靶向Caspase-3蛋白酶药物体内外筛选及评价体系的建立
|
摘要
目的：
针对目前缺乏合适的可以实时观测细胞凋亡的体内外实验模型,本课题旨在建立一种通过报告基因实时反映体内外肝细胞凋亡的细胞及小鼠模型,该模型的建立将有助于方便研究各种因素造成肝细胞凋亡的机制以及进行抗凋亡药物的体内外评价。
方法：
第一部分靶向Caspase-3蛋白酶药物筛选及评侩细胞模型的建立
采用荧光素酶重组及互补策略,构建能用于指示细胞凋亡的表达载体attB-ANluc(ΔDEVD) BCluc,其具体方法是：用萤火虫荧光素酶(Fluc)作为细胞内凋亡发生的报告基因,把Fluc分成单独没有活性两段N-Fluc和C-Fluc, N-Fluc和C-Fluc的N端分别连接上已知具有高亲和力的A肽和B肽,在N-Fluc和B肽间插入caspase-3的酶切位点DEVD,并在报告基因前插入噬菌体整合酶的识别位点attB。未发生细胞凋亡时,Fluc的两端被隔开,Fluc几乎没有活性；当细胞发生凋亡时激活caspase-3蛋白酶,caspase-3识别其酶切位点DEVD,切开A肽-NFluc与B肽-CFluc之间的连接,由于A肽和B肽具有高亲和力结合的特性,使得Fluc的N段和C段相互靠近而恢复其活性。
将已构建载体通过双酶切鉴定及基因测序鉴定后,完全正确的产物大提质粒通过脂质体共转染Hepal-6细胞,阿霉素诱导后,应用活体荧光成像技术监测荧光素酶活性的变化来指示细胞内凋亡的发生,同时应用Western blot检测有活性的Caspase-3蛋白酶的表达,从而建立一种通过监测荧光素酶的活性来反映Caspase-3蛋白酶的活性的可视化的细胞模型。
第二部分靶向Caspase-3蛋白酶药物筛查和评价细胞模型的应用
1.为了证实所建立的细胞模型能够根据荧光素酶活性变化特异性指示不同浓度药物诱导细胞凋亡的效果,指示载体转染Hepal-6细胞,转染24h后以不同浓度的阿霉素诱导细胞凋亡,48h后收集细胞并用活体荧光成像监测荧光素酶活性变化同时应用Western blot检测Caspase-3蛋白酶的活性变化。
2.为了验证所建立的细胞模型是否能够根据荧光素酶活性变化特异性指示不同浓度凋亡抑制剂对药物诱导细胞凋亡的效果做出评价,指示载体质粒与shRNA质粒共转染Hepal-6细胞,转染24h后以一定浓度的阿霉素诱导细胞凋亡,48h后收集细胞并用活体荧光成像监测荧光素酶的活性同时应用Western blot检测Caspase-3蛋白酶的活性。
第三部分靶向Caspase-3蛋白酶药物评价和筛查瞬时表达模型的建立及活体荧光成像条件的优化
为了建立可视化的小鼠模型,首先要对活体荧光成像的条件进行优化,建立目的基因瞬时表达小鼠模型,既快速又能准确地对Caspase-3蛋白酶药物作出评价和筛查,以及对诱导肝细胞凋亡的药物或病理模型进行评价。通过对表达载体质粒attB-ANluc-DEVD-BCluc应用水动力技术转染至小鼠肝脏,在不同荧光背景下,通过活体荧光成像技术优化最佳成像条件、Western blot监测小鼠肝脏Fluc的表达,建立Caspase-3蛋白酶指示载体瞬时表达小鼠模型。
第四部分靶向Caspase-3蛋白酶药物筛选和评价长期稳定表达小鼠模型的建立
将含OC31整合酶识别位点attB的Caspase-3蛋白表达载体attB-ANluc-DEVD-BCluc10μg与编码OC31整合酶的质粒Pphic31φ20μg,通过水动力技术共转染至小鼠肝脏,Pphic31φ表达的整合酶识别attB-ANluc-DEVD-BCluc的attB位点,并介导质粒attB-ANluc-DEVD-BCluc与小鼠染色体上attP的同源序列(mp11、mp12)间进行位点特异性整合,将attB-ANluc-DEVD-BCluc整合入小鼠肝细胞染色体,巢式PCR对目的基因是否发生整合以及整合位点做鉴定,通过DNA电泳以及基因测序来佐证；应用活体荧光成像技术监测小鼠体内荧光素酶活性的表达,结合Western blot监测小鼠肝脏Fluc的表达以及Caspase-3蛋白酶活性的变化,建立一种通过可视化手段监测小鼠体内荧光素酶活性来反映Caspase-3蛋白酶活性的长期稳定表达小鼠模型。
第五部分靶向Caspase-3蛋白酶药物筛查和评价可视化小鼠模型的应用
1.ConA诱导的免疫性肝损伤模型中的应用
为了验证所建立的小鼠模型能否根据荧光素酶活性变化特异性指示药物诱导细胞凋亡的效果,小鼠尾静脉注射ConA诱导肝细胞凋亡；通过活体荧光成像监测不同浓度及同一浓度不同时间的ConA诱导肝细胞凋亡的变化,同时应用血清学及组织学评价肝脏的损伤程度。
2. LPS/D-GalN诱导的爆发性肝炎模型中的应用
为了验证所建立的小鼠模型能否根据荧光素酶活性变化特异性指示药物诱导细胞凋亡的效果,用LPS/D-GALN诱导肝细胞凋亡；通过活体荧光成像监测了不同浓度及同一浓度不同时间的LPS/D-GALN诱导肝细胞凋亡的变化,同时应用血清学及组织学评价肝脏的损伤程度。
3.抗调亡药物(shRNA)的体外评价试验(不同浓度梯度)
为了验证所建立的小鼠模型是否能够根据荧光素酶活性变化特异性指示凋亡抑制剂(shRNA)对药物诱导细胞凋亡的抑制效果做出评价,我们构建了靶向caspase-3的shRNA,通过水动力转染技术分别将caspase-3干扰RNA及空载对照质粒转染到整合了attB-ANluc-DEVD-BCluc的小鼠模型体内,并给于LPS/D-GALN诱导凋亡,活体荧光成像监测小鼠肝脏中荧光素酶活性变化。
4.小鼠模型活体荧光成像评价凋亡抑制剂(Z-VAD-FMK)
为了验证所建立的小鼠模型是否能够根据荧光素酶活性变化特异性指示凋亡抑制剂对药物诱导细胞凋亡的抑制效果做出评价,将整合了attB-ANluc-DEVD-BCluc的小鼠模型,给于LPS/D-GALN诱导肝细胞凋亡,2h后尾静脉给予凋亡抑制剂Z-VAD-FMK,9h后活体荧光成像技术监测荧光素酶的活性变化。
5.活体荧光成像监测MHV-3诱导的肝细胞凋亡
肝脏内发生的炎症及各种因素引起的感染常诱导肝细胞凋亡的发生,为了验证该模型能否用于监测肝脏内炎症及感染状态,用致死剂量的鼠肝炎病毒MHV-3感染整合attB-ANluc-DEVD-BCluc的小鼠模型,并于感染后不同时间点监测荧光素酶活性变化。
结果：
第一部分在我们建立的细胞体系中经阿霉素处理后24h,细胞出现显著凋亡,阿霉素诱导荧光素酶活性的升高与细胞凋亡相关,并呈现剂量依赖效应,为了证实荧光素酶活性变化特异性依赖于Caspase-3蛋白酶活性对报告基因attB-ANluc (ΔDEVD)BCluc的切割,将上述处理后的细胞进行Western blot检测。结果显示：阿霉素药物诱导组可检测到attB-ANluc (ΔDEVD) BCluc的N端切割条带,而对照组未见attB-ANluc (ΔDEVD) BCluc的切割,并且对attB-ANluc (ΔDEVD) BCluc的切割特异性依赖于有活性的Caspase-3蛋白酶。
第二部分细胞模型的应用
1.活体荧光成像结果显示,随着药物浓度的升高荧光素酶活性逐渐升高,其特异性依赖于有活性的Caspase-3蛋白酶的变化。
2.活体荧光成像结果显示,靶向Caspase-3shRNA在24h后的抑制效果大于48h后的抑制效果,与Western blot所示结果一致。
第三部分瞬时表达小鼠模型的建立及活体荧光成像的条件优化
1.成功建立了活体荧光成像Caspase-3蛋白酶药物筛查和评价的瞬时表达小鼠模型；
2.活体荧光成像的条件优化,优化了活体荧光成像Caspase-3蛋白酶药物筛查和评价的小鼠模型。
3.水动力转染后指示载体在小鼠肝脏特异性表达,经LPS/DalN诱导肝细胞凋亡后24h萤光素酶表达达到高峰,随着时间的推移萤光素酶表达逐渐降低,10天左右降至本底；
4.应用Western blot对有活性的Caspase-3蛋白酶进行了检测,结果与应用活体荧光成像监测到的萤光素酶活性表达具有正相关性；
第四部分长期稳定表达小鼠模型的建立
水动力转染后指示载体在小鼠肝脏特异性表达,1d后达到高峰,随后逐渐降低,10天内降至本底。通过巢式PCR对小鼠是否发生整合以及整合位点做进一步鉴定,结果显示,在生成的两个杂合位点attR和attL扩增出了大小约250bp的特异性片段,将PCR产物凝胶回收后连接T载体,进行测序,BLAST比对分析发现,表达载体和小鼠第2号染色体的mpsL1位点发生整合,整合铰链区(core area)为TTG,且有数个碱基的丢失,至此证实成功建立了Caspase-3指示载体长期稳定表达小鼠模型。
第五部分可视化长期稳定表达小鼠模型的应用
1. ConA诱导的免疫性肝损伤模型中的应用
结果显示：所建立的小鼠模型能够通过监测荧光素酶活性的变化来指示不同浓度及同一浓度不同时间的ConA诱导肝细胞凋亡,并且与血清学和组织学所示结果一致。
2. LPS/D-GalN诱导的爆发性肝炎模型中的应用
结果显示：所建立的小鼠模型能够通过监测荧光素酶活性的变化来指示不同浓度及同一浓度不同时间的LPS/D-GALN所诱导的肝细胞凋亡,并且与血清学和组织学所示结果一致。
3.抗调亡药物(shRNA)的体外评价试验(不同浓度梯度)
结果证实：所建立的小鼠模型能够通过监测荧光素酶活性的变化来指示抗调亡药物(shRNA)抑制凋亡发生的效果,24h后的抑制效果明显大于48h后的抑制效果。
4.小鼠模型活体荧光成像评价凋亡抑制剂(Z-VAD-FMK)
结果显示,凋亡抑制剂(Z-VAD-FMK)与对照组相比荧光素酶活性下降了大约150倍左右,说明Z-VAD-FMK抑制了LPS/D-GALN诱导的肝细胞凋亡。
5.活体荧光成像监测MHV-3诱导的肝细胞凋亡
结果显示,感染后48h荧光素酶活性升高,72h最强,96h小鼠死亡,说明MHV-3感染诱导了肝细胞凋亡的发生,这与文献报道一致,说明该模型能够用于指示肝脏内的炎症及感染。
结论：
成功建立了一种监测Caspase-3蛋白酶活性的细胞及小鼠模型,通过可视化监测荧光素酶的活性来反应Caspase-3蛋白酶活性的表达水平,从而为抗凋亡药物的体内外筛查、评价以及抗凋亡机制的研究提供了一定的技术平台。
Objective:
To establish an imaging system for evaluating the Caspase-3proteinase activity and monitoring the anti-caspase-3inhibitors by bioluminescence both in vitro and in vivo.
