CN106676057A - Serum-free medium for inducing differentiation of umbilical cord mesenchymal stem cells into insulin secretory-like cells and preparation method and application thereof - Google Patents
Serum-free medium for inducing differentiation of umbilical cord mesenchymal stem cells into insulin secretory-like cells and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a novel serum-free medium. The medium includes high-glucose DMEM (glucose content is 4.5g/L), B27, recombinant human basic fibroblast growth factor (b-FGF), nicotianamine, N-2, vinblastine III (Conophylline), non-essential amino acid (NEAA), heparin (Heparin), epidermal growth factor (EGF), hepatocyte growth factor (HGF), serum replacement (SR), an insulin-transferrin-selenium compound (ITS), and pentagastrin. By the utilization of the medium of the invention, mesenchymal stem cells can be differentiated into insulin secretory-like cells within 6 days by a one-step method.
Description
Technical field
The present invention relates to the research field of stem cell, more particularly to a kind of new, efficiently dry thin suitable for mesenchyme
Born of the same parents induce serum-free medium of differentiation and its production and use to insulin secretion like cell.
Background technology
Diabetes have become a kind of 21 century worldwide epidemic diseases, are the third-largest after tumor, vascular lesion
The Chronic Non-Communicable Diseasess of serious threat human health, the feature with high fatality rate, high disability rate and high health care costs.Mesh
Up to 1.14 hundred million, quantity reaches the first in the world to front diabetes mellitus in China patient numbers, account for global diabetes patient sum three/
One.Therefore, the severe public health problem that diabetes have become our times various countries, particularly China faces.
After 20 middle of century, with rise and the development of human organ transplant technology, pancreas transplantation is used as diabetes
A kind of Therapeutic Method is introduced into medical field.But it is possible to for the organ transplanted taking needed for limited amount, and organ transplantation after all
With also usually allowing people to hang back, and organ transplantation often causes stronger immunologic rejection, and these problems all perplex always
Doctor and patient.
In recent years, the fast development of theory of stem cell and technology brings new hope to the treatment of diabeticss.Especially
It is that (i.e. adult stem cell can be for the transdifferentiationof ability of adult stem cell with transdifferentiationof under certain microenvironment effect
Specific cell and tissue) confirmation, provide another alternative cell derived for cellular transplantation therapy diabetes.Grind
Study carefully and show, it is thin that medulla mesenchyma cell, adult fat's cell, navel blood stem cell can be divided under given conditions islets of langerhans sample
Born of the same parents, these cells in vitro can excreting insulin, it is expected to become another cell derived of diabetes cellular transplantation therapy.
Umbilical cord mesenchymal stem cells originate from mesoderm, with sub- totipotential differentiation potential, in specific inside and outside microenvironment
Under the conditions of, can be to the histiocyte differentiation of three germinal layers.Compared with other adult stem cells, umbilical cord mesenchymal stem cells are drawn materials
Convenient, abundance uses the umbilical cord tissue for being generally viewed as Biohazard Waste, without problems of morals principles.Additionally, umbilicuss
Band mescenchymal stem cell proliferation rates in vitro is fast, and immunogenicity is relatively low, and exogenous pollution is few, thus with more research valency
Value.
The content of the invention
The present inventor's research obtains a kind of new, efficient suitable for mesenchyma stem cell to pancreatic islet element secretion sample
The serum-free medium of cell induction differentiation, can be by umbilical cord mesenchymal stem cells serum-free environment in vitro using this culture medium
Under, insulin secretion like cell was induced to differentiate into through 6-7 days.
It is an object of the present invention to provide a kind of nothing that umbilical cord mesenchymal stem cells are induced to differentiate into insulin secretion like cell
Blood serum medium, using the serum-free medium can rapid induction be insulin within 6 days by umbilical cord mesenchymal stem cells
Secretion like cell.
It is a further object of the present invention to provide the preparation method of the culture medium.
The a further object of the present invention is to provide a kind of method using the culture medium induction differentiation umbilical cord mesenchymal stem cells.
It is yet another object of the invention to provide the purposes of the culture medium.
Technical scheme is as follows.
