CN110090227A

CN110090227A - Purposes of the human amnion membrane in treatment graft versus host disease(GVH disease)

Info

Publication number: CN110090227A
Application number: CN201910310688.6A
Authority: CN
Inventors: 余路阳; 张传宇; 杨棚捷; 袁惟芯; 李金英; 郭礼和; 邵小燕; 刘佳
Original assignee: Sino-American iCELL (Shanghai) Biotechnology Co Ltd; Zhejiang University ZJU
Current assignee: Sino-American iCELL (Shanghai) Biotechnology Co Ltd; Zhejiang University ZJU
Priority date: 2018-04-18
Filing date: 2019-04-17
Publication date: 2019-08-06
Anticipated expiration: 2039-04-17
Also published as: CN110090227B

Abstract

The present invention relates to purposes of the human amnion membrane (hAECs) in treatment graft versus host disease(GVH disease) (aGVHD).Cell preparation the invention discloses the amniotic epithelial cells for using effective dose or containing amniotic epithelial cells is independent or the method for being treated and/or being improved graft versus host disease(GVH disease) is used in combination with other medicines, amniotic epithelial cells can be given to patient using the method for intravenous injection or spinal cord intracavitary administration, the dosage range given every time is about 10³‑10⁹Cell, can effectively mitigate infiltration of the inflammatory cell caused by aGVHD to target organ, while also substantially reduced target organ lesion, while also found that hAECs has apoptosis-promoting effect to multiple Leukemia Cell Lines, can be used in treating graft versus host disease(GVH disease).

Description

Purposes of the human amnion membrane in treatment graft versus host disease(GVH disease)

Technical field

The invention belongs to field of biotechnology, and in particular to human amnion membrane is in treatment graft versus host disease(GVH disease) Purposes.

Background technique

Hematopoietic stem cell transplantation (Hematopoietie stem cell transplantation, HSCT) is earliest by beauty State scientist Thomas professor started to introduce clinic the 1950s, and after decades of development, HSCT is big for treating Malignant hematologic diseases and metabolism birth defect of amount etc., are existing only tumor radical cure means.

In hematopoietic transplant, Graft versus leukemia (graft-versus-leukaemia, GVL) or graft antitumor (graft-versus-tumour, GVT) is the dominant mechanism of its treating cancer.In contrast, the master after hematopoietic stem cell transplantation Complication graft versus host disease(GVH disease) (graft-versus-host disease, GVHD) is wanted, is main original dead after transplanting Cause, according to statistics, about 50% patient for receiving hematopoietic transplant will appear GVHD symptom, therefore seriously limit this important therapy Generally carry out.

GVHD is a kind of immunological disorders for influencing multiple tracts, including gastrointestinal tract, liver, skin and lung, seriously Quality of life and life span after threatening patient's transplanting.General foundation clinical symptoms time of origin length, GVHD can be divided into Two classes: Acute GVHD (aGVHD) and chronic GVHD (cGVHD).

The cause of GVHD is: (mainly HLA human body is white for genetic determinants albumen in donor's T cell docking donee's cells Cellular antigens, histocompatibility antigen) response caused by immune response, therefore the frequency of disease development of universal aGVHD and HLA It mismatches horizontal directly related.And ideal donor and recipient are that HLA-A/B/C/DRB1 is matched entirely.

GVHD morbidity may be summarized to be three processes: one, antigen presenting cell (APCs, antigen-presenting Cells activation)；Two, the activation of donor's T cell, proliferation, differentiation and migration；Three, the damage of target organ.

Experimentally from animal, T cell plays the role of a nucleus in the morbidity of GVHD.Now there are three types of the strategies of removal T cell: It is transplanted after external removal T cell；External sorting CD34+ stem cell carries out；T cell is removed by antibody specificity in vivo. These strategies all show good curative effect for GVHD (no matter aGVHD or cGVHD), unfortunately these treatment sides Case is but using high graft failure rate, transplanting postoperative infection and high relapse rate as cost.Furthermore other main methods of GVHD are prevented and treated It is that immunosupress processing is carried out to recipient, such as cyclosporin A (cyclosporin-A, CsA), mycophenolate/wheat is replaced to examine phenol Ester (mycophenolate mofetil, MMF), steroids (steroids) etc., but these schemes also reduce GVL effect simultaneously It answers, increases cancer return probability as the result is shown.In recent years, using such as Treg, modulability gamma delta T cells (gamma delta T reg) fill The cell therapy protocols such as matter stem cell (MSC) show preferable curative effect in experimental animal and clinic.

In each subgroup of T cell, CD4+ subgroup is closely related with GVHD by wide coverage, four major subpopulations Th1, Influence of the polarization balance to the disease incidence and coincident with severity degree of condition of GVHD between Th2, Th17 and Treg is very big.Clinically, It was reported that influencing the basic reason of CD4+ differentiation is, when transplanting between donor and recipient MHC and MiHA distribution type degree. Distribution type degree influences CD4+ and CD8+ effector cell by influencing the polarization of CD4+ subgroup, is finally reached to GVHD even The regulation of GVL.In the Acute GVHD as an inflammatory process, detection discovery CD4+ polarizes toward Th1.Recent study is newly sent out Existing one kind can be with the CD4+ cell subsets of yield IL-17, i.e. Th17.Subsequent mouse disease model and clinical research find it in urgency Property GVHD in, to inflammation starting and tissue damage play an important role.And have studies have shown that Th2 in GVHD, with injury of lungs And chronic GVHD is related.And in 4 major subpopulations of CD4+, only Treg from the beginning of identified to finding itself and GVHD phase Association is widely believed that it has inhibition to GVHD and improves effect, while still retaining the efficiency of GVL.To sum up, to CD4+ subgroup The research that polarization changes seems increasingly important.

Due to donor selection optimization, pretreating scheme improve, the optimization of graft versus host disease(GVH disease) (GVHD) prevention scheme and The raising of supportive treatment means, the clinical application of Allo-HSCT has made much progress at present, but acute graft versus host disease (aGVHD) it is still one of major causes of death after transplanting, needs to develop new clinical treatment.

Summary of the invention

The technical problem to be solved by the present invention is to treat problem for existing graft versus host disease(GVH disease), new treatment is provided Drug or method.

On the one hand, human amnion membrane (human amniotic epithelial is utilized the present invention provides a kind of Cells, hAECs) treating the purposes in graft versus host disease(GVH disease).

Allogeneic hematopoietic stem cell transplantation is the only resource for curing hematological malignancies now, but graft-versus-host Disease is the main side effect of hematopoietic stem cell transplantation, is the bottleneck for limiting hematopoietic stem cell transplantation.Since amniotic epithelial cells have Have and inhibit lymphocyte hybrid reaction, inhibits the characteristics such as autoimmune disease and solid tumor resisting, therefore present inventor Amniotic epithelial cells These characteristics are desirable with, seek whether it has response to treatment to graft versus host disease(GVH disease).

