中国癌症杂志
中國癌癥雜誌
중국암증잡지
CHINA ONCOLOGY
2013年
1期
10-16
,共7页
樊全荣%史俊文%贾俊双%高飞%张余琴%赵尊兰%姚开泰%肖东
樊全榮%史俊文%賈俊雙%高飛%張餘琴%趙尊蘭%姚開泰%肖東
번전영%사준문%가준쌍%고비%장여금%조존란%요개태%초동
萤火虫荧光素酶%绿色荧光蛋白%慢病毒%293T细胞%诱导性多潜能干细胞%畸胎瘤
螢火蟲熒光素酶%綠色熒光蛋白%慢病毒%293T細胞%誘導性多潛能榦細胞%畸胎瘤
형화충형광소매%록색형광단백%만병독%293T세포%유도성다잠능간세포%기태류
Firefly luciferase%Green fluorescent protein%Lentivirus%293T cells:Induced pluripotent stem cells%Teratoma
背景与目的:诱导性多潜能干细胞(induced pluripotent stem cells,iPS细胞)在修复受损组织器官和治疗人类疾病方面已展示了良好的应用前景,但iPS细胞具有形成畸胎瘤的作用,这是其安全应用于临床的巨大障碍之一.为此本研究拟利用慢病毒体外感染的方法,建立携带萤火虫荧光素酶(firefly luciferase,Fluc)和绿色荧光蛋白(green fluorescent protein,ZsGreen)双报告基因的小鼠iPS细胞系,为活体动态监测畸胎瘤形成、定植和形成畸胎瘤所需最小细胞剂量等提供实验基础.方法:构建含Fluc和ZsGreen双报告基因的慢病毒载体.以pLH2BmRFP质粒为模板,PCR扩增获得Fluc基因片段,将其克隆至利用BamHⅠ酶切的pHAGE-fullEF1a-MCS-IZsGreen质粒中,挑选一个阳性克隆质粒进行测序,最终获得携带双报告基因的慢病毒载体pEF1a-Fluc-IRES-ZsGreen(pELZG).采用脂质体介导的瞬时转染生产携带Fluc和ZsGreen基因的病毒,收集病毒上清,并感染iPS细胞,数天后荧光显微镜下观察是否有绿色荧光的iPS细胞克隆,不断挑取ZsGreen阳性的iPS细胞克隆以进一步纯化.将标记好的iPS细胞移植入裸鼠皮下,观察成瘤情况.结果:pELZG载体分别经XbaⅠ、PvuⅡ、SacⅠ单酶切,得到的片段大小分别为10.005、3.282和6.723 kb,2.441、3.401和6.604 kb,预示成功构建了Fluc和ZsGreen双报告基因的慢病毒载体.利用其生产的病毒上清成功感染iPS细胞,经过不断的纯化,最终获得含稳定表达Fluc和ZsGreen双报告基因的小鼠iPS细胞系.将标记好的iPS细胞植入裸鼠皮下后,观察到畸胎瘤的形成.结论:成功建立了携带Fluc和ZsGreen双报告基因的小鼠iPS细胞系,为相关后续研究打下了良好的基础.标记后的iPS细胞对畸胎瘤的形成和发展具有很好的示踪作用.
揹景與目的:誘導性多潛能榦細胞(induced pluripotent stem cells,iPS細胞)在脩複受損組織器官和治療人類疾病方麵已展示瞭良好的應用前景,但iPS細胞具有形成畸胎瘤的作用,這是其安全應用于臨床的巨大障礙之一.為此本研究擬利用慢病毒體外感染的方法,建立攜帶螢火蟲熒光素酶(firefly luciferase,Fluc)和綠色熒光蛋白(green fluorescent protein,ZsGreen)雙報告基因的小鼠iPS細胞繫,為活體動態鑑測畸胎瘤形成、定植和形成畸胎瘤所需最小細胞劑量等提供實驗基礎.方法:構建含Fluc和ZsGreen雙報告基因的慢病毒載體.以pLH2BmRFP質粒為模闆,PCR擴增穫得Fluc基因片段,將其剋隆至利用BamHⅠ酶切的pHAGE-fullEF1a-MCS-IZsGreen質粒中,挑選一箇暘性剋隆質粒進行測序,最終穫得攜帶雙報告基因的慢病毒載體pEF1a-Fluc-IRES-ZsGreen(pELZG).採用脂質體介導的瞬時轉染生產攜帶Fluc和ZsGreen基因的病毒,收集病毒上清,併感染iPS細胞,數天後熒光顯微鏡下觀察是否有綠色熒光的iPS細胞剋隆,不斷挑取ZsGreen暘性的iPS細胞剋隆以進一步純化.將標記好的iPS細胞移植入裸鼠皮下,觀察成瘤情況.結果:pELZG載體分彆經XbaⅠ、PvuⅡ、SacⅠ單酶切,得到的片段大小分彆為10.005、3.282和6.723 kb,2.441、3.401和6.604 kb,預示成功構建瞭Fluc和ZsGreen雙報告基因的慢病毒載體.利用其生產的病毒上清成功感染iPS細胞,經過不斷的純化,最終穫得含穩定錶達Fluc和ZsGreen雙報告基因的小鼠iPS細胞繫.將標記好的iPS細胞植入裸鼠皮下後,觀察到畸胎瘤的形成.結論:成功建立瞭攜帶Fluc和ZsGreen雙報告基因的小鼠iPS細胞繫,為相關後續研究打下瞭良好的基礎.標記後的iPS細胞對畸胎瘤的形成和髮展具有很好的示蹤作用.
