체외에서 생쥐배아 발생에 있어서 Vero 세포와의 공동 배양이 생쥐배아의 bax와 bcl-2 유전자의 발현에 미치는 영향
The Effects of Vero Cell Co-culture on the Expression of Bax and Bcl-2 Genes in the Mouse Embryo Development
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목적: Vero 세포와의 공배양이 체외에서 생쥐 배아 발생에 미치는 영향과 이들 배아의 bax와 bcl-2 유전자 발현에 미치는 영향을 알아보고자 한다. 연구 방법: 5-6주령의 ICR (institute of cancer research, 이하 ICR로 약함)계 암컷 생쥐를 과배란 유도하고 수컷과 합사하여 2-세포기 배아를 회수하였다. 대조군은 10% fetal bovine serum (Gibco BRL, FBS)이 포함된 Ham’s F-10 (Gibco BRL)을 기본 배양액으로 사용하여, 37℃, 5% CO2 하에서 배양하였으며 Vero 세포의 효과를 보기 위하여 기본 배양액에 Vero 세포를 단층으로 깐 후 동일한 배양 조건으로 배양하여 배아의 발생을 24시간 간격으로 3일 동안 비교 관찰하고, 각 실험 군별로 배아를 모아서 total RNA를 뽑고 reverse transcription-polymerize chain reaction (RT-PCR)을 수행하여 bax와 bcl-2 유전자 발현을 확인하였다. 결과: Vero 세포와의 공동배양을 하였을 경우 발생 1일에서 3일째까지의 배아 발생률은 각각 78.3%, 50.7%, 27.2%로 Vero 세포와의 공동배양을 하지 않은 군의 배아 발생률 60.5%, 29.3%, 20.7%에 비하여 높았다. 한편, 발생 24시간에 배아의 bax 유전자 발현은 Vero 세포와 공동배양을 하지 않은 군이 Vero 세포와의 공동배양 한 군보다 유의하게 높았다. Bcl-2 유전자 발현은 발생 72시간에 Vero 세포와의 공동배양 한 군이 Vero 세포와의 공동배양을 하지 않은 군보다 유의하게 높았다. 결론: 본 연구 결과 Vero 세포 공배양이 2-세포기 생쥐배아의 발생에 유익하며 생쥐배아에 있어 세포자멸 조절 유전자인 bax와 bcl-2 유전자의 발현에 영향을 미친다는 것을 알 수 있었다.
Abstract
Objective: To investigate the effects of Vero cell co-culture on the development of mouse embryo in vitro and the expression of bax and bcl-2 genes in the mouse embryo. Methods: The 2-cell mouse embryos were obtained from oviduct of 5-6 weeks old mated female ICR mice superovulated with pregnant mare’s serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). The 2-cell embryos in Ham’s F-10 medium supplemented with 10% FBS with or without Vero cells, were incubated at 37℃ in a 5% CO2 humidified air chamber respectively and we observed mouse embryo development and collected each group of embryos for the purpose of extraction of total RNA every 24 hours for 3 days. We carried out the RT-PCR to assess mRNA levels for bax and bcl-2 gene. Results: The rate of embryo development of with Vero cell co-cultured group was 78.3%, 50.7%, 27.2% and that of without Vero cell co- cultured group was 60.5%, 29.3%, 20.7% respectively. Bax mRNA expression level of without Vero cell co-cultured group was significantly higher than that of with Vero cell co-cultured group at 24 hours. Bcl-2 mRNA expression level of Vero cell co-cultured group was significantly higher than that of without Vero cell co-cultured group at 72 hours. Conclusion: These findings suggested that Vero cell co-culture is beneficial in the development of mouse embryos and stimulates bax and bcl-2 gene expression.
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