CN104419660B - Prepare the method for autologous stem cell, test kit, stem cell and application - Google Patents
Prepare the method for autologous stem cell, test kit, stem cell and application Download PDFInfo
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Abstract
The invention provides a kind of method preparing autologous stem cell, the method includes: somatic cell cultivated 3-6 days in cell culture fluid, collects the autologous stem cell generated, wherein, described cell culture fluid is RPMI1640 culture fluid, wherein contains the Y-27632(C of 1-100 μM14H21N3O 2HCl)；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 M-CSF (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 dextran sulfate of 1-100 μ g/mL；The AMD3100(1,1 ' of 1-100nM-[1,4-phenylene two (methylene)]-two-1,4,8,11-tetraazacyclododecane tetradecanes)；The M-Alendronate sodium (Malendronate sodium trihydrate) of 1-100 μM；The Pamidronate Disodium of 1-100 μM.Thus, the invention provides a kind of the prepare method of autologous stem cell, 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 yield, purity, speed of production and safety.
Description
Technical field
The present invention relates to biomedical sector, it particularly relates to prepare the method for autologous stem cell, test kit, stem cell and application.
Background technology
Stem cell is the pluripotent cell that a class has the of self-replication capacity, and under certain condition, it can be divided into several functions cell.Stage of development 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 renewal and differentiation potential, is the cell of internal layer cell pattern when germ cell division develops into blastaea, and it has can to the ability of the cell differentiation of each germinal layer category.Adult cell refers to and is present in the stem cell with reparation and regeneration capacity (including bone marrow, nerve, muscle, epidermis, testis or kidney etc.) in people and mammal 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, so that tissue and organ keep growth and the dynamic equilibrium of decline, with embryonic stem cell the difference is that, adult stem cell may only break up to its organization type settled down.Stem cell is that one is not fully broken up, still jejune cell, has the potential function regenerating various histoorgans and human body, and medical circle is called " general-purpose cell ".
In decades, hematopoietic 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.Hematopoietic stem cell is also proved to have 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 extensively used for the reproducibility repairing and treating of the multiple disease in regenerative medicine and repair medical science field.
Owing to allosome or xenogenic stem cells transplanting can be inevitably generated immunological rejection, autologous stem cells has become as one of most important field of modern life science development.After humans and animals birth, the hematopoietic stem cell quantity retained in body is extremely rare.Therefore, the source difficulty of autologous stem cells and production quantity are not enough, are always up annoying the further development of modern regenerative medicine and autologous tissue's organ engineering.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 technique, it has been proved that somatic cell can be re-coded and reverse differentiation, produces to be referred to as the autologous pluripotent stem cell (inducedpluripotentstemcell of inductivity, iPS stem cell), for the production technology of autologous stem cells provide new by way of.Therefore, utilize the reverse differentiation of somatic cell to produce autologous pluripotent stem cell and have become as the focus of stem cells technology research and development.
The source of current hematopoietic 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 mobilization general number of nucleated cells of Peripheral blood stem cell only reach 5-10 × 108/ Kg, CD34+ cell only reaches 1-5 × 106/Kg；80ml cord blood stem cell quantity is less, and CD34+ cell only reaches 1-5 × 106.Research display, in the disease in non-blood disease field and the repairing and treating of wound, the consumption of hematopoietic stem cell becomes positive correlation with the effect size of histoorgan disease and injury repairing.The quantity of three of the above hematopoietic stem cell, also can not meet far away great substantive tissue organ disease, and such as the needs of the regeneration of heart disease, cerebral nerve disease and damage, hepatopathy, renal failure etc. disease and repairing and treating, its curative effect is substantially not enough.
Therefore, it is necessary to the derived from hematopoietic precursor cells that research and development makes new advances.Additionally, 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 autologous stem cell source problem that quantity is few and safety is low not enough, retrievable in prior art, according to an aspect of the invention, it is provided a kind of method preparing autologous stem cell, the method includes:
Gather the somatic cell of human or animal.The somatic cell obtained is with 5 × 106Density be inoculated in Tissue Culture Dish, change cell culture fluid.
Preferably, gather peripheral blood and/or the bone marrow fluid of human or animal, the conventional Ficoll centrifugal separation technology of application, it is centrifuged, separates and obtain blood mononuclear cells.The mononuclearcell obtained is with 5 × 106Density be inoculated in Tissue Culture Dish, change cell culture fluid.
