CN108486153A - The application and method of FGF2 and 1 fusion of TGF-β in promoting silk cell-proliferation activity and anti-inflammatory properties - Google Patents
The application and method of FGF2 and 1 fusion of TGF-β in promoting silk cell-proliferation activity and anti-inflammatory properties Download PDFInfo
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- CN108486153A CN108486153A CN201810214294.6A CN201810214294A CN108486153A CN 108486153 A CN108486153 A CN 108486153A CN 201810214294 A CN201810214294 A CN 201810214294A CN 108486153 A CN108486153 A CN 108486153A
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Abstract
The application and method that the present invention relates to a kind of FGF2 and 1 fusions of TGF β in promoting silk cell-proliferation activity and anti-inflammatory properties, by expressing FGF2 and TGF β 1 simultaneously in Cocoon silk, and it cultivates and can secret out of the silk material containing the two functional proteins simultaneously, silk is assigned to promote the activity of cell Proliferation and adjust immunoinflammatory, and no cytotoxicity, it can be applied in field of medicaments as wound dressing raw material.
Description
Technical field
The invention belongs to biotechnologies, are related to FGF2 and are promoting silk cell-proliferation activity with 1 fusion of TGF-β
With the application in anti-inflammatory properties；It further relates to prepare the method for promoting cell-proliferation activity and anti-inflammatory properties silk.
Background technology
Post-traumatic wound healing is a long-term, complicated process.Wound healing phase is broadly divided into inflammatory stage, increases
Stage and structure remodelling phase are grown, many growth factors are required in each stage and participate in regulation and control, such as epidermal growth factor, blood
Platelet derivative growth factor, fibroblast growth factor, metastatic cells growth factor, interleukins etc..For example, research report
Road points out, basic fibroblast growth factor (Basic fibroblast growth factor, FGF2,16~18.5kD)
Can ERK accesses in active cell, the expression of enhancing transcription factor ElK1, to accelerate cell Proliferation, meanwhile, FGF2 also has
The effect that scar is formed is reduced, to promoting wound healing to play an important roll；On the other hand, metastatic cells growth factor
(Transforming cell growth factor, TGF-β 1 ,~12.5kD) take part in inflammatory in wound healing process,
In each stage such as proliferation and structure remodeling, the proliferation of cell can not only be promoted, moreover it is possible to adjust immune in wound healing process
Reaction, prevents wound inflammation.Therefore, TGF-β 1 is added to and helps effectively slow down biology in regenerating bone or cartilage biomaterial
The immunological rejection of material in vivo, and then accelerate soft symphysis.
Silk is protein fibre, has good biocompatibility, biodegradability and morphological plasticity, is good
Good biomaterial, is with a wide range of applications in medical tissue engineering field.From silkworm transgeneic procedure Establishing it
Afterwards, researcher develops in succession to turn in the specific expressed foreign protein including Bombyx mori posterior silkgland, middle division of silkgland
Gene expression system, and carried out the genetic improvement research of silk.For example, by natural silk layer specifically expressing spider silk-
The improvement of silk fusion protein obtains the stronger new silk of mechanical property；By in natural silk layer specifically expressing red fluorescence egg
In vain, the fluorescins such as green fluorescent protein improvement obtains colored new silk；And by silk specifically expressing people at
Fibroblast growth factor FGF1 improvement obtain can promote cell Proliferation, accelerating wound healing new function silk.At us
In the research of early period, the multi-gene expression system based on 2A cleavage of peptide (2A) is established, which can be in domestic natural silk gland and cocoon
The domestic natural silk gland 2A multi-gene expression systems for expressing multiple target genes in silk simultaneously, can simply and efficiently recombinantly express
Multiple target genes, and then the performance of silk is transformed.But it has no be in using domestic natural silk gland 2A multi-gene expression systems at present
The report of FGF2 and TGF-β 1 are expressed in silk cocoon silk simultaneously.
