CN110184234A - It can promote the method for the Nano silver grain of Human Periodontal Ligament Fibroblasts Osteoblast Differentiation - Google Patents
It can promote the method for the Nano silver grain of Human Periodontal Ligament Fibroblasts Osteoblast Differentiation Download PDFInfo
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
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- C—CHEMISTRY; METALLURGY
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
It can promote the method for the Nano silver grain of Human Periodontal Ligament Fibroblasts Osteoblast Differentiation, it is characterized by: specifically: the primary Human Periodontal Ligament Fibroblasts of normal isolation culture carry out cellular identification by immunocytochemical stain cytokeratin and vimentin；During Orthodontic Treatment, apply mechanical force appropriate in tooth, causes the reconstruction of periodontal membrane tissue first, and then alveolar bone remodeling occurs, be finally subjected to displacement tooth and achieve the purpose that rescue.AgNPs can promote the Osteoblast Differentiation of mammal mesenchymal stem cell.In addition, AgNPs promotes fibroblast to be divided into myofibroblast, so that contraction of wounds be promoted to heal.Advantages of the present invention: studying influence and specific molecular mechanism of the AgNPs to hPDLFs Osteoblast Differentiation using In vitro cell experiment, improves the efficiency of alveolar bone remodeling, and then accelerate Orthodontic Tooth Movement, the course for the treatment of is rescued in shortening.
Description
Technical field
The present invention relates to orthodontic fields, in particular to can promote the silver of Human Periodontal Ligament Fibroblasts Osteoblast Differentiations
The method of nanoparticle.
Background technique
During Orthodontic Treatment, applies mechanical force appropriate in tooth, cause periodontal membrane tissue first
Reconstruction, and then alveolar bone remodeling occurs, it is finally subjected to displacement tooth and achievees the purpose that rescue.Parodontium is to be located at alveolar bone
A kind of special connective tissue between cementum plays tooth support, nutrition, perception and periodontium repair function.Tooth
All film fibroblasts (periodontal fibroblast cells, PDLFs) are most important cells in periodontal membrane tissue,
With stem cell properties, osteoblast, cementum like cell and fat cell can be divided into.PDLFs is to receive at first just
The cell of abnormal power, by some biochemical signals (cell factor, prostanoid and neurotransmitter) induce PDLFs skeletonization to
Differentiation assists the reconstruction of bone tissue during correction, it has also become correction scholar institute question of common concern.
Summary of the invention
The purpose of the invention is to improve the efficiency of alveolar bone remodeling under the premise of safe and effective, and then accelerate just
Abnormal tooth is mobile, and the course for the treatment of is rescued in shortening.Studied using In vitro cell experiment influence of the AgNPs to hPDLFs Osteoblast Differentiation and
Specific molecular mechanism is how the efficiency of alveolar bone remodeling to be improved under the premise of safe and effective, and then accelerate correction tooth
Tooth is mobile, and the course for the treatment of is rescued in shortening.
The present invention provides the method for the Nano silver grain that can promote Human Periodontal Ligament Fibroblasts Osteoblast Differentiation, features
It is: the method for the Nano silver grain of Human Periodontal Ligament Fibroblasts Osteoblast Differentiation can be promoted, specially
The primary Human Periodontal Ligament Fibroblasts of normal isolation culture, pass through immunocytochemical stain cytokeratin and wave
Shape silk-fibroin carries out cellular identification；Chemical reduction method prepares AgNPs, with different concentration (0 μM, 25 μM, 50 μM, 100 μM, 250
μM, 500 μM) AgNPs acts on hPDLFs, MTT detects cell viability, determines the appropriate activity range of AgNPs；
(0 μM, 25 μM, 50 μM, 100 μM) processing hPDLFs of AgNPs of various concentration, and osteogenic induction is carried out, alkaline phosphorus
Sour enzyme (ALP) Activity determination determines that various concentration AgNPs acts on ALP activity change in lower hPDLFs；Alizarin red staining identification is each
Group cell Mineral nodules formational situation；Real-time PCR and Western blot detection osteoblast differentiation marker (ALP,
Collagen-I, Runx2, OCN, OSX) mRNA and protein expression level；
Nano silver grain (AgNPs), which refers to, accomplishes nanoscale metallic silver simple substance for partial size, and usual partial size is in 1~100nm
Between；It is reported that AgNPs has broad-spectrum antibacterial action, it is still effective to antibiotic antibody-resistant bacterium；It is imitated in view of its significant antibacterial
Fruit, AgNPs has been widely used for medical field, especially on orthopaedic implants；In addition, it is some research shows that
AgNPs can promote the Osteoblast Differentiation of mammal mesenchymal stem cell；The plan of this paper inquire into AgNPs to hPDLFs at
Whether the influence and specific molecular mechanism of bone differentiation, this part content influence parodontium into fiber finer first against AgNPs
The vigor of born of the same parents is analyzed；
Experimental material:
It is primary to be separately cultured Human Periodontal Ligament Fibroblasts (hPDLFs)；
Laboratory prepares Nano silver grain (AgNPs)
Experimental method:
The synthesis of AgNPs:
Chemical reduction method is prepared Nano silver grain (AgNPs): the 0.1mM silver nitrate of Xiang Hanyou 0.7mM sodium citrate
(AgNO3) 10mg sodium borohydride (NaBH is added in solution4), then solution is stirred overnight, until passing through UV/Vis optical spectroscopy
It is further increased less than absorbance at 400nm, it is final to obtain the AgNPs that concentration is 1mM；JEOL company high-resolution is used simultaneously
Rate transmission electron microscope JEM-1400Flash and its highly sensitive sCMOS camera of outfit carry out morphology and size to the AgNPs of synthesis
Detection, operating voltage is set as 120kv；
The primary separation and culture of periodontal ligament fibroblasts in vitro:
People's parodontium have drawn from Chinese Medical Sciences University's stomatological hospital Shengang outpatient clinic it is medical because of correction need to have tooth pulled out
20 patients, the age was in 12~18 years old range；After linking up, Written informed consent is signed, materials and experimental implementation scheme are logical
Cross the approval of Ethics Committee, Chinese Medical Sciences University；The dental health of selection, no saprodontia or periodontal disease, patient gargles before having tooth pulled out；
After tooth extraction after, first using physiological saline repeated flushing root surface to surface without blood after, put into it is sterile, contain low concentration
In dual anti-PBS liquid, and draw materials as early as possible in laboratory；Rear cell activity extends at any time and reduces tooth in vitro, generally
It is advisable with being no more than 6 hours；First carry out tissue block scraping, in superclean bench by tooth move into sterile petri dish in,
PBS is rinsed 5 times, removes blood stains；A small amount of DMEM culture solution is added dropwise to keep diseased Root Surfaces wet, is carefully scraped with sterile razor blade back
1/3 position parodontium in root of the tooth, the tissue block that will be scraped do not shred, and are transferred directly in 15ml centrifuge tube, are added containing quality point
0.05%I Collagen Type VI enzyme solution 4ml is counted to digest tissue block, 37 DEG C of water-baths is placed in and is incubated for；Every 5min shakes 1 time, digests altogether
30min, 156g are centrifuged 5min, remove supernatant, collect tissue block；4ml PBS is washed 2 times, and centrifugation removal supernatant collects tissue block；It connects
Kind is added DMEM complete medium in 6 orifice plates (10%FBS+DMEM+1% is dual anti-)；It is placed in 37 DEG C, 5%CO2Saturated humidity training
Case culture is supported, liquid is changed within the 2nd day, retains supernatant renewed vaccination into new hole, continues to cultivate；It is covered with to cell, 1mlPBS cleaning 1
It is secondary, 1min is digested with 0.25% pancreatin of 1ml, complete medium terminates digestion, gently blows and beats cell, cell suspension is collected into
In 15ml centrifuge tube, 100g is centrifuged 3min, removes supernatant, and 1:2 passage is inoculated into 6 orifice plates；Take 4-6 for cell for this research；
Cell processing and grouping:
4-6 replaces osteogenic for cell, carries out Osteoblast Differentiation induction and (fills in rice comprising 100nM in osteogenic
Pine, 50 μM of vitamin Cs and 10mM sodium β-glycerophosphate), and AgNPs (0 μM, 25 μM, 50 μM, 100 of various concentration are given simultaneously
μM, 250 μM, 500 μM) intervened, replacement in osteogenic every 3 days is primary in experimentation；
Immunocyte dyeing:
Pass through immunocytochemical stain detection cytokeratin (cytokeratin) and vimentin (vimentin) mirror
Determine hPDLFs；HPDLFs, which is seeded on 12 orifice plates for be covered with glass slide, carries out cell climbing sheet, and 4% paraformaldehyde is added and fixes 15 points
It removes, is added 0.01M PBS buffer solution soaking and washing 3 times, each 5min continuously adds 0.1%Triton after outwelling PBS after clock
X-100 removes Triton X-100 and PBS in the static incubation 20min of room temperature, and 3%H is added2O2It is incubated at room temperature 15min, in elimination
Endogenous peroxidase enzymatic activity rejoins PBS soaking and washing later, and normal serum is added, and blood is removed in incubation at room temperature 15min hypsokinesis
Clearly, without cleaning；Primary antibody cytokeratin and vimentin antibody are diluted with PBS according to 1:200 respectively, have been added dropwise to
All standing cell, 4 DEG C overnight, is added PBS soaking and washing 3 times, removes primary antibody；PBS is removed, by biotin labeling goat antirabbit two
It is anti-to dilute 200 times with PBS, it is added dropwise to and cell is completely covered, 37 DEG C of incubation 30min are added PBS soaking and washing 3 times, remove two
It is anti-；PBS is removed, HRP labelled streptavidin PBS is diluted 200 times, is added dropwise to and cell is completely covered, be incubated at room temperature
30min is added PBS soaking and washing 3 times, removes HRP labelled streptavidin；PBS is removed, colour reagent is added dropwise into 12 orifice plates
(being made of DAB colour reagent A, B mixed in equal amounts) sops up DAB when color just deepens, to 12 orifice plates to cell is completely covered
Middle addition distilled water impregnates 5min；It removes, haematoxylin is added to cell is completely covered, dyes 5min, immerses flowing water in tap water
Rinse 2min；1% hydrochloride alcohol differentiation 2s is added dropwise, immerses in tap water immediately, the anti-indigo plant 20min of flowing water；50% glycerol (glycerol:
Distilled water=1:1) mounting microscopy；It takes pictures under microscopically observation dyeing effect, 200 × mirror (100 μm of length of the scale)；
MTT detects cell viability:
Cell viability is measured with mtt assay, culture Human Periodontal Ligament Fibroblasts to density are 90% or so, and appropriate bulk is added
0.25% long-pending trypsin digestion cell is added complete medium and terminates reaction after cell rounding；Mixed liquor is collected to 15ml examination
Pipe, 90g are centrifuged 3 minutes；Supernatant is removed, the complete medium of 1ml is added, cell count is carried out, with 4 × 103Cells/well density to
Inoculating cell in 96 orifice plates is co-cultured according to the AgNPs of experimental group and various concentration (0,25,50,100,250,500 μM)
Carry out MTT detection afterwards for 24 hours, culture medium of the replacement containing 0.5mg/mL MTT continues culture 4 hours；150 μ are added after sucking supernatant
LDMSO is protected from light microplate reader after standing 10min and measures its OD value at 570nm, counted to dissolve plastidogenetic purple crystal
According to analysis.
