CN101643719B

CN101643719B - Simplified method for isolation and culture of umbilical mesenchymal stem cells and application thereof in treatment of rheumatoid arthritis

Info

Publication number: CN101643719B
Application number: CN2009100896329A
Authority: CN
Inventors: 栗占国; 穆荣; 刘燕鹰
Original assignee: Peking University Peoples Hospital
Current assignee: Peking University; Peking University Peoples Hospital
Priority date: 2009-07-27
Filing date: 2009-07-27
Publication date: 2013-07-24
Anticipated expiration: 2029-07-27
Also published as: CN101643719A

Abstract

The invention develops a simplified method for the in-vitro isolation and expansion of umbilical mesenchymal stem cells (MSCs) and confirms that the MSCs obtained by the method have the effects of immunoregulation and inflammation inhibition in the treatment of rheumatoid arthritis.

Description

A kind of method for isolation and culture of umbilical mesenchymal stem cells of simplification and the application in treating rheumatoid arthritis thereof

Technical field

The present invention relates to a kind of umbilical cord mesenchymal stem cells (umbilical cord mesenchymal stem cells, UCMSCs) structure of isolation cultivation method and the immunomodulatory in treating rheumatoid arthritis thereof and but scorching effect of simplification.

Background technology

Mescenchymal stem cell (mesenchymal stem cells, MSCs) be the tissue stem cell that a class has multidirectional differentiation potential, can obtain from multiple tissue such as blood, liver, marrow, fat, skin, be a group multipotential cell, can be to differentiation such as multiple histocyte such as bone, cartilage, muscle, ligament, tendon, fat.At present, MSCs is mainly derived from marrow, but because there is the possibility of height virus pollution in bone marrow derived MSCs, and obvious downtrending appears in its cell quantity and amplification, differentiation capability with age, substitute bone marrow MSCs so seek a kind of energy, and can remedy the MSCs source of its defective, more and more receive various countries scholar's concern.Additive method has certain difficulty as cultivate MSCs from Cord blood and peripheral blood, and MSCs quantity in the peripheral blood of the Cord blood of term birth or mobilization remains in dispute.Therefore, separation and Culture UCMSCs from puerperal rejected material umbilical cord is for the application of MSCs provides new material source and basic data.Yet existing UCMSCs adopts the traditional method of protease digestion mostly, and this method operating time is long, the isolated cells state difference, and increase easily the chance of polluting, therefore, the UCMSCs isolation cultivation method of seeking to simplify is the bottleneck that solves the clinical and laboratory requirement of MSCs at present.Based on above requirement, the inventor through explore for many years invented a kind of simplification the UCMSCs isolation cultivation method, original 5-6 hour operating time is foreshortened to 30 minutes.And studies confirm that the isolating UCMSCs of this method has phenotype and the immunoloregulation function identical with ordinary method isolated M SCs.

(rheumatoid arthritis RA) is a kind of general autoimmune disease that is feature with chronic destructive arthropathy to rheumatoid arthritis.But, even cause joint deformity without the RA protracted course of disease of correct treatment.And the methods of treatment of existing RA comprises traditional immunosuppressor and biotechnological formulation, has drawbacks such as side effect is big, price height, need constantly to seek for this reason more economical effectively and the methods of treatment of non-evident effect.Existing studies confirm that, MSCs has immunoloregulation function, and in vitro study shows that marrow and adipose-derived MSCs can suppress the propagation of RA patient T cell and the secretion of inflammatory cytokine.For this reason, this seminar utilize UCMSCs that the aforesaid method separation and Culture obtains external with RA patient T cell and become fiber-like synovial cell (FLSs) to cultivate altogether, observe its effect in the RA treatment.But find the immunologic function of UCMSCs suppressor T cell and inflammatory reaction and the invasive ability of FLSs.The clinical application for the treatment of RA for UCMSCs provides foundation.

Summary of the invention

The objective of the invention is to set up the UCMSCs isolation cultivation method of a simplification, and be applied to the treatment of RA.

This research is on the basis of existing UCMSCs isolation cultivation method, flow process simplifies the operation, remove unnecessary operation stepss such as protease digestion, original 5-6 hour operating time is foreshortened to 30 minutes, reduced the chance of cell contamination, and through identifying that the isolating UCMSCs of this method has phenotype and the immunoloregulation function same with the isolating UCMSCs of ordinary method.

The UCMSCs that utilizes the aforesaid method separation and Culture to obtain cultivates with RA patient T cell and FLSs altogether external, observes its effect in the RA treatment.But find the propagation of UCMSCs suppressor T cell and FLSs, can suppress the secretion of inflammatory cytokine (TNF-α, IL-6), can reduce the invasive ability of FLSs, raise CD4 ⁺FOXP3 ⁺The expression of Treg cell.The clinical application for the treatment of RA for UCMSCs provides foundation.

