CN110038160B

CN110038160B - Scaffold material with function of delaying cell aging and application thereof

Info

Publication number: CN110038160B
Application number: CN201910272795.4A
Authority: CN
Inventors: 尤蓉; 关燕清; 石璐; 陈素容
Original assignee: South China Normal University
Current assignee: South China Normal University
Priority date: 2019-04-04
Filing date: 2019-04-04
Publication date: 2021-09-24
Anticipated expiration: 2039-04-04
Also published as: CN110038160A

Abstract

The invention discloses a scaffold material with the function of delaying cell aging and application thereof, wherein the scaffold material is a polylactic acid-caprolactone scaffold material grafted with resveratrol. The characteristics and cell culture researches of the scaffold material provided by the invention show that the modified scaffold material not only overcomes the defects that the scaffold body is hydrophobic and the pores are too small to be beneficial to cell growth due to the addition of hydrophilic groups, but also can obviously delay cell aging. The material modification method is simple and easy to implement, has obvious effect, is used for nourishing liver seed cells, has the effects of delaying cell senescence and improving the activity of the seed cells, and lays a good foundation for preparation of liver tissue engineering. Provides a new method for solving the problem of cell life in the research of liver tissue engineering.

Description

Scaffold material with function of delaying cell aging and application thereof

Technical Field

The invention relates to the technical field of tissue engineering, in particular to a scaffold material with a function of delaying cell senescence and application thereof.

Background

China is a region with high incidence of liver diseases, and various liver diseases seriously harm human health. Liver transplantation is currently the major approach to treat acute and chronic liver failure as well as other severe liver diseases. However, limited by the source of the liver donor, clinical application of the liver donor is extremely difficult. As an ideal way for sustainably providing liver tissues, tissue engineering is expected to become an effective scheme for solving the clinical dilemma of liver diseases. Liver tissue engineering is an emerging discipline generated by combining the principles of biology and engineering, and the core of the liver tissue engineering is to establish a three-dimensional space complex of cells and biological materials, namely a vital living tissue, so as to reconstruct the form, structure and function of a damaged tissue and achieve permanent replacement.

In the tissue engineering research for constructing transplantable liver, in the in vitro culture process of seed cells, due to the influence of factors such as long-term culture, passage, nutrition, dissolved oxygen and the like, the seed cells planted on the scaffold material are easy to age and stop secreting matrix, so that the phenomena of cell aging and subsequent death occur in a large area, and the calcification is caused by the death of the large area of seed cells and the constructed tissue engineering is prevented from being applied to living bodies. Therefore, the method is a key problem to be solved urgently in the research of liver tissue engineering.

Cellular senescence is a cellular response program in which cells are activated after being subjected to certain specific stimuli. There has been long-term effort to study various phenotypic characteristics of cells after senescence and the molecular mechanisms and signaling pathways that can activate the senescence process. Research shows that cell senescence can be induced by shortening telomerase, genome damage, activation of tumor inhibition signals and the like. Once the cell is senescent, the cell morphology changes: the cells become flattened, increased in volume, and accompanied by an increase in lysosomal activity (SA- β -Gal). Meanwhile, cell cycle arrest is at the stage of G0 or G1, and cell proliferation ability is reduced. The main signal pathways inducing cellular senescence include the Ras-p53 pathway, the p16-Rb pathway, and the p53/p 16-independent signal pathway, such as the SKP2-p 27-related signal pathway. Cell senescence plays an important role in limiting tumor development and treating fibrotic diseases, but influences seed cell viability and preparation of transplantable tissue engineering materials in tissue engineering research, and becomes an important bottleneck for in vitro preparation of in vitro liver tissue engineering.

In order to solve the problem of cell aging in tissue engineering, various research works for delaying cell aging and enhancing the activity of seed cells are continuously carried out, which mainly comprise: 1) the growth factors are utilized to promote cell proliferation and delay cell aging, for example, Haojie and the like cover Wharton jelly on a culture medium for delaying the aging of stem cells, wherein the Wharton jelly contains rich growth factors; 2) the application of the acellular scaffold, the acellular scaffold is a three-dimensional scaffold which is subjected to acellular treatment and retains a complete vascular network structure, and the scaffold structure provides a suitable microenvironment for the attachment and proliferation of cells and has the effects of promoting the growth of the cells and delaying the senescence of the cells; 3) the use of extracellular matrix (ECM) is a network of macromolecules synthesized and secreted by cells extracellularly, distributed on the cell surface or between cells, to provide a suitable environment for cell growth, promote cell proliferation and delay cell senescence. Therefore, the effect of delaying cell aging in the current research work is mainly realized by the cell growth promoting effect of the cell factor, but the practical application is limited by factors such as high cost, complex manufacturing and the like.