Influence of the AgNPs to hPDLFs Osteoblast Differentiation:
Influence and correlating markings developed by molecule level of the various concentration range AgNPs to hPDLFs cell Osteoblast Differentiation become
Change；
Material and method:
Experimental material:
It is primary to be separately cultured Human Periodontal Ligament Fibroblasts (hPDLFs)；
Laboratory prepares 2.2 experimental method of Nano silver grain (AgNPs)
Cell culture and grouping:
4-6 replaces osteogenic for cell, continues culture 7 or 21 days, carries out Osteoblast Differentiation induction (in osteogenic
Include 100nM dexamethasone, 50 μM of vitamin Cs and 10mM sodium β-glycerophosphate), and AgNPs (0 μ of various concentration is given simultaneously
M, 25 μM, 50 μM, 100 μM) to be intervened, replacement in osteogenic every 3 days is primary in experimentation；
Alkaline phosphatase (ALP) Activity determination:
ALP activity in the 7th day detection hPDLFs of Osteoblast Differentiation；Group of cells is collected, ultrasound is broken under condition of ice bath
Broken, 420g is centrifuged 10 minutes, takes supernatant, carries out determining the protein quantity by BCA kit；Firstly, by the BSA egg of 0.5mg/ml
White titer is added on each hole of ELISA Plate according to 0,1,2,4,8,12,16,20 μ L, residual volume PBS buffer solution polishing to 20 μ L,
1 μ L of sample to be tested and 19 μ LPBS buffers mix；According to A liquid: B liquid volume ratio 50:1 prepares BCA working solution, and BCA is added in every hole
200 μ L of working solution, is placed in 37 DEG C of reaction 20min, and microplate reader 570nm wavelength surveys OD value；Each sample egg is calculated according to standard curve
White concentration, and with normal saline dilution at uniform concentration 2mg/ml；
Alizarin red staining；
After Osteoblast Differentiation 21 days, group of cells fixes 20 minutes with 4% paraformaldehyde, then with 0.1% alizarin red S solution
Dyeing, microscopically observation record coloration result (100 ×)；
Real-time PCR detects skeletonization marker mRNA expression:
1ml TRIpure lysate is added into sample, mixes well, stands 5min at room temperature；Add 200 μ L chlorine
It is imitative, it mixes, stands 3min at room temperature；4 DEG C of 10000g are centrifuged 10min, and upper strata aqueous phase is taken to be transferred in new centrifuge tube；Be added etc.
The isopropanol of volume mixes, and is placed in -20 DEG C overnight；After 15h, 4 DEG C of 10000g are centrifuged 10min, abandon supernatant；1ml 75% is added
Ethyl alcohol, 4 DEG C of 3400g are centrifuged 3min, abandon supernatant；5-6min is stood at room temperature；Add 30 μ L RNase-free ddH2O, places
2min is mixed well, and is completely dissolved to its precipitating, gained is sample total serum IgE；It is surveyed using ultraviolet specrophotometer NANO2000
The concentration of RNA in fixed each sample；
Reverse transcription is carried out into cDNA referring to RNA sample of the kit specification to collection；Firstly, dense according to each sample rna
Degree, RNA volume need to be added by calculating every reaction system, guarantee that the RNA concentration of loading is consistent, successively to after processing without nuclease
1 μ L oligo (dT) is added in ice bath PCR pipe15, 1 μ L random (random primer) and respective volume RNA sample are finally mended
Fill ddH2O to overall reaction system be 12.5 μ L；70 DEG C of heating are after five minutes, 2 minutes cooling rapidly on ice, of short duration centrifugation aggregation
Reaction solution continuously adds 45 × Buffer of μ L and 0.5 μ LRnase inhibitor (RNase inhibitor), 2 μ into system
L dNTP (2.5mM each), 1 μ L (200U) reverse transcriptase (M-MLV), stirs and evenly mixs；Setting program: 25 DEG C of 10min, 42 DEG C
50min, 80 DEG C of 10min finally obtain 20 μ L cDNA templates；
1 μ L cDNA template is taken to carry out PCR detection；Primer is first diluted to 10 μM, every reaction system upstream and downstream primer adds
0.5 μ L adds 10 μ L SYBR GREEN mastermix, and residual volume is with ddH2O complements to 20 μ L.
AgNPs synthesis detection
The Nano silver grain (AgNPs) of 1mM, transmission electron microscope (TEM) are prepared using above-mentioned chemical reduction method
Lower observation AgNPs is rounded, and average particle diameter is 10nm (5-15nm), and distribution uniform has good stability, and nothing is significantly built up
Phenomenon.
HPDLFs morphological observation
Under the microscope, the periodontal ligament fibroblasts in vitro after passage, cell cytoplasm retraction, cytoplasmic process disappearance, cell space is in ball to light
Shape is in spindle shape.After 24 hours, cell is substantially all adherent, and to surrounding growth, cell body is plentiful, and kernel is clear, can
See, nuclear division is as after passage, cell distribution is uniform, and the speed of growth is accelerated, 3 to 5 days available 90% or more cells
Fusion.
Immunocyte dyeing identification hPDLFs
Immunocytochemistry result is that keratin stain is negative, and shows the cell contamination of not ectodermal origin, waveform
Silk-fibroin dyeing is positive cell, and brown color is presented in endochylema, it was demonstrated that cell origin is in mesoderm, according to above-mentioned coloration result, this
A little cells can be accredited as fibroblast (hPDLFs).
MTT detects influence of the various concentration AgNPs to cell viability
MTT detects influence of the various concentration AgNPs to hPDLFs cell viability, the results show that compared with cellular control unit,
AgNPs processing has no significant effect hPDLFs cell viability within the scope of 25-100 μM, 250 μM and 500 μM of AgNPs thin to hPDLFs
Born of the same parents' vigor, which exists, to be significantly inhibited.
In orthodontic treatment, after mild and lasting Orthodontic force acts on tooth body, parodontium side is towed, the other side by
Compressing, parodontium generate metabolism and change.It is squeezed and tightens on the pressure side periodontal membrane tissue, periodontal spaces narrow, blood vessel constriction
Blood flow is reduced, and collagenous fibres and substrate degradation absorb, and differentiate osteoclast, these variations are reinforcing 48~72h
Occur.The periodontal membrane fiber stretching of tension side is elongated, and periodontal spaces are broadening, collagenous fibres and matrix hyperplasia, and fibroblast increases
It grows, osteoblast differentiation, tooth has certain loosening, and parodontium direction also changes.After pressure releases tooth is stablized, periodontal is fine
Dimension is adjusted rearrangement and adheres to again, is held dental branches in the new position by the parodontium of reshaping, and restore normal periodontal
The width in gap.It can be seen that the skeletonization of PDLFs controls the update of periodontium and the most important and correction of regeneration to differentiation
The key of alveolar bone remodeling during treatment.The research of Jacobs et al.] just confirm that the intensity of mechanical stress can be changed PDLFs's
Skeletonization is to differentiation degree, after applying static mechanical stress 12h to it, when researcher observes that stress is 5%,
The expression quantity of the osteoblasts in vitro such as collagen-I, ALP, OCN is significantly increased.Therefore, people's parodontium is selected in this research
Fibroblast (human periodontal fibroblast cells, hPDLFs) is used as research object, probes into its biology
Characteristic is learned, and finds to have and hPDLFs skeletonization is promoted to intervene to the material of differentiation characteristic it, to reach quickening correction
Rate of tooth movement reduces the purpose for the treatment of time.HPDLFs in this project is to pass through primary point using traditional enzyme digestion
It is obtained from cultivating and carrying out immunocytochemical stain identification, ensure that the purity of experimental cell.
