Use skin mesenchymal stem cells treatment system sclerosis
Technical field
The invention belongs to field of immunology, and in particular to use skin mesenchymal stem cells treatment system sclerosis.
Background technique
Systemic sclerosis (SSc) is also referred to as chorionitis, is one kind with limitation or diffusivity pachyderma and fibrosis
The systemic autoimmune diseases being characterized.Lesion characteristic is that skin fiber hyperplasia and blood vessel onion-skin sample change, and eventually leads to skin
Skin hardening, blood vessel ischemic.This disease clinically characterized by limitation or diffusivity pachyderma and fibrosis, removes skin involvement
Outside, it can also influence internal organ (organs such as the heart, lung and alimentary canal), as a kind of autoimmunity disease, often with antinuclear antibodies, resist
The autoantibodies such as silk point antibody, anti-Scl-70.Feature outstanding is sclerosis of the skin, most common with hand, skin of foot hardening, when serious
It is stiff can to there is whole skin.Other than skin, systemic sclerosis can also cause viscera disease, most commonly swallow
The clinical manifestations such as difficulty, esophageal reflux, pulmonary interstitial fibrosis, hydropericardium and renal insufficiency.
This main disease of the treatment of systemic sclerosis there is no specific medicament at present.Skin involvement range and lesion degree are to examine
Disconnected and assessment prognosis important evidence, and popularity and severity that important organ involves determine its prognosis.Early treatment mesh
Be prevent new skin and internal organs involvement, and the purpose in advanced stage is to improve existing symptom.The treatment atmosphere one of the disease
As treat and symptomatic treatment.
General treatment includes: (1) glucocorticoid and immunosuppressor: generally speaking glucocorticoid to this disease curative effect not
Significantly, but to the inflammatory phase of inflammatory myopathy, interstitial lung disease there is certain curative effect;Also have in oedema phase early stage, arthralgia and myalgia
Curative effect.Dosage is 30~40mg/d of prednisone, is used in conjunction 3~4 weeks, decrescence to maintenance dose l0~15mg/d.Immunosuppressor is to skin
The Research Literature of the treatment of skin hardening is few.Common Ciclosporin A, cyclophosphamide, imuran etc., have been reported that skin joint
There is certain curative effect with renal lesions, application is merged with glucocorticoid, curative effect often can be improved and reduces glucocorticoid dosage.
(2) penicillamine: during procollagen is transformed into collagen, monoamine oxidase is needed to participate in polymerization and cross link.Penicillamine
Copper ion in monoamine oxidase can be complexed, to inhibit new collagen maturation, and clostridiopetidase A can be activated, make established collagen
Fiber degradation.Common adverse reaction have fever, anorexia, Nausea and vomiting, canker sore, parageusia, fash, leucocyte and
Decrease of platelet, albuminuria and blood urine etc..(3) methotrexate (MTX): the treatment guidelines of European antirheumatic Union Recommendation are hard for skin
The only treatment recommendation changed is methotrexate (MTX), the randomized controlled trials of 2 high quality the result shows that, methotrexate (MTX) is oral or muscle
Injection can improve the relevant cutaneous lesions progress of systemic sclerosis, therefore, it is recommended that being used for the skin disease of systemic sclerosis
Become.(4) other: in recent years, have document report a variety of new using relaxain, Imatinib, CD20 monoclonal antibody, TGF-β antibody etc.
Treatment method treatment sclerosis of the skin obtains good curative effect, but is not yet received is widely used to promote at present, it may be considered that is used for
Intractable patient.
Other treatment: it in recent years, both at home and abroad using the peripheral stem cell transplantation treatment through CD34 cell sorting, obtains
Certain effect, but somewhat expensive, transplanting adverse reaction risk is higher, only recommends to be used for intractable patient.Domestic Sun Lingyun religion
It is beheaded and first obtains preferable effect early period with mescenchymal stem cell treatment system sclerosis, confirm it up for more studying
Curative effect.
Bone marrow interstital stem cell answering in preparation treatment autoimmune disease drug is disclosed in CN101485685A
With.Active constituent of the bone marrow MSCs as treatment autoimmune disease drug, has unique immunological regulation and repairs in the application
Multiple effect has the characteristics that " treatment is convenient, curative effect is lasting " for treating autoimmune disease, patient can be helped to reduce drug
The type and quantity and the various side effects of drug bring for the treatment of, in some instances it may even be possible to deactivate the drug therapies such as immunosuppressor, drop
Low actual and disability rate improve patients ' life quality;Meanwhile using bone marrow MSCs as the treatment autoimmune disease of active constituent
The drug of disease is low compared with other novel biological agent expenses, and most of patients has corresponding economic capability.Therefore bone marrow MSCs exist
Application in preparation treatment autoimmune disease drug has huge social and economic benefit.But the application still has
Functioning efficiency it is low, it is still necessary to continue improve could large-scale promotion use.Especially the invention is since announcement, and there is no quilts
It promotes the use of, this also absolutely proves that this method remains certain defect.
