CN103191155A - Application of mesenchymal stem cells in preparation of multiple sclerosis treatment medicines, and extraction method of mesenchymal stem cells - Google Patents
Application of mesenchymal stem cells in preparation of multiple sclerosis treatment medicines, and extraction method of mesenchymal stem cells Download PDFInfo
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- CN103191155A CN103191155A CN2012100027503A CN201210002750A CN103191155A CN 103191155 A CN103191155 A CN 103191155A CN 2012100027503 A CN2012100027503 A CN 2012100027503A CN 201210002750 A CN201210002750 A CN 201210002750A CN 103191155 A CN103191155 A CN 103191155A
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Abstract
The invention aims to provide an application of mesenchymal stem cells in the preparation of multiple sclerosis treatment medicines. The mesenchymal stem cells can effectively inhibit the excess activated effect T cell immune-response in the central nervous system of a multiple sclerosis patient, induce the generation of regulative T cells having a specific function and differentiate to form new nerve cells, so the damaged nerve tissues are repaired, thereby the multiple sclerosis is treated. The skin-derived mesenchymal stem cells used in the invention have the advantages of easy obtaining, no immune repellency, high curative effect, difficult recurrence after disease heal, and small side effects.
Description
Technical field
The present invention relates to mesenchymal stem cells and have application, particularly skin mescenchymal stem cell (the Skin-derived Mesenchymal Stem Cells) application in the medicine of preparation multiple sclerosis in the medicine of immunoloregulation function in preparation.
Background technology
Multiple sclerosis (multiple sclerosis) is chronic, struvite, the autoimmune disease of a kind of central nervous system of betiding (brain and spinal cord), it is characterized in that unusual neurological pathological changes (neurodegeneration) initiation is based on the inflammatory cell infiltration of T cell, B cell, antigen presenting cell in the central nervous system, thereby demyelination (demyelination) phenomenon appears in the nerve that causes many places, and in the process of myelin tissue repair, harden along axonal loss.The definite pathogenic factor of multiple sclerosis is not clear, composite factors such as envirment factor (such as infection, wound etc.) and genetic factor have determined individual susceptibility, the incomplete symptom (Hauser and Oksenberg, 2006) of function of nervous system such as that the patient may occur is handicapped, visual impairment, pain.
Multiple sclerosis does not still have the medicine of radical cure at present, IFN-β and glatiramer acetate are the first line medicine of common treatment recurrence type multiple sclerosis (relapsing-remitting multiple sclerosis), it is generally acknowledged this two kinds of medicines energy inflammation-inhibiting T cellullar immunologic responses, yet its mechanism of action is still indeterminate, also only can reach the releive probability (Hauser and Oksenberg, 2006) of palindromia of appropriateness.The medicine that another kind is used for the treatment of serious multiple sclerosis patients is Natalizumab, its mechanism of action is to see through to suppress the expressed cell adhesion molecule α of pathogenic lymphocytic cell surface 4 integration elements, reach the effect (Hauser and Oksenberg, 2006) that stops lymphocyte to migrate to the central nervous system.Though Natalizumab has curative effect preferably, because lymphocyte is indispensable in the body normal immunoreaction, do not stop optionally that lymphocyte migrates to the central nervous system and causes the sufferer of very high percentage to produce brain infection symptom (Martin, 2010) in the back of receiving treatment.In addition, antibody class drug manufacture cost height, expensive, common patient are difficult to and can bear.Duo Hair property sclerosis is one of topmost disease that causes function of nervous system's incompleteness, and the whole world has up to ten million people ill, and patient's daily life often is subjected to serious Pull Even.Based on this point, the new therapy of independently researching and developing efficient and low side effect is imperative.