In view of these, we have carried out MTT test first, living to hPDLFs cell with the AgNPs for assessing various concentration
The influence of power.AgNPs particle used in this research is spherical in shape, and average diameter is 10nm (5-15nm).It is observed that making
After being handled hPDLFs cell 24 hours with the AgNPs that concentration reaches 250 μM, cell survival rate i.e. presentation significant decrease (69.00 ±
1.49% compared with the control, P < 0.001).Therefore, in research later, we select the AgNPs of 25-100 μM of concentration, into
One step assesses influence of the AgNPs to hPDLFs Osteoblast Differentiation.
Influence of the AgNPs to hPDLFs Osteoblast Differentiation:
Some researches show that, AgNPs can promote mesenchymal stem cell (bone mesenchymal stem cells,
BMSCs in-vitro multiplication and Osteoblast Differentiation), and promote union in vivo[31].The formation of bone there are two types of form, in film at
Bone and entochondrostosis, in the osteogenetic process of both forms, osteoblast all during bone matrix secretion and mineralising,
It played an important role[32].According to research report before, alkaline phosphatase (ALP) activity and osteocalcin (OCN) are inspections
Test the important indicator of osteoblast cultured in vitro[33-35].Runt associated transcription factor 2 (Runx2) is generally acknowledged at present most important
The control gene of Osteoblast Specific transcription factor, referred to as skeleton development and reconstruction[36].With the exception of Runx2 gene
And the hereditary disease occurred prompts us, the gene is steady in the development, reconstruction and interior environment of osteoblast differentiation maturation and bone
In a series of physiology courses such as fixed, complicated and accurate regulating and controlling effect is all played[37].Osterix (OSX) is located at Runx2's
Downstream is simultaneously regulated and controled by it, is played an important role in Osteoblast Differentiation latter stage and bon e formation[38].I-type collagen
(collagen-I) it is acted in osteoblast differentiation Late Effect Osteoblast Differentiation, to affect the Mineralization process of bone tissue[39]。
Influence and correlating markings developed by molecule water of research and inquirement various concentration range AgNPs in this part to hPDLFs cell Osteoblast Differentiation
Flat variation.
Experimental material
It is primary to be separately cultured Human Periodontal Ligament Fibroblasts (hPDLFs)；
Laboratory prepares Nano silver grain (AgNPs)
Experimental method
Cell culture and grouping
4-6 replaces osteogenic for cell, continues culture 7 or 21 days, carries out Osteoblast Differentiation induction (in osteogenic
Include 100nM dexamethasone, 50 μM of vitamin Cs and 10mM sodium β-glycerophosphate), and AgNPs (0 μ of various concentration is given simultaneously
M, 25 μM, 50 μM, 100 μM) to be intervened, replacement in osteogenic every 3 days is primary in experimentation.
Alkaline phosphatase (ALP) Activity determination
ALP activity in the 7th day detection hPDLFs of Osteoblast Differentiation.Group of cells is collected, ultrasound is broken under condition of ice bath
Broken, 420g is centrifuged 10 minutes, takes supernatant, carries out determining the protein quantity by BCA kit.Firstly, by the BSA egg of 0.5mg/ml
White titer is added on each hole of ELISA Plate according to 0,1,2,4,8,12,16,20 μ L, residual volume PBS buffer solution polishing to 20 μ L,
1 μ L of sample to be tested and 19 μ LPBS buffers mix.According to A liquid: B liquid volume ratio 50:1 prepares BCA working solution, and BCA is added in every hole
200 μ L of working solution, is placed in 37 DEG C of reaction 20min, and microplate reader 570nm wavelength surveys OD value.Each sample egg is calculated according to standard curve
White concentration, and with normal saline dilution at uniform concentration 2mg/ml.
Alizarin red staining
After Osteoblast Differentiation 21 days, group of cells fixes 20 minutes with 4% paraformaldehyde, then with 0.1% alizarin red S solution
Dyeing, microscopically observation record coloration result (100 ×).
Real-time PCR detects skeletonization marker mRNA expression
1ml TRIpure lysate is added into sample, mixes well, stands 5min at room temperature；Add 200 μ L chlorine
It is imitative, it mixes, stands 3min at room temperature；4 DEG C of 10000g are centrifuged 10min, and upper strata aqueous phase is taken to be transferred in new centrifuge tube；Be added etc.
The isopropanol of volume mixes, and is placed in -20 DEG C overnight；After 15h, 4 DEG C of 10000g are centrifuged 10min, abandon supernatant；1ml 75% is added
Ethyl alcohol, 4 DEG C of 3400g are centrifuged 3min, abandon supernatant；5-6min is stood at room temperature；Add 30 μ L RNase-free ddH2O, places
2min is mixed well, and is completely dissolved to its precipitating, gained is sample total serum IgE.It is surveyed using ultraviolet specrophotometer NANO2000
The concentration of RNA in fixed each sample.
Reverse transcription is carried out into cDNA referring to RNA sample of the kit specification to collection.Firstly, dense according to each sample rna
Degree, RNA volume need to be added by calculating every reaction system, guarantee that the RNA concentration of loading is consistent, successively to after processing without nuclease
1 μ L oligo (dT) is added in ice bath PCR pipe15, 1 μ L random (random primer) and respective volume RNA sample are finally mended
Fill ddH2O to overall reaction system be 12.5 μ L；70 DEG C of heating are after five minutes, 2 minutes cooling rapidly on ice, of short duration centrifugation aggregation
Reaction solution continuously adds 45 × Buffer of μ L and 0.5 μ LRnase inhibitor (RNase inhibitor), 2 μ into system
L dNTP (2.5mM each), 1 μ L (200U) reverse transcriptase (M-MLV), stirs and evenly mixs.Setting program: 25 DEG C of 10min, 42 DEG C
50min, 80 DEG C of 10min finally obtain 20 μ L cDNA templates.
1 μ L cDNA template is taken to carry out PCR detection.Primer is first diluted to 10 μM, every reaction system upstream and downstream primer adds
0.5 μ L adds 10 μ L SYBR GREEN mastermix, and residual volume is with ddH2O complements to 20 μ L.Primer entrusts Shanghai
The synthesis of Sheng Gong bioengineering Co., Ltd,
2.2.5Western blot detects skeletonization marker protein expression
Group of cells is collected, with the RIPA buffer cracking culture cell containing 1%PMSF, BCA method measures protein compression
Degree.It is shut being clamped after two pieces of clean glass plate alignment, is perpendicularly fixed on table top, prepares encapsulating.According to destination protein molecule
Measure size, this experiment need to prepare 5% concentration glue and 8%, 10%, 13% separation gel (formula see the table below).Separation gel position
It is preferential to prepare below gel, it is eventually adding APS, TEMED, encapsulating after mixing rapidly is sealed, static 30min with aqueous；Removal
APS, TEMED, encapsulating, comb sealing is added in hydraulic layer in prepared concentration glue.Static 30min extracts comb, in preparation
Sample.
Loading volume is 20 μ L, contains 40 μ g albumen, therefore with 5 × loading buffer and PBS diluted protein sample, it boils
It is denaturalized 5min, it is stand-by to obtain sample solution.Electrophoresis liquid is injected to the positive and negative electrode of electrophoresis tank, the above-mentioned electrophoresis sample solution of 20 μ L is added in every hole,
5 μ L albumen Marker are eventually adding, electrophoresis apparatus is connected, starting voltage 80V carries out constant pressure electrophoresis about 2.5h.
4 DEG C of buffer pre-coolings of transfer, sponge, filter paper transfer to soak in buffer, to the pvdf membrane progress side of appropriate size
It to label, is put into anhydrous methanol and soaks, then transfer buffer and infiltrate 20min.Electrophoresis is completed to take out gel, reference protein
Remaining blob of viscose is placed in transfer buffer and impregnates according to the molecular weight shear gel of destination protein and internal reference albumen by Marker
5-10min；Sponge, filter paper, gel, pvdf membrane (front face gel), filter paper and sea are successively completed since cathode is pressed from both sides in transfer
It is continuous.Transfer folder is fixed, is inserted into transfer groove, supplements enough transfer buffers, set maximum current, voltage 80V, under low temperature
Transfer 1.5h.
Taking-up pvdf membrane puts shake 5min in TBST buffer and transfers in confining liquid (the 5% of TBST buffer
Skimmed milk power solution) closing is immersed, shaking table 1h completes capping.Antibody is diluted to antibody working solution with 5% skimmed milk power and (incubates
The condition of educating see the table below).It takes out the pvdf membrane closed and antibody working solution is put into togerther in hybrid belt, after sealing, 4 DEG C were incubated for
Night.It is same to dilute secondary antibody working solution (incubation conditions see the table below) with 5% skimmed milk power, the pvdf membrane being incubated for after primary antibody is taken out,
It is added in TBST, shaking table elutes repeatedly, 5min × 4 time, is put into togerther in hybridization bag with secondary antibody working solution, seals, and carries out secondary antibody
It is incubated for, incubation conditions are 37 DEG C, 45 minutes.It takes out pvdf membrane to immerse again in TBST liquid, shaking table shakes elution, 5min × 6 time
The pvdf membrane is put into magazine afterwards.Two kinds of reagents in ECL luminescence reagent box are mixed in the ratio of 1:1, are added in magazine
On pvdf membrane and it is exposed.The time for exposure can be adjusted according to the protein band on film.
After exposure, pvdf membrane, distilled water flushing are taken out, shaking table elutes 2 times repeatedly.Cleared distilled water, addition are stripped off liquid and are flooded
No pvdf membrane, shaking table 15min, liquid is stripped off in evacuation, and distilled water flushing is clean.Shaking table in pvdf membrane immersion TBST buffer is taken out to shake
Dynamic 5min.It is transferred to confining liquid (5% skimmed milk power solution of TBST buffer) and immerses closing, shaking table shakes 1h and completes envelope
Close reaction.
