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Method for inducing differentiation of human mesenchymal stem cells to osteoblasts and application thereof

An osteoblast differentiation and stem cell technology, applied in the field of biomedicine, can solve the problems of restricting clinical application, side effects of bone tissue, complex induction system, etc., and achieve the effect of promoting osteoblast differentiation and avoiding osteoblast difficulties

Active Publication Date: 2019-11-05
AFFILIATED HOSPITAL OF ZUNYI MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are disadvantages such as complicated induction system and low osteogenic differentiation efficiency, and dexamethasone is a hormone drug, which has serious side effects on bone tissue, which seriously restricts its clinical application

Method used

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  • Method for inducing differentiation of human mesenchymal stem cells to osteoblasts and application thereof
  • Method for inducing differentiation of human mesenchymal stem cells to osteoblasts and application thereof
  • Method for inducing differentiation of human mesenchymal stem cells to osteoblasts and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Isolation, culture and identification of human mesenchymal stem cells

[0033] Mesenchymal stem cells were isolated from amniotic membrane using fresh placenta of healthy pregnant women who were delivered by full-term cesarean section by mechanical method combined with two enzymes (trypsin and type II collagenase) digestion. Resuspend the cells in low-sugar DMEM medium containing 10% fetal bovine serum, plant them in T25 cell culture flasks, and culture at 37°C, containing 5% CO2 and air saturation humidity above 95%, until the cell fusion degree reaches 80% or more. Subcultured, the 2nd-5th passage cells were used for subsequent experiments. When the second generation cells were cultured to a confluence of 80%, the cells were collected to identify cell surface molecules by flow cytometry. The cells highly expressed CD90, CD105, CD73, CD44 and CD29 and other mesenchymal cell surface molecules, but did not express CD34 , CD11b, CD19, CD45 and HLA-DR and other...

Embodiment 2

[0034] Embodiment 2: Isolation and identification of GD-A natural compound

[0035] The dried Ganoderma lucidum fruiting bodies were crushed by micro-breaking the wall, extracted three times with 10 times the volume of 90-95% methanol under reflux, each time for 2 hours, and the extract was concentrated and dried under reduced pressure to obtain the extract. After being suspended in warm water, it was extracted with ethyl acetate. The ethyl acetate extraction part was chromatographed on a silica gel column and eluted with a gradient of dichloromethane-methanol (1:0→0:1). Compound GD-A was obtained by gradient elution with petroleum ether-ethyl acetate (1:0→0:1), and the eluted fraction was separated by medium-pressure liquid chromatography and reversed-phase semi-preparative chromatography. The compound is 95% pure through HPLC analysis, as figure 2 As shown, its mass spectrometry results are shown in image 3 As shown, its molecular weight is 436, and its molecular formula...

Embodiment 3

[0037] Example 3: Cytotoxicity analysis of GD-A on human mesenchymal stem cells

[0038] In order to illustrate the safety problem of using the compound of the present invention, that is, whether it is safe for human mesenchymal stem cells, a corresponding test is carried out:

[0039] Using the MTT method, different doses (0.001 μM, 0.01 μM, 0.1 μM, 1.0 μM, 10 μM and 100 μM) of GD-A were measured, and the human mesenchymal stem cells were continuously affected for 24 hours, 48 ​​hours and 72 hours respectively. The result is as Figure 6 As shown, compared with the control group, after GD-A was treated for 24h, 48h and 72h, in the dose range of 0.001μM-10μM, GD-A had no cytotoxicity to human mesenchymal stem cells, and even promoted cell proliferation, especially After 24 hours, it showed obvious effect of promoting cell proliferation. However, when the dose of GD-A was 100μM, the three time points all inhibited cell proliferation and had obvious cytotoxicity.

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Abstract

The invention relates to a method for inducing differentiation of human mesenchymal stem cells to osteoblasts and application thereof. By adopting the method of the invention, differentiation of humanmesenchymal stem cells to osteoblasts can be significantly promoted, the cellular morphology becomes oval to polygon, and osteoblast-related gene protein and osteoblast marker alkaline phosphatase are highly expressed. Thereby, alkaline phosphatase activity is remarkably increased, and a large number of mineralized calcium nodules are finally formed. The osteoblasts formed by the osteogenic induction method are applied to seed cells for bone related disease cell transplantation and bone tissue engineering, bone lesions are replaced with osteoblasts, and the difficulty of obtaining osteoblastsin vitro and the problem of high immunogenicity after cell transplantation can be avoided.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method and application for inducing differentiation of human mesenchymal stem cells into osteoblasts. Background technique [0002] Bone defects, osteoporosis, avascular necrosis of the femoral head and other bone diseases have a high incidence rate, even exceeding cardiovascular and cerebrovascular diseases. The disability caused by bone diseases seriously affects the quality of life of patients. Moreover, traditional therapy has little effect, which is a major problem in the medical field and brings a heavy burden to the country and society, becoming a major challenge in the field of public health (Dimitriou R, et al. Bone regeneration: current concepts and future directions. BMC Med. 2011,9: 66; Wang Y, et al . Small molecules and their controlled release that induce the osteogenic / chondrogenic commitment of stem cells. Biotechnology Advances, 2015, 33(8):1626-40.). ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077A61L27/38A61K35/32A61P19/08C07J9/00
CPCC12N5/0654A61L27/3821A61K35/32A61P19/08C07J9/00C12N2506/1392C12N2501/999
Inventor 肖建辉王怡晴
Owner AFFILIATED HOSPITAL OF ZUNYI MEDICAL COLLEGE
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