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BMP composition for synergistic induction of ossification differentiation, and application thereof

A technology of osteogenic differentiation and composition, applied in the field of genetic engineering

Inactive Publication Date: 2011-10-05
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, whether BMPs have a synergistic induction of osteogenic differentiation has not been reported

Method used

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  • BMP composition for synergistic induction of ossification differentiation, and application thereof
  • BMP composition for synergistic induction of ossification differentiation, and application thereof
  • BMP composition for synergistic induction of ossification differentiation, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Construction and Identification of BMP Gene Recombinant Adenovirus

[0037] 1. Construction of recombinant adenovirus with BMP gene

[0038] The construction process of BMP gene recombinant adenovirus is as follows: figure 1 shown, including the following steps:

[0039] 1. Amplification of BMP gene

[0040] According to the coding gene sequences of 5 kinds of human BMP reported by GenBank: BMP2 (accession number is NM-001200.2, SEQ ID No.1), BMP4 (accession number is NM-001202.3, SEQ ID No.2), BMP6 (accession number is Design and synthesis of 5 pairs of primers: BMP2F / BMP2R, BMP4F / BMP4R, BMP6F / BMP6R, BMP7F / BMP7R, BMP9F / BMP9R were used for PCR amplification of BMP2, 4, 6, 7, and 9 genes respectively, and the primer sequences were as follows:

[0041] BMP2F: 5'-cgcggtacc ggatcc accaccatggtggccgggacccgctgtcttc-3' (SEQ ID No. 6),

[0042] BMP2R: 5'-tgctctaga aagctt ctagcgacacccacaaccctccac-3' (SEQ ID No. 7);

[0043] BMP4F: 5'-cgcggtacc ggatcc accacc...

Embodiment 2

[0062] Example 2. Construction and identification of BMP double gene co-expression recombinant adenovirus

[0063] 1. Construction of BMP double-gene co-expression recombinant adenovirus

[0064] The construction process of BMP double gene co-expression recombinant adenovirus is as follows: figure 1 shown, including the following steps:

[0065] 1. Amplification of BMP double genes

[0066]According to the coding gene sequences of 5 human BMPs reported by GenBank, 5 pairs of primers were designed and synthesized: BMP2F / BMP2R, BMP4F / BMP4R, BMP6F / BMP6R, BMP7F / BMP7R, BMP9F* / BMP9R*, which were used for PCR amplification of BMP2, 4, 6, 7, 9 genes, wherein the sequences of primers BMP2F / BMP2R, BMP4F / BMP4R, BMP6F / BMP6R, BMP7F / BMP7R are the same as those described in Example 1, and the sequences of primers BMP9F* / BMP9R* are as follows:

[0067] BMP9F*: 5'cgc ggtacc accaccatgggcatgtgtcctggggcactgtgggtg-3' (SEQ ID No. 16),

[0068] BMP9R*: 5'-tgc tctaga ctacctgcacccaacactctgccac...

experiment example 1

[0081] Experimental Example 1: Detection of BMP9 and BMP2, 4, 6 or 7 synergistically inducing osteogenic differentiation of C3H10 cells

[0082] Early osteogenic differentiation marker - ALP activity detection

[0083] Seed C3C10 cells to 100mm 2 In the culture dish, after the cells adhered to the wall, the BMP double gene co-expressed recombinant adenoviruses Ad-BMP9 / BMP2, Ad-BMP9 / BMP4, Ad-BMP9 / BMP6 or Ad-BMP9 / BMP7 were transferred at an MOI of 50-200 rpm. C3C10 cells were transfected, and on the 9th day after transfection, the cells were lysed with cell culture lysate (Lot#27134901, Promega Company), centrifuged to get the supernatant, and alkaline phosphatase chemiluminescence detection kit (SEAP Chemiluminescent Assay Kit, BD Clontech company) to detect ALP activity, and operate according to the instructions of the kit.

[0084] The result is as image 3 As shown, the ALP activity of C3C10 cells transfected with BMP double gene co-expression recombinant adenovirus Ad-BM...

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Abstract

The invention discloses a BMP composition for synergistic induction of ossification differentiation. The BMP composition comprises of a BMP 9 and a MBP 2, 4, 6 or 7, and has a stronger induction effect on ossification differentiation than a single BMP. The invention also discloses a BMP gene recombinant virus composition and a BMP double-gene coexpression recombinant virus, which express the BMP composition for synergistic induction of ossification differentiation in eukaryotic cells, and can introduce the BMP 9 gene and the BMP2, 4, 6 or 7 gene into target cells and enable high efficient and stabilized expression, without in vitro translation and BMP separation and purification, so as to simplify BMP preparation process and reduce costs. The BMP composition, BMP gene recombinant virus composition and the BMP double-gene coexpression recombinant virus of the present invention can be applied to preparation of tissue engineering artificial bones and medicaments for treating bone defect, delayed unions of bones, nonunion of fractures and spinal diseases.

Description

technical field [0001] The present invention relates to the technical field of genetic engineering, in particular to bone morphogenetic protein (bone morphogenetic protein, BMP) composition synergistically inducing osteogenic differentiation, BMP gene recombinant virus composition and BMP double gene co-expression recombinant virus, and also relates to the above-mentioned BMP combination The application of BMP gene recombination virus composition and BMP double gene co-expression recombination virus. Background technique [0002] Bone defects and fracture nonunions caused by trauma, tumor, infection or improper fracture treatment are very common in clinical practice. Its conventional treatment is bone grafting. Traditional bone graft materials include autologous bone, allograft bone and xenograft bone. Autologous bone has the same bone scaffold as the recipient bone, retains osteoblasts, cytokines and other biologically active substances, has osteoinduction, osteoconductio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61K48/00A61P19/08C12N15/86C12N15/861A61L27/54
Inventor 何通川邓忠良康权罗进勇陈亮何百成罗小辑
Owner CHONGQING MEDICAL UNIVERSITY
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