CN102399740A - Method and kit allowing reverse differentiation of human somatic cells for generation of autologous pancreatic stem cells and autologous pancreas islet, and application thereof - Google Patents
Method and kit allowing reverse differentiation of human somatic cells for generation of autologous pancreatic stem cells and autologous pancreas islet, and application thereof Download PDFInfo
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Abstract
The invention provides a method allowing reverse differentiation of human somatic cells for generation of autologous pancreatic stem cells and autologous pancreas islet. The method is as follows: culture solutions containing extractives of different plants and different protein components are used to culture human somatic cells step by step, which enables the somatic cells to generate autologous pancreatic stem cells through reverse differentiation, and autologous pancreas islet is generated through further culture. The method provided in the invention enables the first generation of autologous pancreatic stem cells and autologous pancreas islet at a magnitude order of tens of billions to be generated within two to three weeks. The plant and protein-induced human autologous pancreatic stem cells have similar cell specific phenotypes and a plurality of cell differentiation capability as those of human natural pancreatic stem cells; the autologous pancreatic stem cells and autologous pancreas islet cells have immense potential both in the application field of treatment of pancreatic diseases like insulin deficiency type diabetes and in the engineering field of autologous pancreatic tissue; the invention is also applicable to establishment of novel autologous pancreatic stem cell libraries and long-term permanent preservation of autologous pancreatic stem cells.
Description
Technical field
The present invention relates to field of biomedicine technology, particularly, the present invention relates to the reverse differentiation of a kind of people's of making somatocyte and produce from the body pancreatic stem cells with from the cell culture processes and the application thereof of body pancreas islet.
Background technology
In embryo's pancreas in period genesis and development, multipotential stem cell progressively breaks up several kinds of endocrine cells of generation, and what occur the earliest is the cell that produces hyperglycemic-glycogenolytic factor and Regular Insulin, i.e. pancreas islet (Langerhans island).Arrived birth back 2-3 week, the profile of pancreas islet is clearly demarcated, ripe; At this moment, almost can't see the activity of pancreas multipotential stem cell in the pancreas, the activity that has stopped producing new pancreas islet; Moreover; Since produce in the pancreas Regular Insulin islet cells regenerative power very a little less than, cause the various causes of disease of insulin-dependent diabetes (IDDM), losing of pancreas islet degeneration necrosis that causes and pancreas islet is a terminal procedures; Therefore, IDDM patient relies on exogenous insulin throughout one's life.(diabetes mellitus DM) becomes the third-largest disease after cardiovascular disorder and tumour to mellitus.The Along with people's growth in the living standard, environmental factors, the increase of aging population and fat incidence, the sickness rate of mellitus is ascendant trend year by year.Onset diabetes mechanism is complicated, prepares Regular Insulin so far from nineteen twenty-one, and treatment of diabetes is main with medicine, insulin injection.But these methods are the interior glucose level of control agent accurately, can not stop the generation of diabetic complication.
On principle, pancreas or pancreatic islets transplantation are the methods of radical cure insulin-dependent diabetes (IDDM).In decades, American-European countries's research and development allosome pancreas or allosome/Islets Xenotransplantation treatment insulin-dependent diabetes has been obtained the short-term clinical effectiveness.But lack because of the donor tissue source is serious deficient, and lose clinical effectiveness after the immunological rejection that occurs after allosome pancreas or the allosome/Islets Xenotransplantation.Research and development become the task of top priority of solution radical cure insulin-dependent diabetes from the body pancreatic stem cells with from the production technology of body pancreas islet.In recent years, the applying gene recombinant technology is verified, and somatocyte can be by recompile and reverse differentiation, produce to be called as inducibility from body multipotential stem cell (iPS stem cell), for the production technology of autologous stem cells provide new by way of.
Somatocyte is that stem cell forward breaks up generation, has the daughter cell of some concrete function.The chromosomal DNA of somatic chromosomal DNA and stem cell not there are differences on the quantity of gene structure and gene.Topmost difference between somatocyte and the stem cell possibly be that some functional gene is in the different activity expression status.The functional gene expression difference has determined the concrete function of cell and the difference of form.We infer that stem cell is behind the program start that somatocyte forward breaks up, and the switch of the stem cell characteristic of stem cell decision gene itself just is closed, and subsequently, stem cell just has been differentiated to form the somatocyte with certain specific function; In other words, in the somatic chromosome dna sequence dna, still there is pancreatic stem cells characteristic decision gene, for example Pdx-1, Nkx2.2 and Pax7 gene etc., these genes just are in closes or stationary state.Utilize some material to open the gene switchings such as pancreatic stem cells characteristic decision gene Pdx-1, Nkx2.2 and Pax7 in the somatocyte dna sequence dna, these somatocyte just maybe reverse again differentiation and form new pancreatic stem cells, and then is differentiated to form from the body pancreas islet.
