Preparation and application of bionic medical fibroblast

A technology of byssin and protein, applied in the field of medical material preparation, can solve the problems of insolubility in neutral solution, application limitation, fast degradation speed, etc., to avoid the spread of terrestrial biological diseases, good adaptability and stability, biological good compatibility

Active Publication Date: 2018-12-07
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the natural collagen hydrogel has weak mechanical properties, rapid degradation and easy to cause immune reactions and other shortcomings; natural hyaluronic acid is sensitive to strong acid, strong alkali, heat, free radicals and hyaluronidase, has poor stability, and is prone to Degradation; Chitosan is insoluble in neutral solutions and most organic solvents due to its tight crystal structure, so its application is greatly limited

Method used

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  • Preparation and application of bionic medical fibroblast
  • Preparation and application of bionic medical fibroblast
  • Preparation and application of bionic medical fibroblast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Screening of Sbp5-2 protein

[0024] 1. Cloning of the whole gene of Sbp5-2

[0025] 1.1 Combined with the sequence information of mass spectrometry identification results, design RACE primers

[0026] Using Primer Premier 5 software to design primers for RACE (Rapid Amplification of cDNA Ends) Sbp-5-2

[0027] 3' RACE primer: TCCCAACAATGGAGTATGTGAAGATGCTG,

[0028] 5' RACE primer: GCCTAGTGTAACAGTCATTCTCCAGCCAAG;

[0029] 1.2 RNA extraction

[0030] Three patellofrerio scallops in the adaptation period (0-2h), the growth period of silk secretion (2-16h) and the balance period of silk secretion (16-24h) were dissected and their feet were taken according to the RNA was extracted by the method of Hu et al. (2006) and mixed in equal amounts. Using SMARTer™ RACE cDNA Amplification Kit (Clontech, CA, USA), according to the instructions, construct 5'RACE and 3'RACE libraries.

[0031] 1.3 Cloning the 3' and 5' ends of the target gene respectively

[0032] Add...

Embodiment 2

[0041] Example 2: Preparation of silk-forming fibers

[0042] 1. RM 6 Clonal expression of CT

[0043] According to the amino acid sequence analysis of Sbp5-2, it was found that the part except the signal peptide sequence had multiple functional modules formed by paired cysteines, and a C-terminal (C Terminal) sequence different from the functional modules. The functional module contains a large number of hydrophilic amino acids, and the subclone RM of six functional modules connected after the C-terminal (C Terminal) sequence is selected by screening 6 CT (amino acid sequence number: 160-324) was purified and expressed. ( figure 1 ), the subclone RM 6 The sequence of CT is SEQ ID NO:2;

[0044]NKYYGCPNNGVCEDAEATRNNSCTSYMCVLSKRRTLMKWEILKTGCKTDEGCKYDTDEWTDPDASSCVTRRCDVTFNPTDKTYNSVNRVARHGCRASNGTCYYNSETWLENDCYTRRCDVTTTDKGESMAAIKIESGICKDADGSCKGYGEAMQYRSGAATLNCV;

[0045] 1.1RM 6 CT primer design

[0046] 5' Primer: GGGGTACCAACAGTTGCACCTCATATATGTGTA

[0047] 3' Primer: CCG...

Embodiment 3

[0070] Example 3: Properties of Filamented Fibers

[0071] 1. Characterization of the basic properties of silk-forming fibers:

[0072] 1) Material wet stability and water absorption detection

[0073] Soak the macroscopically formed wet fiber filaments (about 0.1g) in PBS solution, take them out at 0, 12, 24, 36, 48, 60, 72, 84, 96, and 108 hours, and blot the surface moisture with filter paper. Call the mass M wet . After freezing at -80°C, freeze-drying is carried out, and its mass is called M after freeze-drying. dry , do three parallel sets.

[0074] Swelling rate = (M wet -M dry ) / M dry ×100%.

[0075] Water absorption = (M wet -M dry ) / M wet ×100%.

[0076] Figure 5 for RM 6 The test results of CT protein fiber swelling rate (left picture) and water absorption rate (right picture); the test results show that it maintains a relatively stable swelling performance in a wet environment, and the material swelling rate maintains at about 400% within 108 hours. ...

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Abstract

The invention provides a bionic medical fibroblast with a sequence of SEQ ID NO:1. On the one hand, the invention provides a preparation method for preparing a protein fragment of the fibroblast. Theprotein fragment is 160-324-locus amino acid of a scallop byssus protein with a sequence of SEQ ID NO:1, and the sequence of the protein fragment is SEQ ID NO:2. The protein prepared into the fibroblast is prepared by virtue of a genetic engineering method from the material source, is low in cost and environment-friendly and can be produced in a large scale. A protein-base material is derived fromocean scallop byssus, is formed through polymerization in a similar seawater environment and has good adaptability and stability to a humidity environment. The bionic medical fibroblast has relatively good development prospects in the medical material field. According to a marine organism derived material, the transmission risk of terrestrial biological diseases is avoided in the aspect of the biological safety of materials, and an examination result shows that the material has no cytotoxicity and has good biocompatibility.

Description

technical field [0001] The invention belongs to the technical field of medical material preparation, and in particular relates to a silk-forming fiber and its application. Background technique [0002] Biomedical materials, also known as biomaterials, are special functional materials that can diagnose, treat, replace, repair, induce regeneration or enhance the functions of cells, tissues and organs in the body. The biological body contains a large amount of water-based extracellular fluid, and the body's cell tissue is always in a "wet" environment composed of interstitial fluid, plasma and lymph. Therefore, for in vivo repair and plugging, support materials must first have good underwater stability and swelling, and at a higher level, excellent biocompatibility and controllable biodegradation and absorption are also required. At present, the popular research materials mainly used in the in vivo environment mainly include some extracellular matrix (extracellular matrix, ECM...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435A61L27/22A61L27/50
CPCA61L27/227A61L27/50C07K14/43504C08L89/00
Inventor 刘伟治王硕硕徐平平
Owner OCEAN UNIV OF CHINA
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