By series of studies, inventor is surprisingly had found, method of the present invention not only alleviates inflammation caused by aGVHD Infiltration of the disease cell to target organ, while also substantially reduced target organ lesion.Simultaneously it has also been found that hAECs is thin to multiple leukaemia Born of the same parents system has apoptosis-promoting effect.It therefore, can the present inventors considered that hAECs is the effective ways for treating graft versus host disease(GVH disease) Using the candidate sources treated as such disease cells, therefore complete the present invention.

Human amnion membrane (human amniotic epithelial cells, hAECs) be from placenta near The cell separated on the amnion of fetus side.Placenta belongs to the waste after pregnant woman's production, so the cell in source is not deposited In ethics problem.

Placenta anatomically, can be divided mainly into three-layer weave structure: amnioic epithelium layer, chorion from inside to outside (chorion), and uterine decidua (decidua) and the source of each layer tissue is completely different.Uterine decidua comes from parent, suede Trichilemma is originated from trophoderm (trophoblast), and amnioic epithelium layer is from eight days epiblasts of after fertilization (epiblast), i.e., with Embryonic stem cell the same (embryonic stem cells, ESCs) all derives from embryo inner cell mass (inner cell Mass), and hAECs can keep multipotency after being divided into amnioic epithelium for a long time.It is more that Miki etc. confirms that hAECs can be expressed Energy stem cell (such as embryonic stem cell) main surface maker (such as Oct4, Sox2, Nanog, SSEA-3, SSEA-4 etc.), shows It has the specific potential broken up to three embryonic tissues of embryonic stem cell.Vitro differentiation experiment display, no matter from transcription Horizontal or expression shows that hAECs has external triploblastica differentiation potential.However unlike ESCs, hAECs body Interior to test at teratoma but at feminine gender, main cause is can not to be divided into because of its no telomerase activity with triploblastica The institutional framework of differentiation.Also hAECs is used as cell therapy just because of this, does not have oncogenicity (including benign tumor, sarcoma and cancer).

In addition, hAECs cell surface hardly expresses MHCII type molecule, inflammation, allergy and immune response will not be caused, Therefore immunogenicity is very low, is suitble to transplanted cells treatment.HAECs separation process is relatively simple, removes on scraping later amnion A small amount of blood cell agglomerate there's almost no other types cell contamination, and blood cell belongs in unprovoked situation Suspension cell, thus can be by cell culture after, change liquid removal.According to inventor and external scientist's experiment statistics, every There are about 8,000 ten thousand to 3 hundred million hAECs cells for the amnion of people.Under the conditions of existing for the EGF, hAECs has stronger proliferative capacity, It can be proliferated within about 36 hours a generation, and vigorous proliferative capacity can be kept in preceding generation (in about 10 generations).These advantages are protected Sufficient amount single cell type can be obtained by having demonstrate,proved the application, to meet the requirement of clinical treatment.

In addition to stemness, hAECs is exactly immune regulative there are also a kind of important properties.Early in leaf early 20th century, begin to There is researcher to attempt using amnion as the skin of interim graft materials substitution patient oneself to repair damage successively, and takes Obtained good curative effect.In such phenomenon behind, researcher has found that amnion has anti-repulsion and inhibits bacterial wound growth Effect, this prompt amnion may have certain immune regulative.Ueta et al. discovery, amnion, which has, inhibits lymphocyte mixing anti- The property answered.Li et al. is found later, and the main composition cell hAECs itself of amnion can be pressed down with secretory immune inhibiting factor The chemotaxis of neutrophil leucocyte and macrophage processed, and inhibit T, B cell is by the post-stimulatory proliferation of mitogen.Separately there is science Family's report, amnion also may cause to the macrophage apoptosis of IFN γ activation.Recently, it has been found that hAECs has immune regulative, it can It is obviously improved the autoimmune disease state of an illness.It is interesting that hAECs also has the property of anticancer.Kang etc. has found hAECs in body It can inhibit the proliferation of breast cancer cell line outside, the increase of the upper breast cancer knurl of nude mice can be inhibited in vivo and extend depositing for mouse Live time.Discovery is found by a series of experiment to solid tumor cell systems recently, and amnion, which has, inhibits a variety of solid tumor cells System's proliferation.About tumour is inhibited, primarily now think that hAECs is by inhibiting Tumor Angiongesis and promoting apoptosis of tumor cells Two aspects work.

To sum up, hAECs has stemness and low immunogenicity, therefore can be used as and repair hematopoietic transplant patient because of irradiation damage Natural material be immunoreacted without causing, this can help patient accelerate physical recovery.It is with immune regulative, Ke Yigai The GVHD of maximum seondary effect after kind hematopoietic transplant.It can help host to resist outer to a certain extent with antibiotic property Carry out microbiological attack, prevents transplanting postoperative infection.And its cancer resistance, the function of GVL can be had both while treating GVHD.

Therefore, human amnion membrane hAECs is applied in the treatment of graft versus host disease(GVH disease) by the present invention for the first time, and With good effect, the treatment for such current disease provides a kind of scheme.

On the one hand, the invention discloses treated and/or improved in preparation by human amnion membrane or its cell preparation to move Purposes in the drug of graft versus host disease.Using the human amnion membrane of effective dose or its cell preparation can individually or It is used in combination with other medicines and is treated and/or improved graft versus host disease(GVH disease).Effective dose, which refers to, to be enough to improve or prevent The symptom of medical conditions or the amount of illness.The visual many factors of the effective quantity of specific subject are changed, such as to be treated Disease, the holistic health of patient, the method and approach of administration and dosage and the seriousness of side effect.Effective quantity can be to avoid The maximum dose or dosage regimen of significant side effect or toxic effect.

In another embodiment of the present invention, the animal with graft versus host disease(GVH disease) refers to mammal.? In highly preferred embodiment, the animal is ox, horse, sheep, monkey, dog, rat, mouse, rabbit or the mankind.Optimal In the embodiment of choosing, the animal with graft versus host disease(GVH disease) refers to the mankind.

In the another embodiment of invention, the cell preparation includes human amnion membrane and pharmaceutically acceptable Carrier.Pharmaceutically acceptable carrier of the present invention refers to suitable for people and/or animal, without excessive adverse side effect The substance that there is suitable beneficial/relative risk of (such as toxicity, irritation and allergic reaction), such as pharmaceutically acceptable solvent, Suspending agent or excipient are conducive to cell survival, the prepared cell of transmissibility to human or animal.Carrier is according to suitably planning Administration mode and select.Carrier of the invention includes but is not limited to various physiological buffers, as physiological saline, phosphate buffer, Artificial cerebrospinal fluid or whole serum, cord serum etc..It may also include various man-made supports, including but not limited to gelfoam, de- Calcium bone, polyglycolic acid (PGA), polylactic acid (PLA) and their copolymer.

