CN108660110A - A method of broken up using the aquagel evoked stem cell of bacteria cellulose piezoelectricity - Google Patents
A method of broken up using the aquagel evoked stem cell of bacteria cellulose piezoelectricity Download PDFInfo
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Abstract
The invention discloses a kind of methods using the aquagel evoked stem cell differentiation of bacteria cellulose piezoelectricity, are as follows：Sheet bacteria cellulose aquagel SDS deionized water solutions are handled, then are handled with NaOH deionized water solutions, then with the abundant soaking and washing of deionized water；Sheet bacteria cellulose aquagel small pieces are positioned in orifice plate；Disperseed with cell culture medium to obtain stem cell suspension after stem cell of the culture in Tissue Culture Flask is digested, the sheet bacteria cellulose aquagel surface of inoculation in the orifice plate, after culture 1 day~90 days, the content and expression with the relevant specific proteins of Neural Differentiation and gene into the cell is detected.Present invention firstly discovers that and disclose with the aquagel evoked stem cell of bacteria cellulose piezoelectricity to nerve or spongiocyte break up method, and by experiment prove sheet bacteria cellulose aquagel induce stem cell to nerve or spongiocyte differentiation effect it is good.
Description
Technical field
The present invention relates to a kind of methods more particularly to one using the aquagel evoked stem cell differentiation of bacteria cellulose piezoelectricity
The method that kind is broken up with the aquagel evoked stem cell of sheet bacteria cellulose piezoelectricity to nerve or spongiocyte, belongs to biomaterial
Technical field.
Background technology
With the development of society, Tissue Engineering Study field receives extensive attention and develops, CO2 laser weld and regeneration are
How one of most important one research contents realizes that stem cell becomes the important of people's research to the rapid induction of Neural Differentiation
One of content.The existing method for inducing neural differentiation of stem cells including the use of biochemical factors, inducing culture or
Biomaterial etc. come induce or promote stem cell to nerve or spongiocyte differentiation.But these method induction times are about 2~4
In week, the period used is longer, and this strongly limits CO2 laser welds and regenerated efficiency.
Numerous studies show that the Neural Differentiation of electro photoluminescence and stem cell is contacted with close, and electro photoluminescence can significantly improve
Stem cell and the relevant protein content of nerve and gene level.But existing electrical stimulus patterns are required for greatly real by external circuits
Existing, system is huge and complicated, it has not been convenient to apply.
Based on this, needing not rely on external circuits there is an urgent need for one kind can induce stem cell quickly to nerve cell or colloid
The method of cell differentiation.
Invention content
Induce stem cell to nerve or spongiocyte point using flexible piezoelectric material the purpose of the present invention is to provide a kind of
The method of change, the development for pushing CO2 laser weld and regeneration techniques.
To achieve the above object, the present invention adopts the following technical scheme that：
A method of broken up using the aquagel evoked stem cell of bacteria cellulose piezoelectricity, is as follows：
(1) by the sheet bacteria cellulose aquagel bought in Guilin Qi Hong Science and Technology Ltd.s with 1%~10% SDS
Deionized water solution handles 1h~for 24 hours at 30 DEG C~180 DEG C, and in 40 in 1%~10% NaOH deionized water solutions
DEG C~160 DEG C at handle 1h~8h, to be purified to sheet bacteria cellulose aquagel；It is fully impregnated with deionized water clear
It washes, the sheet bacteria cellulose aquagel after cleaning is cut into 0.5 × 0.5cm2~2 × 2cm2Small pieces it is spare；
(2) sheet bacteria cellulose aquagel small pieces are positioned in orifice plate, 75% alcohol is added and impregnates 12h wherein
~48h, while sterilization is carried out with ultra violet lamp 30min~180min in aseptic operating platform, with PBS by sheet bacterium
The alcohol of the small on piece of cellulose aquagel is cleaned spare；
(3) disperse to obtain stem cell with cell culture medium after being digested stem cell of the culture in Tissue Culture Flask and hang
Liquid, is 0.01ml~5ml by volume, and number of cells is 2 × 103~1 × 106The sheet of a cell suspension inoculation in the orifice plate
Bacteria cellulose aquagel surface after standing 10min~120min, supplements 0ml~5mL cell culture mediums, will do in the orifice plate
Cell is cultivated on sheet bacteria cellulose aquagel substrate, is ultrasonically treated 0min~60min daily, and replace within every 2 days primary
Cell culture medium；
(4) after cultivating 1 day~90 days, using immunofluorescence staining and real time fluorescent quantitative nucleic acid amplification, detection is thin
The content and expression of intracellular and the relevant specific proteins of Neural Differentiation and gene Tuj1 and GFAP.
