Preparation method of sheepskin decellularized protein matrigel

Abstract

The invention discloses a preparation method of sheepskin acellular protein matrigel. The preparation method comprises the following steps: pretreating sheepskin, extracting an extracellular matrix and forming matrigel. The matrigel can simulate an in-vivo extracellular matrix, a matrigel with a growth condition similar to an in-vivo environment is provided for cells, the sheepskin matrigel has low immunogenicity, the risk of immunological rejection is reduced, and the sheepskin matrigel is relatively wide in source, low in cost and good in cell compatibility.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to a method for preparing a decellularized sheepskin protein matrix glue. Background Art

[0002] In the biomedical field, matrix glue, as a commonly used research tool, plays an important role in cell biology research and drug screening. Matrix glue not only has physical functions such as connection, support, water retention, pressure resistance and protection, but also plays a full range of biological effects on the basic declarative activity of cells. Including, affecting cell survival, growth and death; affecting biological processes such as cell differentiation, proliferation, adhesion, morphogenesis and phenotypic expression; determining the shape of cells; controlling cell differentiation; participating in cell migration. It can simulate the extracellular matrix environment in the body and provide cells with a three-dimensional spatial structure, thereby promoting cell growth and function maintenance. Especially in tumor research, matrix glue can provide support similar to the tumor microenvironment, making the study of cell interactions closer to the physiological state, greatly promoting the progress of disease model construction, cell proliferation and migration.

[0003] At present, there are two main methods for preparing matrix glue: one is to extract it from animal tissues, and the other is to obtain it by decellularization through cell culture. Although the matrix glue prepared by decellularization after cell culture has better control of its composition and the obtained matrix glue has good consistency, the culture medium used contains animal-derived components such as fetal bovine serum, resulting in high raw material costs and poor biocompatibility of the obtained matrix glue, which is not conducive to clinical use. Summary of the invention

[0004] In view of this, it is necessary to address the above problems. The present application provides a matrix gel based on sheepskin extracts to simulate the structure and function of the extracellular matrix in vivo, providing cells with a growth condition similar to the in vivo environment.

[0005] In order to solve the problems existing in the prior art, The present application provides a method for preparing acellular sheepskin protein matrix glue, comprising the following steps: (1) Pre-treat the sheepskin by rinsing with Triton X-100, PBS, and deionized water in sequence; (2) Digestion and enzymatic hydrolysis with pepsin-added hydrochloric acid solution to extract the extracellular matrix; (3) Dissolve with PBS and let stand at 37°C to obtain matrix gel; The matrix glue remains liquid at 0-4°C, solidifies at 35-40°C, and has the ability to promote the healing of wounds in diabetic mice.

[0006] Furthermore, the pH value of the PBS in step (1) and step (3) is 7.0-7.6, preferably 7.4.

[0007] Furthermore, the volume fraction of Triton X-100 in step (1) is 0.2% to 0.7%, preferably 0.5%.

[0008] Furthermore, in step (1), the sheepskin is washed 1 to 3 times with Triton X-100, PBS, and deionized water, respectively, and the amount of each substance used in each wash is 15 to 25 times the area of ​​the sheepskin.

[0009] Furthermore, before the step (1), the fresh sheepskin is firstly soaked in povidone-iodine for preliminary disinfection, the wool is removed, the sheepskin is washed with physiological saline to remove the sand and impurities on the surface of the sheepskin, the subcutaneous fat is cleaned off, and the sheepskin is cut into small cubes with a length and width of 1 cm.

[0010] Furthermore, the rinsing in step (1) is performed at a rotation speed of 120-180 rpm for 30-60 minutes, preferably at a rotation speed of 150 rpm for 30 minutes.

[0011] Furthermore, the specific steps of step (2) are: freeze-drying the sheepskin pretreated in step (1), digesting and hydrolyzing the freeze-dried sample with a 0.05-0.1M hydrochloric acid solution added with pepsin, the pH value of the hydrochloric acid solution is 1-1.5, and then using 1M NaOH to adjust the pH of the system to neutral to obtain a mixed solution, and freeze-drying again to obtain an extracellular matrix, wherein the mass ratio of pepsin to the volume of hydrochloric acid is 0.03-0.05g:50ml.