Method:
Based on the different parts of this project.
First of all, in order to establish the in vitro system, which is the cellular-based model, using a reporter assay for imaging of Caspase-3protease activity in vitro.Taking split firefly luciferase complementation strategy, the reporter vector attB-ANluc (DEVD) BCluc was been constituted by the split N-and C-terminal fragments of luciferase, and fused to highly affinity peptides, pepA and pepB, respectively, and then inserted the Caspase-3cleavage sit of DEVD. While activating the Caspase-3proteinase, activated Caspase-3will cleave the reporter, enabling separation of ANLuc and BCLuc. Highly-affinity peptide A and peptide B lead to NLuc and CLuc complementation, and therefor restores luciferase activity.
We cotransfected attB-ANluc (DEVD) BCluc into Hepal-6cell linage,48h later, cells were harvested and the luciferase activity was detected by bioluminescence imaging. The results proved that both exogenous and active Caspase-3protease cleaved the attB-ANluc (DEVD) BCluc reporter plasmid, which lead to an increased luciferase activity. In living animals, we cotransfected attB-ANluc (DEVD) BCluc through hydrodynamic tail vein injection and the luciferase activity was detected by bioluminescence imaging. We obtained identical results with that in cellular based model.
Secondly, in order to establish the system in vivo, this is considered the live animal-based model.
We tried to integrate the reporter expression cassette attB-ANLuc (DEVD) BCLuc into mouse liver chromosome and thus established a reporter mouse model that allows noninvasive detection of caspase-3activity in liver. The reporter plasmid attB-ANLuc (DEVD) BCLuc that contains fragment of attB and ANLuc (DEVD) BCLuc was codelivered with φpC31integrase plasmids specifically to mouse liver by hydrodynamic injection procedure.(?)C31integrase mediated integration of the reporter gene into mouse liver chromosome.
Thirdly, we were thinking about how to apply for the cellular-based model and live mouse model to evaluate the different induced apoptosis conditions and scan different anti-apoptosis inhibitors. Specifically to say what we tested as follows:
1. Under cellular-based model we tested one DEVD-targeted siRNA, one of anti-apoptosis inhibor (Z-VAD-FMK) and various concentrations of Dox to treat Hepal-6cells.
2. Under mouse-based model we tested various liver damage conditions which can indicate the different clinicals.We used these mice to characterize in vivo activation of caspase-3upon treatment with ConA, LPS/GalN and infection with MHV; and then we tested the DEVD-targeted siRNA and one inhibor (Z-VAD-FMK).
Results:
The first part proved that both exogenous and active Caspase-3protease cleaved the attB-ANluc (DEVD) BCluc reporter plasmid, which leads to an increase in luciferase activity, in consistent with the results from Western blot; we saw the active Caspase-3protein cleaved the fused flue protein.
Thesecond part is through nested PCR assay, bioluminescent technique and western blot assay showed that the established mouse model can be used for monitering various liver damaged conditions and as well for screening anti-apoptosis compounds.
The third part reached the results as follows:
1. The result showed, the reporter assay system using split firefly luciferase complementation strategy proved useful for evaluating Caspase-3protease activity in cells level.
2. Our data showed that liver apoptosis can be directly monitored by our mouse model through imaging the activity of luciferase; and shRNA targeting caspase-3protein and anti-apoptosis inhibitors were also been effectively evaluated.
Conclusion
Our data showed that liver apoptosis could be evaluated by the activity of luciferase in vitro and in vivo model. The models we set up can be used for screening anti-apoptosis compounds targeted to liver cell apoptosis.
针对目前缺乏合适的可以实时观测细胞凋亡的体内外实验模型,本课题旨在建立一种通过报告基因实时反映体内外肝细胞凋亡的细胞及小鼠模型,该模型的建立将有助于方便研究各种因素造成肝细胞凋亡的机制以及进行抗凋亡药物的体内外评价。
方法：
第一部分靶向Caspase-3蛋白酶药物筛选及评侩细胞模型的建立
采用荧光素酶重组及互补策略,构建能用于指示细胞凋亡的表达载体attB-ANluc(ΔDEVD) BCluc,其具体方法是：用萤火虫荧光素酶(Fluc)作为细胞内凋亡发生的报告基因,把Fluc分成单独没有活性两段N-Fluc和C-Fluc, N-Fluc和C-Fluc的N端分别连接上已知具有高亲和力的A肽和B肽,在N-Fluc和B肽间插入caspase-3的酶切位点DEVD,并在报告基因前插入噬菌体整合酶的识别位点attB。未发生细胞凋亡时,Fluc的两端被隔开,Fluc几乎没有活性；当细胞发生凋亡时激活caspase-3蛋白酶,caspase-3识别其酶切位点DEVD,切开A肽-NFluc与B肽-CFluc之间的连接,由于A肽和B肽具有高亲和力结合的特性,使得Fluc的N段和C段相互靠近而恢复其活性。
将已构建载体通过双酶切鉴定及基因测序鉴定后,完全正确的产物大提质粒通过脂质体共转染Hepal-6细胞,阿霉素诱导后,应用活体荧光成像技术监测荧光素酶活性的变化来指示细胞内凋亡的发生,同时应用Western blot检测有活性的Caspase-3蛋白酶的表达,从而建立一种通过监测荧光素酶的活性来反映Caspase-3蛋白酶的活性的可视化的细胞模型。