Mesenchymal stem cells (mesenchymal stem cell) is a kind of not differentiation, the self-hypertrophy of energy and the multipotential cell (Pittenger et al., 1999) that can be divided into multiple mescenchymal tissue cell.In early days, mescenchymal stem cell is many to be obtained from bone marrow, mesenchymal stem cells MSCs has the immunne response of inhibition and induces speciality (the Xu et al. of periphery immunologic tolerance, 2007), the transplant that causes when once being applied to suppressing bone marrow transplantation is to host's rejection (graft-versus-host disease, GVHD) (Le Blanc et al., 2004).Except bone marrow, mescenchymal stem cell also is present in other adult tissues.Be different from mesenchymal stem cells MSCs, mescenchymal stem cell in dermal layer of the skin, except being divided into mesoderm (mesodermal) linked groups cell (as adipose cell, smooth muscle cell), also have the histiocytic characteristics of multiple non-mesoderm such as the neurocyte of being divided into, hematopoietic cell, hepatocyte, great using value (Sellheyer and Krahl, 2010) is therefore arranged clinically.Because a large amount of dermatological specimens is easy to obtain than bone marrow, from dermatological specimens extract treatment with mescenchymal stem cell belong to the feasibility height in fact, purposes is wide, invasive is little and the selection of tool economic benefit.
Yet the immunoloregulation function of skin mescenchymal stem cell is still undiscovered at present.The invention provides a kind of skin mescenchymal stem cell that sees through suppresses pathogenic effector T cell selectively and replys in the inflammation environment, impel nervous cell regenerating and reach the multiple sclerosis cell therapy of therapeutic effect, and according to the application that the application of medicine of immunoloregulation function particularly prepares the medicine for the treatment of multiple sclerosis of getting everything ready of this legal system.
Summary of the invention
Technical problem to be solved by this invention provides a kind of mesenchymal stem cells and has application in the medicine of immunoloregulation function and the extracting method of relevant mesenchymal stem cells in preparation:
Mesenchymal stem cells is preparing the application for the treatment of in the medicine with immunoloregulation function.
The application of mesenchymal stem cells in the medicine of preparation treatment multiple sclerosis.
Described mesenchymal stem cells is preparing the application for the treatment of in the medicine with immunoloregulation function, and wherein said mesenchymal stem cells derives from the dermal layer of the skin.
The application of described mesenchymal stem cells in the medicine of preparation treatment multiple sclerosis, wherein said mesenchymal stem cells derives from the dermal layer of the skin.
Mesenchymal stem cells described in the described application is extracted by following method: the clip skin of back places the dispase enzyme, uses PBS liquid to clean de-epithelization, place collagenase to digest corium, add DNase I digestion after finishing again, wash one time with PBS again, blow and beat into single cell suspension and filtration with dropper, be inoculated in the culture dish, discard suspension cell, attached cell continue is cultivated, at the bottom of waiting to cover with bottle after the major part, trypsinization, routine goes down to posterity.
From mouse skin corium, separate and obtain the corium mescenchymal stem cell: get newborn mice, put to death back clip skin of back and place the dispase enzyme, use PBS liquid to clean, de-epithelization, place collagenase to digest corium, add DNase I digestion after finishing again, wash one time with PBS again, blow and beat into single cell suspension and filtration with dropper, be inoculated in the culture dish, discard suspension cell, attached cell continue is cultivated, at the bottom of waiting to cover with bottle after the major part, trypsinization, routine goes down to posterity, and gets the cell that reaches the third generation and studies.It is identified: be inoculated in the different induction systems after the corium mescenchymal stem cell trypsinization with In vitro culture, cultivate by the row tissue staining, identify the differentiation situation.Dye by alizarin red to osteoblast differentiation; Break up by morphological observation and oil red dyeing (Fig. 1) to adipose cell.Observe the skin mescenchymal stem cell that from the mouse skin skin corium, extracts and have immunoregulatory characteristic, effective depression effect T cellullar immunologic response (Fig. 2) when In vitro culture: with the mouse T cell that cell marker labelled immune magnetic bead separates, cultivate altogether with the skin mescenchymal stem cell; In cultivating altogether, stimulate the T cell with antibody.The division growth situation of the cell that labelling is crossed can be detected by flow cytometer, this description of test the same consubstantiality skin mescenchymal stem cell with mesenchymal stem cells MSCs all can be under vitro inhibition antibody stimulates T cell division propagation.In the therapeutic effect experiment of test skin mescenchymal stem cell to multiple sclerosis, the skin mescenchymal stem cell is injected a kind of experimental autoimmune encephalomyelitis(EAE very similar to human multiple sclerosis on histology, immunology, polygenes characteristic and therapeutic response) in the mice body, the EAE mice the treatment of skin mescenchymal stem cell after 30 days by basic cure (Fig. 3).
