell produced from the reverse differentiation of mouse blood mononuclearcell (PMBC).From Fig. 2 B and Fig. 2 C, between the incubation period of the 1st day to the 5th day, autologous stem cell transformation efficiency has the trend of rising, and at about the 5th day, transformation efficiency peaked, and now, the transformation efficiency of sample is all between 20% to 40%.It can also be seen that from figure, be identical substantially, and the transformation efficiency of PMBC, MBC is all between 20% to 40% by the transformation efficiency of the sample of CD34, CD133 antibody characterization.
Test the security authentication of 3 autologous stem cells
Get 1 monthly age Balb/C nude mice 50, male and female half and half, raise in regular grade environment.Mouse is divided into three groups at random, wherein autologous stem cell group 20, tumour cell group 20, blank group 10.
20 of autologous stem cell group mouse are divided at random two groups (first groups and second group), often organize 10.First group of mouse takes the mode of tail vein transplantation: by the protein induced property self-generation hemopoietic stem cell of preparation in embodiment 2 with 1 × 10
7individual cell/metering only enters in Mice Body from tail vein injections.The mode that second group of mouse takes tail vein injection and subcutaneous injection to carry out simultaneously: by the protein induced property self-generation hemopoietic stem cell prepared in embodiment 2 with 1 × 10
7individual cell/metering is only injected in Mice Body by tail vein injection+hypodermic mode in nape portion.
20 of tumour cell group mouse are divided at random two groups (the 3rd groups and the 4th group), often organize 10.3rd group of mouse adopts the mode of tail vein transplantation: by melanoma (B16) cell with 1 × 10
7individual cell/metering only enters in Mice Body from tail vein injections.4th group of mouse is in hypodermic mode: by melanoma (B16) cell with 1 × 10
7individual cell/metering is only injected in Mice Body from nape portion.
The mouse of blank group is the 5th group of mouse, respectively organizes the equal physiological saline of volume be injected into above in Mice Body in tail vein injection+hypodermic mode in nape portion.
After having injected, the mouse of different group is separately raised, and observe the formation with or without tubercle and tumour every day.Within 21 days, put to death half mouse afterwards, and carry out pathologic finding.Continue to raise residue mouse, observe after 12 weeks and put to death and make pathologic finding.
Test-results is shown in Fig. 3 A to Fig. 3 F, and Fig. 3 A is the photo of after Balb/c nude mice vein transplantation inducibility autologous stem cell 105 days; Fig. 3 B is the Balb/c nude mice vein transplantation B16 tumour cell photo of 105 days; Fig. 3 C is the photo of after Balb/c nude mice by subcutaneous transplanting+vein transplantation inducibility autologous stem cell 105 days; Fig. 3 D is the photo of after Balb/c nude mice by subcutaneous transplanting B16 tumour cell 105 days; Fig. 3 E is the internal anatomy of the lung tumor that Balb/c nude mice vein transplantation B16 tumour cell produces for 21 days afterwards; Fig. 3 F is the Tumor incidence histogram of each test group and control group.
By above-mentioned safety testing, in injection, first group, second group and the 5th group of mouse do not find after 21 days and after 12 weeks that an example has knurl apodization and resembles.And the 3rd group and the 4th group of mouse knurl occurs at position, injection 21 days rear sections become, after injecting 12 weeks, all find that knurl apodization resembles.Meanwhile, engaging histogram can find out, after first group and second group of injection autologous stem cell, tumorigenic probability is 0, and the 3rd group and the 4th group are after injection B16 tumour cell, and tumorigenic probability is 100%.Therefore, the obtained PiHPS of method of the present invention is used to have higher-security.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (14)
1. prepare a method for autologous stem cell, comprising:
Somatocyte is cultivated 3-6 days in cell culture fluid, and collect the autologous stem cell generated, wherein, described cell culture fluid is RPMI1640 nutrient solution, wherein containing the Y-27632(C of 1-100 μM
14h
21n
3o2HCl); The STEM CELL FACTOR of 1-100ng/mL; The interleukin Ⅲ of 1-100ng/mL; The interleukin 6 of 1-100ng/mL; The interleukin-11 of 1-100ng/mL; The macrophage colony stimulating factor (M-CSF) of 1-100ng/mL; The granulocyte colony-stimulating factor (G-CSF) of 1-100ng/mL; The fucoidin of 1-100 μ g/mL; The T 500 of 1-100 μ g/mL; The AMD3100(1 of 1-100nM, 1 '-[Isosorbide-5-Nitrae-phenylene two (methylene radical)]-two-Isosorbide-5-Nitrae, 8,11-tetraazacyclododecane tetradecane); The M-alendronate sodium (Malendronate sodium trihydrate) of 1-100 μM; The Pamidronate Disodium of 1-100 μM.
