CN105087477A - Application of mesenchymal stem cell modified by miR-21 antisense nucleotide - Google Patents

Abstract

The research group selects the optimum method for preparing a product of the invention, namely, enabling 100nM miR-21inhibitor transfected by lipo 2000 RNAimax to enter the stem cell. The stem cell product prepared by the method is enhanced in the capacity of secreting transforming growth factor TPF-beta 1, thus the capacity of inducing the T cell to differentiate towards regulatory T cells after co-culture of the cell and the T cell is enhanced, namely, the immunoregulation capacity of the stem cell is enhanced. In an in-vivo experiment, a medicine is adopted for inducing a mouse for establishing an acute enteritis model, the stem cell and a control cell which are transfected by miR-21 inhibitor are transfused into the body of the mouse through caudal vein by using the dosage of 1X10<6>, the result shows that the clinical symptoms and the immune indices of the mouse with stem cell group transfected by miR-21 inhibitor transfused are superior to those of the control cell group with transfusion, namely, the stem cell transfected by miR-21 inhibitor can be used for more effectively treating mouse enteritis. All in all, the stem cell with strong and stable effect is prepared, and experimental basis is provided for improving the application efficiency of the stem cell in clinic.

Description

The purposes of the mesenchymal stem cells MSCs that miR-21 antisense nucleotide is modified

Technical field

The present invention relates to adult stem cell field, by specific antisense nucleotide sequences silences mescenchymal stem cell (mesenchymalstemcell, MSCs) in, the expression level of endogenous miR-21 enhances the immunoregulation capability of MSCs, and the MSCs purposes after research modification.

Background technology

In traditional concept, the MSCs such as mesenchymal stem cells MSCs is proved to be after being separated first and can be divided into the different tissues cells such as bone, cartilage, fat, pancreas islet, liver and nerve under different induced environment, treat the most frequently used seed cell of skeletal diseases at present, but along with the prolongation of Time in Vitro, it is transplanted osteogenic ability after in ex vivo and obviously declines, and its Multidirectional Differentiation ability is also lost gradually.In addition research shows that mesenchymal stem cells MSCs can by suppressing panimmunity cell as dendritic cell, and T lymphocyte, the function of bone-marrow-derived lymphocyte, plays important immunoregulation effect.MSCs is applied to whole body clinically, treats various systemic disease, and as diabetes, rheumatoid arthritis, sjogren syndrome etc., play an important role in immunity of organism regulation and control.But still need and will develop the new way of infused cells, at present by the method for venoclysis cell.Its problems faced implants efficiency heterogeneity exactly, can not produce obvious and lasting therapeutic action to some case.In addition, along with the time lengthening of vitro culture, MSCs also can lose the biologic activity of part, reduces application efficiency.Increasing seminar all comes to realise again to find and builds a kind of importance improving the implantation efficiency of MSCs, makes it produce more obvious and lasting therapeutic action.

Microrna s (microRNAs, miRNAs) is small molecules, the noncoding single stranded RNA of level modulation after a class participation genetic transcription, and length 19 ~ 25nt, has lapsed to substantial connection with the growth of cell, can regulating cell propagation, differentiation etc.MiRNAs mediates specific gene silencing or activates said target mrna and affect protein synthesis, the genetic expression of level after regulatory transcription, has now proved that miRNAs can regulate and control multiple physiology and pathological process [8-10].MiRNAs has the biological characteristics [11] such as organic evolution conservative property, tissue specificity, gene cluster collection phenomenon, Space-time speciality of height.MiRNAs is by RNA interference channel degraded said target mrna; In animal, miRNAs is combined with 3 '-untranslated region partial sequence reverse complemental of said target mrna, with RNA disturb unlike, the stability of said target mrna is unaffected, and suppressed after translation initiation.Therefore, the expression level of miRNA in cell can be changed by the complementary specificity antisense strand of miRNAs, increase the post-transcriptional level of target gene thus the propagation of change cell, differentiation capability.