In the method preparing autologous stem cell, somatic sources is in the in vitro tissue of human or animal, including one or more in blood, bone marrow fluid, skin and fat.
In the method preparing autologous stem cell, somatic cell includes cord blood cell, placenta cells, Skin Cell, blood nucleated cell and/or adipose cell.
CO at 37 DEG C and 5%2Environment in, somatic cell is cultivated 3-6 days in cell culture fluid, described culture fluid is RPMI1640 culture fluid.Wherein, culture fluid contains:
The Y-27632(C of 1-100 μM14H21N3O 2HCl), it is preferable that value is 10 μMs；
The stem cell factor of 1-100ng/mL, it is preferable that be worth for 10ng/mL；
The interleukin Ⅲ of 1-100ng/mL, it is preferable that be worth for 10ng/mL；
The interleukin-6 of 1-100ng/mL, it is preferable that be worth for 10ng/mL；
The interleukin-11 of 1-100ng/mL, it is preferable that be worth for 10ng/mL；
The M-CSF (M-CSF) of 1-100ng/mL, it is preferable that be worth for 10ng/mL；
The granulocyte colony-stimulating factor (G-CSF) of 1-100ng/mL, it is preferable that be worth for 10ng/mL；
The fucoidin of 1-100 μ g/mL, it is preferable that value is 10 μ g/mL；
The dextran sulfate of 1-100 μ g/mL, it is preferable that value is 10 μ g/mL；
The AMD3100(1 of 1-100nM, 1 '-[Isosorbide-5-Nitrae-phenylene two (methylene)]-two-Isosorbide-5-Nitrae, 8,11-tetraazacyclododecane tetradecanes), it is preferable that it is worth for 10ng/mL；
The M-Alendronate sodium (Malendronatesodiumtrihydrate) of 1-100 μM, it is preferable that value is 10 μMs；
The Pamidronate Disodium of 1-100 μM, it is preferable that value is 10 μMs.
Blow and beat cultured cells gently, after making cell suspend completely, collect cell in centrifuge tube, be centrifuged-normal saline suspend cell cleaning operation, after repeating 3 times, with normal saline suspension protein induced property autologous stem cell, obtain autologous stem cell.
The autologous stem cell freezen protective that will prepare, the method for freezen protective is: by the autologous stem cell that obtains with 1 × 105Individual/ml to 1 × 106The cell density of individual/ml mixes with the mixed liquor of isopyknic dimethyl sulfoxide and the low molecular dextran of 10%, and mixture is cooled to-80 DEG C, is then transferred in the liquid nitrogen of-186 DEG C deep-bed drying.
In the above-mentioned methods, it is preferable that the RPMI164 culture fluid used contains: the Y-27632(C of 10 μMs14H21N3O 2HCl)；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 M-CSF (M-CSF) of 10ng/mL；The granulocyte colony-stimulating factor (G-CSF) of 10ng/mL；The fucoidin of 10 μ g/mL；The dextran sulfate of 10 μ g/mL；The AMD3100(1,1 ' of 10nM-[1,4-phenylene two (methylene)]-two-1,4,8,11-tetraazacyclododecane tetradecanes)；(M-Alendronate sodium) Malendronatesodiumtrihydrate of 10 μMs；The Pamidronate Disodium of 10 μMs.
According to another aspect of the present invention, a kind of test kit for preparing autologous stem cell is additionally provided.Test kit comprises a kind of cell culture fluid (RPMI1640 culture fluid), wherein contains:
The Y-27632(C of 1-100 μM14H21N3O 2HCl), it is preferable that value is 10 μMs；
The stem cell factor of 1-100ng/mL, it is preferable that be worth for 10ng/mL；
The interleukin Ⅲ of 1-100ng/mL, it is preferable that be worth for 10ng/mL；
The interleukin-6 of 1-100ng/mL, it is preferable that be worth for 10ng/mL；
The interleukin-11 of 1-100ng/mL, it is preferable that be worth for 10ng/mL；
The M-CSF (M-CSF) of 1-100ng/mL, it is preferable that be worth for 10ng/mL；
The granulocyte colony-stimulating factor (G-CSF) of 1-100ng/mL, it is preferable that be worth for 10ng/mL；
The fucoidin of 1-100 μ g/mL, it is preferable that value is 10 μ g/mL；
The dextran sulfate of 1-100 μ g/mL, it is preferable that value is 10 μ g/mL；
The AMD3100(1 of 1-100nM, 1 '-[Isosorbide-5-Nitrae-phenylene two (methylene)]-two-Isosorbide-5-Nitrae, 8,11-tetraazacyclododecane tetradecanes), it is preferable that it is worth for 10ng/mL；
The M-Alendronate sodium (Malendronatesodiumtrihydrate) of 1-100 μM, it is preferable that value is 10 μMs；
The Pamidronate Disodium of 1-100 μM, it is preferable that value is 10 μMs.