Using having the beneficial effect that for above-mentioned technical proposal：
Cellulose is a kind of piezopolymer, bacteria cellulose soft texture therein and with higher crystallinity and poly-
Right, microstructure is in hyperfine Nanofiber Network structure, has higher biocompatibility, adaptability and good biology
Degradability, therefore the present invention selects material of the cellulose as inducing neural differentiation of stem cells.Piezoelectric material as it is a kind of by
Will produce the material of electric signal to mechanical force, ultrasonic wave can provide contactless mechanical force for piezoelectric material, so as to
It induces piezoelectric material and generates electric signal, therefore, piezoelectric material can provide contactless electric signal to cell for cell
Carry out electro photoluminescence.For the present invention by stem cell culture on flexible substrate, stem cell, which is prone to pattern, becomes nerve simultaneously
Like cell.Therefore, the sheet bacteria cellulose aquagel that the present invention selects is used for the nerve of stem cell as flexible piezoelectric material
Differentiation has remarkable result.
Further, the fiber bundle diameters of step (1) the sheet bacteria cellulose aquagel are 50nm~100nm, length
It it is 10 μm~100 μm, raw material is acetobacter, and solid content is 1%~5wt%, and has piezoelectricity.
Further, step (1) the sheet bacteria cellulose aquagel is as cell culture substrate, thickness 0.1mm
~5mm.
Preferably, step (2) described orifice plate is 48 orifice plates, 24 orifice plates, 12 orifice plates or 6 orifice plates.
Preferably, step (3) described stem cell is mesenchymal stem cell, adipose-derived mescenchymal stem cell or dental pulp
Mescenchymal stem cell.
Preferably, step (3) described cell culture medium is+10% newborn bovine serum of low sugar DMEM culture mediums or fetal calf serum
+ 1% is dual anti-；+ 10% newborn bovine serum of DMEM in high glucose culture medium or fetal calf serum+1% are dual anti-；Or α-DMEM culture mediums+10%
Newborn bovine serum or fetal calf serum+1% are dual anti-.
The main component of low sugar DMEM culture mediums is 1.0g/L glucose, amino acid, vitamin, inorganic salts and phenol red etc.；
The main component of DMEM in high glucose culture medium is 4.5g/L glucose, amino acid, vitamin, inorganic salts and phenol red etc.；α-DMEM trainings
The main component for supporting base is 1.0g/L glucose, amino acid, vitamin, inorganic salts, ribonucleotide, dezyribonucleoside and phenol red
Deng.Newborn bovine serum is by one kind that will be obtained after plasma removing fibrin after 12-24 hours new born bovine venous blood collections of being born
Nutriment for animal cell culture；Fetal calf serum is taken plasma removing fiber after blood by the puncture of age in August tire cor bovinum
A kind of nutriment for animal cell culture obtained after albumen；Dual anti-is Pen .- Strep solution (100 ×), green
The content of mycin is 10000U/ml, and the content of streptomysin is 10mg/ml.
Preferably, step (3) described condition of culture is 37 DEG C, contains 5%CO2Steam-laden moist environment.
