Acipenser dabryanus spermatogonium culture solution and application thereof

A technology of spermatogonia and Dabry's sturgeon, which is applied in the biological field and can solve the problems of the undiscovered Dabry's spermatogonia separation and culture method, etc.

Active Publication Date: 2016-02-10
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no relevant report on the method of isolating and culturing the spermatogonia of Acipenser dabryi.

Method used

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  • Acipenser dabryanus spermatogonium culture solution and application thereof
  • Acipenser dabryanus spermatogonium culture solution and application thereof
  • Acipenser dabryanus spermatogonium culture solution and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0049] A Dabry's sturgeon spermatogonia culture fluid, comprising:

[0050]

[0051] the rest is SFM, the pH of the culture solution is 8.0.

[0052] The preparation method of described dabry's sturgeon spermatogonia culture fluid comprises the following steps:

[0053] 1. Configuration of the mother liquor of each component:

[0054] (1) Configuration of 10ml500g / LBSA mother solution (100X):

[0055] Use a balance to accurately weigh the required 0.05g of BSA with an accuracy of 0.0001g:

[0056] According to the formula: measuring weight = formula concentration × total volume of BSA mother liquor

[0057] Put the weighed BSA into the beaker, add 6ml SFM basal culture medium, fully dissolve BSA, continue to add For SFM basal culture medium, dilute the dissolved BSA solution to 10ml.

[0058] (2) Configuration of 10ml 200mmol / LL-Glutamine mother solution (100X):

[0059] Accurately weigh 0.3654gL-Glutamine with a balance, with an accuracy of 0.0001g:

[0060] Acco...

Embodiment 2-6

[0081] A Dabry's sturgeon spermatogonia culture fluid, comprising:

[0082]

[0083] The Dabry's sturgeon serum is the serum of 2-year-old male Dabry's sturgeon, and the rest are SFM medium.

Embodiment 8

[0085] A kind of Dabry's sturgeon spermatogonia culture medium is used in cultivating spermatogonia, comprising

[0086] (1) Preparation of Acipenser dabryi testis cell suspension:

[0087] The following operations were all carried out in a sterile biological safety cabinet.

[0088] 1) Move the cleaned testis tissue of Acipenser dabfieldii into a petri dish, cut the testis tissue into pieces, add 0.25% trypsin digestion solution at the ratio of digestion solution:tissue block=4ml:1g (v / w), and culture at 20°C Digest in the box and pipette once every 30min.

[0089] 2) After digesting the testis tissue in step 1) for 3 hours, add L-15 medium + 20% fetal bovine serum to terminate the digestion, and filter through a 40 μm filter. Centrifuge at 200g for 5min at 4°C and resuspend in L-15 medium + 20% fetal bovine serum.

[0090] 3) Cell counting, and staining with trypan blue: cell suspension=1:1 (v / v), after calculating the viable cell rate under a microscope, use L-15+20% fet...

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Abstract

The invention discloses an acipenser dabryanus spermatogonium culture solution and application thereof. According to the culture solution, a low-serum culture medium which does not have fetal calf serum, adopts various growth factors, nonessential amino acid, sodium pyruvate and other additives as substitutes and is assisted by a small amount of male acipenser dabryanus serum is provided so as to guarantee mass reproduction of spermatogonium and low reproduction rate of a spermatic cell; and the spermatic cell 13d which is cultured to the 13rd day are doped with 25mu M BrdU and is marked as a newly reproduced cell. 24h immunohistochemical experiments indicate that the ratio of germ cell marked cells in newly reproduced cells after BrdU is doped for 24 hours is 85.78 percent. According to the culture solution, the current problem that the acipenser dabryanus spermatogonium passage cannot be massively cultured can be solved, and an effective cell platform is provided to acipenser dabryanus reproductive development researches.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a dabry's spermatogonia culture medium and its application. Background technique [0002] Dabry's sturgeon (Acipenserdabryanus) belongs to the order Acipenserdabryanus, Acipenseridae, and the genus Acipenserus, also known as Shalazi and Yangtze River Acipenser. It is mainly distributed in the lower reaches of the Jinsha River and the upper reaches of the Yangtze River, and has high economic and scientific value. In recent years, due to factors such as environmental pollution, habitat destruction, shipping and overfishing, the population of wild Dabry's sturgeon has declined sharply. In the past 20 years, due to the construction of Gezhouba Project, environmental pollution and overfishing in the upper reaches of the Yangtze River, the resources of Dabry's sturgeon have been extremely scarce (ZhuangPingetal1996, Zhanghuietal2011). In 1988, the Dabry's sturgeon was listed as a critica...

Claims

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Application Information

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IPC IPC(8): C12N5/076
Inventor 颉璇厉萍危起伟叶欢杨晓鸽马杰曾令兵李创举
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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