CN102776150A - Method for inducing umbilical cord mesenchymal stem cells to differentiate into endothelial cells - Google Patents

Abstract

Belonging to the field of biomedicine, the invention relates to a method for paeoniflorin to induce umbilical cord mesenchymal stem cells to differentiate into endothelial cells and application thereof. In the invention, the paeoniflorin has a structure as shown in formula (I). The induction differentiation steps of the method comprise: inducing the umbilical cord mesenchymal stem cells respectively in a beta-mercaptoethanol medium with a concentration of 1-100micromoles/L and a retinoic acid 5%DMEM medium with a concentration of 1-100micromoles/L for 24h, then shifting the cells into a paeoniflorin-containing endothelial cell conditioned medium, half of the solution is changed every two days. And after 10-14d of induction, umbilical cord mesenchymal stem cell derived endothelial cells can be obtained. In the invention, the application of the paeoniflorin and the method can promote differentiation of the umbilical cord mesenchymal stem cells, improve the differentiation efficiency, and can be used in cell transplantation treatment of ischemic vascular diseases. The obtained endothelial cells can be further prepared into cell preparations to be used for ischemic vascular disease treatment, and seed cell application of vascular tissue engineering.

Description

A kind of method of inducing umbilical cord mesenchymal stem cells to be divided into endotheliocyte

Technical field

The invention belongs to biomedical sector; Relate to the method that induced dry-cell is divided into endotheliocyte; Be specifically related to induce umbilical cord mesenchymal stem cells to be divided into the method for endotheliocyte, relate in particular to peoniflorin and induce umbilical cord mesenchymal stem cells to be divided into the method and the purposes of endotheliocyte.

Background technology

It is reported that cellular transplantation therapy is started with from cell levels, promote the ishemic part new vessel to form, improve blood supply, is the New Policy of treating ischemic angiopathy at present.Along with the investigator learns updating of deepening continuously of characteristic understanding and separation and purification means to various stem cell biologicals, making therapeutic vasculogenesis methods such as adopting stem cell rebulid effective side Zhi Xunhuan at ishemic part becomes possibility gradually; Therefore, the investigator has produced keen interest to increasing the serious lower limb ischemia of angiogenesis treatment through stem cell transplantation.

At present, stem cell is used to treat ischemic disorders and has become one of focus of basis and clinical study; The research of relevant preclinical phase has demonstrated the challenging achievement of cellular replacement therapy, but still exists many problems to need solution badly, as: cell source, immunoreation and ethics dispute etc.Discover, during the clinical treatment lower limb ischemia, obtain satisfied trouble limb and pour into again, need to transplant heterology stem cell 2x10⁴/ g body weight; Through cultured and amplified in vitro, the peripheric venous blood of 100ml adult healthy volunteers can produce 5.0x10 usually approximately⁶Individual endothelial stem cell by above-mentioned result of study budget, is used to treat the limbs of a severe ischemic; At least need 12 liters of peripheric venous bloods could to obtain the endothelial stem cell of sufficient amount, this is extremely difficulty realization beyond doubt, therefore; To the treatment of above-mentioned illness, the source problem that comes of stem cell is needed solution badly.

There are some researches show that umbilical cord mesenchymal stem cells is anotherly except that marrow, peripheral blood to have a potential source of human stem cell, having a extensive future of fields such as organizational project, Transplanted cells, gene therapy.Umbilical cord is compared following remarkable advantage as the another kind of source of human stem cell of stem cell transplantation with marrow with peripheral blood hematopoietic stem cells: dried, the progenitor cell that can implant for a long time in vivo is rich in (1); When making external colony-forming test, the HSC's of the colony forming unit ratio adult derived from bone marrow that cell produces is bigger; (2) length of the Telomerase of stem cell is longer than adult cell; (3) transplanting back graft versus host disease incidence is low; (4) in external long-term cultivation, still can increase.At present, along with developing rapidly of stem cells technology, induce differentiation that comparatively perfect technology has been arranged by multipotential stem cell to endotheliocyte, but still have defectives such as differentiation efficiency is low.

