CN109674818A - Purposes of the hAMSCs in preparation treatment acute graft versus host disease drug - Google Patents

Abstract

The present invention provides purposes of the human amnion mesenchymal stem cell in preparation treatment acute graft versus host disease drug.HAMSCs had both met adult tissue's mescenchymal stem cell Basic biological characteristics, also expressed embryonic stem cell mark, more stronger than BMSCs proliferative capacity.When the healthy human peripheral blood mononuclearcell (PBMCs) of hAMSCs and PHA activation co-cultures, the ratio of Th1, Tc1 cell subsets reduces, the ratio of Th2, Tc2, Treg cell subsets raises, IL-2 and the decline of the secretion level of IFN-γ, IL-10 level increases, therefore, hAMSCs can be used for treating preparation treatment acute graft versus host disease drug.

Description

Purposes of the hAMSCs in preparation treatment acute graft versus host disease drug

Technical field

The present invention relates to fields of biomedicine, acute in preparation treatment more particularly to a kind of human amnion mesenchymal stem cell Purposes in graft versus host disease(GVH disease) drug.

Background technique

Allogeneic Hematopoietic Stem Cell Transplantation (allogeneic Hematopoietic Stem Cell Transplantation, allo-HSCT) it has been widely used in hematological system and the treatment of non-blood systemic disease, even cure The unique method of certain diseases.Acute graft versus host disease (acute graft-versus-host disease, aGVHD) One of the main reason for being restricting current allo-HSCT curative effect.AGVHD pathogenesis mainly due to donor-recipient HLA do not conform to it is sharp Live donor's T lymphocyte, for donee's tissue, a kind of organogenetic abnormal immune reaction.It is various involved in whole process to exempt from Epidemic disease cell subsets, inflammatory mediator, endocellular transduction signal and gene commitment.T lymphocyte activation be induction GVHD occur, The primary link of development, the damage that the GVHD after receiving chemicotherapy and transplanting before transplanting will cause TEC lead to the center of standard Tolerance mechanism is limited, not can be removed reactive T, bone-marrow-derived lymphocyte, and especially Naive T lymphocyte (not undergoing antigen) induction is tighter The GVHD of weight.

With advancing age, the atrophy of thymus gland of people, functional deterioration, internal T, bone-marrow-derived lymphocyte maturation to pass through whole body Lymphatic system, Tregs are the T cell subgroups of a kind of regulation body's immunity, maintain tolerance of the immune system to self component, Immune homeostasis is kept, is played a significant role in anti-infectious immunity, antineoplastic immune, inhibition are immune.Periphery in Tregs inductor The effect of immune tolerance has defined, but specific mechanism is not clear.Recent study shows that Tregs exempts from body recovery Epidemic disease function is related to Graft-versus-tumor response, adopts before allo-HSCT and is transfused donor T regs, and the incidence of aGVHD is obvious It reducing, it is related with the adjusting of T lymphocyte subgroup to the inflammatory mediator of GVHD that this may relate to JAK/STAT and NK- κ B access, Molecular mechanism therein also needs further to inquire into.The prevention and treatment method of ideal aGVHD is receptor to donor T lymphocyte Immune tolerance is generated, immunologic reconstitution after transplanting is not influenced while retaining Graft-versus-tumor response, cell and small molecule are with good Good application prospect.

AGVHD study of incident mechanism is still insufficient at present, and pathogenesis is intricate.Clinic, which lacks, effectively prevents and controls Treatment measure.In recent years, stem cell is considered as the treatment most promising strategy of GVHD.The MSC main source of clinical application at present Many challenges are faced in the clinical application of marrow, but BMSC, such as adopting marrow has traumatic, and cell viability is by the factors shadow such as donor age It rings, cultured cell in vitro slow growth, is easy aging, cell heterogeneity causes clinical efficacy different.

Summary of the invention

In view of the foregoing deficiencies of prior art, the purpose of the present invention is to provide human amnion mesenchymal stem cells to make Purposes in standby treatment acute graft versus host disease drug, for solve in the prior art Allogeneic Hematopoietic Stem Cell Transplantation by The problems such as acute graft versus host disease restricts.