Description of drawings

Fig. 1. the evaluation of umbilical cord mesenchymal stem cells (A): the third generation UCMSCs (B) of simplified method separation and Culture: the flow cytometry cell phenotype is analyzed the fat of (C): UCMSCs and is induced differentiation-oil red O stain. (D): the osteogenic induction differentiation-alkaline phosphatase staining of UCMSCs

Fig. 2. umbilical cord mesenchymal stem cells is to the influence of FLSs propagation. (A): TNF-α (10ng/ml) but the propagation of obvious stimulation FLSs, on the contrary, under the situation that no TNF-α stimulates, UCMSCs itself can't stimulate the propagation of FLSs, #P＜0.01 (B): UCMSCs can obviously suppress the propagation of FLSs, and is dose-dependently.Compare with the contrast of no UCMSCs, #P＜0.01, ^*P＜0.05 (C): in 5 days co-culture system,, still can suppress the propagation of FLSs even UCMSCs added at the 3rd day.Compare with the contrast of no UCMSCs, ^*P＜0.05

Fig. 3. umbilical cord mesenchymal stem cells is to the influence of FLSs invasive ability: no matter UCMSCs contacts with FLSs is altogether cultivated, or isolate altogether with the transwell system and to cultivate, or cells and supernatant and the FLSs of UCMSCs cultivated altogether, all can obviously reduce the invasive ability of FLSs.Compare #P＜0.01 with the contrast of no UC-MSCs

Fig. 4. umbilical cord mesenchymal stem cells is to the influence (A) of FLSs cytokine secretion: isolate in the co-culture system at transwell, UCMSCs can suppress the secretion (B) of FLSs IL-6: in the co-culture system, the secretion of different time points IL-6.Compare with the FLSs single culture, #P＜0.01, ^*P＜0.05

Fig. 5. umbilical cord mesenchymal stem cells is to the obvious propagation of suppressor T cell of the influence of T cell proliferation: UCMSCs, and is dose-dependently.Compare with the contrast of no UC-MSCs, #P＜0.01, ^*P＜0.05

Fig. 6. umbilical cord mesenchymal stem cells influences T cell cytokine excretory: UCMSCs can obviously suppress the secretion of the T cell TNF α of PHA stimulation, compares #P＜0.01 with the contrast of no UCMSCs

Fig. 7. umbilical cord mesenchymal stem cells can raise the expression of Treg: no matter have or not PHA to stimulate, UCMSCs all can raise CD4 ⁺FOXP3 ⁺The expression of Treg.Compare #P＜0.01 with the contrast of no UCMSCs

Embodiment

Embodiment 1: a kind of structure of method for isolation and culture of umbilical mesenchymal stem cells of simplification.

A. get normal vaginal delivery or c-section umbilical cord, (the ABO/Rh blood group detects through strict trace routine, the HLA somatotype detects, syphilis antibody detects, the HIV immunodetection, CMV antibody test, Australia antigen(AA) antibody test) determine safety after, visible dirt of naked eyes and blood are removed in the stroke-physiological saline solution flushing.

B. aseptic hammer-shears umbilical cord is to the 3-5mm size.

C. with the tissue block that obtains among the step b, be transferred in the 50ml centrifuge tube, add the PBS40ml that contains 1% mycillin, the centrifugal 250g of room temperature 5 minutes abandons supernatant.

D. step tissue block that c obtains is cultivated with 10% α MEM, changes liquid in per 3 days.

E. there were fusiformis and polygon cell to climb out of to 7 days around the tissue block of the left and right sides, and increase gradually.

F. reach about 80% when merging to 10-14 days left and right sides cells, go down to posterity with 0.25% trysinization.

G. went down to posterity once every 3-5 days after.

H. identify to the third generation.

I. the cell after identifying continues to cultivate standby or frozen.

This method is compared with ordinary method, has following characteristics: the step of 1) saving conventional pancreatin and collagenase digesting; 2) the hammer-shears umbilical cord need not littler to the 3-5mm size; 2) can reach 80% in 10-14 days and merge, shift to an earlier date the time in 1 week than ordinary method at least.3) nutrient solution α MEM is with 10% autoserum, AB serum or clinical grade serum-free medium.It is advantageous that: 1) save protease digestion step, can shorten the operating time, and can avoid causing the shortcoming of cell state difference because of protease digestion; 2) tissue block only need be cut to 3-5mm size, need not to operate routinely to cut the size to 1mm, also can obviously shorten the operating time.So this method foreshortens to 30 minutes with operating time of the required 5-6 of routine operation method hour and can finish, and can improve cell state, the generation time also can shift to an earlier date for 1 week first.Reduced the probability that cell contamination takes place.

