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Study on Regulation of Natal Bovine Spermatogonial Stem Cells Proliferation in Vitro

Author: ZhangShiQiang
Tutor: ZhangYong
School: Northwest University of Science and Technology
Course: Developmental Biology
Keywords: Spermatogonial stem cells Cell proliferation Training system Human telomerase reverse transcriptase Cow
CLC: Q813
Type: Master's thesis
Year: 2007
Downloads: 125
Quote: 1
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Abstract


Spermatogonial stem cells (spermatogonial stem cell) is a group of people with a high degree of self-renewal and differentiation potential within the gonads of male animals , the genetic information of the cell can be passed to offspring . Spermatogonial stem cells in medicine , genetics , transgenic animal research has broad application prospects in recent years has become a research focus of many scholars . Spermatogonial stem cells in in vitro difficult to maintain long-term , which become obstacles for further study . The study by the separation , purification and identification of newborn bovine spermatogonial stem cells , and their in vitro culture system has been optimized , and the import of human telomerase reverse transcriptase (human telomerase reverse transcriptase, hTERT) gene , in order to promote the proliferation and extension of its in vitro cell life . The study obtained the following results : an average survival rate of newborn calf testis cells isolated by the two - step enzyme digestion of 89.00 percent spermatogonial stem cells ratio was 21.61% , the adherent cells was 23.34% . Separation of the newborn calf testicular cells after Percoll density gradient centrifugation , results show that : the spermatogonial stem cells were mainly distributed in the 27% ~ 35% Percoll gradient , a purity of 69.27% ??. Newborn calf fine original stem cell sets off the cell chemistry and immune histochemical identification showed that : alkaline phosphatase expression was weakly positive , integrin beta1 and C-kit expression was positive ; Identification of RT-PCR showed that : newborn calf essence of the original stem cells express fine original stem cells the specific gene gfrα1 and c -kit . Through optimization of the newborn calf fine original stem cells in vitro culture system showed that: in the medium supplemented with 2.5 ? S , 10μg / L LIF 20μg / L SCF, 80μg / L of GDNF as well as support cells inoculated as 5.0 × 105 cells / mL density feeder layer , can significantly promote the spermatogonial stem cell proliferation . Newborn calf fine stem cells by liposome transfection pCl-Neo-hTERT carrier 600μg/mL of G418 screened positive cells . Detection of telomerase activity : the not the positive cells OD450/630 of transfected cells 4 times ; growth curves show that : the positive cells in vitro population doubling time 1.9 times . These results prove that , two-step enzyme digestion method combined with Percoll density gradient centrifugation separation purification newborn calf fine original stem cells can meet the need for further research ; optimize the culture system and transfected with hTERT gene can significantly with promoting newborn calf essence of the original stem cell in vitro proliferation .

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CLC: > Biological Sciences > Bioengineering ( Biotechnology ) > Cell Engineering
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