Resveratrol (RSV), a non-flavonoid polyphenolic compound, is a natural phytoalexin produced by plants under stress. It has the functions of preventing cardiovascular diseases, reducing blood fat, delaying senility, resisting cancer, etc. Various preparations, such as liposome, nanoparticle, micelle and other biological preparations, are developed to improve the bioavailability of resveratrol. Meanwhile, resveratrol has been primarily tried in bone tissue engineering due to its functions of delaying senescence and promoting osteoblast differentiation, for example, collagen/resveratrol scaffold material is developed in bone tissue engineering to facilitate bone tissue regeneration, which indicates that resveratrol can be loaded on the scaffold material for release to regulate cell growth. However, current studies show that for endothelial progenitor cells, low concentrations (<50 μmol/L) of resveratrol have a regulatory function in delaying cellular senescence, while high concentrations (>50 μmol/L) of resveratrol inhibit cell growth. Meanwhile, for mesenchymal stem cells, low-concentration resveratrol is proved to be capable of effectively delaying cell senescence through a SIRT1 pathway, is a natural activator of a senescence-associated protein SIRT1, and can act on an SIRT1 amino terminal activation domain in vivo to change a sequence of conservative glutamic acid to lysine or alanine in the domain, so that the expression of non-histidine proteins such as FOXO1/3, nuclear transcription factor (NF) -Kb and the like is increased, and the expression of senescence-associated proteins such as p53, p21 and the like is reduced.

However, there is no report on the application of the compound in liver tissue engineering research, and it is unclear whether the compound can also promote cell growth and delay cell senescence for liver seeds.

The natural material or the artificial high molecular polymer can provide a matrix environment for cells, and P (CL-DLLA) is used as a blending material of polylactic acid and polycaprolactone, has good toughness, degradation performance and the like, and is a proper scaffold material for liver tissue engineering. The scaffold P (CL-DLLA) has strong hydrophobicity and small pores, so that the scaffold P has the defect of being not beneficial to cell growth.

In summary, a suitable degradable scaffold which utilizes resveratrol and controls the release of the resveratrol is absent at present, the cell aging can be delayed, the cost is low, the method is simple, and the concentration is controllable, which is the first problem to be solved when the resveratrol is used for culturing liver seed cells.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a high molecular polymer scaffold material capable of promoting cell growth and delaying hepatocyte senescence and application thereof.

The first purpose of the invention is to provide a scaffold material for delaying cell aging.

The second purpose of the invention is to provide a preparation method of the scaffold material for delaying cell aging.

The third purpose of the invention is to provide the stent material prepared by the preparation method.

The fourth purpose of the invention is to provide the application of the scaffold material in liver tissue engineering and/or delaying cell aging.

In order to achieve the purpose, the invention is realized by the following technical scheme:

as shown in figure 1, in the invention, Benzophenone (BP) is used as an initiator, Acrylic Acid (AA) is modified on polylactic acid-caprolactone scaffold material (P (CL-DLLA)) through an ultraviolet light grafting method, the AA-P (CL-DLLA) with-COOH groups is dissolved in 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) ethanol solution, and Resveratrol (RSV) with the effect of delaying cell aging is grafted on the AA-P (CL-DLLA) through an esterification reaction to form the RSV-AA-P (CL-DLLA) scaffold material. The three-dimensional scaffold material is used for culturing hepatic seed cells, and the result shows that the scaffold material can obviously delay the senescence of the hepatic seed cells.

The infrared structural map shows that the stretching vibration peak of a benzene ring C ═ C exists in the RSV-AA-P (CL-DLLA stent material, the XPS structure shows that the C, O element proportion of the RSV-AA-P (CL-DLLA) is obviously different from that of the P (CL-DLLA) and the AA-P (CL-DLLA), initially shows that the RSV is successfully grafted on the AA-P (CL-DLLA), and the scanning electron microscope shows that the RSV-AA-P (CL-DLLA) stent has a complex surface/internal microenvironment and pores are enlarged, so that the defects that the adhesion growth of cells is not facilitated due to the fact that the raw material P (CL-DLLA) is hydrophobic, too small in pores and the like are overcome, and a good matrix environment can be provided for cell culture.

