Recent studies have shown that phosphatase and tensin homolog-deleted on chromosome ten （PTEN） gene plays an important role in ischemic brain damage and synaptic plasticity. The AdEasy system, which has been widely ...Recent studies have shown that phosphatase and tensin homolog-deleted on chromosome ten （PTEN） gene plays an important role in ischemic brain damage and synaptic plasticity. The AdEasy system, which has been widely used, greatly simplifies preparation of recombinant adenovirus. Therefore, recombinant defective adenovirus vector carrying human PTEN tumor suppressor gene （Ad-PTEN） was constructed using the AdEasy-1 system and was transfected into HEK293 cells for packaging and amplification. Infection efficiency and expression intensity were observed in primary cultured rat hippocampal neurons infected with Ad-PTEN in vitro. Results revealed a cytopathic effect in green fluorescent protein expression, which increased with prolonged time. After three cycles of amplification, the adenovirus titer was increased to an adequate titer for infecting hippocampal neurons. The entire process typically requires 4-5 weeks for completion. Results suggested that recombinant defective adenovirus vector carrying the PTEN gene was successfully and rapidly constructed using the AdEasy system.展开更多
目的构建稳定表达成熟miRNA-30a和miRNA-30e腺病毒表达载体。方法小鼠基因组中分别扩增出带有酶切位点的mmu-miR-30a、mmu-miR-30e目的基因,目的基因连接到穿梭质粒pSES-HUS,带有目的基因的穿梭质粒重组到骨架质粒AdEasy1上,重组质粒转...目的构建稳定表达成熟miRNA-30a和miRNA-30e腺病毒表达载体。方法小鼠基因组中分别扩增出带有酶切位点的mmu-miR-30a、mmu-miR-30e目的基因,目的基因连接到穿梭质粒pSES-HUS,带有目的基因的穿梭质粒重组到骨架质粒AdEasy1上,重组质粒转染到Hek-293细胞中包装成腺病毒载体,反复扩增之后获得高滴度的pAd-mmu-miR-30a、pAd-mmu-miR-30e腺病毒。用目的腺病毒分3组(RFP对照组,miR-30a组,miR-30e组)感染小鼠Mefs细胞,用荧光定量PCR方法检测mmu-miR-30a、mmu-miR-30e表达情况。结果分别扩增出带有酶切位点的357 bp mmu-miR-30a,324 bp mmu-miR-30e,并成功连接到pSES-HUS上,进一步与AdEasy1重组,包装得到腺病毒表达载体,通过荧光定量PCR检测,Mefs细胞中mmu-miR-30a升高26.46±7.46倍,mmu-miR-30e升高2.76±0.25倍。结论成功构建了成熟miRNA的腺病毒表达载体。展开更多
目的 探讨表达c FLIPs重组腺病毒诱导淋巴细胞抗凋亡作用。方法 采用RT PCR方法,从人T淋巴细胞株的总RNA中克隆c FLIPs基因;通过pAdeasy系统,构建表达c FLIPs的重组腺病毒Ad c FLIPs;Hochest染色法及PI染色流式细胞仪检测anti Apo 1...目的 探讨表达c FLIPs重组腺病毒诱导淋巴细胞抗凋亡作用。方法 采用RT PCR方法,从人T淋巴细胞株的总RNA中克隆c FLIPs基因;通过pAdeasy系统,构建表达c FLIPs的重组腺病毒Ad c FLIPs;Hochest染色法及PI染色流式细胞仪检测anti Apo 1诱导感染Ad c FLIPs后的H9细胞凋亡。结果 采用RT PCR方法从人T淋巴细胞株中扩增出c FLIPs基因;构建重组腺病毒Ad c FLIPs ;经Hoechst染色,荧光显微镜下观察细胞核的形态,结果发现,经anti Apo 1诱导凋亡处理2 4h后,未经Ad c FLIPs感染的H9细胞有大量的细胞核呈浓染致密的固缩形态或颗粒状荧光的凋亡细胞;Ad c FLIPs感染的H9细胞中细胞核呈浓染致密的固缩形态或颗粒状荧光的细胞数明显减少,大部分细胞染色质呈弥漫均匀低强度荧光;流式细胞仪分析经PI染色后的H9细胞,未经Ad c FLIPs预处理的H9细胞在anti Apo 1作用2 4h后,细胞的凋亡率分别为4 8 33%±7 4 1% ;经Ad c FLIPs预处理1d的H9细胞,其细胞凋亡率分别下降到3 6 0 %±0 2 1%。结论 重组腺病毒Ad c FLIPs可有效地诱导淋巴细胞的抗凋亡作用。展开更多