Summary of the invention

The purpose of this invention is to provide a kind of method that induced dry-cell is divided into endotheliocyte that relates to; Be specifically related to induce umbilical cord mesenchymal stem cells to be divided into the method for endotheliocyte, relate in particular to peoniflorin and induce umbilical cord mesenchymal stem cells to be divided into the method for endotheliocyte.

Further purpose of the present invention provides the purposes of described method in preparation endotheliocyte preparation.

Among the present invention, described umbilical cord mesenchymal stem cells derives from stripped fetal cord, and the umbilical cord mesenchymal stem cells of acquisition is through separating, increase and inducing atomization to make umbilical cord mesenchymal stem cells source endotheliocyte.

Of the present invention inducing in the method that umbilical cord mesenchymal stem cells is divided into endotheliocyte; After umbilical cord mesenchymal stem cells induced in the substratum that contains beta-mercaptoethanol and vitamin A acid 5%DMEM respectively; Change the endotheliocyte conditioned medium that contains peoniflorin over to, obtain required endotheliocyte.This method utilizes peoniflorin to induce umbilical cord mesenchymal stem cells to be divided into endotheliocyte, can effectively solve the difficult problem that defective such as low because of cytodifferentiation efficient in the transplanted endothelial cell treatment, that the cytodifferentiation cycle is long causes.

Particularly, method of inducing umbilical cord mesenchymal stem cells to be divided into endotheliocyte of the present invention is characterized in that, may further comprise the steps:

(1) aseptic condition obtains the umbilical cord that exsomatizes down, cleans through containing two anti-PBS, and chopping digests respectively through collagenase and pancreatin again, collects single cell suspension, splitting erythrocyte, inoculation culture then;

(2) containing 20% foetal calf serum, 1-50ng/Cultivate in the mescenchymal stem cell substratum of mlbFGF, change liquid every other day, obtained tire umbilical cord mesenchymal stem cells after going down to posterity in 3-4 days;

(3) after the above-mentioned umbilical cord mesenchymal stem cells that makes is induced 24 hours in containing beta-mercaptoethanol 1-100 μ mol/L substratum, in the substratum of 1-100 μ mol/L 5%DMEM, induced 24 hours;

(4) with the umbilical cord mesenchymal stem cells after above-mentioned the inducing, change the endotheliocyte conditioned medium that contains peoniflorin over to, half amount is changed liquid every three days, induces 10-14 days, obtains umbilical cord mesenchymal stem cells source endotheliocyte;

In the described endotheliocyte conditioned medium, contain the substratum of peoniflorin 1-100 μ mol/L, ECGF100 μ mol/L, VEGF 50-100ng/ml, bFGF 10-100ng/ml 5%DMEM.

In one embodiment of the present of invention, the described endotheliocyte conditioned medium that contains peoniflorin is: the substratum of peoniflorin 0.25mg/L-50mg/L, ECGF1-100ng/ml, VEGF 50-100ng/ml, bFGF 10-100ng/ml 5%DMEM.

Among the present invention, described peoniflorin is the monomeric compound that extracts in Chinese herbaceous peony and the river radix paeoniae rubrathe root, is the main effective constituent of the root of herbaceous peony and the radix paeoniae rubrathe, its molecular formula: C23H28O11, and molecular weight: 480.45, have the structure of formula I:

（Ⅰ） ；

Described peoniflorin can be prepared by the routine techniques method by commercially available or those skilled in the art;

The common described radix paeoniae rubrathe and the root of herbaceous peony are tcm clinical practice medicines commonly used: the radix paeoniae rubrathe is mainly used in the treatment cardiovascular and cerebrovascular diseases, and the root of herbaceous peony is mainly used in some immunological diseases of treatment and gynaecopathia; Described peoniflorin is aspect metabolism, but regulating blood lipid and reducing blood sugar; At immunology, but the inflammation-inhibiting generation has therapeutic action to rheumatoid arthritis.