In order to achieve the above objects and other related objects, it is anxious in preparation treatment to provide human amnion mesenchymal stem cell by the present invention Purposes in property graft versus host disease(GVH disease) drug.

In some embodiments of the invention, the human amnion mesenchymal stem cell expresses embryonic stem cell mark.

In some embodiments of the invention, the human amnion mesenchymal stem cell and phytolectin (PHA) are activated When healthy human peripheral blood mononuclearcell (PBMCs) co-cultures, the ratio of Th1, Tc1 cell subsets is reduced.Therefore, in co-cultivation The content reduction of IL-4, IFN-γ in clear, then inflammatory factor is reduced in environment, and inflammatory symptom mitigates, it was demonstrated that human amnion mesenchymal The ion vitro immunization adjustment effect of stem cell.

In some embodiments of the invention, the ratio up-regulation of Th2, Tc2, Treg cell subsets, IL-2 and IFN-γ Secretion level decline.Treg cell is immunity regulatory cell, subset proportions up-regulation, it was demonstrated that inhibit inflammation occurrence and development in environment Cytokine secretion increases, and inflammatory symptom mitigates, it was demonstrated that the ion vitro immunization adjustment effect of human amnion mesenchymal stem cell.

In some embodiments of the invention, IL-10 level increases.IL-10 is the factor for inhibiting inflammation, and expression increases It is high, it was demonstrated that immunoregulation effect enhances in cultivating system.

As described above, human amnion mesenchymal stem cell of the invention is in preparation treatment acute graft versus host disease drug Purposes, have the advantages that hAMSCs had both met adult tissue's mescenchymal stem cell Basic biological characteristics, also express Embryonic stem cell mark, it is more stronger than BMSCs proliferative capacity.The healthy human peripheral blood mononuclearcell of hAMSCs and PHA activation (PBMCs) when co-culturing, the ratio of Th1, Tc1 cell subsets is reduced, the ratio up-regulation of Th2, Tc2, Treg cell subsets, IL-2 Decline with the secretion level of IFN-γ, IL-10 level increases, and therefore, hAMSCs can be used for treating preparation treatment acute graft Anti- host's medicine.

Detailed description of the invention

Fig. 1 is shown as the technical research route map of the embodiment of the present invention 1.

Fig. 2 is shown as hAMSCs and hBMSCs cellular morphology figure in the embodiment of the present invention 1.

Fig. 3 is shown as the growth characteristics figure of hAMSC, hBMSCs originally culture in the embodiment of the present invention 1.

Fig. 4-1, Fig. 4-2, Fig. 4-3, Fig. 4-4 are shown as the differentiation of hAMSC, hBMSCs and mark in the embodiment of the present invention 1 Object expression characteristic figure.

Fig. 5-1, Fig. 5-2 are shown as in the embodiment of the present invention 1 hAMSC, hBMSCs to the adjustment effect of T lymphocyte subgroup Figure.

Fig. 6-1, Fig. 6-2, Fig. 6-3 are shown as in the embodiment of the present invention 1 hAMSC, hBMSCs to the immune tune of cell factor Save action diagram.

Specific embodiment

Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification Other advantages and efficacy of the present invention can be easily understood for disclosed content.The present invention can also pass through in addition different specific realities The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints and application, without departing from Various modifications or alterations are carried out under spirit of the invention.

Human amnion mesenchymal stem cell (human amniotic mesenchymal stem cell, hAMSC) derives from Healthy pregnant women Celiotomy Wean waste-placenta amnion, is easily obtained, and quantity is abundant；Low immunogenicity with MSC and immune The essential characteristics such as adjustment effect, and without oncogenicity in transplanting animal body, it is ideal immune privilege cell.

2004, Le Blanc etc. reported that 1 MSC treats the case that intractable aGVHD succeeds first.Then, Le The clinical research of 55 application MSC treatment steroid-resistant aGVHD of report again in 2008 such as Blanc, as a result, it has been found that: 30 are obtained Complete incidence graph (complete response, CR), 9 be improved significantly, prompt MSC may be a kind of very promising Prevent and treat aGVHD product.