Preferably, the cell culture fluid in mentioned reagent box (RPMI1640 culture fluid) contains: the Y-27632(C of 10 μMs14H21N3O 2HCl)；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 M-CSF (M-CSF) of 10ng/mL；The granulocyte colony-stimulating factor (G-CSF) of 10ng/mL；The fucoidin of 10 μ g/mL；The dextran sulfate of 10 μ g/mL；The AMD3100(1,1 ' of 10nM-[1,4-phenylene two (methylene)]-two-1,4,8,11-tetraazacyclododecane tetradecanes)；The M-Alendronate sodium (Malendronatesodiumtrihydrate) 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 mice, produce protein induced property autologous stem cell, we term it: protein induced property autologous stem cell (ProteininducedHematologicStemCell, 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 antiaging agent or health product and preparation relate to the medicine of 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 the prepare method of autologous stem cell, 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 yield, purity, speed of production and safety.
Accompanying drawing explanation
Figure 1A to Fig. 1 D is form and CD34 and the CD133 specific phenotypes qualification figure of the people's autologous stem cell from the reverse differentiation production of human blood mononuclearcell；
Fig. 1 E to Fig. 1 H is form and CD34 and the CD133 specific phenotypes qualification figure of the mice autologous stem cell from the reverse differentiation production 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, mice autologous stem cell；
Fig. 2 A is the flow cytometer survey view of the people's autologous stem cell produced from the reverse differentiation of human blood mononuclearcell (PMBC)；
Fig. 2 B is the curve chart of the conversion ratio of the people's autologous stem cell from the reverse differentiation production of human blood mononuclearcell (PMBC)；
Fig. 2 C is the curve chart of the conversion ratio of the mice autologous stem cell from the reverse differentiation production of mouse blood mononuclearcell (PMBC)；
Fig. 3 A is Balb/c nude mice vein transplantation inductivity autologous stem cell photo after 105 days；
Fig. 3 B is Balb/c nude mice vein transplantation B16 tumor cell photo after 105 days；
Fig. 3 C is the photo of 105 days after Balb/c nude mice by subcutaneous transplanting+vein transplantation inductivity autologous stem cell；
Fig. 3 D is that Balb/c nude mice by subcutaneous transplants B16 tumor cell photo after 105 days；
Fig. 3 E is the internal anatomy of the lung tumor that Balb/c nude mice vein transplantation B16 tumor cell produced after 21 days；
Fig. 3 F is the Tumor incidence block diagram of each test group and matched group.
Detailed description of the invention
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is only a part of embodiment of the present invention, rather than whole embodiments.Based on the embodiment in the present invention, the every other embodiment that those of ordinary skill in the art obtain, broadly fall into 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 cryogenic conditions of 4-10 degree.In the RPMI640 culture fluid of 500ml, it is separately added into: the Y-27632(C14H21N3O 2HCl 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 M-CSF (M-CSF) of 10ng/mL；The granulocyte colony-stimulating factor (G-CSF) of 10ng/mL；The fucoidin of 10 μ g/mL；The dextran sulfate of 10 μ g/mL；The AMD3100(1,1 ' of 10nM-[1,4-phenylene two (methylene)]-two-1,4,8,11-tetraazacyclododecane tetradecanes)；The M-Alendronate sodium (Malendronatesodiumtrihydrate) of 10 μMs；The Pamidronate Disodium of 10 μMs.
It is sufficiently mixed, dissolves, then through the frit of 0.22 micron pore size, sterilization, be configured to cell culture fluid.