At present by using the expression level of endogenous miRNA in the special silenced cell of the mode of liposome transfection in cell.The genetic modification mode that this kind changes miRNA expression has compared with traditional genetic modification: do not need virus vector, solves the problem of virus uncontrollability in vivo; Transfection efficiency is high; The feature such as easy to operate.By using the specific antisense chain of liposome transfection miRNA can the endogenous expression of miRNA in silenced cell efficiently in cell.Find in the research of this seminar, in the expression level negative regulation MSCs of miR-21, change the expression level of growth factor-beta 1 (transforminggrowthfactor-β 1, TGF-β 1).TGF-β 1 as a kind of immunosuppressor can Immunosuppression active cells propagation and suppress lymphopoiesis, thus affect the immunoregulation capability of MSCs.Therefore content of the present invention is the ability utilizing miR-21 antisense strand transfection MSCs to strengthen emiocytosis TGF-β 1, and then the immunoregulation capability of strengthen continuously MSCs.Again the MSCs of miRNA antisense strand modified is applied in stem-cell therapy DeGrain or invalid systemic disease afterwards, improves stem cell within working lipe for the result for the treatment of of systemic disease.

Summary of the invention

Goal of the invention

1) propose a kind of easy, with low cost, do not need virus vector and stem cell modifying method to human non-toxic's side effect;

2) preparation method of the stem cell after above-mentioned modification is proposed;

3) optimization method strengthening stem cell immunoregulation capability characteristic through above-mentioned modification is proposed;

4) propose modified after the clinical disease that may treat of stem cell.

Invention thinking

Anti-sense-miRNA, as the complete complementary chain of corresponding miRNA, is all the small RNA moleculars through 2 '-0-methyl stability chemically modified.By interacting with corresponding miRNA specificity, the degraded of this miRNA can be promoted, thus realize the suppression to specific miRNA function, block the effect of miRNA in cell.Find that miR-21 can the immunoregulation capability of negative regulation MSCs by early-stage Study.Therefore the antisense strand miR-21inhibitor transfection of miR-21 is entered cell for the mode by liposome transfection by this patent content, the expression level of miR-21 in specificity silenced cell, blocks miR-21 downstream passages in cell and improves the immunoregulation capability of MSCs.Therefore will be how an important consideration content by the mode of this nucleotide sequence transfered cell.The concentration importing miR-21inhibitor in addition is also need careful selection, just can play best effect under best concentration, finally finds the preparation method of stem cell and the indication of application after modifying to be all the contents of patent of the present invention.

Beneficial effect of the present invention

1. the stem cell methods prepared by the present invention is easy, quick, product is harmless.

Microscopic observation finds, MSCs prepared by the present invention maintains basic cellular form, maintain Cell tracking, and save the support that extracellular matrix is connected to form, do not destroy the function transfection miR-21inhibitor such as the adhesion proliferation and growth of cell surface protein not affect the proliferation and apoptosis of stem cell, illustrate that transfection miR-21inhibitor does not have cytotoxicity to MSCs.

2. the stem cell products biology performance prepared by the present invention is stablized, and by screening the optimization of its performance in vitro, clear and definite prepared product has more significant immunological competence

Detect from gene level and find, people MSCs in week age, mouse BMMSCs equal Absorbable organic halogens ground maintain the expression effect of reticent miR-21 after transfection, and sustainablely increase substantially the secretion level of cell to TGF-β 1; Simultaneously by promoting that T cell breaks up ratio to regulatory T cells (regulatoryTcells, Tregcells) with T cell Dual culture.Namely the stem cell products prepared by the present invention enhances the immunoregulation capability of stem cell.

3. stem cell products involved in the present invention under inflammatory environment, has more lasting and obvious effect to the systemic immune such as autoimmune disease and immunologic derangement disease compared with the cell do not modified in Mice Body.

After Mice Body innerlich anwenden thing induction enteritis, BMMSCs after being modified by tail vein input miR-21inhibitor, find that the result for the treatment of of input transfection miR-21inhibitorBMMSCs group is obviously better than transfection control group and non-transfection group, and improve detection of plasma index of correlation, recover immunity in body.Namely the stem cell products prepared by the present invention has more obvious result for the treatment of in vivo.

Embodiment part is shown in specific experiment data and explanation.

Accompanying drawing explanation

1. Fig. 1 is embodiment 1: used stem cell biology CHARACTERISTICS IDENTIFICATION in experiment;

2. Fig. 2 is embodiment 2: miR-21 transfection reagent and transfection concentrations screening in cell;

3. Fig. 3 is the method that miR-21inhibitor-MSCs is prepared in the reticent best transfection concentrations screening of embodiment 3:miR-21;

4. Fig. 4 is embodiment 4: transfection miR-21inhibitor does not affect the proliferation and apoptosis of mouse BMMSCs;

5. Fig. 5 is the expression level of TGF-β 1 in cell in embodiment 5:miR-21 negative regulation;

6. Fig. 6 is the immunoregulation capability of embodiment 6:miR-21 negative regulation BMMSCs;

7. Fig. 7 be embodiment 7:TGF-beta 1 antibodies can in and miR-21 regulate and control BMMSCs immunoregulation capability effect;

