CN104474558A - Application of microRNA (ribonucleic acid) in preparing biological agent for controlling apoptosis ofjoint fibroblast-like synoviocytes - Google Patents
Application of microRNA (ribonucleic acid) in preparing biological agent for controlling apoptosis ofjoint fibroblast-like synoviocytes Download PDFInfo
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Abstract
The invention discloses an application of microRNA (ribonucleic acid) in preparing a biological agent for controlling the apoptosis ofjoint fibroblast-like synoviocytes. The microRNA adopts ggo-miR-192 or hme-miR-34, the sequence of ggo-miR-192 is shown as the SEQ ID NO.1, and the sequence of hme-miR-34 is shown as the SEQ ID NO.2.
Description
Technical field
The invention belongs to field of molecular biotechnology, be specifically related to the application of microRNA in preparation regulation and control synovium of joint apoptosis of fibroblasts biological preparation.
Background technology
Rheumatoid arthritis (Rheumatoid arthritis, RA) be a kind of cytokine mediated autoimmune disease being dominant phenotype with joint chronic inflammatory disease, destroy as feature with the Progressive symmetric erythrokeratodermia in the paraplasm of synovial tissue and joint, RA apoptosis of synoviocytes mechanism impediment, cause synovioblast (Fibroblast-like Synoviocytes, FLS) infiltration of hyper-proliferative and a large amount of inflammatory cell,, thus there is pathological change in apoptosis of synoviocytes and propagation unbalance.For the pathogenic factors of RA, research and develop multiple medicine both at home and abroad.
MicroRNA (microRNA) is the regulation and control small RNA molecular that in non-coding RNA family, length is about 18-32 nucleotide, and it is by the mode negative regulation target gene that mRNA shears and Profilin matter is translated.Up to now, ten hundreds of microRNA molecule has been found at more than 200 species such as nematicide, arabidopsis, fruit bat, mice and people.Further research shows, microRNA can regulate the protein coding gene of about 50%, and microRNA molecule take part in the various physiological processes comprising growth, cell differentiation, apoptosis, lipid metabolism and hormone secretion etc., and comprise the multiple pathological process such as immunity, inflammation of RA.
MiRNA (microRNA) is the important non-coding RNA of a class, translates or degraded target gene mRNA plays a role by suppressing the mRNA of target gene on post-transcriptional level.According to miRBase data base, up to the present, had been found that the microRNA gene of 678 mankind, and evidence suggests still have a large amount of microRNA to remain to be discovered in the mankind, the sum of microRNA may be more.Think that microRNA participates in much important biological process at present, as transcription factor regulated and control network, the sequencing contro in growth course, nerve synapse is formed, cell proliferation, cell death, cell differentiation etc.
Summary of the invention
The present invention is intended to overcome the deficiencies in the prior art, provides the application of microRNA in preparation regulation and control synovium of joint fibroblast (FLS) apoptosis biological preparation.
Jiangxi genuine traditional Chinese medicine rattan Lycopodium clavatum (Lycopodiastrum casuarinoides) has good therapeutic effect to RA patients clinically.The present inventor tests the component analysis at each position of rattan Lycopodium clavatum and extraction and active function, not yet finds effective chemical composition.Inventor, by extracting the microRNA in rattan Lycopodium clavatum body, carries out the animal model effect experiment of apoptosis of synoviocytes regulating and controlling effect and RA mice.To the microRNA order-checking of extracting, Testing and appraisal, to 2519 kinds of microRNAs of 153 species, therefrom screens 2 microRNAs to the synovioblast transfection of RA, finds to have regulating effect to the apoptosis of FLS.With microRNA ggo-miR-192 and the process of hme-miR-34 transfection FLS cell, pass through the FCM analysis to FLS apoptosis and DAPI and TUNEL marker detection, the apoptotic regulating and controlling effect successful of FLS strengthens, and both performances are to the apoptotic positive regulating and controlling effect of FLS.Ggo-miR-192 sequence is as shown in SEQ ID NO.1, and hme-miR-34 sequence is as shown in SEQ ID NO.2.