Cell culture fluid is placed in test kit, thus making the test kit for producing protein inductivity autologous stem cell.
The preparation of protein induced property autologous stem cell (M-PiHPS) of embodiment 2 mice
The Balb/C male mice selecting the 3-5 monthly age (is bought from radiological medicine institute of the Chinese Academy of Medical Sciences (Tianjin), the animal quality certification number: SCXK(Tianjin) 2010-0002), gather its whole blood, use conventional Ficoll centrifugal separation technology blood to be centrifuged, separating thus obtaining blood mononuclear cells.By the mononuclearcell of acquisition with 5 × 106The density of individual/ml, is inoculated in Tissue Culture Dish, and is added thereto in embodiment 1 cell culture fluid of preparation, at the CO of 37 DEG C and 5%2Incubator in cultivate 5 days thus obtaining protein induced property autologous stem cell.Blow and beat cultured cells gently, after making cell suspend completely, collect cell in centrifuge tube, be centrifuged-normal saline suspend cell cleaning operation, after repeating 3 times, with the normal saline protein induced property autologous stem cell of suspension, obtain protein induced property autologous stem cell.By the autologous stem cell that obtains with 1 × 105Individual/ml to 1 × 106The cell density of individual/ml mixes with the mixed liquor of isopyknic dimethyl sulfoxide and the low molecular dextran of 10%, and mixture is cooled to-80 DEG C, is then transferred in the liquid nitrogen of-186 DEG C deep-bed drying.
The preparation of 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 technology blood to be centrifuged, separating thus obtaining blood mononuclear cells.By the mononuclearcell of acquisition with 5 × 106The density of individual/ml, is inoculated in Tissue Culture Dish, and is added thereto in embodiment 1 cell culture fluid of preparation, at the CO of 37 DEG C and 5%2Incubator in cultivate 5 days thus obtaining protein induced property autologous stem cell.Blow and beat cultured cells gently, after making cell suspend completely, collect cell in centrifuge tube, be centrifuged-normal saline suspend cell cleaning operation, after repeating 3 times, with the normal saline protein induced property autologous stem cell of suspension, obtain protein induced property autologous stem cell.By the protein induced property autologous stem cell that obtains with 1 × 105Individual/ml to 1 × 106The cell density of individual/ml mixes with the mixed liquor of isopyknic dimethyl sulfoxide and the low molecular dextran of 10%, and mixture is cooled to-80 DEG C, is then transferred in the liquid nitrogen of-186 DEG C deep-bed drying.
PiHPS embodiment 2 and embodiment 3 prepared by tests below carries out type qualification and secure authentication.
Test 1 cell-specific phenotypic evaluation
Microscope slide with the poly-D-Lys prepared in advance, select the best cell suspending liquid concentration of the non-male self-generation stem cell of testis endogenous binding protein inductivity of preparation in embodiment 2 and embodiment 3, under 1800RCF, rotate throwing sheet with sectioning cells centrifuge make slide and carry out labelling for 2 minutes, throw cell density on sheet (cell is uniform, and density is suitable).Throwing sheet is deposited in-20 degree refrigerators frozen after drying.Spend taking-up Cell sheet glass refrigerator from-20 to dry in room temperature, use marking pen defined area at reverse side.Then protein induced property autologous stem cell normal saline suspension embodiment 2 prepared uses 4% paraformaldehyde (to configure with PBS respectively, purchased from solarbio company, production code member is P1010) fixing, punch 10 minutes and after the cleaning of phosphate buffer (PBS) through 0.2%TritonX-100, use 10% Normal Goat Serum to close 1 hour when room temperature, obtain sample.Repeat sample making step, use the protein induced property autologous stem cell obtained in embodiment 2 to prepare sample A and sample B, use the protein induced property autologous stem cell obtained in embodiment 3 to prepare 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；Anti-human for first antibody reagent rabbit CD133 is added in sample D.Make the antigen in four samples react 12 hours (or overnight) respectively with first antibody when 4 DEG C.Then after using phosphate buffer (PBS) to clean sample, second antibody being added separately in four samples, second antibody is use the goat anti-rabbit igg (1:200 of FITC labelling；Buying from Chemicon company, production code member is AP132F).Make the first antibody in four samples react with second antibody 1 hour respectively when room temperature.Then, with DAPI(1:1000) carry out dye core, carry out under room temperature condition 1 minute.After using phosphate buffer PBS, sample carrying out glycerol mounting operation, used edge sealing agent is glycerol/PBS mountant (art designing biotech firm manufactures for glycerol: PBS=1:9, pH8.0-8.5).Finally use Nikon fluorescence microscope to take pictures, in 6-8 hour, complete various taking pictures.
