CN103191154B - Mesenchymal stem cells and the application of extracting method in the psoriatic medicine of preparation thereof - Google Patents
Mesenchymal stem cells and the application of extracting method in the psoriatic medicine of preparation thereof Download PDFInfo
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Abstract
The object of the present invention is to provide mesenchymal stem cells to have the application in the psoriatic medicine for the treatment of in preparation, the skin mesenchymal stem cells related in this application obtains easily, do not have that immunologic rejection, curative effect are high, disease not easily recurs and side effect is little after treatment.
Description
Technical field
The present invention relates to mesenchymal stem cells and prepare the application in immunoloregulation function medicine, particularly skin mesenchymal stem cells (the Skin-derived Mesenchymal Stem Cells) application in the psoriatic medicine of preparation treatment.
Background technology
Psoriasis (psoriasis) is a kind of skin chronic inflammatory disease of common immune-related, recurrent, it is characterized in that intradermal dense based on the inflammatory cell infiltration of T cell, DC, natural killer T cells and macrophage.The epidermal hyper-proliferative of psoriatic's skin and cannot normal differentiation, makes its epidermis significantly thicken, forms a scab and increase (Nestle et al., 2009).Psoriatic definite pathogenic factor is not clear, and the composite factors such as envirment factor (such as infection, wound, pressure etc.) and genetic factor determine individual susceptibility.Psoriasis there is no the medicine (Nestle et al., 2009) of radical cure at present, and the immunosuppressant of the inanimates such as early treatment's many uses steroid, this type of non-specific immunosuppressant has the side effect of certain degree.In recent years, biological preparation is as tumor necrosis factor preparation Etanercept(Embrel) etc. be also used to curing psoriasis, this type of medicine mostly is humanized antibody, for intercepting the effect between psoriasis related inflammation cytokine and receptor.Because many inflammatory cytokine are indispensable in body normal immunoreaction, optionally stop that inflammatory cytokine also can cause the side effect after medication, after drug withdrawal, disease easily rebounds.In addition, antibody class drug manufacture cost is high, expensive, common patient is very difficult can bear.China has about had from China since 1984 and is diagnosed as psoriasis more than 4,000,000 patients, and autonomous research and development China has the psoriatic novel drugs of the treatment of intellectual property imperative.
Mesenchymal stem cells (mesenchymal stem cell) be a kind ofly not break up, can self-hypertrophy and the multipotential cell (Nestle et al., 2009) of multiple mescenchymal tissue cell can be divided into.In early days, mescenchymal stem cell is many to be obtained from bone marrow, mesenchymal stem cells MSCs has speciality (the Nestle et al. of Immunosuppression response and inducing peripheral immunologic tolerance, 2009), once the transplant being applied to causing when suppressing bone marrow transplantation is to host rejection reaction (graft-versus-host disease, GVHD) (Nestle et al., 2009).In addition to bone marrow, mescenchymal stem cell is also present in other adult tissues.Be different from mesenchymal stem cells MSCs, mescenchymal stem cell in dermal layer of the skin, except mesoderm (mesodermal) linked groups cell (as adipose cell, smooth muscle cell) can be divided into, also there is the characteristic being divided into the multiple non-mesoderm tissues cells such as neurocyte, hematopoietic cell, hepatocyte, therefore great using value (Nestle et al., 2009) is had clinically.Due to a large amount of dermatological specimens comparatively bone marrow be easy to obtain, extract from dermatological specimens that treatment mescenchymal stem cell is real belongs to that feasibility is high, purposes is wide, invasive is little and the selection of tool economic benefit.But the immunoloregulation function of skin mesenchymal stem cells is not yet found at present, the present invention will study on this question.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of mesenchymal stem cells and is preparing the application had in the medicine of immunoloregulation function, and the extracting method of relevant mesenchymal stem cells:
Mesenchymal stem cells treats the application had in the medicine of immunoloregulation function in preparation.
The application of mesenchymal stem cells in the psoriatic medicine of preparation treatment.
Described mesenchymal stem cells treats the application had in the medicine of immunoloregulation function in preparation, wherein said mesenchymal stem cells derives from dermal layer of the skin.
The application of described mesenchymal stem cells in the psoriatic medicine of preparation treatment, wherein said mesenchymal stem cells derives from dermal layer of the skin.
Mesenchymal stem cells described in described application is extracted by following method: clip skin of back is placed in dispase enzyme, and digestion is cleaned after spending the night, de-epithelization, corium is placed in collagenase digest, adds DNase I after having digested again and digest, wash one time with PBS, single cell suspension is blown and beaten into dropper, be inoculated in after filtration in culture dish, after cultivation, discard suspension cell, attached cell is continued to cultivate, after major part at the bottom of covering with bottle, trypsinization, routine passage.
