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Method for extracting unsaturated fatty acid composition from humen amniotic epithelial cells, and application thereof

A technology for unsaturated fatty acids and epithelial cells, which is applied in the field of extracting unsaturated fatty acid compositions in free state, can solve the problems that the output of PUFAs cannot meet the needs of human beings, there is no data showing the function of human amniotic membrane epithelial cells, and the price is expensive. Reasonable, broad medicinal prospect, low cost effect

Active Publication Date: 2011-11-02
广西南宁灵康赛诺科生物科技有限公司
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Problems solved by technology

[0006] 3. [Title] Characteristics of human amniotic epithelial cells differentiated into osteoblasts [Author] Song Xiujun, Chen Daixiong, Fang Ning Zhangtao, Liu Zulin, Liu Jinwei and Wan Weihong [Institution] Affiliated Hospital of Zunyi Medical College, Key Laboratory of Cell Engineering of Guizhou Province, [Title] China Tissue Engineering Research and Clinical Rehabilitation. 2008, 12(38).-7518-7521 [Abstract] Background: It was found that human amniotic epithelial cell lines WISH and FC implanted in nude mice could differentiate into cartilage and osteogenesis. Whether the amniotic epithelial cells of human fetal membranes delivered at term have osteogenic properties is rarely reported at home and abroad
However, there is no data at home and abroad to show the role of human amniotic epithelial cells in this process
[0017] At present, PUFAs sold in the form of commercial products are mainly extracted from deep-sea fish, which are expensive and have limited yields. Microalgae and yeast strains that produce PUFAs through microbial genetic engineering also face many problems that need to be solved, making the production of PUFAs difficult. It has been unable to meet the needs of human beings, and it is urgent to develop new resources

Method used

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  • Method for extracting unsaturated fatty acid composition from humen amniotic epithelial cells, and application thereof
  • Method for extracting unsaturated fatty acid composition from humen amniotic epithelial cells, and application thereof
  • Method for extracting unsaturated fatty acid composition from humen amniotic epithelial cells, and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1: Morphological Observation and Lipid Staining of Amniotic Epithelial Cells of Normal Full-term Cesarean Section

[0037] The amniotic membrane was taken from a normal full-term cesarean section, fixed in 10% formaldehyde solution for 24 hours, rinsed in running water, dehydrated in gradient alcohol, transparent in xylene, embedded in wax. Slice with a LEICA microtome with a thickness of 7 μm. After spreading in warm water at 45°C, pick up the slides with poly-lysine-coated glass slides. Let dry and set aside.

[0038] Paraffin sections were routinely dewaxed to water. After washing with distilled water, dip in hematoxylin and lightly dye for 1-2 minutes, then wash with tap water. Inject 70% alcohol for 5 seconds. Into Sudan III staining solution for 30 minutes. Wash with 70% alcohol for 5-10 seconds. Wash with distilled water, and cover with glycerin gelatin.

[0039] After the sections were stained in Nile blue sulfate solution for 30 minutes, they wer...

Embodiment 2

[0043] Example 2: Acquisition, isolation, cultivation and identification of human amniotic epithelial cells

[0044] The amniotic membrane was torn off from the placenta discarded after cesarean section, and placed in 1×PBS containing penicillin and streptomycin double antibodies and washed repeatedly until there was no blood. Spread the epithelium face down in a culture dish, and use a cell scraper to remove the connective tissue under the amnion dense layer. After repeated washing in 1×PBS, the amniotic membrane was cut into small pieces of about 1mm×1mm with ophthalmic scissors, added with newly prepared 0.01% collagenase IV, and placed in a 37°C incubator for 2-3 hours to digest. Collect the liquid in a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Rinse once with 1×PBS. Add 0.25% trypsin to digest at room temperature for 0.5-1 hour. Serum-containing medium terminates the digestion. Centrifuge at 1000 rpm for 5 minutes and discard ...

Embodiment 3

[0048] Example 3: Detection of the type and content of unsaturated fatty acids in free state extracted from human amniotic epithelial cells by gas chromatography / mass spectrometry

[0049] In order to reduce the interference of non-free fatty acids during the experiment, the cells were broken using a low-temperature ultra-high pressure continuous flow cell breaker, and 1×PBS 25ml was added to each 1.8L cell sample for dilution and washing. The whole process of breaking was in a low-temperature water bath at 4°C-6°C To maintain the activity and performance of the original substances in the cells. Add 75 ml of n-hexane to the crushed sample, and vibrate with an oscillator at 500 rpm for 12 hours. After 25 ml of the organic phase was dried by nitrogen blowing, the dried fatty acid was derivatized with boron trifluoride methanol solution. After adding 10ml of n-heptane for extraction, take 5ml of it and dry it with nitrogen. Use 1ml of n-hexane to reconstitute and measure on the...

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Abstract

The invention relates to a method for extracting an unsaturated fatty acid composition from humen amniotic epithelial cells (HAECs), and an application thereof. The unsaturated fatty acid composition mainly comprises polyunsaturated fatty acids (PUFAs) which comprise DHA, arachidonic acid, dihomo-gamma-linolenic acid, gama-linolenic acid and linoleic acid. The unsaturated fatty acid composition can be provided for preparing drugs for treating nervous system degenerative diseases (such as Parkinson's disease), ocular surface diseases, cardiovascular and cerebrovascular diseases and neural injury diseases. In the prior art, the commercial PUFAs are mainly extracted from deep-sea fishes or algaes, price of the PUFAs is expensive and output of the PUFAs is limit. According to the present invention, the free-state PUFAs composition extracted from the HAECs has advantages of sufficient source, low cost, high content, reasonable distribution ratio of n-6 PUFAs / n-3 PUFAs, and has a wide medicinal prospect.

Description

technical field [0001] The invention relates to a method for isolating useful substances from human amniotic epithelial cells, in particular to a method and application for extracting unsaturated fatty acid composition in a free state from the cytoplasm of human amniotic epithelial cells. Background technique [0002] Human amniotic epithelial cells are derived from placental amniotic membrane tissue. They are a kind of adult stem cells with obvious plasticity and multipotential differentiation potential. Under the regulation of different growth factors, they can differentiate into different tissue cell types derived from the three germ layers, such as liver Like cells, cardiomyocytes, glial cells, neurons and islet-like cells. During the early development of human embryos, human amniotic epithelial cells (HAECs) originate from the inner cell mass during blastocyst development, and are formed from some cells of the epiblast (amnioblasts) at the beginning of the second week a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07C57/03C07C57/12C07C51/48A61K31/202A61K31/201A61P25/28A61P9/00
Inventor 朱梅陈彪
Owner 广西南宁灵康赛诺科生物科技有限公司
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