In view of the type of disease to be treated, those skilled in the art can suitably select the conjunction of amniotic epithelial cells Suitable state: the cell (slightly mentioning part) of the collection without any processing；Partially purified cell；The cell of purifying is then through cultivating Amplification.

In a preferred embodiment of invention, a kind of side that amniotic epithelial cells are separated from amnion tissue is provided Method the described method comprises the following steps:

(1) amnion is obtained from placenta tissue by mechanically decoupled；

(2) amnion after cleaning is digested with digestive ferment, and postdigestive liquid is centrifuged, can be obtained people's amnion Epithelial cell.

Amniotic epithelial cells of the present invention derive from the mankind.Amnion can be separated from vitro Human plactnta, using physiology Buffer, which rinses, removes haemocyte, and machinery rejects residual chorion and blood vessel.Separation refers to the emigrated cells from tissue sample And it is separated with other tissue.Isolated using any routine techniques or method from intact human amniotic membrane's epithelium layer tissue it is unicellular, These techniques or methods include mechanical force (shred-ability or shearing force), with a kind of or combined protease such as clostridiopetidase A, pancreas egg White enzyme, lipase, release enzyme (liberase) and pepsin carry out enzymic digestion or mechanical and enzyme method combination.

In a preferred embodiment of the present invention, after the acquisition of people's amnion answers multipara to authorize agreement, healthy production is taken The postcesarean placenta tissue of woman obtains whole amnion by mechanically decoupled.

In another preferred embodiment of the present invention, can continue to cultivate to human amnion membrane is obtained in step 2, Preferred condition of culture are as follows: with 1 × 10⁶-1×10⁸Cell inoculation in culture dish, is placed in two by the density of a cell/plate It is cultivated in carbonoxide incubator, changes culture solution after human amnion membrane is adherent, by cell dissociation after cell covers with plate Get off to be frozen.

Active cell population is concentrated those skilled in the art's available known other methods.Washed after these processing/dense Contracting step can be implemented individually or simultaneously.In addition to the method described above, can also cell washing after or culture after, be further purified or Deposition activity cell colony reduces heteroproteose cell and dead cell.Cell in suspended liquid can be realized by following technology: floating Force density sedimentation centrifugation, the differentiated adhesion with solid phase and elution, immune magnetic pearl, Fluorescence laser cell sorting from solid phase (FACS) or other technologies.These different technologies and the example of device for carrying out these technologies can be found in the prior art and listing Commodity.

There is no limit as long as can be used for the culture medium of cell culture i.e. for the type of basal medium used the present invention It can.Preferred culture medium includes DMEM culture medium and NPBM culture medium.To what may be contained in basal medium above-mentioned There is no limit preferred ingredient includes F-12, FCS and neuronal survival factors etc. to other component types.

In another preferred embodiment of the present invention, bFGF (alkalinity is added in the basal medium upward mentioned Fibroblast growth factor) or EGF (epidermal growth factor).All add in such a case, it is possible to which one kind or both is added Enter.The citing concentration of bFGF or EGF above-mentioned are 1ng/ml to 100ng/ml, and preferred concentration is 10ng/ml.To addition Time and method there is no limit.Preferably, every when cultivating amniotic epithelial cells above-mentioned in basal medium Reagent is added in it.

Amniotic epithelial cells can be given in patient, such as intravenous injection or spinal cavity using any suitable method Injection etc..These usual cells are included in pharmaceutically acceptable fluid nutrient medium.Cell is given can be repeatedly or continuous Carry out (for example, by continuously implantation injection cerebrospinal fluid).In general, multiple administrations mode will be usually spaced at least 7-10 days It uses respectively.Another method is to be had kind using operation by cell seeding in bioabsorbable material such as gelfoam Position needed for the bioabsorbable material implantation of cell.Above two method can obtain better curative effect in combination with application.

The suitable amount of amniotic epithelial cells will be according to the age of patient, gender, weight, health status and other factors And change.In general, the dosage range given every time is about 10³-10⁹Cell, typically about 10⁶-10⁷Cell.

In some embodiments of the present invention, amniotic epithelial cells are administered to trouble together with one or more drugs Person, the drug be selected from methylprednisolone, cyclosporine, tacrolimus, Mycophenolate Mofetil, methotrexate (MTX), glucocorticoid, Imuran, Thalidomide, anti-T cell monoclonal antibody (anti-CD49d McAb), -2 receptor of anti-IL-8 antibody etc. in One kind or combinations thereof.

The present invention using the amniotic epithelial cells of effective dose or cell preparation containing amniotic epithelial cells individually or It is used in combination with other medicines and is treated and/or improved graft versus host disease(GVH disease).Being proved using animal model of the invention should Method can effectively mitigate infiltration of the inflammatory cell caused by aGVHD to target organ, while also substantially reduced target organ lesion, together When also found hAECs to multiple Leukemia Cell Lines have apoptosis-promoting effect, can be used in treat graft versus host disease(GVH disease), facing Bed application will above have extensive prospect.Human amnion membrane is used for the treatment of graft versus host disease(GVH disease) by the present invention, sufficiently The advantages of having played human amnion membrane, wherein human amnion membrane mainly has the advantage of the following aspects:

(1) multipotency can be kept for a long time and has embryonic stem cell is specific to dive to what three embryonic tissues broke up Energy；

(2) cell surface hardly expresses MHCII type molecule, therefore will not cause inflammation, allergy and immune response, transplanting Distribution type requires also corresponding reduce；

(3) have and adjust the ability that is immunoreacted in vivo and in vitro, can be secreted when cultivating in vitro panimmunity adjust because Son, anti-angiogenic proteins or anti-inflammatory factors GAP-associated protein GAP；

(4) there is low immunogenicity, can be regarded as immune privilege cell, the function of nonantigenic presentation can subtract after transplanting Few immunocyte source, avoids the generation of immunological rejection；

(5) reverse transcriptase of telomere is not expressed, without oncogenicity (including benign tumor, sarcoma and cancer)；

(6) there is stronger proliferative capacity, and vigorous proliferative capacity can be kept in preceding generation (in about 10 generations)；

(7) from a wealth of sources, materials are easy, not application limitation, and ethics problem is not present.

