Application of cell factor-loaded methacrylated gelatin in liver tissue regeneration

Abstract

The invention discloses application of cell factor loaded methacrylated gelatin in liver tissue regeneration, and relates to the field of application of liver tissue regeneration, the cell factor loaded methacrylated gelatin comprises cell factor EGF and FGF loaded methacrylated gelatin, and under the help of phenyl-2, 4, 6-trimethylbenzoyl lithium phosphinate, the cell factor EGF and FGF loaded methacrylated gelatin can be used for preparing the cell factor loaded methacrylated gelatin. And gelling through 405nm UV irradiation to form the methacrylated gelatin loaded with the cell factors. According to the methacrylated gelatin loaded with the cell factors EGF and FGF, provided by the invention, the liver tissue regeneration effect is greatly improved. Test results show that the capacity of the methacrylated gelatin loaded with the cytokines EGF and FGF in liver tissue regeneration is enhanced, and the 2-day liver tissue regeneration promotion rate is greater than 20%.

Description

Technical Field

[0001] This invention relates to the field of liver tissue regeneration, and particularly to the application of methacrylamide gelatin loaded with cytokines in liver tissue regeneration. Background Technology

[0002] To address the problem that existing methacrylamide gelatin is ineffective in promoting liver tissue regeneration, this invention provides methacrylamide gelatin loaded with cytokines EGF and FGF. With methacrylamide gelatin as the main agent and small amounts of cytokines EGF and FGF, in synergy with the cross-linking agent described in this invention, the effect of promoting liver regeneration is enhanced. Summary of the Invention

[0003] The purpose of this invention is to provide the application of cytokine-loaded methacrylamide gelatin in liver tissue regeneration, in order to solve the problems mentioned in the background art.

[0004] To achieve the above objectives, the present invention provides the following technical solution: the application of cytokine-loaded methacrylated gelatin in liver tissue regeneration, comprising methacrylated gelatin loaded with cytokines EGF and FGF, wherein the methacrylated gelatin loaded with cytokines EGF and FGF is gelled by 405nm UV irradiation with the aid of lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid to form cytokine-loaded methacrylated gelatin; This cytokine-loaded methacrylamide gelatin is used in liver tissue regeneration.

[0005] Preferably, the specific preparation method of the cytokine-loaded methacrylamide gelatin includes: S1: Weigh 2.5 parts by weight of phenyl-2,4,6-trimethylbenzoyl lithium phosphinate, 300 parts by weight of methacrylamide gelatin solid and 1000 parts by weight of phosphate buffer solution, and mix the three together. S2: Shake and mix thoroughly to ensure that the methacrylamide gelatin solid and phenyl-2,4,6-trimethylbenzoyl lithium phosphinate are fully mixed; Heat in a light-protected water bath at 65℃ for 30 minutes, removing and shaking every 10 minutes during the process; S3: After the 30-minute water bath, take it out and observe to confirm that all solid parts have completely dissolved and the resulting solution is uniform and transparent; The centrifuge tubes were transferred to a 37 ℃ water bath and cooled to isothermal. Then, EGF at a working concentration of 200 ng / mL and FGF at a working concentration of 100 ng / mL were added. After mixing by pipetting, the mixture was kept at 37 ℃ for later use, thus obtaining the liver tissue regeneration scaffold solution. S4: Draw up the liver tissue regeneration scaffold solution and drip it onto the wound. After dripping, irradiate it with a 405 nm UV light source for 10 seconds to solidify it until the entire wound surface is completely covered by the gel solid.

[0006] The technical effects and advantages of this invention are as follows: 1. The methacrylamide gelatin loaded with cytokines EGF and FGF provided by this invention significantly improves the effect of liver tissue regeneration. Experimental results show that the methacrylamide gelatin loaded with cytokines EGF and FGF provided by this invention has enhanced ability to promote liver tissue regeneration, with a liver tissue regeneration rate of >20% after 2 days. Attached Figure Description

[0007] Figure 1 This is a schematic diagram of the Ki67-positive hepatocyte experimental results of the present invention. Detailed Implementation

[0008] This invention provides, for example Figure 1 The application of cytokine-loaded methacrylamide gelatin in liver tissue regeneration is shown.

[0009] This invention relates to a methacrylamide gelatin loaded with the cytokines EGF and FGF. The material is gelled by 405 nm UV irradiation with the aid of lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid. Compared with methacrylamide gelatin without cytokines, this material exhibits better liver tissue regeneration promotion function.

[0010] To address the problem that existing methacrylamide gelatin is ineffective in promoting liver tissue regeneration, this invention provides methacrylamide gelatin loaded with cytokines EGF and FGF. Methacrylamide gelatin is used as the main agent, with small amounts of cytokines EGF and FGF added, synergistically with the cross-linking agent described in this invention, to enhance the liver regeneration-promoting effect.

