CN104800243A - Use of recombinant mesenchymal stem cell in preparation of immunosuppressant - Google Patents
Use of recombinant mesenchymal stem cell in preparation of immunosuppressant Download PDFInfo
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Abstract
The invention discloses a use of a recombinant mesenchymal stem cell in preparation of an immunosuppressant. The invention relates to the recombinant mesenchymal stem cell capable of expressing a chemokine receptor CCR7 on a cytomembrane. After infusion, the recombinant mesenchymal stem cells can be specifically transferred to a secondary lymphatic organ, and a large amount of the recombinant mesenchymal stem cells are gathered in a T cell enrichment region in the secondary lymphatic organ and can efficiently inhibit T cell immunoreaction. Through use of the recombinant mesenchymal stem cell in treatment on graft-versus-host disease after bone marrow transplantation, immunological rejection after organ transplantation and autoimmune diseases, a cost is reduced, a cell use amount is reduced and side-effects are relieved. In treatment, the recombinant mesenchymal stem cell retains tumor cell killing effects and provides a high efficiency and beneficial novel approach for clinical treatment on graft-versus-host disease after bone marrow transplantation, immunological rejection after organ transplantation and autoimmune diseases of tumor high-risk groups.
Description
Technical field
The present invention relates to stem-cell therapy field, specifically about the application of mescenchymal stem cell in preparation immunosuppressant of a kind of endoglin expression chemokine receptors CCR7.
Background technology
Mescenchymal stem cell is the adult stem cell that a group separated from bone marrow at first has self renewal and multi-lineage potential.Research in recent years shows: mescenchymal stem cell has immunoloregulation function widely.Mescenchymal stem cell is to panimmunity cell, and comprising T cell, B cell, NK cell, DC etc. all has immunoregulation effect.Immunoregulation effect in the body that mescenchymal stem cell is mainly played by secreted cytokine is found in the treatment of effective zoopery and clinical disease.
At present, mescenchymal stem cell carries out immunization therapy to clinical disease as the immunologic rejection, clone disease, systemic lupus erythematosus (sle) etc. after hematopoietic stem cell transplantation or graft versus host disease, solid organ transplantation and has entered clinical II-III stage phase in the U.S..On May 17th, 2012, Canadian Health Administration mechanism announces a human mesenchymal stem cell medicine-Prochymal of approval
tMlisting, makes Prochymal
tMbecome the first stem cell drugs truly getting permission to be used for the treatment of systemic disease in the whole world, also indicate that mescenchymal stem cell is opened as the gate of immunomodulator clinical practice.But still problems faced is: 1) show at polycentric III phase clinical effectiveness: in body, immunoregulation effect is very unobvious; 2) large [the Tyndall A.Successes and failures of stem cell transplantation in autoimmune diseases.Hematology Am Soc Hematol Educ Program.2011 of cell consumption; 2011:280-284.] [Martin I, Baldomero H, Tyndall A, Niederwieser D, Gratwohl A.A survey on cell and engineered tissue therapies in Europe in2008.Tissue Eng Part A2010; 16:2419-2427.]
The clinical treatment mode of a lot of mescenchymal stem cells all comprises in-situ injection and intravenous injection two kinds of modes [http://www.clinicaltrials.gov/].But because intravenous injection is simple, whole body system disease, as graft versus host disease, clone disease, systemic lupus erythematosus (sle) etc. or based on intravenous injection.So this type of disease of clinical treatment needs a large amount of cells, if clinical mescenchymal stem cell immunization therapy dosage is 1-9 × 10
6mSCs/Kg, 2 times weekly, continuous surrounding, adds treatment cost in this process undoubtedly, simultaneously also may bring as thromboembolism, the danger such as tumor recurrence.Therefore, in the effective body of mescenchymal stem cell, migration is key [the Karp JM and Leng Teo GS.Mesenchymal Stem Cell Homing:The Devil Is in the Details.Cell Stem Cell.2009 reaching the most efficient, most economical two general objectives of cell therapy; 4 (3): 206-216.Ankrum J, Karp JM.Mesenchymal stem cell therapy:Two steps forward, one step back.Trends Mol Med.2010; 16 (5): 203-9].
