Method for promoting osteoblast differentiation by using Runx2 and Osterix and application thereof

An osteoblast differentiation and protein technology, which is applied in the use of Runx2 and Osterix to promote osteoblast differentiation and its application fields, can solve the problems of ineffective use of osteoblast differentiation and bone formation.

Active Publication Date: 2013-01-30
NORTHEAST NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This in vivo and in vitro discrepancy suggests that Runx2 has not been effectively utilized to promote osteoblast differentiation and bone formation

Method used

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  • Method for promoting osteoblast differentiation by using Runx2 and Osterix and application thereof
  • Method for promoting osteoblast differentiation by using Runx2 and Osterix and application thereof
  • Method for promoting osteoblast differentiation by using Runx2 and Osterix and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Plasmid Construction and Cell Culture

[0047] Cell culture

[0048] Plat-E cells (Cell Biolabs Inc.) were cultured in a medium containing 10% fetal bovine serum (Hyclone), 1 μg / ml puromycin (puromycin, Sigma), 10 μg / ml blasticin (brasticidine, Sigma), 100 U / ml Penicillin (Sigma), 100 μg / ml streptomycin (Sigma) in DMDM ​​medium (Gibco) and adhered to 6-well plates coated with rat tail type I collagen. C3H10T1 / 2 cell lines (ATCC, CCL-226) and other cell lines obtained from C3H10T1 / 2 cells were cultured in DMDM ​​medium containing 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin middle. The above cells were cultured at 37°C, 5% CO 2 in the incubator.

[0049] Plasmid construction

[0050] Insert the cDNA of FLAG-tagged full-length Runx2 (SEQ.ID.No.2, derived from mouse) and FLAG-tagged full-length Osterix (SEQ.ID.No.4, derived from mouse) into pMXs / neo Vector (Cell Biolabs Inc), or insert FLAG-tagged full-length Osterix (SEQ.ID.No....

Embodiment 2

[0052] Example 2: Establishment of C3H10T1 / 2 cell lines stably expressing Runx2 or Osterix respectively and stably expressing Runx2 and Osterix simultaneously

[0053] This example was accomplished by cell transfection, retrovirus infection and immunoblotting.

[0054] Specifically, pMXs / neo (as a negative control), and the Runx2-pMXs / neo constructed in Example 1, and the Osterix-pMXs / neo plasmids were transformed into Plat-E cells with Fugene 6 transfection reagent (Promega) . After 72 hours, the supernatant of the medium (DMEM medium containing 10% fetal bovine serum) contained the retrovirus. After filtering through a 0.45 μm filter membrane (Milipore), the filtrate was mixed with 5 mg / ml polybrene (polybrene, Sigma) at a ratio of 1000:1 and added to the C3H10T1 / 2 cells adhered one day in advance. After 6 hours, fresh medium was changed and culture was continued for 24 hours. The cells were screened with G418 to obtain the cells infected by the retrovirus, and the uninfe...

Embodiment 3

[0057] Example 3: The ability of Osterix to induce osteoblast differentiation is weaker than that of Runx2

[0058] The Ctrl, Runx2 and Osterix cells obtained in Example 2 were differentiated in osteoblast differentiation medium (adding 100 μg / ml ascorbic acid (Sigma) and 5 mM β-glycerophosphate (β-glycerophosphate, Sigma) to normal medium) for 6 Alkaline phosphatase activity staining and von Kossa staining were performed two days later. It was found that the ability of Osterix to induce alkaline phosphatase activity level and calcification level was significantly weaker than that of Runx2. See results figure 1 B and figure 1 c.

[0059] The Ctrl, Runx2 and Osterix cells obtained in Example 2 were also cultured in normal medium until confluent and stained for alkaline phosphatase activity. The results still show that Osterix is ​​weaker than Runx2 in inducing the level of alkaline phosphatase activity. See results figure 1 b.

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Abstract

The invention relates to a method for co-expression of Runx2 and Osterix in mesenchymal stem cells or other non-osteoblasts by a special mode that the expression level of Runx2 is less than the expression level of Osterix, thus speeding up the induction of osteoblast differentiation. The invention provides a pharmaceutical composition for prevention or treatment of osteoblast differentiation related diseases. The pharmaceutical composition contains Runx2 protein and Osterix protein, wherein the Runx2 protein content is smaller than the Osterix protein content. The invention also provides application of the Runx2 protein and the Osterix protein in preparation of the pharmaceutical composition for prevention or treatment of osteoblast differentiation related diseases, and in the pharmaceutical composition, the content of the Runx2 protein is less than that of the Osterix protein. The pharmaceutical composition and the special co-expression mode of Runx2 and Osterix provided in the invention can be used for treatment of osteoporosis, osteogenesis imperfecta, periodontal diseases, fractures and other bone diseases, and also can be used for research, development and production of tissue-engineered bones. The invention also provides a method for screening drugs preventing and treating bone diseases.

Description

technical field [0001] The present invention relates to the application of Runx2 and Osterix in the preparation of a pharmaceutical composition for preventing or treating diseases related to osteoblast differentiation, in the pharmaceutical composition, the content of Runx2 protein is less than that of Osterix protein, preferably, The ratio of Runx2 protein content to Osterix protein content is 1:N, wherein N is greater than 1, such as 1:2, 1:3, to 1:4 or even a lower ratio, and the more preferred ratio is 1:4. Specifically, the present invention accelerates the induction of non-osteoblast-oriented osteoblast differentiation or enhances the function of osteoblasts by co-expressing Runx2 and Osterix in a manner that the expression (or activity) level of Runx2 is lower than that of Osterix. And according to changing the ratio of the expression (or activity) level of Runx2 to the expression (or activity) level of Osterix, thereby causing changes in the differentiation and functio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61K48/00A61P19/08A61P19/10C12Q1/02
Inventor 刘文广孟令仪张忠丽曾宪录
Owner NORTHEAST NORMAL UNIVERSITY
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