배경여목적:유도성다잠능간세포(induced pluripotent stem cells,iPS세포)재수복수손조직기관화치료인류질병방면이전시료량호적응용전경,단iPS세포구유형성기태류적작용,저시기안전응용우림상적거대장애지일.위차본연구의이용만병독체외감염적방법,건립휴대형화충형광소매(firefly luciferase,Fluc)화록색형광단백(green fluorescent protein,ZsGreen)쌍보고기인적소서iPS세포계,위활체동태감측기태류형성、정식화형성기태류소수최소세포제량등제공실험기출.방법:구건함Fluc화ZsGreen쌍보고기인적만병독재체.이pLH2BmRFP질립위모판,PCR확증획득Fluc기인편단,장기극륭지이용BamHⅠ매절적pHAGE-fullEF1a-MCS-IZsGreen질립중,도선일개양성극륭질립진행측서,최종획득휴대쌍보고기인적만병독재체pEF1a-Fluc-IRES-ZsGreen(pELZG).채용지질체개도적순시전염생산휴대Fluc화ZsGreen기인적병독,수집병독상청,병감염iPS세포,수천후형광현미경하관찰시부유록색형광적iPS세포극륭,불단도취ZsGreen양성적iPS세포극륭이진일보순화.장표기호적iPS세포이식입라서피하,관찰성류정황.결과:pELZG재체분별경XbaⅠ、PvuⅡ、SacⅠ단매절,득도적편단대소분별위10.005、3.282화6.723 kb,2.441、3.401화6.604 kb,예시성공구건료Fluc화ZsGreen쌍보고기인적만병독재체.이용기생산적병독상청성공감염iPS세포,경과불단적순화,최종획득함은정표체Fluc화ZsGreen쌍보고기인적소서iPS세포계.장표기호적iPS세포식입라서피하후,관찰도기태류적형성.결론:성공건립료휴대Fluc화ZsGreen쌍보고기인적소서iPS세포계,위상관후속연구타하료량호적기출.표기후적iPS세포대기태류적형성화발전구유흔호적시종작용.
Background and purpose:Induced pluripotent stem cells (iPS cells) have demonstrated a good prospect, which includes repairing damaged tissues, organs and the treatment of human diseases, but iPS cells have the ability to form a teratoma, which is one of the huge obstacles involved in its security used in clinical trials. Thus, this study intended to establish mouse iPS cell lines carrying firefly luciferase (Fluc) and green fluorescent protein (ZsGreen) dual reporter genes to lay the foundation for in vivo monitoring teratoma formation and determining the minimum cell dose required for teratoma formation. Methods:To generate lentiviral expression vector harboring Fluc and zsGreen genes, Fluc gene was amplified by PCR from the template of pLH2BmRFP, and subsequently cloned into the plasmid of pHAGE-fullEF1a-MCS-IzsGreen to obtain the final vector of pEF1a-Fluc-IRES-ZsGreen (referred to as pELZG) which was used to produce lentiviruses carrying Fluc and ZsGreen genes, followed by confirming that lentiviruses harboring Fluc and ZsGreen genes were successfully made through GFP assay under fluorescent stereo microscope after infecting 293T cells. Lentivirus supernatant harboring Fluc and ZsGreen genes were employed to infect iPS cells, followed by GFP assay under fluorescent stereo microscope several days after infection. The labeled iPS cells were transplanted into subcutaneous of nude mice. Results:As expected, the gel electrophoresis of pELZG vector respectively digested with XbaⅠ, PvuⅡand SacⅠ, respectively resulted in a 10.005 kb, 3.282 kb, 6.723 kb, and 2.441 kb, 3.401 kb, 6.604 kb bands. It indicated that the lentiviral vector carrying the Fluc and ZsGreen dual reporter gene was successfully constructed. Mouse iPS cells were successfully infected by lentivirus supernatant, while the complete GFP-positive iPS cell colonies were obtained by picking clones. After iPS cells were transplanted into nude mice, teratoma formation was observed. Conclusion:Mouse iPS cell line stably expressing Fluc and ZsGreen genes was successfully generated, and the Fluc-and ZsGreen-labeled iPS cells can trace the formation and development of the teratoma.