第二部分靶向Caspase-3蛋白酶药物筛查和评价细胞模型的应用
1.为了证实所建立的细胞模型能够根据荧光素酶活性变化特异性指示不同浓度药物诱导细胞凋亡的效果,指示载体转染Hepal-6细胞,转染24h后以不同浓度的阿霉素诱导细胞凋亡,48h后收集细胞并用活体荧光成像监测荧光素酶活性变化同时应用Western blot检测Caspase-3蛋白酶的活性变化。
2.为了验证所建立的细胞模型是否能够根据荧光素酶活性变化特异性指示不同浓度凋亡抑制剂对药物诱导细胞凋亡的效果做出评价,指示载体质粒与shRNA质粒共转染Hepal-6细胞,转染24h后以一定浓度的阿霉素诱导细胞凋亡,48h后收集细胞并用活体荧光成像监测荧光素酶的活性同时应用Western blot检测Caspase-3蛋白酶的活性。
第三部分靶向Caspase-3蛋白酶药物评价和筛查瞬时表达模型的建立及活体荧光成像条件的优化
为了建立可视化的小鼠模型,首先要对活体荧光成像的条件进行优化,建立目的基因瞬时表达小鼠模型,既快速又能准确地对Caspase-3蛋白酶药物作出评价和筛查,以及对诱导肝细胞凋亡的药物或病理模型进行评价。通过对表达载体质粒attB-ANluc-DEVD-BCluc应用水动力技术转染至小鼠肝脏,在不同荧光背景下,通过活体荧光成像技术优化最佳成像条件、Western blot监测小鼠肝脏Fluc的表达,建立Caspase-3蛋白酶指示载体瞬时表达小鼠模型。
第四部分靶向Caspase-3蛋白酶药物筛选和评价长期稳定表达小鼠模型的建立
将含OC31整合酶识别位点attB的Caspase-3蛋白表达载体attB-ANluc-DEVD-BCluc10μg与编码OC31整合酶的质粒Pphic31φ20μg,通过水动力技术共转染至小鼠肝脏,Pphic31φ表达的整合酶识别attB-ANluc-DEVD-BCluc的attB位点,并介导质粒attB-ANluc-DEVD-BCluc与小鼠染色体上attP的同源序列(mp11、mp12)间进行位点特异性整合,将attB-ANluc-DEVD-BCluc整合入小鼠肝细胞染色体,巢式PCR对目的基因是否发生整合以及整合位点做鉴定,通过DNA电泳以及基因测序来佐证；应用活体荧光成像技术监测小鼠体内荧光素酶活性的表达,结合Western blot监测小鼠肝脏Fluc的表达以及Caspase-3蛋白酶活性的变化,建立一种通过可视化手段监测小鼠体内荧光素酶活性来反映Caspase-3蛋白酶活性的长期稳定表达小鼠模型。
第五部分靶向Caspase-3蛋白酶药物筛查和评价可视化小鼠模型的应用
1.ConA诱导的免疫性肝损伤模型中的应用
为了验证所建立的小鼠模型能否根据荧光素酶活性变化特异性指示药物诱导细胞凋亡的效果,小鼠尾静脉注射ConA诱导肝细胞凋亡；通过活体荧光成像监测不同浓度及同一浓度不同时间的ConA诱导肝细胞凋亡的变化,同时应用血清学及组织学评价肝脏的损伤程度。
2. LPS/D-GalN诱导的爆发性肝炎模型中的应用
为了验证所建立的小鼠模型能否根据荧光素酶活性变化特异性指示药物诱导细胞凋亡的效果,用LPS/D-GALN诱导肝细胞凋亡；通过活体荧光成像监测了不同浓度及同一浓度不同时间的LPS/D-GALN诱导肝细胞凋亡的变化,同时应用血清学及组织学评价肝脏的损伤程度。
3.抗调亡药物(shRNA)的体外评价试验(不同浓度梯度)
为了验证所建立的小鼠模型是否能够根据荧光素酶活性变化特异性指示凋亡抑制剂(shRNA)对药物诱导细胞凋亡的抑制效果做出评价,我们构建了靶向caspase-3的shRNA,通过水动力转染技术分别将caspase-3干扰RNA及空载对照质粒转染到整合了attB-ANluc-DEVD-BCluc的小鼠模型体内,并给于LPS/D-GALN诱导凋亡,活体荧光成像监测小鼠肝脏中荧光素酶活性变化。
4.小鼠模型活体荧光成像评价凋亡抑制剂(Z-VAD-FMK)
为了验证所建立的小鼠模型是否能够根据荧光素酶活性变化特异性指示凋亡抑制剂对药物诱导细胞凋亡的抑制效果做出评价,将整合了attB-ANluc-DEVD-BCluc的小鼠模型,给于LPS/D-GALN诱导肝细胞凋亡,2h后尾静脉给予凋亡抑制剂Z-VAD-FMK,9h后活体荧光成像技术监测荧光素酶的活性变化。
5.活体荧光成像监测MHV-3诱导的肝细胞凋亡
肝脏内发生的炎症及各种因素引起的感染常诱导肝细胞凋亡的发生,为了验证该模型能否用于监测肝脏内炎症及感染状态,用致死剂量的鼠肝炎病毒MHV-3感染整合attB-ANluc-DEVD-BCluc的小鼠模型,并于感染后不同时间点监测荧光素酶活性变化。
结果：
第一部分在我们建立的细胞体系中经阿霉素处理后24h,细胞出现显著凋亡,阿霉素诱导荧光素酶活性的升高与细胞凋亡相关,并呈现剂量依赖效应,为了证实荧光素酶活性变化特异性依赖于Caspase-3蛋白酶活性对报告基因attB-ANluc (ΔDEVD)BCluc的切割,将上述处理后的细胞进行Western blot检测。结果显示：阿霉素药物诱导组可检测到attB-ANluc (ΔDEVD) BCluc的N端切割条带,而对照组未见attB-ANluc (ΔDEVD) BCluc的切割,并且对attB-ANluc (ΔDEVD) BCluc的切割特异性依赖于有活性的Caspase-3蛋白酶。
第二部分细胞模型的应用
1.活体荧光成像结果显示,随着药物浓度的升高荧光素酶活性逐渐升高,其特异性依赖于有活性的Caspase-3蛋白酶的变化。
2.活体荧光成像结果显示,靶向Caspase-3shRNA在24h后的抑制效果大于48h后的抑制效果,与Western blot所示结果一致。
第三部分瞬时表达小鼠模型的建立及活体荧光成像的条件优化
1.成功建立了活体荧光成像Caspase-3蛋白酶药物筛查和评价的瞬时表达小鼠模型；
2.活体荧光成像的条件优化,优化了活体荧光成像Caspase-3蛋白酶药物筛查和评价的小鼠模型。
3.水动力转染后指示载体在小鼠肝脏特异性表达,经LPS/DalN诱导肝细胞凋亡后24h萤光素酶表达达到高峰,随着时间的推移萤光素酶表达逐渐降低,10天左右降至本底；
4.应用Western blot对有活性的Caspase-3蛋白酶进行了检测,结果与应用活体荧光成像监测到的萤光素酶活性表达具有正相关性；
第四部分长期稳定表达小鼠模型的建立
水动力转染后指示载体在小鼠肝脏特异性表达,1d后达到高峰,随后逐渐降低,10天内降至本底。通过巢式PCR对小鼠是否发生整合以及整合位点做进一步鉴定,结果显示,在生成的两个杂合位点attR和attL扩增出了大小约250bp的特异性片段,将PCR产物凝胶回收后连接T载体,进行测序,BLAST比对分析发现,表达载体和小鼠第2号染色体的mpsL1位点发生整合,整合铰链区(core area)为TTG,且有数个碱基的丢失,至此证实成功建立了Caspase-3指示载体长期稳定表达小鼠模型。
第五部分可视化长期稳定表达小鼠模型的应用
1. ConA诱导的免疫性肝损伤模型中的应用
结果显示：所建立的小鼠模型能够通过监测荧光素酶活性的变化来指示不同浓度及同一浓度不同时间的ConA诱导肝细胞凋亡,并且与血清学和组织学所示结果一致。
2. LPS/D-GalN诱导的爆发性肝炎模型中的应用
结果显示：所建立的小鼠模型能够通过监测荧光素酶活性的变化来指示不同浓度及同一浓度不同时间的LPS/D-GALN所诱导的肝细胞凋亡,并且与血清学和组织学所示结果一致。
3.抗调亡药物(shRNA)的体外评价试验(不同浓度梯度)
结果证实：所建立的小鼠模型能够通过监测荧光素酶活性的变化来指示抗调亡药物(shRNA)抑制凋亡发生的效果,24h后的抑制效果明显大于48h后的抑制效果。
4.小鼠模型活体荧光成像评价凋亡抑制剂(Z-VAD-FMK)
结果显示,凋亡抑制剂(Z-VAD-FMK)与对照组相比荧光素酶活性下降了大约150倍左右,说明Z-VAD-FMK抑制了LPS/D-GALN诱导的肝细胞凋亡。
5.活体荧光成像监测MHV-3诱导的肝细胞凋亡
结果显示,感染后48h荧光素酶活性升高,72h最强,96h小鼠死亡,说明MHV-3感染诱导了肝细胞凋亡的发生,这与文献报道一致,说明该模型能够用于指示肝脏内的炎症及感染。
结论：
成功建立了一种监测Caspase-3蛋白酶活性的细胞及小鼠模型,通过可视化监测荧光素酶的活性来反应Caspase-3蛋白酶活性的表达水平,从而为抗凋亡药物的体内外筛查、评价以及抗凋亡机制的研究提供了一定的技术平台。
Objective:
To establish an imaging system for evaluating the Caspase-3proteinase activity and monitoring the anti-caspase-3inhibitors by bioluminescence both in vitro and in vivo.
Method:
Based on the different parts of this project.
First of all, in order to establish the in vitro system, which is the cellular-based model, using a reporter assay for imaging of Caspase-3protease activity in vitro.Taking split firefly luciferase complementation strategy, the reporter vector attB-ANluc (DEVD) BCluc was been constituted by the split N-and C-terminal fragments of luciferase, and fused to highly affinity peptides, pepA and pepB, respectively, and then inserted the Caspase-3cleavage sit of DEVD. While activating the Caspase-3proteinase, activated Caspase-3will cleave the reporter, enabling separation of ANLuc and BCLuc. Highly-affinity peptide A and peptide B lead to NLuc and CLuc complementation, and therefor restores luciferase activity.
