A kind of periodontal ligament stem cell proliferation and osteogenic differentiation promoter

A periodontal ligament stem cell and osteogenic differentiation technology, applied in the field of periodontal ligament stem cells, can solve the problems of restoring periodontal support tissue, inability to

Active Publication Date: 2021-04-16
山东中医药大学第二附属医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods can only temporarily relieve the patient's symptoms, but cannot fundamentally restore the periodontal supporting tissue

Method used

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  • A kind of periodontal ligament stem cell proliferation and osteogenic differentiation promoter
  • A kind of periodontal ligament stem cell proliferation and osteogenic differentiation promoter
  • A kind of periodontal ligament stem cell proliferation and osteogenic differentiation promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0039] Silencing RP11-289F5.1 promotes ALPase activity in dental pulp stem cells

[0040] Since the expression of RP11-289F5.1 was gradually down-regulated during the osteogenic differentiation of dental pulp stem cells, we speculated that inhibiting the expression of RP11-289F5.1 could effectively promote the osteogenic differentiation of dental pulp stem cells.

[0041] 1. Design sh-RP11-289F5.1 and verify the suppression effect

[0042] (1) Design and synthesize shRNA of RP11-289F5.1, the sequence of sh-RP11-289F5.1 is as follows (the vector used is pENTR™ / U6):

[0043] sh-RP11-289F5.1 sequence Top Strand 5'-CACCGCTGAAGAACGCAATCCAACAGAGATGTTGGATTGCGTTTCTTCAGC-3', SEQ ID NO.6 Bottom Strand 5'-AAAAGCTGAAGAACGCAATCCAACATCTCTGTTGGATTGCGTTTCTTCAGC-3', SEQ ID NO.7

[0044] (2) Use fluorescent quantitative PCR to detect the inhibitory effect of sh-RP11-289F5.1, the experimental results are as follows figure 2 shown. It can be seen from the figure ...

Embodiment 3

[0052] Silencing RP11-289F5.1 to study its effect on the mineralization of periodontal ligament stem cells

[0053](1) The experiment was divided into three groups. The NC group was cultured with complete medium; the sh-NC group was transfected with sh-NC and cultured with osteogenic differentiation medium; the sh-RP11-289F5.1 group was transfected with RP11-289F5 .1 and use osteogenic differentiation medium for culture;

[0054] (2) Culture according to the experimental grouping in step (1). After 21 days of osteogenic differentiation, wash the cells with PBS, rinse the cells twice, and fix the cells with 4% paraformaldehyde for 30 min;

[0055] (3) Discard the fixative, rinse twice with PBS, prepare alizarin red staining solution, add 1.5ml alizarin red staining solution to each well, and incubate at room temperature for 30 minutes in the dark;

[0056] (4) Remove the alizarin red staining solution, rinse twice with deionized water, and take pictures for records.

[0057] ...

Embodiment 4

[0059] (1) The experiment was divided into three groups. The NC group was cultured with complete medium; the sh-NC group was transfected with sh-NC and cultured with osteogenic differentiation medium; the sh-RP11-289F5.1 group was transfected with RP11-289F5 .1 and use osteogenic differentiation medium for culture;

[0060] (2) After 14 days of osteogenic differentiation induction, the culture medium was removed and washed 3 times with PBS;

[0061] (3) Add cell lysate to lyse the cells, collect the lysate into a 1.5ml EP tube, ultrasonically crush and extract protein for 5min, 2s ultrasonication / 3s pause cycle;

[0062] (4) Centrifuge at 12000r / min for 20min at 4°C to collect the precipitate;

[0063] (5) Use the BCA method to measure the protein concentration, and add 5×SDS loading buffer;

[0064] (6) Configure 12% separating gel, load 20ug of sample, stacking gel 80V, 30min, separating gel 120V until bromophenol blue runs to the bottom of the separating gel;

[0065] (7...

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Abstract

The invention provides a periodontal ligament stem cell proliferation and osteogenic differentiation accelerator, which belongs to the technical field of stem cells. The present invention finds for the first time that the expression level of non-coding RNA RP11-289F5.1 is down-regulated during the osteogenic differentiation of periodontal ligament stem cells. Secondly, the present invention designs and synthesizes shRNA for silencing RP11‑289F5.1, and proves that silencing RP11‑289F5.1 can effectively promote osteogenic differentiation and proliferation of periodontal ligament stem cells.

Description

technical field [0001] The invention belongs to the technical field of periodontal ligament stem cells, in particular to a periodontal stem cell proliferation and osteogenic differentiation accelerator. Background technique [0002] Periodontitis is one of the common oral diseases, which is a chronic inflammatory disease of periodontal supporting tissues. Clinically, the main characteristics of patients with periodontitis are: bleeding on probing, redness and swelling of the gums, periodontal formation, and resorption of grooved bone, and eventually tooth loosening and loss. At present, the conventional treatment methods for periodontitis include basic treatment such as periodontal scaling and scaling, periodontal surgery such as periodontal flap surgery and root planing, and combined photodynamic therapy and other methods. However, these methods can only temporarily relieve the patient's symptoms, but cannot fundamentally restore the periodontal supporting tissue. [0003...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/113C12N5/077C12N5/0775
CPCC07K14/4705C12N5/0654C12N15/113C12N2310/14C12N5/0668C12N2501/999C12N2310/531
Inventor 杨桂梅孙玉梅颜淑云张瑜刘京群
Owner 山东中医药大学第二附属医院
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