Rat Hepatic Stellate Cells (HSC) are inoculated on RSV-AA-P (CL-DLLA) stent materials and cultured for 4 days, and the content of RSV released on the stent materials is analyzed by HPLC, so that the RSV-AA-P (CL-DLLA) can be slowly released, and the maximum concentration of RSV released is about 6.0 mu mol/L; MTT results show that HSC cells on three materials can grow and proliferate normally, but the survival rate of the cells on RSV-AA-P (CL-DLLA) is the highest; ROS analysis of reactive oxygen species showed a lower number of senescent cells grown on RSV-AA-P (CL-DLLA) scaffold material. The comprehensive result shows that the RSV-AA-P (CL-DLLA) scaffold material has the function of delaying the senescence of hepatic seed cells.

Therefore, the invention claims a scaffold material for delaying cell aging, which is a polylactic acid-caprolactone scaffold material grafted with resveratrol.

Preferably, the support material is resveratrol grafted to the polylactic acid-caprolactone support material through carbonyl.

Meanwhile, the invention also claims a preparation method of the scaffold material for delaying cell aging, which grafts resveratrol on the polylactic acid-caprolactone scaffold material.

Preferably, the method comprises the following steps:

s1, performing acrylic acid modification on a polylactic acid-caprolactone support material;

s2, grafting resveratrol.

Wherein the structural formula of the polylactic acid-caprolactone is as follows:

the acrylic acid has the structural formula:

the polylactic acid-caprolactone scaffold material is prepared by acrylic acid through the following steps:

the product obtained after the minor-branch resveratrol is as follows:

in step S1, the polylactic acid-caprolactone scaffold material is modified by acrylic acid to introduce carbonyl,

in step S2, resveratrol is grafted to the polylactic acid-caprolactone scaffold material by esterification with the introduced carbonyl group.

Preferably, the polylactic acid-caprolactone scaffold material is subjected to acrylic acid modification by using an ultraviolet light grafting method.

More preferably, benzophenone is used as an initiator, and the polylactic acid-caprolactone scaffold material is subjected to acrylic acid modification by an ultraviolet light grafting method.

More preferably, the method comprises the following steps:

s11, soaking the polylactic acid-caprolactone support material in a benzophenone aqueous solution;

s12, after drying, placing the mixture under a high-pressure mercury lamp for irradiation;

s13, placing the ethanol solution soaked in the acrylic acid in an ultraviolet lamp for irradiation;

s14, washing and soaking by using a phosphate buffer solution, and drying to obtain the product.

Preferably, in step S11, the polylactic acid-caprolactone scaffold material is washed and then soaked in the benzophenone aqueous solution.

More preferably, in step S11, the washing step includes soaking the polylactic acid-caprolactone scaffold material in an aqueous solution of hydrogen peroxide (V/V) with a mass concentration of 2-5%, soaking for 4-6 min, and drying.

More preferably, in step S11, the washing is performed by immersing the polylactic acid-caprolactone scaffold material in an aqueous solution of hydrogen peroxide (V/V) having a mass concentration of 3%, immersing for 5min, and drying.

Preferably, in step S11, the concentration of benzophenone in the benzophenone aqueous solution is 0.30 to 0.50 mol/L.

More preferably, in step S11, the concentration of benzophenone in the aqueous solution of benzophenone is 0.40 mol/L.

Preferably, in step S11, the soaking time is 0.5-1.5 h.

More preferably, in step S11, the soaking time is 1 h.

Preferably, in step S12, the sample is irradiated by 10cm under a high-pressure mercury lamp.

Preferably, in step S12, the power of the high-pressure mercury lamp is 100-150W.

More preferably, in step S12, the power of the high-pressure mercury lamp is 125W.

Preferably, in step S12, the high-pressure mercury lamp is irradiated for 10-20 min.

More preferably, in step S12, the high-pressure mercury lamp is irradiated for 15 min.

Preferably, in the step S13, the concentration of the ethanol solution of acrylic acid is 3-5% (V/V).