Invention content
In view of this, one of the objects of the present invention is to provide a kind of FGF2 and 1 fusion of TGF-β to promote silk thin
Application in born of the same parents' proliferation activity and anti-inflammatory properties；The second object of the present invention is that providing preparation promotes cell-proliferation activity and resist
The method of scorching function silk；The third object of the present invention is to provide promotes cell-proliferation activity and anti-inflammatory properties by the preparation
Silk made from the method for silk；The fourth object of the present invention is that providing silk is preparing promotion cell Proliferation and anti-inflammatory wound
Application in mouth dressing.
For achieving the above object, the present invention provides the following technical solutions：
Applications of the FGF2 with 1 fusion of TGF-β in promoting silk cell-proliferation activity and anti-inflammatory properties, the FGF2
Nucleotide sequence with 1 fusion of TGF-β is as shown in SEQ ID NO.1.
Preferably, the amino acid sequence of the FGF2 and 1 fusion of TGF-β is as shown in SEQ ID NO.2.
2. preparing the method for promoting cell-proliferation activity and anti-inflammatory properties silk, include the following steps：By FGF2 and TGF-β
1 fusion is connected into silkworm transgene expression vector, obtains injection plasmid, then injects silkworm seed, cultivates simultaneously screening transgenic man
Silkworm；The nucleotide sequence of the FGF2 and 1 fusion of TGF-β is as shown in SEQ ID NO.1.
Preferably, the preparation method of the recombinant vector is as follows：FGF2 and 1 fusion of TGF-β are connected into through BamHI and
The transition vector pSL1180 [Hr3Ser1DsRedSer1PA] of NotI digestions after then using AscI single endonuclease digestions, recycles target fragment
After be connected to expression vector Piggybac [3xp3DsRed SV40], obtain injection plasmid, be named as Piggybac [3xp3
DsRed SV40；Hr3Ser1FGF2-2A-TGF_β1Ser1PA].
Preferably, the method for the injection silkworm seed is：Mass ratio 1:1 configures plasmid to be injected and contains Piggybac swivel bases
Plasmid mixed liquor is injected into silkworm by the Help plasmid mixed liquors of enzyme using micro-manipulator and micro syringe under the microscope
In ovum.
Preferably, the culture and screening transgenic silkworm are that the silkworm seed after injection is placed in 25 DEG C and 90% relative humidity
Under hasten the hatching of silkworms 11 days after obtain G0 and wait for newly-hatched silkworm, raised with fresh mulberry leaf to being placed on small straw bundles to spin cocoons, after changing moth selfing obtain G1 to silkworm, wait for G1
After embryonic development 6 days, the silkworm for expressing DsRed marker gene in the eyes and nerve of silkworm embryos is screened, that is, obtains and turns base
Because of silkworm.
3. promoting silk made from the method for cell-proliferation activity and anti-inflammatory properties silk by described prepare.
4. the silk is preparing the application in promoting cell Proliferation and anti-inflammatory biomaterial.
The beneficial effects of the present invention are：The invention discloses FGF2 and 1 fusion of TGF-β, and silk cell to be promoted to increase
Grow activity and anti-inflammatory properties in application, the present invention will utilize domestic natural silk gland 2A multi-gene expressions system in Cocoon silk simultaneously
FGF2 and TGF-β 1 are expressed, and cultivates and can secret out of the silk material containing the two functional proteins simultaneously, assigns silkworm
Silk promotes the activity of cell Proliferation and adjusts immunoinflammatory, which can be used as wound dressing raw material in field of medicaments
It is applied.
Description of the drawings
In order to keep the purpose of the present invention, technical solution and advantageous effect clearer, the present invention provides following attached drawing and carries out
Explanation：
Fig. 1 is the cultivation and identification for turning FGF2 and 1 fusion silkworms of TGF_ β；(A：Transgene expression vector structure chart；
Hr3 is the enhancer of bombyx mori nuclear polyhydrosis virus, and Ser1-P is the endogenous sericin 1 gene promoter of silkworm, and Ser1PA is silkworm
Endogenous sericin 1 gene 3-UTR sequences, 3xp3DsRedSV40 are transgene carrier screening-gene；B：Transgenosis the selection result； C
With D：The normal silkworm of quantitative PCR detection and FGF2 in transgenic bombyx mori middle division of silkgland and 1 expressions of TGF_ β；E：Weight in cocoon shell
The detection of expression of histone FGF2 and TGF_ β 1).