On the one hand, the present invention provides a kind of inducing umbilical cord mesenchymal stem (UC-MSCs) that is used for insulin secretion sample
The serum-free medium of cell differentiation, the serum-free medium is included：DMEM in high glucose, serum substitute (SR), B27 serum-frees
Additive, Insulin-Transferrin-selenium compound (ITS), non essential amino acid (NEAA), N-2 serum-free additives, and
Heparin (Heparin), vincaleucoblastine III (Conophylline), Vitamin B3 (Nicotinamide) and recombination human basic are into fibre
Dimension somatomedin (b-FGF), epithelical cell growth factor (EGF), hepatocyte growth factor (HGF) and pentagastrin
(Pentagastrin)。
Preferably, in terms of 100 parts by volume, the serum-free medium is included：The glucose content of 85-95 parts by volume is
The DMEM in high glucose of 4.5g/L, the serum substitute (SR) of 5-8 parts by volume, B27 serum-free additives (50 ×) of 1-4 parts by volume,
Insulin-Transferrin-the selenium compound (ITS) of 1-1.5 parts by volume, non essential amino acid (NEAA) water of 0.5-2 parts by volume
N-2 serum-free additives (100 ×) of solution, 0.5-2 parts by volume, and the final concentration of 0.5- in the serum-free medium
It is the heparin (Heparin) of 2ug/ml, the vincaleucoblastine III (Conophylline) of final concentration of 50-200ng/ml, final concentration of
The Vitamin B3 (Nicotinamide) of 5-20mmol/L and final concentration are respectively the recombination human basic fibroblast life of 5-20ng/ml
The long factor (b-FGF), epithelical cell growth factor (EGF), hepatocyte growth factor (HGF) and pentagastrin
(Pentagastrin)。
It is highly preferred that in terms of 100 parts by volume, the serum-free medium is included：The glucose content of 89 parts by volume is
The DMEM in high glucose of 4.5g/L, the serum substitute (SR) of 5 parts by volume, B27 serum-free additives (50 ×) of 2 parts by volume, 1 volume
Part Insulin-Transferrin-selenium compound (ITS), non essential amino acid (NEAA) aqueous solution of 1 parts by volume, 1 parts by volume
N-2 serum-free additives (100 ×), and in the serum-free medium heparin (Heparin) of final concentration of 1ug/ml,
The vincaleucoblastine III (Conophylline) of final concentration of 100ng/ml, the Vitamin B3 of final concentration of 10mmol/L
And final concentration is respectively recombination human basic fibroblast somatomedin (b-FGF), the epidermis cell of 10ng/ml (Nicotinamide)
Somatomedin (EGF), hepatocyte growth factor (HGF) and pentagastrin (Pentagastrin).
Wherein, the non-essential amino aqueous acid is respectively glycine, alanine, the L- Tianmens of 8-12mM comprising concentration
Amide, L-Aspartic acid, glutamic acid, proline and serine.
According to the specific embodiment of the application, the non-essential amino aqueous acid can adopt Gibco companies article No.
For 11140 product.
According to the specific embodiment of the application, the serum substitute can adopt KnockOutTMSerum
Replacement (Gibco Products, article No. 10828-010).
According to the specific embodiment of the application, the B27 serum-frees additive (50 ×) can adopt Gibco companies goods
Number for 17504044 product.
According to the specific embodiment of the application, the Insulin-Transferrin-selenium compound (ITS) can adopt
Sigma companies article No. is the product of I3146.
According to the specific embodiment of the application, the N-2 serum-frees additive (100 ×) can adopt Gibco companies
Article No. is 17502048 product.
Counted as 100mL with the total amount of the serum-free medium, its composition can be situation shown in table 1 below.
Table 1
Preferably, the composition of the serum-free medium can be situation shown in table 2 below.
Table 2
| Composition | Consumption |
| DMEM in high glucose culture medium (glucose content 4.5g/L) | 89ml |
| SR | 5ml |
| B27(50X) | 2ml |
| ITS | 1ml |
| N-2(100X) | 1ml |
| NEAA | 1ml |
| Heparin | 100ug |
| Conophylline(100μg/ml) | 10μg |
| Nicotinamide | 1mmol |
| b-FGF(1μg/ml) | 1μg |
| EGF | 1μg |
| HGF | 1μg |
| Pentagastrin | 1μg |
For the serum-free medium of the present invention, the umbilical cord mesenchymal stem cells are dry thin for the umbilical cord mesenchyma of people source
Born of the same parents (hUC-MSCs), between the people's umbilical cord preferably isolated from spontaneous labor or the fresh umbilical cord tissue of caesarean delivered healthy newborn
Mesenchymal stem cells.
On the other hand, the present invention provides the preparation method of above-mentioned serum-free medium, and the preparation method includes：By each
Composition mix homogeneously.
Further aspect, the present invention provides a kind of inducing umbilical cord mesenchymal stem that is used for and breaks up to insulin secretion like cell
Method, methods described includes：Using serum-free medium culture umbilical cord mesenchymal stem cells of the present invention.