8. Fig. 8 is the mouse experiment enteritis that the reticent BMMSCs of embodiment 8:miR-21 more effectively can treat DSS induction;

9. Fig. 9 is that the reticent BMMSCs of embodiment 9:miR-21 raises enteritis mice serum TGF-β 1 and lowers IL-17 level.

Embodiment

Cell cultures

Experiment material Healthy People marrow comes from wound or orthognathic surgery needs fibular autograft patient, Healthy People periodontal tissue comes from because correction needs FFI premolar teeth or third molar, and Healthy People deciduous teeth pulp tissue comes from the clinical deciduous teeth needing to pull out because of delay.

1. the cultivation of people's Odontogenic cysts MSCs

Type I collagen is cultivated

The fresh tooth pulled out is put into immediately and aseptic PBS and antibiotic centrifuge tube is housed, separation and Culture dental pulp stem cell in 12 hours.With sterile gauze wraps around tooth sclerous tissues, and knock tooth body open obtain pulp tissue with alcohol-pickled hammer of sterilizing, repeatedly clean with PBS, shred as far as possible, put containing in 3mg/mlI Collagenase Type and 4mg/mlDispase solution, 37 DEG C of water-bath digestion 0.5-1 hour, cross 70 μm of cell sieve collecting cells, the centrifugal 10min of 1000rpm, with the resuspended one-tenth single cell suspension of appropriate substratum.Adjustment cell concn to 1 × 10 ²/ ml, is inoculated in 96 orifice plates by cell, in 37 DEG C, 5%CO in α-MEM substratum (containing 20% foetal calf serum, 2mmol/L glutamine, 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates) ₂cultivate, every 3-5 days changes liquid once.Every day is observation of cell upgrowth situation under inverted microscope.After 1-2 week, the cell getting Clonal growth goes down to posterity with 0.25% tryptic digestion.

2. the cultivation of the non-Odontogenic cysts MSCs of people

Human marrow mesenchymal stem cell is cultivated

Healthy People marrow comes from wound or orthognathic surgery needs fibular autograft patient.The fresh tooth pulled out is put into immediately and aseptic PBS and antibiotic centrifuge tube is housed, separation and Culture periodontal ligament stem cell in 12 hours.Peel off 1/3 periodontium tissue in the middle part of root of the tooth gently, repeatedly clean with PBS, shred as far as possible, be placed in containing 3mg/mLI Collagenase Type and 4mg/mLDispase solution, 37 DEG C of water-bath digestion 0.5-1 hour, cross 70 μm of cell sieve collecting cells, the centrifugal 10min of 1000rpm, with the resuspended one-tenth single cell suspension of appropriate substratum.Adjustment cell concn to 1 × 10 ²/ mL, is inoculated in 96 orifice plates by cell, in 37 DEG C, 5%CO in α-MEM substratum (containing 20% foetal calf serum, 2mmol/L glutamine, 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates) ₂cultivate, every 3-5 days changes liquid once.Every day is observation of cell upgrowth situation under inverted microscope.After 1-2 week, the cell getting Clonal growth goes down to posterity with 0.25% tryptic digestion.

3. the cultivation of mouse BMMSCs

The all female C57/BL6 mouse of 6-8, the skin of hind leg is peelled off after de-neck is lethal, draw α-MEM perfect medium (containing bovine serum 10%) with 1mL syringe after exposing femur and shin bone and go out marrow from the broken ends of fractured bone, the marrow of every mouse is cultivated in the culture dish of four ware 10cm.10 days later half changes liquid, can go down to posterity after 15 days; Treat that cell clone grows to 80% fusion and can go down to posterity, Secondary Culture is to the third generation;

Stem cell is identified

1. flow cytomery surface marker

1) 20 minutes are fixed under the centrifugal rear use 4% paraformaldehyde room temperature of cell dissociation;

2) wash once with PBS, then use PBS suspendible cell, adjustment cell concn is 10 ⁶individual cell/100 μ L

3) 100 μ L cell suspensions are respectively got, add anti-Stro-1 antibody (1: 10), CD146 antibody (1.0 μ g), CD90 antibody (2 μ g) respectively, SSEA-4 antibody (10 μ L), at 4 DEG C, lucifuge hatches 1 hour; Rupture of membranes process should be done before dyeing;

4) 1000 revs/min centrifugal 5 minutes, wash once with PBS;

5) abandon supernatant, add 100 μ LPBS suspendible cells, Stro-1 antibody staining adds the anti-mouse IgM antibody (1: 200) of FITC mark again, room temperature lucifuge 30 minutes;

6) flow cytometer loading, detects the expression of associated molecule.