Among the present invention, described peoniflorin is used to prepare substratum, has to promote the effect of navel mescenchymal stem cell to endothelial cell differentiation, can improve differentiation efficiency, shorten between differentiation phase.

Among the present invention, the metamorphosis that has carried out in the umbilical cord mesenchymal stem cells culturing process is observed, and the result shows that after cell attachment grew to 3 days, spindle cell occurred; The cell attachment growth had cloning cluster to form after 7 days; P3 is for cell, and cellular form is homogeneous very, and cell is all tall and thin, is fusiformis, type one-tenth fiber-like.

Among the present invention, the surperficial important sign thing of the umbilical cord mesenchymal stem cells that the analysis of employing Flow Cytometry is obtained, the result shows that the cell that is obtained is the cell with mesenchyme characteristic in placenta source.

Among the present invention, identify the endotheliocyte of inducing differentiation through two fluorescence, the result shows, wherein, the DIL-LDL stained positive FITC-UEA-1 stained positive that takes on a red color is green, and two stained positive cytodifferentiation are endotheliocyte.

Among the present invention, described peoniflorin induces the endotheliocyte of differentiation to can be used for the cellular transplantation therapy and the intravascular tissue engineering of ischemic angiopathy;

Or, process the endotheliocyte preparation, be used to prepare the medicine of treating ischemic angiopathy or be used for the tissue repair of the blood vessel injury that other reason causes;

Said medicine is mainly used in the cellular transplantation therapy of ischemic angiopathy, and described ischemic angiopathy comprises ischemic cardiovascular, ischemic cerebrovascular and ischemia lower limb vascular disease.

For the ease of understanding, below will describe in detail method and purposes of inducing umbilical cord mesenchymal stem cells to be divided into endotheliocyte of the present invention through concrete accompanying drawing and embodiment.What need particularly point out is that these descriptions only are exemplary descriptions, do not constitute limitation of the scope of the invention.According to the argumentation of this specification sheets, many variations of the present invention, change all are conspicuous concerning one of ordinary skill in the art.

Description of drawings

Fig. 1 has shown the metamorphosis in the umbilical cord mesenchymal stem cells culturing process, and wherein, A: after cell attachment grew to 3 days, spindle cell occurred; B: the cell attachment growth had cloning cluster to form after 7 days; C:P3 is for cell, and cellular form is homogeneous very, and cell is all tall and thin, is fusiformis, type one-tenth fiber-like.

Fig. 2 is the surperficial important sign thing that Flow Cytometry is identified umbilical cord mesenchymal stem cells, wherein, adopts Flow Cytometry to analyze the umbilical cord mesenchymal stem cells that this institute obtains; The result shows: CD44, CD73, CD105; Marker expression such as CD29 are respectively 98.5%; 97.8%, 99.4%, 97.2%; CD29 and CD44 are the specific signs of stem cell, and the prompting cell is the cell with mesenchyme characteristic in placenta source.

Fig. 3 is DIL-LDL and the two fluorescence evaluation figure of FITC-UEA-1 that are induced to differentiate into endotheliocyte; Fluorescent microscope detects the attached cell surface marker; Wherein, the DIL-LDL stained positive FITC-UEA-1 stained positive that takes on a red color is green, and two stained positive cytodifferentiation are endotheliocyte.

Embodiment

Embodiment 1 preparation contains the peoniflorin cell culture medium

By ordinary method, preparation contains the endotheliocyte conditioned medium of peoniflorin, wherein:

Peoniflorin 0.25mg/L-50mg/L, ECGF1-100ng/ml, VEGF 50-100ng/ml, bFGF10-100ng/ml 5%DMEM.