Detailed description of the invention

CD3 and CD45 accounting illustrates in each group of Fig. 1

Each group mouse appearance diagram of Fig. 2

Each group mouse weight of Fig. 3 changes over time diagram

Each group mouse survival rate of Fig. 4 changes over time diagram

Fig. 5 flow cytometry shows Th1 in each group Mice Body, Th17 and Treg accounting diagram.AGVHD=PBMC group, AGVHD preventing=PBMC+hAECs group, aGVHD treating=PBMC+hAECs (Day7) group.

Each group mouse endothelial adhesion molecule expression diagram of Fig. 6 histogenic immunity fluorescence

Each group mouse target organ slice HE dyeing diagram of Fig. 7

Each group mouse organs pathological score diagram of Fig. 8

Each group mouse lung Masson dyeing diagram of Fig. 9

Main aGVHD target organ paraffin section CD3 (rufous) immunohistochemical staining of each group mouse of Figure 10

Figure 11 hAECs slow-virus infection diagram

Figure 12 hAECs shows in the default bitmap of Mice Body

Specific embodiment

The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional method in that art and condition, or It is selected according to product manual.

The primary amniotic epithelial cells isolation and culture of embodiment 1

1, the source of people's amnion

In order to avoid birth canal microbial contamination, caesarean birth fetal placenta has been selected.Due to mature rear signal stimulus of giving a birth, sheep Apoptosis can occur for film, therefore preferably use prematurity of fetus placenta (before 38 weeks).Multipara authorize agree to after, take healthy puerpera (HIV, The serological reactions such as syphilis, hepatitis A, hepatitis B, hepatitis are illustrated as feminine gender) postcesarean placenta tissue, cross-shaped knife cuts tire Disk obtains whole amnion by mechanically decoupled.

2, the separation of hAECs (whole process requires sterile working)

Surgical neonate placenta before obtaining 39 weeks, from placenta inner face denuded amniotic membrane, by immersion contain F12/DMEM and (contain 1X Pen .- Strep and anphotericin) basal medium centrifuge tube in.4 DEG C of cold chains are transported between laboratory cell.

Amnion is taken out, and every amnion is placed in 40ml CMF-HBSS (Pen .- Strep containing 1X and anphotericin) Mucus is gone in middle cleaning, and scrapes off the mesenchyma layer and mucus close to chorionic membrane with tweezers, is repeated 3 times, and washing renews every time Container and new HBSS liquid.

Clean amnion is transferred to new container, adds 0.05% pancreatin of 10ml/EDTA, overturns 30s, abandons liquid.

Amnion is transferred to new container, adds 0.05% pancreatin of 20ml/EDTA, 37 DEG C of water-baths are incubated for 10min and abandon liquid.

Amnion is transferred to new container, and 25ml pancreatin/37 DEG C of EDTA water-bath is incubated for 40min, saves digestive juice.

Amnion is transferred to new container after first digestion, and 25ml pancreatin/37 DEG C of EDTA water-bath is incubated for 40min, saves digestive juice.

Isometric digestion terminate liquid (F12/DMEM contains 5%FBS, 1xL- glutamic acid, 1x pyruvic acid), 400g centrifugation is added 10min.Liquid is abandoned, with amnion complete medium: F12/DMEM (KnockOut Serum Replacement) containing 5%KSR, 1x Precipitating is resuspended in L-Glutamine, 1x pyruvic acid, 10ng/ml hEGF, 1Xps (Penicillin-Streptomycin).

Isometric digestion terminate liquid is added, 400g is centrifuged 10min.Liquid is abandoned, precipitating is resuspended in complete medium.

100um sieve is crossed, is counted, with 10⁵Cells/cm2 be seeded to culture dish or be placed in frozen stock solution (90%FBS, 10% DMSO liquid nitrogen cryopreservation is spare in).

3, it the inoculated and cultured of hAECs and freezes

Cell culture: inoculation 1 × 10⁷After a cell, culture solution is changed after hAECs is adherent, changes primary culture within three days later Liquid.

Cell dissociation gets off to freeze after cell covers with plate: 15cm dish adds 5ml pancreatin, after 10min under mirror Cell becomes that equivalent digestion terminate liquid is added to terminate digestion when suspended state full when observation, cell rounding and plane shake plate.With Micropipettor at same direction blows down the cell on culture dish, moves into 15ml centrifuge tube, and 300g is collected carefully after being centrifuged 3min Then born of the same parents carry out cell count.Frozen stock solution is added in cryopreservation tube, indicates cell after freezing date, batch and cell quantity It is put into cryopreservation tube, then cryopreservation tube is put into freezing storing box at once and puts freezing storing box into -80 DEG C of refrigerators, takes out and freezes after 12h Cell is moved into liquid nitrogen container and is saved by box.

The separation of 2 human peripheral blood single nucleus cell of embodiment is cultivated and is frozen

In aseptic aspiration donor peripheral blood to the collection belt containing anti-coagulants.

In 50ml centrifuge tube be added 5ml Ficoll liquid, with sterile Pasteur pipet draw blood be softly added dropwise in On Ficoll liquid.

Without centrifugal acceleration, room temperature 400g is centrifuged 30min, until obvious layering occurs in blood.

New pasteur pipet carefully draws the tunica albuginea layer in layering, and it is equal that mixing in the DPBS containing 10ml (containing 1xPS) is added It is even.Room temperature 300g is centrifuged 5min, abandons liquid.

(containing 1xPS), precipitating was resuspended in the DPBS of 10ml, and room temperature 300g is centrifuged 5min, abandons liquid.

It is spare with complete medium (1640,10%FBS, PS) resuspension, or freeze after being resuspended with frozen stock solution liquid in liquid nitrogen.

3 Establishment of mouse model of embodiment and aGVHD mouse standards of grading

1, NOD/SCID mouse irradiation damage model foundation

6~8 week old NOD/SCID mouse, which are bought, from company is transferred to animal house raising 1 week to adapt to environment.Drinking water adds Enter gentamicin (32x10^4U/L) and erythromycin (250mg/L).

Mouse carries out systemic sublethal dose irradiation with the X-ray of 2.7Gray dosage after a week.

Secondary Nikkei tail vein injects hAECs 2X10^6 every mouse to every mouse.Control group then injects corresponding body Long-pending PBS.

2, NCG chmice acute GVHD model foundation

6~8 week old NCG mouse, which are bought, from company is transferred to animal house raising 1 week to adapt to environment.It is big that celebrating is added in drinking water Mycin (32x10^4U/L) and erythromycin (250mg/L).

10^7 human peripheral blood single nucleus cell (PBMC), disease prevention group are injected to every mouse through tail vein after a week Then in addition inject hAECs 2X10^6 every mouse.Disease treatment group then after a week, then is every other day infused having injected PBMC Penetrate 2X10^6 hAECs, 6x10^6 hAECs of every mouse co-injection.Control group then injects the PBS of corresponding volume.