More preferably, in step S13, the concentration of the ethanol solution of acrylic acid is 4% (V/V).

Preferably, in step S13, the ultraviolet lamp is irradiated for 30-60 min.

More preferably, in step S13, the ultraviolet lamp is irradiated for 45 min.

Preferably, in step S14, the pH of the phosphate buffer is 6.5-7.5.

More preferably, in step S14, the phosphate buffer has a pH of 7.

Preferably, the grafting of resveratrol comprises the following steps:

s21, softening the support material;

s22, placing the mixture into a 30-50% ethanol water solution (V/V) for reaction, wherein the 30-50% ethanol water solution contains resveratrol and EDC, the total concentration of the resveratrol and the EDC is 5-10 g/L, and the mass ratio of the resveratrol to 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 0.8-1.2: 4-6;

and S23, respectively washing with 30-50% ethanol water solution and deionized water, and drying in vacuum to obtain the water-soluble glass.

The total concentration of resveratrol and EDC represents the mass sum of resveratrol and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in relation to the solvent (aqueous ethanol).

Preferably, the concentration of the ethanol aqueous solution is 40%.

Preferably, in step S22, the mass ratio of resveratrol to 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 1: 5.

Preferably, in step S22, the total concentration of resveratrol and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 7.5 g/L.

Preferably, in step S23, the reaction condition is shaking at room temperature for 12-36 h.

More preferably, in step S23, the reaction condition is shaking at room temperature for 24 h.

The stent material prepared by any one of the preparation methods also belongs to the protection scope of the invention.

The application of the bracket material in liver tissue engineering and/or delaying cell aging also belongs to the protection scope of the invention.

Preferably, the cell is a hepatocyte seed cell.

Preferably, the number of cells seeded is about 1.5X 10³～2.0×10³cm2。

More preferably, the optimal number of cells to be seeded is about 4000/2.25cm²。

Compared with the prior art, the invention has the following beneficial effects:

the characteristics and cell culture researches of the scaffold material provided by the invention show that the modified scaffold material not only overcomes the defects that the scaffold body is hydrophobic and the pores are too small to be beneficial to cell growth due to the addition of hydrophilic groups, but also can obviously delay cell aging. The material modification method is simple and easy to implement, has obvious effect, is used for nourishing liver seed cells, has the effects of delaying cell senescence and improving the activity of the seed cells, and lays a good foundation for preparation of liver tissue engineering. Provides a new method for solving the problem of cell life in the research of liver tissue engineering.

Drawings

FIG. 1 is a schematic diagram of RSV-AA-P (CL-DLLA) preparation; a is a reaction process; and B is a schematic diagram of the effect of RSV-AA-P (CL-DLLA) material after cell inoculation.

Fig. 2 is a structural representation of the composite material. The method comprises the following steps of A, infrared representation of various material structures, and B, Raman representation of various material structures; a is P (CL-DLLA); b is AA-P (CL-DLLA); c is RSV-AA-P (CL-DLLA)).

FIG. 3 is an SEM image of three materials; a is₁、a₂Is P (CL-DLLA); b₁、b₂AA-P (CL-DLLA), and RSV-AA-P (CL-DLLA) at c1 and c 2.

FIG. 4 is an analysis of the RSV content released on the scaffold material; a is the relation between the number of cultured cells on an RSV-AA-P (CL-DLLA) bracket and the concentration of released resveratrol; and b is the relation between the time for culturing cells on an RSV-AA-P (CL-DLLA) bracket and the concentration of released resveratrol.

FIG. 5 is a scanning electron microscope image of cells growing on the scaffold; a is₁、a₂Is P (CL-DLLA); b₁、b₂Is AA-P (CL-DLLA), c₁、c₂Is RSV-AA-P (CL-DLLA).

FIG. 6 is a graph of DAPI staining vs. β -galactosidase staining; DAPI staining on the left and β -galactosidase staining on the right; a is₁、a₂Is P (CL-DLLA), b₁、b₂Is AA-P (CL-DLLA), c₁、c₂Is RSV-AA-P (CL-DLLA).

FIG. 7 is a MTT assay for cell viability; wherein a is P (CL-DLLA), b is AA-P (CL-DLLA), and c is RSV-AA-P (CL-DLLA); indicates significant difference in t-test (p < 0.05).