Summary of the invention
Technical problem to be solved by this invention is: the reverse differentiation control technique of somatocyte of researching and developing a kind of new people; Do not changing people's somatic chromosome dna sequence dna; Do not insert under the situation of any alien gene or dna fragmentation; Application contains certain plants extract and proteic a series of prescription, at the external pancreatic stem cells characteristic decision gene switching that activates again in the somatocyte dna sequence dna, makes the reverse again differentiation of these somatocyte and forms new pancreatic stem cells and from the body pancreas islet.
In order to solve the problems of the technologies described above, to an object of the present invention is to provide the reverse differentiation of the somatocyte that makes the people and produce method from the body pancreatic stem cells.Another object of the present invention provides and adopts this to produce the method from the body pancreas islet from the body pancreatic stem cells.Another purpose of the present invention provide through aforesaid method obtain from the body pancreatic stem cells with from the body pancreas islet, with and application in the medicine of the multiple disease of preparation treatment.The application that contains plant milk extract and proteic various kinds of cell nutrient solution and this nutrient solution that a further object of the present invention provides in aforesaid method to be adopted.In addition, the present invention also provides and is used to accordingly to prepare from the body pancreatic stem cells with from the test kit of body pancreas islet.
The objective of the invention is to realize through following technical scheme:
On the one hand, the present invention provides the reverse differentiation of a kind of people's of making somatocyte to produce the method from the body pancreatic stem cells, and said method comprises:
People's somatocyte was cultivated 48-72 hour in cell culture fluid C1; Said cell culture fluid C1 is RPMI 1640, and contains Fructus Momordicae charantiae extract 5-100mg/ml, Y-27632 (Sigma company) 1-25 μ M, STEMCELLFACTOR (stem cell factor) 1-100ng/ml, interleukin (IL-3) 1-100ng/ml, interleukin 6 (IL-6) 1-100ng/ml; Using cell culture fluid C2 then instead cultivated 9-15 days; Said cell culture fluid C2 is RPMI 1640; And contain Fructus Momordicae charantiae extract 5-100mg/ml, Rhizoma Gastrodiae extract 5-100mg/ml, Y-276321-25 μ M, STEMCELLFACTOR 1-100ng/ml, interleukin 1-100ng/ml, interleukin 6 1-100ng/ml, thereby acquisition plant and protein induced property are from the body pancreatic stem cells.
Preferably, at 5%CO
2With carry out cell cultures under 37 ℃ of conditions.
In aforesaid method, said people's somatocyte includes but not limited to people's peripheral blood cell, cord blood cell, placenta cells, skin cells, adipocyte.
Preferably, before people's somatocyte is cultivated with cell culture fluid C1, earlier with 2-5 * 10
6Density, at 5%CO
2With cultivation under 37 ℃ of conditions 24-48 hour.
Preferably; Earlier cell was cultivated 72 hours in cell culture fluid C1, said cell culture fluid C1 is the RPMI 1640 that contains Fructus Momordicae charantiae extract 50mg/ml, Y-27632 10 μ M, STEMCELLFACTOR 10ng/ml, interleukin 10ng/ml, interleukin 6 10ng/ml; Using cell culture fluid C2 then instead cultivated 9-12 days; Said cell culture fluid C2 is the RPMI 1640 that contains Fructus Momordicae charantiae extract 50mg/ml, Rhizoma Gastrodiae extract 50mg/ml, Y-27632 10 μ M, STEMCELLFACTOR 10ng/ml, interleukin 10ng/ml, interleukin 6 10ng/ml, thereby acquisition plant and protein induced property are from the body pancreatic stem cells.
On the other hand; The present invention provides the method for a kind of generation from the body pancreas islet; Said method comprises: adopt cell culture fluid C3 above-mentioned plant that obtains of cultivation and protein induced property from the body pancreatic stem cells; Cultivate and formed pancreas islet in 9-12 days; Said cell culture fluid C3 is RPMI 1640, and contains Rhizoma Gastrodiae extract 5-100mg/ml, vitamin PP (Nicotinamide) 1-100ng/ml, Prostatropin (bFGF) 1-100ng/ml, Regular Insulin 1-100ng/ml, transforming growth factor-beta (TGF-β) 1-100ng/ml, type-1 insulin like growth factor (IGF-1) 1-100ng/ml.
Preferably, at 5%CO
2With carry out cell cultures under 37 ℃ of conditions.
Preferably; Plant that obtains and protein induced property are cultivated 9-12 days formation pancreas islet from the body pancreatic stem cells in cell culture fluid C3, said cell culture fluid C3 is the RPMI 1640 that contains Rhizoma Gastrodiae extract 50mg/ml, vitamin PP 10ng/ml, Prostatropin 10ng/ml, Regular Insulin 10ng/ml, transforminggrowthfactor-0ng/ml, type-1 insulin like growth factor 10ng/ml.
