中华生物医学工程杂志
中華生物醫學工程雜誌
중화생물의학공정잡지
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
2011年
3期
220-224
,共5页
王银芬%李伟强%方榕%刘佳%杨达雅%胡承恒%伍贵富%项鹏
王銀芬%李偉彊%方榕%劉佳%楊達雅%鬍承恆%伍貴富%項鵬
왕은분%리위강%방용%류가%양체아%호승항%오귀부%항붕
慢病毒属%Isl1基因%入骨髓间充质干细胞%转染%遗传载体
慢病毒屬%Isl1基因%入骨髓間充質榦細胞%轉染%遺傳載體
만병독속%Isl1기인%입골수간충질간세포%전염%유전재체
Lentivirus%Isl1 gene%Human mesenchymal stem cells%Transfection%Genetic vector
研究慢病毒介导的Isl1基因在人骨髓间充质干细胞(hMSC)中的表达情况,并检测其下游基因Nkx2.5和Flk-1的表达。方法 多位点Gateway载体构建技术构建2K7puro/EF 1α-Isl1慢病毒表达载体(由EF1α启动子启动Isl1基因表达)。包装细胞293FT细胞获得慢病毒,再转染至hMSC。利用嘌呤霉素抗性筛选出Isll阳性细胞。应用RT-PCR、Western blotting检测hMSC中Isl1基因在转染后1～6周的mRNA和1～4周的蛋白表达情况。结果 成功构建了慢病毒载体EF1α-Isl1,转染后hMSC中检测出Isl1基因mRNA和蛋白的表达,其mRNA表达随着培养时间延长而上调,第3周表达量(0.65±0.14)较1、2周增多(0.36±0.09,0.37±0.05,均P＜0.05),3周后表达趋于稳定。转染后在无任何诱导剂的情况下,检测到Isl1下游基因Nkx2.5的表达,但尚未见Flk-1的表达。结论通过慢病毒载体将Isl1基因转染hMSC后,可获得长期稳定的表达,并在无任何诱导剂的情况下,能促进下游基因Nkx2.5的表达。
研究慢病毒介導的Isl1基因在人骨髓間充質榦細胞(hMSC)中的錶達情況,併檢測其下遊基因Nkx2.5和Flk-1的錶達。方法 多位點Gateway載體構建技術構建2K7puro/EF 1α-Isl1慢病毒錶達載體(由EF1α啟動子啟動Isl1基因錶達)。包裝細胞293FT細胞穫得慢病毒,再轉染至hMSC。利用嘌呤黴素抗性篩選齣Isll暘性細胞。應用RT-PCR、Western blotting檢測hMSC中Isl1基因在轉染後1～6週的mRNA和1～4週的蛋白錶達情況。結果 成功構建瞭慢病毒載體EF1α-Isl1,轉染後hMSC中檢測齣Isl1基因mRNA和蛋白的錶達,其mRNA錶達隨著培養時間延長而上調,第3週錶達量(0.65±0.14)較1、2週增多(0.36±0.09,0.37±0.05,均P＜0.05),3週後錶達趨于穩定。轉染後在無任何誘導劑的情況下,檢測到Isl1下遊基因Nkx2.5的錶達,但尚未見Flk-1的錶達。結論通過慢病毒載體將Isl1基因轉染hMSC後,可穫得長期穩定的錶達,併在無任何誘導劑的情況下,能促進下遊基因Nkx2.5的錶達。
연구만병독개도적Isl1기인재인골수간충질간세포(hMSC)중적표체정황,병검측기하유기인Nkx2.5화Flk-1적표체。방법 다위점Gateway재체구건기술구건2K7puro/EF 1α-Isl1만병독표체재체(유EF1α계동자계동Isl1기인표체)。포장세포293FT세포획득만병독,재전염지hMSC。이용표령매소항성사선출Isll양성세포。응용RT-PCR、Western blotting검측hMSC중Isl1기인재전염후1～6주적mRNA화1～4주적단백표체정황。결과 성공구건료만병독재체EF1α-Isl1,전염후hMSC중검측출Isl1기인mRNA화단백적표체,기mRNA표체수착배양시간연장이상조,제3주표체량(0.65±0.14)교1、2주증다(0.36±0.09,0.37±0.05,균P＜0.05),3주후표체추우은정。전염후재무임하유도제적정황하,검측도Isl1하유기인Nkx2.5적표체,단상미견Flk-1적표체。결론통과만병독재체장Isl1기인전염hMSC후,가획득장기은정적표체,병재무임하유도제적정황하,능촉진하유기인Nkx2.5적표체。
Objective To explore the expression of lentivirus- mediated Isl1 gene in human mesenchymal stem cells (hMSC) and to detect the expressions of its downstream genes Nkx2.5 and Flk- 1.Methods Lentiviral expression vector 2K7puro/EF1α-Isl1 was constructed with multi-site Gateway vector construction technique (promotor EF1α driving Isl1 gene expression). After packaging in 293FT cells, the lentivirus was then transfected into hMSC. The Isl1-positive hMSC were screened by resistance to puromycin.Reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were then used to detect the Isl1 mRNA expression in hMSC within 1-6 weeks and protein expression within 1-4 weeks after transfection,respectively. Results Lentiviral vector EF1α-Isl1 was successfully constructed, and mRNA and protein expressions of Isl1 gene in hMSC were detected after transfection. Notably, the mRNA expression of Isl1 increased over time, which was higher at week 3 (0.65±0.14) compared to weeks 1 and 2 (0.36±0.09 and 0.37±0.05, both P＜0.05) and remained stable after week 3. In the absence of inducer, expression of Isl1 downstream gene Nkx2.5 but not Flk-1 was detected in Isl1-positive hMSC. Conclusion Long-term stable expression profile of Isl1 genes may be obtained by transfection into hMSC through lentiviral vectors, which can also result in expression of downstream Nkx2.5 gene in absence of inducer.