We cotransfected attB-ANluc (DEVD) BCluc into Hepal-6cell linage,48h later, cells were harvested and the luciferase activity was detected by bioluminescence imaging. The results proved that both exogenous and active Caspase-3protease cleaved the attB-ANluc (DEVD) BCluc reporter plasmid, which lead to an increased luciferase activity. In living animals, we cotransfected attB-ANluc (DEVD) BCluc through hydrodynamic tail vein injection and the luciferase activity was detected by bioluminescence imaging. We obtained identical results with that in cellular based model.
Secondly, in order to establish the system in vivo, this is considered the live animal-based model.
We tried to integrate the reporter expression cassette attB-ANLuc (DEVD) BCLuc into mouse liver chromosome and thus established a reporter mouse model that allows noninvasive detection of caspase-3activity in liver. The reporter plasmid attB-ANLuc (DEVD) BCLuc that contains fragment of attB and ANLuc (DEVD) BCLuc was codelivered with φpC31integrase plasmids specifically to mouse liver by hydrodynamic injection procedure.(?)C31integrase mediated integration of the reporter gene into mouse liver chromosome.
Thirdly, we were thinking about how to apply for the cellular-based model and live mouse model to evaluate the different induced apoptosis conditions and scan different anti-apoptosis inhibitors. Specifically to say what we tested as follows:
1. Under cellular-based model we tested one DEVD-targeted siRNA, one of anti-apoptosis inhibor (Z-VAD-FMK) and various concentrations of Dox to treat Hepal-6cells.
2. Under mouse-based model we tested various liver damage conditions which can indicate the different clinicals.We used these mice to characterize in vivo activation of caspase-3upon treatment with ConA, LPS/GalN and infection with MHV; and then we tested the DEVD-targeted siRNA and one inhibor (Z-VAD-FMK).
Results:
The first part proved that both exogenous and active Caspase-3protease cleaved the attB-ANluc (DEVD) BCluc reporter plasmid, which leads to an increase in luciferase activity, in consistent with the results from Western blot; we saw the active Caspase-3protein cleaved the fused flue protein.
Thesecond part is through nested PCR assay, bioluminescent technique and western blot assay showed that the established mouse model can be used for monitering various liver damaged conditions and as well for screening anti-apoptosis compounds.
The third part reached the results as follows:
1. The result showed, the reporter assay system using split firefly luciferase complementation strategy proved useful for evaluating Caspase-3protease activity in cells level.
2. Our data showed that liver apoptosis can be directly monitored by our mouse model through imaging the activity of luciferase; and shRNA targeting caspase-3protein and anti-apoptosis inhibitors were also been effectively evaluated.
Conclusion
Our data showed that liver apoptosis could be evaluated by the activity of luciferase in vitro and in vivo model. The models we set up can be used for screening anti-apoptosis compounds targeted to liver cell apoptosis.
引文
[1]Susan Elmore. Apoptosis:A Review of Programmed Cell Death[J]. Toxicol Pathol. 2007,35(4):495-516.
[2]Taylor RC, Cullen SP, Martin SJ. Apoptosis:controlled demolition at the cellular level[J]. Nat Rev Mol Cell Biol.2008,9(3):231-241.
[3]Mukherjee B, Kessinger C, Kobayashi J, et al. DNA-PK phosphorylates histone H2AX during apoptotic DNA fragmentation in mammalian cells[J]. DNA Repair(Amst)2006,5:575-590.
[4]Oberst A, Pop C, Tremblay AG, et al. Inducible dimerization and inducible cleavage reveal a requirement for both processes in caspase-8 activation[J]. J Biol Chem. 2010,285(22):16632-16642.
[5]Lamkanfi M, Festjens N, Declercq W, et al. Vandenabeele P. Caspases in cell survival, proliferation and differentiation[J]. Cell Death Differ.2007,14(1):44-55.
[6]Festjens N, Cornelis S, Lamkanfi M, et al. Caspase-containing complexes in the regulation of cell death and inflammation[J]. Biol Chem.2006,387(8):1005-1016.
[7]Oberst A, Pop C, Tremblay AG, et al. Inducible dimerization and inducible cleavage reveal a requirement for both processes in caspase-8 activation[J]. J Biol Chem. 2010,285(22):16632-16642.
[8]Pop C, Salvesen GS. Human caspases:activation, specificity, and regulation[J]. J Biol Chem.2009,284(33):21777-21781.
[9]Choi BH, Kim W, Wang QC, et al. Kinetin riboside preferentially induces apoptosis by modulating Bcl-2 family proteins and caspase-3 in cancer cells[J]. Cancer Lett., 2008,261 (1):37-45.
[10]mackenzie SH, Clark AC. Death by caspase imerization. [J]. Adv Exp Med Biol. 2012,747:55-73.
[11]Keller N, Mares J, Zerbe O, Gruttermg. Structural and Biochemical Studies on Procaspase-8:New Insights on Initiator Caspase Activation[J]. Structure,2009, 17(3):438-448.
[12]Mrudula Donepudi, Markus G Grutter. Structure and zymogen activation of caspases[J]. Biophysical Chemistry.2002,101-102:145-53.
[13]Walters J, Schipper JL, Swartz P, et al. Allosteric modulation of caspase 3 through mutagenesis[J]. Biosci Rep.2012,32(Pt 4):401-411.
[14]Virag L. Curr Vasc Pharmacol. Structure and Function of Poly (ADP-ribose) Polymerase-1:Role in Oxidative Stress-Related Pathologies[J]. Current Vascular Pharmacology.2005,3(3):209-14.
[15]Dell'Acquaml, Smith KE, Gorski JA, Regulation of neuronal PKA signaling through AKAP targeting dynamics[J]. Eur J Cell Biol.2006,85(7):627-33.
[16]Wang CZ, Johnson KM. The Role of Caspase-3 Activation in Phencyclidine-Induced Neuronal Death in Postnatal Rats[J]. Neuropsychopharmacology,2007, 32(5):1178-1194
[17]Abdul-Ghani M, Megeney LA. Rehabilitation of a Contract Killer:Caspase-3 Directs Stem Cell Differentiation[J]. Cell Stem Cell.2008,2(6):515-516.
[18]Mazumdar B, Meyer K, Ray R. N-Terminal Region of Gelsolin Induces Apoptosis of Activated Hepatic Stellate Cells by a Caspase-Dependent Mechanism [J].Plos One.2012;7(8):e44461.
[19]Woo DH, Han IS, Jung G. Mefenamic acid-induced apoptosis in human liver cancer cell-lines through caspase-3 pathway[J]. Life Sci.2004,75(20):2439-49.
[20]Zender L, Hutker S, Liedtke C, et al. Caspase 8 small interfering RNA prevents acute liver failure in mice[J]. Proc Natl Acad Sci,2003,100(13):7797-802.
[21]Piekarska A, Kubiak R, Omulecka A, et al. Expression of tumour necrosis factor-related apoptosis inducing ligand and caspase-3 in relation to grade of inflammation and stage of fibrosis in chronic hepatitis C. Histopathology 2007;51 (5),597-604.
[22]Malene Hvid, Claus Johansen, Bent Deleuran, et al. Regulation of caspase 14 expression in keratinocytes by inflammatory cytokines-a possible link between reduced skin barrier function and inflammation?[J]. Experimental Dermatology, 2011,20(8):633-636.
[23]Howard C. Masuoka, Maria Eugenia Guicciardi. Caspase Inhibitors for the Treatment of Hepatitis C[J]. Clin Liver Dis.2009,13(3):467-475.
[24]Kalamvoki M, Georgopoulou U, Mavromara P. The NS5 A protein of the hepatitis C virus genotype 1a is cleaved by caspases to produce C-terminal-truncated forms of the protein that reside mainly in the cytosol[J]. J Biol Chem 2006,281:3449-13462.
[25]Kalamvoki M, Mavromara P. Calcium-dependent calpain proteases are mplicated in processing of the hepatitis C virus NS5A protein[J]. J Virol,2004,78:11865-11878.
[26]Rust C, Gores GJ. Apoptosis and liver disease[J]. Am J Med.2000,108(7):567-574.
[27]Canbay A, Friedman S, Gores GJ. Apoptosis:the nexus of liver injury and fibrosis[J]. Hepatology,2004,39(2):273-278.
[28]Jaeschke M, Gujral JS, Bajtml. Apoptosis and necrosis in liver disease. Liver Int. 2004,24(2):85-89.
[29]Grassi A, Susca M, Ferri S, et al. Detection of the M30 neoepitope as a new tool to quantify liver apoptosis:timing and patterns of positivity on frozen and paraffin-embeddedsections[J]. Am J Clin Pathol.2004,121(2):211-219.
[30]Wieckowska A, Zein NN, Yerian LM, et al. In vivo assessment of liver cell apoptosis as a novel biomarker of disease severity in nonalcoholic fatty liver disease. Hepatology[J].2006,44(1):27-33.
[31]Maria Eugenia Guicciardi and Gregory J. Gores. Apoptosis as a Mechanism for Liver Disease Progression[J]. Semin Liver Dis,2010,30(4):402-410.
[32]Canbay A, Feldstein A, Baskin-Bey E, et al. The caspase inhibitor IDN-6556 attenuates hepatic injury and fibrosis in the bile duct ligated mouse[J]. J Pharmacol Exp Ther.2004,308(3):1191-1196.