Preferably, the umbilical cord mesenchymal stem cells are the umbilical cord mesenchymal stem cells of people source, more preferably from nature
The human umbilical cord mesenchymal stem cells that childbirth or the fresh umbilical cord tissue of caesarean delivered healthy newborn are isolated.
Preferably, the method comprising the steps of：
(1) by umbilical cord mesenchymal stem cells with 2-6 × 104Individual cell/cm2Density be inoculated in the serum-free medium
In, then in CO25%th, cultivate at 37 DEG C, such as in CO2Concentration is in 5% 37 DEG C of constant incubators；
(2) 4min is centrifuged under 300-800rpm per the 2-3 days cell cultures by step (1), old culture medium is abandoned in suction, more
It is changed to the fresh serum-free medium；For example, old culture medium cell suspension is slowly drawn in centrifuge tube with pipet,
Low-speed centrifugal 4min under 300-800rpm, then inhales and abandons old culture medium, fresh inducing culture of the present invention is replaced by, per 2-3 days
Change liquid once；
(3) cultivate 6-7 days, harvesting；
(4) the induction differentiation effect of institute's harvesting of detecting step (3).
Wherein, the step (4) includes one or more (preferred whole item) in detection following items：Unit cell pancreas
Island element burst size；The expression of insulin releasing related gene PDX-1, INSULIN and NGN-3；Nuclear extract PDX-1 and endochylema
The presence of protein I nsulin；The positive expression rate of insulin secretion like cell specificity marker PDX-1, NKX6.1, Insulin.
It is highly preferred that the method comprising the steps of：
(1) by umbilical cord mesenchymal stem cells with 5 × 104Individual cell/cm2Density be inoculated in it is ultralow absorption six well culture plates
In the serum-free medium in, then in CO25%th, cultivate at 37 DEG C, such as in CO2Concentration is 5% 37 DEG C of constant temperature training
In foster case；
(2) cell culture of step (1) is centrifuged into 4min under 500rpm per 2 days, old culture medium is abandoned in suction, is replaced by new
The fresh serum-free medium；For example, with pipet old culture medium cell suspension is slowly drawn in centrifuge tube, in 500rpm
Lower low-speed centrifugal 4min, then inhales and abandons old culture medium, is replaced by fresh inducing culture of the present invention, and liquid was changed once per 2 days；
(3) cultivate 6-7 days, harvesting；
(4) the induction differentiation effect of institute's harvesting of detecting step (3).
In step (1), umbilical cord mesenchymal stem cells (UC-MSCs) are preferably the umbilical cord mesenchymal stem cells of people source
(hUC-MSCs) between the people's umbilical cord, more preferably isolated from spontaneous labor or the fresh umbilical cord tissue of caesarean delivered healthy newborn
Mesenchymal stem cells.
Another aspect, the present invention provides purposes of the serum-free medium in stem cell culture medium is prepared.
Wherein, the stem cell is isolated from mammal, the tissue of such as people or organ, the tissue or organ
For one or more in bone marrow, umbilical cord and fat；Preferably, the stem cell does for the umbilical cord mesenchyma of mammal source
The umbilical cord mesenchymal stem cells of cell, more preferably people source；It is further preferred that the stem cell is from spontaneous labor or cuts open
The human umbilical cord mesenchymal stem cells that the fresh umbilical cord tissue of healthy newborn that is produced from palace is isolated.
Inventor's research finds that the new mesenchyma stem cell to pancreatic islet element secretion like cell induction for providing of the invention is broken up
Serum-free medium do not contain serum composition, it is to avoid because serum batch difference causes cell growth mistake in cell cultivation process
The unstable situation of journey, also eliminates and propagates the dangerous possibility of xenogenesis pathogen；Also, the serum-free medium is realized quickly
It is insulin secretion sample stem cell (only needing 6 day time) by mesenchyma stem cell differentiation induction, is the In vitro culture of zooblast
There is provided a kind of efficient solution.
Description of the drawings
Hereinafter, with reference to accompanying drawing describing embodiment of the present invention in detail, wherein：
Fig. 1 be culture medium constitute screening process in cell mass quantity (50 μm of diameter ＞) with incubation time variation tendency
Figure.
Fig. 2 is the comparison of the unit cell insulin release in the culture medium of variable concentrations pentagastrin.
Fig. 3 be variable concentrations ITS culture medium in cellular morphology comparison, wherein Fig. 3 A be 0.5 volume ITS induce 6 days
Cellular morphology, Fig. 3 B are that 1.0 volumes ITS induce the cellular morphology of 6 days, and Fig. 3 C are the cell shape that 2.0 volumes ITS are induced 6 days
State.