[0012] Pepsin is an enzyme that works under acidic conditions. The hydrochloric acid in gastric juice is secreted by gastric parietal cells, which makes the environment in the stomach acidic and provides a suitable pH condition for pepsin. Pepsin can activate pepsinogen under acidic conditions and convert it into active pepsin. Pepsin has a high efficiency in decomposing proteins in an acidic environment and can break down proteins into smaller peptides for subsequent digestion and absorption. Therefore, hydrochloric acid is used to simulate the environment of gastric acid. When hydrochloric acid is prepared into a 0.05~0.1M hydrochloric acid solution, its pH is 1~1.5, which is more in line with the pH value of gastric acid, thereby enabling pepsin to work better in a 0.05~0.1M hydrochloric acid solution.

[0013] Furthermore, the freeze-drying temperature for both times is -60 to -70°C, and the freeze-drying time is 12 to 24 hours. Preferably, the freeze-drying temperature for both times is -60°C, and the freeze-drying time is 20 hours.

[0014] Furthermore, the digestion and enzymatic hydrolysis time is 2 to 3 days, and the operation of adjusting the pH of the system with NaOH is carried out in an ice bath environment.

[0015] Furthermore, in step (3), the concentration of PBS is maintained at 0.01 M, and the resulting matrix gel concentration is 10-20 mg / ml, preferably 10 mg / mL.

[0016] Furthermore, the standing time in step (3) is 8 to 15 minutes, preferably 10 minutes.

[0017] This application uses sheepskin extract to simulate the structure and function of the extracellular matrix in the body, and provides cells with a matrix glue with growth conditions similar to the in vivo environment. Compared with the low cost of existing matrix glue, sheepskin, as a common animal by-product, has a wide source and low cost; secondly, the matrix glue of this application has good biocompatibility and safety, is rich in protein compounds such as collagen, and has a high similarity with the extracellular matrix of the human body; compared with some other animal-derived matrix glues, it may have lower immunogenicity. Reduced the risk of inducing immune response in cell culture and tissue engineering; the gel structure of the matrix glue of this application is relatively stable and can resist changes in the external environment to a certain extent. This stability helps to maintain the physical properties of the matrix glue during cell culture, thereby providing a stable growth environment for cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Schematic diagram of the finished product prepared in Example 1; Figure 2 : The morphology of the matrix gel prepared in Example 1 solidified at 37°C and dissolved at 4°C; Figure 3 : Schematic diagram of Matrigel pressure stress-strain in Example 2; Figure 4 : Result diagram of CCK8 experiment of L929 and 3T3 cell lines inoculated with matrix gel in Example 3; Figure 5 : The results of the live-death staining of L929 and GL261 cell lines inoculated with matrix gel in Example 3; Figure 6 : Result diagram of the matrix gel cell proliferation experiment in Example 3; Figure 7 : Result diagram of Example 4: Effect of matrix gel on wound healing ability in diabetic mice.

[0019] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0020] the term Unless otherwise stated, each of the following terms shall have the meaning set forth below.

[0021] In this article, the terms "comprises", "comprising" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device that includes a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of more restrictions, an element defined by the sentence "comprising a ..." does not exclude the presence of other identical elements in the process, method, article or device that includes the element.

[0022] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0023] It should be noted that, as used herein and in the claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, a nucleic acid molecule refers to one or more nucleic acid molecules. Thus, the terms "a", "a", "one or more", and "at least one" can be used interchangeably. Similarly, the terms "comprising", "including", and "having" can be used interchangeably and should generally be understood to be open-ended and non-restrictive, e.g., not excluding other unlisted elements or steps. DETAILED DESCRIPTION

[0024] The present invention is described in detail below by means of specific embodiments in combination with the accompanying drawings, but it should not be understood that the above subject matter scope of the present invention is limited to the following embodiments.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0026] The materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources. Animal Source: The required animal materials can be selected from the mainstream Chinese large-scale breeding, no external pathogens, good stress resistance of domestic sheep, preferably those raised in SPF grade environment. PBS is composed of phosphate buffer and physiological concentration of salt. The molecular formula of Triton X-100 is C 34 H 62 O 11 .

