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Application of nanometer synthetase in promoting synthesis of high-molecular hyaluronic acid in cells

A high-molecular transparent and hyaluronic acid technology, which is applied in the application field of nano-synthetic enzymes to promote the synthesis of high-molecular hyaluronic acid in cells, can solve the problems of animal tissue source difficulties, poor biocompatibility, and production difficulties, and achieve Good development and application prospects, relieve anxiety, and reduce the number of times

Active Publication Date: 2019-01-29
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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  • Application of nanometer synthetase in promoting synthesis of high-molecular hyaluronic acid in cells
  • Application of nanometer synthetase in promoting synthesis of high-molecular hyaluronic acid in cells
  • Application of nanometer synthetase in promoting synthesis of high-molecular hyaluronic acid in cells

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Experimental program
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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 synovia, and in particular relates to an application of nanometer synthetase in promoting the synthesis of high-molecular hyaluronic acid in cells. Thenanometer synthetase is a nanometer particle-hyaluronic acid synthetase loading system, according to the application, the nanometer particle-hyaluronic acid synthetase loading system is injected intothe articular cavity, then the nanometer particle-hyaluronic acid synthetase loading system enters synovial cells, and then the synovial cells are promoted for secreting high-molecular hyaluronic acid. According to the application, by transferring the nanometer particles loaded with the hyaluronic acid synthetase into the synovial cells, the synovial cells can secrete more high-molecular hyaluronic acid. The generated endogenous self-synthetized high-molecular hyaluronic acid has very good development and application prospects, the animal experiment proves that the injection of the nanometer particle-hyaluronic acid synthetase loading system into the articular cavity provides a novel means for efficiently treating osteoarthritis, and therefore, a new method and a new thought are provided for clinic treatment.

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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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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