Accompanying drawing explanation
Fig. 1 is human synovial cell form photo, and 10x10 takes pictures;
Fig. 2 is human synovial cell transfection NC matched group form photo, and 10x10 takes pictures;
Fig. 3 is human synovial cell transfection hme--miR-34 form photo, and 10x10 takes pictures;
Fig. 4 is human synovial cell transfection ggo-miR-192 form photo, and 10x10 takes pictures;
Fig. 5 is human synovial cell transfection ggo-miR-192Inhibitor form photo, and 10x10 takes pictures;
Fig. 6 is that human synovial cell cell selects door figure;
Fig. 7 is human synovial cell cell own control figure;
Fig. 8 is human synovial cell stream of cells formula apoptosis detection figure;
Fig. 9 is human synovial cell cell transfecting NC streaming apoptosis detection figure;
Figure 10 is human synovial cell cell transfecting hme--miR-34 streaming apoptosis detection figure;
Figure 11 is human synovial cell cell transfecting hme-miR-34Inhibitor streaming apoptosis detection figure;
Figure 12 is human synovial cell cell transfecting ggo-miR-192 streaming apoptosis detection figure;
Figure 13 is human synovial cell cell transfecting ggo-miR-192Inhibitor streaming apoptosis detection figure;
Figure 15 is human synovial cell Tunel detection figure; A schemes: B figure and C figure overlay chart; B schemes: Tunel detects; C schemes, and: DAPI dyes nuclear location;
Figure 14 is that human synovial cell cell different disposal apoptosis detects normal cell ratio chart;
Figure 16 is human synovial cell transfection NC Tunel detection figure; A schemes: B figure and C figure overlay chart; B schemes: Tunel detects; C schemes, and: DAPI dyes nuclear location;
Figure 17 is human synovial cell transfection ggo-miR-192Tunel detection figure; A schemes: B figure and C figure overlay chart; B schemes: Tunel detects; C schemes, and: DAPI dyes nuclear location;
Figure 18 is human synovial cell transfection hme-miR-34Tunel detection figure;
Figure 19 is the tunel detection figure of different sample;
Figure 20 is Tunel (the Nick End labelling method of deoxyribonucleotide terminal transferase mediation) the testing result figure of different disposal to FLS apoptosis influence.
Detailed description of the invention
1 material
1.1 experiment materials: human colon cancer cell HCT116 (your bio tech ltd provides by Rider, Guangzhou)
The miRNA mimic Ncontrol#24 (NC) that people, mice, rat are general, sequence is: 5 '-YCACAACCYCCYAGAAAGAGYAGA-3 ' (SEQ ID NO.5);
NC Inhibitor: be NC reverse complementary sequence, sequence is: 5 '-YCYACYCYYYCYAGGAGGYYGYGA-3 ' (SEQ ID NO.6);
MicroRNA ggo-miR-192 sequence: 5 '-CYGACCYAYGAAYYGACAGCC-3 ' (SEQ ID NO.1);
Ggo-miR-192Inhibitor: be ggo-mir-192 reverse complementary sequence, sequence is: 5 '-GGCYGYCAAYYCAYAGGYCAG-3 ' (SEQ ID NO.3);
MicroRNA hme-miR-34 sequence: 5 '-YGGCAGYGYGGYYAGCYGGYYG-3 ' (SEQ IDNO.2);
Hme-miR-34Inhibitor: be hme-miR-34 reverse complementary sequence, sequence is: 5 '-CAACCAGAGCYAACCACACYGCCA-3 ' (SEQ ID NO.4).
Above sequence is synthesized by Rui Bo bio tech ltd, Guangzhou.
1.2 experiment reagents:
Cell culture reagent: hyclone (HUclone, Cat.No.SH30087.01), DMEM-high glucose medium (HUclone, Cat.No.SH30022.01B), mycillin (HUclone, Cat.No.SH30010), PBS kaliumphosphate buffer (HUclone, Cat.No.SH30256.01B).
1.3 experiment consumptive materials:
6 orifice plate Tissue Culture Plate (CORNING, Cat.No.040810004), 12 orifice plate Tissue Culture Plate (NEST, Cat.No.3524), 24 orifice plate Tissue Culture Plates (CORNING, Cat.No.3548), 48 orifice plate Tissue Culture Plate (CORNING, Cat.No.051010001A), 96 orifice plate Tissue Culture Plates (NEST, Cat.No.PPP-001-030) etc.
2 instruments
The safe and sound clean bench in Suzhou (SW-CJ-IFD), low speed centrifuge (in good, SC3614), inverted light microscope (OLUMPUS CKX41, U-CTR30-2), cell constant temperature incubator (Thermo scientific, HERACELL150i) inverted fluorescence microscope manufacturer: Leica model: DMI6000B.
3. experimental technique
3.1 cell recovery
3.1.1. water-bath is preheated to 37 DEG C
3.1.3. the superclean bench table top of 75% alcohol wipe ultraviolet radiation 30min is used.