Secondary lymphoid organ, comprise spleen white pulp (SPW), lymph node (LN), mesenteric lymph node (MLN), PP knot etc., structure is simple and similar, connects to form by T, bone-marrow-derived lymphocyte, antigen-presenting cell (APC), stromal cell and microtubule.T cell accepts the antigen that APC presents in secondary lymphoid organ, activation and proliferation, and therefore, secondary lymphoid organ is the place that in body, immunoreation starts.Mescenchymal stem cell is widely distributed at whole body after venoclysis, is distributed in lung, intestinal, liver, bone marrow etc., but is seldom present in secondary lymphoid organ.Mescenchymal stem cell at nearly all histoorgan, is made it thin in main battle ground upper body list power by " dilution ", though so that its test in vitro in there is powerful immunoregulation capability, and in vivo in immune modulating treatment effect undesirable.
Clinically after tumor patient hematopoietic stem cell transplantation as carried out a large amount of mesenchymal stem cells infusions, danger [Ning H, the etal.The correlation between cotransplantation of mesenchymal stem cells and higher recurrence rate in hematologic malignancy patients:outcome of a pilot clinical study.Leukemia.2008 of tumor recurrence can be increased; 22:593-599].And results of study a large amount of at present shows, mescenchymal stem cell can promote the generation of tumor, growth, drug resistance, Infiltration and metastasis [Eterno V, et al.Adipose-derived Mesenchymal Stem Cells (ASCs) may favour breast cancer recurrence via HGF/c-Met signaling.Oncotarget.2013; 23.Epub ahead of print] [Bergfeld SA, Blavier L, Declerck YA.Bone Marrow-Derived Mesenchymal Stromal Cells Promote Survival and Drug Resistance in Tumor Cells.Mol Cancer Ther.2014; 6.Epub ahead of print] [Wang D1, Wang S, Shi C.Update on cancer related issues of mesenchymal stem cell-based therapies.Curr Stem Cell Res Ther.2012; 7 (5): 370-80.].
Graft versus host disease after clinical treatment tumour patient hematopoietic stem cell transplantation, in immunological rejection after organ transplantation in tumorigenic high-risk group and treatment of autoimmune diseases, as application has a large amount of infusion of mescenchymal stem cell of immune suppression function widely, except existing, price is high, while the shortcoming that affects blood stable state, larger problem is that a large amount of infusion of mescenchymal stem cell will bring and increases tumor and occur/the danger of recurrence.Therefore, in the treatment in this few class crowd, common mescenchymal stem cell is cautious use of, or even forbidding.
Strong based on immunoregulation capability in the body of common mescenchymal stem cell in clinical treatment, cell consumption is large, the drawback that has the danger of potential increase tumor recurrence/generation, the demand at present for more economical, the efficient cell therapy strategy that can overcome above-mentioned drawback is particularly eager.
Summary of the invention
The invention provides a kind of application of mescenchymal stem cell in preparation immunosuppressant of recombinating.By modifying mescenchymal stem cell, make its endoglin expression CCR7, the migration of the mescenchymal stem cell targeting after infusion is guided to enter secondary lymphoid organ, and be gathered in the T cell enrichment region of secondary lymphoid organ, can with the close contact of T lymphocyte, inhibit the immunoreation that T cell mediates efficiently.More particularly, restructuring mescenchymal stem cell of the present invention remains body antineoplastic ability simultaneously.
For achieving the above object, the present invention includes following technical scheme:
A kind of application of mescenchymal stem cell in preparation immunosuppressant of recombinating, this restructuring mescenchymal stem cell is by expressing chemokine receptors CCR7 to obtain on the cell membrane of mescenchymal stem cell, and this immunosuppressant is applied to the graft versus host disease after the hematopoietic stem cell transplantation of tumor high-risk, the immunological rejection after organ transplantation and/or autoimmune disease.
Application as above, preferably, described mescenchymal stem cell is autologous, xenogenic origin or commercial source.
Application as above, preferably, the described method expressing chemokine receptors CCR7 on the cell membrane of mescenchymal stem cell comprises: the channel genes of CCR7 in mescenchymal stem cell, is made mescenchymal stem cell endoglin expression CCR7 by the method for employing gene transfection; By vector transfectants system, make mescenchymal stem cell endoglin expression CCR7; Adopt the method for Trogocytosis, by antigen presenting cell, CCR7 is offered on the cell membrane of mescenchymal stem cell; Or change structure by existing epicyte protein or external albumen realizes.
Application as above, preferably, the method for described gene transfection, comprises viral vector rotaring redyeing system and non-viral transfection system.