5% (M/V) skimmed milk power is used to work according to the anti-β-actin antibody of 1:500 dilution proportion mouse as internal reference antibody
Liquid.It takes out the pvdf membrane closed and antibody working solution is put into togerther in hybrid belt, seal, 4 DEG C of overnight incubations.Equally with 5%
(M/V) pvdf membrane being incubated for after primary antibody is taken out in skimmed milk power dilution secondary antibody working solution (goat anti-mouse igg-HRP, 1:5000),
It after TBST elution, being put into togerther in hybridization bag, seals with secondary antibody working solution, carry out secondary antibody incubation, incubation conditions are 37 DEG C,
45min.After being incubated for internal reference antibody, takes out pvdf membrane and re-use TBST elution, shaking table shakes 5min × 6 time.ECL luminescence-producing reaction
Pvdf membrane is transferred in magazine afterwards, and carries out darkroom exposure.
Film after exposure is taken pictures scanning, is measured with gel images processing system (Gel-Pro-Analyzer software) each
Semi-quantitative analysis is carried out to image after band specificity gray value, with the ratio between purpose band and internal reference protein band light splitting density value
The reference frame of expressing quantity variation as a purpose.
Statistical analysis
The every group of experiment of this paper is at least repeated 3 times.All experimental datas are indicated with mean ± SD, are utilized SPSS (20.0)
Software carries out one-way ANOVA and Bonfroni parameter and is analyzed.P value is considered that there are significant differences less than 0.05.
Experimental result
HPDLFs alkaline phosphatase activities after AgNPs processing
By to different groups of hPDLFs cells, osteogenic induction culture after normal group and AgNPs intervene, ALP Activity determination
The results show that ALP activity dramatically increases (P < 0.01) in hPDLFs after being incubated for 7d jointly with 100 μM of AgNPs；In addition, we
It observes that ALP activity is increased in hPDLFs after 25 μM of AgNPs and 50 μM of AgNPs are incubated for, but there is no statistical difference.
Alizarin red staining detects hPDLFs Osteoblast Differentiation situation
By to different groups of hPDLFs cells, after normal group and AgNPs intervene after osteogenic induction culture 21d, 0.1% is alizarin
Plain red colouring liquid observes the formational situation of various cellular matrix mineralisings and calcium tubercle after dyeing to it.Microscopically observation result
It shows, after Alizarin red staining, compared with without AgNPs processing group, after 25 μM of -100 μM of AgNPs are incubated for 21d, in hPDLFs
The enhancing of calcium mineralising, it is consistent with ALP testing result
Real-time PCR detects skeletonization Research of predicting markers mRNA expression
By to different groups of hPDLFs cells, after normal group and AgNPs intervene after osteogenic induction culture 7d, Real-
Time PCR detects skeletonization Research of predicting markers mRNA expression, and experimental result is shown, is incubated for 7d through 25 μM of -100 μM of AgNPs
Afterwards, the expression of ALP, Runx2, OCN, OSX and collagen-I mRNA are raised in hPDLFs.
Western blot detects skeletonization marker protein expression
By to different groups of hPDLFs cells, after normal group and AgNPs intervene after osteogenic induction culture 7d, Western
Blot detects skeletonization associated marker proteins expression, and experimental result is shown, compared with without AgNPs processing group, through 25 μ
After M-100 μM of AgNPs is incubated for 7d, the protein expression level up-regulation of ALP, Runx2, OCN, OSX and collagen-I in hPDLFs.
Immunocytochemical stain display culture cytokeratin dyeing is negative, and vimentin stained positive can
It is accredited as fibroblast (hPDLFs)；MTT testing result shows that 250 μM and 500 μM of AgNPs have hPDLFs cell viability
It significantly inhibits.25-100 μM of AgNPs does not influence cell viability.Compared with without AgNPs processing group, 100 μM of AgNPs make
After 7d, ALP activity dramatically increases (P < 0.01) in hPDLFs；After 25 μM of -100 μM of AgNPs are incubated for 7d, ALP in hPDLFs,
The expression of Runx2, OCN, OSX and collagen-I mRNA are increased in concentration dependent, and Western blot testing result is therewith
Unanimously.
Advantages of the present invention:
Influence and specific molecular mechanism of the AgNPs to hPDLFs Osteoblast Differentiation are studied using In vitro cell experiment,
How to improve under the premise of safe and effective the efficiency of alveolar bone remodeling, and then accelerate Orthodontic Tooth Movement, shortening rescues
The course for the treatment of.
Specific embodiment
Embodiment 1
The present invention provides the method for the Nano silver grain that can promote Human Periodontal Ligament Fibroblasts Osteoblast Differentiation, features
It is: the method for the Nano silver grain of Human Periodontal Ligament Fibroblasts Osteoblast Differentiation can be promoted, specially
The primary Human Periodontal Ligament Fibroblasts of normal isolation culture, pass through immunocytochemical stain cytokeratin and wave
Shape silk-fibroin carries out cellular identification；Chemical reduction method prepares AgNPs, with different concentration (0 μM, 25 μM, 50 μM, 100 μM, 250
μM, 500 μM) AgNPs acts on hPDLFs, MTT detects cell viability, determines the appropriate activity range of AgNPs；
(0 μM, 25 μM, 50 μM, 100 μM) processing hPDLFs of AgNPs of various concentration, and osteogenic induction is carried out, alkaline phosphorus
Sour enzyme (ALP) Activity determination determines that various concentration AgNPs acts on ALP activity change in lower hPDLFs；Alizarin red staining identification is each
Group cell Mineral nodules formational situation；Real-time PCR and Western blot detection osteoblast differentiation marker (ALP,
Collagen-I, Runx2, OCN, OSX) mRNA and protein expression level；
Nano silver grain (AgNPs), which refers to, accomplishes nanoscale metallic silver simple substance for partial size, and usual partial size is in 1~100nm
Between；It is reported that AgNPs has broad-spectrum antibacterial action, it is still effective to antibiotic antibody-resistant bacterium；It is imitated in view of its significant antibacterial
Fruit, AgNPs has been widely used for medical field, especially on orthopaedic implants；In addition, it is some research shows that
AgNPs can promote the Osteoblast Differentiation of mammal mesenchymal stem cell；The plan of this paper inquire into AgNPs to hPDLFs at
Whether the influence and specific molecular mechanism of bone differentiation, this part content influence parodontium into fiber finer first against AgNPs
The vigor of born of the same parents is analyzed；
Experimental material:
It is primary to be separately cultured Human Periodontal Ligament Fibroblasts (hPDLFs)；
Laboratory prepares 2.2 experimental method of Nano silver grain (AgNPs)
The synthesis of AgNPs:
Chemical reduction method is prepared Nano silver grain (AgNPs): the 0.1mM silver nitrate of Xiang Hanyou 0.7mM sodium citrate
(AgNO3) 10mg sodium borohydride (NaBH is added in solution4), then solution is stirred overnight, until passing through UV/Vis optical spectroscopy
It is further increased less than absorbance at 400nm, it is final to obtain the AgNPs that concentration is 1mM；JEOL company high-resolution is used simultaneously
Rate transmission electron microscope JEM-1400Flash and its highly sensitive sCMOS camera of outfit carry out morphology and size to the AgNPs of synthesis
Detection, operating voltage is set as 120kv；
The primary separation and culture of periodontal ligament fibroblasts in vitro:
People's parodontium have drawn from Chinese Medical Sciences University's stomatological hospital Shengang outpatient clinic it is medical because of correction need to have tooth pulled out
20 patients, the age was in 12~18 years old range；After linking up, Written informed consent is signed, materials and experimental implementation scheme are logical
Cross the approval of Ethics Committee, Chinese Medical Sciences University；The dental health of selection, no saprodontia or periodontal disease, patient gargles before having tooth pulled out；
After tooth extraction after, first using physiological saline repeated flushing root surface to surface without blood after, put into it is sterile, contain low concentration
In dual anti-PBS liquid, and draw materials as early as possible in laboratory；Rear cell activity extends at any time and reduces tooth in vitro, generally
It is advisable with being no more than 6 hours；First carry out tissue block scraping, in superclean bench by tooth move into sterile petri dish in,
PBS is rinsed 5 times, removes blood stains；A small amount of DMEM culture solution is added dropwise to keep diseased Root Surfaces wet, is carefully scraped with sterile razor blade back
1/3 position parodontium in root of the tooth, the tissue block that will be scraped do not shred, and are transferred directly in 15ml centrifuge tube, are added containing quality point
0.05%I Collagen Type VI enzyme solution 4ml is counted to digest tissue block, 37 DEG C of water-baths is placed in and is incubated for；Every 5min shakes 1 time, digests altogether
30min, 156g are centrifuged 5min, remove supernatant, collect tissue block；4ml PBS is washed 2 times, and centrifugation removal supernatant collects tissue block；It connects
Kind is added DMEM complete medium in 6 orifice plates (10%FBS+DMEM+1% is dual anti-)；It is placed in 37 DEG C, 5%CO2Saturated humidity training
Case culture is supported, liquid is changed within the 2nd day, retains supernatant renewed vaccination into new hole, continues to cultivate；It is covered with to cell, 1ml PBS cleaning