3.1.3. in superclean bench, sterile centrifuge tube, suction pipe, culture bottle etc. is well placed in order.
3.1.4. taking-up cryopreservation tube:
3.1.5. thaw rapidly, rapidly cryopreservation tube is put in the water-bath of preheating and thaw rapidly, and will constantly shake, the liquid in pipe is melted rapidly, also exist in cryopreservation tube when little by little not melting, take out.
3.1.6. use the outer wall of cotton ball soaked in alcohol wiping cryopreservation tube, then take in super-clean bench.
3.1.7. prepare cell suspension, cell is transferred in a 15ml centrifuge tube, drop by drop adds preheated culture medium, rock centrifuge tube simultaneously; The amount adding culture medium will reach more than 10ml.
3.1.8. centrifugal, on low speed centrifuge, with 800rpm centrifugal 5 minutes; Suck supernatant, then use 1ml culture medium re-suspended cell;
3.1.9. cell suspension is distributed in culture dish, culture dish is put into 37 DEG C containing CO
2incubator in cultivate, the time of changing liquid is determined by cell settlement speed conditions.
3.2 cell culture
3.2.1. water-bath is preheated to 37 DEG C
3.2.3. the superclean bench table top of 75% alcohol wipe ultraviolet radiation 30min is used.
3.2.3. in superclean bench, sterile centrifuge tube, suction pipe, culture bottle etc. is well placed in order.
3.2.4. Tissue Culture Flask is taken out, sterile working.
3.2.5. open bottle cap, sop up old culture fluid.
3.2.6. PBS washed cell one to secondary is used.
3.2.7. trUpsin-EDTA solution (1ml/25cm2,2ml/75cm2) is added, bottom fine laundering cell ware.Sop up trUpsin-EDTA solution, put into 37 DEG C of incubator 2-3 minute, pat culture bottle wall and most cells is come off, observe under inverted microscope, when cell will be separated and present round shaped grain shape, add the appropriate fresh culture containing serum and stop trUpsin effect.
3.2.8. inhale up and down with suction pipe and put for several times to break up cell mass, after being mixed evenly, supplying 3n (n is bottle number that goes down to posterity) ml culture medium (MEM), be transferred in new culture bottle according to dilution ratio.
3.2.9. CO2 incubator (condition of culture 5%CO2, saturated humidity, 37 DEG C) is put into
3.3 cell transfectings (RNA)
3.3.1 day before transfection goes down to posterity to cell strain, makes its degree of converging be 30%-50%, and transfection adopts Lipofectamine
tMrNAiMAX (invitrogen, Cat.No.13778075) transfection reagent, micoRNA mimcs working concentration is 50nM (adding separately microRNA ggo-miR-192 or microRNA hme-miR-34), Inhibitor (adding ggo-miR-192Inhibitor or Hme-miR-34Inhibitor respectively accordingly with microRNA) working concentration is 100nM, Opti-MEM (invitrogen, Cat.No.31985070) culture medium is used during transfection.
3.3.2 such as using is 24 orifice plates, every aperture surface area is 1.9cm
2, culture medium consumption is 500ul, and namely absorption miRNA mother solution (20uM) 1.25ul is dissolved into 100ul Opti-MEM culture medium is A liquid mimcs, gets Lipofectamine
tMit is B liquid that RNAiMAX is dissolved in Opti-MEM culture medium, and after B liquid mixes 5 minutes, A liquid and the mixing of B liquid, be added in Tissue Culture Plate after static 20 minutes.More than operation is every hole consumption.
3.3.3 cell growth medium is changed to after hatching 4 hours.
After the microRNA-NC of 3.3.4 transfection band FAM labelling 6 hours, take pictures at fluorescence microscopy Microscopic observation transfection efficiency.
3.3.5 gene level receives sample after being detected as 24-36 hour, protein level receives sample after being detected as 36-48 hour, and cell function cell proliferation, cell cycle, apoptosis, Edu detect, stick, move, attack, cut, mitochondria potential JC-1, cell surface antigen detection etc. are detect after 48 hours.
3.4 cell climbing sheet
3.4.1. according to oneself need be cut into suitable size, in order to being placed in 6 orifice plates, 12 orifice plates or 24 orifice plates
3.4.2. the creep plate will cut out, is placed in concentrated sulphuric acid and soaks and spend the night, and within second day, first uses tap water 5 times, then uses tri-distilled water ultrasonic cleaning 3 times, is placed in lunch box or culture dish and dries the sterilization of laggard horizontal high voltage.
