CN105087468B - A kind of method of efficient induction stem cell to pancreatic islet beta sample cell differentiation - Google Patents
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Abstract
The invention discloses a kind of methods of efficiently induction stem cell to pancreatic islet beta sample cell differentiation.This method comprises: handling the cell mass using MET agonist first under the premise of luring aggregation into, reach the method for efficiently induction stem cell to pancreatic islet beta sample cell differentiation.This method helps to improve the efficiency and vigor of stem cell to pancreatic islet element secretory cell differentiation, and then treats because of β cell damage, hyperglycemia caused by insulin secretion absolute magnitude is reduced.
Description
Technical field
The present invention relates to fields of biomedicine, and in particular to after one kind lures that stem cell is reunited into, is handled using MET agonist
The cell mass reaches the method for efficiently induction stem cell to pancreatic islet beta sample cell differentiation.
Background technique
Type-1 diabetes mellitus is the β cell due to excreting insulin because receiving damage when poisoning or autoimmune attack,
Caused by caused hypoinsulinism.Diabetes patient subjects obesity, hyperglycemia, limb movement disorder, injury of kidney
With the pain of eyes lesion.For many years, to eradicate diabetes, people have been look for the regeneration replacement therapy based on tissue and cell
Method.Wherein, stem-cell therapy attracts attention in recent years.It is believed that stem cell induction is become into insulin secreting cell,
And carry out the reliable approach that transplanting is stem cells in treatment of diabetes.There are many method induction stem cell to pancreatic islet beta sample at present
Cell differentiation.But most methods have all suffered from that differentiation efficiency is low, the problem of cell activity difference after differentiation.These problems resistance
The application of stem cells in treatment of diabetes is hindered.
It is that vitro cannot be provided as vivo environment that stem cell cultivates the faced main problem of differentiation in vitro
Whole necessary condition required for stem cell normal growth.The differentiation of stem cell in vivo is also to pass through the letter with its position
After number interaction of molecules, differentiation program can be correctly carried out.The vivo environment of stem cell is by various cell factors, sugar
The multiple extracellular matrix components such as albumen, lipoprotein and various hormones etc. adjust molecule and constitute.Obvious vitro has been difficult to
Stem cell living environment in full analogue body.Which results in stem cell ahead of time become feeble and die, differentiation efficiency it is low and differentiation after cell viability
Phenomena such as bad.Stem cell aggregate structure is the three-dimensional structure that stem cell concentrates and formed, it provides one to stem cell
A relatively natural support environment, and cause the environment of regional hypoxia.Research has shown that under this configuration, stem cell is expressed
Higher stem cell labeling object such as Met has bigger differentiation potential.Studies have shown that Met can regulate and control the life of stem cell
It is long, self-renewing and differentiation.Met is necessary during hepatocyte differentiation.The β cell of Met gene knockout is to dexamethasone
Stimulation it is extremely sensitive, while hindering the response that β cell stimulates glucose.Our research has shown that, activates MET access,
Be conducive to promote mesenchyma stem cell to pancreatic islet cell differentiation.But more natural differentiation is undoubtedly provided after aggregation
Environment, for using MET agonist, effective stimulus stem cell is rolled into a ball to islet beta-like cell differentiation, has no report at present.
Based on principles above, the present invention handles the cell mass using MET agonist, reaches by luring that stem cell is reunited into
Efficiently induction stem cell to pancreatic islet beta sample cell differentiation.
Summary of the invention
Goal of the invention: one kind of the invention efficiently induces the method for stem cell to pancreatic islet beta sample cell differentiation dry by luring into
After aggregation, MET/HGF signal path is activated using MET agonist, can efficiently induce stem cell to pancreatic islet beta sample cell,
And its vigor can be improved.
Technical solution: in order to solve the above-mentioned technical problems, the present invention provides a kind of efficiently induction stem cell to pancreatic islet beta sample
The method of cell differentiation, method includes the following steps: step a: by luring aggregation into；
Step b: using MET agonist activation processing cell mass, stem cell to pancreatic islet beta sample cell differentiation is efficiently induced.
Wherein, above-mentioned stem cell is embryonic stem cell, fat mesenchymal stem cell, mesenchymal stem cells in umbilical cord blood or navel
One or more of band mescenchymal stem cell.
Wherein, above-mentioned steps a lures the method that aggregation is taken into, refers to and suspends on the coated culture plate of gelatin
Culture, to make aggregation.
Wherein, above-mentioned MET agonist refers to the various molecules that can activate MET albumen, including HGF, active peptide, chemistry
One or more of molecule or engineered protein molecule.