3, aGVHD mouse standards of grading

AGVHD mice clinical phenotype standards of grading (according to KR.Cook points-scoring system, totally 10 points, every other day observe one It is secondary.)

Weight loss: mouse weight mitigation regards as normal 0 point less than 10%；Weight loss is between 10%~25% Obtain 1 point；Degree of alleviation is greater than 25% and obtains 2 points.

Attitudes vibration: mouse posture is without exception to regard as normal 0 point；Only 1 point is obtained in hunchbacked state during the break；Seriously The back of a bow obtains 2 points so that influencing normal activity.

Hair texture variation: mouse hair texture is without exception to regard as normal 0 point；Slight gauffer obtains 1 point；Serious gauffer Obtain 2 points.

Energy: mouse energy is normal to obtain 0 point；Energy slightly weakens to obtain 1 point；It is inactive if non-stimulated to obtain 2 points.

Skin integrity: mouse skin is without exception to regard as normal 0 point；There is exfoliating skin and obtain 1 point in tail portion or sole； There is obvious area of spall and obtains 2 points in skin

AGVHD mouse organs' pathological score standard (totally 4 points)

Lung: normal 0 point normally is regarded as without lesion；Blood vessel week has a small amount of leukocyte recruitment to obtain 0.5 point；Blood vessel week is white thin Born of the same parents' aggregation extent is 1-2 cells thick (being related to 15% blood vessel week), obtains 1 point；Blood vessel week leukocyte recruitment degree is 1-2 cells thick (being related to 15% blood vessel week), and infiltrate into parenchymal tissue, obtain 1.5 points；Blood vessel week leukocyte recruitment degree is 2-3 cells thick (being related to 15% blood vessel week), and infiltrate into parenchymal tissue, obtain 2 points；Blood vessel week leukocyte recruitment degree is that 2-3 cells thick (relates to And 25%-50% blood vessel week), and infiltrate into parenchymal tissue, obtain 2.5 points；Blood vessel week leukocyte recruitment degree is 4-5 cells thick (being related to 25%-50% blood vessel week), and infiltrate into parenchymal tissue, obtain 3 points；Blood vessel week leukocyte recruitment degree is 6-7 cell Thick (being related to 50% blood vessel week), and infiltrate into parenchymal tissue, serious tissue normal configuration of destroying obtains 3.5 points；Blood vessel week is white thin Born of the same parents' aggregation extent is 6-7 cells thick (being related to more than 50% blood vessel week), and is infiltrated into parenchymal tissue, and serious destruction tissue is normal Structure obtains 4 points.

Intestines: normal 0 point normally is regarded as without lesion；Occur drawing nest meronecrosis once in a while, few inflammatory cell infiltration is extremely Submucosa and intestinal villi obtain 0.5 point；Draw nest meronecrosis and reach 15%, inflammatory cell infiltration to 20% submucosa and Intestinal villi obtains 1 point；Draw nest meronecrosis and reach 15%, inflammatory cell infiltration is obtained to 1/3rd submucosas and intestinal villi 1.5 point；Draw nest meronecrosis and reach 25%, inflammatory cell infiltration to 1/3rd submucosas and intestinal villi obtains 2 points；Draw nest Meronecrosis reaches 25%-50%, and inflammatory cell infiltration to 1/3rd submucosas and intestinal villi obtains 2.5 points；Draw nest cell Necrosis is greater than 50%, and inflammatory cell infiltration to 1/3rd submucosas and intestinal villi obtains 3 points；Draw nest meronecrosis to be greater than 50%, inflammatory cell infiltration to 50% submucosa and intestinal villi obtains 3.5 points；Draw nest meronecrosis greater than 50%, inflammation is thin Born of the same parents' infiltration obtains 4 points to 50% submucosa and intestinal villi is greater than.

Spleen: normal 0 point normally is regarded as without lesion；Every square millimeter of group is woven with 10 necrosis or apoptotic cell obtains 1 point； Every square millimeter of group is woven with 10 necrosis or apoptotic cell and occasionally has haemolysis, obtains 1.5 points；Every square millimeter of group is woven with 10-20 Necrosis or apoptotic cell occasionally have haemolysis and damage with institutional framework, obtain 2 points；Every square millimeter of group be woven with 10-20 necrosis or Apoptotic cell has haemolysis and damages with 25% institutional framework below, obtains 2.5 points；It is a bad that every square millimeter of group is woven with 20-40 Dead or apoptotic cell has haemolysis and damages with the institutional framework of 25%-50%, while having lower than 25% tissue fibrosis, obtains 3 Point；Every square millimeter of group is woven with 20-40 necrosis or apoptotic cell, has haemolysis and damages with 50% institutional framework, simultaneously There is 25%-50% tissue fibrosis, obtains 3.5 points；Large area (be greater than 50%) necrosis or apoptotic cell are organized, there is haemolysis and adjoint Institutional framework damage greater than 50%, while having greater than 50% tissue fibrosis, obtain 4 points.

Liver: normal 0 point normally is regarded as without lesion；The every 0.5cm of 1-2 mononuclearcell assembles in focal zone parenchymal tissue Organize to obtain 1 point；Every 0.5cm tissue is there are an endothelitis blood vessel, and at least 2 inflammatory cell infiltrations obtain under every blood vessel endothelium 2 points；Every 0.5cm tissue is there are 3 endothelitis blood vessels, and at least 3 inflammatory cell deep invasions obtain 3 under every blood vessel endothelium Point；There is endothelitis in nearly all blood vessel, at least 3 inflammatory cell deep invasions obtain 4 points under every blood vessel endothelium.

The grouping of 4 aGVHD Experimental model of small mice of embodiment

Using in vivo without mature T cells, B cell, the NOD-Prkdcem26Cd52Il2rgem26Cd22/Nju of NK cell Mouse (NCG mice) is used as model mouse, and implantation human peripheral blood single nucleus cell (PBMC) establishes humanization aGVHD mouse mould Type.Experiment is divided into four groups: 1. using the independent transplantation group of PBMC as aGVHD disease model group；2.hAECs and PBMC is transplanted simultaneously As aGVHD disease prevention group；3. first transplanting PBMC, hAECs is transplanted again after a week as aGVHD disease treatment group；4. injection The training liquid of respective volume is as blank control group.

The results show that FACS is the result shows that in aGVHD model mouse, and (CD45 divides by CD3 and CD45 after PBMC is implanted into 2 weeks Son has expression on all leucocytes and chimeric rate is up to 50% or more (Fig. 1).And it is implanted into hAECs simultaneously and plants after a week Enter hAECs and is all not apparent from the implantation (Fig. 1) for influencing PBMC.Furthermore, it was found that all CD45+ cells of implantation nearly all co-express CD3 (Fig. 1).