文摘Recent studies have shown that phosphatase and tensin homolog-deleted on chromosome ten （PTEN） gene plays an important role in ischemic brain damage and synaptic plasticity. The AdEasy system, which has been widely used, greatly simplifies preparation of recombinant adenovirus. Therefore, recombinant defective adenovirus vector carrying human PTEN tumor suppressor gene （Ad-PTEN） was constructed using the AdEasy-1 system and was transfected into HEK293 cells for packaging and amplification. Infection efficiency and expression intensity were observed in primary cultured rat hippocampal neurons infected with Ad-PTEN in vitro. Results revealed a cytopathic effect in green fluorescent protein expression, which increased with prolonged time. After three cycles of amplification, the adenovirus titer was increased to an adequate titer for infecting hippocampal neurons. The entire process typically requires 4-5 weeks for completion. Results suggested that recombinant defective adenovirus vector carrying the PTEN gene was successfully and rapidly constructed using the AdEasy system.
文摘目的构建稳定表达成熟miRNA-30a和miRNA-30e腺病毒表达载体。方法小鼠基因组中分别扩增出带有酶切位点的mmu-miR-30a、mmu-miR-30e目的基因,目的基因连接到穿梭质粒pSES-HUS,带有目的基因的穿梭质粒重组到骨架质粒AdEasy1上,重组质粒转染到Hek-293细胞中包装成腺病毒载体,反复扩增之后获得高滴度的pAd-mmu-miR-30a、pAd-mmu-miR-30e腺病毒。用目的腺病毒分3组(RFP对照组,miR-30a组,miR-30e组)感染小鼠Mefs细胞,用荧光定量PCR方法检测mmu-miR-30a、mmu-miR-30e表达情况。结果分别扩增出带有酶切位点的357 bp mmu-miR-30a,324 bp mmu-miR-30e,并成功连接到pSES-HUS上,进一步与AdEasy1重组,包装得到腺病毒表达载体,通过荧光定量PCR检测,Mefs细胞中mmu-miR-30a升高26.46±7.46倍,mmu-miR-30e升高2.76±0.25倍。结论成功构建了成熟miRNA的腺病毒表达载体。
文摘目的 探讨表达c FLIPs重组腺病毒诱导淋巴细胞抗凋亡作用。方法 采用RT PCR方法,从人T淋巴细胞株的总RNA中克隆c FLIPs基因;通过pAdeasy系统,构建表达c FLIPs的重组腺病毒Ad c FLIPs;Hochest染色法及PI染色流式细胞仪检测anti Apo 1诱导感染Ad c FLIPs后的H9细胞凋亡。结果 采用RT PCR方法从人T淋巴细胞株中扩增出c FLIPs基因;构建重组腺病毒Ad c FLIPs ;经Hoechst染色,荧光显微镜下观察细胞核的形态,结果发现,经anti Apo 1诱导凋亡处理2 4h后,未经Ad c FLIPs感染的H9细胞有大量的细胞核呈浓染致密的固缩形态或颗粒状荧光的凋亡细胞;Ad c FLIPs感染的H9细胞中细胞核呈浓染致密的固缩形态或颗粒状荧光的细胞数明显减少,大部分细胞染色质呈弥漫均匀低强度荧光;流式细胞仪分析经PI染色后的H9细胞,未经Ad c FLIPs预处理的H9细胞在anti Apo 1作用2 4h后,细胞的凋亡率分别为4 8 33%±7 4 1% ;经Ad c FLIPs预处理1d的H9细胞,其细胞凋亡率分别下降到3 6 0 %±0 2 1%。结论 重组腺病毒Ad c FLIPs可有效地诱导淋巴细胞的抗凋亡作用。