Application as above, preferably, described viral vector rotaring redyeing system is any one or several in adeno-associated virus AA-V, slow virus, adenovirus Ad-V, baculovirus, herpesvirus and Retroviral Transfer system.
Application as above, preferably, described non-viral transfection system is plamid vector transfection system, chitosan nano rotaring redyeing system or uncorrected gene expression rotaring redyeing system.
Application as above, preferably, described tumor high-risk comprises: the 1) mid-aged population of more than 45 years old; 2) high-risk group that causes of inherited genetic factors: the direct line within three generations or collaterals suffer from the medical history of malignant tumor; 3) have the crowd of bad life habits: long-term a large amount of smoking, chronic alcoholism, drug dependence, for a long time overworked, stay up late, life stress is large, operating pressure has unhealthy emotion, personality depressed or use the crowd that mobile phone frequency is high greatly, for a long time; 4) crowd of Bad Eating Habit is had: monophagia, malnutrition, unhygienic diet, diet are irregular, eating speed is slow, overheated mistake of often taking food is pickled as hard as peppery food or frequent feed and smoked fried food; 5) crowd of occupational factor Long Term Contact poisonous and harmful substance; 6) crowd that polluted of living environment: water source and/or aerochemistry pollution, heavy metal pollution, nuclear pollution, microbial contamination or the crowd lived in for a long time in haze weather; 7) chronic or repeatability inflammation crowd is had: chronic bronchitis, chronic hepatitis B virus infecton, HIV patient, chronic cervicitis, chronic gastritis, polyp of stomach, gastric ulcer, mastitis, mammary gland chronic proliferative, polyp intestinal, cystic polyp, prostatitis, sinusitis or long-term functionality metrorrhagia; 8) the innocent and malignant tumour patient after treatment.
Application as above, preferably, described autoimmune disease is systemic lupus erythematosus (sle), mouth xerophthalmia scheorma syndrome, rheumatoid arthritis, ankylosing spondylitis, scleroderma, polyarteritis nodosa, Wegener granulomatosis, ulcerative colitis, autoimmune hemolytic anemia, spontaneous aleukia purpura disease, dermatomyositis, mixed connective tissue disease, myasthenia gravis, Autoimmune Thyroid Diseases, insulin dependent diabetes mellitus (IDDM), pernicious anemia companion chronic atrophic gastritis, goodpasture syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis or acute idiopathic polyneuritis.
Application as above, preferably, described restructuring mescenchymal stem cell and general immunosuppressant conbined usage.
Application as above, preferably, described restructuring mescenchymal stem cell is 1 × 10 for the dosage of human body
4~ 1 × 10
6mSCs/kg.
Beneficial effect of the present invention is:
1. by the endoglin expression CCR7 of mescenchymal stem cell, can targeting secondary lymphoid organ migration in body after infusion, in a large amount of gathering in secondary lymphoid organ local, especially it can be gathered in the T cell enrichment region of secondary lymphoid organ, can with the close contact of T lymphocyte, inhibit the immunoreation that T cell mediates efficiently, cell consumption can be reduced when the graft versus host disease after clinical treatment hematopoietic stem cell transplantation, the immunological rejection after solid organ transplantation and autoimmune disease, reduce cost, reduce the side effect such as thromboembolism.
2. restructuring mescenchymal stem cell of the present invention remains body antineoplastic action simultaneously.Therefore, the danger of tumor recurrence can be reduced during graft versus host disease after this kind of cell clinical treatment tumour patient hematopoietic stem cell transplantation; Also tumorigenic danger can be reduced in immunological rejection after organ transplantation in tumorigenic high-risk group and treatment of autoimmune diseases.
Immunosuppressant prepared by restructuring mescenchymal stem cell of the present invention is when the immunoreation occurred frequently for the treatment of clinical patients, the drawback that in the body overcoming common mescenchymal stem cell, immunoregulation capability is strong, cell consumption is large, have the danger of potential increase tumor recurrence/generation is obviously better than common mescenchymal stem cell.This invention provides efficient and useful New Policy for suppressing patient's immunoreation clinically.
Accompanying drawing explanation
Fig. 1: the CCR7 of mescenchymal stem cell (MSCs) expresses situation.