It is implanted into after two weeks in PBMC, observes that the mouse for being separately implantable PBMC starts to gradually appear a series of aGVHD typical cases Clinical signs: weight loss, energy decline is apathetic, the serious back of a bow, depilation, diarrhea etc., and co-transplantation simultaneously Every disease characterization is readily apparent that improve hAECs (i.e. prevention group) later.Though and transplanting the treatment group of hAECs after a week Therapeutic effect still has more apparent change not as good as prevention group, but compared with disease group.However in the back of a bow, diarrhea (this and posterior tissues Dyeing is consistent), treatment group and prevention group are not much different (Fig. 2, Fig. 3) in energy.Inventor carries out clinical score to this phenotype, As a result prevention group and treatment group and disease group show significant difference.

Life quality of the implantation of hAECs in addition to improving disease mice, it is often more important that extend the life of aGVHD mouse It deposits the time and significantly improves its survival rate, the effect of same prevention group is more preferable than treatment group (Fig. 4).

5 flow cytometry of embodiment

1, cell surface marker dyes

If sample is that tissue or blood cell need splitting erythrocyte.3ml erythrocyte cracked liquid is added in Xiang Guanzhong cell (ACK) cell is resuspended, is incubated for 5~20min on ice.

10ml cell dyeing liquid (PBS containing 2%FBS) is added to terminate ACK cracking in Xiang Guanzhong.The centrifugation of 350g room temperature 5min abandons supernatant.If it is desired, ACK, which can be added, repeats cracking one time.

Step 2 is repeated to wash cell one time.

After viable count, add cell dyeing liquid adjustment cell Particle density in 5-10x10^6cells/ml, with every pipe The cell liquid of 100ul is dispensed into streaming pipe.

It is added in suitable primary antibody to the cell suspension got ready for thering is fluorescent marker to be coupled.It is protected from light on ice and is incubated for 20- 30min。

The cell dyeing liquid that 2ml is added is washed 2 times, and 350g room temperature centrifugation, 5min abandons supernatant.

Cell, flow cytometry analysis is resuspended in the Cell Staining Buffer that 0.5ml is added.

2, factor dyeing intracellular

The cell of stimulation activation is collected, these cells can be the tissue stimulated in vivo, be also possible to through stimulated in vitro Cultivate cell.At last 4-6 hours of stimulating course, albumen transport inhibitors brefeldin A or monensin is added, with suppression Cytokine secretion processed is to extracellular.It is in charge of dyeing as needed.

Before fixed rupture of membranes and dyeing intracellular, padding is carried out to living cells.

Before carrying out dyeing intracellular, cell, every pipe 0.5m l fixer are fixed with Fixation Buffer, room temperature is kept away Light is incubated for fixed 20min.350g is centrifuged 5minutes, abandons supernatant.

If there is stopping the needs of experiment, pause can be walked herein and is saved, continues operation experiments in case of after.Use Cell Staining Buffer washes a cell, and 350g is centrifuged 5minutes, abandons supernatant.Cell Staining Buffer is resuspended thin Born of the same parents, can be in 4 DEG C of short-term preservations.Or it is saved with 90%FCS/10%DMSO in -80 degree longer-terms.

It is resuspended fixed cell with Permeabilization Wash Buffer, 350g is centrifuged 5-10minutes, in abandoning Clearly.

Repeat step 6.

The cell after fixed rupture of membranes is resuspended in 100ul Permeabilization Wash Buffer, and specific fluorescence is added Labelled antibody (amount of antibody adds according to specification), room temperature is protected from light 20min.

Cell is washed twice with 2ml Permeabilization Wash Buffer.350g is centrifuged 5minutes, in abandoning Clearly.

The cell after fixed dyeing, flow cytometer analysis is resuspended with 0.5ml Cell Staining Buffer.

HAECs transformation T cell subgroup is studied by flow cytometry to divide group in vivo and influence CD4+T cell-stimulating, It studies hAECs transformation T cell subgroup by flow cytometry to divide group in vivo and influence CD4+T cell-stimulating, as the result is shown CD4+T cell-stimulating suppressed (Fig. 5-1), Th1 subset proportions reduce (Fig. 5-2), Treg in aGVHD mouse after hAECs treatment Subset proportions increase (Fig. 5-3), but have no significant effect to Th17 subgroup (Fig. 5-4) and Th2 subgroup (Fig. 5-5) ratio.It is above-mentioned HAECs is by inhibiting t cell activation and transformation each subset proportions of T cell to improve aGVHD as the result is shown.

6 immunofluorescence of embodiment

1, cellular immunofluorescence

Every hole cell (24 orifice plate) suction takes training liquid, is added after 1ml PBS rolling is washed and abandons liquid.

It is added 4% paraformaldehyde of 1ml (PBS dilution), is placed in incubation at room temperature 30min.

Liquid is abandoned, and is washed 2 times with PBS rolling, liquid is abandoned.

Triton X-100 (PBS dilution) the incubation at room temperature 15min of 500ul 0.2% is added (if antigen is memebrane protein Skip this step).

10%FBS (or 3% lowlenthal serum or 2% horse serum) is added and closes 1~2h.

Liquid is abandoned, after PBS is added, horizontal shaker 60rpm room temperature is placed in and washes 5min and be repeated 3 times

10%FBS or 3% horse serum dilute primary antibody, and every hole adds 250ul.It is incubated at room temperature 1h (or 4 ° overnight).

Liquid is abandoned, 500ul wash buffer is added, is placed in horizontal shaker 60rpm room temperature and washes 5min, wash altogether 3 times.

10%FBS or 3% horse serum dilution fluorescence are coupled secondary antibody, and every hole adds 250ul.Room temperature, which is protected from light, is incubated for 1h.(this step is opened The subsequent all steps that begin are protected from light operation.)

The diluted DAPI of PBS is added, every hole adds 250ul, is incubated at room temperature 5min.

Liquid is abandoned, 500ul wash buffer is added, is placed in horizontal shaker 60rpm room temperature and washes 5min, wash altogether 2 times.

Liquid is abandoned, microscopy after 250ul PBS is added in every hole.If cell climbing sheet, then mounting drop is added on coverslip, it will Creep plate taking-up places cell facing towards coverslip, this step operation mainly tries not to generate bubble, uses if generating bubble Tweezers carefully squeeze out bubble, microscopy.

2, histogenic immunity fluorescence

It after being taken out in tissue auto, is immediately placed in blood stains are washed away in PBS, is carefully suitable size (note by tissue cutting Meaning not pinch firmly and take tissue).Tissue is placed in the embedded box equipped with frozen section embedding medium (OCT).Embedded box is placed in In the isopentane of dry ice pre-cooling, until OCT and tissue fully charge.