FIG. 8 is a reactive oxygen species ROS staining pattern; the left side is the observation result under the fluorescent visual field; the right side is the observation result under the bright field view; in (a)₁、a₂Is P (CL-DLLA), b₁、b₂Is AA-P (CL-DLLA), c₁、c₂Is RSV-AA-P (CL-DLLA).

FIG. 9 is an intracellular ROS content analysis; a is P (CL-DLLA); b is AA-P (CL-DLLA); c is RSV-AA-P (CL-DLLA); there were significant differences under the x t test (0.01< p <0.05) and very significant differences under the x t test (p < 0.01).

Detailed Description

The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

1. Cell line

Rat hepatic stellate cells (HSC-T6 cell line): purchased from Changsheng biotechnology Limited liability company of Beijing Ding Guo and subcultured in the laboratory. The cell culture conditions are as follows: high-glucose DMEM medium containing 10% fetal calf serum at 37 deg.C and 5.0% CO₂。

2. Experimental reagents and materials

The main reagents and materials used in the study are shown in table 1 below:

table 1:

example 1

1. Acrylic modification of polylactic acid-caprolactone (P (CL-DLLA))

The acrylic acid modified bracket material is prepared by adopting an ultraviolet grafting method, and the specific operation is as follows:

(1) soaking a polylactic acid-caprolactone (P (CL-DLLA)) scaffold material in a hydrogen peroxide solution with the mass concentration of 3% for 5 min;

(2) placing the soaked P (CL-DLLA) stent into 0.40mol/L Benzophenone (BP) solution, and standing for 1 h;

(3) taking out the P (CL-DLLA) bracket, drying, and then placing under a 125W high-pressure mercury lamp for short-distance irradiation for 15 min;

(4) adding 4% acrylic acid ethanol solution (volume ratio) into the P (CL-DLLA) bracket, and irradiating under an ultraviolet lamp for 45 min;

(5) and (3) after irradiation, taking out, washing with a phosphate buffer solution with the pH value of 7, soaking overnight in the phosphate buffer solution with the pH value of 7 at room temperature, and drying to obtain the acrylic acid modified scaffold material AA-P (CL-DLLA).

2. Preparation of graft resveratrol support material

(1) Placing the acrylic acid modified scaffold material AA-P (CL-DLLA) obtained in the last step into water for soaking for 2 hours to soften the scaffold material;

(2) taking out the soaked bracket material AA-P (CL-DLLA), and putting into a 40% ethanol solution containing resveratrol and EDC with the total amount of 7.5g/L, wherein the weight ratio of resveratrol: EDC is 1:5, and the mixture is shaken for 24h by a magnetic stirrer at room temperature;

(3) after being taken out, the mixture is respectively washed by 40 percent ethanol solution and deionized water, and is dried in vacuum to obtain the RSV-AA-P (CL-DLLA) stent material.

Example 2 characterization of RSV-AA-P (CL-DLLA) scaffold Material

Fourier infrared spectroscopy (FTIR)

1. Experimental methods

The P (CL-DLLA), AA-P (CL-DLLA) and RSV-AA-P (CL-DLLA) in example 1 were dried, and then appropriate amounts of each sample were put on a slide glass and measured by an on-machine.

2. Results of the experiment

The result is shown in FIG. 2-A, 1723cm in FIG. 2-A-a^-1And 3439cm^-1Two strong absorption peaks are located, namely C-O and O-H stretching vibration peaks in the bulk material P (CL-DLLA); 1730cm in FIG. 2-A-b^-1The absorption peak is reflected by the stretching vibration of C ═ O on the acrylic acid modified bracket, 3441cm^-1The absorption peak is the stretching vibration of O-H on the material; the absorption peak of 1638cm-1 in the spectrogram 2-A-C is the C ═ O stretching vibration on the material, 3305cm^-1The absorption peak is the stretching vibration of O-H,1421cm^-1、1467cm^-1、1516cm^-1the stretching vibration of RSV-AA-P (C, benzene ring on CL-DLLA scaffold) can prove that the resveratrol is successfully grafted on AA-P (CL-DLLA).

Second, Raman spectrum (Raman)

1. Experimental methods

The P (CL-DLLA), AA-P (CL-DLLA) and RSV-AA-P (CL-DLLA) in example 1 were dried, and then appropriate amounts of the respective samples were put on glass slides and tested on a machine.