On the other hand, the present invention provide by method for preparing from the body pancreatic stem cells with from the body pancreas islet.
It is said from the body pancreatic stem cells with from the application of body pancreas islet that the present invention also provides, and preferably, the present invention provides said and is used for treating the application that relates to the disease medicament that neomorph reproduces and repair in preparation from the body pancreatic stem cells with from the body pancreas islet; Preferably, said disease is the downright bad and various degenerations of pathological lesion, wound of cell, tissue and organs such as system of endocrine tissue, nervous tissue system, liver organization system, renal tissue system; Further preferably, said disease is pancreatic disease and mellitus; More preferably, said disease is acute and chronic pancreatitis, chronic pancreatic gland fibrosis and insulin deficit type mellitus.。
Another aspect; The present invention is provided for making the cell culture fluid of people's the reverse differentiation of somatocyte; Wherein said nutrient solution is selected from cell culture fluid C1, C2 and C3; Said nutrient solution C1 is RPMI 1640; And contain Fructus Momordicae charantiae extract 5-100mg/ml, Y-27632 1-25 μ M, STEMCELLFACTOR 1-100ng/ml, interleukin 1-100ng/ml, interleukin 6 1-100ng/ml; Said cell culture fluid C2 is RPMI 1640; And containing Fructus Momordicae charantiae extract 5-100mg/ml, Rhizoma Gastrodiae extract 5-100mg/ml, Y-27632 1-25 μ M, STEMCELLFACTOR 1-100ng/ml, interleukin 1-100ng/ml, interleukin 6 1-100ng/ml, said cell culture fluid C3 is RPMI 1640, and contains Rhizoma Gastrodiae extract 5-100mg/ml, vitamin PP 1-100ng/ml, Prostatropin 1-100ng/ml, Regular Insulin 1-100ng/ml, transforminggrowthfactor--100ng/ml, type-1 insulin like growth factor 1-100ng/ml.
In addition, the present invention provides above-mentioned cell culture fluid C1 and C2 to make it the application of reverse differentiation generation in the body pancreatic stem cells at the somatocyte of culturing human.
The present invention also provides cell culture fluid C1, C2 and C3 to produce from the body pancreatic stem cells in the reverse differentiation of the somatocyte of culturing human, and then differentiation produces the application in the body pancreas islet.
On the one hand, the present invention is provided for preparing the test kit from the body pancreatic stem cells again, and wherein, said test kit comprises above-mentioned cell culture fluid C1 and C2; Preferably, said test kit further comprises people's somatocyte; Said somatocyte includes but not limited to peripheral blood cell, cord blood cell, placenta cells, skin cells, adipocyte.
The present invention also is provided for preparing the test kit from the body pancreas islet, and wherein, said test kit comprises above-mentioned cell culture fluid C1, C2 and C3; Preferably, said test kit further comprises people's somatocyte; Said somatocyte includes but not limited to peripheral blood cell, cord blood cell, placenta cells, skin cells, adipocyte.
The present invention also provides said test kit to be used for treating the application that relates to the disease medicament that neomorph reproduces and repair from the body pancreatic stem cells with from body pancreas islet and preparation in preparation;
Preferably, said disease is the downright bad and various degenerations of pathological lesion, wound of cell, tissue and organs such as system of endocrine tissue, nervous tissue system, liver organization system, renal tissue system;
Further preferably, said disease is pancreatic disease and mellitus;
More preferably, said disease is acute and chronic pancreatitis, chronic pancreatic gland fibrosis and insulin deficit type mellitus.
Below be detailed description of the present invention:
For completion the present invention relates to following technical problem:
(1) select for use what kind of person somatocyte to produce the raw cell of people from the body pancreatic stem cells as reverse differentiation;
(2) how to handle and cultivate various raw cells;
(3) how to make reverse differentiation production plant of raw cell and protein induced property people from the body pancreatic stem cells;
(4) plant and the protein induced property people that how to detect production are from the body pancreatic stem cells;
(5) how to produce from the body pancreas islet from the differentiation of body pancreatic stem cells from plant and protein induced property;
(6) how to detect production from the body pancreas islet.
Therefore, goal of the invention of the present invention can be achieved through following method:
The people's who selects for use raw material somatocyte includes but not limited to people's people's skin cells, blood nucleated cell, the adipocyte of various somatocyte strains, blood that conventional blood bank preserves and leukocyte suspension, fresh separated preparation of non-immortalization and the immortalization of people's cord blood cell, placenta cells, commercially available acquisition, can be peripheral blood cell, cord blood cell, placenta cells, skin cells, adipocyte.