A kind of method of efficient induction stem cell to pancreatic islet beta sample cell differentiation, the specific steps are as follows:
1) mesenchymal stem cells in umbilical cord blood is incubated at the coated culture plate of 2% gelatin, in the DMEM containing 20% fetal calf serum
After suspending culture 7 days, start to induce when cell mean diameter of a ball is up to 100 microns；
2) cell is cleaned twice with PBS, is cultivated 24 hours using culture medium I；
3) after 24 hours, PBS cleans cell, and is changed to medium ii, continues culture 48 hours；
4) after 48 hours, PBS cleans cell, and is changed to medium ii I, continues culture 48 hours；
5) after 48 hours, PBS cleans cell, and is changed to culture medium IV, continues culture 24 hours；
6) through ELISA immunological quantitative determining；
7) 160 mg/kg streptozotocins are injected to 7 week old mouse peritoneals to induce generation diabetes；
8) when blood sugar concentration reaches 16.7 mmol/L, every mouse peritoneal injection into liver 2.5 × 106Islet beta-like cell
Detect blood sugar concentration every two weeks later.
The ingredient of the culture medium I are as follows: DMEM/F12,1mM sodium butyrate and 50ng/mL activin A.
The ingredient of the medium ii are as follows: DMEM/F12,0.1-0.2% fetal calf serum and 50ng/mL Activin A.
The ingredient of the medium ii I are as follows: DMEM/F12,50ng/mL KGF/FGF, 10ng/mL RA, 10ng/mL HGF
With 1% fetal calf serum.
The ingredient of the culture medium IV are as follows: DMEM/F12,10ng/mL HGF, 1% B27 and 2% fetal calf serum.
The utility model has the advantages that compared with the existing technology, the invention has the following advantages that the present invention is by luring that stem cell is reunited into
Afterwards, stem cell group is handled using MET agonist, can efficiently induces stem cell to pancreatic islet beta sample cell, and its vigor can be improved,
Realize the present invention.
Detailed description of the invention
Fig. 1 is quantitative determined through ELISA, intracellular (A, B) and extracellular (C, D) outer insulin and C- after breaking up successfully
The content of peptide (C-peptide) dramatically increases；
Successful islet beta-like cell is broken up in Fig. 2 application can make the hyperglycemia of hyperglycemia mouse model be eased.
Specific embodiment
Embodiment 1
A kind of method of efficient induction stem cell to pancreatic islet beta sample cell differentiation, method includes the following steps:
1, mesenchymal stem cells in umbilical cord blood is incubated at the coated culture plate of 2% gelatin, is containing 20% fetal calf serum
The DMEM(Life Technologies of (Ultrapure agarose, Invitrogen, Carlsbad, CA),
Gaithersburg, MD, USA) suspend culture 7 days after, start to induce when cell mean diameter of a ball is up to 100 microns.
2, cell is cleaned twice with PBS, is cultivated 24 hours using culture medium I, the ingredient of culture medium I are as follows: DMEM/F12
(Life Technologies), 1mM sodium butyrate (Sigma, St. Louis, MO, USA), 50ng/mL Activin A
(Sigma)。
3, after 24 hours, PBS cleans cell, and is changed to medium ii, continues culture 48 hours, the ingredient of medium ii
Are as follows: DMEM/F12,0.1-0.2% fetal calf serum, 50ng/mL Activin A.
4, after 48 hours, PBS cleans cell, and is changed to medium ii I, continues culture 48 hours, medium ii I at
It is divided into: DMEM/F12,50ng/mL KGF/FGF (Life Technologies), 10ng/mL RA (Life
Technologies), 10ng/mL HGF, 1% fetal calf serum.
5, after 48 hours, PBS cleans cell, and is changed to culture medium IV, continues culture 24 hours, the ingredient of culture medium IV
Are as follows: DMEM/F12,10ng/mL HGF (Life Technologies), 1% B27 (Life Technologies), 2% tire ox blood
Clearly.
6, through ELISA immunological quantitative determining (Ultrasensitive ELISA kits；Merocodia, Uppsala,
Sweden)), the content of intracellular and extracellular outer insulin and C- peptide dramatically increases (Fig. 1) after breaking up successfully.
7,160 mg/kg streptozotocins (STZ, Sigma, St-Louis, MO, USA) are injected to 7 week old mouse peritoneals to come
Induction generates diabetes.
8, when blood sugar concentration reaches 16.7 mmol/L, every mouse peritoneal injection into liver 2.5 × 106After islet beta-like cell
Blood sugar concentration is detected every two weeks.
9, the hyperglycemia of hyperglycemia mouse model can be made to be eased using the successful islet beta-like cell of differentiation (to scheme
2).