2. method according to claim 1, wherein, containing the Y-27632(C of 10 μMs in RPMI1640 nutrient solution
14h
21n
3o2HCl); The STEM CELL FACTOR of 10ng/mL; The interleukin Ⅲ of 10ng/mL; The interleukin 6 of 10ng/mL; The interleukin-11 of 10ng/mL; The macrophage colony stimulating factor (M-CSF) of 10ng/mL; The granulocyte colony-stimulating factor (G-CSF) of 10ng/mL; The fucoidin of 10 μ g/mL; The T 500 of 10 μ g/mL; The AMD3100(1 of 10nM, 1 '-[Isosorbide-5-Nitrae-phenylene two (methylene radical)]-two-Isosorbide-5-Nitrae, 8,11-tetraazacyclododecane tetradecane); The M-alendronate sodium (Malendronate sodium trihydrate) of 10 μMs; The Pamidronate Disodium of 10 μMs.
3. method according to claim 1 and 2, the culture condition wherein in described cell culture fluid is: temperature is the CO of 37 DEG C and 5%
2environment.
4. method according to claim 1, described somatic sources in the in vitro tissue of human or animal, comprise in blood, bone marrow fluid, skin and fat one or more.
5. method according to claim 1, wherein, described somatocyte comprises cord blood cell, placenta cells, skin cells, blood karyocyte and/or adipocyte.
6. method according to claim 1 and 2, comprises further: by the autologous stem cell freezen protective prepared.
7. method according to claim 6, wherein, described freezen protective comprises: by the autologous stem cell that obtains with 1 × 10
5individual/ml to 1 × 10
6the cell density of individual/ml and isopyknic dimethyl sulfoxide (DMSO) and 10% the mixed solution of low molecular dextran mix, be cooled to-80 DEG C, then transfer to deep-bed drying in the liquid nitrogen of-186 DEG C.
8., for the preparation of a test kit for autologous stem cell, comprising:
Cell culture fluid, described cell culture fluid is RPMI1640 nutrient solution, wherein containing the Y-27632(C of 1-100 μM
14h
21n
3o2HCl); The STEM CELL FACTOR of 1-100ng/mL; The interleukin Ⅲ of 1-100ng/mL; The interleukin 6 of 1-100ng/mL; The interleukin-11 of 1-100ng/mL; The macrophage colony stimulating factor (M-CSF) of 1-100ng/mL; The granulocyte colony-stimulating factor (G-CSF) of 1-100ng/mL; The fucoidin of 1-100 μ g/mL; The T 500 of 1-100 μ g/mL; The AMD3100(1 of 1-100nM, 1 '-[Isosorbide-5-Nitrae-phenylene two (methylene radical)]-two-Isosorbide-5-Nitrae, 8,11-tetraazacyclododecane tetradecane); The M-alendronate sodium (Malendronate sodiumtrihydrate) of 1-100 μM; The Pamidronate Disodium of 1-100 μM.
