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생쥐 모델을 이용한 배아의 할구 생검법과 할구가 생검된 배아의 배양시 공배양 효과에 관한 연구: 인간에서의 착상 전 유전진단 기술 개발을 위한 동물실험 모델의 개발
김석현,류범용,지병철,최성미,김희선,방명걸,오선경,서창석,최영민,김정구,문신용,이진용,채희동,김정훈,Kim, S.H.,Ryu, B.Y.,Jee, B.C.,Choi, S.M.,Kim, H.S.,Pang, M.G.,Oh, S.K.,Suh, C.S.,Choi, Y.M.,Kim, J.G.,Moon, S.Y.,Lee, J.Y.,Chae, H.D.,Kim, C.H. 대한생식의학회 1999 Clinical and Experimental Reproductive Medicine Vol.26 No.1
The genetic defects in human gametes and embryos can cause adverse effects on overall reproductive events. Biopsy of embryos for preimplantation genetic diagnosis (PGD) offers a new possibility of having children free of the genetic disease. In addition, advanced embryo culture method may enhance the effectiveness of embryo biopsy for the practical application of PGD. This experimental study was undertaken to evaluate the effects of coculture on the development in vitro of biopsied mouse embryos as a preclinical model for PGD of human embryos. Embryos were obtained after in vitro fertilization (IVF) from F1 hybrid mice (C57BLfemale/CBAmale). Using micromanipulation, 1, 2, 3 or 4 blastomeres of 8-cell stage embryos were aspirated through a hole made in the zona pellucida by zona drilling (ZD) with acidic Tyrode's solution (ATS). After biopsy of blastomeres, embryos were cultured in vitro for 110 hours in Ham's F-10 supplemented with 0.4% BSA or cocultured on the monolayer of Vero cells in the same medium. The frequence of blastocyst formation were recorded, and the embryos beyond blastocyst stage were stained with 10% Giemsa to count the total number of nuclei in each embryo. There was no significant difference in the blastocyst formation between the zona intact control group and the zona drilling (ZD) only, or biopsied groups. The hatching rate of all the treatment groups except 4/8 group was significantly higher than that of control group. In all the treatment groups, there was a significant reduction in the mean cell number of embryos beyond blastocyst stage ($50.2{\pm}14.0$ in control group vs. $41.2{\pm}7.9$ in ZD, $39.3{\pm}8.8$ in 7/8, $29.7{\pm}6.4$ in 6/8, $25.1{\pm}5.7$ in 5/8, and $22.1{\pm}4.3$ in 4/8 groups, p<0.05). When the same treatments were followed by coculture with Vero cells, a similar pattern was seen in the blastocyst formation and the hatching rate. However, in all the treatment groups, there was a significant increase in the mean cell number of embryos beyond blastocyst stage with coculture, compared with the parallel groups without coculture. In the cleavage rate of biopsied blastomeres cultured for 110 hours after IVF, there was no significant difference between coculture and non-coculture groups (87.2% vs. 78.7%). However, the mean cell number of embryos developed from the biopsied blastomeres was significantly higher in coculture group ($11.5{\pm}4.7\;vs.\;5.9{\pm}1.9$, p<0.05). In conclusion, biopsy of mouse embryos after ZD with ATS is a safe and highly efficient method for PGD, and coculture with Vero cells showed a positive effect on the development in vitro of biopsied mouse embryos and blastomeres as a preclinical model for PGD of human embryos.
김정훈 ( Kim J. H. ),성환인 ( H. I. Sung ),최규성 ( K. S. Choi ),김종진 ( J. J. Kim ),원창오 ( C. O. Won ),송기선 ( K. S. Song ) 강원대학교 농업생명과학연구원(구 농업과학연구소) 2020 강원 농업생명환경연구 Vol.32 No.1
This study was conducted to survey the germination characteristics of the seeds of Larix kaempferi, which is a major plantation species in Korea, according to the storage period. The germination experiment was conducted at 25°C, targeting seeds collected in 1996, 2004, 2008, 2009, 2010, and 2011. Prior to the germination experiment, seeds were subjected to different soaking treatments for 1, 2, 3, and 7 days including non-treatment. The germination rate in L. kaempferi seeds was observed to be higher in those that were collected recently; that is, the seeds that were stored for a shorter period of time. The germination rate of the seeds collected in 2011, which were stored immediately after collection, was 20.7%. The seeds from 1996 and 2004 did not germinate at all. The seeds from the year 2008 showed the lowest germination rate at 2.7%. The whole T50 of the test-subjected seeds was observed to be 3-7 days. Germination uniformity tended to decrease with shorter storage period. Soaking treatment lowered the mean germination time and uniformity, except for the stored seeds (collected in 1996 and 2004), which did not germinate. Based on the results of these experiments, such as a reduction in germination rate according to the seed storage period, it seems likely that more detailed investigations into the changes in seed viability and storage management are needed with respect to L. kaempferi seeds.