2.CFSE mixes and Flow cytometry cell proliferation experiment

1) after cell dissociation, with 0.1%BSA re-suspended cell, adjustment cell concn is 1 × 10 ⁶/ ml;

2) add 2 μ L5mMCFSE, make CFSE final concentration be 10 μMs;

3) hatch 10 minutes for 37 DEG C, hatch 5 minutes on ice;

4) centrifugal 10 minutes of 1500rpm, removes supernatant, after washing 3 times, puts into 5cm culture dish with DMEM substratum, 37 DEG C, 5%CO2 cultivation;

5) after 3 days, peptic cell, centrifugal 10 minutes of 1500rpm, 500 μ L0.1%BSA are resuspended;

6) flow cytomery (488 passage), uses Modfit software analysis proliferation index.

3. Osteoinductive differentiation

Be mixed with self-bone grafting nutrient solution: add 2mmol/L glutamine containing in 10% foetal calf serum α-MEM substratum, 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates, 10mM sodium β-glycerophosphate, 10nM dexamethasone, 50mg/L vitamins C.3-4 for cell with 2 × 10 ³/ cm ²concentration be inoculated in 6 orifice plates, wait Growth of Cells to 80% merge after, be replaced by osteogenic induction nutrient solution, within every 2 days, change liquid 1 time, light Microscopic observation calcium tubercle formational situation.In induction after 2 weeks, carry out Alizarin red staining and extract total serum IgE carrying out real-timePCR experiment.

4. Alizarin red staining

1) remove substratum, PBS washes 2 times;

2) 70% ethanol is fixed, 4 DEG C, 1h;

3) distilled water washes 2 times;

4) 40mM alizarin red aqueous solution (pH4.2) room temperature dyeing 1-10 minute, visual inspection coloring case;

5) distilled water washes 5 times, blows and beats gently;

6) Microscopic observation gather image;

7) get 15 visuals field at random, use ImageProPlus6.0 computed in software Alizarin red staining area.

5. adipogenic induction differentiation

Be mixed with fat induction broth: add 2mmol/L glutamine containing in 10% foetal calf serum α-MEM substratum, 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates, 1 μM of dexamethasone, 0.5mMIBMX, 10mg/L Regular Insulin, 10mM indomethacin.

6. oil red dyeing

1) oil red storage liquid: 150mg oil red O is dissolved in 50ml Virahol, 37 DEG C, 30 minutes; 4 DEG C of storages;

2) oil red working fluid: dilute storage liquid with distilled water, ratio is water: storage liquid=4: 6 filtrations make solution be that limpid orange can use;

3) formaldehyde calcipexy liquid: 40% formaldehyde 10ml, CaCl ₂2.0g, ultrapure water 90ml;

4) after cell induction, remove substratum, wash 2 times with PBS;

5) formaldehyde-calcium room temperature fixes 15 minutes;

6) PBS washes 2 times;

7) 60% Virahol mordant dyeing 1 minute;

8) remove Virahol, oil red working fluid room temperature dyes 30 minutes;

9) 60% Virahol embathes once;

10) PBS washes 2 times;

11) Microscopic observation, triglyceride fat drips dyes redness;

12) get 15 visuals field at random, counting positive cell number accounts for total cell count ratio.

7.RealtimePCR

Primer sequence

1)Actin

Forward：5’-TGACGTGGACATCCGCAAAG-3’，

Reverse：5’-TGGAAGGTGGACAGCGAGG-3’

2)ALP

Forward：5’-GCCTCAGCCTTCCATCTGTAAA-3’，

Reverse：5’-GTCCCCACGGTTGTTAATACCA-3’

3)pparγ

Forward：5′-CTCAGACAGATTGTCACGGAACAC-3′，

Reverse：5′-AGTGCAACTGGAAGAAGGGAAAT-3′

8. experimental procedure

RNA extracts

A) sample preparation: cell 1mlTrizol lysate cracking;

B) homogenised sample concuss is mixed, placement 10 minutes, nucleic acid-protein mixture is separated completely on ice;

C) centrifugal 5 minutes of 4 DEG C of 12000rpm, get supernatant, proceed to one new in the centrifuge tube of RNase;

D) every 1ml sample adds 0.2ml chloroform, builds pipe lid, thermal agitation more than 15 seconds, is positioned over 3 minutes on ice;