[33]Wieckowska A, Zein NN, Yerian LM, et al. In vivo assessment of liver cell apoptosis as a novel biomarker of disease severity in nonalcoholic fatty liver disease[J]. Hepatology.2006,44(1):27-33.
[34]Taylor RC, Cullen SP, Martin SJ. Apoptosis:controlled demolition at the cellular level[J]. Nat Rev Mol Cell Biol.2008,9(3):231-241.
[35]Malhi H, Guicciardi ME, Gores GJ. Hepatocyte death:a clear and present danger. Physiol Rev[J].2010,90(3):1165-1194.
[36]Malhi H, Gores GJ. Cellular and molecular mechanisms of liver injury [J]. Gastroenterology.2008,134(6):1641-1654.
[37]Maria Eugenia Guicciardiand Gregory J. Gores. Apoptosis as a Mechanism for Liver Disease Progression[J]. Semin Liver Dis.2010,30(4):402-410.
[38]Po w er CP, W ang J H, M anni ng B. Bacterial li poprotein delays apoptosis in human neutrophils through inhibition of caspase-3 activity:regulatory roles for CD14 and TLR-2[J]. J. Immuno 1.,2008,180(l):664.
[39]Cheung ZH, Leung M CP, Yip HK. A neuroprotective herbal mixture inhibits caspase-3-independent apoptosis in retinal ganglion cells[J]. Cell Mol. Neurobiol., 2008,28(1):137-155.
[40]de Oca J, Azuara D, Sanchez-Santos R, et al. Caspase-3 activity, response to chemotherapy and clinical outcome in patients with colon cancer[J]. Int J Colorectal Dis.2008,23(1):21-27.
[41]James KE, Asgian JL, Li ZZ, et al. Design, synthesis, and evaluation of aza-peptide epoxides as selective and potent inhibitors of caspases-1,-3,-6, and-8[J]. J Med Chem.2004;47(6):1553-1574.
[42]Motchkiss RS, Nicholson DW. Apoptosis and caspases regulate death and inflammation in sepsis[J]. Nature Rev Immunol.2006;6(11):813-822.
[43]Fleischer A, Ghadiri A, Dessauge F, et al. Modulating apoptosis as a target for effective therapy[J]. Mol Immunol.2006;43(8):1065-1079.
[44]Alkhalaf M, El-Mowafy A, Renno W, Resveratrol induced apoptosis in human breast cancer cells is mediated primarily through the caspase-3-dependent path w ay[J]. Arch Med Res.2008,39(2):162-168.
[45]Johansen C, Moeller K, Kragballe K, et al. The activity of caspase-1 is increased in lesional psoriatic epidermis [J]. J Invest Dermatol.2007,127(12):2857-2864.
[46]Joo JH, Yoon SY, Kim JH, et al. S100A6(calcyclin)enhances the sensitivity to apoptosis via the upregulation of caspase-3 activity in Hep3B cells[J]. J Cell Biochem,2008,103(4):1183-1197.
[47]Degterev A, Boyce M, Yuan J. A decade of caspases[J]. Oncogene,2003;22(53): 8543-8567.
[48]Ekici OD, Gotzmg, James KE, et al. Aza-peptide Michael acceptors:A new class of inhibitors specific for caspases and other clan CD cysteine proteases[J]. J Med Chem.2004,47(8):1889-1892.
[49]Ullman BR, Aja T, Deckwerth TL, et al. Structure-activity relationships within a series of caspase inhibitors:Effect of leaving group modifications [J]. Bioorg Med Chem Lett.2003;13(20):3623-3626.
[50]Rudolphi K, Gerwin N, Verzijl N, et. al, an inhibitor of interleukin-1β converting enzyme, reduces joint damage in two murine models of osteoarthritis[J]. Osteoarthritis Cartilage.2003,11(10):738-746.
[51]Yunxia Tao, Iram Zafar, Jun Kim, et al.Caspase-3 Gene Deletion Prolongs Survival in Polycystic Kidney Disease[J]. J Am Soc Nephrol,2008,19(4):749-755.
[52]Graczyk PP. Caspase inhibitors as anti-inflammatory and anti apoptotic agents[J]. Prog Med Chem.2002;39:1-72.
[53]Meier M, Nitschke M, Hocke C, et al. Insulin Inhibits Caspase-3 Activity in Human Renal Tubular Epithelial Cells via the PI3-Kinase/Akt Pathway [J]. Cell Physiol Biochem,2008,21(4):279-286.
[54]Toulmond S, Tang K, Bureau Y, et al. Neuroprotective effects of M826, a reversible caspase-3 inhibitor, in the rat malonate model of Huntington's disease[J]. Br J Pharmacol.2004;141(4):689-697.
[55]Akdemir O, Berksoy I, Karaoglan A, et al. Therapeutic efficacy of Ac-DMQD-CHO, a caspase 3 inhibitor, for rat spinal cord injury[J]. J Clin Neurosci,2008, 15(6):672-678.
[56]Han BH, Xu D, Choi J, et al. Selective, reversible caspase-3 inhibitor is neuroprotective and reveals distinct pathways of cell death after neonatal hypoxic-ischemic brain injury. J Biol Chem.2002;277(33):30128-30136.
[57]Janzen V, Fleming HE, Riedt T, et al. Hematopoietic stem cell responsiveness to exogenous signals limited by Caspase-3 [J]. Cell Stem Cell,2008,2(6):584-594.
[58]Cheng YJ, Lee CH, Lin YP, et al. Caspase-3 enhances lung metastasis and cell migration in a protease-independent mechanism through the ERK pathway [J]. Int J Cancer.,2008,123(6):1278-1285.
[59]Wannamaker W, Davies R, Namchuk M, et al. (S)-1-((S)-2-{[1-(4-Amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide(VX-765), an orally available selective interleukin(IL)-converting enzyme/caspase-1 inhibitor, exhibits potent anti-inflammatory activities by inhibiting release of IL-1β andil-18[J]. J Pharmacol Exp Ther.2007,321(2):509-516.
[60]Puga I, Rao A, Macian F. Targeted cleavage of signaling proteins by caspase 3 inhibits T cell receptor signaling in anergic T cells[J]. Immunit y.2008, 29(2):193-204.
[61]Yao K, Wang K, Xu W, et al. Caspase-3 and its inhibitor Ac-DEVD-CHO in rat lens epithelial cell apoptosis induced by hydrogen in vitro [J]. Chin Med J.2003, 116(7):1034-1038.
[62]Knoblach SM, Alroy DA, Nikolaeva M, et al. Caspase Inhibitor z-DEVD-fink Attenuates Calpain and Necrotic Cell Death in Vitro and After Traumatic Brain Injury[J]. J Cereb Blood Flow Metab.2004,24(10):1119-1132.
[63]Graessmann M, Berg B, Fuchs B, et a. Chemotherapy resistance of mouse WAP-SVT/t breast cancer cells is mediated by osteopontin, inhibiting apoptosis downstream of caspase-3 [J]. Oncogene,2007,26(20):2840-2850.
[64]Grassi A, Susca M, Ferri S, et al. Detection of the M30 neoepitope as a new tool to quantify liver apoptosis:timing and patterns of positivity on frozen and paraffin-embeddedsections[J]. Am J Clin Pathol.2004;121(2):211-219.
[65]D'Amelio M, Cavallucci V, Cecconi F. Neuronal caspase-3 signaling:not only cell death[J]. Cell Death and Differentiation.2010,17:1104-1114.
[66]Fischer U, Schulze-Osthoff K. Apoptosis-based therapies and drug targets[J]. Cell Death Differ.2005,12(Suppl 1):942-961.
[67]Linton SD, Aja T, Armstrong RA, et al. First-in-class pan caspase inhibitor developed for the treatment of liver disease. J Med Chem.2005,48(22):6779-6782.
[68]Pockros P, Schiff E, Shiffman M, et al. Oral IDN-6556, an antiapoptotic caspase inhibitor, may lower aminotransferase activity in patients with chronic hepatitis C[J]. Hepatology.2007,46(2):324-329.
[69]Hardy JA, Lam J, Nguyen JT, et al. Discovery of an allosteric site in the caspases[J]. Proc Natl Acad Sci USA.2004,101(34):12461-12466.
[70]Scheer JM, Romanowski MJ, Wells JA. A common allosteric site and mechanism in caspases[J]. Proc Natl Acad Sci USA.2006,103(20):7595-7600.
[71]Laxman B, Hall DE, Bhojani MS, et al. A Noninvasive real-time imaging of apoptosis[J]. PNAS,2002,99(26):16551-16555.
[72]Coppola JM, Ross BD, Rehemtulla A, et al. Non-invasive imaging of apoptosis and its application in cancer therapeutics[J]. Clin Cancer Res.2008,14(8):2492-2501.
[73]Li S, Tang Z, Yu H, et al. Administration of Naked Plasmid Encoding Hepatic Stimulator Substance by Hydrodynamic Tail Vein Injection Protects Mice from Hepatic Failure by Suppressing the Mitochondrial Permeability Transition[J]. J Pharmacol Exp Ther.2011,338(3):750-7.