The somatic cultivation of the raw material of selecting for use comprises: with 2-5 * 10
6Density, with corresponding cell culture fluid at 5%CO
2With cultivation under 37 ℃ of conditions 24-48 hour;
Making the reverse differentiation of raw cell produce the people comprises from the body pancreatic stem cells: with corresponding raw material somatocyte with corresponding cell culture fluid at 5%CO
2After cultivating 24-48 hour under 37 ℃ of conditions, use cell culture fluid C1 (RPMI 1640, Fructus Momordicae charantiae extract 50mg/ml, Y-27632 10 μ M, STEMCELLFACTOR 10ng/ml, interleukin 10ng/ml, interleukin 6 10ng/ml) instead and continue at 5%CO
2With cultivation under 37 ℃ of conditions 72 hours, use cell culture fluid C2 (RPMI1640, Fructus Momordicae charantiae extract 50mg/ml, Rhizoma Gastrodiae extract 50mg/ml, Y-27632 10 μ M, STEMCELLFACTOR 10ng/ml, interleukin 10ng/ml, interleukin 6 10ng/ml) then instead and continue at 5%CO
2Formed plant and protein induced property in 9-15 days from the body pancreatic stem cells with cultivating under 37 ℃ of conditions.
According to one embodiment of the invention; The people who is obtained can detect through following method from the body pancreatic stem cells: specific cell phenotype Pdx-1, Pax7 and the c-Met etc. that utilize pancreatic stem cells are as detecting index; After using cell culture fluid C2 instead and continue cultivating the the the 6th, the 9th, the 12nd and the 15th day; The formation speed, production rate, generation quantity and the purity that adopt flow cytometer, cellular immunofluorescence technology and Western blot western blotting technique to detect pancreatic stem cells respectively (adopt Pdx-1, Pax7 and c-Met etc. as detecting index and detection thereof; " Isolation of Mouse Pancreatic Ductal Progenitor Cells Expressing CD 133and c-Met by Flow Cytometric Cell Sorting " with reference to people such as Yuji Oshima; Gastroenterology; 132 (2), the 720-732 page or leaf; " pancreatic stem cell molecule marker ", the sick magazine of international digestion, 2006,26 (4), General Hospital, Shenyang Military Command's Digestive System Department (110016), Tian Hong, Guo Xiaozhong).
According to another embodiment of the invention; Comprise from the body pancreas islet from body pancreatic stem cells differentiation generation from plant and protein induced property: cultivate plant and the protein induced property pancreatic stem cells that forms through cell culture fluid C2; Cell culture liquid C3 (RPMI1640, Rhizoma Gastrodiae extract 50mg/ml, vitamin PP 10ng/ml, Prostatropin 10ng/ml, Regular Insulin 10ng/ml, transforminggrowthfactor-0ng/ml, type-1 insulin like growth factor 10ng/ml) continues to cultivate 9-12 days, forms pancreas islet.
According to another embodiment of the present invention, can the detecting through following method of production: adopt cellular immunofluorescence technology and the proteic expression of Western blot western blotting technique detection Regular Insulin from the body pancreas islet.
Plant that obtains according to the present invention and protein induced property are from the body pancreatic stem cells; Can be used as the good seed cell that neomorph is reproduced and repaired, to the pathological lesion of system of endocrine tissue, nervous tissue system, liver organization system, renal tissue system or the like cell, tissue and organ, wound is downright bad and various degenerations have the potential that reproduces and repair; Especially to various pancreatic diseases, as: acute and chronic pancreatitis, chronic pancreatic gland fibrosis or the like have the good curing application prospect; Also can be applicable to vitro tissue and organ engineering, produce from body pancreas with from the body pancreas islet.
In addition; Plant that obtains according to the present invention and protein induced property from the body pancreatic stem cells and from the body pancreas islet to various mellitus; Especially insulin deficit property mellitus have the good prospect of essence treatment like type i diabetes, part type ii diabetes and extraordinary mellitus.
Compared with prior art, the present invention has following advantage at least:
One, the present invention is different from the existing reverse differentiation generation of the recombinant gene inductor cell stem cell (iP stem cell) that utilizes; Provide a kind of production from the body pancreatic stem cells with from the new technique method of body pancreas islet; Stem cell (the Plants and Proteins induced pluripotent stem cell that utilizes the reverse differentiation of plant milk extract and protein prescription technological guide somatocyte to produce; The PPiPS stem cell), make the cell result approach the nature stem cell, safe.Particularly; The present invention is not changing the somatic chromosome dna sequence dna through the somatocyte of employing people's routine, does not insert under the situation of any alien gene or dna fragmentation; The plant milk extract of application process screening and optimizing design and protein prescription make the reverse again differentiation of somatocyte and form from the body pancreatic stem cells; And and then be divided into to from the body pancreas islet, the technology original creation is novel, for insulin deficit type treatment of diabetes provides brand-new originating from the body pancreatic stem cells with from the body pancreas islet; Solved the serious deficient scarce problem of field donor tissue such as present pancreas or pancreas islet and other organ transplantations, also be applicable to set up novel from body pancreatic stem cells storehouse.