E) the superiors are transferred in new pipe by centrifugal 10 minutes of 4 DEG C of 12000rpm;

F) slowly add 1 times of volume isopropanol, put upside down mixing, hatch 10 minutes on ice, RNA is precipitated;

G) centrifugal 10 minutes of 4 DEG C of 12000rpm, discard supernatant;

H) add 1ml75% ethanol, put upside down mixing with washing precipitation, centrifugal 5 minutes of 4 DEG C of 12000rpm, discard waste liquid;

I) open wide lid mouth air drying 5-10 minute, 50 μ L ultrapure water dissolution precipitations, hatching at 56 DEG C 10 minutes can hydrotropy.

L) RNAOD value is detected

Calculate RNA concentration and OD260/280 ratio, be normal usually between 1.6-2.0

Reverse transcription PCR

A) RNA is made into 0.3 μ g/mL concentration;

B) prepare template ribonucleic acid/primer mixed solution in Microtube pipe, delivery plate RNA10 μ L, add each 1 μ L of Oligo-(dT), dNTP (mix);

C) 65 DEG C of insulations after 5 minutes rapidly at chilling more than 2 minutes on ice;

D) 4 μ L5 × PCR reaction buffers are added in sample, 2 μ L0.1MDTT, 1 μ LRNAseInhibit, centrifugal 10 seconds;

E) 37 DEG C of insulations are after 2 minutes, and room temperature adds 1 μ LM-MLV reversed transcriptive enzyme, centrifugal 10 seconds;

F) 37 DEG C are incubated 50 minutes, and 70 DEG C are incubated 15 minutes, 4 DEG C of coolings.

RealtimePCR

A) Real-timePCR reaction system is configured as follows:

B) Real-timePCR reaction system is as follows:

The expression level of miR-21 in silenced cell in above-mentioned stem cell

1. adopt lipo2000 and lipoRNAimax two kinds common transfection reagent different concns to be carried out to the screening of best rotaring transfecting mode and transfection conditions

1) specific miR-21inhibitor has been synthesized from company.Paving 1 × 10 ⁶in BMMSCs cell to 24 orifice plate of/hole, having 150 μ L substratum in every hole, is α-MEM perfect medium;

2) second day, the OPTI-MEM in 50 μ l/ holes mixed with 1 μ LLiopfectamin2000 or 1 μ LLiopfectamin2000RNAimax, hatches 5min;

3) by 2.5 μ L after optimizing, 5Ml, the small RNA molecular that 7.5 μ L synthesize mixes with the OPTI-MEM in other 50 μ/hole; (final concentration of miR-21inhibitor is 50 μMs respectively, 100 μMs, 150 μMs);

4) above-mentioned two kinds of mixed solutions mixing, room temperature lucifuge hatches 20min;

5) sop up original perfect medium, mixed solution is slowly added drop-wise in Tissue Culture Plate;

6) transfection is after 6 hours, sops up mixed solution, and change the complete culture solution of the antibiotic-free of preheating in advance, after three days, Trisol collects BMMSCs, detects the expression of miR-21 in cell;

Reverse transcription after extraction RNA, system is as follows

In order to experimental data is convenient to monitoring and standardization, each 384 orifice plates detected all are provided with two reverse transcription internal references (namely same RNA sample repeats reverse transcription and splice at every turn) and two amplification internal references (sample with a reverse transcription repeats splice at every turn).Reverse transcription program is 16 DEG C, 30min; 42 DEG C, 30min; 85 DEG C, 5min; 4 DEG C, ∞;

TaqmanReal-time method detects miRNA expression level: each miRNA to be measured arranges two multiple holes;

Application of sample system:

Put into 7900ABI quantitative real time PCR Instrument, reaction conditions is as follows:

50 DEG C, 2min; 95 DEG C, 10min; 95 DEG C, 15sec; 60 DEG C, 1min; Latter two stage is arranged to 40 circulations.The in vitro study of stem cell in vitro immunoregulation capability after modifying

The CD4 that mouse bone marrow cells MSCs cell and mouse spleen are originated ⁺t cell Dual culture

1) get Primary mouse bone marrow MSCs to cultivate, third generation BMMSCs is with 5 × 10 ⁵density be seeded to 6 orifice plates; RPMI-1640 perfect medium is cultivated;