Its molecular formula of described peoniflorin: C23H28O11, molecular weight: 480.45, have the structure of formula I:

（Ⅰ） 。

 

Embodiment 2 induces umbilical cord mesenchymal stem cells to be divided into the endotheliocyte test

(1) separates umbilical cord mesenchymal stem cells

Get the umbilical cord tissue that exsomatizes about 5cm under the aseptic condition, use that to contain penicillin concn be after the PBS of 100 μ g/ml washes repeatedly, to shred as the 100U/ml Streptomycin sulphate; Add 0.1% collagenase; In 37 ℃ of digestion 1h, cross 100 eye mesh screens, the collecting cell suspension; With the centrifugal 10min of 100Or/min, abandon supernatant under the room temperature; Remaining tissue adds and contains in 0.05% pancreatin of 0.02%EDTA, in 37 ℃ of digestion 1h, crosses 100 eye mesh screens, and the collecting cell suspension with the centrifugal 10min of 100Or/min, is abandoned supernatant under the room temperature; Collecting cell behind erythrocyte cracked liquid removal red corpuscle, with the mescenchymal stem cell substratum re-suspended cell that contains 20%FBS and bFGF, is processed single cell suspension; Be inoculated in the culturing bottle, place 37 ℃, 5%CO2 is hatched in the incubator of 95% humidity, behind the 3d, changes liquid the 1st time, removes not adherent cell, and every afterwards 48h changes nutrient solution.

(2) mescenchymal stem cell is cultivated with frozen

Treat culturing cell when confluxing 80-90%, digest, with the cultivation of going down to posterity of the moderate ratio of 1:3 with 0.25% trypsinase-0.02%EDTA; Get third generation cell and identify its surperficial important symbol thing.

Frozen employing is slowly freezing.Each component proportions is DMEM:FBS in the frozen storing liquid: DMSO 99.8MIN. (DMSO)=4:5:1, cell concn are 5 * 10⁵～1 * 10⁶After mL/1, cell wrapped up with cotton, through-20 ℃ of 1h ,-80 ℃ were spent the night, and placed the medium-term and long-term preservation of liquid nitrogen at last.Recovery then adopts speed to melt method; Cell is melted rapidly in 37 ℃ of water-baths, with the LG-DMEM washed cell that contains 20%FBS, 1000 rmin^-1, centrifugal 5min, with cell inoculation in the DMEM that contains 20%FBS.

(3) Flow Cytometry detects umbilical cord mesenchymal stem cells surface important symbol thing

1. different generation cells are collected in 0.25% pancreatin-0.02%EDTA digestion, and every routine sample cell count is 2 * 10⁵About individual;

2. cell is resuspended among the 1ml 0.01M PBS and washs 1500rmin 2 times^-1, 10min;

3. discard PBS, and hatch 30min under an anti-Incubating Solution (an anti-extent of dilution the is 1:100) normal temperature;

4. after 0.01M PBS washs 2 times, hatch 30min with FITC-two anti-(extent of dilution is 1:50) 4 ℃ of lucifuges respectively;

5. after 0.01M PBS washs 2 times, discard PBS, add 300 μ l 0.01M PBS re-suspended cells again, be used for facs analysis; As can not go up machine testing immediately, and available 4% Paraformaldehyde 96 or 70% alcohol fixation, 4 ℃ are to be measured.

(4) umbilical cord mesenchymal stem cells is induced to differentiate into endotheliocyte

Get P3 for umbilical cord mesenchymal stem cells, 0.1% gelatin encapsulates orifice plate, according to the inoculation of 2 * 103/cm2 cell density, serum-free incubated overnight; Umbilical cord mesenchymal stem cells was induced 24 hours in the substratum that contains beta-mercaptoethanol 1-100 μ mol/L and vitamin A acid 1-100 μ mol/L 5%DMEM respectively, changed the endotheliocyte conditioned medium that contains peoniflorin then over to;

Wherein, The described endotheliocyte conditioned medium that contains peoniflorin is: the substratum of peoniflorin 0.25mg/L-50mg/L, ECGF1-100ng/ml, VEGF 50-100ng/ml, bFGF10-100ng/ml 5%DMEM; Half amount is changed liquid every three days, induces to obtain umbilical cord mesenchymal stem cells source endotheliocyte in 10-14 days.