[0027] Example 1 Preparation of Matrigel Sheepskin pretreatment: Soak fresh sheepskin in povidone iodine for preliminary disinfection, remove wool, wash with physiological saline to remove gravel impurities on the sheepskin surface, clean subcutaneous fat, and cut into small pieces of 1*1 cm. Place 20 pieces of fresh sheepskin in 200 ml of 0.5% Triton X-100, rinse at 150 rpm for 30 minutes, then rinse with 200 ml of PBS with pH 7.4 at 150 rpm for 30 minutes, wash twice, and finally rinse with 200 ml of deionized water at 150 rpm for 30 minutes, wash twice.

[0028] Extracellular matrix extraction: The pretreated sheepskin was freeze-dried at -60°C for 20 hours to obtain freeze-dried decellularized sheepskin powder. The freeze-dried sample was digested and hydrolyzed in 0.1M hydrochloric acid solution for 2 days, and then the pH of the system was adjusted to neutral using 1M NaOH in an ice bath environment to obtain a mixed solution, which was freeze-dried again at -60°C for 20 hours.

[0029] Matrigel formation: The freeze-dried sample was dissolved in PBS at pH 7.4, and the concentration of the obtained matrigel was maintained at 10 mg / ml. The matrix gel was allowed to stand at 37°C for 10 min to obtain a matrix gel such as Figure 1 The matrix gel can remain liquid at 4°C and quickly gel at 37°C. Figure 2 shown.

[0030] Example 2 Mechanical properties test of matrix gel The matrix gel prepared in Example 1 was mixed with the cell culture medium to obtain a mixed gel culture solution, and then the L929 cell line was inoculated and cultured at 37°C for 1, 3, and 5 days, respectively. At the same time, a blank group without inoculated cells was used for comparison. A microcomputer-controlled electronic universal testing machine (Jinan Wenteng Testing Instrument Co., Ltd. Model: WDW-A) was used to place a 1x1 circular matrix gel in the tester to test the mechanical properties of the matrix gel, and the obtained stress-strain curve was obtained. The results are shown in FIG. Figure 3 As shown, the mechanical properties of the matrix gel containing cells are better and the longer the culture time, the better the mechanical properties of the matrix gel. The longer the blank group is placed, the worse the mechanical properties of the matrix gel.

[0031] Example 3 Cytocompatibility test of matrix gel The matrix gel prepared in Example 1 was subjected to CCK8 detection, and the cell lines used were 3T3 and L929 cell lines. The cells were mixed with the matrix gel in a ratio of 1:1 and inoculated into a 96-well plate for overnight culture. On the second day, 10 μL of CCK-8 reagent (MeilunBio) was added to each well without removing the culture medium. The 96-well plate was gently shaken to mix, and then incubated at 37°C for 1 hour. After the incubation was completed, the absorbance (OD value) of each well was detected at a wavelength of 450 nm using an ELISA reader. 600 nm can be selected as the reference wavelength. After deducting the background value, the absorbance is proportional to the survival rate of the cells. The CCK8 results of L929 and 3T3 cell lines are shown in Figure 2. Figure 4 As shown, from left to right are CCK8 of L929 and 3T3 cell lines, indicating that 15 mg / ml and 10 mg / ml of matrix gel have a certain effect of promoting cell proliferation.

[0032] The matrix gel prepared in Example 1 was stained for cell death and viability, and the cell lines used were GL261 and L929 cell lines. The cells were mixed with the matrix gel in a 1:1 ratio and inoculated into a 48-well plate for overnight culture. The next day, a mixed staining solution (AM+PI) was prepared using a death and viability staining kit (meilunbio item number: MA0361-2) in a dark environment. 1 μL of AM and PI were added to each 2 mL of PBS, and after mixing evenly, it was added to a 48-well plate, 200 μL per well, and then placed in an incubator at 37 °C for 5 minutes, and photographed in the dark. The two cell lines were inoculated into the gel for culture. The results of L929 and GL261 cell death and viability staining are shown in the figure. Figure 5 As shown, different concentrations of matrix gel have good cell compatibility.