Two, method of the present invention can directly adopt separation from patient's somatic cell; The reverse differentiation of this somatocyte is produced from the body pancreatic stem cells with from the body pancreas islet; Use this and avoided the immunological rejection that adopts allosome pancreas or allosome/Islets Xenotransplantation to bring, for clinical application provides significant convenience from the body pancreatic stem cells with from the body pancreas islet.
Three, adopt the karyotype that has normal 2 times of bodies from the body pancreatic stem cells with from the body pancreas islet of method preparation of the present invention.In two groups of nude mice transplant experiments, nude mice subcutaneous transplantation injection 1 * 10
8Individual plant and protein induced property pancreatic stem cells or 1 * 10
8The natural embryonic stem cell of individual; Observe inoculation position not tumour formation is arranged; Observe after 90 days continuously; Find to adopt none example of 10 nude mices of test group of plant of the present invention and protein induced property pancreatic stem cells to produce tumour, 10 nude mices of people's nature embryonic stem cell test group all produce tumour, and therefore plant of the present invention and protein induced property pancreatic stem cells have higher security.The present invention still is a kind of in the individual human at any age, all can production from the body pancreatic stem cells with from the novel technical method of body pancreas islet, can be widely used in suffering from the different ages stratum audient colony of relative disease.
Four, proved adopt method of the present invention can make 200ml people's peripheral blood production rate is fast from the body pancreatic stem cells with from the body pancreas islet generation hundreds of hundred million orders of magnitude other the 1st generations in 2 weeks, output reaches 1-1.5 * 10
11Utilization from the body pancreatic stem cells and from the special phenotype of body pancreas islet as detecting index; Plant and protein induced property people from the body pancreatic stem cells and from the technological process of production of body pancreas islet can be by strictly, repeatedly, systematically carry out Flow Cytometry and immunocytochemical technique and detect; Therefore; As from body seed cell and Regeneration and Repair cell; In regenerative medicine, autologous tissue's organ engineering and the industrialization field that produces from the body pancreatic stem cells, plant and protein induced property people autologous stem cells possibly have good potential application foreground, and have heavy industrialization and industrialization production from body pancreas with from the possibility of body pancreas islet.
Description of drawings
Below, specify embodiments of the invention in conjunction with accompanying drawing, wherein:
Among Fig. 1,1-A is blood mononuclearcell (being raw cell); 1-B be the plant that produces from the reverse differentiation of raw cell and protein induced property pancreatic stem cells (Plants and Proteins induced pancreatic pluripotent stem cell, PPiPPS).
Fig. 2 shows that for using the immunofluorescence chemical detection plant and protein induced property pancreatic stem cells have the special phenotype of Pax7, Pdx-1 and C-Met.
Fig. 3 shows that for using the western blotting technique detection plant and protein induced property pancreatic stem cells have Pax7 and the special phenotype of Pdx-1.
Fig. 4 has the special phenotype of Pax7, Pdx-1 and C-Met for detected by flow cytometry shows plant and protein induced property pancreatic stem cells.
Fig. 5 is for inducing the islet cells of generation from plant and protein induced property pancreatic stem cells.
Fig. 6 has shown from plant and protein induced property pancreatic stem cells and induces the islet cells of generation can synthesis secretion Regular Insulin.
Embodiment
Through following detailed description; To be described further working method of the present invention and characteristics and advantage, certainly, although should be noted that described herein below several kinds of embodiment; People also can be according to basis of the present invention, and the form that proposes various variations and individual character also is possible.
The nutrient solution that adopts among the embodiment is:
Cell culture fluid C1:RPMI 1640 contains Fructus Momordicae charantiae extract 50mg/ml, Y-27632 10 μ M, STEMCELLFACTOR 10ng/ml, interleukin 10ng/ml, interleukin 6 10ng/ml;
Cell culture fluid C2:RPMI1640 contains Fructus Momordicae charantiae extract 50mg/ml, Rhizoma Gastrodiae extract 50mg/ml, Y-27632 10 μ M, STEMCELLFACTOR 10ng/ml, interleukin 10ng/ml, interleukin 6 10ng/ml;
Cell culture fluid C3:RPMI1640 contains Rhizoma Gastrodiae extract 50mg/ml, vitamin PP 10ng/ml, Prostatropin 10ng/ml, Regular Insulin 10ng/ml, transforminggrowthfactor-0ng/ml, type-1 insulin like growth factor 10ng/ml.
Reagent that adopts among the embodiment and source thereof:
Fructus Momordicae charantiae extract and Rhizoma Gastrodiae extract are available from Henan natural plant raw material factory and Zhengzhou litchi promise bio tech ltd; Y-27632 and vitamin PP are available from Sigma company; STEMCELLFACTOR, interleukin, interleukin 6, Prostatropin, Regular Insulin, transforming growth factor-beta and type-1 insulin like growth factor are available from R&D company.
