Application of raloxifene in mesenchymal stem cell in-vitro osteoblast differentiation

A technology of osteogenic differentiation and mesenchymal stem cells, which is applied in the field of cell biotechnology and bone tissue engineering, can solve the problem of low efficiency of directional osteogenic induction and differentiation, achieve the effect of promoting expression and calcium ion deposition, and improving efficiency

Inactive Publication Date: 2012-03-14
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on mesenchymal stem cells has made great progress, but there are still many problems in front of people, one of the important points is: the efficiency of mesenchymal stem cells to induce osteogenic differentiation is low

Method used

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  • Application of raloxifene in mesenchymal stem cell in-vitro osteoblast differentiation
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  • Application of raloxifene in mesenchymal stem cell in-vitro osteoblast differentiation

Examples

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Embodiment 1

[0026] Example 1: Extraction, culture and osteogenic differentiation of rat adipose stem cells

[0027] 1. Extraction of Rat Adipose Stem Cells

[0028] 1) The rats were killed by neck dislocation, fixed on the experimental table in the supine position, and sterilized with 70% ethanol;

[0029] 2) Cut the skin along the direction of the groin, and carefully separate the white adipose tissue of the groin;

[0030] 3) Immediately put the adipose tissue into ice-cold PBS, and rinse it repeatedly 3 times to remove tissue debris;

[0031] 4) Cell separation is carried out in the ultra-clean bench within 30 minutes after sampling;

[0032] 5) Carefully remove the fascia and small blood vessels on the surface of the adipose tissue again, and rinse with ice-cold PBS 3 times to remove the red blood cells adhered to the surface of the adipose tissue;

[0033] 6) Mince the adipose tissue and add twice the volume of 0.075% type I collagenase;

[0034] 7) 5% CO 2 、 Digest with constan...

Embodiment 2

[0042] Example 2: Extraction, culture and osteogenic differentiation of rat bone marrow stem cells

[0043] 1. Extraction of Rat Bone Marrow Stem Cells

[0044] 1) Take 4-week-old SD rats and kill them by neck dislocation

[0045] 2) Soak in 75% ethanol for 10 minutes for disinfection, take out the femur and tibia under aseptic conditions, and thoroughly remove the muscle tissue attached to them, remove the epiphysis at both ends of the femur and tibia, and expose the bone marrow cavity.

[0046]3) Flush the bone marrow cavity with PBS containing heparin (200U / ml) to make a single cell suspension

[0047] 4) Centrifuge at 1500rpm for 10min, remove the upper layer of fat and supernatant, blow and beat the precipitate with PBS, and make bone marrow single cell suspension.

[0048] 5) Use L-DMEM complete medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin and pipette evenly to form a single cell suspension. Transfer the cell suspension to a culture flask...

Embodiment 3

[0051] Example 3: Effect of Raloxifene on Osteogenic Differentiation of Adipose Stem Cells

[0052] Adipose-derived stem cells were divided into 5×10 4 The density of cells / well was seeded in a six-well plate, and after the cells were full, it was replaced with an osteogenic induction medium for osteogenic induction. Experimental grouping: control group (Con, adding dimethyl sulfoxide of corresponding concentration), Ral group (raloxifene group, 10 -8 M, 10 -7 M and 10 -6 M), Ral (10 -7 M)+L-NAME (1 mM) group, 1 mM L-NAME group. After 14 days of induction, the culture medium was discarded, washed three times with PBS, total RNA was extracted by Trizol method, reverse transcribed, and the mRNA levels of BMP2 and ALP were identified by quantitative PCR.

[0053] The results are shown in the figure. We used quantitative PCR to identify the effects of raloxifene on the mRNA expression of osteogenic differentiation marker proteins alkaline phosphatase (ALP) and bone morphogene...

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Abstract

The invention belongs to the technical field of cytobiology and bone tissue engineering, relates to a compound used for treating osteoporosis, and especially relates to an application of raloxifene in mesenchymal stem cell in-vitro osteoblast differentiation. According to the invention, raloxifene is added during a mesenchymal stem cell in-vitro osteoblast differentiation culturing process. With the application, mRNA and protein level expressions of osteoblast differentiation marker genes ALP and BMP2, and calcium ion sedimentation can be increased; and mesenchymal stem cell differentiation towards osteoblast can be promoted. The invention relates to the application of raloxifene in mesenchymal stem cell in-vitro osteoblast differentiation, and in treatments of diseases such as fractures,bone defects, and bone wounds.

Description

1. Technical field [0001] The invention belongs to the technical fields of cell biotechnology and bone tissue engineering, and specifically relates to a compound for treating osteoporosis: the application of raloxifene in the osteogenic differentiation of mesenchymal stem cells in vitro. 2. Background technology [0002] Bone defects caused by bone trauma, tumors and inflammation are common and refractory diseases in orthopedic clinics, and the only way to repair them is through bone transplantation. Bone graft materials can be roughly divided into autologous bone, allogeneic bone, and artificial bone material according to their source. Bone harvesting surgery for autologous bone transplantation increases the trauma of patients, and the amount of bone harvested is limited, which often cannot meet the needs of bone grafting; both allogeneic bone and heterogeneous bone have rejection. Bone tissue engineering can not only repair large areas of bone defects, but also can be sha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K31/4535A61P19/08
Inventor 张峻峰卢翔郭继刚陈江宁
Owner NANJING UNIV
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