The intractable aGVHD that it is invalid for second line treatment that research finds the MSC of third party's derived from bone marrow has certain curative effect, Repairing receptor's thymus function simultaneously improves aGVHD inflammatory conditions.Crucial composition portion of the BMSCs as candidate stem cell microenvironment Point, inside and outside can rebuild hematopoieticmicroenviron-ment, promote hematopoietic cell amplification, shorten the Radiation in jury time.But the source BMSCs, number Amount, cell senescence etc. constrain its application prospect.Currently, the mesenchyma that amnion mesenchymal stem cell (hAMSCs) is placenta source is done Cell, in addition to expression MSCs mark, also expression embryonic stem cell transcription factor OCT-4 draws materials noninvasive, is easily obtained, no ethics Limitation is learned, quantity is abundant, and proliferative capacity is stronger, has potential clinical value.HAMSCs can be in, inside and outside three embryo Layer differentiation, such as liver, muscle, pancreas, blood vessel and neurogenic cell and tissue, treat in regenerative medicine skin, Blood vessel, cornea, neurologic defict reparation, and obtain good curative effect.HAMSCs low expression HLA-ABC and constructive expression HLA-G, expression of HLA-DR surface antigen, does not show low immunogenicity and immunosuppressive action, and in animal body without tumorigenesis Property, it is that comparatively ideal be immunized remits cell.

Research finds that hAMSCs intravenous transplantation ALS transgenic mice can protect motor function, mitigates the mind of its spinal cord Through inflammatory reaction, and can be migrated to pathological tissues.OLLE RINGDE ' N etc. is by people's Fetal Membranes Cells (fetal membrane Cells, FMCs) 9 intractable aGVHD patient's bodies are migrated to, complete reactivity guesses FMCs inflammation ring in vivo up to 75% It can be migrated under border to damaged tissues and carry out immunological regulation or participation blood coagulation system adjusting bleeding.In addition, research shows that moving Into microenvironment locating for the intracorporal MSCs of receptor, soluble factor, extracellular matrix components and the memebrane protein of (niche) can for plant The immune function of MSCs is adjusted, whether receptor applies that immunosuppressor, intracorporal inflammatory conditions are related to MSCs is before transplanting Anti-inflammatory or proinflammatory.Arianna Bonomi etc. is research shows that hAMSCs is able to suppress the swollen of hematopoiesis and non-hemopoietic system Tumor proliferation, and the cytotoxic effect of chemotherapeutics can be resisted, it can be used as the carrier targeted therapy lesion of drug.HAMSCs is used When treating aGVHD, aGVHD severity, pathologic condition and index of correlation wait to study.HAMSC inhibits CD4+T cell in vitro Proliferation, inhibit Th1/Th17 cell differentiation proliferation, induction Tregs generate, induced maturation type DCs be transformed into tolerance type DCs and to M2 type macrophage differentiation proliferation, possible mechanism be related to cell-cell contact inhibit and hAMSC secretion IDO, HLA-G, The immunological regulations medium such as TGF-β, IL-10.

In short, amnion stem cell, which may pass through following mechanism, adjusts immunization inflammatory reaction: 1. low immunogenicity: can inhibit anti- The generation and maturation of former presenting cells；2. adjustable T cell phenotype, regulates and controls ion vitro immunization effect, allosome leaching especially can inhibit Bar cell Proliferation；3. can inhibit the generation of inflammatory factor and inhibit local inflammation microenvironment.