The beneficial effects of the invention are as follows：(1) present invention firstly discovers that and disclosing and being lured with bacteria cellulose piezoelectricity hydrogel
The method that stem cell breaks up to nerve or spongiocyte is led, with the Neural Differentiation that flexible piezoelectric material is stem cell while being provided
Flexible cell culture substrate and contactless stimulating electrical signal have merged the flexibility and the spy of piezoelectricity of biomaterial itself
Point has pushed application of the flexible piezoelectric material in CO2 laser weld；It is experimentally confirmed that the bacteria cellulose piezoelectricity hydrogel of the present invention
The pattern of stem cell can be made Godwards through direction change after only 48h, and after continuing culture 14 days, compared to cell climbing sheet, carefully
Tuj1 the and GFAP contents of intracellular are all significantly improved on albumen and gene level；In the condition for not applying ultrasonic mechanical force
Under, the content of the stem cell Tuj1 genes on sheet bacteria cellulose aquagel has almost no change compared to cell climbing sheet, and
The content of GFAP genes increases 70 times compared to cell climbing sheet；Under conditions of applying ultrasonic mechanical force, sheet bacterial fibers
The content of stem cell Tuj1 genes on hydrogel increases to 1.7 times of cell climbing sheet, and the content of GFAP genes increases to carefully
The thirtyfold of born of the same parents' creep plate；Prove that sheet bacteria cellulose aquagel induces stem cell good to nerve or spongiocyte differentiation effect
It is good.
(2) method that induction stem cell disclosed by the invention breaks up to nerve or spongiocyte, compared to common biology
Chemokines and at nerve-inducing liquid, without the culture medium prescription of expensive growth factor and antibody and complexity, utilize market
Upper common sheet bacteria cellulose aquagel, can be by the method rapid induction stem cell of simple cell inoculation to nerve
Cell or spongiocyte differentiation.
(3) bacteria cellulose aquagel is generally acknowledged excellent bio-medical material, cheap, is readily produced, simultaneously
Since the method for the inducing neural differentiation of stem cells is simple and efficient, thus the method in the present invention can induce stem cell in batches
Neural Differentiation, to push the application of bio-compatible and biodegradable flexible piezoelectric material in CO2 laser weld organizational project.
In short, application of the bacteria cellulose piezoelectricity hydrogel disclosed by the invention in Stem Cells Neural reparation is repaiied for nerve
Multiple and regeneration provides a kind of more satisfactory method, with important application prospects；Meanwhile as cell culture substrate
Sheet bacteria cellulose aquagel derives from a wealth of sources, cheap, and production is convenient, is suitable for the production and application of batch, has wide
Development prospect.
Description of the drawings：
It in order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, below will be in embodiment 1
Attached drawing be briefly described, it should be apparent that, the accompanying drawings in the following description is only the embodiment of the present invention, for this field
For those of ordinary skill, without creative efforts, it can also be obtained according to the attached drawing of offer other attached
Figure.
1. Fig. 1 is scanning electron microscope (SEM) photo after sheet bacteria cellulose aquagel of the present invention freeze-drying；
2. Fig. 2 is observed after being freeze-dried for sheet bacteria cellulose aquagel of the present invention with piezoelectricity force microscope (PFM)
Surface topography map；
3. Fig. 3 is cell of the present invention culture on Solarbio cell climbing sheets；
4. Fig. 4 is stem cell of the present invention cultivates the cell morphology figure after 48h on sheet bacteria cellulose aquagel substrate；
5. Fig. 5 is stem cell of the present invention after being cultivated 14 days under the conditions of not applying ultrasonic mechanical force on cell climbing sheet
Tuj1 and GFAP protein immunization fluorescent staining results；
6. Fig. 6 be stem cell of the present invention on sheet bacteria cellulose aquagel substrate in not applying ultrasonic mechanical force condition
It is lower culture 14 days after Tuj1 and GFAP protein immunization fluorescent staining results；
7. Fig. 7 is Tuj1 of the stem cell of the present invention after being cultivated 14 days under the conditions of applying ultrasonic mechanical force on cell climbing sheet
With GFAP protein immunization fluorescent staining results；
8. Fig. 8 is stem cell of the present invention under the conditions of on sheet bacteria cellulose aquagel substrate in application ultrasonic mechanical force
Tuj1 and GFAP protein immunization fluorescent staining results after cultivating 14 days；
9. Fig. 9 be stem cell of the present invention on cell climbing sheet and sheet-like fiber hydrogel substrate in application or do not apply
The test result of Tuj1 gene contents after being cultivated 14 days under the conditions of ultrasonic mechanical force；
10. Figure 10 be stem cell of the present invention on cell climbing sheet and sheet-like fiber hydrogel substrate in application or do not apply
Add the test result of the GFAP gene contents after cultivating 14 days under the conditions of ultrasonic mechanical force.
