CN101974483A - Application of mesenchymal stem cell in sicca syndrome - Google Patents
Application of mesenchymal stem cell in sicca syndrome Download PDFInfo
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- CN101974483A CN101974483A CN2010105036798A CN201010503679A CN101974483A CN 101974483 A CN101974483 A CN 101974483A CN 2010105036798 A CN2010105036798 A CN 2010105036798A CN 201010503679 A CN201010503679 A CN 201010503679A CN 101974483 A CN101974483 A CN 101974483A
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Abstract
The invention confirms that mesenchymal stem cell can be used for the treatment of sicca syndrome. The mesenchymal stem cell is characterized by being capable of inhibiting the abnormal immune response of sicca syndrome, thus fundamentally controlling the development of sicca syndrome.
Description
Technical field
This research relate to human umbilical cord mesenchymal stem cells (umbilical cord mesenchymal stem cells, UCMSCs) suppress sjogren syndrome (
Syndrome, SS) immunoloregulation function of patient T cell and the therapeutic action in the SS mouse thereof.
Background technology
MSCs is the tissue stem cell that a class has multidirectional differentiation potential, can obtain from multiple tissue such as blood, liver, marrow, fat, skin, be a group multipotential cell, can be to differentiation such as multiple histocyte such as bone, cartilage, muscle, ligament, tendon, fat.At present, MSCs is mainly derived from marrow, but because there is the possibility of height virus pollution in bone marrow derived MSCs, and obvious downtrending appears in its cell quantity and amplification, differentiation capability with age, substitute bone marrow MSCs so seek a kind of energy, and can remedy the MSCs source of its defective, more and more receive various countries scholar's concern.Additive method has certain difficulty as cultivate MSCs from Cord blood and peripheral blood, and MSCs quantity in the peripheral blood of the Cord blood of term birth or mobilization remains in dispute.Therefore, separation and Culture UCMSCs from puerperal rejected material umbilical cord is for the application of MSCs provides new material source and basic data.
SS is a kind of to invade and exocrine glands such as lachrymal gland and sialisterium are the general autoimmune disease of feature.The modal clinical manifestation of this disease is that eye is done, dry, and can involve organs such as lung, kidney, lymphsystem, blood vessel and liver.This sick no tangible regional distributional difference can betide any age, and with 40-60 year, particularly postclimacteric women is in the majority, and men and women's ratio is 1:9.Morbidity in China is 0.77%, and the morbidity in elderly population is up to 4%, and there are nearly 1,000 ten thousand patients in the whole nation.The methods of treatment of existing SS comprises traditional immunosuppressor and biotechnological formulation, has drawbacks such as side effect is big, price height, need constantly to seek for this reason more economical effectively and the methods of treatment of non-evident effect.The NOD mouse is the spontaneous mouse model of SS of relatively generally acknowledging in the world at present.Existing studies confirm that, MSCs has immunoloregulation function, and the multinomial MSCs of studies show that can suppress the propagation of various autoimmune patient T cell and the secretion of inflammatory cytokine.For this reason, this seminar utilizes UCMSCs in external and SS patient T co-culture of cells, and UCMSCs is transplanted in the NOD body, observes the effect of UCMSCs in the SS treatment.But the immunologic function of discovery UCMSCs suppressor T cell has also obviously been improved saliva flow rate and the histopathology of NOD.The clinical application for the treatment of SS for UCMSCs provides foundation.
Summary of the invention
This research and utilization UCMSCs is in external and SS patient T co-culture of cells, and UCMSCs is transplanted in the NOD mouse body, observes its effect in the SS treatment.But find the propagation of UCMSCs suppressor T cell, suppress the secretion of inflammatory cytokine (TNF-α, IL-6, IFN-γ), raise the expression of IL-10, raise CD4
+FOXP3
+The expression of Treg cell, and obviously improved saliva flow rate and the histopathology of NOD.The clinical application for the treatment of SS for UCMSCs provides foundation.