Allogeneic Hematopoietic Stem Cell Transplantation (allogeneic Hematopoietic Stem Cell Transplantation, allo-HSCT) it is widely used in hematological system and the treatment of non-blood systemic disease, even cure certain The unique method of a little diseases.Acute graft versus host disease (acute graft-versus-host disease, aGVHD) is One of the principal element of restricting current allo-HSCT curative effect.Currently, clinically the method for preventing and treating aGVHD is joined with steroids It closes based on immunosuppressor, the incidence that too strong immunosupress can lead to tumor recurrence rate, infection and the second tumour increases. Therefore, the smaller treatment aGVHD means of new, more efficiently and adverse reaction are sought, are that current clinical position is urgently to be resolved The problem of.Donor T lymphocyte be activation transplant recipient aGVHD an important factor for, however grow up after thymus gland functional atrophy and Chemicotherapy before transplanting causes thymic epithelial cells (thymic epithelial cell, TEC) to damage, and body cannot pass through chest The Solid phase of gland central tolerance removes simplified reaction lymphocyte, and the immunological regulation after transplanting relies on substantially thymus-independent How can the periphery immune system of approach improve the periphery immunoloregulation function of transplant recipient or repair the thymus gland induction of damage Central tolerance reduces aGVHD incidence, mitigates aGVHD inflammatory conditions, becomes important research topic.

Mescenchymal stem cell (mesenchymal stem cells, MSC) is to be present in marrow, fat, umbilical cord and placenta A kind of adult stem cell of equal Various Tissues, has the function such as multi-lineage potential, immunological regulation, hematopoiesis support and repair tissue Can, certain curative effect is shown in the treatment of regenerative medicine field and immunity disease, it is especially the most aobvious to the effect of GVHD It writes.MSC is mainly derived from marrow (BMSCs) at present, has been used to treatment clinically intractable aGVHD.Research in recent years show with In vitro culture algebra increase BMSCs cell quantity and vigor decline, the cell of BMSCs is heterogeneous, leads to clinical efficacy not One, and acquisition mode is invasive bone marrow puncture, limits the following BMSCs in clinical application.From people's amnion Mescenchymal stem cell (human amniotic mesenchymal stem cells, hAMSCs) has mescenchymal stem cell base Eigen shows low immunogenicity and immunosuppressive action, is ideal be immunized without oncogenicity in transplanting animal body Cell is absolved, on the other hand, amnion system healthy pregnant women Celiotomy Wean waste, no ethnics Problem, and amnion cell is by disappearing Change partition method to be easily obtained, quantity is abundant, provides new selection for the cell therapy of various diseases.In recent years, people's amnion is dry thin Born of the same parents achieve good result in the subclinical and clinical treatments such as corneal restoration, burn disease, orthopaedic disease, spinal cord injury. This prompt hAMSC may provide one kind new seed cell source for the treatment of aGVHD, but hAMSC to the curative effect of aGVHD such as What, how is inside and outside mechanism of action, and there is presently no the research of this respect reports.

This seminar was the study found that hAMSCs had both met adult tissue's mescenchymal stem cell Basic biological characteristics, also table It is more stronger than BMSCs proliferative capacity up to embryonic stem cell mark.The healthy human peripheral blood mononuclearcell of hAMSCs and PHA activation (PBMCs) when co-culturing, the ratio of Th1, Tc1 cell subsets is reduced, the ratio up-regulation of Th2, Tc2, Treg cell subsets, IL-2 Decline with the secretion level of IFN-γ, IL-10 level increases.

Embodiment 1

1, the separation, culture, identification of hAMSCs and label

It chooses healthy Cesarean esction puerpera and volunteers the fresh amnion of donation (in the case where puerpera's informed consent, through hospital's ethics Committee's approval)；The fresh human placenta for taking postpartum to throw aside under aseptic condition, by amnion and tapetum blunt separation；After rinsing Rete layer is shredded with scissors, is climbed out of cultivation using tissue block cell and is gradually isolated and purified amnion mesenchymal stem cell；Select the 3rd Surface marker identification is carried out for amnion mesenchymal stem cell and is identified to skeletonization, at the Multidirectional Differentiations ability such as rouge, nerve cell；

2, hAMSCs is to T lymphocyte subgroup and relevant cell factor immunoregulation effect, compared with BMMSCs；

It establishes and optimizes hAMSCs and PBMCs co-culture system (directly contact co-culture system), hAMSCs and PBMCs's Ratio is 1:10.1. Culture in vitro: 3. with FCM detection CD4, CD25, Foxp3 (Tregs cell surface marker), Gata3 (Th2 cell transcription factor), Tbx21 (Th1 cell transcription factor), CCK-8 detect PBMC proliferative conditions, specify hAMSC The influence of effect and hAMSC to T cell proliferation, differentiation with effector T cell；2. immune inflammatory factor is adjusted: ELISA inspection The levels such as IFN-γ, IL-2, IL-10 in survey cultivating system.