The invention has the beneficial effects as follows: mesenchymal stem cells suppresses pathogenic effector T cell to be replied selectively and induces regulatory T cells occur and reach therapeutic effect in inflammatory environment.Be separated from mouse skin corium and obtain dermal mesenchymal stem cells: get newborn mice, after putting to death, clip skin of back is placed in dispase enzyme, digestion is cleaned after spending the night, de-epithelization, corium is placed in collagenase digest, add DNase I after having digested again to digest, wash one time with PBS, blow and beat into single cell suspension with dropper, be inoculated in after filtration in culture dish, after cultivation, discard suspension cell, attached cell is continued to cultivate, after major part at the bottom of covering with bottle, trypsinization, routine passage, gets the cell reaching the third generation and studies.It is identified, by being inoculated in different induction systems after the dermal mesenchymal stem cells trypsinization of In vitro culture, through row tissue staining after cultivation, qualification differentiation situation.Alizarin red staining is passed through to osteoblast differentiation; To Adipocyte Differentiation by morphological observation and oil red dyeing.Observe the skin mesenchymal stem cells be separated from mouse skin corium and have immunoregulatory characteristic, when cultivating in vitro, heterogenous skin dermal mesenchymal stem cells (Allogeneic Skin-derived Mesenchymal Stem Cells) and consubstantiality dermis of skin mescenchymal stem cell (Autologous Skin-derived Mesenchymal Stem Cells) all can effective depression effect T cell immunne response.Find in test skin mesenchymal stem cells is to the experiment of psoriatic therapeutic effect, allosome and consubstantiality skin mesenchymal stem cells tail vein inject suffer from " psoriasis " Mice Body in after 21 days, improve " psoriasis " symptom of mice significantly.Skin mesenchymal stem cells disclosed by the invention easily obtains, do not have immunologic rejection, can the response of special suppression pathogenic effector T cell, curative effect is high, side effect is little.
Accompanying drawing explanation
Fig. 1: the isolation identification of mice dermal mesenchymal stem cells:
A. the dermal mesenchymal stem cells of In vitro culture;
B. the Osteoinductive differentiation of dermal mesenchymal stem cells;
C. the adipogenic induction differentiation of dermal mesenchymal stem cells.
Fig. 2: the heterogenous skin mescenchymal stem cell of mice and consubstantiality skin mesenchymal stem cells depression effect T cell immunne response:
A. consubstantiality mesenchymal stem cells MSCs and the thin Dual culture of T 3 days, have 11.5% T cell division growth;
B. body skin mesenchymal stem cells and the thin Dual culture of T 3 days, have 12.8% T cell division growth;
C. consubstantiality skin mesenchymal stem cells and the thin Dual culture of T 3 days, have 19.6% T cell division growth;
D.T cell single culture has the division growth of 85.0%;
E. almost increment is not divided by the T cell that anti-CD3 and anti-CD28 antibody stimulates.This preliminary experiment describe heterogenous skin mescenchymal stem cell the same as mesenchymal stem cells MSCs and consubstantiality skin mesenchymal stem cells antibody all can be suppressed in vitro to stimulate under T cell division growth.
Fig. 3: by PASI index, mice heterogenous skin mescenchymal stem cell and the therapeutic effect of consubstantiality skin mesenchymal stem cells to " psoriasis " of mice obvious:
A. before and after the treatment of heterogenous skin mescenchymal stem cell;
B. before and after the treatment of consubstantiality skin mesenchymal stem cells;
C.6 before and after a disease mice heterogenous skin mescenchymal stem cell treatment;
D.6 before and after only ill consubstantiality skin mesenchymal stem cells is treated.
PASI: " psoriasis " mice " state of an illness " Scoring Index.PASI (Psoriasis Area and Severity Index) i.e. psoriasis area and severity index is the method for evaluation severity of psoriasis general in the world at present.The thickness of plaque psoriasis, erythema, squama 3 indexs are all by 0-4 totally 5 grades of score (0=without, 1=is slight, 2=moderate, 3=severe, 4=pole severe), and it is these three index score sums that the order of severity is divided.Skin lesion area presses 1-6 totally 6 grades of score, and skin lesion area <10% is 1 point, and >10% is 2 points, >30% is 3 points, >50% is 4 points, and >70% is 5 points, and >90% is 6 points.Finally calculate PASI total score by formula, PASI total score=0.1X(head surface integration X head order of severity is divided)+0.2X(upper limb Line Integral X upper limb the order of severity divides)+0.3X(trunk Line Integral X trunk the order of severity divides)+0.4X(lower limb Line Integral X severe lower extremity degree divides).
Detailed description of the invention
1. the separation of mice dermal mesenchymal stem cells:
Take out the newborn mice of raw about 3 days, to crane one execution, alcohol disinfecting, clip skin of back is placed in the dispase enzyme of 0.25%, 4 DEG C of digestion are spent the night, PBS liquid cleans 3 times, de-epithelization, and collagenase corium being placed in 0.2% digests one hour, add DNase I after having digested again to digest 10 to 15 minutes, wash one time with PBS, blow and beat into single cell suspension with dropper, cross and filter bulk tissue and fragment, be inoculated in culture dish, condition of culture is DMEM(low glucose)+10% hyclone, 37 DEG C, 5%CO2.Cultivate after 6 hours, discard suspension cell gently, attached cell is continued to cultivate, after major part at the bottom of covering with bottle, trypsinization, routine passage.Get the cell reaching the third generation to study.
