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Dentin non-collagen programmed preparation method

A non-collagen and dentin technology, which is applied in the programmed preparation of dentin non-collagen and the extraction of hard tissue proteins, can solve the problems of difficult experimental design and difficult quantification of natural dentin, so as to improve the scientific nature and reduce the The effect of time cost and economic cost

Inactive Publication Date: 2018-02-09
AFFILIATED STOMATOLOGICAL HOSPITAL OF NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So as to solve the bottleneck problem of difficult quantification and experimental design of natural dentin in inducing tissue regeneration

Method used

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  • Dentin non-collagen programmed preparation method
  • Dentin non-collagen programmed preparation method
  • Dentin non-collagen programmed preparation method

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Example 1 Preparation of dentin non-collagen

[0041] In this embodiment, the programmed preparation method of dentin non-collagen protein, the main preparation steps are as follows:

[0042] 1) Collect fresh non-carious teeth and store them in 15mmol / L sodium azide for several days after cleaning. Sodium azide is used as a preservative and a variety of enzyme inhibitors;

[0043] 2) Rinse with running water for several hours and store at -80°C for later use;

[0044] 3) Thaw teeth at room temperature, remove all soft tissues, and wash;

[0045] 4) Remove the root cementum with a dental drill or a scalpel;

[0046] 5) Cut the teeth into small pieces with a diamond bone saw or a slow cutter;

[0047] 6) Remove pulp tissue and cells;

[0048] 7) Remove the enamel with a bone knife or bone saw;

[0049] 8) Grinding the dentin sheet into a fine powder; the fine powder obtained by grinding the dentin sheet can pass through a 60-mesh sieve.

[0050] 9) After weighing, p...

Embodiment 2

[0054] Example 2 Detecting the odontogenic / osteogenic induction of the dentin non-collagen protein prepared in Example 1 on bone marrow mesenchymal stem cells of the jaw

[0055] Test method: test method: real-time quantitative PCR and protein immunoblotting detection

[0056] Test process: Add the dentin non-collagen protein prepared in Example 1 to the culture medium in an amount of 10 μg / mL to stimulate bone marrow mesenchymal stem cells for 0, 3, 7, and 14 days, extract total RNA and total protein, and reverse the total RNA Quantitative PCR detection was performed after recording, and the total protein was detected by Western blotting to determine the main indicators of bone marrow mesenchymal stem cells odontogenic / osteogenic differentiation after DNCPs induction for different time OCN / OCN, RUNX2 / RUNX2, OSX / OSX, DSPP / DSP, BSP changes. The result is as Figure 1a — Figure 1b shown.

[0057] Experimental results: The experimental results show that under the i...

Embodiment 3

[0059] Example 3 Detecting the Activation of the MAPK Signaling Pathway During the Odontogenesis / Osteogenesis Induction Effect of Dentin Non-Collagen Protein Prepared in Example 1 on Bone Marrow Mesenchymal Stem Cells

[0060] Test method: Western blot detection

[0061] Test process: Add the dentin non-collagen protein prepared in Example 1 into the culture medium in an amount of 10 μg / mL to stimulate the bone marrow mesenchymal stem cells for 0, 30, 60, and 90 minutes, extract the total protein, and perform protein immunoblotting detection. Phosphorylated P38, phosphorylated ERK, phosphorylated JNK and the total content of P38, ERK, JNK were measured, and gray value analysis was performed.

[0062] Experimental results: The experimental results showed that under the induction of DNCPs, the contents of phosphorylated P38 and phosphorylated ERK in rat long bone marrow mesenchymal stem cells were significantly increased at 30, 60, and 90 minutes, which was significantly higher ...

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Abstract

The invention provides a dentin non-collagen programmed preparation method. The method comprises the following steps: 1) collecting fresh non-dental caries tooth, cleaning the tooth and preserving thetooth in sodium azide with concentration being 15 mmol / L for several days, wherein sodium azide is taken as an antiseptic and a plurality of enzyme inhibitors; 2) flushing the tooth with running water for several hours and preserving the tooth at the temperature of -80 DEG C for standby; 3) unfreezing the tooth at room temperature, removing all soft tissues, cleaning the material; 4) using a dental drill or an operating knife to remove root cementum; 5) using a diamond bone saw or a low-speed cutting machine for cutting the tooth to small sheets; 6) removing pulp tissue and cells; 7) using abone knife or the bone saw to remove enamel; and 8) grinding the dentin sheet to fine powder. The method has the following beneficial results that 1) through a simple making technology, a lot of non collagen used for regenerating the subsequent related dentin induced tissues can be produced; and 2) a prepared dentin non-collagen mixture can simulate the induction action of the natural dentin in the pulp tissue regeneration.

Description

technical field [0001] The invention relates to a method for extracting mixed proteins, in particular to a method for extracting hard tissue proteins, in particular to a programmed preparation method for dentin non-collagen proteins. Background technique [0002] Clinically, root canal treatment is required when the pulp tissue is exposed, infected or inflamed, and the apical area is inflamed, that is, the pulp tissue and infected dentin are removed, and most of the affected teeth after root canal treatment have reduced mechanical strength and are easily shortened. lifespan of the tooth. With the development of tissue engineering technology, inducing pulp tissue regeneration has gradually become a research hotspot. Studies have shown that teeth with apical foramen destruction can be induced to continue to develop and form viable pulp tissue using revascularization methods, which suggests that the dentin of the root canal wall plays a key role in the process of pulp regenera...

Claims

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Application Information

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IPC IPC(8): C07K1/14
CPCC07K1/145
Inventor 于金华雷港周洲张光东庄颖许涛闫明景双林
Owner AFFILIATED STOMATOLOGICAL HOSPITAL OF NANJING MEDICAL UNIV
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