Result of the test is shown in that Figure 1A to Fig. 1 P, 1A to Fig. 1 P illustrate that cell-specific phenotypic evaluation figure, 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 from the reverse differentiation production 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 mice autologous stem cell from the reverse differentiation production of mouse blood mononuclearcell；Fig. 1 I to Fig. 1 P illustrates mouse blood mononuclearcell, CD34 and the CD133 specific phenotypes of mice autologous stem cell identifies comparison diagram.
Wherein, Figure 1A and Figure 1B illustrates unconverted human blood mononuclearcell.
Fig. 1 C illustrates that cell that embodiment 3 prepares is after reacting with primary antibodie (rabbit antihuman CD 34) and two anti-(goat anti-rabbit iggs of FITC labelling), can in fluorescence microscope to fluorescing fractions (light tone region), thus obtained cell behaviour autologous stem cell is described in embodiment 3.
Fig. 1 D illustrates that cell that embodiment 3 prepares is after reacting with primary antibodie (the anti-human CD133 of rabbit) and two anti-(goat anti-rabbit iggs of FITC labelling), can in fluorescence microscope to fluorescing fractions (light tone region), thus obtained cell behaviour autologous stem cell is described in embodiment 3.
Fig. 1 E and Fig. 1 illustrates unconverted mouse blood mononuclearcell.
Fig. 1 G illustrates that cell that embodiment 2 prepares is after reacting with primary antibodie (rabbit antihuman CD 34) and two anti-(goat anti-rabbit iggs of FITC labelling), can in fluorescence microscope to fluorescing fractions (light tone region), thus illustrating in embodiment 2 that obtained cell is mice autologous stem cell.
Fig. 1 H illustrates that cell that embodiment 2 prepares is after reacting with primary antibodie (the anti-human CD133 of rabbit) and two anti-(goat anti-rabbit iggs of FITC labelling), can in fluorescence microscope to fluorescing fractions (light tone region), thus illustrating in embodiment 2 that obtained cell is mice autologous stem cell.
Test the mensuration of 2 autologous stem cell conversion ratios
Method one: flow cytometer measures the conversion ratio of autologous stem cell
Test principle: use immunofluorence technic to carry out labelling CD34, CD133 positive cell, determines autologous stem cell conversion ratio (productivity ratio) by detecting CD34, CD133 positive cell of labelling.
Test procedure:
1) defrosting cell: will make in embodiment 2 and embodiment 3 and the human blood mononuclearcell (PBMC) of cryopreservation and 4 parts of samples of people's autologous stem cell and mouse blood mononuclearcell (PBMC) and mice autologous stem cell are respectively put in 37 degree of water-baths and thaw.
2) PiHPS of human body and the PBMC of mouse, human body and mouse is transferred in EP pipe respectively, centrifugal 5min under room temperature under 2500RCF.
3) removing supernatant, often pipe adds confining liquid (2%FCS, 1%SA PBS dilutes) the resuspension cell precipitation of 400 μ L.
4) under 2500RCF, 5min under room temperature, it is centrifuged.
5) removing supernatant, often pipe adds the confining liquid of 50 μ L, resuspension cell precipitation.
6) pipe adds the CD34 antibody reagent of 20 μ L.It is placed on dark place after mixing and hatches 30min, carry out flow cytometer (U.S. BDFACSCalibur) afterwards and analyze, automatically calculate the CD34 positive cell quantity in specimen and percentage ratio.
Result of the test is shown in Fig. 2 A, and as shown in Figure 2 A, Fig. 2 A is the flow cytometer survey view of the people's autologous stem cell produced from the reverse differentiation of human blood mononuclearcell (PMBC).The conversion ratio of the autologous stem cell measured by flow cytometer is up to 38%.