3, investigative technique route

Investigative technique route is as shown in Figure 1.

4, conclusion

1) hAMSC meets the Basic biological characteristics of MSC, and expresses embryonic stem cell mark；

2) hAMSC ratio BMSC proliferative capacity is stronger；HAMSCs and mononuclearcell adjust Th1/ when directly contacting co-cultivation The ratio of Th2 cell subsets is biased to Th2, and the ratio of Treg cell subsets increases；IL-2 and the decline of the secretion level of IFN-γ, IL-10 level increases.

5, result explanation

Separation, culture, purifying, phenotypic evaluation and the label of 5.1hAMSCs

5.1.1hAMSCs morphology and growth characteristic

Cell, about 1 week or so tissue block week are cultivated in 6 orifice plates Tissue Culture Dish with the α-MEM culture medium containing 10%FBS It encloses and begins with cell and climb out of, hereafter, cell quantity gradually increases, microscopically observation, and cell is in shuttle shape and polygonal, a small number of more Liquid is changed by full dose in star and circle, gradually removes not adherent amnion, cellular morphology is gradually uniform after 10d, occurs two Kind of cell clone: fibroblast sample clone, cell are in spindle shape, and culture to 14-16d cell is up to 80%-90% fusion, in putting Penetrate shape or circinate distribution；Epithelial cell sample clone is in polygonal epithelial cell form.With the extension of incubation time, at Fibroblast-like cells are in dominant growth state, and cell is completely adherent in for 24 hours after passage, and epithelial cell like cell is rare, 5 days left sides The right side reaches 80%-90% fusion.Reached for 3 generations, cellular morphology is in uniform fibroblast-like cells.When cell was passaged to for 6 generation, cell Spindle shape is still maintained, good proliferative capacity is kept.The morphology of hAMSCs and growth characteristic are as shown in Figure 2,3.In Fig. 2, A: The cellular morphology of primary hAMSC；Cellular morphology of the B:P6 for hAMSC；C: the cellular morphology of primary hBMSC；D:P4 is for hBMSC's Cellular morphology.In terms of growth curve from P3 for hAMSC and hBMSC, 1-3d is incubation period after inoculation by hAMSC and hBMSC, 3-8d cell that hAMSC is cultivated in vitro, which is grown, to be accelerated, into logarithmic growth phase, and the 3-7d that hBMSC is cultivated in vitro Cell growth is accelerated, and 7-8d growth and proliferation of cell speed slows down, and the growth rate of hAMSC is significantly faster than that the proliferation speed of hBMSC Degree.In Fig. 3, A: after the culture of people's amnion 7 days, it is seen that there is cell to climb out of around tissue block；B: after the culture of people's amnion 10 days, it is seen that thin Born of the same parents' number of modalities increases, form is gradually uniform；C:P1 was for hAMSCs 3 days, and cell is predominantly at fiber-like, a small amount of epithelial cell It is mingled with therebetween；For D:P3 for hAMSCs 3 days, cellular morphology was in uniform fibroblast-like cells, scale=100 μm.

5.1.2hAMSCs differentiation potential and surface markers is identified

Through flow cytomery, hAMSCs high express CD29, CD44, CD73, CD90 without express CD14, CD34, CD45 and HLA-DR；In addition, hAMSCs also expresses embryonic stem cell ESCs surface marker OCT-3/4.At induced medium After managing 2 weeks, hAMSCs can be to fat cell, osteoblast differentiation, and has skeletonization, at rouge and Neural Cell Phenotypic, this shows HAMSCs has multi-lineage potential, as shown in Fig. 4-1, Fig. 4-2, Fig. 4-3, Fig. 4-4.