Moreover, the materials of bone marrow interstital stem cell are constant, and it is also big to human injury, it is unfavorable for conventional treatment.
In addition, early period the study found that inflammation locally and systemically in the generation of systemic sclerosis, development and concurrent
It plays an important role in the generation of disease.Leucocyte across endothelial migration to vascular wall gap be tissue damage and inflammatory reaction must
Step is wanted, leucocyte and sticking for blood vessel endothelium are this most important rings in the process, and adhesion molecule then mediates this overall process.
It was found that the morbidity of systemic sclerosis and the inflammatory reaction that adhesion molecule mediates are closely related.Especially study the peptide for inhibiting of discovery
It can be significantly reduced the expression of neutrophil leucocyte CD11b/CD18, and then inhibit the generation of systemic sclerosis, promote dry
The synergistic therapeutic effect of cell.
Summary of the invention
The object of the present invention is to provide application of the peptide for inhibiting in preparation treatment system hardening disease drug.
The object of the present invention is to provide peptide for inhibiting with synergy and epidermis interstital stem cell in preparation treatment system
Property hardening disease drug in application.
Further, the systemic sclerosis is diffusivity chorionitis.
Further, the peptide for inhibiting is peptide for inhibiting shown in SEQ ID NO:1.
The epidermis interstital stem cell is prepared in the following manner:
Foreskin or other skin histologies are taken, epidermis is obtained with II type tissue enzymic digestion, epidermis is shredded, then use trypsase
Jin mono- Walk digestion, obtains epidermal cell.
The human epidermal cell is cultivated in above-mentioned cell culture medium, preferably condition of culture are as follows: 36 native 2 DEG C, 5 ± l%
CO2,90-100% humidity, every 2-3 days replacement cell culture mediums.
Wherein when cell grows to 60-90% degrees of fusion, the trypsase and quality for being 0.25% with mass fraction divide
Number mixes for 0.02% EDTA solution according to volume ratio 1:1 and the digestive juice of formation digests, and dispels the people's table for not digesting
Chrotoplast collects the mescenchymal stem cell like cell digested, that is, is people's epidermal derived mesenchymal stem cell-like pluripotent cells.
The method of people's epidermal derived mesenchymal stem cell-like pluripotent cells routine is prepared into Chromosome spreads, carries out caryogram
Check: caryogram is 46 chromosomes of normal male, and banding pattern is normal.
People's epidermis source property mescenchymal stem cell sample multipotency of the invention is detected using flow cytometry and immunohistochemistry technique
(detection method is known skill wood to the surface marker of cell strain, and commercial reagents company can provide kit, provide according to Reagent Company
Technical solution implement), express mescenchymal stem cell specificity marker: CD73, CD90, CD105, Vimentin and
Nestin, positive cell are in brown or brownish black.
The present invention proposes epidermis MSCs and peptide for inhibiting answering in the drug of preparation treatment system hardening disease together
With.There are unique immunological regulation and repair using epidermis MSCs, patient can be helped to reduce the type and quantity of drug therapy
And the various side effects of drug bring, peptide for inhibiting can cooperate with the therapeutic effect of promotion system sclerosis, the two preparation side
Just, low in cost.
Specific embodiment
1 epidermis interstital stem cell in-vitro separation of embodiment, culture
1. preparing cell culture medium:
Glucose content be 5g/L DMEM culture medium in add fetal calf serum, hbFGF, hSCF, nonessential amino acid,
L-Glutamine, gentamicin, and keep the final concentration of above-mentioned various adding ingredients as follows: fetal calf serum: volume fraction
25%, 40ng hbFGF/mL, 20ng hSCF/mL, 2mL 100X nonessential amino acid/100mL, 0.1mL are containing mass fraction
The PBS solution of 3%L- glutamine/1OOmL and 8000U gentamicin/1OOmL.The cell training of the present embodiment is formed therefrom
Support base.The nonessential amino acid of the 100X is purchased from GIBCO company, article No. 11140.