Description of drawings
The evaluation of Fig. 1 .UC-MSCs (A): UCMSCs morphological specificity (third generation) (B): the flow cytometry cell phenotype is analyzed the osteogenic induction differentiation-alkaline phosphatase staining that the fat of (C): UCMSCs is induced differentiation-oil red O stain (D): UCMSCs
Fig. 2 .UC-MSCs is to the therapeutic action (A) of NOD mouse: no matter in 4 week and 8 all administrations, UC-MSCs all can obviously suppress the decline of NOD mouse salivary flow, * P＜0.05, * P＜0.01 (B): no matter in 4 week and 8 all administrations, UC-MSCs all can obviously reduce the expression of SS associated antibodies in the NOD mice serum, * P＜0.05, * * P＜0.01 (C): histopathology shows that UC-MSCs can obviously improve the lymphocytic infiltration of NOD mouse submandibular gland kitchen range
Fig. 3 .UC-MSCs is to the immunoloregulation function (A) of the spleen mononuclearcell (MNCs) of NOD mouse: the multiplication capacity of UCMSCs treatment group MNCs obviously reduces than the PBS group, * P＜0.01 (B): the expression of regulatory T cells (Treg) is obviously raised than the PBS group among the UCMSCs treatment group MNCs, * P＜0.05 (C): external, with do not intervene 16 age in week NOD mouse MNCs and UCMSCs cultivate altogether, as seen UCMSCs can obviously raise the expression of Treg, * * P＜0.01
The expression that Fig. 4 .UC-MSCs can reduce IL-6 in the NOD mice serum to NOD mouse cell factor excretory influence (A): UC-MSCs, * P＜0.01 (B): UCMSCs treatment group is compared with the PBS group, IL-6 down-regulated expression in the MNCs cell conditioned medium, the IL-10 up-regulated, * P＜0.01 (C): external, with do not intervene 16 age in week NOD mouse MNCs and UCMSCs cultivate altogether, as seen UCMSCs can obviously reduce TNF-α, the expression of IFN-γ, raise the expression of IL-10, * P＜0.01UC-MSCs is to the influence (A) of SS patient T cell: but the propagation of UC-MSCs suppressor T cell, and be dose-dependently, * P＜0.05, * P＜0.01 (B): anti-IL-10 monoclonal antibody can reverse the restraining effect of UC-MSCs to T cell proliferation, * P＜0.01 (C): UCMSCs and T co-culture of cells, the expression that can reduce TNF-α, the expression of rise IL-10, * P＜0.05 (D): UCMSCs can obviously raise the expression of SS patient Treg, * P＜0.05
Embodiment
Embodiment 1: the separation of umbilical cord mesenchymal stem cells, cultivation.
A. get normal vaginal delivery or c-section umbilical cord, (the ABO/Rh blood group detects through strict trace routine, the HLA somatotype detects, syphilis antibody detects, the HIV immunodetection, CMV antibody test, Australia antigen(AA) antibody test) determine safety after, visible dirt of naked eyes and blood are removed in the stroke-physiological saline solution flushing.
B. aseptic hammer-shears umbilical cord is to the 3-5mm size.
C. with the tissue block that obtains among the step b, be transferred in the 50ml centrifuge tube, add the PBS40ml that contains 1% mycillin, the centrifugal 250g of room temperature 5 minutes abandons supernatant.
D. step tissue block that c obtains is cultivated with 10% α MEM, changes liquid in per 3 days.
E. there were fusiformis and polygon cell to climb out of to 7 days around the tissue block of the left and right sides, and increase gradually.
F. reach about 80% when merging to 10-14 days left and right sides cells, go down to posterity with 0.25% trysinization.
G. went down to posterity once every 3-5 days after.
H. identify to the third generation.
I. the cell after identifying continues to cultivate standby or frozen.
See Fig. 1.
Embodiment 2:UCMSCs is to the therapeutic action of NOD mouse
This experiment is divided into prevention group and treatment group.The prevention group choose 4 ages in week 20 of female NOD mouse, the treatment group is chosen 20 of female NOD mouse in 8 ages in week.Each is organized interior 20 mouse and is divided into 2 groups at random.Give UC-MSCs and PBS respectively.Abdominal injection 1 * 10
6/ 100ul cell, altogether once.Carrying out salivary flow per two weeks measures.Finish to observe to 16W.Leave and take mice serum, submandibular organization and splenocyte, ELISA detect mice serum SS associated antibodies, and the submaxillary gland pathological analysis is done in HE dyeing.The result shows treatment group and prevention group NOD mouse, and UC-MSCs all can obviously increase its salivary flow; Can obviously reduce the expression of antibody in the NOD mice serum; Histopathology shows that UC-MSCs can obviously improve the lymphocytic infiltration of NOD mouse submandibular gland kitchen range.See Fig. 2.
Embodiment 3:UCMSCs is to the immunoloregulation function of NOD mouse MNCs
(A): get UCMSCs treatment group and PBS group MNCs, 4 * 10
5/ hole is inoculated in 96 orifice plates, and every Kong Jun adds ConA (2 μ g/ml) to be stimulated.120 hours,
3The H method of mixing detects T cell proliferation situation.The MNCs multiplication capacity of UCMSCs treatment group obviously reduces (B) than the PBS group: in like manner, get UCMSCs treatment group and PBS group MNCs, 2 * 10
6/ hole is inoculated in 24 orifice plates, after 48 hours, the expression of Flow cytometry mouse boosting cell Treg, the expression of Treg is obviously raised (C) than PBS group among the UCMSCs treatment group MNCs: external, with do not intervene 16 age in week NOD mouse MNCs (1 * 10
6/ hole) with UCMSCs (2 * 10
4/ hole) cultivate altogether, after 48 hours, the expression of Flow cytometry mouse boosting cell Treg, visible UCMSCs can obviously raise the expression of Treg.See Fig. 3.