Fig. 4 is to insulin secretion like cell atomization using the culture medium inducing umbilical cord mesenchymal stem of the present invention
In (in 10 days) cell mass quantity (100 μm of diameter ＞) with incubation time changing trend diagram.
Fig. 5 is to insulin secretion like cell atomization using the culture medium inducing umbilical cord mesenchymal stem of the present invention
In (in 6 days) cell mass metamorphosis.
Fig. 6 is the insulin secretion like cell specific cell that flow cytometry analysis Jing culture medium inductions of the present invention are obtained
Surface molecular result, Fig. 6 A are PDX-1 changes before and after induction, and Fig. 6 B are NKx6.1 changes before and after induction, before and after Fig. 6 C are for induction
Insulin changes, before left figure is for induction, after right figure was for induction 6 days.
Fig. 7 is that the insulin secretion like cell that umbilical cord mesenchymal stem cells Jing culture medium induction differentiation of the present invention is obtained is special
Property gene transcriptional level RT-PCR analyze, Fig. 7 A for induction before, Fig. 7 B for induction 6 days after；Wherein M is nucleic acid molecular weight mark
Standard, 1 is reference gene GAPDH, and 2 is PDX-1, and 3 is INSULIN, and 4 is NGN-3.
Fig. 8 is that the insulin secretion like cell that umbilical cord mesenchymal stem cells Jing culture medium induction differentiation of the present invention is obtained is special
Property gene protein level analysis, wherein Fig. 8 A be nucleoprotein PDX-1 dyeing, Fig. 8 B be plasmosin INSULIN dye, scheme
8C folds figure to merge.
Fig. 9 is that umbilical cord mesenchymal stem cells induce the insulin secretion sample stem cell for obtaining low through culture medium of the present invention
Sugar and high sugar are per 104Cell uelralante amount is detected, wherein negative group is before induction, it is positive right after induction group was for induction 6 days
It is insulinoma cell according to group.
Specific embodiment
The present invention is illustrated referring to specific embodiment.It will be appreciated by those skilled in the art that these embodiments are only
For illustrating the present invention, it limits never in any form the scope of the present invention.
Experimental technique in following embodiments, if no special instructions, is conventional method.Medicine used in following embodiments
Material raw material, reagent material etc., if no special instructions, are commercially available purchase product.
The NEAA adopted in embodiment is non-essential amino aqueous acid, its include concentration be the glycine of 10mM, third
Propylhomoserin, L- Tianmens amide, L-Aspartic acid, glutamic acid, proline and serine.
EmbodimentThe screening of 1 culture medium composition
Basal medium：DMEM (glucose content 4.5g/L)+5%SR+1%NEAA+2%B27 (50X)+1%N-2
(100X)+10ng/ml HGF+10ng/ml EGF+10ng/ml b-FGF+10mmol/L Nicotinamide；
Screening composition：Add 100ng/ml vincaleucoblastines III (Conophylline), 1 parts by volume in basal medium
ITS, final concentration of 1ug/ml heparin (Heparin) and final concentration are 10ng/ml betacellulins
(betacellulin), saliva element 4 (Exendi-4), insulin like growth factor (IGF-1) and pentagastrin.Each packet feelings
Condition is as shown in table 3 below.
Table 3
In Biohazard Safety Equipment, the hUC- in the 3rd generation for being located away from spontaneous labor neonatal umbilical cord China formula glue tissue is taken
MSCs, with 5 × 104Individual cell/cm2Density is inoculated in six orifice plates of ultralow absorption, and per hole the tested training of each group shown in 2ml tables 3 is added
Foster base, observation of cell growing state, the agglomerating quantity of observation of cell.
As a result：In first group of culture medium, cell mass quantity is few；Second and third, with the addition of Herba Catharanthi Rosei successively in four groups of culture medium
Alkali III, pentagastrin and heparin, cell mass quantity increased, but cell mass structure is not compact；5th group of cell mass number
Amount is most, while cell mass Maturity is best；Six, the seven, eight groups are added successively betacellulin, saliva element 4, Insulin-Like
Somatomedin, but these three factors increase impact less in cell mass number.As a result referring to Fig. 1.
Embodiment 2The screening of medium component (vincaleucoblastine III) content
Tested culture medium：The DMEM in high glucose (glucose content 4.5g/L) of 89 parts by volume, the serum substitute of 5 parts by volume
(SR), the B27 (50X) of 2 parts by volume, the ITS of 1 parts by volume, the non essential amino acid (NEAA) of 1 parts by volume, the N-2 of 1 parts by volume
(100X), the heparin (Heparin) of final concentration of 1ug/ml, the Vitamin B3 of 10mmol/L, final concentration are the weight of 10ng/ml
Group human alkaline fibroblast somatomedin (b-FGF), epithelical cell growth factor (EGF), hepatocyte growth factor (HGF), pentapeptide
Gastrin and Concentraton gradient are 1,10,50,100,200, the vincaleucoblastine III (Conophylline) of 300ng/ml.