1 time, 1min is digested with 0.25% pancreatin of 1ml, complete medium terminates digestion, gently blows and beats cell, cell suspension is collected into
In 15ml centrifuge tube, 100g is centrifuged 3min, removes supernatant, and 1:2 passage is inoculated into 6 orifice plates；Take 4-6 for cell for this research；
Cell processing and grouping:
4-6 replaces osteogenic for cell, carries out Osteoblast Differentiation induction and (fills in rice comprising 100nM in osteogenic
Pine, 50 μM of vitamin Cs and 10mM sodium β-glycerophosphate), and AgNPs (0 μM, 25 μM, 50 μM, 100 of various concentration are given simultaneously
μM, 250 μM, 500 μM) intervened, replacement in osteogenic every 3 days is primary in experimentation；
Immunocyte dyeing:
Pass through immunocytochemical stain detection cytokeratin (cytokeratin) and vimentin (vimentin) mirror
Determine hPDLFs；HPDLFs, which is seeded on 12 orifice plates for be covered with glass slide, carries out cell climbing sheet, and 4% paraformaldehyde is added and fixes 15 points
It removes, is added 0.01M PBS buffer solution soaking and washing 3 times, each 5min continuously adds 0.1%Triton after outwelling PBS after clock
X-100 removes Triton X-100 and PBS in the static incubation 20min of room temperature, and 3%H is added2O2It is incubated at room temperature 15min, in elimination
Endogenous peroxidase enzymatic activity rejoins PBS soaking and washing later, and normal serum is added, and blood is removed in incubation at room temperature 15min hypsokinesis
Clearly, without cleaning；Primary antibody cytokeratin and vimentin antibody are diluted with PBS according to 1:200 respectively, have been added dropwise to
All standing cell, 4 DEG C overnight, is added PBS soaking and washing 3 times, removes primary antibody；PBS is removed, by biotin labeling goat antirabbit two
It is anti-to dilute 200 times with PBS, it is added dropwise to and cell is completely covered, 37 DEG C of incubation 30min are added PBS soaking and washing 3 times, remove two
It is anti-；PBS is removed, HRP labelled streptavidin PBS is diluted 200 times, is added dropwise to and cell is completely covered, be incubated at room temperature
30min is added PBS soaking and washing 3 times, removes HRP labelled streptavidin；PBS is removed, colour reagent is added dropwise into 12 orifice plates
(being made of DAB colour reagent A, B mixed in equal amounts) sops up DAB when color just deepens, to 12 orifice plates to cell is completely covered
Middle addition distilled water impregnates 5min；It removes, haematoxylin is added to cell is completely covered, dyes 5min, immerses flowing water in tap water
Rinse 2min；1% hydrochloride alcohol differentiation 2s is added dropwise, immerses in tap water immediately, the anti-indigo plant 20min of flowing water；50% glycerol (glycerol:
Distilled water=1:1) mounting microscopy；It takes pictures under microscopically observation dyeing effect, 200 × mirror (100 μm of length of the scale)；
MTT detects cell viability:
Cell viability is measured with mtt assay, culture Human Periodontal Ligament Fibroblasts to density are 90% or so, and appropriate bulk is added
0.25% long-pending trypsin digestion cell is added complete medium and terminates reaction after cell rounding；Mixed liquor is collected to 15ml examination
Pipe, 90g are centrifuged 3 minutes；Supernatant is removed, the complete medium of 1ml is added, cell count is carried out, with 4 × 103Cells/well density to
Inoculating cell in 96 orifice plates is co-cultured according to the AgNPs of experimental group and various concentration (0,25,50,100,250,500 μM)
Carry out MTT detection afterwards for 24 hours, culture medium of the replacement containing 0.5mg/mL MTT continues culture 4 hours；150 μ are added after sucking supernatant
LDMSO is protected from light microplate reader after standing 10min and measures its OD value at 570nm, counted to dissolve plastidogenetic purple crystal
According to analysis.
Influence of the AgNPs to hPDLFs Osteoblast Differentiation:
Influence and correlating markings developed by molecule level of the various concentration range AgNPs to hPDLFs cell Osteoblast Differentiation become
Change；
Material and method:
Experimental material:
It is primary to be separately cultured Human Periodontal Ligament Fibroblasts (hPDLFs)；
Laboratory prepares 2.2 experimental method of Nano silver grain (AgNPs)
Cell culture and grouping:
4-6 replaces osteogenic for cell, continues culture 7 or 21 days, carries out Osteoblast Differentiation induction (in osteogenic
Include 100nM dexamethasone, 50 μM of vitamin Cs and 10mM sodium β-glycerophosphate), and AgNPs (0 μ of various concentration is given simultaneously
M, 25 μM, 50 μM, 100 μM) to be intervened, replacement in osteogenic every 3 days is primary in experimentation；
Alkaline phosphatase (ALP) Activity determination:
ALP activity in the 7th day detection hPDLFs of Osteoblast Differentiation；Group of cells is collected, ultrasound is broken under condition of ice bath
Broken, 420g is centrifuged 10 minutes, takes supernatant, carries out determining the protein quantity by BCA kit；Firstly, by the BSA egg of 0.5mg/ml
White titer is added on each hole of ELISA Plate according to 0,1,2,4,8,12,16,20 μ L, residual volume PBS buffer solution polishing to 20 μ L,
1 μ L of sample to be tested and 19 μ LPBS buffers mix；According to A liquid: B liquid volume ratio 50:1 prepares BCA working solution, and BCA is added in every hole
200 μ L of working solution, is placed in 37 DEG C of reaction 20min, and microplate reader 570nm wavelength surveys OD value；Each sample egg is calculated according to standard curve
White concentration, and with normal saline dilution at uniform concentration 2mg/ml；
Alizarin red staining；
After Osteoblast Differentiation 21 days, group of cells fixes 20 minutes with 4% paraformaldehyde, then with 0.1% alizarin red S solution
Dyeing, microscopically observation record coloration result (100 ×)；
Real-time PCR detects skeletonization marker mRNA expression:
1ml TRIpure lysate is added into sample, mixes well, stands 5min at room temperature；Add 200 μ L chlorine
It is imitative, it mixes, stands 3min at room temperature；4 DEG C of 10000g are centrifuged 10min, and upper strata aqueous phase is taken to be transferred in new centrifuge tube；Be added etc.
The isopropanol of volume mixes, and is placed in -20 DEG C overnight；After 15h, 4 DEG C of 10000g are centrifuged 10min, abandon supernatant；1ml 75% is added
Ethyl alcohol, 4 DEG C of 3400g are centrifuged 3min, abandon supernatant；5-6min is stood at room temperature；Add 30 μ L RNase-free ddH2O, places
2min is mixed well, and is completely dissolved to its precipitating, gained is sample total serum IgE；Use ultraviolet specrophotometer NANO 2000
Measure the concentration of RNA in each sample；
Reverse transcription is carried out into cDNA referring to RNA sample of the kit specification to collection；Firstly, dense according to each sample rna
Degree, RNA volume need to be added by calculating every reaction system, guarantee that the RNA concentration of loading is consistent, successively to after processing without nuclease
1 μ L oligo (dT) is added in ice bath PCR pipe15, 1 μ L random (random primer) and respective volume RNA sample are finally mended
Fill ddH2O to overall reaction system be 12.5 μ L；70 DEG C of heating are after five minutes, 2 minutes cooling rapidly on ice, of short duration centrifugation aggregation
Reaction solution continuously adds 45 × Buffer of μ L and 0.5 μ LRnase inhibitor (RNase inhibitor), 2 μ into system
L dNTP (2.5mM each), 1 μ L (200U) reverse transcriptase (M-MLV), stirs and evenly mixs；Setting program: 25 DEG C of 10min, 42 DEG C
50min, 80 DEG C of 10min finally obtain 20 μ L cDNA templates；
1 μ L cDNA template is taken to carry out PCR detection；Primer is first diluted to 10 μM, every reaction system upstream and downstream primer adds
0.5 μ L adds 10 μ L SYBR GREEN mastermix, and residual volume is with ddH2O complements to 20 μ L.
AgNPs synthesis detection
The Nano silver grain (AgNPs) of 1mM, transmission electron microscope (TEM) are prepared using above-mentioned chemical reduction method
Lower observation AgNPs is rounded, and average particle diameter is 10nm (5-15nm), and distribution uniform has good stability, and nothing is significantly built up
Phenomenon.
HPDLFs morphological observation
Under the microscope, the periodontal ligament fibroblasts in vitro after passage, cell cytoplasm retraction, cytoplasmic process disappearance, cell space is in ball to light
Shape is in spindle shape.After 24 hours, cell is substantially all adherent, and to surrounding growth, cell body is plentiful, and kernel is clear, can
See, nuclear division is as after passage, cell distribution is uniform, and the speed of growth is accelerated, 3 to 5 days available 90% or more cells
Fusion.
Immunocyte dyeing identification hPDLFs
Immunocytochemistry result is that keratin stain is negative, and shows the cell contamination of not ectodermal origin, waveform
Silk-fibroin dyeing is positive cell, and brown color is presented in endochylema, it was demonstrated that cell origin is in mesoderm, according to above-mentioned coloration result, this
A little cells can be accredited as fibroblast (hPDLFs).
MTT detects influence of the various concentration AgNPs to cell viability
MTT detects influence of the various concentration AgNPs to hPDLFs cell viability, the results show that compared with cellular control unit,
AgNPs processing has no significant effect hPDLFs cell viability within the scope of 25-100 μM, 250 μM and 500 μM of AgNPs thin to hPDLFs
Born of the same parents' vigor, which exists, to be significantly inhibited.