2. the qualification of mice dermal mesenchymal stem cells:
Be inoculated in after the dermal mesenchymal stem cells trypsinization of In vitro culture in different induction systems, cultivate through row tissue staining after 3 weeks, qualification differentiation situation.Alizarin red staining is passed through to osteoblast differentiation; To Adipocyte Differentiation by morphological observation and oil red dyeing.
3. the skin mesenchymal stem cells depression effect T cell immunne response of mice:
The mice CD4+CD25-T cell of labelled immune Beads enrichment is carried out, with skin mesenchymal stem cells Dual culture (skin mesenchymal stem cells: T cell is 1:4) with cell marker CFSE; T cell is stimulated 3 days with anti-CD3 and anti-CD28 antibody in Dual culture.The division growth situation of the cell that CFSE is labeled can by flow cytomery, this preliminary experiment describe heterogenous skin mescenchymal stem cell the same as mesenchymal stem cells MSCs and consubstantiality skin mesenchymal stem cells antibody all can be suppressed in vitro to stimulate under T cell division growth.
4. mouse skin mescenchymal stem cell is to the treatment of " psoriasis " of mice:
Use the 1st one step process to be separated isolate mesenchymal stem cells from mouse skin corium, be cultured to P3 generation.Every disease mice tail vein injection 1 ' 106 skin mesenchymal stem cells, observe and mark after 21 days, can as can be seen from Figure 3 mice heterogenous skin mescenchymal stem cell and the therapeutic effect of consubstantiality skin mesenchymal stem cells to " psoriasis " of mice obvious.
5. skin mesenchymal stem cells is to the treatment of psoriatic:
From the skin of people, isolate skin mesenchymal stem cells, under gnotobasis, be cultured to P3 generation.Every patient's intravenous injection 50 ' 106 skin mesenchymal stem cells are treated.
Claims (2)
1. mesenchymal stem cells treats the application had in the medicine of immunoloregulation function in preparation, wherein said mesenchymal stem cells derives from dermal layer of the skin, wherein said mesenchymal stem cells is extracted by following method: clip skin of back is placed in dispase enzyme, digestion is cleaned after spending the night, de-epithelization, corium is placed in collagenase digest, add DNase I after having digested again to digest, one time is washed with PBS, single cell suspension is blown and beaten into dropper, be inoculated in after filtration in culture dish, after cultivation, discard suspension cell, attached cell is continued to cultivate, after major part at the bottom of covering with bottle, trypsinization, routine passage.
2. the application of mesenchymal stem cells in the psoriatic medicine of preparation treatment, wherein said mesenchymal stem cells derives from dermal layer of the skin, described mesenchymal stem cells is extracted by following method: clip skin of back is placed in dispase enzyme, digestion is cleaned after spending the night, de-epithelization, corium is placed in collagenase digest, add DNase I after having digested again to digest, one time is washed with PBS, single cell suspension is blown and beaten into dropper, be inoculated in after filtration in culture dish, after cultivation, discard suspension cell, attached cell is continued to cultivate, after major part at the bottom of covering with bottle, trypsinization, routine passage.
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| CN107551095B (en) * | 2017-09-11 | 2020-12-25 | 上海亚睿生物科技有限公司 | A skin repairing agent for psoriasis prepared from stem cell extract and Chinese medicinal extract |
| CN108888618A (en) * | 2018-06-21 | 2018-11-27 | 上海交通大学医学院 | Newtype drug and its application for treating psoriasis |
| CN112566644A (en) * | 2018-07-17 | 2021-03-26 | 北京三有利和泽生物科技有限公司 | Odontogenic stem cells and uses thereof |
| CN109432128A (en) * | 2018-12-07 | 2019-03-08 | 卡替（上海）生物技术股份有限公司 | Application of the dental pulp mescenchymal stem cell in curing psoriasis |
| CN109536443A (en) * | 2018-12-10 | 2019-03-29 | 天津长和生物技术有限公司 | Mescenchymal stem cell, cell culture medium and cultural method and application |
| CN112704687A (en) * | 2019-10-08 | 2021-04-27 | 上海赛比曼生物科技有限公司 | Preparation containing mesenchymal stem cells and application thereof in psoriasis treatment |
| CN114504596A (en) * | 2020-10-23 | 2022-05-17 | 厦门大学 | Application of mesenchymal stem cells derived from pluripotent stem cells in treatment of psoriasis |
| CN115089609A (en) * | 2022-06-23 | 2022-09-23 | 领航干细胞再生医学工程有限公司 | Application of Muse cells in preparation of psoriasis treatment medicine |
| CN116725948B (en) * | 2023-07-14 | 2024-03-26 | 黑龙江八一农垦大学 | Adipose-derived stem cell-mediated gel and preparation method and application thereof |
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| US20080095749A1 (en) * | 2004-03-22 | 2008-04-24 | Sudeepta Aggarwal | Mesenchymal stem cells and uses therefor |
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