Embodiment 1: adopt PeV blood/Cord blood as raw cell, produce plant and protein induced property pancreatic stem cells
The aseptic collection of PeV blood and Cord blood (scientific effort personnel's donations, the Five continents, Beijing woman hospital etc.).Should obtain blood supply or lineal relative's agreement before gathering PeV blood/Cord blood, and write down the heredity of blood supply and family and infect medical history and all relevant virus checking results of hospital.
The conventional anti-freezing of PeV blood/Cord blood, 4 ℃ of preservations.Within 24 hours, deliver to stem cell and make the production center.After the center need be done corresponding virus detection again, get into the centralized computer registration procedure, after the corresponding bar code number of record, blood preparation gets into aseptic stem cell production plant.
At the stem cell production plant, blood carries out the mononuclearcell counting after indicating and using Ficoll standard stripping technique (the 288th page of " modern immunological experiment technology " chapter 12) separation mononuclearcell.Use the DMEM nutrient solution with 2-5 * 10
6Density, at 5%CO
2With cultivation under 37 ℃ of conditions 24-48 hour; Using cell culture fluid C1 instead continues at 5%CO
2With cultivation under 37 ℃ of conditions 72 hours, use cell culture fluid C2 then instead and continue at 5%CO
2With cultivation under 37 ℃ of conditions 12 days.
People to producing detects from the body pancreatic stem cells.Specific cell phenotype Pdx-1, c-met and the Pax7 etc. that utilize pancreatic stem cells are as detecting index; Use cell culture fluid C2 continuation cultivation back the the 6th, the 9th and the 12nd day instead, adopting flow cytometer, cellular immunofluorescence technology and Western blot western blotting technique (" modern immunological experiment technology " chapter 6, chapter 7, the 18 chapter) to detect formation speed, production rate, generation quantity and the purity of pancreatic stem cells respectively.The result finds, the mononuclearcell of 200ml PeV blood/80ml Cord blood the 6th day after cell culture fluid C2 cultivates, and the output of plant and protein induced property pancreatic stem cells can reach 1-1.5 * 10
11(other experimental result is asked for an interview accompanying drawing 1-4).
Microscope shows raw cell and the cellular form from the body pancreatic stem cells of inducing raw cell to produce, respectively shown in Figure 1A and 1B.The special phenotype of Pax7, Pdx-1 and c-met of the inducibility pancreatic stem cells of application cell immunofluorescence chemical technology detection production has been shown among Fig. 2.Use the special phenotype of Pax7, Pdx-1 that Western blot western blotting technique detects plant and protein induced property pancreatic stem cells (PPiPPS), and (blood mononuclear cell on every side is PBMC) as contrast with raw cell.Visible by electrophoresis result, the special phenotype of PPiPPS stem cell Pax7 and Pdx-1 is positive, and the special phenotype of PBMC raw cell Pax7 and Pdx-1 is negative.Fig. 4 is the result of Pax7, Pdx-1 and the special phenotype of c-met of the inducibility pancreatic stem cells of application flow cytometer technology for detection production.As shown in the figure, to cultivate the 9th day with the C2 nutrient solution, the formation speed of Pax7, Pdx-1 and the special phenotype of c-Met is respectively 20.05%, 28.05% and 12.88%.
Plant that is produced and protein induced property people can be through freezing prolonged preservation from the body pancreatic stem cells.Inhale and to beat the plant and the protein induced property pancreatic stem cells of suspension adherent growth and collect in the centrifuge tube of 50ml milliliter, in whizzer with 1500rpm, 4 ℃ centrifugal 10 minutes, abandon supernatant.The plant of preparation and protein induced property pancreatic stem cells are with 1 * 10
10/ ml and DMSO mix, and are cooled to-80 ℃ with 10%DMSO and low molecular dextran concentration, transfer to profound hypothermia preservation in liquid nitrogen (186 ℃) liquid phase then.
Embodiment 2: adopt PeV blood/Cord blood as raw cell, produce from the body pancreas islet
Plant and protein induced property that embodiment 1 obtains produce from the body pancreas islet through differentiation from the body pancreatic stem cells.
After cell culture fluid C2 cultivates formation plant and protein induced property pancreatic stem cells, use the C3 cell culture fluid and continue to cultivate 9-12 days, form pancreas islet.The mononuclearcell of 200ml PeV blood/80ml Cord blood 9-12 days after cell culture fluid C3 cultivates, the output of plant and protein induced property pancreatic cell can reach 0.6-1 * 10
11
Detect from the body pancreas islet what produce.Adopt the proteic expression of cellular immunofluorescence technology for detection Regular Insulin (specifically referring to chief editors such as Shen Guanxin, " modern immunological experiment technology ", the 2nd edition, Hubei science tech publishing house, 1998), detected result is asked for an interview accompanying drawing 5-6.