2) second day, add containing 1 × 10 ⁶the RPMI-1640 substratum 2mL of mouse spleen sorting T cell, simultaneously according to every 1 × 10 ⁴individual cell adds 2 μ L mouse anti-CD3/CD28microbeads with activating T cell, adds IL-2020ng/mL simultaneously and promotes T cell survival, Dual culture four days.Experimentally to divide into groups requirement simultaneously, another with one piece of 6 orifice plate, the T cell of only single culture activation, stimulating group is not as negative control group for a part, and a part uses 10ng/mL mouse TGF-β 1 cytokine induction as positive controls;

3) collect liquid after 4 days, liquid is placed in the magnetic frame accompanying blank flow pipe, the anti-CD3/CD28microbeads in liquid can be attached at streaming tube wall because of magnetic-adsorption effect, is placed in new streaming pipe with clean pipette, extract all liquid;

4) centrifugal 5 minutes of 1500rpm, collects supernatant in clean EP pipe and is stored in-80 degree refrigerators or is used for subsequent detection immediately.Cell can be resuspended for subsequent detection.

Flow cytometry mouse regulatory T-cell ratio

1) Anti-CD4 (1.5 μ L/10 are added ⁶individual cell/mL), Anti-CD25 (1.5 μ L/10 ⁶individual cell/mL), hatch 30 minutes on ice, lucifuge;

2) 1500rpm is centrifugal, removes supernatant, and the damping fluid of configuration is resuspended; Centrifugal again, abandon supernatant;

3) add rupture of membranes liquid, 4 DEG C, the rupture of membranes time is 20 minutes;

4) 1500rpm is centrifugal, removes supernatant, and the damping fluid of configuration is resuspended;

5) Anti-Foxp3 (2 μ L/10 are added ⁶individual cell/mL), hatch 30 minutes on ice, lucifuge;

6) centrifugal, remove supernatant, 500 μ L0.1%BSA are resuspended;

7) flow cytomery.

Antibody neutralizes

In order to verify whether miR-21 carrys out mediated cell function by the secretion level of the soluble factor of regulating cell, at stem cell and CD4 ⁺add 50mg/mLTGF-β 1 or 50mg/mLIL-6 antibody in T cell Dual culture process, with in and the expression of TGF-β 1 and IL-6.

MiR-21inhibitor transfection BMMSCs treats mouse immune disease

1. the mouse experiment enteritis of transfection BMMSCs T 500 DSS induction

DSS inducing mouse colitis model:

1) choose the male C57/BL6 mouse of body weight about about 25g, be divided into two groups, normally feed water for one group, another group tap water configuration 3%DSS solution, feeds water, before feeding water, detects body weight, the overall health of patients of gross examination of skeletal muscle mouse;

2) feed the 3rd day of water, digest each group of BMMSCs, counting, then by tail vein injection in Mice Body, 1*10 ⁶/ only every, second group of DSS is drunk group and is grouped as follows: do not inject group, injection transfection controlBMMSCs group, injection transfection miR-21inhibitorBMMSCs group;

3) monitoring index is as follows:

A) weigh to mouse every day, record diarrhoea situation, finally using Mouse Weight change as record index;

B) and with stool blood detection paper ight soil every day bleeding: the Clinical course disease activity index (DAI) of mouse enteritis is assessed ⁴⁹.Calculate the scoring of following three indexs, specific as follows: (1) loses weight (0 point=unchanged, 1 point=1-5% weight loss, 2 points=5-10% weight loss, 3 points=10-15% weight loss, 4 points of > 15% weight losses); (2) situation of defecating (0 point=normal, 2 points=soft stool, 4 points=watery stool); (3) situation of having blood in stool (0 point=nothing is had blood in stool, 2 points=slightly have blood in stool, 4 points=have blood in stool in a large number).DAI is the summation of above 3 indexs, and from 0 point (without enteritis) to 12 points (serious enteritis) scoring is not etc.;

C) mouse was put to death at the tenth day of DSS induction.Before putting to death, eyeball gets blood, room temperature leave standstill 3h after the centrifugal whole blood 10min of 1500rpm, draw supernatant in clean EP pipe, detect the concentration of TGF-β 1 and IL-17 in serum by ELISA kit;

D) get spleen separating Morr. cell after putting to death immediately and analyze Treg, Th17 cell proportion;

E) same area colon is got, specimens paraffin embedding slices (5 μm) after paraformaldehyde 4 DEG C fixedly spends the night, dehydration, transparent, waxdip, embedding, histotome section, HE dyeing, mounting

2. statistical method

Adopt SPSS17.0 statistics software to carry out statistical analysis, many Sets of Measurement Datas compare employing ANOVA and analyze, and saliva flow rate uses replicate measurement methods analyst, and P < 0.05 has statistical significance.