Embedded tissue is fixed on freezing microtome, is cut to the slice of 0.5um, and slice is affixed on adhesiveness and carries glass On piece.

It needs to be placed at room temperature for 30min before slice dyeing, so that slice is sufficiently adhered to slide.

Sample is put in acetone, -20 DEG C, fixed 10min (if group has been fixed before being woven in embedding, is directly carried out subsequent Operation).

Slice takes out, and is washed 3 times with PBS, each 5min.

Tissue is first enclosed with oil pen, after primary antibody the PBS of primary antibody 5%HBS+1%BSA (dilute) is organizationally added dropwise, It is placed in 4 DEG C of wet box overnight；

Slice takes out, and is washed 3 times with PBS, each 5min；

It removes slice and shows moisture, fluorescence coupling secondary antibody (being diluted with the PBS of 5%HBS+1%BSA) is added dropwise, is placed in wet box Room temperature, which is protected from light, is incubated for 1h.(subsequent operation that this step starts is protected from light operation)

Slice takes out, and is washed 3 times with PBS, each 5min.

After DAPI is diluted with PBS, be added dropwise on slice, be incubated at room temperature 3min, after cleaned 2 times with PBS, each 5min；

Mounting fluid-tight piece, fluorescence microscopy.

The aorta pectoralis for collecting each experimental group and control group mice has carried out frozen section to it and adhesion molecule is exempted from Epidemic disease fluorescent staining.As a result, it has been found that being implanted on its blood vessel endothelium of the aGVHD group of PBMC compared to negative control group (i.e. injection PBS group) The expression of adhesion molecule I-CAM1 and V-CAM1 is significantly raised, and with expected from inventor as hAECs and PBMC co-transplantation with The expression (Fig. 6) of endothelium I-CAM1 and V-CAM1 is obviously reduced afterwards.Therefore, it is considered that hAECs can inhibit endothelial adhesion molecule table Up to mitigate blood vessel GVHD.

The embedding of 7 tissue paraffin of embodiment and slice

Tissue is immediately placed in cleaning in 10% formalin and removes blood stains after taking out in vivo.It cleans 3 times altogether.

Tissue is placed in room temperature in 10% formalin or 4%PFA to fix.

Tissue is pulled out, after PBS is cleaned, with 70% ethyl alcohol room temperature dehydration.

After Gradient elution using ethanol, it is placed in permeabilization in dimethylbenzene.

The good tissue of permeabilization is placed in 4 DEG C of solidifications through paraffin embedding.

By paraffin mass in slicer, foundation different experiments need to be cut into 5-10 μm of not equal slice.

Piece is spread out, glass slide fishes out piece, and dry all steps obtain paraffin section finished product.

8 HE of embodiment dyeing

Paraffin section is placed in 65 DEG C of environment 1h to dewax.

Slice dewaxes through xylene solution three times, successively impregnates 10min, 10min, 5min.

Slice is taken out and carries out aquation through 100% ethyl alcohol 5min, 100% ethyl alcohol 5min and 95% ethyl alcohol 2min.By stream Water rinses 5min.

Slice contaminates core 5min through hematoxylin, and rear flowing water rinses 5min and removes excess dyestuff.

Slice is placed in ethanol solution hydrochloride immersion 2s to break up, rinses 5min through flowing water.

Weak aqua ammonia impregnates 6s, and flowing water rinses 5min, enters 95% ethyl alcohol 4min.

Slice contaminates 40s through Yihong.

Cross 95% ethyl alcohol 2min, 95% ethyl alcohol 2min, 100% ethyl alcohol 4min, 100% ethyl alcohol 4min.

Xylene solution, each 5min, 5min, 5min are crossed respectively.

With microscopy after resinene mounting.

The results show that there is large area endothelial inflammation lesion at aGVHD model group mouse liver portal vein.The lung of lung It bubbles out existing inflammatory cell infiltration and downright bad nodule occurs in periendothelial.There is local edema in kidney.Intestinal villi is found in small intestine Passivation.And the prevention group transplanted simultaneously in PBMC and hAECs, discovery hepatic endothelial inflammation foci almost disappear.Alveolar inflammation is thin Born of the same parents' infiltration significantly reduces, and the necrosis area of periendothelial is obviously reduced.And kidney and small intestine do not find obvious lesion.And in PBMC Transplanting replants treatment group's discovery into hAECs to liver after a week, though the treatment of the lesion of lung compares disease not as good as prevention group Organize still substantially reduced lesion degree (Fig. 7, Fig. 8).Inventor has found that treatment group has no obvious shadow for Pathological change It rings；And it is obvious as prevention group to the improvement result of small intestine, this with the treatment group's changes of weight clinically observed it is small with And the symptom without diarrhea is consistent (Fig. 2, Fig. 3).

9 Masson of embodiment dyeing

Paraffin section is placed in 65 DEG C of environment 1h to dewax.

Reagent A contaminates core 5min, and flowing water rinses 5min.

Slice is put into 65 DEG C of baking ovens to surface of glass slide moisture and evaporates, and slice surface whitens.

Slice is lain in a horizontal plane in wet box, and reagent B is added dropwise on slice and covers sample, room temperature contaminates 10-20min.

Reagent C is added dropwise in tangential section, is incubated for 3min, abandons liquid, is repeated 2 times.

Slice surface moisture is removed, reagent D solution is added dropwise and is incubated for 3-5min.

Reagent D is sucked, reagent adding E is incubated for 5-15s.

Reagent C aqueous solution is added dropwise, abandons liquid, keeps dyeing clear.

Slice through Gradient elution using ethanol, dimethylbenzene permeabilization, with microscopy after neutral gum mounting.

The results show that a large amount of positive stained areas (blue) occurs in aGVHD model group, this prompt disease group lung may go out Show fibrosis lesion, and prevention group positive staining significantly reduces.Although it was unexpected that the HE dyeing of lung, treatment group and CD3 Its pathology improves not as good as prevention group immunohistochemical staining as the result is shown, however in the Masson dyeing of indicating fiber, treatment Group is but significantly better than prevention group (Fig. 9,10).

10 immunohistochemical staining of embodiment

Paraffin section is placed in 65 DEG C of environment 1h to dewax.

Slice is placed in the antigen retrieval buffers for being preheated to 98 DEG C, 98 DEG C of constant-temperature incubation 30min.

It is cooled to room temperature, slice embathes 5min through PBS room temperature.

3% dioxygen water incubation 10min is added dropwise in slice.

It is cleaned 2min x 3 times with PBS.

Drying slice, is added dropwise primary antibody, 4 DEG C are incubated overnight.It is cleaned 2min x 3 times with PBS.

Reagent 1 is added dropwise, is incubated at room temperature 20min, is cleaned 2min x 3 times with PBS.