[74]Barbara Bonamassa, Li Hai, and Dexi Liu. Hydrodynamic Gene Delivery and Its Applications in Pharmaceutical Research[J]. Pharm Res.2011,28(4):694-701.
[75]Gao X, Kim KS, Liu D D. Nonviral gene delivery:what we know and what is next[J]. AAPS J.2007,9:E92-E104.
[76]Herweijer H, Wolff JA. Gene therapy progress and prospects:hydrodynamic gene delivery[J]. Gene Ther.2007,14:99-107.
[77]Kobayashi N, Nishikawa M, Takakura Y, et al. The hydrodynamics-based procedure for controlling the pharmacokinetics of gene medicines at whole body, organ and cellular levels[J]. Drug. Adv. Deliv. Rev.2005,57:713-731
[78]Hodges BL, Scheule RK. Hydrodynamic delivery of DNA[J]. Expert Opin. Biol. Ther.2003,3:911-918.
[79]Zhang G, Gao X, Song YK, et al. Hydroporation as the mechanism of hydrodynamic delivery[J]. Gene Ther.2004,11:675-682.
[80]Takehara T, Uemura A, Tatsumi T, et al. Natural killer cell-mediated ablation of metastatic liver tumors by hydrodynamic injection of ifnalpha gene to mice[J]. Int. J. Cancer.2007,120:1252-1260.
[81]Suda T, Gao X, Stolz DB, et al. Structural impact of hydrodynamic injection on mouse liver[J]. Gene Ther.2007,14:129-137.
[82]Kobayashi N, Nishikawa M, Hirata K, et al. Hydrodynamics-based procedure involves transient hyperpermeability in the hepatic cellular membrane:implication of a nonspecific process in efficient intracellular gene delivery [J]. J. Gene Med. 2004,6:584-592.
[83]Hagstrom JE, Hegge J, Zhang G, et al. A facile nonviral method for delivering genes and sirnas to skeletal muscle of mammalian limbs[J]. Mol. Ther.2004,10:386-398.
[84]Alino SF, Crespo F, Dasi A. Long-term therapeutic levels of human alpha-1 antitrypsin in plasma after hydrodynamic injection of nonviral DNA[J]. Gene Ther. 2003,10:1672-1679.
[85]Suda T, Liu D. Hydrodynamic gene delivery:its principles and applications [J]. Mol. Ther.2007,15:2063-2069.
[86]Neal ZC, Bates MK, Albertini MR, et al. Hydrodynamic limb vein delivery of a xenogeneic DNA cancer vaccine effectively induces antitumor immunity [J]. Mol. Ther.2007,15:422-430.
[87]Ortaldo JR, Winkler-Pickett RT, Bere EW, et al. In vivo hydrodynamic delivery of cdna encoding IL-2:rapid, sustained redistribution, activation of mouse NK cells, and therapeutic potential in the absence of NKT cells[J]. J. Immunol.2005, 175:693-699.
[88]Yazawa H, Murakami T, Li HM, et al. Hydrodynamics-based gene delivery of naked DNA encoding fetal liver kinase-1 gene effectively suppresses the growth of pre-existing tumors [J]. Cancer Gene Ther.2006,13:993-1001.
[89]Tada M, Hatano E, Taura K, et al. High volume hydrodynamic injection of plasmid DNA via the hepatic artery results in a high level of gene expression in rat hepatocellular carcinoma induced by diethylnitrosamine[J]. J. Gene Med.2006, 8:1018-1026.
[90].詹林盛,饶林,彭剑淳,等.HCV IRES介导荧光素酶小鼠体内表达模型的建立[J].中华微生物和免疫学杂志.2004,24(3)：40-241.
[91]Thyagarajan B, Olivares EC, Hollis RP, et al. Site-specific genomic integration in mammalian cells mediated by phage phic31 integrase[J]. Mol Cell Biol.2001, 21(12):3926-3934.
[92]Groth AC, Calos MP.Phage integrases:biology and applications[J]. J Mol Biol.2004, 335(3):667-678.
[93]Olivares EC, Hollis RP, Chalberg TW, et al. Site-specific genomic integration produces therapeutic Factor IX levels in mice[J]. Nat Biotechnol.2002,20(11): 1124-1128.
[94]Chen L, Woo SL. Complete and persistent phenotypic correction of phenylketonuria in mice by site-specific genome integration of murine phenylalanine hydroxylase cdna[J]. PNAS.2005,102(43):15581-15586.
[95]Bertoni C, Jarrahian S, Wheeler TM, et al. Enhancement of plasmid-mediated gene therapy for muscular dystrophy by directed plasmid integration[J]. PNAS.2006, 103(2):419-424.
[96]Welsh DK, Kay SA. Bioluminescence imaging in living organisms[J]. Curr Opin Biotechnol.2005,16(1):73-78.
[97]Negrin RS, Contag CH. In vivo imaging using bioluminescence:a tool for probing graft-versus-host disease[J]. Nat Rev.2006,6:484-489.
[98]Keravala A, Chavez CL, Hu G, et al. Long-term phenotypic correction in factor IX knockout mice by using ΦC31 integrase-mediated gene therapy. [J]. Gene Ther. 2011,18(8):842-848
[99]Woodard LE, Hillman RT, Keravala A, Effect of nuclear localization and hydrodynamic delivery-induced cell division on phic31 integrase activity. [J]. Gene Ther.2010,17(2):217-226.
[100]Sun Z, Wang Y, Fu Q, et al. Long-term hepatitis C internal ribosome entry site-dependent gene expression mediated by phage phic31 integrase in mouse model. [J]. Antivir Ther.2009,14(3):393-400.
[101]Fu Q, Jia S, Sun Z, et al. Phic31 integrase and liver-specific regulatory elements confer high-level, long-term expression of firefly luciferase in mouse liver. [J]. Biotechnol Lett.2009,31(8):1151-1157
[102]Lee S, Hong SW, Choi HS, et al. Experimental parathyroid hormone gene therapy using (?)C31 integrase. [J]. Endocr J.2008 Dec;55(6):1033-41
[103]Chen L, Woo SL. Complete and persistent phenotypic correction of phenylketonuria in mice by site-specific genome integration of murine phenylalanine hydroxylase cdna. [J]. PNAS,2005 Oct 25;102(43):15581-6.
[104]Cook S, Griffin D. Luciferase imaging of a neurotropic viral infection in intact animals[J]. J Virol,2003,77:5333-5338.
[105]Gross S, Moss B, Piwnica-Worms D. Veni, vidi, vici:in vivo molecular imaging of immune response[J]. Immunity 2007,27:533-538.
[106]Hutchens M, Luker GD. Applications of bioluminescence imaging to the study of infectious diseases[J]. Cell Microbiol 2007,9:2315-2322.
[107]Hwang S, Wu T-T, Tong L, Kim K, Martinez-Guzman D, Colantonio A, Uittenbogaart C, Sun R. Persistent gammaherpesvirus replication and dynamic interaction with the host in vivo[J]. J Virol,2008,82:12498-12509.
[108]Luker G, Bardill J, Prior J, et al. Noninvasive bioluminescence imaging of herpes simplex virus type 1 infection and therapy in living mice[J]. J Virol 2002, 76:12149-12161.
[109]Luker G, Pica C, Song J, et al. Imaging 26S proteasome activity and inhibition in living mice[J]. Nat Med 2003,9:969-973.
[110]Luker K, Hutchens M, Schultz T, et al. Bioluminescence imaging of vaccinia virus:effects of interferon on viral replication and spread[J]. Virology 2005, 341:284-300.
[111]Luker K, Luker G. Applications of bioluminescence imaging to antiviral research and therapy.multiple luciferase enzymes and quantitation[J]. Antiviral Res 2008, 78:179-187.
[112]Luker K, Schultz T, Romine J, et al. Transgenic reporter mouse for bioluminescence imaging of herpes simplex virus 1 infection in living mice[J]. Virology 2006, 347:286-295.
[113]Mandl S, Schimmelpfennig C, Edinger M, et al. Understanding immune cell trafficking patterns via in vivo bioluminescence imaging[J]. J Cell Biochem 2002, 39:239-248.
[114]Mandl S, Schimmelpfennig C, Edinger M, et al. Understanding immune cell trafficking patterns via in vivo bioluminescence imaging[J]. J Cell Biochem 2002, 39:239-248.
[115]Yuan J, Shaham S, Ledoux S, et al. The C. Elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1β-converting enzyme[J]. Cell.1993, 75(4):641-652.
[116]kuidak, zhengts, nas, et al. Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice[J]. Nature,1996,384:368-372
[117]Fernander-Alnemri T, Litwack G, Alnemrt ES, et al. Cpp32, a novel human apoptotic protein with homology to caenorhabait is elegans cell death protein ced-e and mammalian ICE[J]. J Biol Chem,1994,269(49):30761-30764.
[118]Tis o N, Pall avicini A, Muraro T, et al. Chromosomal localization of the human genes, Cpp32, Mch2, Mch3, and ichi, in volved in cellular apoptosis[J]. Bioch em Biophys Res Commun,1996,225:983-989.
[119]Nicholson DW, Ali A, Thornberry NA, e t a. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis[J]. Nature,1995,376 (6535):37-43.
