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A method of inducing a human fibroblast to reprogram the human fibroblast into an osteoblast by utilization of Runx2 and a low-molecular-weight compound

A technology of small molecule compounds and human fibroblasts, applied in the field of using Runx2 and small molecule compounds to induce the reprogramming of human fibroblasts into osteoblasts, can solve the problems of side effects of surrounding healthy tissues, complex expression and secretion regulation, etc. The effect of bone formation, avoiding the risk of tumorigenesis

Active Publication Date: 2015-07-08
YUNNAN JICI INSITUTE FOR REGENERATIVE MEDICINE CO LTD
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AI Technical Summary

Problems solved by technology

The expression products of these genes are secreted proteins, and their expression and secretion regulation is complex
Since they can cause ectopic bone formation through paracrine effects on surrounding non-osseous tissues, long-term sustained high expression in transdifferentiated cells may lead to side effects on healthy tissues surrounding the transplantation site

Method used

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  • A method of inducing a human fibroblast to reprogram the human fibroblast into an osteoblast by utilization of Runx2 and a low-molecular-weight compound
  • A method of inducing a human fibroblast to reprogram the human fibroblast into an osteoblast by utilization of Runx2 and a low-molecular-weight compound
  • A method of inducing a human fibroblast to reprogram the human fibroblast into an osteoblast by utilization of Runx2 and a low-molecular-weight compound

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Experimental program
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Effect test

Embodiment 1

[0053] 1. Plasmid construction and cell culture

[0054] 1.1 Culture of human skin fibroblasts: collect common human skin fibroblasts first, and culture human skin fibroblasts in a medium containing 10% fetal bovine serum (Hyclone) + 100U / ml penicillin (Sigma) + 100 μg / ml streptomycin ( Sigma) + high-glucose DMDM ​​(Gibco) and adhered to the 6-well plate coated with gelatin; seeded cell density 5 ~ 6x10 3 / cm 2 Cultured at 37°C in an incubator with 5% CO2.

[0055] 1.2 Construction of the Runx2 lentiviral vector: the cDNA of the full-length Runx2 (derived from human) was inserted into the pVSVG vector containing GFP, and an empty vector of pVSVG containing only GFP was also constructed.

[0056] 2. Virus infects cells

[0057] Lentivirus packaging and infection: the target gene Runx2 plasmid and the virus packaging plasmid were co-transfected into HEK293T cells through Lipofectamine 2000, and a high-titer virus was obtained. Ordinary human skin fibroblasts were infect...

Embodiment 2～ Embodiment 8

[0069] Example 2 ~ Example 8: in embodiment 2 to embodiment 8, culture and transdifferentiation method are identical with embodiment 1, no longer emphatically repeat, but its formula and operation time are different, specifically as shown in the table below.

[0070]

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Abstract

The invention relates to a method of inducing a human fibroblast to reprogram the human fibroblast into an osteoblast by utilization of Runx2 and a low-molecular-weight compound, particularly relates to a method of inducing a human fibroblast to transdifferentiate the human fibroblast into an osteoblast under the existence of dexamethasone by utilization of Runx2 gene expression, a low-molecular-weight compound CHIR99021 and / or a low-molecular-weight compound forskolin, and belongs to the fields of cytobiology and tissue engineering. The method includes: constructing a human skin fibroblast infected by a slow virus carrying Runx2-GFP, performing transdifferentiation of the human fibroblast under the existence of the dexamethasone by utilization of the low-molecular-weight compound CHIR99021 having a concentration of 5-10 muM and the low-molecular-weight compound forskolin having a concentration of 10 muM, changing into an osteoblast inducing liquid after 7-15 days, and inducing, and the fibroblast can be induced into the osteoblast after 5-20 days. The method can obtain osteoblasts simply and rapidly and can be used for cell therapy for bone metabolic disease, fracture, and the like. A pharmaceutical composition provided by the invention can be used for research, development and production of tissue-engineered bone, brings convenience to fundamental research and brings a prospect for clinical application.

Description

technical field [0001] The present invention relates to a method of using Runx2 and small molecule compounds to induce human fibroblasts to reprogram osteoblasts, especially a method of using Runx2 gene expression and small molecule compound CHIR99021 and / or small molecule compound forskolin in the presence of dexamethasone The invention discloses a method for inducing transdifferentiation of human fibroblasts into osteoblasts under certain conditions, belonging to the field of cell biology and tissue engineering. Background technique [0002] Bone tissue engineering technology integrates osteoblasts and / or bioactive factors that can guide bone tissue regeneration into a special scaffold structure as a tissue-engineered composite bone, hoping to replace traditional graft materials and techniques. Therefore, bone tissue engineering requires a large number of seeded cells and specialized scaffold materials to support cell adhesion and differentiation. Traditionally used seed ...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/12C12N5/10C12N5/077
Inventor 胡敏李燕皎
Owner YUNNAN JICI INSITUTE FOR REGENERATIVE MEDICINE CO LTD
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