Beneficial effect of the present invention: mesenchymal stem cells suppresses pathogenic effector T cell selectively and replys and induce regulatory T cells to take place and reach therapeutic effect in the inflammation environment.Skin mescenchymal stem cell disclosed by the invention obtain easily, do not have immunologic rejection, can be in the inflammation environment the pathogenic effector T cell of special inhibition reply, impel after nervous cell regenerating, good effect, the treatment disease to be difficult for recurrence and side effect is little.
Description of drawings
Fig. 1: the isolation identification of mice corium mescenchymal stem cell:
A, the corium mescenchymal stem cell of In vitro culture;
B, the osteogenic induction differentiation of corium mescenchymal stem cell;
C, the one-tenth fat of corium mescenchymal stem cell is induced differentiation.
?
Fig. 2: the consubstantiality skin mescenchymal stem cell depression effect T cellullar immunologic response of mice:
A, consubstantiality mesenchymal stem cells MSCs (autologous bone marrow mesenchymal stem cells) was carefully cultivated 3 days altogether with T, and 11.5% T cell division propagation is arranged;
B, consubstantiality skin mescenchymal stem cell (autologous skin-derived mesenchymal stem cells) was carefully cultivated 3 days altogether with T, and 19.6% T cell division propagation is arranged;
D, T cell single culture has 85.0% division growth;
E, the T cell that anti-CD3 of no use and anti-CD28 antibody stimulate be not division increment almost.This preliminary experiment has illustrated the T cell division propagation that the same consubstantiality skin mescenchymal stem cell with mesenchymal stem cells MSCs all can be under vitro inhibition antibody stimulates.
Fig. 3: mice consubstantiality skin mescenchymal stem cell is obvious to the therapeutic effect of the EAE of mice:
Light color point-like lines: 30 days scoring of consubstantiality skin mescenchymal stem cell treatment EAE in mice;
Dark point-like lines: control mice EAE30 days the scoring of only injecting PBS.Adopt 5 fens point systems, the EAE standards of grading are as follows: 0 is divided into and does not fall ill; 1 to be divided into tail unable; 2 are divided into slight back myasthenia of limbs; 3 are divided into serious back acroparalysis; 4 are divided into quadriplegia; 5 are divided into and are at death's door or dead.
The specific embodiment
1. the separation of mice corium mescenchymal stem cell
Take out the newborn mice of giving birth to about 3 days, the execution of craning one, alcohol disinfecting, the clip skin of back places 0.25% dispase enzyme, 4 ℃ of digestion are spent the night, PBS liquid cleans 3 times, and de-epithelization placed 0.2% collagenase digestion one hour with corium, after finishing, digestion adds DNase I digestion 10 to 15 minutes again, wash one time with PBS, blow and beat into single cell suspension with dropper, remove by filter bulk tissue and fragment, be inoculated in the culture dish, condition of culture is DMEM(low glucose)+10% hyclone, 37 ℃, 5%CO
2Cultivate after 6 hours, discard suspension cell gently, attached cell continue is cultivated, at the bottom of waiting to cover with bottle after the major part, trypsinization, routine goes down to posterity.Getting the cell that reaches the third generation studies.
2. the evaluation of mice corium mescenchymal stem cell
Be inoculated in the different induction systems after the corium mescenchymal stem cell trypsinization with In vitro culture, cultivated for 3 weeks by the row tissue staining, identify the differentiation situation.Dye by alizarin red to osteoblast differentiation; Dye by morphological observation and oil red to the adipose cell differentiation.
3. the skin mescenchymal stem cell depression effect T cellullar immunologic response of mice;
Come the mice CD4 of labelled immune magnetic bead separation with cell marker CFSE
+CD25
-The T cell is cultivated (the skin mescenchymal stem cell: the T cell is 1:4) altogether with the skin mescenchymal stem cell; In cultivating altogether, stimulated the T cell 3 days with anti-CD3 and anti-CD28 antibody.The division growth situation of the cell that the CFSE labelling is crossed can be detected by flow cytometer.