2. Results of the experiment

As shown in FIG. 2-B, it can be seen that the peak of CH2 stretching vibration in the material can be seen in each of 2-B-a, 2-B-B and 2-B-c, which is the main skeleton structure peak in the material, but 1680cm in FIG. 2-B-c^-1The stretching vibration peak of the benzene ring C ═ C exists, which is derived from the C ═ C structure in the resveratrol benzene ring structure. It can be seen that resveratrol has been successfully grafted onto the AA-P (CL-DLLA) surface. Therefore, the results of the Raman spectrum and the infrared spectrum jointly prove that the resveratrol is successfully grafted on the modified material.

X-ray photoelectron spectroscopy (XPS)

1. Experimental methods

About 1cm × 1cm of the sample of example 1 was sampled for P (CL-DLLA), AA-P (CL-DLLA) and RSV-AA-P (CL-DLLA), and the sample was scanned by an X-ray photoelectron spectrometer for elemental analysis of the components of the sample.

2. Results of the experiment

As a result, as shown in Table 2, the ratios of carbon elements of the three materials were different. Specifically, the carbon element ratio of P (CL-DLLA) was 73.19%; the carbon element proportion of AA-P (CL-DLLA) is 70.27 percent, which is reduced compared with the carbon element proportion of the bulk material P (CL-DLLA), and the carbon element proportion of acrylic acid is 60 percent, which shows that acrylic acid successfully modifies the material P (CL-DLLA) to reduce the carbon element proportion of the synthetic material. The carbon element proportion in the RSV-AA-P (CL-DLLA) material is 70.36, and the carbon element proportion in the RSV-AA-P (CL-DLLA) material is increased to a smaller extent than that in the AA-P (CL-DLLA), because the carbon element proportion of resveratrol is 82.35%, and the small-extent increase is to ensure that the resveratrol successfully modifies the AA-P (CL-DLLA) material to further cause the carbon element proportion to be changed. Further illustrating the success of the preparation of RSV and AA co-modified scaffold materials.

Table 2 XPS analysis of various material element ratios:

scanning Electron Microscope (SEM)

1. Experimental methods

Appropriate amounts of P (CL-DLLA), AA-P (CL-DLLA) and RSV-AA-P (CL-DLLA) from example 1 were sampled and observed with a scanning electron microscope.

2. Results of the experiment

The results are shown in fig. 3, and it can be seen that the P (CL-DLLA) material has a flat and smooth surface, and has small undulations and small material pores; the modified AA-P (CL-DLLA) and RSV-AA-P (CL-DLLA) stent surface has acrylic acid and-OH, -COOH and other groups of resveratrol, the hydrophilicity of the stent is enhanced, the stent has a complex surface/internal microenvironment, and the pores of the stent are also increased, so that the defects that the P (CL-DLLA) material is hydrophobic and too small in pores, which are not beneficial to cell adhesion growth, are overcome, and a good matrix environment is provided for cells.

Example 3 biological Effect of RSV-AA-P (CL-DLLA) Stent Material

First, RSV Release assay

To further verify whether the RSV-AA-P (CL-DLLA) material synthesized in example 1 had successfully grafted RSV, the synthetic material was used in hepatocyte-HSC cell culture and the RSV levels in the culture broth were analyzed by HPLC.

1. Experimental methods

HSC cells in logarithmic growth phase are prepared into cell suspension, different cell amounts are respectively inoculated on RSV-AA-P (CL-DLLA) material, the RSV-AA-P material is placed in a 96-well culture plate to be cultured for 7 days, and culture medium in the culture plate is collected every day at the same time. Adding 0.8mL ethyl acetate into the culture medium, shaking for extraction for 15min, standing for layering, taking the upper organic phase, rotating at 40 ℃ for evaporation, and metering the volume of the residue to 1mL by using methanol. The detection was carried out after filtration through a 0.45 μm filter. The mobile phase is methanol: the flow rate of 1% acetic acid solution was 1.0mL/min, the temperature of the cell strain was 30 ℃, the sample size was 5. mu.L, and the detection wavelength was 305 nm.

