中华麻醉学杂志
中華痳醉學雜誌
중화마취학잡지
CHINESE JOURNAL OF ANESTHESIOLOGY
2008年
5期
397-400
,共4页
宋小星%尤圣武%彭章龙%于布为
宋小星%尤聖武%彭章龍%于佈為
송소성%우골무%팽장룡%우포위
β内啡肽%腺病毒科%镇痛%米非司酮
β內啡肽%腺病毒科%鎮痛%米非司酮
β내배태%선병독과%진통%미비사동
beta-Endorphin%Adenoviridae%Analgesia%Mifepristone
目的 构建携带米非司酮(RU486)可调控β内啡肽(β-EP)基因的腺病毒(Ad-RUNEP).方法 构建携带RU486可调控β-EP基因的腺病毒穿梭质粒pDC312-RUNEP,重组得到Ad-RUNEP后进行扩增,纯化和滴度测定,并分别在载体水平和病毒水平进行验证.Ad-RUNEP感染人皮肤癌上皮A431细胞株24 h后,将A431细胞株随机分为6个不同浓度RU486组(R0～5组),每组5孔,均加入RU486诱导β-EP表达,终浓度分别为0、10-10、10-9、10-8、10-7和10-6mol/L,应用RU486孵育48 h后更换为不含RU486的培养液,于开始RU486孵育的第1天、第2天和第4天取培养液,离心后收集上清液,采用ELISA法测定上清液β-EP浓度.结果 经酶切鉴定表明RU486调控系统与β-EP均正确插入pDC312-RUNEP;Ad-RUNEP病毒滴度为2.25×1010pfu/ml.与R0组比较,R1～5组β-EP浓度升高(P＜0.05);与R4组比较,R1～3组β-EP浓度降低(P＜0.05);与孵育第1天比较,第2天时β-EP浓度升高(P＜0.05);与孵育第2天比较,第4天时β-EP浓度降低(P＜0.05).结论 成功构建了Ad-RUNEP,RU486对Ad-RUNEP合成β-EP具有良好的调控作用.
目的 構建攜帶米非司酮(RU486)可調控β內啡肽(β-EP)基因的腺病毒(Ad-RUNEP).方法 構建攜帶RU486可調控β-EP基因的腺病毒穿梭質粒pDC312-RUNEP,重組得到Ad-RUNEP後進行擴增,純化和滴度測定,併分彆在載體水平和病毒水平進行驗證.Ad-RUNEP感染人皮膚癌上皮A431細胞株24 h後,將A431細胞株隨機分為6箇不同濃度RU486組(R0～5組),每組5孔,均加入RU486誘導β-EP錶達,終濃度分彆為0、10-10、10-9、10-8、10-7和10-6mol/L,應用RU486孵育48 h後更換為不含RU486的培養液,于開始RU486孵育的第1天、第2天和第4天取培養液,離心後收集上清液,採用ELISA法測定上清液β-EP濃度.結果 經酶切鑒定錶明RU486調控繫統與β-EP均正確插入pDC312-RUNEP;Ad-RUNEP病毒滴度為2.25×1010pfu/ml.與R0組比較,R1～5組β-EP濃度升高(P＜0.05);與R4組比較,R1～3組β-EP濃度降低(P＜0.05);與孵育第1天比較,第2天時β-EP濃度升高(P＜0.05);與孵育第2天比較,第4天時β-EP濃度降低(P＜0.05).結論 成功構建瞭Ad-RUNEP,RU486對Ad-RUNEP閤成β-EP具有良好的調控作用.
목적 구건휴대미비사동(RU486)가조공β내배태(β-EP)기인적선병독(Ad-RUNEP).방법 구건휴대RU486가조공β-EP기인적선병독천사질립pDC312-RUNEP,중조득도Ad-RUNEP후진행확증,순화화적도측정,병분별재재체수평화병독수평진행험증.Ad-RUNEP감염인피부암상피A431세포주24 h후,장A431세포주수궤분위6개불동농도RU486조(R0～5조),매조5공,균가입RU486유도β-EP표체,종농도분별위0、10-10、10-9、10-8、10-7화10-6mol/L,응용RU486부육48 h후경환위불함RU486적배양액,우개시RU486부육적제1천、제2천화제4천취배양액,리심후수집상청액,채용ELISA법측정상청액β-EP농도.결과 경매절감정표명RU486조공계통여β-EP균정학삽입pDC312-RUNEP;Ad-RUNEP병독적도위2.25×1010pfu/ml.여R0조비교,R1～5조β-EP농도승고(P＜0.05);여R4조비교,R1～3조β-EP농도강저(P＜0.05);여부육제1천비교,제2천시β-EP농도승고(P＜0.05);여부육제2천비교,제4천시β-EP농도강저(P＜0.05).결론 성공구건료Ad-RUNEP,RU486대Ad-RUNEP합성β-EP구유량호적조공작용.
Objective To construct and identify adenovirag (Ad-RUNEP) carrying human B-endorphin(B-EP) genes which can be regulated by mifepfistone (RU486)-inducible system and to evaluate the effects of different concentrations of RU486 on the transgene expression in adenovirus in vitro.Methods The shuttle plagmid pDC312一RUNEP carrying B-EP genes which can be regulated by RU486-inducible system was constructed and was combined with adenovims to form a recombinant Ad-RUNEP using AdMAXTM system.The recombinant Ad.RUNEP was then amplified and purified.The titers of the adenovirus were determined and the adenovirus vector was checked.After being infected by Ad-RUNEP for 24 h,A431 cell line was incubated in liquid culture media containing RU4860,10-10,10-9,10-8,10-7,and 10-6 mol/L respectively(group R0-5) for 48h,and Was then transferred to liquid culture media containing no RU486.The liquid culture media were obtained on 1st.2nd and 4th day of incubation and centrifuged.The supematant Was collected for determination of B-EP concentration by ELISA.Results The analysis of enzyme-incision demonstrated that RU486 regulating system and B-EP were cloned directly into pDC312-RUNEP.The titer of Ad-RUNEP was 2.25×1010 pfuml.The expression of B-EP was significantly higher in group R1-s than in group R0 and was significantly lower in group R1-3 than in group R4 (P<O.05).The expression of B-EP wag significantly higher on 2nd than on 1st and 4th day.Conclusion The adenovims carrying B-EP genes which can be regulated by RU486 (Ad-RUNEP) Wag successfully constructed.