4. the mouse skin mescenchymal stem cell is to the treatment of EAE in mice;
At first be to set up the EAE model, add hot deactivation mycobacterium tuberculosis in incomplete Freund's adjuvant, be complete Freund's adjuvant, MOG35-55 is made into final concentration 10mg/ml.Mice is pressed 1:1 mixing and emulsifying antigen respectively at 2 subcutaneous injections in dorsal part center line both sides by MOG35-55 and CFA.Immunity gave mouse tail vein injection pertussis toxin, PT 200ng/ only at the same day and second day, and inducing mouse produces EAE.Use certain method to separate separating mesenchymal stem cell from mouse skin corium, be cultured to P3 generation.Every ill mouse tail vein injection 1 ' 10
6Individual skin mescenchymal stem cell was observed 30 days and scoring every day.
5. the skin mescenchymal stem cell is to the treatment of human multiple sclerosis patients.
From people's skin, isolate the skin mescenchymal stem cell, be cultured to P3 generation under the gnotobasis.Every patient's intravenous injection 50 ' 10
6Individual skin mescenchymal stem cell is treated.
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Claims (5)
1. mesenchymal stem cells is preparing the application for the treatment of in the medicine with immunoloregulation function.
2. the application of mesenchymal stem cells in the medicine of preparation treatment multiple sclerosis.
3. the described mesenchymal stem cells of claim 1 is preparing the application for the treatment of in the medicine with immunoloregulation function, and wherein said mesenchymal stem cells derives from the dermal layer of the skin.
4. the application of the described mesenchymal stem cells of claim 2 in the medicine of preparation treatment multiple sclerosis, wherein said mesenchymal stem cells derives from the dermal layer of the skin.
5. claim 3 or 4 described application, wherein said mesenchymal stem cells is extracted by following method: the clip skin of back places the dispase enzyme, uses PBS liquid to clean, de-epithelization places collagenase to digest corium, adds DNase I digestion after finishing again, wash one time with PBS again, blow and beat into single cell suspension and filtration with dropper, be inoculated in the culture dish, discard suspension cell, attached cell is continued to cultivate, at the bottom of waiting to cover with bottle after the major part, trypsinization, routine goes down to posterity.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108451981A (en) * | 2018-04-21 | 2018-08-28 | 洛阳轩智生物科技有限公司 | Use skin mesenchymal stem cells treatment system sclerosis |
| CN110257353A (en) * | 2019-05-22 | 2019-09-20 | 中国人民解放军第四军医大学 | The method that application on human skin digests complex enzyme and separates skeptophylaxis cell Treg cell from people on a small quantity full pachydermia |
| CN110305857A (en) * | 2019-05-22 | 2019-10-08 | 中国人民解放军第四军医大学 | The method that mouse skin digests complex enzyme and separates skeptophylaxis cell Treg cell from mouse skin |
| CN114984051A (en) * | 2022-06-27 | 2022-09-02 | 广州惠善医疗技术有限公司 | Application of mesenchymal stem cells in preparation of medicine for treating inflammation and immune related diseases |
| EP4094775A4 (en) * | 2020-02-06 | 2023-04-19 | Talengen International Limited | Method and drug for preventing and treating multiple sclerosis |
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| CN1597937A (en) * | 2004-07-20 | 2005-03-23 | 成都军区昆明总医院 | Extracting material of placental villi cell and its application for inducing differentiating of matrax stem cell |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108451981A (en) * | 2018-04-21 | 2018-08-28 | 洛阳轩智生物科技有限公司 | Use skin mesenchymal stem cells treatment system sclerosis |
| CN108451981B (en) * | 2018-04-21 | 2019-07-23 | 江西汉氏联合干细胞科技有限公司 | Use skin mesenchymal stem cells treatment system sclerosis |
| CN110257353A (en) * | 2019-05-22 | 2019-09-20 | 中国人民解放军第四军医大学 | The method that application on human skin digests complex enzyme and separates skeptophylaxis cell Treg cell from people on a small quantity full pachydermia |
| CN110305857A (en) * | 2019-05-22 | 2019-10-08 | 中国人民解放军第四军医大学 | The method that mouse skin digests complex enzyme and separates skeptophylaxis cell Treg cell from mouse skin |
| EP4094775A4 (en) * | 2020-02-06 | 2023-04-19 | Talengen International Limited | Method and drug for preventing and treating multiple sclerosis |
| CN114984051A (en) * | 2022-06-27 | 2022-09-02 | 广州惠善医疗技术有限公司 | Application of mesenchymal stem cells in preparation of medicine for treating inflammation and immune related diseases |
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| CN103191155B (en) | 2015-04-01 |
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