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Long-term culture method for swine testicle mesenchymal progenitor cells

A culture method and technology of progenitor cells, applied in the field of in vitro separation, detection and differentiation, and long-term culture of porcine Leydig cells

Active Publication Date: 2016-06-01
SOUTH CHINA AGRI UNIV
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  • Application Information

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Problems solved by technology

PLCs are a type of intermediate-stage stem cells differentiated from SLCs. They have similar morphological characteristics to SLCs and are spindle-shaped, but the gene expression has undergone great changes, mainly manifested in the appearance of steroid metabolism-related enzymes (CYP11a1, CYP17a1 and StAR) and a small amount of LHR expression, but no 3β-HSD expression, and it also retains the expression of SLCs markers LIFR and PDGFRa, showing the characteristics of strong division ability

Method used

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  • Long-term culture method for swine testicle mesenchymal progenitor cells
  • Long-term culture method for swine testicle mesenchymal progenitor cells
  • Long-term culture method for swine testicle mesenchymal progenitor cells

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Embodiment 1

[0032] Embodiment 1 A kind of in vitro separation and proliferation method of pig PLCs

[0033] A method for separating and proliferating pig PLCs in vitro, the steps are as follows:

[0034] Piglet testis fibroblasts were cultured under specific culture conditions (containing 15% FBS, 55 μM β-mercaptoethanol, 1% ITS, 1% L-glutamine, 1% nucleotides, 1000IU / mLLIF, 10ng / mLEGF, 10ng / mLbFGF, 20ng / mL PDGF-BB and DMEM medium containing 1% Penicilin-Stretomycin), the cell division ability is strong, and a large number of PLCs clones can be produced in 7 days of cultivation (such as figure 2 A), collect clonal colonies for PCR analysis, and compare testicular tissue, showing that they express PLCs molecular markers (Gata4, Pdgfra1, Lifr, Cyp11a1, Cyp17a1 and Star), and mature mesenchymal cells have not yet appeared at this time, so neither expresses and 3β-Hsd (such as figure 2 B), indicating that under specific culture conditions, porcine PLCs can be rapidly expanded in vitro, and ...

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Abstract

The invention discloses a long-term culture method for swine testicle mesenchymal progenitor cells. The long-term culture method includes separation, detection and differentiation in vitro, and specifically includes the steps of culturing piglet testicle fibroblasts for 5-8 days by means of an in-vitro proliferation culture medium of swine PLCs (progenitor leydig cells) to acquire a PLCs clone colony, culturing by means of a differentiation medium of swine PLCs so that PLCs begin to differentiate into mature testicle mesenchymal cells in the third day, then culturing in the proliferation culture medium, and maintaining in vitro for six month at most. According to the long-term culture method, a mature process of separation, culture, detection and differentiation of swine PLCs is created initially, and proliferation and differentiation maintained in vitro of PLCs can be achieved and controlled by different in-vitro culture systems determined finally. The long-term culture method has high application value in research on biological property of PLCs in vitro, research and development of drugs and vaccine production and the like.

Description

technical field [0001] The invention belongs to the technical field of stem cells, and more specifically, the invention relates to a long-term culture method of porcine testicular stromal progenitor cells, including in vitro separation, detection and differentiation methods. Background technique [0002] In the testicular tissue of male animals, it is well known that there is a type of spermatogonial stem cells that continuously produce sperm on the inner wall of the seminiferous tubules to maintain a constant number; however, there is another type of spermatogonial stem cells that maintain a balance between proliferation and differentiation on the outer wall of the seminiferous tubules Leydig stem cells (Leydigstem cells, SLCs) are the stem cells of the testis. Mature Leydig cells (adultleydig cells, ALCs), about 95% of testosterone in male animals is secreted by ALCs, which plays an important role in maintaining the dynamic balance of testosterone levels in male animals. ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0683C12N2500/10C12N2500/40C12N2500/44C12N2500/84C12N2501/11C12N2501/115
Inventor 白银山张守全冯美莹朱翠卫恒习李莉
Owner SOUTH CHINA AGRI UNIV
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