Method for culturing high-purity primary Leydig cells of Guizhou fragrance pigs

A high-purity technology for Leydig cells, applied in the primary culture of high-purity pig Leydig cells, can solve the problems of slow cell growth, difficulty in stable preparation of spin columns, low activity rate, etc., and achieve high purity, Strong activity, simple and efficient operation

Inactive Publication Date: 2019-03-08
GUIZHOU UNIV
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Problems solved by technology

The present inventor also intends to use the Percoll method to cultivate and purify the Leydig cells of the Xiangzhu testis in the early stage. The results show that the Percoll gradient centrifugal column is difficult to stably prepare, and the interstitial cell layer after centrifugation is mainly concentrated in the 50%-70% Percoll concentration layer, but the The viability rate is low after Panpan blue staining, and the cell growth is slow

Method used

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Embodiment

[0022] Example: A method for culturing high-purity Guizhou Xiang pig primary testicular stromal cells: including:

[0023] 1. Choose Guizhou Xiangzhu boars with healthy development, strong body and good mental state as the experimental subjects.

[0024] 2. Put Guizhou pigs under anesthesia, disinfect their whole body, and focus on cleaning and disinfecting the testicles. Surgically remove the testes on both sides of the pigs, carefully peel off the epididymis, blood vessels, accessory gonads and other tissues, and put them at 4℃ containing 3 % Double antibody in PBS (without Ca 2+ ,Mg 2+ , PH 7.2-7.4), clean 3-5 times.

[0025] 3. Carefully peel off the testicular tunica albuginea, obvious blood vessels and seminiferous tubules, use sterile scissors to divide the testicular tissue into four equal parts, put them in 75% pre-cooled medical alcohol for sterilization for 3-5 minutes, and use 4 ℃ containing 3% double antibody PBS (without Ca 2+ ,Mg 2+ ,PH 7.2-7.4) Clean 2-3 times.

[002...

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Abstract

The invention discloses a method for culturing high-purity primary Leydig cells of Guizhou fragrance pigs. The method is characterized by comprising the following steps: (1) digesting testicular tissue blocks of fragrance pigs through collagenase to obtain a cell suspension solution; (2) repeatedly digesting with 0.25 percent Trypsin-EDTA to obtain Leydig cells; (3) enriching the Leydig cells through a differential centrifugation method, wherein the differential centrifugation method at least comprises three times of centrifugation, the speed of the first-time centrifugation is 800 to 1200 rpmand the time is 3 to 8 min, and the speed of the second-time and third-time centrifugation is 600 to 1000 rpm and the time is 3 to 8 min; (4) purifying the Leydig cells through a differential attachment method. Compared with an existing primary culture and purification method of pig Leydig cells, the method has the advantages that the operation is simpler and more efficient, and the Leydig cellsof the Guizhou fragrance pigs, which have a great quantity, strong activity and high purity, are obtained. The method disclosed by the invention can provide effective technological guidance for deeplyresearching sexual precocity properties of the Guizhou fragrance pigs and related functions of the Leydig cells.

Description

Technical field [0001] The invention belongs to the field of primary cell culture and purification, and specifically relates to a method for obtaining high-purity pig testicular stromal cells in primary culture. Background technique [0002] Leydig cell (LC) is distributed in the connective tissue between the seminiferous tubules. The cell body is oval and irregular in shape and is mainly responsible for the synthesis and secretion of testosterone. Testosterone is an important androgen that promotes the development of internal and external reproductive organs, spermatogenesis, and maintenance of secondary sexual characteristics in male animals. 95% of serum testosterone is secreted by testicular stromal cells. Testosterone plays an important physiological role after interacting with the corresponding target organ receptors. Features. Therefore, the establishment of a convenient, fast, stable and efficient primary culture and purification method of testicular stromal cells is of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0683C12N2509/00
Inventor 龚婷王维勇袁超朱晓锋徐永健蒙利洁宋林锦
Owner GUIZHOU UNIV
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