Human umbilical cord mesenchymal is carried out using the culture medium of above-mentioned variable concentrations vincaleucoblastine III (Conophylline) dry thin
The induction differentiation culture of born of the same parents.
As a result：Concentration be 1 and two groups of tested culture medium of 10ng/ml vincaleucoblastines III in, cell mass edge occur trip
From cell attachment, extend with incubation time, cell mass is loose, shows that cell mass Maturity is inadequate；It is 50,100 and in concentration
In three groups of tested culture medium of 200ng/ml vincaleucoblastines III, cell mass is compact, and cell mass gradually increases, and shows that cell mass increases
Grow in order；In concentration is tested group of 300ng/ml vincaleucoblastines III, cell mass color burn, but cell mass border cell
Form generation changes, and shows that differentiated cell there occurs that non-directional breaks up.
EmbodimentThe screening of 3 medium components (pentagastrin) content
Tested culture medium：The DMEM in high glucose (glucose content 4.5g/L) of 89 parts by volume, the serum substitute of 5 parts by volume
(SR), the B27 (50X) of 2 parts by volume, the ITS of 1 parts by volume, the non essential amino acid (NEAA) of 1 parts by volume, final concentration of 1ug/
Ml heparin (Heparin), the N-2 (100X) of 1 parts by volume, the vincaleucoblastine III (Conophylline) of 100ng/ml, 10mmol/
The Vitamin B3 of L, final concentration be the recombination human basic fibroblast somatomedin (b-FGF) of 10ng/ml, epidermal growth because
Sub (EGF), hepatocyte growth factor (HGF) and Concentraton gradient are 1,2,5,10,20,30, the pentagastrin of 50ng/ml.
The induction differentiation training of human umbilical cord mesenchymal stem cells is carried out using the culture medium of above-mentioned variable concentrations pentagastrin
Support, then using sugared stimulation test, analytical unit cell (1 × 104It is individual) each group insulin release, carry out each group cell
Relatively.Wherein sugared stimulation test is carried out as follows：The culture cell mass of 6 days is collected, 2ml 25mM/L glucose DMEM are separately added into
Stimulate liquid, gently dispel mixing cell mass, 37 DEG C, after stimulating 2 hours supernatant is collected, surveyed with insulin ELISA kit and collected
Supernatant, it is final to read OD450 values.
As a result：Pentagastrin concentration be 5,10, the cell that breaks up in the culture medium of 20ng/ml there is higher unit
Cell insulin release effect.As a result referring to Fig. 2.
EmbodimentThe screening of 4 medium components (ITS) content
Tested culture medium：The DMEM in high glucose (glucose content 4.5g/L) of 89 parts by volume, the serum substitute of 5 parts by volume
(SR), the B27 (50X) of 2 parts by volume, the ITS of 1 parts by volume, the non essential amino acid (NEAA) of 1 parts by volume, final concentration of 1ug/
The heparin (Heparin) of ml, the N-2 (100X) of 1 parts by volume, the vincaleucoblastine III (Conophylline) of 100ng/ml,
It is thin that the Vitamin B3 of 10mmol/L, final concentration are the recombination human basic fibroblast somatomedin (b-FGF) of 10ng/ml, epidermis
The intracellular growth factor (EGF), hepatocyte growth factor (HGF), pentagastrin and Concentraton gradient be 0.2,0.5,0.8,1.0,
1.2nd, 1.5, the ITS of 2.0ng/ml.
The induction differentiation culture of human umbilical cord mesenchymal stem cells is carried out using the culture medium of above-mentioned variable concentrations ITS.
As a result：ITS concentration be 0.2,0.5, in the culture medium of 0.8ng/ml, cell occurs loose dead, and cell mass is straight
Footpath is less, and uneven, and culture dish bottom dead cell is more；ITS concentration be 1.0,1.2, in the culture medium of 1.5ng/ml, carefully
Born of the same parents are agglomerating closely；In ITS concentration is for the culture medium of 2.0ng/ml, although cell mass is closely, dead cell quantity occurs again, opens
Begin to increase.As a result referring to Fig. 3.
