New osteoporosis-resisting medicine screening model expressed by bone formation promoting protein

A technology for anti-osteoporosis and protein expression, which is applied to the determination/testing of microorganisms, biochemical equipment and methods, etc., can solve the problems that the screening models for anti-osteoporosis drugs have not been reported, and achieve short screening cycles and high detection rates. The effect of high accuracy and high sensitivity

Inactive Publication Date: 2003-09-10
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Abstract
  • Description
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Problems solved by technology

[0005] So far, there is no report on the anti-osteo

Method used

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  • New osteoporosis-resisting medicine screening model expressed by bone formation promoting protein
  • New osteoporosis-resisting medicine screening model expressed by bone formation promoting protein
  • New osteoporosis-resisting medicine screening model expressed by bone formation promoting protein

Examples

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[0021] Example 1: Preliminary screening of 3000 strains of fungal fermentation broth

[0022] For strain fermentation, pick a small piece of cultured species from the slope into a 500ml Erlenmeyer flask containing 100ml of fermentation medium, culture it on a rotating shaker at 26°C and 120 rpm for 6 days, and sample for viability. The fermentation broth was centrifuged at 5000 rpm for 15 minutes, and the supernatant was extracted with paper, placed on a test plate inoculated with 1% SO4, incubated at 26°C for 24 hours, and the results were observed. In the tested samples, the diameters of the inhibition zone of the positive strains 2101, 3586, and 3879 were 27mm, 24mm, and 25mm, respectively, and the inhibition effect was significant.

[0023] Then take the above-mentioned positive strain fermentation supernatant and place it on a test plate inoculated with 1% SO4. Place the MVA-containing paper (8mg / piece) next to the paper containing the fermentation broth so that the center is...

Example Embodiment

[0025] Example 2: Re-screening of crude fermentation broth of positive strains

[0026] Cell transfection application GIBCO LipofectAMINE 2000 Reagent kit: trypsinize MC3T3-E1 cells, dilute them with serum-containing DMEM medium and quickly and evenly add them to a 96-well cell culture plate. Hemocytometer counts 20,000 cells / well. Transfection after 24 hours: pYJ2 0.8μg / well, pRL-TK 0.08μg / well, add 25μL of pre-warmed serum-free DMEM medium, liposomes at a ratio of 1μl / well, add 25μL of serum-free DMEM medium, and transfect within 5 minutes The plasmids were mixed and left at room temperature for 20 minutes. At the same time, aspirate and discard the medium in the plate and replace it with 100 μL serum-free DMEM medium. Add 50μl of the mixture to each well and incubate at 37°C for 24hr, then add the test substance (0.2μg / ml, 2μg / ml, 20μg / ml control drug lovastatin and crude fermentation broth of positive strain), and continue to incubate for 24hr.

[0027] Fluorescence detection ...

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Abstract

The present invention relates to osteoporosis-resisting medicine screening model expressed by bone formation promoting protein. The model has steps combining microbiological method for initial screening and cytological method for re-screening. In the initial screening, fermented liquid or other natural product is used to screen HMG-CoA reductase inhibitor in biological cholesterin synthesizing route; and in the re-screening, the initial extractive of fermented liquid or other natural product is used, bone formation protein bmp-2 gene promoter is used as target point and bmp-2 expression effect is used as identification index. The screening model has high sensitivity, is suitable for high-flux screening and can provide promising candidate compounds for the development of osteoporosis-resisting medicine.

Description

Technical field: [0001] The invention relates to a novel anti-osteoporosis drug screening model established at the level of microorganisms and mammalian cells, in particular, a novel anti-osteoporosis drug screening model related to cholesterol synthesis and metabolism and expression regulation of bone-forming proteins. Background technique: [0002] Osteoporosis (Osteoporosis) is a disease with significant reduction in bone mass throughout the body, which can easily lead to motor dysfunction and pathological fractures, and greatly affects people's quality of life. According to statistics, about 100 million people worldwide suffer from osteoporosis. my country is the country with the largest elderly population in the world, and the elderly over 60 years old account for 9.7% of the total population of our country. With the acceleration of the aging process in our country, its severity is also increasing year by year. Finding effective drugs and therapeutic methods for the pr...

Claims

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Application Information

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IPC IPC(8): C12Q1/04C12Q1/26C12Q1/68
Inventor 张月琴杨隽尚广东司书毅苏学惠黄明玉魏玉珍关艳
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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