The SEM that Fig. 2 is transgenosis silk FT observes (A：100 times of enlarged drawings；B：500 times of enlarged drawings).
Fig. 3 is normal cocoon shell and turns the infrared spectrum analysis of FT cocoon shells.
Fig. 4 is silk mechanics property analysis (A：Normal cocoon shell and the load-deformation curve for turning FT cocoon shells；B：Normal cocoon shell
With the fracture strength for turning FT cocoon shells；C：Normal cocoon shell and the elasticity modulus for turning FT cocoon shells；D：Normal cocoon shell and the poplar for turning FT cocoon shells
Family name's modulus).
Fig. 5 is the release characteristic (A that FGF2 and 1 albumen of TGF_ β are recombinated in transgenosis silk FT：Weight in transgenosis silk FT
The release of group FGF2；B：The release of TGF_ β 1 is recombinated in transgenosis silk FT).
Fig. 6 is that transgenosis silk promotes NIH3T3 cell Proliferations (A：After transgenosis silk FT is co-cultured with NIH3T3 cells
EdU coloration results；B：Live-Dead coloration results after transgenosis silk FT is co-cultured with NIH3T3 cells；C：CCK-8 is measured
Transgenosis silk FT and the growth rate after the co-cultivation of NIH3T3 cells).
Fig. 7 is that transgenosis silk FT cytotoxicities test (A with inflammatory：Transgenosis silk FT is co-cultured with NIH3T3 cells
Live-Dead coloration results are utilized afterwards；B：CCK-8 measures the growth speed after transgenosis silk FT is co-cultured with NIH3T3 cells
Rate；C：The expression of transgenosis silk FT and iNOS in cell after the co-cultivation of Raw264.7 cells；D：Transgenosis silk FT with
Raw264.7 cells co-culture the expression of TNF-α in wild Oryza species；E：Transgenosis silk FT and Raw264.7 cells are trained altogether
Support the expression of NO in wild Oryza species).
Fig. 8 is transgenosis silk FT anti-inflammatory activities (A and B：Quantitatively detect the expression of iNOS and IL-1 β；C and D：Carefully
The content of TNF-α and NO in born of the same parents' culture medium；E and F：The expression of iNOS and its quantitative analysis results in cell).
Specific implementation mode
Below in conjunction with attached drawing, the preferred embodiment of the present invention is described in detail.
The experiment material that the embodiment of the present invention uses is as follows：
Cell line：Mouse embryo cell NIH3T3, mouse macrophage Raw264.7.NIH3T3 cells and Raw264.7 are thin
Born of the same parents are cultivated using the DMEM culture mediums (Gibico) containing 10% fetal calf serum (Gibico).
Target gene：Human alkaline fibroblast growth factor (FGF2) and people's metastatic cells growth factor (TGF_ β 1).
Silkworm strain：Non- diapause breed variety D9L.
Embodiment 1, vector construction
According to people's bFGF genes (NCBI：NM_002006.4) with 1 gene (GenBank of people TGF_ β：E03028.1) amino
Acid sequence, after GSG-P2A connection bFGF and TGF_ β 1, and the alkali of the codon optimization fusion by silkworm specificity
Shown in basic sequence SEQ ID NO.1, amino acid sequence is as shown in SEQ ID NO.2；Fusion FGF2-2A-TGF_ β 1 are by gold
Si Rui companies provide synthesis.The segment got off using BamHI, NotI digestion is subcloned to transition vector pSL1180
[Hr3Ser1DsRed Ser1PA].Ser1 is the promoter of the specifically expressed sericin 1 gene of silkworm middle division of silkgland, and Hr3 is silkworm
Enhancer in NPV viruses, Ser1PA are the termination sequence of silkworm sericin 1 gene.And then AscI single endonuclease digestions are utilized, recycling
It is connected to final transgene expression vector Piggybac [3xp3DsRed SV40] after target fragment, obtains Piggybac
[3xp3DsRed SV40；Hr3Ser1FGF2-2A-TGF_ β 1Ser1PA], in carrier structure such as Fig. 1 shown in A.