[120]Matteo Scabini, Fabio Stellari, Enrico Pesenti et al. In vivo imaging of early stage apoptosis by measuring real-time caspase-3/7 activaiton[J]. Apoptosis,2011,16: 198-207.
[121]Michitaka Ozaki, Sanae Haga and Takeaki Ozawa. In vivo monitoring of liver damage using caspase-3 probe[J]. Theranostics,2012 2(2):207-214.
[122]Leu JH, Chen LL, Lin YR, et al. Molecular mechanism of the interactions between white spot syndrome virus anti-apoptosis protein AAP-1(WSSV449)and shrimp effector caspase[J]. Dev Comp Immunol 2010,34:1068-1074.
[123]Karanewsky DS, Bai X, Linton SD, et al. Conformationally constrained inhibitors of caspase-1(interleukin-1β converting enzyme)and of the human ced-3 homologue caspase-3(cpp32, apopain). Bioorg Med Chem Lett.1998;8(19):2757-2762.
[124 Ullman BR, Aja T, Deckwerth TL, etal, et al. Structure-activity relationships within a series of caspase inhibitors:Effect of leaving group modifications. Bioorg Med Chem Lett.2003;13(20):3623-3626.
[125]付秋霞.靶向HCV IRES和NS3蛋白抑制剂筛选及评价小鼠模型的建立.[D][博士学位论文].北京：中国人民解放军军事医学科学院,2006：15
[126]Joseph Sambrook, David W. Russell.分子克隆试验指南,下册,黄培堂等译,第三版,科学出版社,2002,1571.
[127]Joseph Sambrook, David W. Russell.分子克隆试验指南,下册,黄培堂等译,第三版,科学出版社,2002,1570.
[128]Joseph Sambrook, David W. Russell.分子克隆试验指南,下册,黄培堂等译,第三版,科学出版社,2002,1595.
[129]Joseph Sambrook, David W. Russell.分子克隆试验指南,上册,黄培堂等译,第三版,科学出版社,2002,1599.
[130]Putt KS,Chen GW, Pearson JM, et al. Small-molecule activation of procaspase-3 to caspase-3 as a personalized anticancer strategy. Nat Chem Biol.2006;2:543-550.
[131]Kwo PY, Vinayek R. The therapeutic approaches for hepatitis C virus:protease inhibitors and polymerase inhibitors[J]. Gut Liver,2011,5(4):406-17.
[132]Shiryaev SA, Thomsen ER, Strongin AY, et al. New details of HCV NS3/4A proteinase functionality revealed by a high-throughput cleavage assay[J]. Plos One, 2012,7(4):e35759.
[133]Shiryaev SA, Cheltsov AV, Strongin AY. Probing of Exosites Leads to Novel Inhibitor Scaffolds of HCV NS3/4A Proteinase[J]. Plos One,2012,7(7):e40029.
[134]Iro M, Witteveldt J, Patel AH, et al. A reporter cell line for rapid and sensitive evaluation of hepatitis C virus infectivity and replication[J]. Antiviral Res,2009, 83(2):148-55.
[135]Chou CF, Shen S, Tan YJ, et al. The use of hepatitis C virus NS3/4A andsecreted alkaline phosphatase to quantitate cell-cell membrane fusion mediated by severe acute respiratory syndrome coronavirus S protein and the receptor angiotensin-converting enzyme[J]. Anal Biochem,2007,366(2):190-6.
[136]Cole MJ, Pirity M, Hadjantonakis AK, et al. Shedding light on bioscience[J]. EMBO reports,2003,4(9):838
[137]Jeon YH, Choi Y, Kang JH, et al. In vivo monitoring of DNA vaccine gene expression using firefly luciferase as a naked DNA[J]. Vaccine,2006,24:3057
[138]Pianko S, Patella S, Ostapowicz G, et al. Fas-mediated hepatocyte apoptosis is increased by hepatitis C virus infection and alcohol consumption, and may be associated with hepatic fibrosis:mechanisms of liver cell injury in chronic hepatitis C virus infection[J]. J Viral Hepat 2001,8:406-413.
[139]Harmeet Malhi, Gregory J. Gores and John J. Lemasters. Apoptosis and Necrosis in the Liver:A Tale of Two Deaths?[J]. HEPATOLOGY 2006,43(2):31-44.
[140]Fu QX, Wang LC, Zhan LS, et al. Screening compounds against HCV based on MAVS/IFN-β pathway in a replicon model[J]. World J Gastroenterol,2010, 16(44):5582-7.
[141]田粉梅.持续表达HCV全基因组小鼠模型的建立[D][硕士学位论文].北京：中国人民解放军军事医学科学院,2008.
[142]Picazo F., Domingo B., Franco S., et al. Fluorescence resonance energy transfer-based assay for characterization of Hepatitis C Virus NS3-4A protease activity in live cells[J]. Antimicrob Agents Chemother.2009,53:728-734.
[143]王志强.报告基因标记的HCV复制/感染模型的建立[D][硕士学位论文].北京：中国人民解放军军事医学科学院,2009.
[144]樊炜.报告基因标记的HCV长期表达小鼠模型的优化及在疫苗评价中的应用 [D][硕士学位论文].北京：中国人民解放军军事医学科学院,2011.
[145]高博.荧光素酶标记的HCV细胞模型的建立及其在药物评价等方面的应用[D][博士学位论文].北京：中国人民解放军军事医学科学院,2011.
[146]孙志东.HCV IRES小鼠体内长期表达模型的建立及其在药物评价中的应用[D][博士学位论文].北京：中国人民解放军军事医学科学院,2007.
[147]闫虎.树突状细胞的标记及活体荧光成像示踪其在体内的分布归巢[D][硕士学位论文].北京：中国人民解放军军事医学科学院,2011.
[148]Laxman B, Hall DE, Rehemtulla A, et al. Noninvasive real-time imaging of apoptosis[J]. Proc Natl Acad Sci USA 2002 99(26):16551-16555.
[149]Fan F., Wood K., Engineered luciferases for molecular sensing in living cells[J]. Curr Opion Biotechnol.2009,20:14-18.
[150]Zhang N, Weber A, Li B, et al. An inducible nitricoxide synthase-luciferase reporter system for in vivo testing of anti-inflammatory compounds in transgenic mice[J]. J Immunol,2003,170:6307
[151]Juan Du, Fang Zhao, Lin-sheng Zhan, et al. Bioluminescence Imaging allows monitoring hepatitis C virus core protein inhibitors in mice. Plos ONE[J].,2010, 5(11).
[152]Licui Wang, Qiuxia Fu, Linsheng Zhan, et al. Bioluminescence imaging of Hepatitis C virus NS3/4A serine protease activity in cells and living animals[J]. Antiviral Research 2010 87:50-56.
[153]Deroose CM, Reumers V, Gijsbers R, et al. Noninvasive monitoring of long-term lentiviral vector-mediated gene expression in rodent brain with bioluminescence imaging[J]. Mol Ther,2006,14(3):423
[154]Du J, Zhao F, Zhan LS, et al. Bioluminescence Imaging allows monitoring hepatitis C virus core protein inhibitors in mice[J]. Plos One,2010,5(11):e14043.
[155]Tseng J C, Levin B, Hurtado A, et al. Systemic tumor targeting and killing by sindbisviral vectors[J]. Nat Biotechnol,2004,22:70
[156]Sato M, Johnson M, Zhang L Q, et al. Optimization of adenoviral vectors to direct highly amplified prostate-specific expression for imaging and gene therapy[J]. Mol Ther,2003,8(5):726
[157]Gao X, Cui Y, Levenson RM, et al. In vivo cancer targeting and imaging with semiconductor quantum dots[J]. Nat Biotechnol,2004,22(8):969
[158]Wang LC, Fu QX, Zhan LS, et al. Bioluminescence imaging of Hepatitis C virus NS3/4 serine protease activity in cells and living animals[J]. Antiviral Research, 2010,8(7):50-56.
[159]Khalid Shah, Yi Tang, Xandra Breakefield, et al. Real-time imaging of TRAIL-induced apoptosis of glioma tumors in vivo[J].Oncogene 2003,22:6865-6872.
[160]Steinmann E., Wedemeyer H., Manns M. P., et al. Cyclosporine A inhibits Hepatitis C Virus nonstructural protein 2 through cyclophilin A[J]. Hepatology.2009,50: 1638-1645.
[161]Hosseinzadeh L, Behravan J, Mosaffa F, et al. Curcumin potentiates doxorubicin-induced apoptosis in H9c2 cardiac muscle cells through generation of reactive oxygen species[J]. Food Chem Toxicol.2011,49(5):1102-9
[162]Halestrap AP, Clarke SJ, Javadov SA. Mitochondrial permeability transition pore opening during myocardial reperfusion-a target for cardioprotection[J]. Cardiovasc Res 2004,61:372-385.
[163]Baines CP, Kaiser RA, Purcell NH, et al. Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death[J]. Nature 2005, 434:658-662.
[164]Reinehr R, Becker S, Keitel V, et al. Bile salt-induced apoptosis involves NADPH oxidase isoform activation[J]. Gastroenterology 2005,129:2009-2031.
[165]Deng T, Zhang Y. Possible involvement of activation of P53/P21 and demethylation of RUNX 3 in the cytotoxicity against Lovo cells induced by 5-Aza-2'-deoxycytidine[J]. Life Sci.2009,27;84(9-10):311-20.