Embodiment 5Induction time is screened
It is prepared by serum-free inductive differentiation medium：
Formula：The DMEM in high glucose (glucose content 4.5g/L) of 89 parts by volume, the serum substitute (SR) of 5 parts by volume, 2 bodies
Accumulate the B27 (50X), the ITS of 1 parts by volume, the non essential amino acid (NEAA) of 1 parts by volume, the N-2 (100X) of 1 parts by volume, end of part
Concentration be 1ug/ml heparin (Heparin), 100ng/ml vincaleucoblastines III (Conophylline), 10mmol/L Vitamin B3s,
And final concentration is 10ng/ml recombination human basic fibroblast somatomedin (b-FGF), epithelical cell growth factor (EGF), liver
Cell growth factor (HGF), pentagastrin.
Culture medium is made after each composition mix homogeneously.
Cell culture：
In Biohazard Safety Equipment, the hUC- in the 3rd generation for being located away from spontaneous labor neonatal umbilical cord China formula glue tissue is taken
MSCs, by hUC-MSC with 5 × 104Individual cell/cm2Density be inoculated in six well culture plates of ultralow absorption, add the present invention to lure per hole
Culture medium 2ml is led, CO is moved into2Concentration is in 5% 37 DEG C of constant incubators；Old culture basal cell is slowly drawn with pipet to hang
Liquid 500rpm low-speed centrifugals 4min in centrifuge tube inhales and abandons old culture medium, is replaced by fresh inducing culture of the present invention, changes per 2 days
Liquid is once；Successive induction 10 days, observation of cell group number change, and count,
As a result：Cell mass quantity gradually increases in the first six day after cell self-induction, and maximum (223) was reached at the 6th day,
After 6 days, cell mass quantity is gradually decreased.As a result referring to Fig. 4.
Embodiment 6The preparation and application of serum-free inducing culture
It is prepared by serum-free inductive differentiation medium：
Formula：The DMEM in high glucose (glucose content 4.5g/L) of 89 parts by volume, the serum substitute (SR) of 5 parts by volume, 2 bodies
Accumulate the B27 (50X), the ITS of 1 parts by volume, the non essential amino acid (NEAA) of 1 parts by volume, the N-2 (100X) of 1 parts by volume, end of part
Concentration be 1ug/ml heparin (Heparin), 100ng/ml vincaleucoblastines III (Conophylline), 10mmol/L Vitamin B3s,
And final concentration is 10ng/ml recombination human basic fibroblast somatomedin (b-FGF), epithelical cell growth factor (EGF), liver
Cell growth factor (HGF), pentagastrin (Pentagastrin).
Culture medium is made after each composition mix homogeneously.The cell culture reference method of embodiment 5, continuous six days, observation of cell
Group's metamorphosis.
As a result：After inducing the 1st day, aggregation forms small cell cluster, and quantity is more and light transmission is good, with incubation time
Extend, small cell cluster gradually accumulates maxicell group, small cell cluster quantity is reduced, maxicell group quantity gradually increases, with cell
Group's diameter becomes big, and the light transmission of cell mass is gradually reduced；After culture 6 days, maxicell group (mature cell group) occupies major part
The visual field.As a result referring to Fig. 5.
Embodiment 7Flow cytometry analysis insulin secretion like cell specific surfaces mark
The induction differentiation cell mass of 6 days of embodiment 6 is collected in centrifuge tube, then 500rpm is centrifuged under the conditions of 4 DEG C
4min, abandons supernatant and collects cell mass, plus 0.25% pancreatin is gently blown and beaten and mixes 20min simultaneously, treats that cell mass is loosely organized, without meat
Eye visible cell group suspends, and adds fresh culture to terminate digestion, and then 1200rpm is centrifuged 6 minutes, abandons supernatant and collects cell,
PBS twice, by every pipe 1 × 10 of cell5It is individual to be transferred to streaming pipe, add the paraformaldehydes of 500 μ l 4%, room temperature to fix
30min, then 1200rpm be centrifuged 6 minutes, abandon supernatant collect cell, often pipe add 5 μ l PDX-1, NKX6.1, Insulin,
Lucifuge is incubated 30 minutes at IgG1-PE (Isotype control) antibody mixes 4 DEG C, and once, supernatant, plus 500 μ L are removed in centrifugation to PBS
The resuspended mixing of PBS, upper machine testing (flow cytometer XL, Beckman company), each sample collection 1 × 104It is individual thin
Born of the same parents.
As a result：Mescenchymal stem cell expression of insulin secretory cell specificity marker, flow cytometer detection after inducing six days
PDX-1- positive expression rate 51.4%, NKx6.1 positive expression rate 15.36%, insulin positive expression rate 24.03%.Induction
Front cell does not express this mark.As a result referring to Fig. 6.