Interpretation of result

1. can the expression of miR-21 in best silenced cell with 75nM transfection miR-2linhibitor with lipo2000RNAimax;

First to be separated and after identifying stem cell (Fig. 1), the best mode selecting lipo2000 and lipo2000RNAimax transfection reagent and different transfection concentrations to filter out transfection miR-21inhibitor is with lipo2000RNAimax transfection 100nM (Fig. 2, Fig. 3), and this rotaring transfecting mode and concentration can not cause stem cells hyperplasia and Apoptosis (Fig. 4).

2. the ability of transfection miR-21inhibitor sustainable enhancing emiocytosis TGF-β 1, strengthens the immunoregulation capability of BMMSCs;

In mouse BMMSCs and people BMMSCs, people DPSCs, transfection miR-21mimics or miR-21inhibitor realizes miR-21 in cell and, after process LAN and silence, finds the expression level negative correlation (Fig. 5) of TGF-β 1 in miR-21 and cell respectively; And because enhancing the ability of emiocytosis TGF-β 1 after reticent miR-21, and then promote the ability that T cell is broken up to regulatory T cells after enhancing cell and T cell Dual culture, i.e. the miR-21 also negative regulation immunoregulation capability of stem cell (Fig. 6); And this regulating effect can be eliminated by TGF-β 1 neutralizing antibody, illustrate that miR-21 has regulated and controled the immunoregulation capability (Fig. 7) of stem cell really by TGF-β 1.

3. the mouse enteritis effect of transfection miR-21inhibitorBMMSCs treatment DSS induction is better than control group

After setting up acute enteritis model with medicine mouse, by transfection miR-21inhibitorBMMSCs and compared with control cells 1*10 ⁶dosage by tail venous re-transfusion in Mice Body, find that clinical symptom and the immune indexes of feedback transfection miR-21inhibitorBMMSCs group mouse are all better than feeding back compared with control cells group (Fig. 8, Fig. 9), illustrate that miR-21inhibitorBMMSCs more effectively can treat the mouse enteritis of DSS induction.

The advantage of the inventive method and application prospect

Stem cell products prepared by the present invention not only has and more continues stronger immunoregulation capability, and in vivo under inflammatory conditions stable continuing play regulating power, treatment immune correlated disease.And preparation method simple to operation, nontoxic, can carry out on a large scale.

Expect that the human stem cell prepared by the present invention is applied to the DeGrain of stem-cell therapy or does not just have effect at all, improve stem-cell therapy efficiency, for the clinical application of stem cell strengthens practical technique.

After BMMSCs separation and Culture three generations, flow cytometry (A) STRO-1 is positive, and (B) CD146 is positive, and (C) CD90 is positive, and (D) CD-34 is negative.(E) cell raises through osteogenic induction hystazarin red colouring and (F) ALP expression level PCR quantitative expression, and cell has obvious Osteoblast Differentiation ability.(G) cell is through oil red dyeing and (H) PCR quantitative analysis PPAR γ expression level, and cell has significantly one-tenth fat differentiation capability (* P < 0.05).

With lipo2000 (Lipo2000) and lipoRNAimax (RNAimax) transfection 40nM or 75nMmiR-21mimics and contrast miRNAs (NC) in mouse BMMSCs, 50nM or 100nMmiR-21inhibitor (I-miR-21) and corresponding miRNAinhibitorcontrol (I-NC), detects the expression level of miRNAs in cell for four days afterwards.Find that lipoRNAimax can more effective transfection RNA (* P < 0.05).

50nM is marked with lipoRNAimax (RNAimax) transfection FAM in mouse BMMSCs, 100nMmiR-21inhibitor (I-miR-21) and corresponding miRNAinhibitorcontrol (I-NC), detects the expression level of miRNAs in cell for four days afterwards.(A-C) form after optical microphotograph Microscopic observation 50nM, 100nMI-miR-21 transfection; (D) fluorescence intensity after Flow cytometry 50nMFAM mark I-miR-21 transfectional cell; (E) fluorescence intensity after Flow cytometry 100nMFAM mark I-miR-21 transfectional cell; (F) miR-21 expression level after quantifying PCR method detection 50nMI-miR-21 transfectional cell; (G) miR-21 expression level (* P < 0.05) after quantifying PCR method detection 100nMI-miR-21 transfectional cell.

(A-B) in mouse BMMSCs, the apoptosis of stem cell after reticent miR-21, can not be caused, and (C) miR-21 endogenous expression levels be changed after also can not affect the multiplication capacity (* P < 0.05) of stem cell.

