CN106676056A

CN106676056A - Method for inducing differentiation from umbilical cord mesenchymal stem cells to insulin secretion cells

Info

Publication number: CN106676056A
Application number: CN201610979689.6A
Authority: CN
Inventors: 郭镭
Original assignee: Holy Interpretation (beijing) Biological Engineering Co Ltd
Current assignee: Holy Interpretation (beijing) Biological Engineering Co Ltd
Priority date: 2016-11-08
Filing date: 2016-11-08
Publication date: 2017-05-17

Abstract

The invention discloses a method for inducing differentiation from umbilical cord mesenchymal stem cells to insulin secretion cells by adopting a novel serum-free medium. The medium contains DMEM (with the glucose content of 4.5 g/L) containing high glucose, B27, recombinant human basic fibroblast growth factors (b-FGF), Nicotinamide, N-2, Conophylline III (Conophylline), non-essential amino acids (NEAA), Heparin (Heparin), epidermal growth factors (EGF), hepatocyte growth factors (HGF), serum replacement (SR), insulin-transferrin-selenium compounds (ITS) and pentagastrin (Pentagastrin). The method using the medium can realize induced differentiation of mesenchymal stem cells to the insulin secretion cells in one step within six days.

Description

The method that inducing umbilical cord mesenchymal stem breaks up to insulin secretion like cell

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 The method that born of the same parents induce differentiation 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 umbilical cord mesenchymal stem cells are induced to differentiate into islets of langerhans by one kind using serum-free medium The method of element secretion like cell, using the method can rapid induction be insulin within 6 days by umbilical cord mesenchymal stem cells Secretion like cell.

Technical scheme is as follows.

The present invention provides a kind of inducing umbilical cord mesenchymal stem (UC-MSCs) that is used for and breaks up to insulin secretion like cell Method, methods described is included using serum-free medium culture umbilical cord mesenchymal stem cells, wherein the serum-free medium Comprising：It is DMEM in high glucose, serum substitute (SR), B27 serum-free additives, Insulin-Transferrin-selenium compound (ITS), non- Essential amino acids (NEAA), N-2 serum-free additives, and heparin (Heparin), vincaleucoblastine III (Conophylline), Vitamin B3 (Nicotinamide) and recombination human basic fibroblast 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 KnockOut^TM Serum 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

The above-mentioned serum-free medium that the present invention is provided is obtained by the method for comprising the following steps：By each composition mixing Uniformly.

In the method for the invention, it is preferable that between umbilical cord of the umbilical cord mesenchymal stem cells (UC-MSCs) for people source Mesenchymal stem cells (hUC-MSCs), more preferably isolate from spontaneous labor or the fresh umbilical cord tissue of caesarean delivered healthy newborn Human umbilical cord mesenchymal stem cells.

Preferably, the side for breaking up to insulin secretion like cell provided by the present invention for inducing umbilical cord mesenchymal stem Method is comprised the following steps：

(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；

In step (1), the umbilical cord mesenchymal stem cells (UC-MSCs) are 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.

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

1. a kind of method broken up to insulin secretion like cell for inducing umbilical cord mesenchymal stem, methods described includes adopting With serum-free medium culture umbilical cord mesenchymal stem cells, wherein the serum-free medium is included：DMEM in high glucose, blood serum substituting Thing, B27 serum-free additives, Insulin-Transferrin-selenium compound, non essential amino acid, N-2 serum-free additives, and Heparin, vincaleucoblastine III, Vitamin B3 and recombination human basic fibroblast somatomedin, epithelical cell growth factor, hepatocyte life The long factor and pentagastrin.

2. method according to claim 1, it is characterised in that in terms of 100 parts by volume, the serum-free medium 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 parts by volume of 4.5g/L B27 serum-free additives (50 ×), the Insulin-Transferrin-selenium compound of 1-1.5 parts by volume, the non-of 0.5-2 parts by volume must Need N-2 serum-free additives (100 ×) of amino acid solution, 0.5-2 parts by volume, and the end in the serum-free medium Concentration is heparin, the vincaleucoblastine III of final concentration of 50-200ng/ml, the Buddhist nun of final concentration of 5-20mmol/L of 0.5-2ug/ml It is thin that ancient amide and final concentration are respectively the recombination human basic fibroblast somatomedin of 5-20ng/ml, epithelical cell growth factor, liver The intracellular growth factor and pentagastrin.

3. method according to claim 1 and 2, it is characterised in that in terms of 100 parts by volume, the serum-free medium bag Contain：The glucose content of 89 parts by volume is the DMEM in high glucose of 4.5g/L, the B27 of the serum substitute of 5 parts by volume, 2 parts by volume without Serum additive (50 ×), the Insulin-Transferrin-selenium compound of 1 parts by volume, 1 parts by volume non essential amino acid it is water-soluble N-2 serum-free additives (100 ×) of liquid, 1 parts by volume, and in the serum-free medium final concentration of 1ug/ml liver The vincaleucoblastine III of plain, final concentration of 100ng/ml, the Vitamin B3 of final concentration of 10mmol/L and final concentration are respectively 10ng/ The recombination human basic fibroblast somatomedin of ml, epithelical cell growth factor, hepatocyte growth factor and pentagastrin.

4. according to the method in any one of claims 1 to 3, 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.

5. method according to any one of claim 1 to 4, it is characterised in that the serum-free medium is by including The method of following steps is obtained：By each composition mix homogeneously.

6. method according to any one of claim 1 to 5, it is characterised in that the umbilical cord mesenchymal stem cells are behaved The umbilical cord mesenchymal stem cells in source, preferably isolate from spontaneous labor or the fresh umbilical cord tissue of caesarean delivered healthy newborn Human umbilical cord mesenchymal stem cells.

7. method according to any one of claim 1 to 6, it is characterised in that 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；

8. method according to any one of claim 1 to 7, it is characterised in that the step (4) includes the following item of detection One or more in mesh：Unit cell insulin release；Insulin releasing related gene PDX-1, INSULIN and NGN-3 Expression；The presence of Nuclear extract PDX-1 and plasmosin Insulin；Dithizone is dyeed；Insulin secretion like cell is special The positive expression rate of property mark PDX-1, NKX6.1, Insulin.

9. method according to any one of claim 1 to 8, 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；