Embodiment 2:UCMSCs is to the influence of RA-FLSs multiplication capacity

Leave and take RA patient's knee prosthesis postoperative synovial tissue, separation and Culture FLSs uses the cell after 3 generations to test.Be divided into two groups, one group is UCMSCs and RA-FLSs cultivation group altogether, and one group is simple RA-FLSs control group, and two groups all add TNF-α (10ng/ml) and stimulate.120 hours, ³The H method of mixing detects FLSs propagation situation.Found that common cultivation group FLSs propagation is subjected to obvious inhibition, and be dose-dependently,, added UCMSCs on the 3rd day, also can suppress the propagation of FLSs even in 5 days culture system.See Fig. 2.

Embodiment 3:UCMSCs is to the influence of RA-FLSs invasive ability

With 24 holes invasion and attack detection kit, UCMSCs and RA-FLSs are directly contacted common cultivation, the Transwell system is cultivated altogether or cells and supernatant and the FLSs of UCMSCs is cultivated altogether, after 48 hours, the cell count of Transwell cell film is passed in detection, dyeing, and microplate reader is measured the OD560 value.The result as seen, no matter UCMSCs contacts with FLSs altogether and cultivates, and isolates altogether with the transwell system and cultivates, or cells and supernatant and the FLSs of UCMSCs cultivated altogether, all can obviously reduce the invasive ability of FLSs.See Fig. 3.

Embodiment 4:UCMSCs is to the influence of RA-FLSs secretion inflammatory factor

Use the RA patient FLSs after 3 generations, 2 * 10 ⁴/ hole is inoculated in 12 orifice plates, single culture, or cultivate altogether with UCMSCs (1: 1), and directly contact is cultivated or the Transwell system is cultivated altogether altogether.Different time points, the ELISA method detects the secretion level of culture supernatant IL-6.The result shows that in the Transwell system, UCMSCs can suppress the IL-6 secretion.See Fig. 4.

Embodiment 5:UCMSCs is to the influence of T ability of cell proliferation

Separate RA patient's peripheral blood PBMC, magnetic bead sorting CD3 ⁺The T cell.Be divided into two groups, one group is UCMSCs and T co-culture of cells group, and one group is simple T cell control group, and two groups all add PHA (2 μ g/ml) and stimulate.120 hours, ³The H method of mixing detects T cell proliferation situation.Found that common cultivation group T cell proliferation is subjected to obvious inhibition, and be dose-dependently.See Fig. 5.

Embodiment 6:UCMSCs is to the influence of T emiocytosis inflammatory factor

Separate RA patient's peripheral blood PBMC, magnetic bead sorting CD3 ⁺The T cell.2 * 10 ⁶/ hole is inoculated in 24 orifice plates, single culture, or with UCMSCs 2 * 10 ⁴Cultivate altogether in/hole, directly contacts cultivation altogether or Transwell system and cultivate altogether.Different time points, the ELISA method detects the secretion level of culture supernatant TNF-α.The result shows, no matter contacts to cultivate or the Transwell system is cultivated altogether, and UCMSCs all can suppress TNF-α secretion, and is more obvious to add the test group that PHA stimulates especially.See Fig. 6.

Embodiment 7:UCMSCs can raise the expression of Treg

Separate RA patient's peripheral blood PBMC, magnetic bead sorting CD3 ⁺The T cell.2 * 10 ⁶/ hole is inoculated in 24 orifice plates, single culture, or with UCMSCs (2 * 10 ⁴/ hole) cultivate altogether, direct contact is cultivated altogether or the Transwell system is cultivated altogether.Flow cytometry CD4 after 72 hours ⁺FOXP ₃ ⁺The expression of Treg cell finds no matter to have or not PHA to stimulate, and UCMSCs all can raise the expression of Treg.

Claims

The in-vitro separation of a simplification, the amplification umbilical cord mesenchymal stem cells method

It is characterized in that:

A. get normal vaginal delivery or c-section umbilical cord, visible dirt of naked eyes and blood are removed in the stroke-physiological saline solution flushing;

B. aseptic hammer-shears umbilical cord is to the 3-5mm size;

C. the centrifugal 250g5 of room temperature minute, abandon supernatant;

D. tissue block is cultivated with 10% α MEM, changes liquid in per 3 days;

Go down to posterity when e. cell reached 80% fusion to 7-10 days;

F. went down to posterity once every 3-5 days after;

G. identify to the third generation.