3.4.3. under aseptic conditions, the slide of drying is put in suitable culture dish.Coverslip can use the tweezer of metal tweezers, and circular lid slide can be placed in the hole of 24 well culture plates, and the culture dish of diameter 90mm can be put into 10-15 and open coverslip, or puts into the microscope slide of a standard.
3.4.4. microscope slide adds 0.5% gelatin bag by slide.
3.4.5. will handle each group of passage well on slide, and expect a good result, when adding cell, density should be low, in case cell density is too high.
If 3.4.6. cell number is limited, cell suspension directly can be dropped on slide, leave standstill and add culture fluid gently again after 4 hours.In this way, most cells will adhere on slide, and then cultivate about 24 hours, and cell is suitably increased.Now, taking-up microscope slide or coverslip can be fixed.Use reagent and consumptive material: slide is provided by Shanghai Baihuishen Biotechnology Co., Ltd.; Gelatin is by sigma company article No.: G1393.
3.5 cell Tunel detect
3.5.1 cultured cell PBS washes away culture fluid.
3.5.24% formalin 4 DEG C fixes 25 minutes.
3.5.3PBS wash 5 minutes, 2 times.
3.5.40.2%Triton X-100 incubated at room 5 minutes.
3.5.5PBS wash 5 minutes, 2 times.
3.5.6 100 μ l Equilibration Buffer equilibrium at room temperature 10 minutes are added.
3.5.7 add 50 μ l TdT working solutions, 37 DEG C of wet box lucifuges and hatch 60 minutes.
3.5.82X SSC solution washing 15 minutes.
3.5.9PBS solution chamber's temperature laundering 5 minutes, 3 times.
3.5.10DAPI the dye liquor room temperature box lucifuge that wets hatches 10 minutes.
3.5.11 deionized water room temperature washing 5 minutes, 3 times.
3.5.12 the anti-quencher mounting of fluorescence.
3.5.13 fluorescence microscopy (lucifuge, 4 DEG C of preservations).
Use reagent: TUNEL test kit (manufacturer: Promega article No.: G3250)
3.6 flow cytometer detection apoptosis
3.6.1. the culture medium respectively organized by Tissue Culture Plate to be transferred to respectively in the conical pipe of 15ml and is placed on ice.
Attention: may include in these culture medium in process of cell death from the cell that cultivation wooden partition comes off.
3.6.2. use 2ml PBS solution rinse culture plate inner cell gently, remove PBS solution.
3.6.3. add 0.5ml 0.25% pancreatin not containing EDTA, hatch, until basis of microscopic observation starts to come off from cultivation wooden partition to cell.
3.6.4. patting continuously gently makes cell come off completely from cultivation wooden partition.
3.6.5. being resuspended in gently by cell in 1 × binding buffer liquid of culture medium in step 1 or pre-cooling makes its density be approximately 1 × 10
6cell/ml.
3.6.6. by 0.5ml cell suspension from Tissue Culture Plate (5 × 10
5individual cell) transfer in a clean centrifuge tube.
3.6.7. 1.25 μ l Annexin V-FITC are added.
3.6.8. room temperature (18-24 DEG C) lucifuge reacts 15 minutes.
3.6.9. centrifugal 5 minutes of room temperature 1000 × g, removes supernatant.
3.6.10. by resuspended gently for 1 × binding buffer liquid of cell 0.5ml pre-cooling.
3.6.11. 10 μ l Propidium Iodide are added.
3.6.12. sample is placed on and keeps in Dark Place on ice.
3.6.13. analyze with flow cytomery immediately.
Use test kit: Annexin V-FITC cell apoptosis detection kit (keUgen, article No. KGA106)
Use instrument: flow cytometer BD calibur.
With flow cytomery, excitation wavelength Ex=488nm; The green fluorescence suggestion of emission wavelength Em=578nm, Annexin V-FITC uses FL2 Air conduct measurement; Excitation wavelength Ex=546nm; The suggestion of emission wavelength Em=647nm, PI red fluorescence uses FL3 Air conduct measurement.
Fluorescence Compensation Regulation: use the normal cell without apoptosis induction process, carries out the position that fluorescence Compensation Regulation removes spectra overlapping and setting cross door in contrast.