2. obtaining epidermal tissue and epidermal cell:
Clinical discarded prepuce tissues are taken, are immersed in gentamicin containing 1000U/mL PBS solution, 4 DEG C of preservations;4 is small
When interior be handled as follows: sufficiently washed with PBS, dispel haemocyte, cut off sub-surface porosity connective tissue, with II type histaminase
(2U/mL) impregnates digestion overnight, obtains epidermis, then shreds epidermis, and the trypsase for being 0.25% with mass fraction is into one
Walk digests 30 minutes, obtains epidermal cell, is washed with PBS, be then centrifuged for dispelling digestive ferment, with the cell culture medium of the present embodiment
Again suspension cell.
3. cultivating epidermal cell:
The epidermal cell that each sample obtains is inoculated in the T25 culture bottle 3-4 of the cell culture medium containing the present embodiment
It in a, is cultivated in 36 DEG C, 5%C02,90-100% humidified incubator, every 2-3 days replacement cell culture mediums.
4. screening and enrichment people's epidermal derived mesenchymal stem cell-like pluripotent cells:
When cell is grown toDegrees of fusion when, with mass fraction be 0.25% trypsase add mass fraction
Digestive juice for 0.02% EDTA (1:1 is mixed by volume) composition carries out stage digestion;Collect the mesenchyma digested
Stem cell-like cell dispels the human epidermal cell for not digesting;The mescenchymal stem cell like cell being collected into is placed in centrifugation
In pipe,Digestive juice is dispelled in 5min centrifugation;Then with the cell culture medium of the present embodiment again suspension cell,
The cell is people's epidermal derived mesenchymal stem cell-like pluripotent cells of the invention, which is counted.
5. vitro culture of human epidermal derived mesenchymal stem cell-like pluripotent cells strain:
The people's epidermal derived mesenchymal stem cell-like pluripotent cells counted are inoculated in the density of IO5/mL of about 1X
In the T25 culture bottle of cell culture medium containing the present embodiment, in 36 ± 1 DEG C, 5 ± 1%C02、Humidified incubator
Inside cultivated, everyIt, primary with the passage of 1:3 ratio, until 30 is more than generation, and it is dry to obtain people's epidermis source property mesenchyma
Cell sample pluripotent cell strain.
Detection discovery is carried out to people's epidermal derived mesenchymal stem cell-like pluripotent cells strain of the present embodiment, cell strain tool
There is the table shape of mescenchymal stem cell sample, caryogram is normal, express the specificity marker of mescenchymal stem cell and neural precursor:
CD73, CD90, CD105, Vimentin and Nestin.
In addition, with every mouse about 1X 107It is a that cell is injected intraperitoneally and is subcutaneously injected in SCID mice body 3 months
It is generated afterwards without discovery tumour, it is good to be indicated above the present inventor's epidermal derived mesenchymal stem cell-like pluripotent cells biological safety
It is good.
6, appearance and microscopic observation
Color observation is carried out to the supernatant in culture bottle on the day of infusion experiment, no bacterium, fungus growth.
7, live cell fraction
The infusion same day accounts for 95% or more to the cell suspension sampling check live cell fraction of collection.
8, karyotype
Skin MSCs is transfused sampling row karyotype inspection in first 3 days: normal chromosomal caryogram.
The medication of 2 skin MSCs+ peptide for inhibiting of embodiment
1. device therefor, material, reagent
Superclean bench centrifuge disposable syringe injection physiological saline human serum albumin
2. operation before infusion
When total number of cells amount reaches (1-2) X IO6A/kg weight, it is contemplated that infusion prepares:
(1) sampling leaves and takes that cell culture supernatant makees bacterium, fungal culture, detection of mycoplasma and endotoxin are surveyed before infusion
It is fixed;
3 infusion operations
(1) pancreatin digests all cells (operation is the same), is centrifuged (room temperature lOOOrpm, 5min), abandons supernatant, repeatedly sufficiently
Washing;The physiological saline 1OOml for containing 5% human serum albumin is added, only remains insufficient cell O.5% in the cell suspension of infusion
Culture solution.O.1ml cell suspension is extracted to count.It indicates patient's name, prepares infusion.
(2) half an hour gives patient's vein methylprednisolone 40mg before cell infusion.
(3) open hand back vein inputs a small amount of physiological saline with blood transfusion apparatus, connects the separation bag that cell is housed after unobstructed,
Infusion velocity is adjusted, observes patient's reaction, timely anti symptom treatment in infusion process.Whole process time-consuming about 15 minutes.