Embodiment 4:UCMSCs influences NOD mouse cell factor excretory
(A): when experiment finishes, leave and take mice serum, ELISA detects cytokine secretion in the mice serum, finds the expression (B) that UC-MSCs can reduce IL-6 in the NOD mice serum: get UCMSCs treatment group and PBS group MNCs, 2 * 10
6/ hole is inoculated in 24 orifice plates, after 48 hours, collects cell conditioned medium, detect cytokine, the result shows that UCMSCs treatment group compares IL-6 down-regulated expression in the MNCs cell conditioned medium with the PBS group, IL-10 up-regulated (C): external, with do not intervene 16 age in week NOD mouse MNCs (1 * 10
6/ hole) with UCMSCs (2 * 10
4/ hole) cultivates altogether, after 48 hours, detect cytokine in the cell conditioned medium, the expression that visible UCMSCs can obviously reduce TNF-α, IFN-γ, the expression of rise IL-10.See Fig. 4.
Embodiment 5:UC-MSCs is to the influence of SS patient T cell
(A): separate SS patient's peripheral blood PBMC, magnetic bead sorting CD3
+The T cell.Be divided into two groups, one group is UCMSCs and T co-culture of cells group, and one group is simple T cell control group, and two groups all add CD3/CD28 (5 μ g/ml) and stimulate.120 hours,
3The H method of mixing detects T cell proliferation situation.Found that no matter whether UCMSCs contacts with the T cell, cultivation group T cell proliferation is subjected to obvious inhibition altogether, and is dose-dependently.(B): in UCMSCs and T co-culture of cells system, add anti-IL-10 monoclonal antibody (10 μ g/ml), can reverse the restraining effect (C) of UC-MSCs: with SS patient T cell (1 * 10 to T cell proliferation
6/ hole) with UCMSCs (2 * 10
4/ hole) cultivates altogether, after 48 hours, detect cytokine in the cell conditioned medium, the expression that visible UCMSCs can reduce TNF-α, the expression (D) of rise IL-10: SS patient T cell (1 * 10
6/ hole) with UCMSCs (2 * 10
4/ hole) cultivate altogether, after 48 hours, the expression of Flow cytometry Treg, UCMSCs can obviously raise the expression of SS patient Treg.See Fig. 5.
Claims (5)
1. the mescenchymal stem cell in various sources.
2. the mescenchymal stem cell of claim 1 is in the purposes of treatment in the sjogren syndrome.
3. the mescenchymal stem cell of claim 1 is administered to Patients with Sjogren Syndrome.
4. the mescenchymal stem cell of claim 1 is in the purposes of treatment in other autoimmune disease.
5. the mescenchymal stem cell of claim 1 is administered to other autoimmunization patient.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113980896A (en) * | 2021-10-27 | 2022-01-28 | 中国人民解放军军事科学院军事医学研究院 | Application of IRF1 in regulating and controlling immune regulation effect of mesenchymal stem cells and product |
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| US20090324609A1 (en) * | 2007-08-09 | 2009-12-31 | Genzyme Corporation | Method of treating autoimmune disease with mesenchymal stem cells |
| CN101643719A (en) * | 2009-07-27 | 2010-02-10 | 北京大学人民医院 | Simplified method for isolation and culture of umbilical mesenchymal stem cells and application thereof in treatment of rheumatoid arthritis |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090324609A1 (en) * | 2007-08-09 | 2009-12-31 | Genzyme Corporation | Method of treating autoimmune disease with mesenchymal stem cells |
| CN101643719A (en) * | 2009-07-27 | 2010-02-10 | 北京大学人民医院 | Simplified method for isolation and culture of umbilical mesenchymal stem cells and application thereof in treatment of rheumatoid arthritis |
Non-Patent Citations (3)
| Title |
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| 《中国优秀硕士学位论文全文数据库（医药卫生科技辑）》 20100415 肖娜 脐带间充质干细胞联合脐血干细胞治疗Ⅰ型糖尿病的初步研究 , 2 * |
| 《全国临床免疫检验研讨会暨第六届全国临床免疫学术会议论文汇编》 20091231 刘燕鹰等 《 利用脐带间充质干细胞抑制类风湿关节炎免疫异常的实验研究 》 , 2 * |
| 《国际口腔医学杂》 20100731 吴斯媛 成体干细胞在涎腺组织工程中的应用 第37卷, 第4期 2 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113980896A (en) * | 2021-10-27 | 2022-01-28 | 中国人民解放军军事科学院军事医学研究院 | Application of IRF1 in regulating and controlling immune regulation effect of mesenchymal stem cells and product |
| CN113980896B (en) * | 2021-10-27 | 2023-10-20 | 中国人民解放军军事科学院军事医学研究院 | Application of IRF1 in regulation and control of mesenchymal stem cell immunoregulation and product |
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Application publication date: 20110216 |