Method two: immunofluorescence technique detection CD34, CD133 positive cell and counting
Test procedure: 1) prepare PiHPS cell according to the preparation method of embodiment 2, it is different in that, incubation time respectively 1 day, 2 days, 3 days, 4 days, 5 days.Prepare the PiHPS sample of 5 groups of mices.Prepare PiHPS cell according to the preparation method of embodiment 3, be different in that, incubation time respectively 1 day, 2 days, 3 days, 4 days, 5 days.Prepare the PiHPS sample of 5 groups of people.
2) then according to the packet of test 1 and method carry out cell-specific phenotypic evaluation.Under Nikon fluorescence microscope, FITC is labeled as green and DAPI is labeled as blueness, calculate the average of CD34, CD133 specific staining cell of ten border downward views except the average of the nucleus (DAPI colouring) giving ten border downward views, equal to positive cell percentage.
Result of the test
As shown in fig. 2 b and fig. 2 c, Fig. 2 B is the curve chart of the conversion ratio of the people's autologous stem cell from the reverse differentiation production of human blood mononuclearcell (PMBC)；Fig. 2 C is the curve chart of the conversion ratio of the mice autologous stem cell from the reverse differentiation production of mouse blood mononuclearcell (PMBC).By Fig. 2 B and Fig. 2 C it can be seen that during the cultivation of the 1st day to the 5th day, autologous stem cell conversion ratio has the trend of rising, at about the 5th day, conversion ratio peaked, and now, the conversion ratio of sample is between 20% to 40%.From this figure it can be seen that be substantially identical by the conversion ratio of the sample of CD34, CD133 antibody characterization, and the conversion ratio of PMBC, MBC is between 20% to 40%.
Test the security authentication of 3 autologous stem cells
Take 1 monthly age Balb/C nude mice 50, male and female half and half, raise in regular grade environment.Mice is randomly divided into three groups, wherein autologous stem cell group 20, tumor cell group 20, blank group 10.
The 20 of autologous stem cell group mices are randomly divided into two groups (first groups and second group), often group 10.First group of mice takes the mode of tail vein transplantation: by the protein induced property self-generation hematopoietic stem cell of preparation in embodiment 2 with 1 × 107The metering of individual cell/only enters in Mice Body from tail vein injections.The mode that second group of mice takes tail vein injection and subcutaneous injection to carry out simultaneously: by the protein induced property self-generation hematopoietic stem cell of preparation in embodiment 2 with 1 × 107The metering of individual cell/only is injected in Mice Body by tail vein injection+hypodermic mode in nape portion.
The 20 of tumor cell group mices are randomly divided into two groups (the 3rd groups and the 4th group), often group 10.3rd group of mice adopts the mode of tail vein transplantation: by melanoma (B16) cell with 1 × 107The metering of individual cell/only enters in Mice Body from tail vein injections.4th group of mice is in hypodermic mode: by melanoma (B16) cell with 1 × 107The metering of individual cell/only is injected in Mice Body from nape portion.
The mice of blank group is the 5th group of mice, is injected in Mice Body in tail vein injection+hypodermic mode in nape portion by the normal saline equal with above each group volume.
After having injected, the mice of different groups is separately raised, and every day observes the formation with or without tuberosity and tumor.Put to death half mice after 21 days, and carry out pathologic finding.Continue to raise residue mice, put to death after observing 12 weeks and make pathologic finding.
Result of the test is shown in that Fig. 3 A to Fig. 3 F, Fig. 3 A is the photo of 105 days after Balb/c nude mice vein transplantation inductivity autologous stem cell；Fig. 3 B is the Balb/c nude mice vein transplantation B16 tumor cell photo of 105 days；Fig. 3 C is the photo of 105 days after Balb/c nude mice by subcutaneous transplanting+vein transplantation inductivity autologous stem cell；Fig. 3 D is the photo of 105 days after Balb/c nude mice by subcutaneous transplanting B16 tumor cell；Fig. 3 E is the internal anatomy of the lung tumor that Balb/c nude mice vein transplantation B16 tumor cell produced after 21 days；Fig. 3 F is the Tumor incidence block diagram of each test group and matched group.