The preparation of embodiment 2, transgenic bombyx mori
Transgenosis is injected plasmid and is prepared：Single bacterial plaque is chosen after the above-mentioned plasmid built is converted again and shakes bacterium 12h, is taken
10mL bacterium solutions extract plasmid using Qiagen extra pure regent boxes, and plasmid purity is measured using DNA concentration instrument, and 260/280 ratio exists
1.8~1.9, concentration 400~500ng/ μ l, it is spare.
It is prepared by the silkworm egg for being used to prepare transgenic bombyx mori：High-quality mulberry leaf raise silkworm to being placed on small straw bundles to spin cocoons, and after pupa time moth, are selfed
Mode mate 6~8 hours after, female moth through 4 ° refrigerate, it is spare.
The cultivation and screening of transgenic bombyx mori：With mass ratio 1:1 configures plasmid to be injected and contains Piggybac transposases
Help plasmid mixed liquors.Female moth is taken out, after its silkworm seed within 2h, under SZX16 microscopes (Olympus), is used
TransferMan NK2 micro-manipulators and 5247 micro syringes of Femto Jet (Eppendorf) inject plasmid mixed liquor.
Silkworm seed after injection is placed under 25 DEG C and 90% relative humidity hasten the hatching of silkworms 11 days after obtain G0 and wait for newly-hatched silkworm, raised meticulously with fresh mulberry leaf
It supports to set and be placed on small straw bundles to spin cocoons, be selfed after change moth and obtain G1 to silkworm.After G1 was for embryonic development 6 days, Olympus SZX12 fluorescent and stereos are used
Microscope (Olympus) screens the DsRed marker gene in the eyes of silkworm embryos, nerve, and the results are shown in Table 1.
1. transgenic bombyx mori of table injection statistics
The results show that 400 non-diapause silkworm seeds of silkworm polyvoltinism kind D9L of microinjection, after hastening the hatching of silkworms, 95 silkworms
Ovum normal incubation, hatching rate 24%, meticulously after raising to five ages, through pupa time, in the adult stage by obtaining 35 after being selfed oviposition
A G1 has been sieved to the positive moth circle (as shown in B in Fig. 1) that 18 eyes send out red fluorescence, positive rate to moth circle by screening
It is 51%.By Preliminary Identification, we are by the highest strain of exogenous protein expression amount (FT, Transgenic FT silkworm)
Subsequent research has been carried out as research object.
Embodiment 3, transgenic bombyx mori detection
1, quantitative PCR
The normal silkworms of WT and transgenic bombyx mori five ages middle division of silkgland are dissected, total serum IgE is carried out using total serum IgE extraction agent box
Extraction.2 μ g total serum IgEs are taken, reverse transcription is carried out using reverse transcription reagent box.Utilize SYBR Premix exTaq kits
(Takara) in 7000 quantitative PCR apparatus of ABI Fast (Applied Biosystems) to the endogenous sericin 1 gene in sample
(Ser1), the detection of the mRNA of 1 genes of FGF2 and TGF_ β.Using sw22934 as reference gene.RT-PCR primer table 1.
The quantitative primer that table 1. uses in testing
In quantitative PCR result such as Fig. 1 shown in C and D, the results show that relative to WT silkworms, at the middle part of transgenic bombyx mori FT
In sericterium, containing a large amount of FGF2mRNA and TGF_ β 1mRNA, be respectively relative to the expression quantity of endogenous Ser1 genes 8.25%,
7.74%.
2, Protein Detection
The extraction of cell protein sample：Culture medium is discarded, 1mL PBS (135mM NaCl, 2.7mM KCl, 1.5mM are utilized
KH2PO4With 8mM K2HPO4, pH7.4) cleaning cell 3 times, be added that 150 μ l newly match containing protease inhibitors PSPF's
RIPA lysates are placed in 30min on ice, and 13400rpm centrifuges 5min after collecting cell, and it is cell protein sample to take supernatant.
The extraction of silkworm middle division of silkgland protein sample：Dissection silkworm middle division of silkgland shreds, and 4 spend night extraction in immersion PBS.
Extract centrifuges 5min through 13400rpm, and it is silkworm middle division of silkgland protein sample to take supernatant；
The extraction of cocoon glutelin sample：Pulverizer crushes cocoon shell, takes 20mg cocoon powder, utilizes the protein extract (8M of 1mL
Urea, 25mM Tris-HCl, pH7.0) 30min is extracted in 80 degree, every 10min fully shakings are primary.Extract is through 13400rpm
5min is centrifuged, it is cocoon glutelin sample to take supernatant；
SDS-PAGE and Western Blotting：Above-mentioned cell sample, silkworm middle division of silkgland sample and cocoon shell sample are adopted
Protein concentration (Beyotime) is measured with enhanced BCA protein determination kits.The protein sample of quality such as take to carry out SDS-
PAGE electrophoresis, is detected using coomassie brilliant blue staining.After the completion of electrophoresis, protein sample is gone to by PVDF by transferring film instrument
Film washes film by the closing of 5% skimmed milk power, 5 PBST, applies primary antibody (anti-FGF2 antibody, anti-TGF-b1 antibody, anti-
GADPH antibody and anti-iNOS antibody), after 5 PBST wash film, apply the operations such as secondary antibody (anti-mouse or rabbit igg antibody), utilize
ECL Western Blotting Detection System (Amersham Biosciences) show the band on film.It exposes
Light mode uses automatic exposure.
In SDS-PAGE and Western blotting such as Fig. 1 shown in E, recombination is also detected in transgenosis cocoon shell FT
1 albumen of bFGF and TGF_ β of expression, content are respectively 1.79 μ g/g, 0.25 μ g/g.Show FGF2 and 1 fusions of TGF_ β
It is successfully transcribed, and has translated two independent albumen, this shows bioactive for it and provides the foundation.
3, Electronic Speculum is observed
It takes cocoon shell middle layer cocoon piece sample to carry out metal spraying processing, utilizes surface sweeping Electronic Speculum (Supra 55sapphire, Zeiss)
It is observed and is taken pictures.Process of taking pictures carries out at room temperature, voltage 3.0kV；Three independent samples clap 10 results.As a result
As shown in A in Fig. 2.The results show that in the structure of cocoon shell and gap, silk between transgenosis silk FT and normal silkworm WT
Size (WT with FT silk diameters be respectively 18.5 ± 0.4 μm, 19.3 ± 0.4 μm) etc. no significant difference.
4, infrared spectrum measurement
It takes silk cocoon cocoon shell middle layer cocoon piece as detection sample, then utilizes infrared spectrum measurement instrument (Thermo
Fisher scientific) it is measured；Each sample measures 30 times, carry out interpretation of result is averaged, as a result such as Fig. 3 institutes
Show.The results show that the infrared spectrum of transgenosis silk FT and normal silkworm WT have height between wave-length coverage 800~4000
The similitude of degree is shown in the secondary structures such as α spirals, β-pleated sheet inside silk also without significant difference.
5, mechanics performance determining
Silk sample is extracted from cocoon shell, is fixed on the paper tape of about 2cm notches, and mechanical property is carried out using universl tester
Measurement.Rate of extension is 1mm/min, 25 degree of test temperature, humidity 60%, each sample measurement 40 times.Measurement result is aobvious
Show, maximum fracture strength and the elasticity modulus of transgenosis silk FT are slightly less than normal silk, but between the two without conspicuousness
Difference (as shown in C in Fig. 2).The maximum fracture strength of WT and FT silks be respectively 158.4 ± 7.32Mpa, 139.0 ±
9.78Mpa；The maximum fracture elasticity modulus of WT and FT silks are respectively 14.89 ± 1.0%, 13.45 ± 1.02%；WT and FT
The Young's modulus of silk is respectively 37.98 ± 2.75Mpa, 38.77 ± 2.67Mpa (as shown in Figure 4).Show bright, transgenic silkworm
Silk FT is with natural silk no significant difference, the advantageous property with natural silk.
6, the characterization of transgenic bombyx mori silk FT
The FGF2 recombinantly expressed in transgenosis silk FT and 1 protein delivery features of TGF_ β are had detected using elisa technique,
The results are shown in Figure 5, the results show that the FGF2 and TGF_ β 1 in transgenosis silk FT effectively can be released slowly from silk
It releases.Within preceding 30h, only a small amount of FGF2 and TGF_ β 1 are released from silk, and rate of release is slow；
After 30h, the FGF2 and TGF_ β 1 in silk are gradually discharged with relative constant rate, finally, from 30mg transgenosis FT silks
In can release 1 recombinant proteins of TGF_ β of FGF2 and 4.2 μ g more than 11.9 μ g.
7, silk NIH3T3 cell proliferation experiments
Cocoon shell cuts the cocoon piece as a diameter of 0.5cm after layering, is sterilized by ultraviolet direct irradiation 6h.
The DMEM culture mediums of 0.25% serum of NIH3T3 cells are layered on 96 orifice plates, and per 500, hole cell, 100 μ l systems were cultivated
Night.It is put into after cocoon piece is soaked using culture medium in 96 orifice plates after continuously cultivating 1 day, 2 days, 3 days, it is careful to take out cocoon piece, with new
100 fresh μ l culture mediums replace old culture medium.The cell hole for containing FGF1 or FGF2 standard items albumen is set as positive right
According to group.The cell being proliferated is dyed using Click-iT EdU kits (Invitrogen)；Utilize Live-
Dead staining kits (Molecular ProbesTM) living cells and dead cell are dyed respectively；Utilize CCK-8 reagents
Box (Beyotime) analyzes the quantity of cell.It is handled 1 day using total serum IgE extraction agent box (Sigma) extraction each sample
RT-PCR of the total serum IgE of NIH3T3 cells afterwards for the later stage is tested, and the results are shown in Figure 6.
EdU coloration results are shown, compared to Null and WT group cells, the thin of danger signal occur in transgenosis cocoon piece FT groups
Born of the same parents' showed increased, it is close with the cell quantity of red fluorescent is showed in FGF2 standard item groups, illustrate to pass through transgenosis
The cell that cell division proliferation is occurring in cell after the FT co-cultivations of cocoon piece is more.Live-Dead staining kits can
Living cells is dyed into green, dead cell dyes red.It is found after NIH3T3 cell dyeings after being co-cultured to transgenosis cocoon piece FT,
It is presented the quantity of the living cells of green and is significantly more than the quantity of living cells in normal WT cocoons piece group, substantially and positive controls
Cell quantity after FGF2 standard items cultures is identical.Meanwhile the quantity that the dead cell of danger signal is showed between each group have no it is bright
Significant difference is different.In addition, we also have detected the proliferative conditions of each group cell using CCK-8 kit quantifications.The results show that altogether
1 day after culture, the cell quantity in transgenosis cocoon piece FT groups is all remarkably higher than Null and WT groups or even slightly higher with after 3 days within 2 days
In the cell quantity of positive control standard items FGF2 groups.The result of comprehensive EdU dyeing, Live-Dead dyeing and CCK-8 kits
It is found that transgenosis cocoon piece FT can significantly enhance proliferation and the growth of NIH3T3 cells.
8, silk is anti-inflammatory and cytotoxicity experiment
It is in order to detect the biological safety of transgenosis silk FT, the silk cocoon cocoon piece and NIH3T3 cells, Raw264.7 is thin
Born of the same parents are co-cultured, and whether observation transgenosis silk is enough to will produce cytotoxicity and inflammation inducing can react.Specific side
Method is：The DMEM culture mediums of 10% serum of Raw264.7 cells are layered on 24 orifice plates, per 500, hole cell, 500ul systems, and training
It supports overnight.The Escherichia coli 0111 of 25ng/ml are added:After the lipopolysaccharides (Sigma) of B4, put after cocoon piece is soaked using culture medium
Enter in 24 orifice plates, 8/hole, using standard items TGF-b1 as sample controls, continuous culture carefully takes out cocoon piece after 1 day, 2 days, directly
It is for use to pick up cell culture fluid.By the cocoon piece of transgenosis silk FT and NIH3T3 cell co-culture a couple of days, Live-Dead dyes
Color reagent box coloration result shows, the NIH3T3 cell quantities after the cocoon piece of FT co-cultures one day are less, and dead cell is less, works as training
After supporting the time to 3 days, 5 days, the quantity of NIH3T3 cells significantly increases, and at especially the 5th day, NIH3T3 cells have covered with
Entire culture plate, and there is not the case where a large amount of cells (as shown in A in Fig. 7).In addition, being tried using CCK-8 cell quantifications
Agent box detects the quantity of the cell after FT silks co-culture, it is found that is had no between its quantity and the NIH3T3 normally cultivated significantly
The quantity of difference, also, the extension of the incubation time with the time, cell increases sharply (as shown in B in Fig. 7), illustrates to turn base
Because silk FT has no cytology toxicity.After the cocoon piece of transgenosis silk FT and Raw264.7 cell co-culture a couple of days, detection
The expression of pro-inflammatory signal molecule nitrogen monoxide synthase iNOS in Raw264.7 cells, the results show that in transgenic silkworm
After 1 day, 2 days, a large amount of inflammatory signal, the training of FT silks are produced compared to LPS induction group cells with cell co-culture by silk FT
Group cell is supported as the cell normally cultivated, inducing macrophage does not generate serious inflammatory signal (such as C institutes in Fig. 7
Show).Content by detecting proinflammatory molecular weight tumor cell necrosis factor TNF-α and NO in cell culture medium also demonstrates
This viewpoint.Compared to the macrophage group normally cultivated, transgenosis silk FT co-culture 1 day, 2 days after culture in do not examine
A large amount of TNF-α is measured to generate with NO, and LPS induction group cells induce a large amount of TNF-α with NO (such as D and E institutes in Fig. 7
Show), illustrate that transgenosis silk FT will not the generation of inducing cell inflammatory.
Generally speaking, transgenosis silk FT does not have cytology toxicity and will not induce serious Cellular inflammatory reaction,
With the good biological safety of biology.
9, Cellular inflammatory is tested
After normal WT cocoons piece, transgenosis cocoon piece FT and standard items TGF_ β 1 are handled Raw2647 cells 1 day, detection is thin
The expression of pro-inflammatory signals molecule iNOS and IL-1 β in born of the same parents' inflammatory molecule access, the results are shown in Figure 8.RT-PCR results are aobvious
To show, LPS inductions group significantly makes Raw264.7 cells produce a large amount of inflammatory reaction, after 1 β standard items TGF_ is added,
INOS mRNA and IL-1 β mRNA expressions are significantly lower, and description standard product TGF_ β 1 have anti-inflammatory activity.WT cocoons piece is not
The iNOS mRNA that LPS inductions generate can be effectively reduced, and transgenosis FT cocoon pieces significantly reduce what LPS inductions generated
INOS mRNA, about 0.6 times；For IL-1 β, WT can effectively reduce the IL-1 β that LPS inductions generate with transgenosis FT cocoons piece,
But transgenosis FT cocoons piece has stronger activity compared to WT cocoon pieces.Further, we also have detected in cell culture also
The content of proinflammatory molecule TNF-α and NO.The results show that the Raw64.7 cells in LPS inductions are total to silk or standard items
After cultivating 6h, 12h, the content of the TNF-α in transgenosis cocoon piece FT group culture mediums is markedly inferior to normal cocoon piece WT groups and is lured with LPS
Lead the content in group culture medium, respectively the 0.64 of WT groups times, 0.78 times, and the content of NO is also significantly reduced, only WT
0.49 times, 0.66 times of group.In addition, I also has detected the expression of iNOS in Raw64.7 cells.The results show that when turning base
After 6h being co-cultured because of cocoon piece FT altogether with the Raw264.7 cells after induction, the iNOS in cell between WT and transgenosis cocoon piece FT
Expression and no significant difference, it is significantly low with the expression of the iNOS in the cell of transgenosis cocoon piece FT cultures after 12h
In normal WT groups, only its 0.59 times, illustrate that transgenosis cocoon piece FT can also significantly decrease LPS induction Raw264.7 cells and generate
INOS contents.To sum up result illustrates, transgenosis cocoon piece FT can significantly decrease LPS inductions as standard items TGF_ β 1
The expression for the iNOS, IL-1 β, TNF-α and NO that Raw264.7 cells generate illustrates that transgenosis silk FT has anti-inflammatory biology
Activity.
Finally illustrate, preferred embodiment above is merely illustrative of the technical solution of the present invention and unrestricted, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Sequence table
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<120>The application and method of FGF2 and 1 fusion of TGF-β in promoting silk cell-proliferation activity and anti-inflammatory properties
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Claims (8)
- Applications of the 1.FGF2 with 1 fusion of TGF-β in promoting silk cell-proliferation activity and anti-inflammatory properties, feature exist In：The nucleotide sequence of the FGF2 and 1 fusion of TGF-β is as shown in SEQ ID NO.1.
- 2. application according to claim 1, it is characterised in that：The amino acid sequence of the FGF2 and 1 fusion of TGF-β As shown in SEQ ID NO.2.
- 3. preparing the method for promoting cell-proliferation activity and anti-inflammatory properties silk, which is characterized in that include the following steps：By FGF2 It is connected into silkworm transgene expression vector with 1 fusion of TGF-β, obtains injection plasmid, then inject silkworm seed, cultivates and screen and turn Gene silkworm；The nucleotide sequence of the FGF2 and 1 fusion of TGF-β is as shown in SEQ ID NO.1.
- 4. preparing the method for promoting cell-proliferation activity and anti-inflammatory properties silk according to claim 3, it is characterised in that：Institute The preparation method for stating recombinant vector is as follows：FGF2 and 1 fusion of TGF-β are connected into the transition through BamHI and NotI digestions to carry After then using AscI single endonuclease digestions, gene expression is connected to after recycling target fragment by body pSL1180 [Hr3Ser1DsRedSer1PA] Carrier Piggybac [3xp3 DsRed SV40] obtains injection plasmid, is named as Piggybac [3xp3 DsRed SV40；Hr3 Ser1 FGF2-2A-TGF_β1 Ser1PA]。
- 5. preparing the method for promoting cell-proliferation activity and anti-inflammatory properties silk according to claim 3, which is characterized in that institute Stating the method for injecting silkworm seed is：Mass ratio 1:1 configuration plasmid to be injected is mixed with the Help plasmids containing Piggybac transposases Plasmid mixed liquor is injected into silkworm seed using micro-manipulator and micro syringe by liquid under the microscope.
- 6. preparing the method for promoting cell-proliferation activity and anti-inflammatory properties silk according to claim 3, it is characterised in that：Institute State culture and screening transgenic silkworm be the silkworm seed after injection is placed under 25 DEG C and 90% relative humidity hasten the hatching of silkworms 11 days after obtain G0 waits for newly-hatched silkworm, is raised with fresh mulberry leaf to being placed on small straw bundles to spin cocoons, and is selfed after change moth and obtains G1 to silkworm, after G1 was for embryonic development 6 days, sieved The silkworm for expressing DsRed marker gene in the eyes and nerve of silkworm embryos is selected, that is, obtains transgenic bombyx mori.
- 7. being promoted made from the method for cell-proliferation activity and anti-inflammatory properties silk by described prepare of any one of claim 3~6 Silk.
- 8. silk described in claim 7 is preparing the application in promoting cell Proliferation and anti-inflammatory wound dressing.
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