Embodiment 8RT-PCR analyzes insulin secretion like cell specific gene
The induction differentiation cell mass of 6 days of embodiment 6 is collected in centrifuge tube, by total RNA extraction reagent box (R6834-01,
OMRGA Products) extracting RNA, the RNA of extracting is obtained with reverse transcription reagent box (RR014A, TAKARA product) reverse transcription
CDNA samples, performing PCR of going forward side by side amplification moves to gel imaging instrument observation after agarose gel electrophoresiies.
As a result：Expression of insulin secretion like cell marker gene after induction, PDX-1, INSULIN and NGN-3 have bright
The different band of degree.As a result referring to Fig. 7.
Embodiment 9Immuning fluorescent dyeing analysis hUC-MSCs specific proteinses
The induction differentiation cell mass of 6 days of embodiment 6 is collected in centrifuge tube, is used after fixing 15 minutes with 4% paraformaldehyde
0.25%TritonX-100 punchings are processed 20 minutes, and after lowlenthal serum closing anti-human (the anti-Insulin of red Mus for having diluted is added
Antibody), the overnight lucifuge incubation at 4 DEG C；Afterwards and then to contaminate PDX-1 nucleoprotein, the overnight lucifuge incubation at 4 DEG C, fluorescence shows
Micro- Microscopic observation.
As a result：Induce differentiation to obtain insulin secretion like cell in protein expression INSULIN in the process of the present invention and
PDX-1 specific proteinses.As a result referring to Fig. 8.
Embodiment 10Unit cell amount of insulin secretion is detected
The induction differentiation cell mass of 6 days of embodiment 6 is collected in centrifuge tube, supernatant is abandoned, PBS re-suspended cells group is light and slow to blow
Inhale, 500rpm is centrifuged 3 minutes；Abandon after PBS and be separately added into 5.5mM/L glucoses DMEM stimulations liquid (low sugar group) and 25mM/L Fructus Vitis viniferaes
Sugared DMEM stimulates liquid (high sugar group), gently dispels mixing cell mass, 37 DEG C, under the conditions of 5%CO2, stimulates 2 hours；500rpm from
The heart 3 minutes, collects the standby survey of supernatant；ELISA detects amount of insulin secretion；Positive controls are islet cell tumor.
As a result：Induce the insulin secretion sample stem cell for obtaining in the case where high sugar stimulates per 10 through culture medium of the present invention4Carefully
Born of the same parents uelralante 4.393uIU/ml, positive controls are in the case where high sugar stimulates per 104Cell uelralante 6.9828uIU/
ml.Induction group=62.91% positive group.As a result referring to Fig. 9.
Above specific description of embodiments of the present invention is not intended to limit the present invention, and those skilled in the art can be according to this
Invention is variously modified or deforms, and without departing from the spirit of the present invention, all should belong to the model of claims of the present invention
Enclose.
Claims (10)
1. a kind of serum-free medium broken up to insulin secretion like cell for inducing umbilical cord mesenchymal stem, the nothing
Blood serum medium is included：DMEM in high glucose, serum substitute, B27 serum-free additives, Insulin-Transferrin-selenium compound,
Non essential amino acid, N-2 serum-free additives, and the life of heparin, vincaleucoblastine III, Vitamin B3 and recombination human basic fibroblast
The long factor, epithelical cell growth factor, hepatocyte growth factor and pentagastrin.
2. serum-free medium according to claim 1, it is characterised in that in terms of 100 parts by volume, the serum-free culture
Base is included：The glucose content of 85-95 parts by volume is DMEM in high glucose, the serum substitute of 5-8 parts by volume, the 1-4 body of 4.5g/L
B27 serum-free additives (50 ×), the Insulin-Transferrin-selenium compound of 1-1.5 parts by volume, 0.5-2 parts by volume of product part
Non-essential amino aqueous acid, N-2 serum-free additives (100 ×) of 0.5-2 parts by volume, and in the serum-free culture
The heparin of final concentration of 0.5-2ug/ml, the vincaleucoblastine III of final concentration of 50-200ng/ml, final concentration of 5- in base
The Vitamin B3 and final concentration of 20mmol/L is respectively recombination human basic fibroblast somatomedin, the epidermis cell of 5-20ng/ml
Somatomedin, hepatocyte growth factor and pentagastrin；
Preferably, in terms of 100 parts by volume, the serum-free medium is included：The glucose content of 89 parts by volume is 4.5g/L's
DMEM in high glucose, the serum substitute of 5 parts by volume, B27 serum-free additives (50 ×) of 2 parts by volume, 1 parts by volume insulin-
Transferrinss-selenium compound, the non-essential amino aqueous acid of 1 parts by volume, the N-2 serum-frees additive (100 of 1 parts by volume
×), and the heparin of final concentration of 1ug/ml, the vincaleucoblastine of final concentration of 100ng/ml in the serum-free medium
IIIth, the Vitamin B3 and final concentration of final concentration of 10mmol/L be respectively 10ng/ml recombination human basic fibroblast somatomedin,
Epithelical cell growth factor, hepatocyte growth factor and pentagastrin.
