Extracting method of bovine pituitary extract

An extraction method and extract technology, applied in the field of biomedicine, can solve the problems of low extraction efficiency, inability to large-scale production, complex extraction method, etc., and achieve the effects of high extraction efficiency, suitable for large-scale production, and saving pretreatment time.

Active Publication Date: 2017-10-27
GUANGDONG BOXI BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of the above problems, the purpose of the present invention is to provide a method for extracting bovine pituitary gland extract, wh

Method used

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  • Extracting method of bovine pituitary extract
  • Extracting method of bovine pituitary extract
  • Extracting method of bovine pituitary extract

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] BPE extraction: Collect 40 fresh bovine pituitary glands, place them in RPMI-1640 medium, and transport them back to the operation room at low temperature. Brush with 4°C pre-cooled PBS for 3 to 5 times. After cleaning the blood stains, remove the external adhesion tissue and white fascia, expose the whole pituitary gland, then cut it into pieces with scissors, add PMSF with a final concentration of 10mmol / L, ultrasonic break, and ultrasonic The frequency is 300W, 5s / time, with an interval of 10s between each two times, until the tissue becomes a paste, and the diameter of the particles in the tissue does not exceed 0.05cm. Transfer the above treatment to a clean beaker, add 4°C pre-cooled PBS at a ratio of 1g / ml, then stir in a 4°C refrigerator at 300rpm for 150min, then transfer the mixture to a centrifuge tube, centrifuge at 8000rpm for 10min and collect The supernatant was subjected to secondary centrifugation at 10,000 rpm for 15 min, and the supernatant was collec...

Embodiment 2

[0031] BPE extraction: Collect 40 fresh bovine pituitary glands, place them in RPMI-1640 medium, and transport them back to the operation room at low temperature. Scrub with 4°C pre-cooled PBS for 3 to 5 times. After cleaning the blood stains, remove the external adhesion tissue and white fascia, expose the whole pituitary gland, then cut it into pieces with scissors, add PMSF with a final concentration of 50mmol / L, ultrasonic break, and ultrasonic The frequency is 500W, 5s / time, with an interval of 10s between each two times, until the tissue becomes a paste, and the diameter of the particles in the tissue does not exceed 0.05cm. Transfer the above treatment to a clean beaker, add 4°C pre-cooled physiological saline at a ratio of 2g / ml, then stir in a 4°C refrigerator at a speed of 300rpm for 100min, then transfer the mixture to a centrifuge tube, centrifuge at 6000rpm for 15min The supernatant was collected for secondary centrifugation at 10,000 rpm for 15 min, and the super...

Embodiment 3

[0033]BPE extraction: Collect 30 fresh bovine pituitary glands, place them in DMEM medium, and transport them back to the operation room at low temperature. Brush with 4°C pre-cooled PBS for 3 to 5 times. After cleaning the blood stains, remove the external adhesion tissue and white fascia, expose the whole pituitary gland, add PMSF with a final concentration of 100mmol / L, and then use a homogenizer at 2000rpm, Start for 3 seconds for the first time, and perform jogging and crushing at intervals of 1-2 seconds until the tissue becomes a paste, and the diameter of the particles in the tissue does not exceed 0.05cm. Transfer the above treatment to a clean beaker, add 4°C pre-cooled PBS at a ratio of 0.3g / ml, then stir in a 4°C refrigerator at a speed of 400rpm for 90min, then transfer the mixture to a centrifuge tube, and centrifuge at 6000rpm for 10min The supernatant was collected for secondary centrifugation at 13000 rpm for 10 min, and the supernatant was collected. Use a c...

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Abstract

The invention discloses an extracting method of a bovine pituitary extract. The method comprises the following steps: obtaining a fresh bovine pituitary, removing blood stains and impurities on a tissue surface, removing external adhesive tissue and a white envelope, and exposing the whole pituitary tissue; adding a protease inhibitor into the pituitary tissue, crushing into paste, then adding a protein extracting liquid, performing stirring and centrifuging sequentially at low temperature of 0-4 DEG C, and collecting supernate; and sterilizing the supernate to obtain the bovine pituitary extract. With the adoption of the method, the bovine pituitary extract contains more than 15g/ml of protein; the bovine pituitary extract can be applied to cell culture and tissue engineering culture. The extracting method is simple, high in extracting efficiency, and suitable for mass production, thereby providing a strong foundation for mass production of the bovine pituitary extract.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for extracting bovine pituitary gland extract. Background technique [0002] As a substitute for bovine serum, bovine pituitary extract (BPE) is an important additive in serum-free medium, which can effectively remove the adverse effects of complex components in serum on cells, especially for stem cells and stem cells. Pro-differentiation and pro-aging effects of cells, so as to ensure the biological characteristics of cells. It is often used in the cultivation of keratinocytes, melanocytes, oral mucosa cells and other epithelial cells, mesenchymal cells and other cells and in the production of tissue engineering products containing the above cells. The main components of its function are ethanolamine, fatty acids and various protein factors and hormones contained in it, such as growth hormone (GH), thyroid stimulating hormone (TSH), adrenocorticotropic ...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0037C12N5/0629C12N2500/84
Inventor 支旭勃卢永波
Owner GUANGDONG BOXI BIO TECH CO LTD
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