A:RT-PCR method detects the CCR7 expression of Balb/C mice and C57BL/6 bone marrow derived MSCs;
B:RT-PCR method detects the expression of three kinds of cell eGFP and CCR7;
C: real-time quantitative PCR detects the mrna expression level of three kinds of cell eGFP genes;
D: real-time quantitative PCR detects the mrna expression level of three kinds of cell CCR7 genes;
E: the situation of the film expression CCR7 of flow cytomery three kinds of cells.
Fig. 2 A: three groups of mice spleen (SP), lymph node (LN), mesenteric lymph node (MLN), PP knot (PP) cut into slices fluorescent microscopy images after DAPI nuclear marker.
Fig. 2 B:eGFP
+the frequency of MSCs each histoorgan in vivo.
Fig. 3: the MSCs/CCR7-eGFP after tail venoclysis in spleen and lymph node and CD3
+t cell, CD11c
+dendritic cell and B220
+the microphotograph of B cell.
The experimental result of the anti-host disease of Fig. 4: MSCs/CCR7-eGFP efficient inhibition of transplant (GvHD).
In Fig. 5: GvHD mouse model, MSCs/CCR7-eGFP is for body antineoplastic aptitude tests result.
The immunoregulation of Fig. 6: in murine heterotropic skin transplantation model, MSCs/CCR7-eGFP and the test result on the impact of body tumor growth ability.
The immunoregulation of Fig. 7: in mice inflammatory bowel disease models, MSCs/CCR7-eGFP and the test result on the impact of body tumor growth ability.
Detailed description of the invention
On the cell membrane of mescenchymal stem cell, express chemokine receptors CCR7 in the present invention realize by following methods: the channel genes of CCR7 in mescenchymal stem cell, is made mescenchymal stem cell endoglin expression CCR7 by the method for employing gene transfection; By vector transfectants system, make mescenchymal stem cell endoglin expression CCR7; Adopt the method for Trogocytosis, by antigen presenting cell, CCR7 is offered on the cell membrane of mescenchymal stem cell; Or change structure by existing epicyte protein or external albumen realizes.
Wherein, the method for described gene transfection, comprises viral vector rotaring redyeing system and non-viral transfection system; Described viral vector rotaring redyeing system can be any one or several in adeno-associated virus AA-V, slow virus, adenovirus Ad-V, baculovirus, herpesvirus and Retroviral Transfer system; Described non-viral transfection system can be plamid vector transfection system, chitosan nano rotaring redyeing system or uncorrected gene expression rotaring redyeing system.
Below in conjunction with specific embodiment, the invention will be further described, but the present invention is not limited to following examples.
In following embodiment, if no special instructions, conventional method is.
Embodiment 1 prepares the restructuring mescenchymal stem cell of expressing chemokine receptors CCR7
1. mouse bone marrow cells MSCs separation and Culture
Sample is 2-3 C57BL/6 mice in age in week (Military Medical Science Institute's Experimental Animal Center), disconnected neck puts to death mice, separating mouse tibia and femur under 75% alcohol-pickled sterilization mice → aseptic condition, be placed in plate → rub attachment removal muscle with the hands with sterile gauze, removal metaphysis → repeatedly rinse medullary cavity with PBS+10%FCS (Hyclone), go out medullary cell → be inoculated in culture bottle, at the bottom of osteocomma is uniformly distributed bottle, cultivating system is containing α t-MEM (Gibco)+10%FCS (Hyclone)+100U/ml penicillin+100U/ml streptomycin, 37 DEG C, 5%CO
2saturated humidity condition under cultivate → within every 3 days, change liquid 1 time → cell when converging close to 70-80%, 0.25% pancreatin (Hyclone) had digestive transfer culture, passage cell inoculum density is 4000 cell/cm
2.
2. adeno-associated virus infecting mouse MSCs
EGFP-gland relevant viral vector and the CCR7-eGFP-gland relevant viral vector of the customization of Invitrogen company proceed to mice MSCs respectively.Method is: MSCs is spread 48 orifice plates, 2 × 10
4/ hole.Change new culture medium next day into, wherein add virus (MOI=20), polybrene (8 μ g/ml), infects and changes normal cultivating system after 6 hours.The MSCs called after MSCs/eGFP of eGFP will be imported; The MSCs called after MSCs/CCR7-eGFP of CCR7-eGFP carrier will be imported.