[0011] This invention provides a formulation for methacrylated gelatin loaded with the cytokines EGF and FGF to promote liver tissue regeneration. 2.5 parts by weight of phenyl-2,4,6-trimethylbenzoyl lithium phosphine oxide, 300 parts by weight of methacrylated gelatin solid, and 1000 parts by weight of phosphate buffer solution are weighed and mixed. The mixture is shaken to ensure thorough mixing of the methacrylated gelatin solid and phenyl-2,4,6-trimethylbenzoyl lithium phosphine oxide. The mixture is heated in a 65°C water bath in the dark for 30 min, shaking every 10 min. After the 30-min water bath, the mixture is observed to confirm that all solids have completely dissolved and the resulting solution is homogeneous and transparent. Subsequently, the centrifuge tube is transferred to a 37°C water bath and cooled to isothermal. EGF at a working concentration of 200 ng / mL and FGF at a concentration of 100 ng / mL are added. The mixture is then mixed by pipetting and held at 37°C for later use to obtain the liver tissue regeneration scaffold solution. A liver tissue regeneration scaffold solution was aspirated and dripped into the wound. After application, the wound was irradiated with a 405 nm UV light source for 10 seconds to solidify it until the entire wound surface was completely encapsulated in the gel solid. The methacrylamide gelatin formulation loaded with cytokines EGF and FGF can enhance the regenerative capacity of liver tissue.

[0012] The methacrylamide gelatin loaded with cytokines EGF and FGF provided by this invention significantly improves the effect of liver tissue regeneration. Experimental results show that the methacrylamide gelatin loaded with cytokines EGF and FGF provided by this invention has enhanced ability to promote liver tissue regeneration, with a liver tissue regeneration rate of >20% after 2 days.

[0013] Specifically, 8-week-old healthy male C57 / B6 mice (20-25 g) were randomly divided into two groups (no cytokine group and cytokine-containing group), with 5 mice in each group. Anesthesia was administered via intraperitoneal injection of 1.25% aphthylamine solution at a dosage of 20 μL per gram of body weight. After anesthesia took effect, a liver laceration model was established. In short, an incision was made in the midline of the abdomen, and the skin and abdominal wall were sequentially cut open. Gentle pressure was applied to the abdomen to expel the left lobe of the liver through the incision. A 10 mm transverse incision was then made in the left lobe of the liver using a scalpel blade to simulate a liver laceration.

[0014] After causing a liver laceration during surgery, the incision site and surrounding tissue were quickly and thoroughly disinfected with alcohol swabs, and excess blood was aspirated to expose the wound. Approximately 100 μL of liver tissue regeneration scaffold gel was slowly dripped into the wound. After dripping, the wound was irradiated with a 405 nm UV light source for 10 seconds to solidify it until the entire wound was completely encapsulated in the gel. Subsequently, the gel-coated liver was carefully pushed back into the abdominal cavity (if the gel was too large or its shape was unsuitable for placement back into the abdominal cavity, a portion should be appropriately removed without compromising the wound protection). The abdominal wall and skin were sutured layer by layer, and the wound was disinfected. After surgery, the mouse was placed on a warming blanket until it regained consciousness. Subsequently, the experimental animals were housed under SPF conditions, provided with ample food and water, and kept under a regular 12-hour light / dark cycle for 2 days.

[0015] Experimental animals were sacrificed on day 2, and liver samples were collected. After rinsing away blood in PBS, the callus area (approximately 1 cm × 1 cm) was surgically removed; simultaneously, the gel adhering to the liver was carefully peeled off in the liver tissue regeneration scaffold group. The tissue was fixed with 4% PFA for 24 h and then embedded in paraffin. Paraffin sections were prepared, and the Ki67 amplification marker was stained with immunofluorescence. The level of liver regeneration promoted by the liver tissue regeneration scaffold was determined by statistically analyzing the number of Ki67-positive cells in the sections. The number of Ki67-positive cells in the PBS group was N0, and the number of Ki67-positive cells in the liver tissue regeneration scaffold group was N1.

[0016] The rate of hepatocyte regeneration (%) = ((N1-N0)) / N0 × 100%.

Claims

1. The application of methacrylated gelatin loaded with cytokines in liver tissue regeneration, including methacrylated gelatin loaded with cytokines EGF and FGF, characterized in that: The methacrylamide gelatin loaded with cytokines EGF and FGF was gelled by 405 nm UV irradiation with the aid of lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid to form methacrylamide gelatin loaded with cytokines. This cytokine-loaded methacrylamide gelatin is used in liver tissue regeneration.

2. The application of the cytokine-loaded methacrylamide gelatin according to claim 1 in liver tissue regeneration, characterized in that: The specific preparation method of the cytokine-loaded methacrylamide gelatin includes: S1: Weigh 2.5 parts by weight of phenyl-2,4,6-trimethylbenzoyl lithium phosphinate, 300 parts by weight of methacrylamide gelatin solid and 1000 parts by weight of phosphate buffer solution, and mix the three together. S2: Shake and mix thoroughly to ensure that the methacrylamide gelatin solid and phenyl-2,4,6-trimethylbenzoyl lithium phosphinate are fully mixed; Heat in a light-protected water bath at 65℃ for 30 minutes, removing and shaking every 10 minutes during the process; S3: After the 30-minute water bath, take it out and observe to confirm that all solid parts have completely dissolved and the resulting solution is uniform and transparent; The centrifuge tubes were transferred to a 37 ℃ water bath and cooled to isothermal. Then, EGF at a working concentration of 200 ng / mL and FGF at a working concentration of 100 ng / mL were added. After mixing by pipetting, the mixture was kept at 37 ℃ for later use, thus obtaining the liver tissue regeneration scaffold solution. S4: Draw up the liver tissue regeneration scaffold solution and drip it onto the wound. After dripping, irradiate it with a 405 nm UV light source for 10 seconds to solidify it until the entire wound surface is completely covered by the gel solid.

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