At mouse BMMSCs, people BMMSCs, with lipoRNAimax (RNAimax) transfection 50nM in people DPSCs, 75nMmiR-21mimics (miR-21), control (NC) or 50nM, 100nMmiR-21inhibitor (I-miR-21) miRNAinhibitorcontrol (I-NC), realizes the process LAN of miR-21 in cell and reticent effect respectively.After four days, ELISA method detects the level expressing TGF-β 1 in cell conditioned medium.(A) TGF-β 1 down-regulated expression after process LAN miR-21 in mouse BMMSCs; (B) TGF-β 1 up-regulated after reticent miR-21 in mouse BMMSCs; (C) in people BMMSCs, after process LAN and reticent miR-21, TGF-β 1 is in harmonious proportion down rise respectively; (D) in people BMMSCs, after process LAN and reticent miR-21, TGF-β 1 is in harmonious proportion down rise respectively, (* P < 0.05).

The endogenous expression levels of process LAN and reticent miR-21 in mouse BMMSCs, cultivated after four days, and ELISA method detects the expression of TGF-β 1 in supernatant.(A) miR-21 negative regulation BMMSCs secretes TGF-β 1 level, and (B) IL-6 level also raises, and (C) does not affect IL-17; By the T cell Dual culture of the BMMSCs of reticent miR-21 and activation after four days, (D-E) compared with transfection control Dual culture group, the ratio of Tregs obviously raises, (F) TGF-β 1 expression level in supernatant also obviously raises, and in (G) miR-21 silenced cell, TGF-β 1 messenger RNA(mRNA) expression level raises; (* P < 0.05).

Get mouse primary BMMSCs to go down to posterity, by BMMSCs transfection miR-21inhibitor (I-miR-21) and miRNAcontrol (I-NC) and the CD4 activated respectively ⁺t cell Dual culture, 50mg/mLTGF-beta 1 antibodies (anti-TGF-β 1antibody), (anti-IL-6antibody) or isotype control Ab (isotypeantibody) is added in culture system, after four days, ELISA method detects expression and the Treg cytodifferentiation ratio of TGF-β 1 in supernatant.(A-C) to add in co-culture system in TGF-β 1 Idiotype antibody and after TGF-β 1, miR-21 silenced cell Dual culture group Treg differentiation effect is eliminated, and in (D) supernatant, TGF-β 1 is neutralized.(E-G) differentiation that IL-6 specific antibody can not eliminate Treg is added.(*P＜0.05)。

Induce C57BL/6 mouse 9 days at 3%DSS, within the 3rd day, feed back the reticent BMMSCs (DSSI-miR-21) of miR-21 and transfection control BMMSCs (DSSI-NC), detect Mouse Weight and Diseases Inflammatory index every day.(B) comparatively feed back DSSI-NCBMMSCs, feed back DSSI-miR-21BMMSCs and more effectively recovered body weight gain at the 4th day, (C) reduces mouse disease activity index simultaneously; Within 9th day, put to death the ratio that mouse detects regulatory T cells in mouse mesenteric lymph nodes.(D-E) feed back DSSI-miR-21BMMSCs and comparatively feed back the ratio that DSSI-NCBMMSCs can raise Tregs in mesenteric lymph nodes; (F-G) feed back DSSI-miR-21BMMSCs and comparatively feed back the ratio that DSSI-NCBMMSCs can lower Th17 in mesenteric lymph nodes; (F) get distance distal colon 5cm position holostrome intestinal tissue and carry out HE dyeing.Result display feeds back DSSI-miR-21BMMSCs more effectively can reduce the complete of inflammatory cell infiltration degree maintenance epithelium of intestinal mucosa, (* P < 0.05).

C57BL/6 mouse is induced 9 days at 3%DSS, within 3rd day, feed back the reticent BMMSCs (DSSI-miR-21) of miR-21 and transfection control BMMSCs (DSSI-NC), within 9th day, put to death after eyeball of mouse gets blood and get supernatant, detect the level of TGF-β 1 and IL-17 in Mice Body, feed back the level that DSSI-miR-21BMMSCs can raise TGF-β 1 (A) in serum and lower IL-17 (B), and degree comparatively to feed back WTBMMSCs high.(*p＜0.05)

Claims (3)

1. the side of stem cell products prepared by claimed the present invention.

2. the claimed optimization method shining stem cell products immunoregulation capability prepared by news the present invention.

3. the clinical disease type that may treat of stem cell prepared by claimed the present invention.