3.RT-PCR detects MSCs and expresses at the CCR7 of mRNA level in-site
Collect the MSCs that BalB/c mice is separated with C57BL/6 mice, with the spleen mononuclearcell (SPMNC) of strain as positive control, the MSCs/eGFP after collection transfection simultaneously, MSCs/CCR7-eGFP.The mRNA level in-site of each group of cellular expression CCR7 is detected by RT-PCR method.Primer is as follows:
Table 1
As shown in Figure 1, the MSCs in Figure 1A: RT-PCR result display BalB/C mice and C57BL/6 mice source does not all express CCR7 to result, and the splenocyte (SPMNC) with strain source is then positive.The expression of house-keeping gene HPRT is as internal reference; Figure 1B: normal MSCs is blank, and the MSC proceeding to empty carrier is transfection control (MSCs/eGFP), CCR7 gene is proceeded to MSCs (MSCs/CCR7-eGFP).RT-PCR testing result shows: MSC/eGFP and MSC/eGFP-CCR7 expresses eGFP; In three kinds of cells, the mRNA of MSC/CCR7-eGFP successful expression CCR7, the expression of house-keeping gene HPRT is as internal reference.This result shows that CCR7 gene successfully proceeds to MSCs.
4. real time quantitative PCR method detects the mRNA level in-site that MSCs expresses CCR7
Collect MSCs, MSCs/eGFP, MSCs/eGFP-CCR7 tri-groups of cells respectively.Conventional method extracts RNA, detects the mRNA level in-site of three kinds of cell CCR7 with real time quantitative PCR method.Primer is as follows:
Table 2
Result is see Fig. 1 C, D: real-time quantitative PCR detects the mrna expression level of MSCs, MSCs/eGFP, MSCs/eGFP-CCR7 tri-kinds of cell eGFP genes and CCR7 gene; Further demonstrate that the result of Figure 1B.
5. the situation of flow cytomery MSCs film expression CCR7
MSCs, MSCs/eGFP, MSCs/CCR7-eGFP tri-groups of cells use mice CD16/32 antibody (ebioscience company) to close respectively, then CCR7-PE antibody (ebioscience company) labeled cell is used, Fig. 1 E flow cytomery result shows: MSCs, MSCs/eGFP do not express membrane molecule CCR7, and MSCs/CCR7-eGFP has 93.0-98.4% to express membrane molecule CCR7.
Embodiment 2 detects in MSCs body to secondary lymphoid organ transfer ability and rich region
1. detect in MSCs body to secondary lymphoid organ transfer ability
By infusion MSCs, MSCs/eGFP or MSCs/CCR7-eGFP (1 × 10
6) three groups of mices (often organizing 4); Get after 5 days after infusion spleen (SP), lymph node (LN), mesenteric lymph node (MLN), PP knot cook frozen section, DAPI carries out nuclear marker.Fluorescence microscopy Microscopic observation green cells.Fig. 2 A result shows: MSCs/eGFP does not have the ability to secondary lymphoid organ migration, and MSCs/eGFP-CCR7 has a large amount of distributions in SP, LN, MLN, PP.The eGFP of each organ is calculated shown in Fig. 2 B
+the frequency (number/high power field of green fluorescence) of MSCs each Histological and organic distribution in vivo, the MSCs/eGFP showing infusion quantitatively does not have the ability of moving to secondary lymphoid organ in vivo, and the MSCs/eGFP-CCR7 of infusion just has the ability of secondary lymphoid organ targeting migration in body.
2.MSCs/eGFP-CCR7 is present in T cell enrichment region in secondary lymphoid organ
By the SP of each MSCs/eGFP-CCR7 infusion mice, the frozen section of LN is cooked immunofluorescence dyeing, with fluorescence microscopy Microscopic observation MSCs/CCR7-eGFP (green) and CD3 after the CD3 antibody of phycoerythrin labelling (PE) or B220 antibody incubation
+t lymphocyte (red, Fig. 3 A), CD11c
+dendritic cell (red, Fig. 3 B) or B220
+the relation of bone-marrow-derived lymphocyte (red, Fig. 3 C), DAPI (blueness) dyeing showed cell core.In secondary lymphoid organ, have various kinds of cell, MSCs/CCR7-eGFP is present in T cell enrichment region in secondary lymphoid organ, and this result has established the basis immunoreation of T cell mediation in its body of MSCs/CCR7-eGFP to powerful immunoregulation capability.