(5) the umbilical cord mesenchyma cell induction is divided into endotheliocyte

Umbilical cord mesenchymal stem cells is after endothelium is induced 14d, and cell (8 * 10/ hole) in 96 orifice plates is heavily spread in digestion, identifies after heavily spreading 36h.Attached cell and Dil-acLDL (2.4 μ g/mL) are hatched l hour to detect the picked-up of cell to acLDL for 37 ℃; With 2% Paraformaldehyde 96 fixed cell 1Omin, embathe 3 times each 5min, then with PBS with the Ulex europaeus lectin I (FITC-UEA-I of FITC mark; 10 μ g/ml) be added on above-mentioned sample and under 37 ℃, hatch lh, PBS embathes 3 times, each 5min; Through the laser confocal microscope identification of cell to Di-acLDL (redness; Excitation wavelength 543nm) picked-up combines (green, excitation wavelength 477nm) two stained positive cells (being yellow) with FITC-UEA-I.

Test-results confirms that the method that peoniflorin of the present invention induces umbilical cord mesenchymal stem cells to be divided into endotheliocyte can obviously promote the umbilical cord mesenchymal stem cells differentiation, and improves and separately win efficient; The endotheliocyte that is broken up can be used for the cellular transplantation therapy of ischemic angiopathy; The endotheliocyte that is obtained can further be prepared into cell preparation, be used for ischemic angiopathy treatment, and the seed cell of intravascular tissue engineering use.

Claims (8)

1. method of inducing umbilical cord mesenchymal stem cells to be divided into endotheliocyte; It is characterized in that; After umbilical cord mesenchymal stem cells induced in the substratum that contains beta-mercaptoethanol and vitamin A acid 5%DMEM respectively; Change the endotheliocyte conditioned medium that contains peoniflorin over to, obtain required endotheliocyte, may further comprise the steps:

(1) aseptic condition obtains the umbilical cord that exsomatizes down, cleans through containing two anti-PBS, and chopping digests respectively through collagenase and pancreatin again, collects single cell suspension, splitting erythrocyte, inoculation culture then;

(2) containing 20% foetal calf serum, 1-50ng/Cultivate in the mescenchymal stem cell substratum of mlbFGF, change liquid every other day, obtained tire umbilical cord mesenchymal stem cells after going down to posterity in 3-4 days;

(3) after the above-mentioned umbilical cord mesenchymal stem cells that makes is induced 24 hours in containing beta-mercaptoethanol 1-100 μ mol/L substratum, in the substratum of 1-100 μ mol/L 5%DMEM, induced 24 hours;

(4) with the umbilical cord mesenchymal stem cells after above-mentioned the inducing, change the endotheliocyte conditioned medium that contains peoniflorin over to, half amount is changed liquid every three days, induces 10-14 days, obtains umbilical cord mesenchymal stem cells source endotheliocyte.

2. by the described method of claim 1, it is characterized in that, in the described endotheliocyte conditioned medium, contain peoniflorin 1-100 μ mol/L, ECGF100 μ mol/L, VEGF 50-100ng/ml, bFGF 10-100ng/ml 5%DMEM.

3. by the described method of claim 1, it is characterized in that described peoniflorin molecular formula is C23H28O11, molecular weight is 480.45, has the structure of formula I:

（Ⅰ） 。

4. the purposes of peoniflorin in preparation endotheliocyte conditioned medium.

5. by the described purposes of claim 4, it is characterized in that, in the described endotheliocyte conditioned medium, contain peoniflorin 1-100 μ mol/L, ECGF100 μ mol/L, VEGF 50-100ng/ml, bFGF 10-100ng/ml 5%DMEM.

6. the purposes of the prepared endotheliocyte of the method for claim 1 in preparation endotheliocyte preparation.

7. by the described purposes of claim 6, it is characterized in that the purposes of described endotheliocyte preparation in the tissue repair medicine of the medicine of preparation treatment ischemic angiopathy or the blood vessel injury that other reason causes.

8. by the described purposes of claim 7, it is characterized in that described ischemic angiopathy is selected from ischemic cardiovascular, ischemic cerebrovascular or ischemia lower limb vascular disease.

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