3. serum-free medium according to claim 1 and 2, it is characterised in that the non-essential amino aqueous acid bag
Glycine, alanine, L- Tianmens amide, L-Aspartic acid, glutamic acid, proline and the silk ammonia of 8-12mM are respectively containing concentration
Acid.
4. serum-free medium according to any one of claim 1 to 3, it is characterised in that the umbilical cord mesenchyma is done
Cell is the umbilical cord mesenchymal stem cells of people source, more preferably from spontaneous labor or the fresh umbilical cord of caesarean delivered healthy newborn
The human umbilical cord mesenchymal stem cells that separate tissue goes out.
5. the preparation method of the serum-free medium any one of Claims 1-4, the preparation method includes：Will be each
Individual composition mix homogeneously.
6. a kind of method broken up to insulin secretion like cell for inducing umbilical cord mesenchymal stem, methods described includes：
Using the serum-free medium culture umbilical cord mesenchymal stem cells any one of Claims 1-4.
7. method according to claim 6, it is characterised in that between umbilical cord of the umbilical cord mesenchymal stem cells for people source
Mesenchymal stem cells, between the people's umbilical cord more preferably isolated from spontaneous labor or the fresh umbilical cord tissue of caesarean delivered healthy newborn
Mesenchymal stem cells；
Preferably, the method comprising the steps of：
(1) by umbilical cord mesenchymal stem cells with 2-6 × 104Individual cell/cm2Density be inoculated in the serum-free medium, so
Afterwards in CO25%th, cultivate at 37 DEG C；
(2) 4min is centrifuged under 300-800rpm per the 2-3 days cell cultures by step (1), suction is abandoned old culture medium, is replaced by
The fresh serum-free medium；
(3) cultivate 6-7 days, harvesting；
(4) the induction differentiation effect of institute's harvesting of detecting step (3)；
Preferably, the step (4) includes one or more in detection following items：Unit cell insulin release；Pancreas
The expression of island element release related gene PDX-1, INSULIN and NGN-3；Nuclear extract PDX-1's and plasmosin Insulin
Exist；Dithizone is dyeed；The positive expression rate of insulin secretion like cell specificity marker PDX-1, NKX6.1, Insulin.
8. the method according to claim 6 or 7, it is characterised in that the method comprising the steps of：
(1) by umbilical cord mesenchymal stem cells with 5 × 104Individual cell/cm2Density be inoculated in it is ultralow absorption six well culture plates in
In the serum-free medium, then in CO25%th, cultivate at 37 DEG C；
(2) cell culture of step (1) is centrifuged into 4min under 500rpm per 2 days, suction is abandoned old culture medium, is replaced by fresh
The serum-free medium；
(3) cultivate 6-7 days, harvesting；
(4) the induction differentiation effect of institute's harvesting of detecting step (3).
9. the serum-free medium any one of Claims 1-4 prepare stem cell culture medium in purposes.
10. purposes according to claim 9, it is characterised in that the stem cell from mammal, the tissue of such as people or
It is isolated in organ, it is described tissue or organ be bone marrow, umbilical cord and fat in one or more；
Preferably, the stem cell is the umbilical cord mesenchymal stem cells of mammal source, more preferably between the umbilical cord of people source
Mesenchymal stem cells；
It is further preferred that the stem cell is isolated from spontaneous labor or the fresh umbilical cord tissue of caesarean delivered healthy newborn
Human umbilical cord mesenchymal stem cells.
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| CN201610980017.7A CN106676057A (en) | 2016-11-08 | 2016-11-08 | Serum-free medium for inducing differentiation of umbilical cord mesenchymal stem cells into insulin secretory-like cells and preparation method and application thereof |
| PCT/CN2017/082791 WO2018086319A1 (en) | 2016-11-08 | 2017-05-03 | Serum-free medium inducing differentiation of umbilical cord mesenchymal stem cell into insulin-secretion-like cell and preparation method and use thereof |
| US16/346,762 US11339372B2 (en) | 2016-11-08 | 2017-05-03 | Serum-free medium inducing differentiation of umbilical cord mesenchymal stem cell into insulin-secretion-like cell and preparation method and use thereof |
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