生物技术通讯
生物技術通訊
생물기술통신
LETTERS IN BIOTECHNOLOGY
2014年
4期
511-514
,共4页
李章华%赵强%唐欢%许海甲%廖文%潘峰%刘农乐
李章華%趙彊%唐歡%許海甲%廖文%潘峰%劉農樂
리장화%조강%당환%허해갑%료문%반봉%류농악
核心结合因子α1%腺病毒%间充质干细胞
覈心結閤因子α1%腺病毒%間充質榦細胞
핵심결합인자α1%선병독%간충질간세포
core binding factor alpha 1%adenovirus%mesenchymal stem cell
目的:构建成骨细胞特异性转录因子--核心结合因子α1(Cbfa1)重组腺病毒载体,为后期应用Cbfa1基因治疗股骨头坏死、骨折和骨缺损等疾病奠定基础。方法:以目的基因Cbfa1全长cDNA为模板进行PCR扩增,将扩增产物克隆到pShuttle-CMV载体的相应酶切位点,获得目的基因载体pShuttle-CMV-Cbfa1,在大肠杆菌BJ5183中和pAdEasy-1同源重组,筛选阳性克隆,经酶切、PCR及测序鉴定,线性化后用脂质体法转染HEK293细胞进行包装、扩增,用报告基因GFP对病毒滴度和感染效率进行监测,酚氯仿抽提纯化病毒,AdEasy1/Cbfa1感染间充质干细胞(MSC)后检测Cbfa1的表达。结果:测序、酶切及PCR证实Cbfa1基因重组腺病毒载体构建成功,AdEasy1/Cbfa1感染MSC后Cbfa1的表达显著升高。结论:构建了含Cbfa1基因的重组腺病毒载体,该基因能促进MSC向成骨细胞分化,预示其可作为治疗基因应用于股骨头坏死、骨折和骨缺损的治疗。
目的:構建成骨細胞特異性轉錄因子--覈心結閤因子α1(Cbfa1)重組腺病毒載體,為後期應用Cbfa1基因治療股骨頭壞死、骨摺和骨缺損等疾病奠定基礎。方法:以目的基因Cbfa1全長cDNA為模闆進行PCR擴增,將擴增產物剋隆到pShuttle-CMV載體的相應酶切位點,穫得目的基因載體pShuttle-CMV-Cbfa1,在大腸桿菌BJ5183中和pAdEasy-1同源重組,篩選暘性剋隆,經酶切、PCR及測序鑒定,線性化後用脂質體法轉染HEK293細胞進行包裝、擴增,用報告基因GFP對病毒滴度和感染效率進行鑑測,酚氯倣抽提純化病毒,AdEasy1/Cbfa1感染間充質榦細胞(MSC)後檢測Cbfa1的錶達。結果:測序、酶切及PCR證實Cbfa1基因重組腺病毒載體構建成功,AdEasy1/Cbfa1感染MSC後Cbfa1的錶達顯著升高。結論:構建瞭含Cbfa1基因的重組腺病毒載體,該基因能促進MSC嚮成骨細胞分化,預示其可作為治療基因應用于股骨頭壞死、骨摺和骨缺損的治療。
목적:구건성골세포특이성전록인자--핵심결합인자α1(Cbfa1)중조선병독재체,위후기응용Cbfa1기인치료고골두배사、골절화골결손등질병전정기출。방법:이목적기인Cbfa1전장cDNA위모판진행PCR확증,장확증산물극륭도pShuttle-CMV재체적상응매절위점,획득목적기인재체pShuttle-CMV-Cbfa1,재대장간균BJ5183중화pAdEasy-1동원중조,사선양성극륭,경매절、PCR급측서감정,선성화후용지질체법전염HEK293세포진행포장、확증,용보고기인GFP대병독적도화감염효솔진행감측,분록방추제순화병독,AdEasy1/Cbfa1감염간충질간세포(MSC)후검측Cbfa1적표체。결과:측서、매절급PCR증실Cbfa1기인중조선병독재체구건성공,AdEasy1/Cbfa1감염MSC후Cbfa1적표체현저승고。결론:구건료함Cbfa1기인적중조선병독재체,해기인능촉진MSC향성골세포분화,예시기가작위치료기인응용우고골두배사、골절화골결손적치료。
Objective: To construct the recombinant adenovirus expressing the osteoblast specific transcription fac-tor Cbfa1(core binding factor alpha 1) for gene therapy of osteonecrosis of the femeral head, fracture and bone de-fect. Methods: The Cbfa1 gene fragment was amplified by PCR according to the cDNA of Cbfa1, then the frag-ment was cloned to pShuttle-CMV. The linearized shuttle plasmid was homogenously recombined with AdEasy-1 in E.coli BJ5183. The candidate clone was further analyzed by restriction endonuclease digestion, PCR, and se-quence scan. Then the recombined adenovirus was transfected into HEK293 cells for packaging and amplifying. Ad-Easy1/Cbfa1 was purified by CsCl banding. Infection titer and rate were monitored by green fluorescent protein expression. The protein expression of Cbfa1 was detected by Western-blotting after AdEasy1/Cbfa1 infected mesen-chymal stem cell(MSC). Results: Sequence scan, restriction endonuclease, and PCR confirmed that Cbfa1 was cloned to the adenovirus vector successfully. The protein expression of Cbfa1 was detected after AdEasy1/Cbfa1 in-fected MSC by Western-blotting. Conclusion: The recombined adenovirus AdEasy1/Cbfa1 was constructed success-fully, which could enhance the ability of MSC differentiation to osteoblast, and will be benefit for treatment of os-teonecrosis of femoral head, fracture and bone defect in the future.