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Method for efficiently promoting proliferation of primary cell of nucleus pulposus and secretion of extracellular matrix based on biomechanical effect

A technology of primary cells and nucleus pulposus cells, which is applied in the field of efficiently promoting the proliferation of nucleus pulposus primary cells and secreting extracellular matrix, which can solve the problems of difficulty in in vitro culture and the small number of nucleus pulposus cells in the nucleus pulposus tissue in the intervertebral disc, and achieve biological The effect of strong academic function, promotion of nucleus pulposus cell proliferation, and efficient culture

Active Publication Date: 2020-06-19
SHANDONG FIRST MEDICAL UNIV & SHANDONG ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although patients with lumbar disc herniation are relatively common, they are older, the nucleus pulposus tissue in the intervertebral disc is aging and the number of nucleus pulposus cells is small, and it is difficult to culture in vitro

Method used

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  • Method for efficiently promoting proliferation of primary cell of nucleus pulposus and secretion of extracellular matrix based on biomechanical effect
  • Method for efficiently promoting proliferation of primary cell of nucleus pulposus and secretion of extracellular matrix based on biomechanical effect
  • Method for efficiently promoting proliferation of primary cell of nucleus pulposus and secretion of extracellular matrix based on biomechanical effect

Examples

Experimental program
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Effect test

Embodiment 1

[0056] A method for efficiently promoting the proliferation of primary nucleus pulposus cells based on biomechanical effects, comprising the following steps:

[0057] (1) After the primary cells of the nucleus pulposus obtained from the nucleus pulposus tissue are cultivated until the culture flask is overgrown, subculture is performed, and the second generation nucleus pulposus cells are taken;

[0058] (2) After assembling the MechanoCulture, a mechanical loader for cells, install the added force silica gel plate (such as figure 1 shown), ultraviolet sterilization;

[0059] (3) Digest each bottle of second-generation nucleus pulposus cells with 1ml of 0.25% trypsin solution. After digestion, place them in a centrifuge for centrifugation at 1500rpm for 5min, and remove the supernatant after centrifugation. Add 1 ml of DMEM culture medium containing 10v / v% FBS (Gibco) to resuspend the nucleus pulposus cell pellet until no tissue clumps can be seen to form a cell suspension, w...

experiment example

[0075] Fluorescence quantitative method was used to detect the mRNA content of type Ⅰ collagen (COL Ⅰ), type Ⅱ collagen (COL Ⅱ), type Ⅲ collagen (COL Ⅲ), elastin and aggrecan (Aggrecan) expression in nucleus pulposus cells, and the mRNA sequence of each gene was used The NCBI database was searched, and primers were designed using Primer-5 software; reaction conditions: pre-denaturation at 95°C for 30s, denaturation at 95°C for 5s, annealing at 55°C for 30s, and amplification for 40 cycles. The relative expression level of the gene was calculated after measuring the CT value of the cell samples cultured for 7 days in Example 1 and the control group 1 by using a fluorescence quantitative gene amplification instrument.

[0076] Table 1 Relative mRNA expression of secretion in nucleus pulposus cells

[0077] Gene Example 1 Control group 1 COLI 0.9 1.5 COL II 1.8 1.0 COLⅢ 0.9 0.5 Elastin (ELN) 1.3 0.8 Aggrecan 2.9 0.9

[0078] T...

Embodiment 2

[0080] A method for efficiently promoting the proliferation of primary nucleus pulposus cells based on biomechanical effects, comprising the following steps:

[0081] (1) After the primary cells of the nucleus pulposus obtained from the nucleus pulposus tissue are cultivated until the culture flask is overgrown, subculture is performed, and the second generation nucleus pulposus cells are taken;

[0082] (2) Assemble the MechanoCulture, a mechanical loader for cells, install the afterburner silica gel plate, and sterilize it by ultraviolet light;

[0083] (3) Digest each bottle of second-generation nucleus pulposus cells with 1ml of 0.25% trypsin solution. After digestion, place them in a centrifuge for centrifugation at 1500rpm for 5min, and remove the supernatant after centrifugation. Add 1 ml of DMEM culture medium containing 10v / v% FBS (Gibco) to resuspend the nucleus pulposus cell pellet until no tissue clumps can be seen to form a cell suspension, which is counted with a...

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Abstract

The invention relates to a method for efficiently promoting the proliferation of a primary cell of a nucleus pulposus and a secretory extracellular matrix based on a biomechanical effect. The method includes the following step: performing mechanical loading on a cell in a process of culturing the primary cell of the nucleus pulposus. A periodic mechanical stress may promote the proliferation of anucleus pulposus cell on the basis that original characteristics of the primary cell of the nucleus pulposus are retained, the biological function of the secretory extracellular matrix is stronger, and the activity and growth state of the nucleus pulposus cell of tissue engineering are remarkably improved.

Description

technical field [0001] The disclosure relates to a method for efficiently promoting the proliferation and secretion of extracellular matrix of primary nucleus pulposus cells based on biomechanical effects. Background technique [0002] The information disclosed in this Background section is only intended to increase the understanding of the general background of the disclosure, and is not necessarily to be taken as an acknowledgment or any form of suggestion that the information constitutes prior art that is already known to those skilled in the art. [0003] Low back pain is the most common disease in orthopedics. It not only causes mental pain to patients, but also causes huge economic burden. However, there is still no effective treatment in clinical practice. Studies have reported that about 40% of patients' low back pain is caused by intervertebral disc degeneration, and the most obvious changes exist in the nucleus pulposus tissue. The latest research has found that t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0655C12N2527/00
Inventor 王丹丹曹盛楠师浩钧刘凡杰李华忠陈元振王磊磊王涛任鹏程孟岩周军
Owner SHANDONG FIRST MEDICAL UNIV & SHANDONG ACADEMY OF MEDICAL SCI
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