Specific implementation mode
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.It should be understood that described, examples are merely exemplary, does not constitute any restrictions to the scope of the present invention.This field
Technical staff should be understood that without departing from the spirit and scope of the invention can to the details of technical solution of the present invention and
Form is modified or is replaced, but these modifications or substitutions each fall within protection scope of the present invention.
Embodiment 1
1. by the sheet bacteria cellulose aquagel bought in Guilin Qi Hong Science and Technology Ltd.s with 1% SDS deionizations
Aqueous solution handles 1h at 30 DEG C, is used in combination in 1% NaOH deionized water solutions and handles 1h at 40 DEG C with fine to sheet bacterium
Dimension hydrogel is purified；Sheet bacteria cellulose aquagel after purification is cut into after the abundant soaking and washing of deionized water
0.5×0.5cm2Small pieces it is spare.
To observe the microscopic appearance of sheet bacteria cellulose aquagel, after sheet bacteria cellulose aquagel is freeze-dried
It is observed with scanning electron microscope (SEM), photo is as shown in Figure 1, illustrate that bacteria cellulose is fibrous-network structure.
For the piezoelectric response of detection bacterium cellulose, by the bacteria cellulose after freeze-drying with piezoelectricity force microscope into
Row test, obtained piezoelectric signal figure is as shown in Fig. 2, show that bacteria cellulose has good piezoelectric property.
2. cell climbing sheet and sheet bacteria cellulose aquagel small pieces are positioned in orifice plate respectively, 75% alcohol is added simultaneously
12h is impregnated wherein, while carrying out sterilization with ultra violet lamp 30min in aseptic operating platform, is cleaned alcohol with PBS
It is spare；
It is hanged 3. disperseing to obtain stem cell with cell culture medium after stem cell of the culture in Tissue Culture Flask is digested
Liquid, by volume be 0.01ml, including number of cells be about 2 × 103The cell that is inoculated with respectively in the orifice plate of cell suspension climb
Piece and sheet bacteria cellulose aquagel surface after standing 10min, supplement 5mL cell culture mediums, stem cell are existed in the orifice plate
37 DEG C, contain 5%CO2Moist environment in cultivated on cell climbing sheet and sheet bacteria cellulose aquagel substrate, cultivate 48h
Afterwards, cytoskeleton dyeing observation cell morphology is carried out, obtained immunofluorescence dyeing result is as shown in Figure 3 and Figure 4, as a result table
Bright, it is in fusiformis to cultivate the cell on cell climbing sheet still, and the cell cultivated on sheet bacteria cellulose aquagel has been in
The pattern of existing nerve or spongiocyte illustrates that cellulose aquagel is likely to induction stem cell and Godwards breaks up through direction.