Fig. 5 shows by the plant of embodiment 1 and protein induced property pancreatic stem cells through inducing the islet cells of generation, and Fig. 6 has shown that this islet cells can synthesis secretion Regular Insulin.
Embodiment 3
Present embodiment carries out the safety research that nude mice is transplanted to the plant and the protein induced property of preparation among the embodiment 1 from the body pancreatic stem cells, and is specific as follows.
In two groups of nude mice transplant experiments, nude mice subcutaneous transplantation injection 1 * 10
8The pancreatic stem cells of individual plant and protein induced property or 1 * 10
8Individual's natural pancreatic stem cells (Hainan Medical College's reproductive center is given), observing inoculation position has not tumour formation.Observed 90 days continuously.
The result shows that none example of plant and 10 nude mices of protein induced property pancreatic stem cells test group produces tumour, has higher security; And 10 nude mices of people's nature embryonic stem cell test group all produce tumour.
Claims (13)
1. one kind makes people's the reverse differentiation of somatocyte produce the method from the body pancreatic stem cells, and wherein, said method comprises:
People's somatocyte was cultivated 48-72 hour in cell culture fluid C1; Said cell culture fluid C1 is RPMI 1640, and contains Fructus Momordicae charantiae extract 5-100mg/ml, Y-27632 1-25 μ M, STEMCELLFACTOR 1-100ng/ml, interleukin 1-100ng/ml, interleukin 6 1-100ng/ml; Using cell culture fluid C2 then instead cultivated 9-15 days; Said cell culture fluid C2 is RPMI 1640; And contain Fructus Momordicae charantiae extract 5-100mg/ml, Rhizoma Gastrodiae extract 5-100mg/ml, Y-27632 1-25 μ M, STEMCELLFACTOR 1-100ng/ml, interleukin 1-100ng/ml, interleukin 6 1-100ng/ml, thereby acquisition plant and protein induced property are from the body pancreatic stem cells;
Preferably, at 5%CO
2With carry out cell cultures under 37 ℃ of conditions.
The method of claim 1, wherein said people's somatocyte include but not limited to the people around hemocyte, cord blood cell, placenta cells, skin cells, adipocyte;
Preferably, before people's somatocyte is cultivated with cell culture fluid C1, earlier with 2-5 * 10
6Density, at 5%CO
2With cultivation under 37 ℃ of conditions 24-48 hour;
Preferably; Earlier cell was cultivated 72 hours in cell culture fluid C1, said cell culture fluid C1 is the RPMI 1640 that contains Fructus Momordicae charantiae extract 50mg/ml, Y-27632 10 μ M, STEMCELLFACTOR 10ng/ml, interleukin 10ng/ml, interleukin 6 10ng/ml; Using cell culture fluid C2 then instead cultivated 9-12 days; Said cell culture fluid C2 is the RPMI 1640 that contains Fructus Momordicae charantiae extract 50mg/ml, Rhizoma Gastrodiae extract 50mg/ml, Y-27632 10 μ M, STEMCELLFACTOR 10ng/ml, interleukin 10ng/ml, interleukin 6 10ng/ml, thereby acquisition plant and protein induced property are from the body pancreatic stem cells.
3. a generation is from the method for body pancreas islet; Wherein, Said method comprises: adopt cell culture fluid C3 to cultivate 9-12 days formation pancreas islet from the body pancreatic stem cells according to claim 1 or claim 2 plant and protein induced property; Said cell culture fluid C3 is RPMI 1640, and contains Rhizoma Gastrodiae extract 5-100mg/ml, vitamin PP 1-100ng/ml, Prostatropin 1-100ng/ml, Regular Insulin 1-100ng/ml, transforminggrowthfactor--100ng/ml, type-1 insulin like growth factor 1-100ng/ml;
Preferably, at 5%CO
2With carry out cell cultures under 37 ℃ of conditions;
Preferably; Plant as claimed in claim 1 and protein induced property are cultivated 9-12 days formation pancreas islet from the body pancreatic stem cells in cell culture fluid C3, said cell culture fluid C3 is the RPMI1640 that contains Rhizoma Gastrodiae extract 50mg/ml, vitamin PP 10ng/ml, Prostatropin 10ng/ml, Regular Insulin 10ng/ml, transforminggrowthfactor-0ng/ml, type-1 insulin like growth factor 10ng/ml.
According to claim 1 or claim 2 method produce from the body pancreatic stem cells.
As the said method of claim 3 produce from the body pancreas islet.
6. be used for treating the application that relates to the disease medicament that neomorph reproduces and repair as claim 4 is said in preparation from the body pancreatic stem cells;
Preferably, said disease is the downright bad and various degenerations of pathological lesion, wound of cell, tissue and organs such as system of endocrine tissue, nervous tissue system, liver organization system, renal tissue system;
Further preferably, said disease is pancreatic disease and mellitus;
More preferably, said disease is acute and chronic pancreatitis, chronic pancreatic gland fibrosis and insulin deficit type mellitus.