[166]Deng T, Zhang Y.5-Aza-2'-deoxycytidine reactivates expression of RUNX3 by deletion of DNA methyltransferases leading to caspase independent apoptosis in colorectal cancer Lovo cells[J]. Biomed Pharmacother.2009,63(7):492-500.
[167]Zender L, Hutker S, Liedtke C, et al. Caspase-8 small interfering RNA prvents acute liver failure in mice[J]. PNAS,2003,100(13):7797-7802
[168]Walter J. Wurzer, Oliver Planz, Christina Ehrhardt. Caspase 3 activation is essential for efficient influenza virus propagation[J]. The EMBO Journal,2003,22(11):2717-2728
[169]W ang J, liw, M i n J, et a 1. Fas sirna reduces apoptotic cell death of al logeneic transplanted hepatocytes in mouse spleenl[J]. Transplant Proc,2003,35(4):1594-51
[170]Song E, Lee SK, Wang J, et al. RNA interferencet argeting Fas protects mice from fulminant hepatitis[J]. Nat Med,2003,9:347-511
[171]Eszter Kapusi, Katja Kempe, Myroslava Rubtsova, et al. Phic31 Integrase-Mediated Site-Specific Recombination in Barley[J]. plos One.2012,7(9):e45353.
[172]Kittiwongwattana C, Lutz K, Clark M, et al. Plastid marker gene excision by the phic31 phage site-specific recombinase[J]. Plant Mol Biol,2007,64:137-143.
[173]Thomason LC, Calendar R, Ow DW. Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage phic31 site-specific recombination system[J]. Mol Genet Genomics,2001,265:1031-1038.
[174]Allen BG, Weeks DL. Transgenic Xenopus laevis embryos can be generated using phic31 integrase[J]. Nat Methods,2005,2:975-979.
[175]Liu F, Song Y, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA[J]. Gene Ther.1999,6:1258-1266.
[176]Zhang G, Gao X, Song YK, et al. Hydroporation as the mechanism of hydrodynamic delivery[J]. Gene Ther.2004,11:675-682.
[177]Suda T, Gao X, Stolz DB, et al. Structural impact of hydrodynamic injection on mouse liver[J]. Gene Ther.2007,14:129-137.
[178]Kobayashi N, Nishikawa M, Hirata K, et al. Hydrodynamics-based procedure involves transient hyperpermeability in the hepatic cellular membrane.implication of a nonspecific process in efficient intracellular gene delivery[J]. J. Gene Med. 2004,6:584-592.
[179]Maruyama H, Higuchi N, Nishikawa Y, et al. High-level expression of naked DNA delivered to rat liver via tail vein injection[J]. J. Gene Med.2002,4:333-341.
[180]Eastman SJ, Baskin KM, Hodges BL, et al. Development of catheter-based procedures for transducing the isolated rabbit liver with plasmid DNA[J]. Hum. Gene Ther.2002;13:2065-2077.
[181]Dooley CT, Dore TM, Hanson GT et al. Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators. J Biol Chem.2004, 279:22284-93.
[182]Kanno A, Yamanaka Y, Hirano H et al. Cyclic luciferase for real-time sensing of caspase-3 activities in living mammals[J]. Angew Chem Int Ed.2007,46:7595-9.
[183]Zhu L, Xie J, Swierczewska M et al. Real-time video imaging of protease expression in vivo[J]. Theranostics.2011,1:18-27.
[184]Lexman B, Hall DE, Bhojani MS et al. Noninvasive real-time imaging of apoptosis[J]. PNAS.2002,99:16551-5.
[185]Jiequn Wu, Qinglin Zhang, Wei Deng, et al.Toward Improvement of Erythromycin A Production in an Industrial Saccharopolyspora erythraea Strain via Facilitation of Genetic Manipulation with an Artificial attb Site for Specific Recombination[J]. Appl Environ Microbiol.2011,77(21):7508-7516.
[186]Oliynyk M. Complete genome sequence of the erythromycin-producing bacterium Saccharopolyspora erythraea NRRL23338[J]. Nat. Biotechnol.2007,25:447-453.
[187]Rodriguez E. Rapid engineering of polyketide overproduction by gene transfer to industrially optimized strains[J]. J. Ind. Microbiol. Biotechnol.2003 30:480-488.
[188]Lister JA. Chapter 11-Use of Phage phic31 Integrase as a Tool for Zebrafish Genome Manipulation J]. Methods Cell Biol.2011,104:195-208.
[189]Karow M, Calos MP. The therapeutic potential of phic31 integrase as a gene therapy system[J]. Expert Opin Biol Ther.2011,11(10):1287-96. Epub 2011 Jul 8.
[190]Ginsburg DS, Calos MP. Site-specific integration with phic31 integrase for prolonged expression of therapeutic genes. Adv Genet.2005,54:179-87
[191]Sung CM, Yeh CT, Shiau SS, Hydrodynamics-Based Transfection of the Combination of Betacellulin and Neurogenic Differentiation 1 DNA Ameliorates Hyperglycemia in Mice with Streptozotocin-Induced Diabetes[J]. Diabetes Technol Ther.2011,3(5):519-25
[192]Tsung-Hsing chen, chau-Ting YEH, Yu-Pin HO, et al. Hydrodynamics-based Transfection of Pancreatic Duodenal Homeobox 1 DNA Improves Hyperglycemia and is Associated with Limited Complications in Diabetic Mice[J]. Endocrine Journal 2009,56(6),783-790
[193]Changml, Chen JC, Yeh CT, et al. Gene gun Bombardment with dna-Coated gold Particles is a Potential alternative to Hydrodynamics-Based Transfection for delivering genes into superficial Hepatocytes[J]. Hum Gene Ther,2008,19:391-5.
[194]Huang LR, Wu HL, Chen PJ, at al. An immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection[J]. PNAS,2006,103: 17862-7.
[195]Allen BG, Weeks DL. Using phic31 integrase to make transgenic Xenopus laevis embryos[J]. Nat Protoc,2006,1:1248-1257.
[196]Bateman JR, Lee AM, Wu CT. Site-specific transformation of Drosophila via phic31 integrase-mediated cassette exchange[J]. Genetics,2006,173:769-777.
[197]Venken KJ, He Y, Hoskins RA, et al. P[acman]:a BAC transgenic platform for targeted insertion of large DNA fragments in D. Melanogaster[J]. Science,2006, 314:1747-1751.
[198]Zhang G, Budker V, Wolff JA. High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA[J]. Hum. Gene Ther. 1999,10:1735-1737.
[199]Hodges BL, Scheule RK. Hydrodynamic delivery of DNA[J]. Expert Opin. Biol. Ther.2003,3:911-918.
[200]. Hagstrom JE, Hegge J, Zhang G, et al. A facile nonviral method for delivering genes and sirnas to skeletal muscle of mammalian limbs[J]. Mol. Ther.2004, 10:386-398.
[201]Zhang G, Budker V, Williams P, et al. Efficient expression of naked DNA delivered intraarterially to limb muscles of nonhuman primates [J]. Hum. Gene Ther.2001, 12:427-438.
[202]mccaffrey AP, Ohashi K, Meuse L, et al. Determinants of hepatitis C translational initiation in vitro, in cultured cells and mice[J]. Mol. Ther.2002,5:676-684.
[203]Zhang G, Song YK, Liu D. Long-term expression of human alphal-antitrypsin gene in mouse liver achieved by intravenous administration of plasmid DNA using a hydrodynamics-based procedure[J]. Gene Ther.2000,7:1344-1349.
[204]Ozaki M, Haga S, Ozawa T. Theranostics. In Vivo Monitoring of Liver Damage Using Caspase-3 Probe [J]. Theranostics,2012,2(2):207-14.
[205]Ray P, De A, Patel M, Gambhir SS. Monitoring caspase-3 activation with a multimodality imaging sensor in living subjects[J]. Clin Cancer Res.2008,14: 5801-9.
[206]Yhee JY, Kim SA, Koo H et al. Optical imaging of cancer-related proteases using near-infrared fluorescence matrix metallopro-teinase-sensitive and cathepsin B-sensitive probes[J]. Theranostics.2012,2:179-89.
[207]Kim GB, Kim Y.-P. Analysis of protease activity using quantum dots[J]. Theranostics.2012;2:127-38.
[208]Jiang N, Zhang X, Zheng X, A Novel In Vivo sirna Delivery System Specifically Targeting Liver Cells for Protection of cona-Induced Fulminant Hepatitis[J]. plos One.2012,7(9):e44138.
[209]Zheng X, Lian D, Wong A, Bygrave M, Ichim TE, et al. (2009)Novel small interfering RNA-containing solution protecting donor organs in heart transplanta-tion[J]. Circulation 120:1099-1107
[210]Gabizon A, Horowitz AT, Goren D, et al. Targeting folate receptor with folate linked to extremities of poly(ethylene glycol)-grafted liposomes:in vitro studies[J]. Bioconjugate Chem,2009,10:289-298.
[211]Takeda K, Hayakawa Y, Van Kaer L, et al. OCritical contribution of liver natural killer T cells to a murine model of hepatitis[J].2000,97:5498-5503
[212]Li WW, