CN106700099A - Colorless and transparent silk sericin hydrogel, stent prepared therefrom and application thereof - Google Patents
Colorless and transparent silk sericin hydrogel, stent prepared therefrom and application thereof Download PDFInfo
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Abstract
The invention relates to a colorless and transparent sericin hydrogel, which is prepared by utilizing HRP and H2O2 to catalyze silk sericin cross-linking. The invention also provides application of the silk sericin hydrogel in biomedical materials. The invention also provides a stent obtained by freeze-drying of the silk sericin hydrogel. The invention also provides the role of the stent in in-vitro construction of a three-dimensional tumor model, in-vitro construction of a cell culture model, study of drug resistance of tumors, and screening of new drugs. The silk sericin hydrogel provided by the invention and the stent prepared therefrom can be used for carrying cells, especially tumor cells, and establishing a three-dimensional tumor model, and because of the colorless and transparent characteristics, the sericin hydrogel and the stent provided by the invention are conducive to observation and tracing of the constructed three-dimensional tumor model, thus better studying tumor cells and the development biological behaviors of tumor tissues.
Description
Technical field
The present invention relates to tissue engineering material field, more particularly, it is related to a kind of sericin hydrogel of water white transparency
And its support and the application of preparation.
Background technology
Hydrogel (Hydrogel) is the gel with water as decentralized medium, the water soluble polymer with cross-linked network
In there are hydrophobic grouping and hydrophilic radical, hydrophilic radical and water molecules, hydrone is connected to netted inside, and hydrophobic group
The cross-linked polymer of group's water-swellable.
Sericin (Silk Sericin) is wrapped around a kind of natural macromolecular viscous protein on fibroin fiber top layer, about
Account for the 20-30% of silk cocoon content, by molecular weight for the polypeptide of 24-400kDa is constituted, its molecule by serine, asparatate and
18 kinds of amino acid compositions such as glycine.In recent years, due to the good biocompatibility of sericin, to cell there is adhesion and protects
The biology performances such as shield effect, as the emerging material of biomedical sector.
The crosslinking of protein is broadly divided into two class methods, chemical crosslinking and enzyme crosslinking.Conventional chemical cross-linking agent has penta 2
Aldehyde and Geniposide.Being usually used in the enzyme of protein cross has TGase (TG) and polyphenol oxidase (PPO).
At present, sericin is mainly cross-linked into water using chemical cross-linking agent glutaraldehyde and natural biological crosslinking agent Geniposide
Gel stent, has no using the method for enzyme-catalyzed cross-linking to prepare hydrogel scaffold.
In China, cancer has turned into first of the disease cause of the death, and morbidity and mortality have turned into very in lasting rising, cancer
Important public health problem.China is pre- in respect of 429.2 million news hair tumor cases and 281.4 ten thousand deaths within 2015.
The growing multiplication of study tumor cell, invasion and attack, transfer and resistance, are the important means for finding cancer target and medicine.
Two dimension (2D) research model that traditional research institute for tumour mechanism of causing a disease and oncotherapy uses is to be based on
Two dimensional surface, it is difficult to the stereochemical structure and internal environment of true tumour in analogue body.If external three-dimensional (3D) tumour can be set up
Model, and stimulate for study tumor cell behavior and to external world and medicine response, then can more accurately reflect it is true in vivo
The situation of tumour.However, external 3D tumor models can be built currently without one kind, and suitable for such tumor model
Observed, the branch carrier material of spike and research.
The content of the invention
To solve problem above, the invention provides a kind of sericin hydrogel of water white transparency, it is by using HRP
And H2O2Catalysis sericin crosslinking obtains the sericin hydrogel and prepares.
Preferably, the working concentration of the HRP is 0.625-25U/mL, the H2O2Working concentration be
0.00075%-0.03%, the concentration of the sericin is 2.5-40mg/mL.
Preferably, the method that the sericin hydrogel passes through to comprise the following steps is obtained：
1) it is prepared into the sericin aqueous solution using the silk without bombyx mori silk fibroin；
2) H for being to addition concentration in the sericin aqueous solution2O2And HRP, obtain preformation glue；
3) by the preformation glue in 37 DEG C of plastics, the sericin hydrogel is obtained.
Preferably, the silk is the silk produced by bombyx mori silk fibroin deletion form silkworm.
Present invention also offers application of the above-mentioned sericin hydrogel in medical material.
Present invention also offers a kind of support obtained by above-mentioned sericin hydrogel freeze-drying.
The effect in three-dimensional nodule model is built in vitro present invention also offers above-mentioned support.
The effect in cell culture model is built in vitro present invention also offers above-mentioned support.
Present invention also offers effect of the above-mentioned support in drug resistance of tumor is studied.
Present invention also offers effect of the above-mentioned support in new medicament screen.
Sericin hydrogel of the invention and support prepared therefrom, are a kind of brand-new Biocomposite materials, no
It is same as the sericin hydrogel of other crosslinking agents formation, the silk gum that the present invention is formed using brand-new crosslinking agent and crosslinking method
Protein hydrogel is water white transparency.Based on this good characteristic, it can be used for following purposes：
1. set up 3D tumor models, more truly and relevantly in analogue body true tumour structure, be applied to tumour and cause a disease
Mechanism and the research of oncotherapy；
2., as cell culture model, the characteristic of its water white transparency can apply to the direct observation and spike of cell；
3. as tumor drug resistance model, more preferable simulation tumor drug resistance；
4., as medicine and the new screening for synthesizing compound and research platform, its water white transparency characteristic can be preferably direct
The corresponding medicine of spike, newly synthesize compound.
Sericin hydrogel of the invention and its prepared support can preferably in analogue body true tumour it is vertical
Body structure and tumor microenvironment, for the research of tumour mechanism of causing a disease, tumor drug resistance, oncotherapy is provided closer to true tumour feelings
The research model of condition, can also be used to 3D cell culture in addition, it is also possible to be applied to medicine and new synthesis compound screening and
Spike.
Brief description of the drawings
Fig. 1 is the schematic diagram that silk gum concentration influences on sericin hydrogel gelation time；
Fig. 2 is the schematic diagram that HRP concentration influences on sericin hydrogel gelation time；
Fig. 3 is H2O2The schematic diagram that concentration influences on sericin hydrogel gelation time；
Fig. 4 is the expansion rate of water absorption under sericin hydrogel scaffold condition of different pH；
Fig. 5 is the degraded situation schematic diagram of sericin hydrogel scaffold under condition of different pH；
Fig. 6 is the ESEM microstructure schematic diagram of the sericin hydrogel scaffold that different temperatures is freezed；
Fig. 7 is the aperture statistics block diagram of the sericin hydrogel scaffold that different temperatures is freezed；
Fig. 8 is the schematic appearance of sericin hydrogel prepared by different crosslinking agents；
Fig. 9 is the results of IR of sericin and sericin hydrogel；
Figure 10 is the fluorescence spectrum of sericin and sericin hydrogel；
Figure 11 is the light microscopic photo that sericin hydrogel carries cell；
Figure 12 is the adherent cell number statistical block diagram of sericin hydrogel；
Figure 13 is the cell viability statistics block diagram carried on sericin hydrogel；
Figure 14 is that phalloidine carries the immunofluorescence photograph that the β-actin on cell are dyeed to sericin hydrogel
Piece；
Figure 15 is the column statistical chart of the cell bulk area that sericin hydrogel is carried；
Figure 16 is the column statistical chart of the cell body girth that sericin hydrogel is carried；
Figure 17 is that the Laser Scanning Confocal Microscope of repopulating cell on sericin hydrogel scaffold is taken pictures schematic diagram；
Figure 18 carries electromicroscopic photograph, the HE stained photographs of the 3D sericin hydrogel scaffold ultra microstructures of HT-29 cells
With the HE stained photographs of Colorectal Carcinoma；
Figure 19 is the 1st day, the 5th day, the light of the 10th day of the 3D sericin hydrogel scaffold cultures for carrying HT-29 cells
Mirror photo and HE stained photographs；
Figure 20 is the dimensional structure diagram of 3D tumor models；
Figure 21 is the schematic diagram of the 3D sericins hydrogel scaffold and Colorectal Carcinoma for carrying HT-29 cells；
Figure 22 is the cell of 2D plane cultures and the cell of 3D sericin hydrogel scaffold cultures in nude mice by subcutaneous into knurl
The signal of experiment；.
Figure 23 is that 2D planes and 3D sericin hydrogel scaffold culture HT-29 cells show 5 FU 5 fluorouracil resistance situation
It is intended to；
Figure 24 is that 2D planes and 3D sericin hydrogel scaffold culture HT-29 cells show celecoxib resistance situation
It is intended to.
Specific embodiment
Principle of the invention and feature are described below in conjunction with example and accompanying drawing, example is served only for explaining this hair
It is bright, it is not intended to limit the scope of the present invention.
The preparation of sericin hydrogel and hydrogel scaffold
Sericin hydrogel of the invention and hydrogel scaffold are prepared by the following method：
1. the silk gum aqueous solution is prepared
Silk cocoon (be purchased from Inst. of Silkworm, Chinese Academy of Agricultural Sciences) of the silk from bombyx mori silk fibroin deletion mutation kind.Pass through
Following methods prepare the silk gum aqueous solution from the silk cocoon：
1) weigh silkworm mutating variety silk cocoon 1g and be cut into 1cm2Fragment, after be placed in cleaning beaker in, use ultra-pure water
Cleaning 3 times, 3500rpm is centrifuged 5 minutes removal moisture content；
2) to step 1) in add the concentration of 55mL for the LiBr aqueous solution of 6mol/L in the silk cocoon fragment that obtains, this is burnt
Cup is put into thermostat water bath 35 DEG C of water-baths 24 hours, dissolves sericin；
3) by step 2) obtain solution and be transferred in centrifuge tube 3500rpm being centrifuged 5 minutes, insoluble substance is removed with filter,
The solution clarified；
4) to step 3) the Tris-HCl buffer solutions (1mol/L, pH 9.0) of 1/4 volume are added in the settled solution that obtains；
5) by step 4) in solution be transferred in the bag filter (MWCO 3500) for having pre-processed, then by bag filter two
End clamp, is positioned in the beaker of the 0.001mol/L Tris-HCl buffer solutions containing pH 9.0；The beaker is placed in
Low rate mixing dialysis, a water was changed every 3 hours on agitator, was dialysed 48 hours altogether, wherein last time is saturating using ultra-pure water
Analysis；
6) in the sericin aqueous solution after dialysis in 5) being gone into centrifuge tube, 4000rpm is centrifuged 5 minutes, removal precipitation；
7) the sericin aqueous solution is reinstalled in bag filter and by bag filter two ends clamp, then by bag filter
It is placed in the PEG6000 solution that mass percentage concentration is 20% and concentrates；The sericin aqueous solution is concentrated to concentration it is about 2% and is
Only；
8) 4 DEG C of Refrigerator stores are placed in standby.
2. H is prepared2O2Working solution：By the H of 30% (v/v)2O2Storing liquid presses 1 with ultra-pure water:100 dilution proportion is
The H of 0.3% (v/v)2O2Working solution.
3. it is the HRP (lucifuge) of 1.25U/ μ L to prepare concentration, and 1mg HRP are equivalent to 250U.
4. sericin hydrogel is prepared
By H2O2Working solution, HRP the and 20mg/mL silk gum aqueous solution of 1.25U/ μ L presses 1:1:100 ratio is placed in after mixing
37 DEG C of plastics, are placed in 37 DEG C of incubators at least more than 30 minutes, wait it to be cross-linked to form hydrogel.
5. above-mentioned sericin hydrogel is put into mould, the cylindrical of thickness 1cm diameters 1cm is made, after plastic
- 80 DEG C are placed in, are taken out after freezing 24h；Sample is placed in into drying in frozen vacuum dryer, and (it is suitable to be determined according to sample size
Drying time), obtain sericin hydrogel scaffold.
Different silk gum concentration, various concentrations HRP, different H2O2Influence of the concentration to gelation time
According to the method described above and parameter, different silk gum working concentration (2.5,5,10,20,28 and 40mg/ are used respectively
ML), different HRP working concentrations (0.625,1.25,1.875,2.5,3.125,6.25,12.5 and 25U/mL) and preparation are not
Same H2O2Working concentration (7.5,15,22.5,37.5,75,150 and 300 μ L/mL), finally prepares different according to the method described above
Sericin hydrogel, determines its gelation time.Result as Figure 1-3, in the concentration of measured silk gum gelation time with
The concentration for sericin is raised and shortened, about 6-80s, and gelation time is with the concentration of HRP in the concentration of measured HRP
Raise and shorten, about 6-130s, in measured H2O2Concentration in gelation time with H2O2Concentration raise and extend, about
It is 44-120s.
37 DEG C of expansion rate of water absorption of support
Support made above is weighed, and is soaked in pH 3 respectively, pH 7.4 in the PBS of pH 11, is placed in 37 DEG C of leachings
Taken out after bubble 24h, determined as follows：
Wherein, Ws is the weight under swelling state, and Wd is dry weight.
Result is as shown in figure 4, the water absorbing properties of support are moderate.
The degraded of support
PH3 is placed a stent into, in the PBS of pH7.4, pH11, pH buffer solutions is changed daily, at shown time point
Sample is taken out, is dried, weighed and compared with initial weight, calculate degradation rate.Result is as shown in figure 5, in the slow of pH7.4 and pH11
It is very fast in degraded in first 30 days or so in fliud flushing, it is degradable at the 100th day or so.
The ultra microstructure of support
By sericin hydrogel respectively at -20 DEG C, -80 DEG C, freezed again after -196 DEG C of freezings and obtain support, using scanning
Electron microscope observation its ultra microstructure, as a result as shown in fig. 6, sericin hydrogel scaffold has a large amount of Micro porosity knots
Structure, these pore space structures can be used as extracellular matrix, there is provided the microenvironment of cell growth, the exchange of promotion nutriment.
The lyophilized influence to hole size of different temperatures
By scanning electron microscopic observation at -20 DEG C, -80 DEG C, the hole of the support freezed after -196 DEG C of freezings is measured and counted
Its hole size.As shown in Figures 6 and 7, with freeze temperature reduction, the aperture of support hole is smaller for result.The hole knot of support
Structure is the condition that nutrition and gas exchanges are carried out as pharmaceutical carrier and cell carrier.
The outward appearance of sericin hydrogel
The outward appearance of the sericin hydrogel synthesized using preceding method is as shown in figure 8, using HRP/H2O2The silk gum of crosslinking
Protein hydrogel appearance colorless is transparent, can be used as the direct observation of cell of cell culture model, it is also possible to be applied to fluorescence or mark
Remember the direct observation and spike of material, and have certain color cell can be influenceed to train using the sericin hydrogel of glutaraldehyde cross-linking
Foster direct observation and spike, the sericin hydrogel being crosslinked using Geniposide cannot carry out the direct sight of cell in black and blue color
Examine and spike, be not suitable for tumour culture model.
The infrared spectrum analysis of sericin and sericin hydrogel
Sericin and silk gum are determined using FTIS (Nexus, Thermal Nicolet, USA)
The characteristic peak of protein hydrogel.
As shown in Figure 9：Use HRP/H2O2Sericin secondary structure in the sericin hydrogel of crosslinking is without substantially change
Change, sericin hydrogel can well keep the conformation of natural silk gum.From characteristic peak Amide I, Amide II, Amide
III, Amide IV can be seen that sericin and the collection of illustrative plates of sericin hydrogel is essentially identical, and features described above peak is not bright
It is aobvious to change, show that the polypeptide secondary structure in sericin hydrogel is similar in pure silk glue protein, show sericin water
Gel can well maintain the native conformation of sericin.
The fluorescence spectrum test of sericin and sericin hydrogel
Using the exciting light of different wave length, the launching light of the full spectrum of sericin and sericin hydrogel is determined respectively
Intensity, as a result as shown in Figure 10, fluorescence intensity weakens after sericin forms sericin hydrogel, and its fluorescing frequencies is slightly
Micro- red shift.
Sericin hydrogel carries C2C12 cells (mouse muscle-forming cell)
Sericin hydrogel is prepared using foregoing method, is spread it evenly in Tissue Culture Dish, treat its plastic
Gently washed with sterile PBS buffer three times afterwards, then with 75% ethanol soak a few hours, sterile PBS buffer is then used again
Wash three times, will cultivate cell collect, it is resuspended, with 1 × 106Cell number plant in above-mentioned pre-coated sericin water
It is control with the culture dish without sericin hydrogel in the culture dish of gel, is placed in 37 DEG C, CO2Concentration is 5% cell training
Culture in case is supported, 0, cell take pictures and counting statistics under general light using microscope within 1,2 day, as a result such as Figure 11 institutes
Show, sericin hydrogel sertoli cell can be adhered to and bred well.
Respectively at the 4th hour, the adherent cell number of the 8th hour statistics control group and sericin hydrogel group, as a result
As shown in figure 12, sericin hydrogel have good biocompatibility and can sertoli cell adhere to.
Respectively at the 2nd day, cell viability was determined using MTT methods in the 3rd day, as a result as shown in figure 13, compared with control group,
The good no difference of science of statistics compared with normal culture dish of sericin hydrogel biocompatibility.
Repopulating cell is adhered to and survived on sericin hydrogel
Using above method by cell seeding in the culture dish of pre-coated sericin hydrogel, not to be coated with silk
The culture dish of glue protein hydrogel is placed in 37 DEG C, CO as control2Concentration be 5% cell culture incubator in culture 10 days after, adopt
Fixed with 4% paraformaldehyde, carry out phalloidin display β-actin, and nucleus is marked with DAPI, using laser copolymerization
Focusing microscope observation is taken pictures, and as a result as shown in figure 14, is counted the cell volume in all visuals field of taking pictures and is measured thin with Photoshop
Born of the same parents' size, statistics is as shown in Figure 15 and Figure 16.Compared with control group, sericin hydrogel sertoli cell can glue well
Echo survival.
Propagation of the repopulating cell on sericin hydrogel scaffold
Lyophilized support is prepared using foregoing method, lyophilized support is cut into thickness for 1mm, the shape of a diameter of 4.8mm,
The cell of culture is collected, with 10 μ L culture medium re-suspended cells, by 2 × 104Cell number plant in support center.That plants is steady
Turn GFP SHY5Y cells laser confocal microscope taken pictures at the 1st day, the 5th day and the 10th day respectively after show cell seeding
Situation.Result cell as shown in figure 17 in the bracket being capable of three-dimensional distribution growth.
Tumor model is built on hydrogel scaffold
During HT-29 cells are planted into above-mentioned sericin hydrogel scaffold as the method previously described, after culture 10 days, adopt
Fixed with 4% paraformaldehyde, FFPE, section, carry out HE dyeing, control group is with 1 × 10 by HT-29 cells6/ only
Cell number is inoculated in nude mice (the male mouse of 6-8 weeks) dorsal sc, as a result as Figure 18 displays use sericin hydrogel scaffold
The tumour cell of plantation is similar to the structure of tumor tissues, has extracellular matrix as tumor tissues, being capable of proper topotype
Intend the stereochemical structure and tumor microenvironment of tumor tissues.Taken pictures at the 1st day, the 5th day and the 10th day respectively, as a result such as Figure 19
It is shown.
The structure of the tumor model for building above
Tumor model established above is compared with the stereochemical structure of tumor tissues, as shown in the ideograph of Figure 20,
Sericin hydrogel scaffold set up 3D tumor models in (Figure 21), inside be necrotic area, outside be breeding blanket, this result with
The structure of tumor tissues is completely the same.
The speed of growth of tumor model
Foregoing method is respectively adopted to be cultivated on ordinary cells culture dish (2D) and 3D sericin hydrogel scaffolds carefully
Born of the same parents, cell viability was determined at the 5th day and the 10th day using mtt assay.By HT-29 cells with 1 × 106/ only cell number inoculation
In nude mice dorsal sc, the nude mice for being used is the male mouse of 6-8 weeks, is calculated as after into knurl the 0th day, and tumour is hereafter measured daily
Size, ratio is made with daily tumor size and the tumor size of the 0th day, calculates the internal propagation of tumour as internal group of (in
Vivo) as shown in figure 22, the tumor proliferation situation of the tumor model reflection that 3D sericins hydrogel scaffold is set up as a result is shown
Closer to the growing state of internal true tumour.
The drug resistance experiment of tumour cell
Foregoing ordinary cells culture dish (2D planes) is respectively adopted and 3D sericin hydrogel scaffold cultures HT-29 is thin
Born of the same parents, and with dosage be respectively that 5 FU 5 fluorouracil (5-FU) shown in Figure 18 and celecoxib process cell, determine thin using mtt assay
Born of the same parents' vigor reflects cells resistance situation, and as a result as shown in figs. 23 and 24, the 5 FU 5 fluorouracil (5-FU) and plug of same dose come
The cell that former times cloth acts on 3D model cultures after cell is higher than the cell survival rate that 2D is cultivated, and illustrates 3D sericin hydrogels
The cell of support culture more resistance, closer to the situation of true drug resistance of tumor cell.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all it is of the invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc. should be included within the scope of the present invention.
Claims (10)
1. the sericin hydrogel of a kind of water white transparency, it is characterised in that by using HRP and H2O2Catalysis sericin is handed over
Connection obtains the sericin hydrogel and prepares.
2. sericin hydrogel according to claim 1, it is characterised in that the working concentration of the HRP is 0.625-
25U/mL, the H2O2Working concentration be 0.00075%-0.03%, the concentration of the sericin is 2.5-40mg/mL.
3. sericin hydrogel according to claim 1 and 2, it is characterised in that the method by comprising the following steps
Prepare：
1) it is prepared into the sericin aqueous solution using the silk without bombyx mori silk fibroin；
2) H for being to addition concentration in the sericin aqueous solution2O2And HRP, obtain preformation glue；
3) by the preformation glue in 37 DEG C of plastics, the sericin hydrogel is obtained.
4. sericin hydrogel according to claim 3, it is characterised in that the silk is by bombyx mori silk fibroin deletion form
The silk that silkworm is produced.
5. application of the sericin hydrogel any one of claim 1-4 in medical material.
6. a kind of support, it is characterised in that the sericin hydrogel freeze-drying as any one of claim 1-4 is obtained
Arrive.
7. the support described in claim 6 builds the effect in three-dimensional nodule model in vitro.
8. the support described in claim 6 builds the effect in cell culture model in vitro.
9. effect of the support described in claim 6 in drug resistance of tumor is studied.
10. effect of the support described in claim 6 in new medicament screen.
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| CN110229530A (en) * | 2019-07-05 | 2019-09-13 | 青岛大学 | A kind of dual network silk fibroin hydrogel and preparation method thereof |
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| CN107840973B (en) * | 2017-10-20 | 2020-02-04 | 江苏科技大学 | Sericin hydrogel and preparation method and application thereof |
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