In Fig. 4-1, (A) undifferentiated hAMSCs is in fibroblast-like cells form；(B) hAMSCs breaks up to osteoblast direction Alizarin red agent coloration result；(C) the oil red dyeing that hAMSCs breaks up to fat cell direction；(D) hAMSCs is to nerve cell side To the nerve fibre-M immunohistochemical staining of differentiation, scale=100 μm.

Streaming result: hAMSCs expresses CD29, CD44, CD73, CD90, OCT-3/4, without expressing CD14, CD34, CD45 And HLA-DR.

5.2hAMSCs and BMMSCs immunoloregulation function compares

This part research mononuclearcell is isolated from peripheral blood, after being incubated overnight in vitro remove attached cell to PBMC is obtained, (as immunocyte, the hAMSC and hBMSC for comparing various concentration respectively make the inhibition of PBMC using PBMC The inhibiting effect having to the PBMC of PHA stimulation respectively with, experimental result prompt hAMSC and hBMSC, and with hAMSC and The increase of hBMSC concentration, the two enhance the inhibiting effect of the PHA PBMC stimulated, prompt the mesenchyma of two kinds of separate sources dry Cell has dose dependent to the inhibiting effect of lymphocyte, and compared with the hBMSC of same concentrations, hAMSC experimental group with HBMSC experimental group inhibits immune cell propagation degree similar, and results of statistical analysis prompts comparison in difference between the two without system Meter learns meaning.This result of study prompts, in experimental group hAMSC+PBMC+PHA (1:1) and hBMSC+PBMC+PHA (1:1) supernatant For IFN-γ concentration compared with IFN-γ concentration in positive controls PBMC+PHA supernatant, hAMSC and hBMSC can inhibit T lymph Cell secretion of gamma-IFN, results of statistical analysis prompt have notable difference (P < 0.01).Thus speculate, hAMSC and hBMSC can It can be to CD4⁺Th1 cells play inflammatory reaction has negative-feedback regu- lation effect.In addition, this research has detected hAMSC+PBMC+PHA The content of IFN-γ in experimental group (1:1) and hBMSC+PBMC+PHA experimental group (1:1) culture supernatant, respectively with individually cultivate IFN-γ content balance in hAMSC and hBMSC supernatant, the mesenchyma of the former IFN-γ level obviously higher than two kinds of separate sources Stem cell individually in culture group (negative control group) cell supernatant IFN-γ content (P < 0.01).Although hAMSC+PBMC The content of IFN-γ and IFN- in hBMSC+PBMC+PHA co-cultivation group (1:1) supernatant in+PHA co-cultivation group (1:1) supernatant The content of γ is compared, both statistical analysis no significant difference (P=0.056), but the former IFN-γ content is lower.Knot Fruit is as follows: (1) ratio of hAMSC+PBMC+PHA, hBMSC+PBMC+PHA co-cultivation group Treg, Th2, Tc2 cell subsets compared with PBMC+PHA group obviously rises (P < 0.05), the ratio of Th1, Tc1 cell subsets be decreased obviously compared with PBMC+PHA group (P < 0.05), and the variation of hAMSC+PBMC+PHA, hBMSC+PBMC+PHA co-cultivation group T cell subgroup is not statistically significant (P>0.05)；(2) ELISA result prompts, and compared with PBMC+PHA group, hAMSC+PBMC+PHA, hBMSC+PHA+PBMC are trained altogether The level for supporting IL-2 in group supernatant is decreased obviously (P < 0.05), and hAMSC+PBMC+PHA, hBMSC+PHA+PBMC are co-cultured IL-10 level obviously rises (P < 0.05) in group supernatant, in hAMSC+PBMC+PHA, hBMSC+PBMC+PHA co-cultivation group The variation no difference of science of statistics (P > 0.05) of IL-2, IL-10 content in clear liquid.This research prompt: hAMSC, hBMSC human peripheral blood Lymphocyte has similar immunoloregulation function.