Immunomodulatory effect and the detection on tumor growth impact in embodiment 3MSCs/CCR7-eGFP body
1.MSCs/CCR7-eGFP is to the suppression of GvHD
GvHD is the immunological diseases of typical T cell mediation.The foundation of mice GvHD model: the BALB/c male mice getting 20-24g, collects splenocyte and medullary cell, counting; 2 × 10
7splenocyte+1 × 10
7the 22-24g C57BL/6 male mice that medullary cell/0.25mlPBS tail vein injection 9Gy irradiates, sets up aGvHD model (GvHD group).Be total to infusion 1 × 10 respectively on this basis
5or 1 × 10
6mSCs/eGFP, 1 × 10
5or 1 × 10
6mSCs/CCR7-eGFP.1 × 10
6the mice that MSCs/eGFP tail vein is total to infusion is called GvHD+MSCs/eGFP mice; 1 × 10
6the mice that MSCs/CCR7-eGFP tail vein is total to infusion is called GvHD+MSCs/CCR7-eGFP mice.
After infusion, every day observes three groups of mouse survival and state, record survival curve, with immunomodulatory effect in the body observing MSCs, and detects serum Cellular inflammatory factor level.As shown in Figure 4 A, MSCs/CCR7-eGFP infusion obviously extends the life span of GvHD mice, and its effect is obviously better than the MSCs/eGFP infusion group of same dose.Modeling is after 2 weeks, and GvHD mice shows as that weight loss, the back of a bow, diarrhoea, hair are fluffy and disorderly, energy reduces.Infusion MSCs/CCR7-eGFP mice GvHD performance then has clear improvement, and its effect is obviously better than the MSCs/eGFP infusion group (Fig. 4 B) altogether of same dose.ELISA result shows, compared with GvHD, GvHD+MSCs/eGFP mice, MSCs/CCR7-eGFP altogether infusion significantly improves anti-inflammatory factors IL-4 level in mice serum (Fig. 4 C), significantly reduces inflammatory factor IFN-γ level (Fig. 4 D).Above results proved that: the MSCs expressing CCR7 has immunoregulation capability in the body more powerful than common MSCs.
2., in mice GvHD model, detect MSCs/eGFP-CCR7 for body antineoplastic capacity
Mice GvHD, GvHD+MSCs/eGFP (1 × 10
6), MSCs/CCR7-eGFP (1 × 10
6) same day of foundation of model, infusion 5 × 10 altogether
5eL4 leukaemia, to set up the GvHD mouse model of lotus tumor.Record the life span of each group of mice, and draw survival curve, observe two kinds of MSCs to the impact of tumor growth.Found that: compared with the tumor-bearing mice of GvHD+EL4, MSCs/eGFP altogether infusion does not extend the life span of mice, and MSCs/eGFP-CCR7 is defeated altogether, obviously extends the life span (Fig. 5 A) of mice; The neoplasm invasiveness situation of each group of mouse liver is observed in modeling after 2 weeks, Fig. 5 B result shows: homology splenocyte and tumor cell altogether infusion group (syno-infusion+EL4) mouse liver have obvious tumor nodule, prove mouse-borne tumor success (Fig. 5 Ba); GvHD+EL4 group mouse liver has significantly hemorrhage and downright bad, illustrates that mice has obvious GvHD that (Fig. 5 Bb) occurs; GvHD+EL4+MSCs/eGFP group mouse liver GvHD change has some improvement, and also still has many tumor nodules (Fig. 5 Bc); GvHD+EL4+MSCs/CCR7-eGFP mice is hemorrhage, downright bad and tumor nodule is all improved, and illustrates that the common infusion of MSCs/CCR7-eGFP is while alleviating GvHD, remains body antineoplastic ability (Fig. 5 Bd);
3. dermatoheteroplasty model (allogeneic skin graft, alloSG) mice load melanoma (B16), detects the capacity of MSCs/eGFP-CCR7 for body tumor growth.
Set up the melanomatous dermatoheteroplasty mouse model (alloSG+B16+PBS) of load: by Balb/C mouse skin (1Gm
2) migrate to C57 mouse back, melanoma cell on the same day (B16) 5 × 10
5side abdominal part hypodermic, sets up load melanoma murine heterotropic skin transplantation model, next day, tail venoclysis 0.2ml PBS; Tail venoclysis 1 × 10 on model basis
6mSCs/eGFP, is defined as alloSG+B16+MSCs/eGFP group; Or tail venoclysis 1 × 10
6mSCs/CCR7-eGFP, is defined as alloSG+B16+MSCs/CCR7-eGFP group.