Embodiment 2
1. by the sheet bacteria cellulose aquagel bought in Guilin Qi Hong Science and Technology Ltd.s with 5.5% SDS go from
Sub- aqueous solution handles 12.5h at 105 DEG C, is used in combination in 5.5% NaOH deionized water solutions and handles 4.5h at 100 DEG C with right
Sheet bacteria cellulose aquagel is purified.With after the abundant soaking and washing of deionized water by sheet bacteria cellulose after purification
Hydrogel is cut into 1.25 × 1.25cm2Small pieces it is spare；
2. cell climbing sheet and sheet bacteria cellulose aquagel small pieces are positioned in orifice plate respectively, 75% alcohol is added simultaneously
30h is impregnated wherein, while carrying out sterilization with ultra violet lamp 105min in aseptic operating platform, is washed alcohol with PBS
It is net spare；
It is hanged 3. disperseing to obtain stem cell with cell culture medium after stem cell of the culture in Tissue Culture Flask is digested
Liquid, by volume be 2.505ml, including number of cells be about 5.01 × 105Cell suspension be inoculated in the orifice plate thin respectively
Born of the same parents' creep plate and sheet bacteria cellulose aquagel surface after standing 65min, supplement 2.5mL cell culture mediums, will do in the orifice plate
Cell contains 5%CO at 37 DEG C2Moist environment in cultivated on cell climbing sheet and sheet bacteria cellulose aquagel substrate, so
After be divided into ultrasound group and non-ultrasonic group：Ultrasound group is ultrasonically treated 30min daily, and non-ultrasound group is without supersound process, and every 2 days
Replace a cell culture medium；After culture 14 days, detected using immunofluorescence staining and real time fluorescent quantitative nucleic acid amplification
Into the cell with the content and expression of the relevant specific proteins of Neural Differentiation and gene Tuj1 and GFAP.Tuj1 and GFAP eggs
As viewed in figures 5-8, the results show that under conditions of not applying ultrasonic mechanical force, sheet bacterium is fine for white immunofluorescence dyeing result
GFAP protein contents on dimension hydrogel are significantly improved, and the content of Tuj1 albumen has almost no change；Applying ultrasound
Under conditions of mechanical force, the Tuj1 protein contents on sheet bacteria cellulose aquagel are significantly improved, and GFAP albumen contains
Amount is reduced compared to the sheet bacteria cellulose aquagel for not applying ultrasonic mechanical force.Tuj1 and GFAP gene contents are tested
As a result as shown in figs. 9-10, the results showed that, under conditions of not applying ultrasonic mechanical force, on sheet bacteria cellulose aquagel
The content of stem cell Tuj1 genes has almost no change compared to cell climbing sheet, and the content of GFAP genes increases compared to cell climbing sheet
50 times；Under conditions of applying ultrasonic mechanical force, the content of the stem cell Tuj1 genes on sheet bacteria cellulose aquagel
Increasing to 1.1 times of cell climbing sheet, the content of GFAP genes increases to 10 times of cell climbing sheet, but compared to not applying ultrasound
Sheet bacteria cellulose aquagel GFAP contents when mechanical force reduce.Therefore, the induction of sheet bacteria cellulose aquagel is dry thin
Born of the same parents are good to nerve or spongiocyte differentiation effect, and can more promote stem cell to star under conditions of not applying ultrasonic mechanical force
Shape spongiocyte breaks up, and can more mesenchymal stem cells into neurons be promoted to break up under conditions of applying ultrasonic mechanical force.
Embodiment 3
1. by the sheet bacteria cellulose aquagel bought in Guilin Qi Hong Science and Technology Ltd.s with 10% SDS deionizations
Aqueous solution handles in the NaOH deionized water solutions for be used in combination for 24 hours 10% at 180 DEG C and handles 8h at 160 DEG C with thin to sheet
Fungin hydrogel is purified；With after the abundant soaking and washing of deionized water by sheet bacteria cellulose aquagel after purification
It is cut into 2 × 2cm2Small pieces it is spare；
2. cell climbing sheet and sheet bacteria cellulose aquagel small pieces are positioned in orifice plate respectively, 75% alcohol is added simultaneously
48h is impregnated wherein, while carrying out sterilization with ultra violet lamp 180min in aseptic operating platform, is washed alcohol with PBS
It is net spare；
It is hanged 3. disperseing to obtain stem cell with cell culture medium after stem cell of the culture in Tissue Culture Flask is digested
Liquid, by volume be 5ml, including number of cells be about 1 × 106Cell suspension be inoculated with respectively cell climbing sheet in the orifice plate and
Sheet bacteria cellulose aquagel surface after standing 120min, supplements 0mL cell culture mediums, by stem cell 37 in the orifice plate
DEG C, contain 5%CO2Moist environment in cultivated on cell climbing sheet and sheet bacteria cellulose aquagel substrate, daily ultrasound at
Manage 60min；Culture 90 days after, using immunofluorescence staining and real time fluorescent quantitative nucleic acid amplification detection into the cell with nerve
Break up the content and expression of relevant specific proteins and gene Tuj1 and GFAP.
