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Method for obtaining salivary gland stem cell mass of hirudo nipponia through enzymolysis and application of salivary gland stem cell mass

A Japanese medical leech and salivary gland technology, applied in the field of cell separation, can solve problems such as poor effect, and achieve the effect of high efficiency and high activity

Pending Publication Date: 2022-06-24
CHINA JILIANG UNIV
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  • Application Information

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Problems solved by technology

However, there is no record in the prior art to obtain the technology of isolating Hirudo salivary gland stem cell clusters, and when the technology of enzymatically obtaining Hirudo salivary gland cells is applied to the separation of Hirudo salivary gland stem cell clusters, the effect is relatively poor

Method used

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  • Method for obtaining salivary gland stem cell mass of hirudo nipponia through enzymolysis and application of salivary gland stem cell mass
  • Method for obtaining salivary gland stem cell mass of hirudo nipponia through enzymolysis and application of salivary gland stem cell mass
  • Method for obtaining salivary gland stem cell mass of hirudo nipponia through enzymolysis and application of salivary gland stem cell mass

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Embodiment

[0044] A method for obtaining Japanese medicinal leech salivary gland stem cell mass by enzymolysis, comprising the following steps:

[0045] (1) Pretreatment: Take 10-20 dried Japanese leeches and place them in purified water at room temperature for 24 hours.

[0046] (2) Material collection: Japanese leech was anesthetized with 10% alcohol, and the mucus on its body surface was wiped with a sterile cotton ball containing 75% alcohol. Dissect in the ultra-clean workbench, fix the head and tail on the foam board with sterile pins, use sterile surgical instruments to take the salivary gland tissue, and place it in pre-cooled D-PBS buffer (the sampling time is controlled at within 1h). Change the D-PBS buffer 3 times and rinse to remove blood stains.

[0047] (3) Enzymatic hydrolysis

[0048] A. Put the obtained salivary gland tissue of Japanese medicinal leech into the digestion bottle, add 5 mL of enzyme solution prepared in advance and sterilized by filtration, and the enz...

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Abstract

The invention discloses a method for obtaining salivary gland stem cell clusters of Hirudo nipponia through enzymolysis and application, and relates to the technical field of cell separation.According to the technical scheme, the method is characterized in that the salivary gland tissue blocks of Hirudo nipponia are subjected to multiple times of enzymolysis, and finally the stem cell clusters with the capacity of being split and differentiated into new salivary gland cells are obtained; according to the method, in-vitro long-term culture of salivary gland cells of Hirudo nipponia is realized, the efficiency is higher than that of primary culture for passage of salivary gland cells of Hirudo nipponia obtained by direct separation, natural hirudin is extracted by separating salivary gland cell metabolites, and compared with recombinant hirudin obtained by genetic engineering means, the method has the advantages that the yield is high, and the cost is low. The hirudin obtained by the method is higher in activity and higher in efficiency.

Description

technical field [0001] The invention relates to the technical field of cell separation, more particularly, to a method and application for obtaining Japanese leech salivary gland stem cell mass by enzymatic hydrolysis. Background technique [0002] The salivary gland secretions of Japanese medicine leeches contain hirudin, which is the substance with the strongest antithrombin activity found so far, while the substances contained in the salivary gland secretions of Japanese medicine leeches have strong antithrombin activity and antiplatelet activity. The effect of aggregation was higher than that of the broad-bodied leech and the tapered leech. As an important biological resource of natural anticoagulant substances, Japanese leech is the key technology to obtain natural hirudin by isolating and culturing salivary gland cells. The use of cell engineering to produce hirudin reduces the capture of wild Japanese leeches in nature and protects Wildlife Resources. [0003] In th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C07K14/815C07K1/14
CPCC12N5/0633C07K14/815C12N2509/00C12N2509/10
Inventor 瞿如钰罗媛媛鲍亮管峰王为民
Owner CHINA JILIANG UNIV
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