Observe the heterodermic time-to-live of three groups of mices, the xenogeneic skin time-to-live of result display alloSG+B16+MSCs/CCR7-eGFP group mice obviously extends (*, p < 0.05), and the time-to-live of the xenogeneic skin of alloSG+B16+MSCs/eGFP group mice and matched group zero difference (Fig. 6 A).Modeling is after 2 weeks, and alloSG+B16+MSCs/eGFP group melanoma is obviously large than matched group volume, and weight obviously increases (*, p < 0.05); But alloSG+B16+MSCs/CCR7-eGFP group melanoma and matched group volume and weight are all without obviously distinguishing (n.s., without significant difference) (Fig. 6 B, C).
4. in load melanoma mice inflammatory bowel (Inflammatory bowel disease, IBD) model, the immunoregulation of MSCs/CCR7-eGFP and body tumor growth ability is tested.
Feed mice with 2%DSS, continuous 7 days, feed the first day melanoma cell (B16) 5 × 10 of water
5side abdominal part hypodermic, tail venoclysis 0.2ml PBS, the melanomatous inflammatory bowel mouse model (IBD+B16) of induction load.Tail venoclysis 1 × 10 on model basis
6mSCs/eGFP, is defined as IBD+B16+MSCs/eGFP group; Or tail venoclysis 1 × 10
6mSCs/CCR7-eGFP, is defined as IBD+B16+MSCs/CCR7-eGFP group.
Observe the life span of three groups of mices, result shows: the IBD+B16+MSCs/CCR7-eGFP group mouse survival time is obviously longer than matched group (Fig. 7 A).Modeling is after 14 days, and IBD+B 16+MSCs/eGFP group melanoma is obviously large than matched group volume, and weight obviously increases (*, p < 0.05); But IBD+B16+MSCs/CCR7-eGFP group melanoma and matched group volume and weight are all without obviously distinguishing (n.s., without significant difference) (Fig. 7 B).
Claims (10)
1. the application of mescenchymal stem cell in preparation immunosuppressant of recombinating, it is characterized in that, this restructuring mescenchymal stem cell is by expressing chemokine receptors CCR7 to obtain on the cell membrane of mescenchymal stem cell, and this immunosuppressant is applied to the graft versus host disease after the hematopoietic stem cell transplantation of tumor high-risk, the immunological rejection after organ transplantation and/or autoimmune disease.
2. apply as claimed in claim 1, it is characterized in that, described restructuring mescenchymal stem cell is autologous, xenogenic origin or commercial source.
3. apply as claimed in claim 1, it is characterized in that, the described method expressing chemokine receptors CCR7 on the cell membrane of mescenchymal stem cell comprises: the channel genes of CCR7 in mescenchymal stem cell, is made mescenchymal stem cell endoglin expression CCR7 by the method for employing gene transfection; By vector transfectants system, make mescenchymal stem cell endoglin expression CCR7; Adopt the method for Trogocytosis, by antigen presenting cell, CCR7 is offered on the cell membrane of mescenchymal stem cell; Or change structure by existing epicyte protein or external albumen realizes.
4. apply as claimed in claim 3, it is characterized in that, the method for described gene transfection, comprise viral vector rotaring redyeing system and non-viral transfection system.
5. apply as claimed in claim 4, it is characterized in that, described viral vector rotaring redyeing system is any one or several in adeno-associated virus AA-V, slow virus, adenovirus Ad-V, baculovirus, herpesvirus and Retroviral Transfer system.
6. apply as claimed in claim 4, it is characterized in that, described non-viral transfection system is plamid vector transfection system, chitosan nano rotaring redyeing system or uncorrected gene expression rotaring redyeing system.