Each embodiment is described by the way of progressive in this specification, the highlights of each of the examples are with other
The difference of embodiment, just to refer each other for identical similar portion between each embodiment.For device disclosed in embodiment
For, since it is corresponded to the methods disclosed in the examples, so description is fairly simple, related place is said referring to method part
It is bright.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest range caused.
Claims (6)
1. a kind of method using the aquagel evoked stem cell differentiation of bacteria cellulose piezoelectricity, which is characterized in that specific steps are such as
Under：
(1) sheet bacteria cellulose aquagel is handled with 1%~10% SDS deionized water solutions at 30 DEG C~180 DEG C
1h~for 24 hours, and 1h~8h is handled at 40 DEG C~160 DEG C in 1%~10% NaOH deionized water solutions；Then spend from
Sheet bacteria cellulose aquagel after cleaning is cut into 0.5 × 0.5cm by the sub- abundant soaking and washing of water2~2 × 2cm2Small pieces
It is spare；
(2) sheet bacteria cellulose aquagel small pieces are positioned in orifice plate, 75% alcohol of addition and wherein immersion 12h~
48h, while sterilization is carried out with ultra violet lamp 30min~180min in aseptic operating platform, it is with PBS that sheet bacterium is fine
The alcohol of the dimension small on piece of hydrogel is cleaned spare；
(3) disperseed with cell culture medium to obtain stem cell suspension after being digested stem cell of the culture in Tissue Culture Flask,
It is 0.01ml~5ml by volume, number of cells is 2 × 103~1 × 106The sheet bacterium of a cell suspension inoculation in the orifice plate
Cellulose aquagel surface after standing 10min~120min, supplements 0ml~5mL cell culture mediums, by stem cell in the orifice plate
It is cultivated on sheet bacteria cellulose aquagel substrate, is ultrasonically treated 0min~60min daily, and every 2 days replace a cell
Culture medium；
(4) after cultivating 1 day~90 days, content and the expression with the relevant specific proteins of Neural Differentiation and gene into the cell is detected
It is horizontal.
2. a kind of application of the bacteria cellulose piezoelectricity hydrogel according to claim 1 in Stem Cells Neural reparation,
It is characterized in that, for step (1) the sheet bacteria cellulose aquagel as cell culture substrate, thickness is 0.1mm~5mm.
3. a kind of method using the aquagel evoked stem cell differentiation of bacteria cellulose piezoelectricity according to claim 1,
It is characterized in that, step (2) described orifice plate is 48 orifice plates, 24 orifice plates, 12 orifice plates or 6 orifice plates.
4. a kind of method using the aquagel evoked stem cell differentiation of bacteria cellulose piezoelectricity according to claim 1,
It is characterized in that, step (3) described stem cell is mesenchymal stem cell, adipose-derived mescenchymal stem cell or dental pulp mesenchyma
Stem cell.
5. a kind of method using the aquagel evoked stem cell differentiation of bacteria cellulose piezoelectricity according to claim 1,
It is characterized in that, step (3) described cell culture medium is+10% newborn bovine serum of low sugar DMEM culture mediums or fetal calf serum+1% pair
It is anti-；+ 10% newborn bovine serum of DMEM in high glucose culture medium or fetal calf serum+1% are dual anti-；Or+10% new born bovine of α-DMEM culture mediums
Serum or fetal calf serum+1% are dual anti-.
6. a kind of method using the aquagel evoked stem cell differentiation of bacteria cellulose piezoelectricity according to claim 1,
It is characterized in that, step (3) described condition of culture is 37 DEG C, contains 5%CO2Steam-laden moist environment.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110269957A (en) * | 2019-07-15 | 2019-09-24 | 天津赛尔康生物医药科技有限公司 | A kind of umbilical cord MSCs modified bacteria cellulose compound support frame material and preparation method thereof |
| CN112029725A (en) * | 2020-09-21 | 2020-12-04 | 山东大学 | Method for promoting macrophage polarization to M1 type by utilizing piezoelectric effect and application |
| WO2024098285A1 (en) * | 2022-11-09 | 2024-05-16 | 深圳先进技术研究院 | Exosome program-controlled tissue repair material and preparation method therefor |
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