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Chromatography용 Paper, $\mu$RPC Column 및 Superose Column을 이용한 정자의 이동을 자극하는 난포액 성분의 분리
박영식 한국수정란이식학회 1998 한국동물생명공학회지 Vol.13 No.3
난포액내 함유되어 있는 단백질성분 중에서 sucrose 층으로부터 정자의 swim-up 이동을 자 극하는 성분을 분리하기 위하여 paper chromatography (PC) 및 reverse phase column (RPC) 과 superose column (SC)를 이용한 액체 chromatography의 분리효과를 조사하였던 바 결과는 다음과 같다. 1. Chromatography용 paper로 분리한 각 band 의 성분은 첨가농도가 증가할수록 정자의 이동과 운동을 자극하였으며, 특히 band 1 성분은 정자의 이동을 유의하게 증가시켰다. 그러나, 동일 첨가수준에서 bands 성분의 정자 이동과 운동자극효과는 난포액의 효과에 비하여 유의하게 낮았다. 2. $\mu$RPC를 이용 2~5mm 난포로 부터 분리한 성분중 RT3.33, RT7.00, RTl3.87 및 RTl6.6A 성분은 정자의 이동을 자극하였으나, 자극효과는 매우 적었다. 3. $\mu$RPC를 이용 10mm 난포로 부터 분리한 성분은 정자의 이동과 활력을 자극하지 않았다. 4. SC를 이용 2-5mm 난포로 부터 분리한 성분 중 RVI.35 성분과 RV0.82 성분은 정자의 이동과 운동을 유의하게 자극하였다. 결론적으로 난포액내 정자의 이동과 운동을 자극하는 단백질 성분은 superose column을 이용하여 효과적으로 분리할 수 있으며, 분리된 RVI.35 성분과 RV0.82 성분은 정자의 swim-up 분리를 자극하였다. To efficiently separate a protein stimulating sperm swim-up migration and movement from follicular proteins, the effect of paper chromatography and liquid chromatography with reverse phase column and superose column on protein separation was examined. And the results obtained were as follows; 1. The band component that was separated with paper chromatography stimulated sperm migration and movement depending on its additional levels. Especially, band I component significantly increased sperm migration. But, all components of bands 1, 2 and 3 showed lower sperm migration and movement, compared to follicular fluid at the same additional level. 2. Among the components separated from follicular protein of 2~5mm follicles with reverse phase column ($\mu$RPC), components at retention time (RT) of 3.33, 7.00, 13.87, and 16.6A minutes stimulated sperm migration within a limited range. 3. All components separated from follicular protein of 10mm follicles with $\mu$RPC column didn't stimulate sperm migration and movement. 4. Among the components separated from follicular protein of 2~5m follicles with superose column, components at retention volume (RV) of 1.35 and 0.82 ml significantly stimulated sperm migration and movement. In conclusion, protein components stimulating sperm migration and movement were efficiently separated with superose column in Smart system. Especially, components of RV 1.35 and RV0.82 stimulated sperm swim-up separation.
비인두 암종에서 Epstein-Barr virus 감염과 p53 유전자 변이의 관계
박영식,최홍란 전남대학교 치과대학 2001 구강과학 Vol.13 No.1
Nasopharyngeal carcinoma is a common neoplasm of the head and neck, and commonly found in Southeast Asia. Epstein Barr virus(EBV) is found within the malignant cells in most cases of nasopharyngeal carcinoma. The p53 gene has been implicated as a tumor suppressing gene in many types of human cancers, but the frequency of mutations varies in different cancers. To find out the correlation between EBV infection and p53 mutation in nasopharyngeal carcinoma, we evaluated the presence of EBV using EBER 1 in situ hybridization and a mutation of p53, using PCR SSCP analysis and DAN sequencing method. We examined nasopharyngeal specimens from 33 patients, histologicaly consisting of 27 cases of nonkeratinizing squamous cell carcinoma (WHO Ⅱ) and 6 cases of undifferentiated carcinoma (WHO Ⅲ) according to the World Health Organization claasification. EBER1 were detected in 14 of 33 (42.4%) cases. Positive hybridization signals were restricted to the nuclei of tumor cells. Of 14 cases 14 cases, 11 out of 27 (40.7%) cases of WHO Ⅱ, and 3 out 6 (50%) cases of WHO Ⅲ were positive. 33 biopsy specimens were analyzed by PCR SSCP for p53 gene mutations. Our study concentrated on exons 5 through 9 of the p53 gene. However, we did not detect any mobility shifts n exon 5/6, 7 and 8/9 of the p5 gene in comparison to the banding patterns of the normal control. The reliability of the SSCP analysis was confirmed by sequencing five randomly chosen specimens for the exon 5 9 region. Among five cases, 4 out 5 cases were EBER1 positive and histologially WHO Ⅱ, and 1 out of 5 cases were EBER1 negative and WHO Ⅲ. We detected point mutation in 3 cases were EBER 1 positive and WHO Ⅱ. No alteration of the exon 7 and 8/9 of the p53 sequence was observed. Alteration occurred in 2 out of 3 cases; GCC was replaced by GTC at codon 131, resulting in a change in amino acid sequence from alanine to valine. In one 3 cases, TCC was replaced by TTC at codon 149, resulting in a change from arginine to arginine. Based on the above results, this study shows correlation between the presence of EBV and mutations of the p53 gene, suggesting contributions to malignant transformation in nasopharynx. We need to futher investigate this situation to understand the stepwise progression of nasopharyngeal carcinogenesis.