7. apply as claimed in claim 1, it is characterized in that, described tumor high-risk is: the 1) mid-aged population of more than 45 years old; 2) high-risk group that causes of inherited genetic factors: the direct line within three generations or collaterals suffer from the medical history of malignant tumor; 3) have the crowd of bad life habits: long-term a large amount of smoking, chronic alcoholism, drug dependence, for a long time overworked, stay up late, life stress is large, operating pressure has unhealthy emotion, personality depressed or use the crowd that mobile phone frequency is high greatly, for a long time; 4) crowd of Bad Eating Habit is had: monophagia, malnutrition, unhygienic diet, diet are irregular, eating speed is slow, overheated mistake of often taking food is pickled as hard as peppery food or frequent feed and smoked fried food; 5) crowd of occupational factor Long Term Contact poisonous and harmful substance; 6) crowd that polluted of living environment: water source and/or aerochemistry pollution, heavy metal pollution, nuclear pollution, microbial contamination or the crowd lived in for a long time in haze weather; 7) chronic or repeatability inflammation crowd is had: chronic bronchitis, chronic hepatitis B virus infecton, HIV patient, chronic cervicitis, chronic gastritis, polyp of stomach, gastric ulcer, mastitis, mammary gland chronic proliferative, polyp intestinal, cystic polyp, prostatitis, sinusitis or long-term functionality metrorrhagia; 8) the innocent and malignant tumour patient after treatment.
8. the application described in claim 1, it is characterized in that, described autoimmune disease is systemic lupus erythematosus (sle), mouth xerophthalmia scheorma syndrome, rheumatoid arthritis, ankylosing spondylitis, scleroderma, polyarteritis nodosa, Wegener granulomatosis, ulcerative colitis, autoimmune hemolytic anemia, spontaneous aleukia purpura disease, dermatomyositis, mixed connective tissue disease, myasthenia gravis, Autoimmune Thyroid Diseases, insulin dependent diabetes mellitus (IDDM), pernicious anemia companion chronic atrophic gastritis, goodpasture syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis or acute idiopathic polyneuritis.
9. the application as described in any one of claim 1-8, is characterized in that, described restructuring mescenchymal stem cell and general immunosuppressant conbined usage.
10. the application as described in any one of claim 1-8, is characterized in that, described restructuring mescenchymal stem cell is 1 × 10 for the dosage of human body
4~ 1 × 10
6mSCs/kg.
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| WO2017214939A1 (en) * | 2016-06-16 | 2017-12-21 | 毛侃琅 | Lentiviral expression vector for improving expression level of ccr7 gene, and applications thereof |
| CN109891238A (en) * | 2016-10-25 | 2019-06-14 | 生物技术Rna制药有限公司 | The dosage of immunotherapeutic agent determines |
| CN110088623A (en) * | 2016-10-17 | 2019-08-02 | 社会福祉法人三星生命公益财团 | Select the method for treating the efficient stem cell of immune disorders |
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| CN105316286A (en) * | 2015-11-26 | 2016-02-10 | 中国人民解放军军事医学科学院基础医学研究所 | Method for preparing recombination mesenchymal stem cell and recombination mesenchymal stem cell prepared through method |
| CN105343894A (en) * | 2015-11-26 | 2016-02-24 | 中国人民解放军军事医学科学院基础医学研究所 | Recombinant mesenchymal stem cell efficient in secreting prostaglandin E2 (PGE2) and its application |
| CN105316286B (en) * | 2015-11-26 | 2018-12-14 | 中国人民解放军军事医学科学院基础医学研究所 | A kind of method of preparation and reorganization mescenchymal stem cell and its obtained recombination mescenchymal stem cell |
| CN105343894B (en) * | 2015-11-26 | 2019-01-29 | 中国人民解放军军事医学科学院基础医学研究所 | The recombination mescenchymal stem cell of efficient secretion prostaglandin E2 (PGE2) and its application |
| WO2017214939A1 (en) * | 2016-06-16 | 2017-12-21 | 毛侃琅 | Lentiviral expression vector for improving expression level of ccr7 gene, and applications thereof |
| CN110088623A (en) * | 2016-10-17 | 2019-08-02 | 社会福祉法人三星生命公益财团 | Select the method for treating the efficient stem cell of immune disorders |
| CN110088623B (en) * | 2016-10-17 | 2022-08-09 | 细胞生命有限公司 | Methods of selecting high-potency stem cells for treating immune disorders |
| CN109891238A (en) * | 2016-10-25 | 2019-06-14 | 生物技术Rna制药有限公司 | The dosage of immunotherapeutic agent determines |
| CN111139222A (en) * | 2019-05-30 | 2020-05-12 | 北京双因生物科技有限公司 | Recombinant mesenchymal stem cell and preparation method and application thereof |
| CN110447601A (en) * | 2019-08-09 | 2019-11-15 | 南京医科大学 | A kind of targeting macrophage is overexpressed the preparation method of genetic mouse model |
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