Reagent 2 is added dropwise, is incubated at room temperature 20min, is cleaned 2min x 3 times with PBS.

DAB colour developing 5-20min is added dropwise.

5min, haematoxylin redyeing, Gradient elution using ethanol, dimethylbenzene permeabilization and with after resinene mounting are rinsed through flowing water Microscopy.

11 MACS of embodiment sorting

PBMC is packed into sterile tube after being resuspended with training liquid to the every 100ul of 10^7 cell, 10ul antibody, ice is added in every 100ul Upper incubation 15min.

Magnetic bead is resuspended to be allowed to mix, 10ul magnetic bead is added in every 100ul, is incubated for 15min on ice.

3ml MojoSort is added^TMBuffer and cell mix.

Cell is placed in magnet absorption 5min.

Liquid is poured out to collecting pipe, with same volume training liquid mixing, 350g is from 5min.It (can be weighed if you need to improve the rate of recovery Multiple 3-5 step)

Liquid is abandoned, cultivates or freezes after being resuspended with complete medium.

12 CFSE of embodiment detection proliferation

10^6-5x10^6 cell is resuspended in 1mlCFDA SE marking fluid.

With CFDA SE marking fluid dilution CFDA SE storing liquid (1000X) to 2X.

1ml CFDA SE storing liquid (2X) is added in 1, soft to mix, 37 DEG C of incubation 10min.

Complete medium (containing serum) is added to terminate reaction, overturns several lower mixings.

300g is centrifuged 5min, abandons liquid.

10ml complete medium is added to be resuspended.

300g is centrifuged 5min, abandons liquid.

10ml complete medium is added to be resuspended, 37 DEG C of incubations 5min, to promote CFDA SE resident in the cell and not The CFDA SE of reaction enters complete cell culture medium.

300g is centrifuged 5min, abandons liquid.

The cell marked is cultivated as needed.

Cell is collected, FACS detects intracellular average fluorescent strength.

The positioning of 13 slow-virus infection of embodiment and hAECs in target organ

Viral related each element plasmid and vector plasmid, after DH5 α expansion is numerous, with endotoxin free plasmid extraction Kit carries out plasmid extraction.

It is long to about 80% or so to 10cm disk to be inoculated with 293T.

By each plasmid of virus be mixed it is even after, turn reagent with water and calcium and mix, incubation at room temperature 2min.

Above two solution is rocked uniformly.

37 DEG C of overnight incubations change liquid and collect virus in right times.

After virus is mixed with appropriate polybrene, after aim cell infection 48-72h is added, label protein table is detected It reaches, to determine efficiency of infection.

Its form of the hAECs of virus infection is more normal, and green fluorescence microscope photo then indicates high infection effect Rate (Figure 11).HAECs with GFP label individually or with PBMC is implanted in NCG Mice Body jointly, takes each mouse after a week Organ, extracting mRNA and reverse transcription are cDNA, have detected GFP in the expression of each tissue with RT-PCR, indicate hAECs with this In the positioning scenarios of each tissue.GFP is in kidney as the result is shown, lung, high expression in liver, and in small intestine, spleen, expresses in lymph node It is extremely low, it does not almost find to express in marrow.This prompt hAECs is primarily located within kidney, lung, in liver (Figure 12).

The present invention has studied human amnion membrane (human amniotic epithelial cells, hAECs) and is moving Potential ability in the treatment of graft versus host disease simultaneously excavates its cure mechanism.The results show that hAECs and PBMC co-transplantation is pre- Anti- group and treatment group significantly improve the clinical phenotypes and pathology of mouse compared to disease group to the preferable inhibitory effect of aGVHD Phenotype, while significantly improving the survival rate of mouse.

Prove that human amnion membrane can effectively mitigate inflammatory cell pair caused by aGVHD using animal model of the invention The infiltration of target organ, while also substantially reduced target organ lesion, while also found that hAECs has multiple Leukemia Cell Lines Apoptosis-promoting effect can be used in treating graft versus host disease(GVH disease), will have extensive prospect in clinical application.

In this description, the present invention is described with reference to specific embodiment, and the explanation of embodiment is only intended to help Assistant solves method and its core concept of the invention.Explanation of the invention is illustrative and be not restrictive, art technology Personnel can easily make improvement and modification without departing from the principle of the present invention, but these improvement and modification are also all fallen Enter the claims in the present invention protection in the range of.

Claims

1. the use of human amnion membrane or its cell preparation in preparation treatment and/or the drug for improving graft versus host disease(GVH disease) On the way.

2. purposes according to claim 1, it is characterised in that: use the human amnion membrane of effective dose or its cell Preparation is used in combination individually or with other medicines and is treated and/or improved graft versus host disease(GVH disease).

3. purposes according to claim 1, it is characterised in that: the proper states of the amniotic epithelial cells are without appointing Then cell, partially purified cell or the cell of purifying of the collection of where reason are expanded through culture.

4. purposes according to claim 1, it is characterised in that: any suitable method can be used amniotic epithelial cells Give patient, such as intravenous injection or spinal cord intracavitary administration etc..

5. purposes according to claim 4, it is characterised in that: cell, which is given, can repeat or be carried out continuously, multiple administrations Mode will usually be spaced at least 7-10 days and use respectively.

6. purposes according to claim 1, it is characterised in that: the dosage range that amniotic epithelial cells are given every time is 10³- 10⁹Cell.

7. purposes according to claim 1, it is characterised in that: apply amniotic epithelial cells together with one or more drugs With patient is given, the drug is selected from methylprednisolone, cyclosporine, tacrolimus, Mycophenolate Mofetil, methotrexate (MTX), sugared skin Matter hormone, imuran, Thalidomide, anti-T cell monoclonal antibody (anti-CD49d McAb), -2 receptor of anti-IL-8 it is anti- One of body etc. or combinations thereof.

8. purposes according to claim 1-7, it is characterised in that: the amniotic epithelial cells are by including following The method of step is prepared:

(1) amnion is obtained from placenta tissue by mechanically decoupled；

(2) amnion after cleaning is digested with digestive ferment, and postdigestive liquid is centrifuged, can be obtained people's amnioic epithelium Cell.

9. purposes according to claim 8, it is characterised in that: continue to cultivate to human amnion membrane is obtained in step 2, Preferred condition of culture are as follows: with 1 × 10⁶-1×10⁸Cell inoculation in culture dish, is placed in two by the density of a cell/plate It is cultivated in carbonoxide incubator, changes culture solution after human amnion membrane is adherent, by cell dissociation after cell covers with plate Get off to be frozen.

10. purposes according to claim 9, it is characterised in that: bFGF (basic fibroblast can be added in basal medium Porcine HGF) or EGF (epidermal growth factor).