[0033] The matrix gel prepared in Example 1 was used for cell proliferation experiment, and the cell line used was L929 cell line. The cells were inoculated into the gel for culture. First, the cells were digested and resuspended in culture medium. According to the amount of cells, the culture medium containing cells and the matrix gel (20 mg / ml) were mixed at a ratio of 1:1. The mixed matrix was then placed at 37°C for 10 minutes. After gelation, it was placed in the culture medium for culture. The L929 cell proliferation results are shown in Figure 1. Figure 6 As shown, different concentrations of Matrigel resulted in better cell proliferation.

[0034] Example 4 Matrigel promotes wound healing in diabetic mice BALB / C mice (5-6 weeks old, male) were fasted for 12h or 16h, and STZ concentration of 50mg / kg was intraperitoneally injected for 5 consecutive days. Fasting was required before each injection. If blood sugar did not rise after one week, 1-2 injections could be given. If it was still unsuccessful, no more administration was allowed. After modeling, tests were performed to distinguish mice with successful modeling from mice with unsuccessful modeling for subsequent experiments.

[0035] The matrix gel prepared in Example 1 was used for experiments. L929 cell line and macrophages were inoculated into the matrix gel. The two were compared with PBS group, zinc oxide group and blank gel group respectively to observe the wound healing effect on diabetic mice. Figure 7 As shown, the matrix glue of the present application has a good ability to repair wounds.

[0036] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for preparing acellular sheepskin protein matrix glue, characterized in that: The following steps are involved: (1) Pre-treat the sheepskin by rinsing with Triton X-100, PBS, and deionized water in sequence; (2) Digestion and enzymatic hydrolysis with pepsin-added hydrochloric acid solution to extract the extracellular matrix; (3) Dissolve with PBS and let stand at 37°C to obtain matrix gel; The matrix glue remains liquid at 0-4°C, solidifies at 35-40°C, and has the ability to promote the healing of wounds in diabetic mice.

2. The method according to claim 1, characterized in that: The pH value of the PBS in step (1) and step (3) is 7.0-7.6; the volume fraction of Triton X-100 is 0.2%-0.7%.

3. The method according to claim 1, characterized in that In step (1), the sheepskin is washed 1 to 3 times with Triton X-100, PBS, and deionized water, respectively, and the amount of each substance used in each wash is 15 to 25 times the area of ​​the sheepskin.

4. The method according to claim 1, characterized in that Prior to step (1), the fresh sheepskin is firstly soaked in povidone-iodine for preliminary disinfection, the wool is removed, the sheepskin is washed with physiological saline to remove the sand and impurities on the surface of the sheepskin, the subcutaneous fat is cleaned off, and the sheepskin is cut into small cubes with a length and width of 1 cm.

5. The method according to claim 1, characterized in that The rinsing in step (1) is performed at a rotation speed of 120 to 180 rpm for 30 to 60 minutes.

6. The method according to claim 1, characterized in that The specific steps of step (2) are: freeze-drying the sheepskin pretreated in step (1), digesting and enzymolyzing the freeze-dried sample with a 0.05-0.1M hydrochloric acid solution added with pepsin, and then adjusting the pH of the system to neutral with 1M NaOH to obtain a mixed solution, and freeze-drying again to obtain an extracellular matrix, wherein the mass ratio of pepsin to the volume of hydrochloric acid is 0.03-0.05g:50ml.

7. The method according to claim 6, characterized in that The freeze-drying temperature for both times was -60~-70℃ and the freeze-drying time was 12~24 hours.

8. The method according to claim 6, characterized in that The digestion and enzymatic hydrolysis time is 2 to 3 days, and the operation of adjusting the pH of the system with NaOH is carried out in an ice bath environment.

9. The method according to claim 1, characterized in that: In step (3), the concentration of PBS is maintained at 0.01 M, and the resulting matrix gel concentration is 10-20 mg / ml.

10. The method according to claim 1, characterized in that The standing time in step (3) is 8 to 15 minutes.

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