Apoptotic cell to all for cytoactive qualification dyestuff as PI has anti-metachromia, non-viable non-apoptotic cell then can not.Cell membrane has the DNA of the cell of damage can dye generation red fluorescence by PI, and the cell that cell membrane remains intact then does not have red fluorescence and produces.Therefore, apoptotic early stage PI can not dye and there is no red fluorescent.Normal live cells similarly.On the scatterplot of bivariate flow cytometer, left lower quadrant display living cells, is (FITC-/PI-); Right upper quadrant is non-living cell, i.e. non-viable non-apoptotic cell, is (FITC+/PI+); And right lower quadrant is apoptotic cell, manifest (FITC+/PI-).After Annexin V-FITC and propidium iodide stain, normal living cells is not by Annexin V-FITC and propidium iodide stain (lower left corner); The early stage cell of apoptosis is only dyeed by Annexin V-FITC, and propidium iodide stain is negative (lower right corner); The cell in non-viable non-apoptotic cell and apoptosis late period can simultaneously by Annexin V-FITC and propidium iodide stain (upper right corner).What the upper left corner occurred is metrical error in tolerance band.
On U-up; Under L-lower; L-left is left; R-right is right
UL upper left; UR upper right; LL lower-left; LR bottom right
4 experimental results
4.1 cellular morphologies (see Fig. 1 to Fig. 5);
Streaming apoptosis detects (see table 1, Fig. 6 to Figure 14); Figure 14 shows the ratio of each process normal live cells; human synovial cell fluidic cell apoptosis testing result; the normal live cells minimum number of hme-miR-34 and ggo-miR-192 process; the normal live cells quantity of other each process is obviously more, and the living cells quantity of display hme-miR-34Inhibitor and ggo-miR-192Inhibitor process is maximum.
4.2tunel detects (see Figure 15 to Figure 20).
The apoptosis of table 1. human synovial cell cell different disposal detects
Table 1 is presented at the detected value of UR door, LR door and UR+LR door, hme-miR-34's and ggo-miR-192 is maximum (3.61 and 4.14), apparently higher than the processed group of normal cell and negative control NC, significantly increase respectively than hme-miR-34Inhibitor and ggo-miR-192Inhibitor process (1.24 and 1.32), difference is extremely remarkable.Show that hme-miR-34 and ggo-miR-192 to the positive regulation effect of people's synovioblast apoptosis clearly.
Figure 19 shows the apoptosis ratio of each process, and difference is extremely remarkable.The apoptotic cell quantity of hme-miR-34 and ggo-miR-192 process in Figure 19 is than hme-miR-34Inhibitor and ggo-miR-192Inhibitor process showed increased, and difference is extremely remarkable; With the apoptotic cells increased of normal cell and negative control process, difference is extremely remarkable.
By FCM analysis and TUNEL cell end labelling testing result, hme-miR-34 and ggo-miR-192 to the regulating and controlling effect of people's synovioblast apoptosis clearly, hme-miR-34 and ggo-miR-192 is to the regulating and controlling effect of people's synovioblast apoptosis in display, the biological preparation of its unique adjusting function.
Claims (1)
1. the application of microRNA in preparation regulation and control synovium of joint apoptosis of fibroblasts biological preparation, described microRNA be ggo-miR-192 or hme-miR-34, ggo-miR-192 sequence as shown in SEQ ID NO.1, hme-miR-34 sequence is as shown in SEQ ID NO.2.
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Citations (2)
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| WO2012092382A2 (en) * | 2010-12-29 | 2012-07-05 | The United States Government As Represented By The Department Of Veterans Affairs | Role of microrna in t cell immune response |
| EP2285960B1 (en) * | 2008-05-08 | 2015-07-08 | Asuragen, INC. | Compositions and methods related to mir-184 modulation of neovascularization or angiogenesis |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2285960B1 (en) * | 2008-05-08 | 2015-07-08 | Asuragen, INC. | Compositions and methods related to mir-184 modulation of neovascularization or angiogenesis |
| WO2012092382A2 (en) * | 2010-12-29 | 2012-07-05 | The United States Government As Represented By The Department Of Veterans Affairs | Role of microrna in t cell immune response |
Non-Patent Citations (3)
| Title |
|---|
| FABIENNE NIEDERER等: "Down-regulation of microRNA-34a* in rheumatoid arthritis synovial fibroblasts promotes apoptosis resistance", 《ARTHRITIS&RHEUMATISM》 * |
| 娄文加 等: "miR-34 家族——肿瘤抑制蛋白p53高度相关的microRNA", 《遗传》 * |
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