By above-mentioned safety testing, first group, second group and the 5th group of mice do not find after injection 21 days and after 12 weeks an example have tumor apodization as.And the 3rd group and the 4th group of mice occur tumor to become at injection position, 21 days rear sections, after injecting 12 weeks, all find tumor apodizations as.Meanwhile, block diagram is engaged it can be seen that after first group and second group injection autologous stem cell, tumorigenic probability is 0, and the 3rd group and the 4th group is being injected after B16 tumor cell, and tumorigenic probability is 100%.Therefore, the PiHPS that the method for the present invention prepares is used to have higher-security.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all within the spirit and principles in the present invention, any amendment of making, equivalent replacement, improvement etc., should be included within protection scope of the present invention.
Claims (14)
1. the method preparing autologous stem cell, particularly as follows:
Somatic cell being cultivated 3-6 days in cell culture fluid, collect the autologous stem cell generated, wherein, described cell culture fluid is RPMI1640 culture fluid, wherein contains the Y-27632 of 1-100 μM；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 M-CSF of 1-100ng/mL；The granulocyte colony-stimulating factor of 1-100ng/mL；The fucoidin of 1-100 μ g/mL；The dextran sulfate of 1-100 μ g/mL；The 1,1 ' of 1-100nM-[1,4-phenylene two (methylene)]-two-1,4,8,11-tetraazacyclododecane tetradecanes；The M-Alendronate sodium of 1-100 μM；The Pamidronate Disodium of 1-100 μM.
2. method according to claim 1, wherein, containing the Y-27632 of 10 μMs in RPMI1640 culture fluid；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 M-CSF of 10ng/mL；The granulocyte colony-stimulating factor of 10ng/mL；The fucoidin of 10 μ g/mL；The dextran sulfate of 10 μ g/mL；The 1,1 ' of 10nM-[1,4-phenylene two (methylene)]-two-1,4,8,11-tetraazacyclododecane tetradecanes；The M-Alendronate sodium of 10 μMs；The Pamidronate Disodium of 10 μMs.
3. method according to claim 1 and 2, wherein the condition of culture 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 is in the in vitro tissue of human or animal, including one or more in blood, bone marrow fluid, skin and fat.
5. method according to claim 1, wherein, described somatic cell includes cord blood cell, placenta cells, Skin Cell, blood nucleated cell and/or adipose cell.
6. method according to claim 1 and 2, wherein, further, the autologous stem cell freezen protective that will prepare.
7. method according to claim 6, wherein, described freezen protective is: by the autologous stem cell that obtains with 1 × 105Individual/ml to 1 × 106The cell density of individual/ml mixes with the mixed liquor of isopyknic dimethyl sulfoxide and the low molecular dextran of 10%, is cooled to-80 DEG C, is then transferred in the liquid nitrogen of-186 DEG C deep-bed drying.
8. for preparing a test kit for autologous stem cell, including: cell culture fluid, described cell culture fluid is RPMI1640 culture fluid, wherein contains the Y-27632 of 1-100 μM；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 M-CSF of 1-100ng/mL；The granulocyte colony-stimulating factor of 1-100ng/mL；The fucoidin of 1-100 μ g/mL；The dextran sulfate of 1-100 μ g/mL；The 1,1 ' of 1-100nM-[1,4-phenylene two (methylene)]-two-1,4,8,11-tetraazacyclododecane tetradecanes；The M-Alendronate sodium of 1-100 μM；The Pamidronate Disodium of 1-100 μM.
9. for preparing a test kit for autologous stem cell, including: cell culture fluid, described cell culture fluid is RPMI1640 culture fluid, wherein contains the Y-27632 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 M-CSF of 10ng/mL；The granulocyte colony-stimulating factor of 10ng/mL；The fucoidin of 10 μ g/mL；The dextran sulfate of 10 μ g/mL；The 1,1 ' of 10nM-[1,4-phenylene two (methylene)]-two-1,4,8,11-tetraazacyclododecane tetradecanes；The M-Alendronate sodium of 10 μMs；The Pamidronate Disodium of 10 μMs.
10. the application in setting up autologous stem cell storehouse of the test kit according to claim 8 or claim 9.
11. autologous stem cell prepared by the method according to claim 1-7 any one.
12. the application that autologous stem cell according to claim 11 is in setting up autologous stem cell storehouse.
13. the application that autologous stem cell according to claim 11 is in preparing antiaging agent or health product.
14. autologous stem cell according to claim 11 relates to the application in the medicine of the disease that neomorph is reproduced and repaired in preparation.
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| 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 |
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| 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 |
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| 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 |
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