4. case inclusion criteria: 1) be diagnosed as systemic sclerosis, 2) signature informed consent form agrees to stem-cell therapy.Row
Except standard: 1) patient is there are serious infection, and 2) the disease terminal phase, there is important organ function and has been badly damaged.
Transplantation Project: the input of skin MSCs vein, cell number are 1X 106A/kg weight.Peptide for inhibiting 100ng/kg weight
It inputs simultaneously.Input in 20 days is primary, totally 2 courses for the treatment of.
It is chosen in December, -2017 in 2016 systemic sclerosis patient 90, wherein male 56, female 34, average year
Year in age (45.8 scholar 2.4), average course of disease (3.2 scholar 1.1) year.All patients meet American Rheumatism Association's examining about SSc
Disconnected standard.According to the parting standard of the disease, hardening period, oedema phase and one hardening period of oedema, there is 28,50 and 12 respectively;Acra type
86, diffuse 4, type.
5. observation item and curative effect determinate standard
To every patient before the treatment, after skin tightly firmly change carry out self-assessment and doctor scoring.Self-assessment is adopted
Simulation ruler method is regarded with 10cm, the above are effective such as affected area skin-bound and hair fold sense disappear substantially or mitigate 75%;Subtract
Light 30% one 75% be effective;Mitigate 30% or less to be considered as in vain.Total effective rate is calculated according to effective and effective number of cases.Simultaneously by referring to
Fixed doctor according to chorionitis skin, tightly score by hard scoring criteria, the total score of more pretherapy and post-treatment affected area skin.
The Liver and kidney function of patient, blood, routine urinalysis and serum electrolyte are checked before and after treatment in EDTA, while recording adverse drug reaction.
Tightly hard scoring criteria is shown in Table 1 to SSc skin.
1 skin of table tightly hard scoring criteria
Criterion: it is normal for can clamping the skin pinched with thumb, is otherwise exception;Middle score=upper and lower extremities score
The score of (abnormal skin proximally being checked from finger (toe) end, score by the boundary of arrival) plus neck surface.
After 1 month, corresponding system curative effect is assessed.The results are shown in Table 2:
The curative effect of 2 stem-cell therapy SSc of table
Wherein effectively: including effective and effective number of cases;Occurs pruitus in b.Erythematous rash, treatment stop.
It can be seen from the results above that using the cell plus peptide for inhibiting treatment acra type case-hardening disease on have compared with
Good curative effect, and it is bad for type therapeutic effect is diffused.In addition, in course of disease treatment, either hardening period, oedema phase or water
Swollen-hardening period, all has preferable therapeutic effect.
In order to detect the therapeutic effect of peptide for inhibiting.Method is the same as in preceding, selection acra type patient 10, individually injection inhibition
Peptide, condition are same as above.As a result such as the following table 3:
In order to detect the individual therapeutic effect of stem cell.Method is the same as in preceding, selection acra type patient 10, individually injection
Stem cell, condition are same as above.As a result such as the following table 4:
As can be seen from tables 3 and 4 that exclusive use peptide for inhibiting or exclusive use stem cell cannot be significant in curative effect
Both achieve the effect that together.
In addition, passing through the flow cytometer measurement blood samples of patients effectively treated neutrophil leucocyte before and after treatment
The expression of CD11b/CD18, as a result (x as shown in table 5 below-± s, wherein p < 0.05):
Group |
CD11b/CD18 |
Normal person (20) |
240±51 |
Only inject the patient (10) of peptide for inhibiting |
282±48 |
The patient for only injecting peptide for inhibiting, before not injecting (10) |
954±84 |
Only inject the patient (10) of stem cell |
468±39 |
The patient for only injecting stem cell, before not injecting (10) |
896±70 |
The patient that the two is injected treats effectively (64) |
921±66 |
The patient (treatment is effective) that the two is injected, before not injecting ((64) |
256±25 |
It can be seen from the results above that peptide for inhibiting provided by the invention can preferably inhibit the expression of CD11b/CD18,
And then inhibit the generation of sclerosis, while promoting stem-cell therapy effect.
Sequence table
<110>Luoyang Xuan Zhi Biotechnology Co., Ltd
<120>skin mesenchymal stem cells treatment system sclerosis is used
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<170> SIPOSequenceListing 1.0
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<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
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Cys Trp Trp Ala Ala Arg Leu Val Ala Ile Cys Phe Glu Trp Gln Arg
1 5 10 15
Trp Ser His Arg Phe Gln Arg Ser Trp Arg Leu
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