7. as claimed in claim 5ly be used for treating the application that relates to the disease medicament that neomorph reproduces and repair in preparation from the body pancreas islet;
Preferably, said disease is the downright bad and various degenerations of pathological lesion, wound of cell, tissue and organs such as system of endocrine tissue, nervous tissue system, liver organization system, renal tissue system;
Further preferably, said disease is pancreatic disease and mellitus;
More preferably, said disease is acute and chronic pancreatitis, chronic pancreatic gland fibrosis and insulin deficit type mellitus.
8. cell culture fluid that is used to make people's the reverse differentiation of somatocyte; Wherein, Said nutrient solution is selected from cell culture fluid C1, C2 and C3; Said nutrient solution C1 is RPMI 1640, and contains Fructus Momordicae charantiae extract 5-100mg/ml, Y-27632 1-25 μ M, STEMCELLFACTOR 1-100ng/ml, interleukin 1-100ng/ml, interleukin 6 1-100ng/ml; Said cell culture fluid C2 is RPMI1640, and contains Fructus Momordicae charantiae extract 5-100mg/ml, Rhizoma Gastrodiae extract 5-100mg/ml, Y-276321-25 μ M, STEMCELLFACTOR 1-100ng/ml, interleukin 1-100ng/ml, interleukin 6 1-100ng/ml; Said cell culture fluid C3 is RPMI 1640, and contains Rhizoma Gastrodiae extract 5-100mg/ml, vitamin PP 1-100ng/ml, Prostatropin 1-100ng/ml, Regular Insulin 1-100ng/ml, transforminggrowthfactor--100ng/ml, type-1 insulin like growth factor 1-100ng/ml.
9. make it the application of reverse differentiation generation in the body pancreatic stem cells like said cell culture fluid C1 of claim 8 and C2 at the somatocyte of culturing human.
10. cell culture fluid C1 as claimed in claim 8, C2 and C3 produce from the body pancreatic stem cells in the reverse differentiation of the somatocyte of culturing human, and then differentiation produces the application in the body pancreas islet.
11. one kind is used to prepare the test kit from the body pancreatic stem cells, wherein, said test kit comprises cell culture fluid C1 as claimed in claim 8 and C2;
Preferably, said test kit further comprises people's somatocyte; Said somatocyte includes but not limited to people's hemocyte, cord blood cell, placenta cells, skin cells, adipocyte on every side.
12. one kind is used to prepare the test kit from the body pancreas islet, wherein, said test kit comprises cell culture fluid C1 as claimed in claim 8, C2 and C3;
Preferably, said test kit further comprises people's somatocyte; Said somatocyte includes but not limited to people's hemocyte, cord blood cell, placenta cells, skin cells, adipocyte on every side.
13. be used for treating the application that relates to the disease medicament that neomorph reproduces and repair from the body pancreatic stem cells with from body pancreas islet and preparation in preparation like claim 11 or 12 described test kits;
Preferably, said disease is the downright bad and various degenerations of pathological lesion, wound of cell, tissue and organs such as system of endocrine tissue, nervous tissue system, liver organization system, renal tissue system;
Further preferably, said disease is pancreatic disease and mellitus;
More preferably, said disease is acute and chronic pancreatitis, chronic pancreatic gland fibrosis and insulin deficit type mellitus.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102890870A CN102399740A (en) | 2010-09-19 | 2010-09-19 | Method and kit allowing reverse differentiation of human somatic cells for generation of autologous pancreatic stem cells and autologous pancreas islet, and application thereof |
| PCT/CN2011/001394 WO2012034353A1 (en) | 2010-09-19 | 2011-08-22 | Method and kit for generating autologous pancreatic stem cell and autologous islet through reverse differentiation of human somatic cell, and use thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102890870A CN102399740A (en) | 2010-09-19 | 2010-09-19 | Method and kit allowing reverse differentiation of human somatic cells for generation of autologous pancreatic stem cells and autologous pancreas islet, and application thereof |
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| CN (1) | CN102399740A (en) |
| WO (1) | WO2012034353A1 (en) |
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|---|---|---|---|---|
| CN111840207A (en) * | 2019-04-10 | 2020-10-30 | 上海交通大学医学院附属上海儿童医学中心 | In-vivo implantable microporous bag and using method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU778929B2 (en) * | 1999-12-06 | 2004-12-23 | General Hospital Corporation, The | Pancreatic stem cells and their use in transplantation |
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- 2010-09-19 CN CN2010102890870A patent/CN102399740A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111840207A (en) * | 2019-04-10 | 2020-10-30 | 上海交通大学医学院附属上海儿童医学中心 | In-vivo implantable microporous bag and using method and application thereof |
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| WO2012034353A1 (en) | 2012-03-22 |
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