2. Results of the experiment

The results are shown in FIG. 4. As can be seen from FIG. 4-a, when different numbers of cells were seeded on RSV-AA-P (CL-DLLA) material and cultured, the amount of released resveratrol increased and then decreased as the number of seeded cells increased, and thus was determined to be 2.25cm in area²The optimal number of cells seeded on the scaffold material of (a) is about 4000. Under the condition, the RSV level released from the RSV-AA-P (CL-DLLA) material is shown in a figure 4-b, and the RSV can be continuously and slowly released when cells grow on the material, the release amount of the RSV is increased along with the increase of culture time, and the RSV is released at a concentration of 6 mu mol/L when cultured for 7 days, and the concentration is consistent with the concentration range of the RSV reported by the current research for delaying cell senescence, so that the RSV-AA-P (CL-DLLA) material can be used for delaying cell senescence.

Second, scanning electron microscope analysis of cells

In order to observe the adhesion, aggregation and growth of HSC cells on the surface of the material, HSC cells growing on the surface of the material are analyzed by a scanning electron microscope.

1. Experimental methods

HSC cells reach logarithmic phase after proliferation and culture in a culture flask to 80%, are inoculated to three materials of P (CL-DLLA), AA-P (CL-DLLA) and RSV-AA-P (CL-DLLA) in example 1 of 0.5cm multiplied by 0.5cm, are placed in a 96-well plate for culture for 4 days, then the culture medium is discarded, and the cells are washed with PBS for 3 times, and are shaken on a shaking table for 5 minutes each time; glutaraldehyde 200. mu.L per well is added to fix the cells for 4 hours, and then washed 3 times with PBS, each time shaking on a shaker for 5 minutes; after discarding PBS, gradient dehydration is carried out by using 50%, 75%, 80%, 90% and 100% ethanol, wherein 50%, 75%, 80% and 90% ethanol is used once for 5 minutes each time, and 100% ethanol is used twice for 3 minutes each time; after ethanol is discarded, the mixture is frozen and dried in vacuum to be completely dry and is placed in a scanning electron microscope for detection.

2. Results of the experiment

The results are shown in FIG. 5. As can be seen from FIG. 5-a, the HSC cells grown on the material P (CL-DLLA) were in a smaller number, and it was seen that HSC cells were more difficult to adhere, aggregate and grow on the material P (CL-DLLA), which is related to the hydrophobicity of the material. The number of cells growing on the modified material AA-P (CL-DLLA) and RSV-AA-P (CL-DLLA) is large, the growth condition is good (as shown in figures 5-b and 5-c), and the modified material is also shown to have obviously increased hydrophilicity, which is beneficial to the growth of cells. While the cells grown on the RSV-AA-P (CL-DLLA material) were significantly aggregated into clusters relative to the cells on the material AA-P (CL-DLLA) (as shown in FIG. 5-c 2), indicating that the material grafted with resveratrol was more favorable for the growth and proliferation of cells.

Third, DAPI staining and beta-galactosidase staining

1. Experimental methods

(1) DAPI staining

HSC cells in logarithmic growth phase were seeded on three materials of P (CL-DLLA), AA-P (CL-DLLA) and RSV-AA-P (CL-DLLA) in example 1 and cultured in 96-well plate for 4 days. Then the medium was discarded, washed 3 times with PBS, and shaken on a shaker for 5 minutes each time; adding 4% paraformaldehyde into each well, and fixing at room temperature for 30 min; washing with PBS for 3 times, adding 0.2% Triton X-100 into each well, and permeabilizing for 30 min; washing with PBS for 3 times; photophobic DAPI (5. mu.g/ml) was added to each well, nuclei were stained for 1min, washed 3 times with PBS, and nuclei exhibiting blue color were observed under an inverted fluorescence microscope.

(2) Staining with beta-galactosidase

HSC cells in logarithmic growth phase were seeded on three materials of P (CL-DLLA), AA-P (CL-DLLA) and RSV-AA-P (CL-DLLA) in example 1 and cultured in 96-well plate for 4 days. Then the medium was discarded, washed 3 times with PBS, and shaken on a shaker for 5 minutes each time; then adding beta-galactosidase staining fixing liquid, and fixing for 10 minutes at room temperature; cell fixative was aspirated off, and cells were washed 3 times with PBS for 3 minutes each; absorbing PBS, adding a dyeing working solution, sealing the pore plate by using a preservative film, and incubating overnight at 37 ℃; the cells appearing blue were observed under a normal light microscope.

