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 technology
Systemic sclerosis (SSc) is also referred to as chorionitis, be 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 from being involved, and the purpose in late period 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, apocleisis, 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 the skin disease for systemic sclerosis
Become.(4) other:In recent years, there is document report a variety of new using relaxain, Imatinib, CD20 monoclonal antibodies, TGF-β antibody etc.
Therapy 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:In recent years, it is obtained both at home and abroad using the peripheral stem cell transplantation treatment through CD34 cell sortings
Certain effect, but somewhat expensive, transplanting adverse reaction risk is higher, only recommends to be used for intractable patient.Domestic Sun Lingyun religions
Being beheaded first uses mescenchymal stem cell treatment system sclerosis to obtain preferable effect early period, it is confirmed up for more studying
Curative effect.
Bone marrow interstital stem cell is disclosed in CN101485685A is preparing answering in treating autoimmune disease drug
With.Active constituent of the bone marrow MSCs as treatment autoimmune disease drug, has unique immunological regulation and repaiies 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 various side effects that the type and quantity and medicine band for the treatment of are come, 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
The application prepared in treatment autoimmune disease drug has huge social and economic benefit.But the application still has
It is low functioning efficiency, it is still necessary to which continuing, which improves ability large-scale promotion, uses.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
The expression of neutrophil leucocyte CD11b/CD18, and then the generation of suppression system sclerosis can be significantly reduced, is promoted dry
The synergistic therapeutic effect of cell.
Invention content
The object of the present invention is to provide application of the peptide for inhibiting in preparing treatment system hardening disease drug.
The object of the present invention is to provide peptide for inhibiting with synergy and epidermis interstital stem cell to prepare treatment system
Property hardening disease drug in application.
Further, the systemic sclerosis is diffusivity chorionitis.
Further, the peptide for inhibiting is SEQ ID NO:Peptide for inhibiting shown in 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 digestions, 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, and preferably condition of culture is:36 soil 2 DEG C, 5 ± l%
CO2,90-100% humidity replaced cell culture medium every 2-3 days.
Wherein when cell growth to 60-90% degrees of fusion, the trypsase and the quality that are 0.25% with mass fraction are divided
The EDTA solution that number is 0.02% is according to volume ratio 1:1 digestive juice for mixing and being formed 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
It checks:Caryogram is 46 chromosomes of normal male, and banding pattern is normal.
People's epidermis source property mescenchymal stem cell sample multipotency of the present 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, be provided 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 for preparing 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 that medicine band is come, peptide for inhibiting can cooperate with the therapeutic effect of promotion system sclerosis, the two preparation side
Just, of low cost.
Specific implementation mode
1 epidermis interstital stem cell in-vitro separation of embodiment, culture
1. preparing cell culture medium:
Glucose content be 5g/L DMEM culture mediums in addition 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 is
25%, 40ng hbFGF/mL, 20ng hSCF/mL, 2mL 100X nonessential amino acids/100mL, 0.1mL are containing mass fraction
The PBS solution of 3%L- glutamine/1OOmL and 8000U gentamicins/1OOmL.The cell training of the present embodiment is formed therefrom
Support base.The nonessential amino acid of the 100X is purchased from GIBCO companies, article No. 11140.
2. obtaining epidermal tissue and epidermal cell:
Clinical discarded prepuce tissues are taken, are immersed in the PBS solution of gentamicin containing 1000U/mL, 4 DEG C of preservations;4 is small
When interior be handled as follows:It is fully washed with PBS, dispels haemocyte, cut off sub-surface porosity connective tissue, with II type histaminases
(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 bottles 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, cell culture medium was replaced every 2-3 days.
4. screening and enrichment people's epidermal derived mesenchymal stem cell-like pluripotent cells:
When cell growth extremelyDegrees of fusion when, with mass fraction be 0.25% trypsase add mass fraction
For 0.02% EDTA (by volume 1:1 mixing) composition digestive juice carry 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 centrifugations;Then the cell culture medium suspension cell again of the present embodiment is used,
The cell is people's epidermal derived mesenchymal stem cell-like pluripotent cells of the present 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 bottles of cell culture medium containing the present embodiment, in 36 ± 1 DEG C, 5 ± 1%C02、Humidified incubator
Inside cultivated, everyIt, with 1:The passage of 3 ratios is primary, until 30 is more than generation, and it is dry to obtain people's epidermis source property mesenchyma
Cell sample pluripotent cell strain.
Discovery is detected 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, expresses 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 by cell through being injected intraperitoneally and being subcutaneously injected in SCID mice body 3 months
Afterwards without finding that tumour generates, 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), centrifuges (room temperature lOOOrpm, 5min), abandons supernatant, repeatedly fully
Washing;The physiological saline 1OOml containing 5% human serum albumin is added, insufficient cell O.5% is only remained 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, the separation bag equipped with cell is connected after unobstructed,
Infusion velocity is adjusted, patient's reaction, timely anti symptom treatment are observed in infusion process.Whole process takes 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) there are serious infection by patient, in 2) Ji Bing Final latter stages, important organ function have occurred and have been badly damaged.
Transplantation Project:Skin MSCs veins input, and cell number is 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 chooses in the December, -2017 in 2016 of systemic sclerosis patient 90, wherein man 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, it 75% or more is effective to disappear or mitigate substantially such as affected area skin-bound and hair fold sense;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 skins.
1 skin of table tightly hard scoring criteria
Criterion:It is normal that can clamp 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, by the boundary score reached) plus neck surface.
After 1 month, corresponding system curative effect is assessed.The results are shown in Table 2:
The 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:
From table 3 and table 4 as can be seen that exclusive use peptide for inhibiting or exclusive use stem cell cannot be notable in curative effect
Both achieve the effect that together.
In addition, measuring the blood samples of patients effectively treated neutrophil leucocyte before and after treatment by flow cytometer
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
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<120>Use skin mesenchymal stem cells treatment system sclerosis
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