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The application of nano-synthetase to promote the synthesis of high-molecular hyaluronic acid in cells

A technology of polymer transparency and hyaluronic acid, which is applied in the application field of nano-synthetic enzymes to promote the synthesis of polymer hyaluronic acid in cells, can solve the problems of difficult animal tissue source, poor biocompatibility and low yield, etc. Good development and application prospects, reducing anxiety, and improving the effect of HA content

Active Publication Date: 2021-10-22
WUHAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The extraction method mainly uses cockscomb, human umbilical cord, vitreous body of bull’s eye, etc. as raw materials for production, and the tissue must be freshly collected, but the source of animal tissue is difficult and expensive, which brings certain difficulties to the production of HA The production of HA by fermentation is to use streptococcus to convert cheap glucose into HA. This method does not depend on animal organs, and the output is not limited, but the output of HA is not high, and the biocompatibility is not as good as the former

Method used

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  • The application of nano-synthetase to promote the synthesis of high-molecular hyaluronic acid in cells
  • The application of nano-synthetase to promote the synthesis of high-molecular hyaluronic acid in cells
  • The application of nano-synthetase to promote the synthesis of high-molecular hyaluronic acid in cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1. Isolation, culture and identification of synoviocytes

[0032] 1. Take the synovial tissue from one side of the articular disc of a human or animal, and culture it by the tissue block method.

[0033] 2. Synoviocytes were typed by single cell cloning method.

[0034] (1) Single cell cloning

[0035] Collect the adaptive medium and cultivate the synoviocytes to a semi-confluent state, suck out all the culture medium and centrifuge, filter through a filter with a diameter of 0.22 μm, and take a bottle of cells in the logarithmic growth phase for routine digestion, centrifugation, and counting. Dilute the cells to 45-50 cells per 100 μL, mix well, and plant. Observe the 96-well plate under an inverted microscope, record the serial number of only a single cell, then add 150-200 μL of culture solution to only a single cell well, and place in CO 2 Incubator cultivation. Observation was performed after culturing for 86-96 hours. Select well-grown single-cell clone well...

Embodiment 2

[0040] Example 2 To study the efficiency of RITC-labeled polyethyleneimine surface-modified mesoporous silicon nanoparticles into synovial fibroblasts in vitro

[0041] 1. Preparation of mesoporous silicon nanoparticles modified on the surface of polyethyleneimine, and labeled with RITC fluorescence:

[0042](1) Disperse the synthesized porous nanoparticles (10-100 mg) into 10-200 ml of water, adjust the pH to about 10, and then add 0.1 ml to 5 ml of 3-(trihydroxysilyl) propane Add 3-(Trihydroxysilyl)propylmethylphosphonate to the solution, stir at 40°C for 4 hours, and obtain nanoparticles by centrifugation (3700 rpm for ten minutes); then the solid particles are resuspended in 50- 500 mg of polyethyleneimine (Polyethyleneimine, PEI, molecular weight 5000-50000 Da) solution, stirred at room temperature for 4 hours. As for the centrifugation in the high-speed centrifuge at 20,000 rpm for ten minutes, the mesoporous silicon nanoparticles modified on the surface of polyethylene...

Embodiment 3

[0051] Example 3 Establishment of nanoparticle-hyaluronan synthase loading system

[0052] 1. Preparation of a mesoporous silicon nanoparticle-hyaluronic acid synthase loading system, specifically including the following steps:

[0053] (1) Take 10ug of hyaluronic acid synthase, add PBS to dissolve and expand the volume to 100ul to obtain a 100ug / ml hyaluronic acid synthase solution;

[0054] (2) Mix 10uL of 100ug / ml hyaluronic acid synthase solution with 10uL of 100ug / ml RITC-labeled polyethyleneimine surface-modified mesoporous silicon nanoparticles DMEM solution, shake gently for 24h, and then centrifuge, Collect the supernatant and pellet after centrifugation.

[0055] Calculate the drug loading of the nanoparticle-hyaluronan synthase loading system, drug loading (%)=(input amount of hyaluronan synthase (ug)-hyaluronan synthase content in the supernatant (ug)) / mass of nanoparticles × 100%;

[0056] Detect the release amount of hyaluronic acid synthase, and draw the re...

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Abstract

The invention belongs to the technical field of synovial fluid, and specifically relates to the application of a nano-synthetic enzyme to promote the synthesis of high-molecular hyaluronic acid in cells. The nanosynthetase is a nanoparticle-hyaluronan synthase loading system, and the application is to inject the nanoparticle-hyaluronan synthase loading system into the joint cavity, and the nanoparticle-hyaluronan synthetase loading system enters the joint cavity. membrane cells, thereby promoting the secretion of high molecular weight hyaluronic acid by synoviocytes. In the present invention, the nanoparticles loaded with hyaluronic acid synthase are transferred into the synoviocytes, so that the synoviocytes can secrete more high-molecular-weight hyaluronic acid. The endogenous, self-synthesized high-molecular-weight hyaluronic acid produced by the present invention Acid has very good development and application prospects. Animal experiments have confirmed that intra-articular injection of nanoparticles-hyaluronic acid synthase loading system provides a new means for highly efficient treatment of osteoarthritis, and provides new methods and methods for clinical treatment. train of thought.

Description

technical field [0001] The invention belongs to the technical field of synovial fluid, and in particular relates to the application of a nano-synthetic enzyme to promote the synthesis of high-molecular hyaluronic acid in cells. Background technique [0002] Hyaluronic acid (HA) is a highly viscous substance first isolated from the bovine vitreous by Meyer and Palmer in 1934. [0003] The synovial fluid in the human body is secreted by the synovial cells of the joints, and the synovial fluid covers the surface of the joints. HA is the main component of joint synovial fluid and plays a vital role in the physiological function of joints. When diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA) occur, the production and metabolism of HA in the joints are abnormal, and the concentration and relative molecular weight (Mr) of HA in the synovial fluid are significantly reduced. Degradation and destruction occur, leading to joint physiological disturbances. At prese...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/45A61K9/51A61K47/32A61K47/04A61P19/02A61P29/00
CPCA61K9/0019A61K9/5115A61K9/5138A61K38/45A61P19/02A61P29/00C12Y204/01212
Inventor 龙星徐纯郭慧琳李慧敏李健蔡恒星孟庆功房维柯金李颖杰江恒华
Owner WUHAN UNIV
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