In orthodontic treatment, after mild and lasting Orthodontic force acts on tooth body, parodontium side is towed, the other side by
Compressing, parodontium generate metabolism and change.It is squeezed and tightens on the pressure side periodontal membrane tissue, periodontal spaces narrow, blood vessel constriction
Blood flow is reduced, and collagenous fibres and substrate degradation absorb, and differentiate osteoclast, these variations are reinforcing 48~72h
Occur.The periodontal membrane fiber stretching of tension side is elongated, and periodontal spaces are broadening, collagenous fibres and matrix hyperplasia, and fibroblast increases
It grows, osteoblast differentiation, tooth has certain loosening, and parodontium direction also changes.After pressure releases tooth is stablized, periodontal is fine
Dimension is adjusted rearrangement and adheres to again, is held dental branches in the new position by the parodontium of reshaping, and restore normal periodontal
The width in gap.It can be seen that the skeletonization of PDLFs controls the update of periodontium and the most important and correction of regeneration to differentiation
The key of alveolar bone remodeling during treatment.The research of Jacobs et al.]Just confirm that the intensity of mechanical stress can be changed PDLFs's
Skeletonization is to differentiation degree, after applying static mechanical stress 12h to it, when researcher observes that stress is 5%,
The expression quantity of the osteoblasts in vitro such as collagen-I, ALP, OCN is significantly increased.Therefore, people's parodontium is selected in this research
Fibroblast (human periodontal fibroblast cells, hPDLFs) is used as research object, probes into its biology
Characteristic is learned, and finds to have and hPDLFs skeletonization is promoted to intervene to the material of differentiation characteristic it, to reach quickening correction
Rate of tooth movement reduces the purpose for the treatment of time.HPDLFs in this project is to pass through primary point using traditional enzyme digestion
It is obtained from cultivating and carrying out immunocytochemical stain identification, ensure that the purity of experimental cell.
In view of these, we have carried out MTT test first, living to hPDLFs cell with the AgNPs for assessing various concentration
The influence of power.AgNPs particle used in this research is spherical in shape, and average diameter is 10nm (5-15nm).It is observed that making
After being handled hPDLFs cell 24 hours with the AgNPs that concentration reaches 250 μM, cell survival rate i.e. presentation significant decrease (69.00 ±
1.49% compared with the control, P < 0.001).Therefore, in research later, we select the AgNPs of 25-100 μM of concentration, into
One step assesses influence of the AgNPs to hPDLFs Osteoblast Differentiation.
Influence of the AgNPs to hPDLFs Osteoblast Differentiation:
Some researches show that, AgNPs can promote mesenchymal stem cell (bone mesenchymal stem cells,
BMSCs in-vitro multiplication and Osteoblast Differentiation), and promote union in vivo[31].The formation of bone there are two types of form, in film at
Bone and entochondrostosis, in the osteogenetic process of both forms, osteoblast all during bone matrix secretion and mineralising,
It played an important role[32].According to research report before, alkaline phosphatase (ALP) activity and osteocalcin (OCN) are inspections
Test the important indicator of osteoblast cultured in vitro[33-35].Runt associated transcription factor 2 (Runx2) is generally acknowledged at present most important
The control gene of Osteoblast Specific transcription factor, referred to as skeleton development and reconstruction[36].With the exception of Runx2 gene
And the hereditary disease occurred prompts us, the gene is steady in the development, reconstruction and interior environment of osteoblast differentiation maturation and bone
In a series of physiology courses such as fixed, complicated and accurate regulating and controlling effect is all played[37].Osterix (OSX) is located at Runx2's
Downstream is simultaneously regulated and controled by it, is played an important role in Osteoblast Differentiation latter stage and bon e formation[38].I-type collagen
(collagen-I) it is acted in osteoblast differentiation Late Effect Osteoblast Differentiation, to affect the Mineralization process of bone tissue[39]。
Influence and correlating markings developed by molecule water of research and inquirement various concentration range AgNPs in this part to hPDLFs cell Osteoblast Differentiation
Flat variation.
Experimental material
It is primary to be separately cultured Human Periodontal Ligament Fibroblasts (hPDLFs)；
Laboratory prepares Nano silver grain (AgNPs)
Experimental method
Cell culture and grouping
4-6 replaces osteogenic for cell, continues culture 7 or 21 days, carries out Osteoblast Differentiation induction (in osteogenic
Include 100nM dexamethasone, 50 μM of vitamin Cs and 10mM sodium β-glycerophosphate), and AgNPs (0 μ of various concentration is given simultaneously
M, 25 μM, 50 μM, 100 μM) to be intervened, replacement in osteogenic every 3 days is primary in experimentation.
Alkaline phosphatase (ALP) Activity determination
ALP activity in the 7th day detection hPDLFs of Osteoblast Differentiation.Group of cells is collected, ultrasound is broken under condition of ice bath
Broken, 420g is centrifuged 10 minutes, takes supernatant, carries out determining the protein quantity by BCA kit.Firstly, by the BSA egg of 0.5mg/ml
White titer is added on each hole of ELISA Plate according to 0,1,2,4,8,12,16,20 μ L, residual volume PBS buffer solution polishing to 20 μ L,
1 μ L of sample to be tested and 19 μ LPBS buffers mix.According to A liquid: B liquid volume ratio 50:1 prepares BCA working solution, and BCA is added in every hole
200 μ L of working solution, is placed in 37 DEG C of reaction 20min, and microplate reader 570nm wavelength surveys OD value.Each sample egg is calculated according to standard curve
White concentration, and with normal saline dilution at uniform concentration 2mg/ml.
Alizarin red staining
After Osteoblast Differentiation 21 days, group of cells fixes 20 minutes with 4% paraformaldehyde, then with 0.1% alizarin red S solution
Dyeing, microscopically observation record coloration result (100 ×).
Real-time PCR detects skeletonization marker mRNA expression
1ml TRIpure lysate is added into sample, mixes well, stands 5min at room temperature；Add 200 μ L chlorine
It is imitative, it mixes, stands 3min at room temperature；4 DEG C of 10000g are centrifuged 10min, and upper strata aqueous phase is taken to be transferred in new centrifuge tube；Be added etc.
The isopropanol of volume mixes, and is placed in -20 DEG C overnight；After 15h, 4 DEG C of 10000g are centrifuged 10min, abandon supernatant；1ml 75% is added
Ethyl alcohol, 4 DEG C of 3400g are centrifuged 3min, abandon supernatant；5-6min is stood at room temperature；Add 30 μ L RNase-free ddH2O, places
2min is mixed well, and is completely dissolved to its precipitating, gained is sample total serum IgE.It is surveyed using ultraviolet specrophotometer NANO2000
The concentration of RNA in fixed each sample.
Reverse transcription is carried out into cDNA referring to RNA sample of the kit specification to collection.Firstly, dense according to each sample rna
Degree, RNA volume need to be added by calculating every reaction system, guarantee that the RNA concentration of loading is consistent, successively to after processing without nuclease
1 μ L oligo (dT) is added in ice bath PCR pipe15, 1 μ L random (random primer) and respective volume RNA sample are finally mended
Fill ddH2O to overall reaction system be 12.5 μ L；70 DEG C of heating are after five minutes, 2 minutes cooling rapidly on ice, of short duration centrifugation aggregation
Reaction solution continuously adds 45 × Buffer of μ L and 0.5 μ LRnase inhibitor (RNase inhibitor), 2 μ into system
L dNTP (2.5mM each), 1 μ L (200U) reverse transcriptase (M-MLV), stirs and evenly mixs.Setting program: 25 DEG C of 10min, 42 DEG C
50min, 80 DEG C of 10min finally obtain 20 μ L cDNA templates.
1 μ LcDNA template is taken to carry out PCR detection.Primer is first diluted to 10 μM, every reaction system upstream and downstream primer adds
0.5 μ L adds 10 μ L SYBR GREEN mastermix, and residual volume is with ddH2O complements to 20 μ L.Primer entrusts Shanghai
The synthesis of Sheng Gong bioengineering Co., Ltd,
2.2.5Western blot detects skeletonization marker protein expression
Group of cells is collected, with the RIPA buffer cracking culture cell containing 1%PMSF, BCA method measures protein compression
Degree.It is shut being clamped after two pieces of clean glass plate alignment, is perpendicularly fixed on table top, prepares encapsulating.According to destination protein molecule
Measure size, this experiment need to prepare 5% concentration glue and 8%, 10%, 13% separation gel (formula see the table below).Separation gel position
It is preferential to prepare below gel, it is eventually adding APS, TEMED, encapsulating after mixing rapidly is sealed, static 30min with aqueous；Removal
APS, TEMED, encapsulating, comb sealing is added in hydraulic layer in prepared concentration glue.Static 30min extracts comb, in preparation
Sample.
Loading volume is 20 μ L, contains 40 μ g albumen, therefore with 5 × loading buffer and PBS diluted protein sample, it boils
It is denaturalized 5min, it is stand-by to obtain sample solution.Electrophoresis liquid is injected to the positive and negative electrode of electrophoresis tank, the above-mentioned electrophoresis sample solution of 20 μ L is added in every hole,
5 μ L albumen Marker are eventually adding, electrophoresis apparatus is connected, starting voltage 80V carries out constant pressure electrophoresis about 2.5h.
4 DEG C of buffer pre-coolings of transfer, sponge, filter paper transfer to soak in buffer, to the pvdf membrane progress side of appropriate size
It to label, is put into anhydrous methanol and soaks, then transfer buffer and infiltrate 20min.Electrophoresis is completed to take out gel, reference protein
Remaining blob of viscose is placed in transfer buffer and impregnates according to the molecular weight shear gel of destination protein and internal reference albumen by Marker
5-10min；Sponge, filter paper, gel, pvdf membrane (front face gel), filter paper and sea are successively completed since cathode is pressed from both sides in transfer
It is continuous.Transfer folder is fixed, is inserted into transfer groove, supplements enough transfer buffers, set maximum current, voltage 80V, under low temperature
Transfer 1.5h.