5.2.1CCK-8 method measures PBMC proliferative conditions

HAMSC+PBMC+PHA group and hBMSC+PBMC+PHA group are respectively compared with PHMC+PHA group and PBMC group, hAMSC It is inhibited (P < 0.05) to the lymphopoiesis that PHA stimulation is added with hBMSC.And with hAMSC and hBMSC Increasing for ratio detects that OD value is smaller, prompts hAMSC and hBMSC ratio with PBMC co-culture system, inhibits with it PBMC proliferative capacity is directly proportional.Two kinds of MSC inhibiting effect of same ratio compare, then no statistical difference difference (P > 0.05).

Table 1.MSC is to PBMC proliferation function

^*P < o.05, compared with PBMC+PHA group.

5.2.2 Flow cytometry T lymphocyte subgroup changes

If Fig. 5-1, Fig. 5-2 show hAMSC, hBMSCs to the adjustment effect figure of T lymphocyte subgroup, two kinds of sources What MSC human peripheral blood T lymphocyte subgroup influenced the results are shown in Table 2.Compared with PBMC+PHA group, two co-cultivation groups can promote Treg, Th2, Tc2 cell subsets ratio of the PBMC of PHA stimulation rises (P < 0.05), reduces Th1, Tc1 cell subsets ratio, The no significant difference (P > 0.05) that the MSC in two kinds of sources influences T lymphocyte subgroup.

Adjustment effect of the table 2.MSC to T lymphocyte subgroup

^*P > 0.05, vs hBMSC+PBMC+PHA group；^ΔP < 0.05,^**P < 0.01, vs PBMC+PHA group；^#P < 0.05,^&P < 0.01.vs hAMSC+PBMC+PHA group.

5.2.3 ELISA detects levels of cytokine secretion variation

As Fig. 6-1, Fig. 6-2, Fig. 6-3 show hAMSC, hBMSCs to the immunoregulation effect figure of cell factor, hAMSC + PBMC+PHA organize supernatant IFN-γ content it is low compared with hBMSC+PBMC+PHA group (P=0.056), the two compared with PBMC+PHA group, IFN-γ content is substantially reduced (P < 0.05).

IL-2 content is slightly lower compared with hBMSC+PBMC+PHA group (P=0.564) in hAMSC+PBMC+PHA group supernatant, the two point Not compared with PBMC+PHA group, IL-2 content is substantially reduced (P < 0.05).

IL-10 content is slightly higher compared with hBMSC+PBMC+PHA group (P=0.773) in hAMSC+PBMC+PHA group supernatant, the two Compared with PBMC+PHA group, IL-10 content is significantly raised (P < 0.05).

In conclusion hAMSCs had both met adult tissue's mescenchymal stem cell Basic biological characteristics, it is dry also to express embryo Cell sign, it is more stronger than BMSCs proliferative capacity.The healthy human peripheral blood mononuclearcell (PBMCs) of hAMSCs and PHA activation is altogether When culture, the ratio of Th1, Tc1 cell subsets is reduced, the ratio up-regulation of Th2, Tc2, Treg cell subsets, IL-2 and IFN-γ Secretion level decline, IL-10 level increase, therefore, hAMSCs can be used for treating preparation treatment acute graft versus host disease Drug.

The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as At all equivalent modifications or change, should be covered by the claims of the present invention.

Claims (5)

1. purposes of the human amnion mesenchymal stem cell in preparation treatment acute graft versus host disease drug.

2. purposes according to claim 1, it is characterised in that: the human amnion mesenchymal stem cell expresses embryonic stem cell Mark.

3. purposes according to claim 1, it is characterised in that: the human amnion mesenchymal stem cell and phytolectin swash When healthy human peripheral blood mononuclearcell (PBMCs) living co-cultures, the ratio of Th1, Tc1 cell subsets is reduced.

4. purposes according to claim 3, it is characterised in that: the ratio of Th2, Tc2, Treg cell subsets raises, IL-2 Decline with the secretion level of IFN-γ.

5. purposes according to claim 3, it is characterised in that: IL-10 level increases.