The above is only a preferred embodiment of the present invention, it should be pointed out that: those skilled in the art are come
It says, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (7)
1. a kind of method of efficiently induction stem cell to pancreatic islet beta sample cell differentiation, which is characterized in that this method includes following step
It is rapid:
Step a: by luring aggregation into；The step a lures the method that aggregation is taken into, refers in the coated training of gelatin
It supports and carries out suspension culture on plate, to make aggregation；
Step b: using MET agonist activation processing cell mass, efficiently inducing stem cell to pancreatic islet beta sample cell differentiation,
The stem cell is fat mesenchymal stem cell, one in mesenchymal stem cells in umbilical cord blood or umbilical cord mesenchymal stem cells
Kind is several.
2. a kind of method of efficiently induction stem cell to pancreatic islet beta sample cell differentiation according to claim 1, feature exist
In the MET agonist refers to the various molecules that can activate MET albumen, including HGF, active peptide, chemical molecular or gene
One or more of engineered protein molecule.
3. a kind of method of efficiently induction stem cell to pancreatic islet beta sample cell differentiation according to claim 1, feature exist
In, the specific steps are as follows:
1) mesenchymal stem cells in umbilical cord blood is incubated at the coated culture plate of 2% gelatin, suspends in the DMEM containing 20% fetal calf serum
After culture 7 days, start to induce when cell mean diameter of a ball is up to 100 microns；
2) cell is cleaned twice with PBS, is cultivated 24 hours using culture medium I；
3) after 24 hours, PBS cleans cell, and is changed to medium ii, continues culture 48 hours；
4) after 48 hours, PBS cleans cell, and is changed to medium ii I, continues culture 48 hours；
5) after 48 hours, PBS cleans cell, and is changed to culture medium IV, continues culture 24 hours；
6) through ELISA immunological quantitative determining；
7) 160 mg/kg streptozotocins are injected to 7 week old mouse peritoneals to induce generation diabetes；
8) when blood sugar concentration reaches 16.7 mmol/L, every mouse peritoneal injection into liver 2.5 × 106It is every after islet beta-like cell
Two weeks detection blood sugar concentrations.
4. a kind of method of efficiently induction stem cell to pancreatic islet beta sample cell differentiation according to claim 3, feature exist
In the ingredient of the culture medium I are as follows: DMEM/F12,1mM sodium butyrate and 50ng/mL activin A.
5. a kind of method of efficiently induction stem cell to pancreatic islet beta sample cell differentiation according to claim 3, feature exist
In the ingredient of the medium ii are as follows: DMEM/F12,0.1-0.2% fetal calf serum and 50ng/mL Activin A.
6. a kind of method of efficiently induction stem cell to pancreatic islet beta sample cell differentiation according to claim 3, feature exist
In the ingredient of the medium ii I are as follows: DMEM/F12,50ng/mL KGF/FGF, 10ng/mL RA, 10ng/mL HGF and 1%
Fetal calf serum.
7. a kind of method of efficiently induction stem cell to pancreatic islet beta sample cell differentiation according to claim 3, feature exist
In the ingredient of the culture medium IV are as follows: DMEM/F12,10ng/mL HGF, 1% B27 and 2% fetal calf serum.
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| KR102141641B1 (en) * | 2020-01-31 | 2020-08-06 | 주식회사 래디안 | Method for differentiation of dermal papilla cells from human adipose mesenchymal stem cells |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1298201A1 (en) * | 2001-09-27 | 2003-04-02 | Cardion AG | Process for the production of cells exhibiting an islet-beta-cell-like state |
| CN101160390A (en) * | 2005-04-15 | 2008-04-09 | 北京大学 | Method of inducing embryonic stem cells into pancreatic cells |
| CN101603027A (en) * | 2008-06-12 | 2009-12-16 | 西北农林科技大学 | External evoked human marrow mesenchymal stem cell is divided into the method for insulin-like cell |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1298201A1 (en) * | 2001-09-27 | 2003-04-02 | Cardion AG | Process for the production of cells exhibiting an islet-beta-cell-like state |
| CN101160390A (en) * | 2005-04-15 | 2008-04-09 | 北京大学 | Method of inducing embryonic stem cells into pancreatic cells |
| CN101603027A (en) * | 2008-06-12 | 2009-12-16 | 西北农林科技大学 | External evoked human marrow mesenchymal stem cell is divided into the method for insulin-like cell |
Non-Patent Citations (2)
| Title |
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| 成人骨髓间充质干细胞体外定向诱导分化为胰岛样细胞团的研究;李艳华等;《自然科学进展》;20030630;第13卷(第6期);全文 * |
| 胚胎干细胞向胰岛细胞诱导分化的研究;王波等;《国外医学生物医学工程分册》;20040430;第27卷(第2期);全文 * |
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