Taking-up pvdf membrane puts shake 5min in TBST buffer and transfers in confining liquid (the 5% of TBST buffer
Skimmed milk power solution) closing is immersed, shaking table 1h completes capping.Antibody is diluted to antibody working solution with 5% skimmed milk power and (incubates
The condition of educating see the table below).It takes out the pvdf membrane closed and antibody working solution is put into togerther in hybrid belt, after sealing, 4 DEG C were incubated for
Night.It is same to dilute secondary antibody working solution (incubation conditions see the table below) with 5% skimmed milk power, the pvdf membrane being incubated for after primary antibody is taken out,
It is added in TBST, shaking table elutes repeatedly, 5min × 4 time, is put into togerther in hybridization bag with secondary antibody working solution, seals, and carries out secondary antibody
It is incubated for, incubation conditions are 37 DEG C, 45 minutes.It takes out pvdf membrane to immerse again in TBST liquid, shaking table shakes elution, 5min × 6 time
The pvdf membrane is put into magazine afterwards.Two kinds of reagents in ECL luminescence reagent box are mixed in the ratio of 1:1, are added in magazine
On pvdf membrane and it is exposed.The time for exposure can be adjusted according to the protein band on film.
After exposure, pvdf membrane, distilled water flushing are taken out, shaking table elutes 2 times repeatedly.Cleared distilled water, addition are stripped off liquid and are flooded
No pvdf membrane, shaking table 15min, liquid is stripped off in evacuation, and distilled water flushing is clean.Shaking table in pvdf membrane immersion TBST buffer is taken out to shake
Dynamic 5min.It is transferred to confining liquid (5% skimmed milk power solution of TBST buffer) and immerses closing, shaking table shakes 1h and completes envelope
Close reaction.
5% (M/V) skimmed milk power is used to work according to the anti-β-actin antibody of 1:500 dilution proportion mouse as internal reference antibody
Liquid.It takes out the pvdf membrane closed and antibody working solution is put into togerther in hybrid belt, seal, 4 DEG C of overnight incubations.Equally with 5%
(M/V) pvdf membrane being incubated for after primary antibody is taken out in skimmed milk power dilution secondary antibody working solution (goat anti-mouse igg-HRP, 1:5000),
It after TBST elution, being put into togerther in hybridization bag, seals with secondary antibody working solution, carry out secondary antibody incubation, incubation conditions are 37 DEG C,
45min.After being incubated for internal reference antibody, takes out pvdf membrane and re-use TBST elution, shaking table shakes 5min × 6 time.ECL luminescence-producing reaction
Pvdf membrane is transferred in magazine afterwards, and carries out darkroom exposure.
Film after exposure is taken pictures scanning, is measured with gel images processing system (Gel-Pro-Analyzer software) each
Semi-quantitative analysis is carried out to image after band specificity gray value, with the ratio between purpose band and internal reference protein band light splitting density value
The reference frame of expressing quantity variation as a purpose.
Statistical analysis
The every group of experiment of this paper is at least repeated 3 times.All experimental datas are indicated with mean ± SD, are utilized SPSS (20.0)
Software carries out one-way ANOVA and Bonfroni parameter and is analyzed.P value is considered that there are significant differences less than 0.05.
Experimental result
HPDLFs alkaline phosphatase activities after AgNPs processing
By to different groups of hPDLFs cells, osteogenic induction culture after normal group and AgNPs intervene, ALP Activity determination
The results show that ALP activity dramatically increases (P < 0.01) in hPDLFs after being incubated for 7d jointly with 100 μM of AgNPs；In addition, we
It observes that ALP activity is increased in hPDLFs after 25 μM of AgNPs and 50 μM of AgNPs are incubated for, but there is no statistical difference.
Alizarin red staining detects hPDLFs Osteoblast Differentiation situation
By to different groups of hPDLFs cells, after normal group and AgNPs intervene after osteogenic induction culture 21d, 0.1% is alizarin
Plain red colouring liquid observes the formational situation of various cellular matrix mineralisings and calcium tubercle after dyeing to it.Microscopically observation result
It shows, after Alizarin red staining, compared with without AgNPs processing group, after 25 μM of -100 μM of AgNPs are incubated for 21d, in hPDLFs
The enhancing of calcium mineralising, it is consistent with ALP testing result
Real-time PCR detects skeletonization Research of predicting markers mRNA expression
By to different groups of hPDLFs cells, after normal group and AgNPs intervene after osteogenic induction culture 7d, Real-
Time PCR detects skeletonization Research of predicting markers mRNA expression, and experimental result is shown, is incubated for 7d through 25 μM of -100 μM of AgNPs
Afterwards, the expression of ALP, Runx2, OCN, OSX and collagen-ImRNA are raised in hPDLFs.
Western blot detects skeletonization marker protein expression
By to different groups of hPDLFs cells, after normal group and AgNPs intervene after osteogenic induction culture 7d, Western
Blot detects skeletonization associated marker proteins expression, and experimental result is shown, compared with without AgNPs processing group, through 25 μ
After M-100 μM of AgNPs is incubated for 7d, the protein expression level up-regulation of ALP, Runx2, OCN, OSX and collagen-I in hPDLFs.
Immunocytochemical stain display culture cytokeratin dyeing is negative, and vimentin stained positive can
It is accredited as fibroblast (hPDLFs)；MTT testing result shows that 250 μM and 500 μM of AgNPs have hPDLFs cell viability
It significantly inhibits.25-100 μM of AgNPs does not influence cell viability.Compared with without AgNPs processing group, 100 μM of AgNPs make
After 7d, ALP activity dramatically increases (P < 0.01) in hPDLFs；After 25 μM of -100 μM of AgNPs are incubated for 7d, ALP in hPDLFs,
The expression of Runx2, OCN, OSX and collagen-I mRNA are increased in concentration dependent, and Western blot testing result is therewith
Unanimously.
Claims (3)
1. the method for the Nano silver grain of Human Periodontal Ligament Fibroblasts Osteoblast Differentiation can be promoted, it is characterised in that: people can be promoted
The method of the Nano silver grain of periodontal ligament fibroblasts in vitro Osteoblast Differentiation, specifically:
The primary Human Periodontal Ligament Fibroblasts of normal isolation culture, pass through immunocytochemical stain cytokeratin and waveform silk
Albumen carries out cellular identification；Chemical reduction method prepares AgNPs, with different concentration (0 μM, 25 μM, 50 μM, 100 μM, 250 μM,
500 μM) AgNPs acts on hPDLFs, and MTT detects cell viability, determines the appropriate activity range of AgNPs；
(0 μM, 25 μM, 50 μM, 100 μM) processing hPDLFs of AgNPs of various concentration, and carry out osteogenic induction, alkaline phosphatase
(ALP) Activity determination determines that various concentration AgNPs acts on ALP activity change in lower hPDLFs；Alizarin red staining identifies that each group is thin
Born of the same parents' Mineral nodules formational situation；Real-time PCR and Western blot detection osteoblast differentiation marker (ALP,
Collagen-I, Runx2, OCN, OSX) mRNA and protein expression level.
2. the method for the Nano silver grain described in accordance with the claim 1 that Human Periodontal Ligament Fibroblasts Osteoblast Differentiation can be promoted,
It is characterized by: Nano silver grain (AgNPs), which refers to, accomplishes nanoscale metallic silver simple substance for partial size, usual partial size 1~
Between 100nm；
It is analyzed first against the AgNPs vigor for whether influencing periodontal ligament fibroblasts in vitro；
Experimental material:
It is primary to be separately cultured Human Periodontal Ligament Fibroblasts (hPDLFs)；
Laboratory prepares Nano silver grain (AgNPs)
Experimental method:
The synthesis of AgNPs:
Chemical reduction method is prepared Nano silver grain (AgNPs): the 0.1mM silver nitrate (AgNO of Xiang Hanyou 0.7mM sodium citrate3) molten
10mg sodium borohydride (NaBH is added in liquid4), then solution is stirred overnight, until by UV/Vis optical spectroscopy less than
Absorbance further increases at 400nm, final to obtain the AgNPs that concentration is 1mM；It is saturating using JEOL company high-resolution simultaneously
Radio mirror JEM-1400Flash and its highly sensitive sCMOS camera of outfit carry out the inspection of morphology and size to the AgNPs of synthesis
It surveys, operating voltage is set as 120kv；
The primary separation and culture of periodontal ligament fibroblasts in vitro:
People's parodontium has drawn from 20 medical for needing to have tooth pulled out because of correction of Chinese Medical Sciences University's stomatological hospital Shengang outpatient clinic
Patient, the age was in 12~18 years old range；After linking up, Written informed consent is signed, materials and during experimental implementation scheme passes through
The approval of Ethics Committee, medical university, state；The dental health of selection, no saprodontia or periodontal disease, patient gargles before having tooth pulled out；To tooth
Tooth extract after, first using physiological saline repeated flushing root surface to surface without blood after, put into it is sterile, containing low concentration it is dual anti-
PBS liquid in, and draw materials as early as possible in laboratory；Rear cell activity extends at any time and reduces tooth in vitro, generally with not
It was advisable more than 6 hours；The scraping for carrying out tissue block first, in moving into tooth in sterile petri dish in superclean bench, PBS is rushed
It washes 5 times, removes blood stains；A small amount of DMEM culture solution is added dropwise to keep diseased Root Surfaces wet, carefully scrapes root of the tooth with sterile razor blade back
In 1/3 position parodontium, the tissue block that will be scraped do not shred, is transferred directly in 15ml centrifuge tube, is added and contains mass fraction
