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Method for constructing machin progenitor leydig cell immortal line

A technology of testicular interstitium and establishment method, applied in the field of culture medium for establishment of immortalized line of interstitial progenitor cells of cynomolgus monkeys

Inactive Publication Date: 2015-05-13
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
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  • Claims
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Problems solved by technology

[0005] At present, rat and mouse Leydig cells immortalized lines have been successfully established, but Leydig cell lines of large animals and humans have not been established, especially SLCs and PLCs with stemness have not been reported.

Method used

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  • Method for constructing machin progenitor leydig cell immortal line
  • Method for constructing machin progenitor leydig cell immortal line
  • Method for constructing machin progenitor leydig cell immortal line

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Embodiment 1

[0030] see figure 1 , is a road map of the establishment method of a cynomolgus monkey testis interstitial progenitor cell immortal line of the present invention, comprising the following specific steps:

[0031] 1) Obtain a relatively high proportion of Leydig cells by differential attachment method

[0032] The cynomolgus monkey testicular tissue obtained by the puncture method was subjected to two-step digestion to obtain a single-cell suspension, and a high proportion of cynomolgus monkey Leydig cells were obtained by differential adhesion. Strong, with prominent nuclei (eg figure 2 A) Complete culture medium until the F3 generation cells begin to differentiate, and the cell morphology also changes, from the early fusiform, triangular growth to oval, to mature Leydig cells, unable to maintain an undifferentiated state (such as figure 2 B and 2C); After the F9 generation, the cells spread out into a leaf shape and cannot continue to be subcultured (such as figure 2 D)...

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Abstract

The invention discloses a method for constructing a machin progenitor leydig cell immortal line. The method comprises the following steps: by taking machin testicular tissues, obtaining leydig cells of a high ratio, culturing in a complete medium for 20-28 hours, replacing fresh culture solution, continuously culturing, and performing cell passage to the leydig cells presenting fibroblast growth in uniform form; infecting the leydig cells by utilizing slow virus solution carrying Large T and Egfp, performing fluorescence labeling cell screening, and performing cell passage to a cell strain for stably expressing Egfp; and screening the cell strain for stably expressing Egfp, identifying the progenitor leydig cells, continuously performing cell passage for over 50 generations, and finally constructing the machin progenitor leydig cell immortal line. The in-vitro cell passage of the progenitor leydig cell immortal line with the transferred Large T exceeds 62 generations, the leydig cell labeled proteins such as CYP11A1, CYP17A1 and STAR are expressed, marker genes of mesenchymal stem cells such as Pdgfral and Lifr are expressed, the cell shape is maintained into the fiber shape, and a characteristic progenitor leydig cell immortal line is obtained.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to a culture medium for establishing an immortal line of cynomolgus monkey testicular interstitial progenitor cells and a method and application of using the culture medium to establish an immortal line of cynomolgus monkey testicular interstitial progenitor cells. Background technique [0002] Leydig cells (LCs), germ cells, supporting cells, myocytes, blood vessels and lymphatic vessels exist in the testes of adult animals. It is still difficult to separate a single type of cells in vitro using various physical, chemical and immunological methods, which has caused certain difficulties in the study of cell biological properties and drug screening. Cell lines are important materials for the study of cell biological properties and drug screening, and the establishment of cell lines is the primary basis for various research. [0003] Leydig cells include Le...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867A61K35/52A61P15/08
Inventor 张守全白银山冯美莹张秀娟卫恒习李莉
Owner SOUTH CHINA AGRI UNIV
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