9., for the preparation of a test kit for autologous stem cell, comprising:
Cell culture fluid, described cell culture fluid is RPMI1640 nutrient solution, wherein containing the Y-27632(C of 10 μMs
14h
21n
3o2HCl); The STEM CELL FACTOR of 10ng/mL; The interleukin Ⅲ of 10ng/mL; The interleukin 6 of 10ng/mL; The interleukin-11 of 10ng/mL; The macrophage colony stimulating factor (M-CSF) of 10ng/mL; The granulocyte colony-stimulating factor (G-CSF) of 10ng/mL; The fucoidin of 10 μ g/mL; The T 500 of 10 μ g/mL; The AMD3100(1 of 10nM, 1 '-[Isosorbide-5-Nitrae-phenylene two (methylene radical)]-two-Isosorbide-5-Nitrae, 8,11-tetraazacyclododecane tetradecane); The M-alendronate sodium (Malendronate sodium trihydrate) of 10 μMs; The Pamidronate Disodium of 10 μMs.
10. test kit is according to claim 8 or claim 9 setting up the application in autologous stem cell storehouse.
Autologous stem cell prepared by 11. methods according to claim 1-7 any one.
12. autologous stem cells according to claim 11 are setting up the application in autologous stem cell storehouse.
13. autologous stem cells according to claim 11 are preparing the application in antiaging agent or healthcare products.
14. autologous stem cells according to claim 11 are preparing the application related in the medicine of the disease that neomorph is reproduced and repaired.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726405A (en) * | 2015-03-19 | 2015-06-24 | 河南中科干细胞基因工程有限公司 | Separation and database establishing method for cord blood hematopoietic stem cells |
| CN107326009A (en) * | 2017-05-02 | 2017-11-07 | 深圳百年干细胞技术研究院有限公司 | Blood mononuclear cells are made inversely to break up method, kit and the purposes for producing the autologous retinal stem cells of people's haematogenous |
| CN109540632A (en) * | 2018-11-12 | 2019-03-29 | 滨州医学院 | A kind of Cell immunohistochemical staining method |
| CN110129257A (en) * | 2019-05-15 | 2019-08-16 | 林柳吟 | Preparation method, kit and the application of haematogenous women self-generation stem cell |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101735979A (en) * | 2009-12-31 | 2010-06-16 | 浙江中赢控股集团有限公司 | Method for in vitro amplification of hemopoietic stem cells and precursor cells |
| CN102399741A (en) * | 2010-09-19 | 2012-04-04 | 林雄斌 | Culture solution and method for producing hemopoietic stem cell by reversely splitting somatic cell and application of culture liquid |
-
2013
- 2013-08-19 CN CN201310361684.3A patent/CN104419660B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101735979A (en) * | 2009-12-31 | 2010-06-16 | 浙江中赢控股集团有限公司 | Method for in vitro amplification of hemopoietic stem cells and precursor cells |
| CN102399741A (en) * | 2010-09-19 | 2012-04-04 | 林雄斌 | Culture solution and method for producing hemopoietic stem cell by reversely splitting somatic cell and application of culture liquid |
Non-Patent Citations (1)
| Title |
|---|
| ZHAO SHENG-MING等: "Targeted expansion and regulation of genetically modified cord blood stem/progenitor cells in vitro", 《中国组织工程研究与临床康复》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726405A (en) * | 2015-03-19 | 2015-06-24 | 河南中科干细胞基因工程有限公司 | Separation and database establishing method for cord blood hematopoietic stem cells |
| CN107326009A (en) * | 2017-05-02 | 2017-11-07 | 深圳百年干细胞技术研究院有限公司 | Blood mononuclear cells are made inversely to break up method, kit and the purposes for producing the autologous retinal stem cells of people's haematogenous |
| CN107326009B (en) * | 2017-05-02 | 2019-03-08 | 深圳百年干细胞技术研究院有限公司 | Blood mononuclear cells are made inversely to break up method, kit and the purposes for generating the self retinal stem cells of people's haematogenous |
| CN109540632A (en) * | 2018-11-12 | 2019-03-29 | 滨州医学院 | A kind of Cell immunohistochemical staining method |
| CN110129257A (en) * | 2019-05-15 | 2019-08-16 | 林柳吟 | Preparation method, kit and the application of haematogenous women self-generation stem cell |
| CN111484971A (en) * | 2019-05-15 | 2020-08-04 | 林柳吟 | Preparation method, kit and application of blood-derived female autologous reproductive stem cells |
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| CN104419660B (en) | 2016-07-06 |
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