2. Results of the experiment

The results are shown in FIG. 6. As can be seen from the figure, the HSC cells grown on the P (CL-DLLA) material are few, and the cells are difficult to survive on the P (CL-DLLA) material, while the cells on the modified AA-P (CL-DLLA) and RSV-AA-P (CL-DLLA) materials are large in number and good in growth condition, and further the modified material is proved to be beneficial to the growth and proliferation of the cells. Meanwhile, beta-galactosidase staining shows that the number of aged HSC cells on the RSV-AA-P (CL-DLLA) scaffold is small, cells growing on the AA-P (CL-DLLA) are aged more, so that the modified scaffold material grafted by the resveratrol has the effect of delaying the senescence of the HSC cells, the number of the cells on the RSV-AA-P (CL-DLLA) scaffold is large, the aged cells are small, and the modified scaffold material can be used for culturing liver seed cells.

Fourth, cell survival rate detection

1. Experimental methods

HSC cells in logarithmic growth phase were inoculated on three materials of P (CL-DLLA), AA-P (CL-DLLA) and RSV-AA-P (CL-DLLA) in example 1, and cultured in 96-well culture plates for 4 days; then adding the prepared MTT dye solution, and putting the mixture into an incubator to incubate for 4 hours in a dark place; absorbing the solution, adding an equal amount of DMSO solution into each hole, and oscillating on a shaking table at a low speed for 10 minutes to fully dissolve crystals; in the microplate reader OD₄₉₀The absorbance of each well was measured and compared.

2. Results of the experiment

The results are shown in FIG. 7, the survival rate of HSC cells on P (CL-DLLA) on the material is the lowest, while the number of cells surviving on AA-P (CL-DLLA) and RSV-AA-P (CL-DLLA) is large, the growth condition is good, which shows that the cells can grow and proliferate normally on the modified material, thus further proving that RSV-AA-P (CL-DLLA) material modified by RSV and AA can promote the cell growth and delay the cell senescence.

Fifth, ROS analysis of reactive oxygen species

To further demonstrate that RSV-AA-P (CL-DLLA) is able to delay HSC cell senescence, intracellular ROS staining assays were performed on cultured material.

1. Experimental methods

HSC cells in logarithmic growth phase were seeded on three materials of P (CL-DLLA), AA-P (CL-DLLA) and RSV-AA-P (CL-DLLA) in example 1 and cultured in 96-well plate for 4 days. Then removing the culture medium, adding DCFH-DA working solution diluted by a proper volume, and incubating for 30 minutes in a incubator at the temperature of 37 ℃ in a dark place; washing the cells with serum-free medium to substantially remove DCFH-DA that has not entered the cells; cells exhibiting fluorescence were observed under 480nm excitation light using a confocal laser microscope.

2. Results of the experiment

The results are shown in FIG. 8. The results show that the fluorescence in HSC cells is minimum on RSV-AA-P (CL-DLLA) scaffold material, namely the generated reactive oxygen species ROS is minimum; cells which fluoresce on the blank scaffold material and the modified material AA-P (CL-DLLA) are obviously increased, namely, the cells generate more ROS. Proves that the target material RSV-AA-P (CL-DLLA) has the function of delaying the senescence of hepatic seed cells.

Claims

1. A preparation method of a scaffold material for delaying cell aging is characterized in that resveratrol is grafted on a polylactic acid-caprolactone scaffold material;

the method comprises the following steps:

s2, grafting resveratrol;

benzophenone is used as an initiator, and the polylactic acid-caprolactone scaffold material is subjected to acrylic acid modification by an ultraviolet light grafting method.

2. The method of claim 1, wherein the grafting of resveratrol comprises the steps of:

s21, softening the support material;

s22, placing the mixture into a 30-50% ethanol water solution (V/V) for reaction, wherein the 30-50% ethanol water solution contains resveratrol and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, the total concentration of the resveratrol and EDC is 5-10 g/L, and the mass ratio of the resveratrol to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 0.8-1.2: 4-6;

3. The scaffold material prepared by the preparation method of any one of claims 1 to 2.

4. Use of the scaffold material according to claim 3 for liver tissue engineering and/or delaying cellular senescence.

5. The use of claim 4, wherein the cell is a hepatocyte seed cell.