0.05%I Collagen Type VI enzyme solution 4ml digests tissue block, is placed in 37 DEG C of water-baths and is incubated for；Every 5min shakes 1 time, digests altogether
30min, 156g are centrifuged 5min, remove supernatant, collect tissue block；4ml PBS is washed 2 times, and centrifugation removal supernatant collects tissue block；It connects
Kind is added DMEM complete medium in 6 orifice plates (10%FBS+DMEM+1% is dual anti-)；It is placed in 37 DEG C, 5%CO2Saturated humidity training
Case culture is supported, liquid is changed within the 2nd day, retains supernatant renewed vaccination into new hole, continues to cultivate；It is covered with to cell, 1mlPBS cleaning 1
It is secondary, 1min is digested with 0.25% pancreatin of 1ml, complete medium terminates digestion, gently blows and beats cell, cell suspension is collected into
In 15ml centrifuge tube, 100g is centrifuged 3min, removes supernatant, and 1:2 passage is inoculated into 6 orifice plates；Take 4-6 for cell for this research
Scheme；
Cell processing and grouping:
4-6 replaces osteogenic for cell, and carrying out Osteoblast Differentiation induction (includes 100nM dexamethasone, 50 in osteogenic
μM vitamin C and 10mM sodium β-glycerophosphate), and give simultaneously various concentration AgNPs (0 μM, 25 μM, 50 μM, 100 μM,
250 μM, 500 μM) to be intervened, replacement in osteogenic every 3 days is primary in experimentation；
Immunocyte dyeing:
Pass through immunocytochemical stain detection cytokeratin (cytokeratin) and vimentin (vimentin) identification
hPDLFs；HPDLFs, which is seeded on 12 orifice plates for be covered with glass slide, carries out cell climbing sheet, and 4% paraformaldehyde is added and fixes 15 minutes
After remove, be added 0.01M PBS buffer solution soaking and washing 3 times, each 5min continuously adds 0.1%Triton after outwelling PBS
X-100 removes Triton X-100 and PBS in the static incubation 20min of room temperature, and 3%H is added2O2It is incubated at room temperature 15min, in elimination
Endogenous peroxidase enzymatic activity rejoins PBS soaking and washing later, and normal serum is added, and blood is removed in incubation at room temperature 15min hypsokinesis
Clearly, without cleaning；Primary antibody cytokeratin and vimentin antibody are diluted with PBS according to 1:200 respectively, have been added dropwise to
All standing cell, 4 DEG C overnight, is added PBS soaking and washing 3 times, removes primary antibody；PBS is removed, by biotin labeling goat antirabbit two
It is anti-to dilute 200 times with PBS, it is added dropwise to and cell is completely covered, 37 DEG C of incubation 30min are added PBS soaking and washing 3 times, remove two
It is anti-；PBS is removed, HRP labelled streptavidin PBS is diluted 200 times, is added dropwise to and cell is completely covered, be incubated at room temperature
30min is added PBS soaking and washing 3 times, removes HRP labelled streptavidin；PBS is removed, colour reagent is added dropwise into 12 orifice plates
(being made of DAB colour reagent A, B mixed in equal amounts) sops up DAB when color just deepens, to 12 orifice plates to cell is completely covered
Middle addition distilled water impregnates 5min；It removes, haematoxylin is added to cell is completely covered, dyes 5min, immerses flowing water in tap water
Rinse 2min；1% hydrochloride alcohol differentiation 2s is added dropwise, immerses in tap water immediately, the anti-indigo plant 20min of flowing water；50% glycerol (glycerol:
Distilled water=1:1) mounting microscopy；It takes pictures under microscopically observation dyeing effect, 200 × mirror (100 μm of length of the scale)；
MTT detects cell viability:
Cell viability is measured with mtt assay, culture Human Periodontal Ligament Fibroblasts to density are 90% or so, and proper volume is added
0.25% trypsin digestion cell is added complete medium and terminates reaction after cell rounding；Mixed liquor is collected to 15ml test tube,
90g is centrifuged 3 minutes；Supernatant is removed, the complete medium of 1ml is added, cell count is carried out, with 4 × 103Cells/well density is to 96
Inoculating cell in orifice plate co-cultures for 24 hours according to the AgNPs of experimental group and various concentration (0,25,50,100,250,500 μM)
MTT detection is carried out afterwards, and culture medium of the replacement containing 0.5mg/mL MTT continues culture 4 hours；150 μ L are added after sucking supernatant
DMSO is protected from light microplate reader after standing 10min and measures its OD value at 570nm, counted to dissolve plastidogenetic purple crystal
According to analysis.
3. the method for the Nano silver grain described in accordance with the claim 1 that Human Periodontal Ligament Fibroblasts Osteoblast Differentiation can be promoted,
It is characterized by: the measuring method that AgNPs influences hPDLFs Osteoblast Differentiation:
Influence and correlating markings developed by molecule level variation of the various concentration range AgNPs to hPDLFs cell Osteoblast Differentiation；
Material and method:
Experimental material:
It is primary to be separately cultured Human Periodontal Ligament Fibroblasts (hPDLFs)；
Laboratory prepares 2.2 experimental method of Nano silver grain (AgNPs)
Cell culture and grouping:
4-6 replaces osteogenic for cell, continues culture 7 or 21 days, carries out Osteoblast Differentiation induction and (includes in osteogenic
100nM dexamethasone, 50 μM of vitamin Cs and 10mM sodium β-glycerophosphate), and AgNPs (0 μM, 25 of various concentration are given simultaneously
μM, 50 μM, 100 μM) intervened, replacement in osteogenic every 3 days is primary in experimentation；
Alkaline phosphatase (ALP) Activity determination:
ALP activity in the 7th day detection hPDLFs of Osteoblast Differentiation；Group of cells is collected, ultrasonication under condition of ice bath,
420g is centrifuged 10 minutes, takes supernatant, carries out determining the protein quantity by BCA kit；Firstly, by the BSA albumen of 0.5mg/ml
Titer is added on each hole of ELISA Plate according to 0,1,2,4,8,12,16,20 μ L, residual volume PBS buffer solution polishing to 20 μ L, to
1 μ L of sample and 19 μ LPBS buffers mix；According to A liquid: B liquid volume ratio 50:1 prepares BCA working solution, and BCA work is added in every hole
Make 200 μ L of liquid, be placed in 37 DEG C of reaction 20min, microplate reader 570nm wavelength surveys OD value；Each sample albumen is calculated according to standard curve
Concentration, and with normal saline dilution at uniform concentration 2mg/ml；
Alizarin red staining；
After Osteoblast Differentiation 21 days, group of cells fixes 20 minutes with 4% paraformaldehyde, is then contaminated with 0.1% alizarin red S solution
Color, microscopically observation record coloration result (100 ×)；
Real-time PCR detects skeletonization marker mRNA expression:
1ml TRIpure lysate is added into sample, mixes well, stands 5min at room temperature；200 μ L chloroforms are added, are mixed
It is even, 3min is stood at room temperature；4 DEG C of 10000g are centrifuged 10min, and upper strata aqueous phase is taken to be transferred in new centrifuge tube；It is added isometric
Isopropanol, mix, be placed in -20 DEG C overnight；After 15h, 4 DEG C of 10000g are centrifuged 10min, abandon supernatant；75% ethyl alcohol of 1ml is added,
4 DEG C of 3400g are centrifuged 3min, abandon supernatant；5-6min is stood at room temperature；Add 30 μ L RNase-free ddH2O, places 2min, fill
Divide and mix, is completely dissolved to its precipitating, gained is sample total serum IgE；Various kinds is measured using ultraviolet specrophotometer NANO 2000
The concentration of RNA in this；
Reverse transcription is carried out into cDNA referring to RNA sample of the kit specification to collection；Firstly, according to each sample rna concentration, meter
RNA volume need to be added by calculating every reaction system, guarantee that the RNA concentration of loading is consistent, successively to the ice bath without nuclease after processing
1 μ L oligo (dT) is added in PCR pipe15, 1 μ L random (random primer) and respective volume RNA sample finally supplement
ddH2O to overall reaction system be 12.5 μ L；70 DEG C of heating are after five minutes, 2 minutes cooling rapidly on ice, and of short duration centrifugation aggregation is anti-
Liquid is answered, 45 × Buffer of μ L and 0.5 μ LRnase inhibitor (RNase inhibitor), 2 μ L are continuously added into system
DNTP (2.5mM each), 1 μ L (200U) reverse transcriptase (M-MLV), stirs and evenly mixs；Setting program: 25 DEG C of 10min, 42 DEG C
50min, 80 DEG C of 10min finally obtain 20 μ L cDNA templates；
1 μ L cDNA template is taken to carry out PCR detection；Primer is first diluted to 10 μM, every reaction system upstream and downstream primer adds 0.5 μ
L adds 10 μ L SYBR GREEN mastermix, and residual volume is with ddH2O complements to 20 μ L.
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| CN110938669A (en) * | 2019-12-20 | 2020-03-31 | 南昌大学第二附属医院 | Method for verifying stimulation of osteogenic differentiation of valve interstitial cells by inorganic phosphate |
| CN114028616A (en) * | 2021-09-28 | 2022-02-11 | 遵义医科大学 | Preparation method of tissue engineering material for alveolar bone defect repair |
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| Title |
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| MIN WEI等: "Nanomaterials modulate stem cell differentiation: biological interaction and underlying mechanisms.", 《JOURNAL OF NANOBIOTECHNOLOGY》 * |
| 张杨珩等: "金纳米对人牙周膜成纤维细胞成骨分化的作用研究", 《2016 牙周病学学术研讨会》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110938669A (en) * | 2019-12-20 | 2020-03-31 | 南昌大学第二附属医院 | Method for verifying stimulation of osteogenic differentiation of valve interstitial cells by inorganic phosphate |
| CN110938669B (en) * | 2019-12-20 | 2023-03-28 | 南昌大学第二附属医院 | Verification method for stimulating osteogenic differentiation of valve interstitial cells by inorganic phosphate |
| CN114028616A (en) * | 2021-09-28 | 2022-02-11 | 遵义医科大学 | Preparation method of tissue engineering material for alveolar bone defect repair |
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