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Accelerant for promoting mesenchymal stem cell osteogenic differentiation

A technology for osteogenic differentiation and stem cells, applied in the field of stem cells

Active Publication Date: 2020-08-14
青岛赛尔斯干细胞库有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Current studies have shown that snail polypeptides have anti-tumor effects, but there is no research on the osteogenic differentiation of snail polypeptides in bone marrow mesenchymal stem cells.

Method used

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  • Accelerant for promoting mesenchymal stem cell osteogenic differentiation
  • Accelerant for promoting mesenchymal stem cell osteogenic differentiation
  • Accelerant for promoting mesenchymal stem cell osteogenic differentiation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] (1) Wash the mud snail, take 100g of soft tissue, freeze-dry and pulverize, add 300g of water to obtain mud snail homogenate;

[0042] (2) Add 8g of papain, adjust the pH to 6, and bathe in a constant temperature water bath at 55°C for 4 hours to obtain the snail hydrolyzate;

[0043] (3) Heat in a water bath at 95°C for 20 minutes, centrifuge at 5000r / min to take the supernatant, and obtain the crude snail extract;

[0044] (4) Pass the coarse mud snail extract through a nanofiltration membrane with a pore size of 2nm to obtain the fine mud snail extract;

[0045] (5) Vacuum freeze-drying the fine mud snail extract in a vacuum freeze dryer to obtain the mud snail polypeptide.

Embodiment 2

[0047] Fluorescent quantitative PCR experiment

[0048] 1. RNA extraction

[0049] (1) Inoculate the bone marrow mesenchymal stem cells in the culture plate, and when the cell density reaches 70%, add the osteogenic induction medium containing 0mg / ml, 25mg / ml, 50mg / ml and 100mg / ml mud snail polypeptide respectively , the medium was changed every 2-3 days, and each group had 3 repetitions.

[0050] (2) After 14 days of culture, remove the medium, add 500 μL Trizol, and repeatedly pipette the cells until a clear and non-viscous liquid is formed;

[0051] (3) Transfer the mixture to a 1.5ml EP tube, add 100μL of chloroform, shake vigorously on the shaker for 15 seconds, and let stand at room temperature for 5 minutes;

[0052] (4) 4°C, low-temperature high-speed centrifuge, 12000r / min centrifuge for 15min, carefully transfer the upper transparent RNA aqueous phase to a new RNase-free EP tube;

[0053] (5) Add an equal volume of isopropanol and mix well, place on ice for 10min,...

Embodiment 3

[0072] Western blot detection

[0073] 1. Protein extraction

[0074] (1) Seed the bone marrow mesenchymal stem cells in the culture plate. When the cell density reaches 70%, add the osteogenic induction medium containing 0mg / ml and 100mg / ml mud snail polypeptide respectively, and carry out once every 2-3 days Medium was changed, and each group had 3 repetitions.

[0075] (2) After 14 days, add 100 μL RIPA lysate to the culture plate, scrape off the cells with a cell scraper, and collect the cell lysate into an EP tube;

[0076] (3) Break the cells with a cell ultrasonic breaker, centrifuge at 4°C, 12000r / min, for 15min;

[0077] (4) Transfer the supernatant to a new EP tube, and use the BCA method to measure the protein concentration;

[0078] (5) Use 5× loading buffer to adjust the protein concentration to 2 μg / μL, and cook in boiling water for 5 minutes to obtain protein samples.

[0079] 2.Western blot experiment

[0080] (1) Configure 12% separating gel and 5% stacki...

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Abstract

The invention belongs to the technical field of stem cells, and particularly relates to an accelerant for promoting mesenchymal stem cell osteogenic differentiation. Fluorescent quantitative PCR experiments, Western Blot experiments and alizarin red staining experiments find that bullacta exarata polypeptide can obviously promote the expression of the mRNA level and the protein level of mesenchymal stem cell osteogenic differentiation related genes ALP, BGLAP and RUNX2, and can obviously promote the mineralization level of bone marrow mesenchymal stem cells, so that the bullacta exarata polypeptide can be used as the accelerant of the mesenchymal stem cell osteogenic differentiation to promote osteogenic differentiation of the mesenchymal stem cells, and more osteoblasts are provided for treatment of osteoporosis, bone injury and other diseases.

Description

technical field [0001] The invention belongs to the technical field of stem cells, and in particular relates to an accelerator for promoting osteogenic differentiation of mesenchymal stem cells. Background technique [0002] In the clinical treatment of bone injuries, the treatment and prevention of large bone defects, postoperative nonunion and osteoporosis have become urgent problems to be solved in orthopedics. The traditional bone grafting surgery has a lot of damage and limited bone mass, so a less traumatic, safe and effective method is needed to replace it. Currently, tissue engineering technology has become an important method to solve bone nonunion and bone defect. Enhanced bone formation is an effective treatment for bone damage. important strategy. [0003] Bone marrow mesenchymal stem cells are a kind of primitive cells with self-renewal and multidirectional differentiation potential, and are the main source of osteoblasts in vivo. The proliferation and osteoge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/077C12P21/06C07K1/14C07K1/34
CPCC12N5/0663C12N5/0654C12P21/06C07K1/145C07K1/34C12N2501/998
Inventor 不公告发明人
Owner 青岛赛尔斯干细胞库有限公司
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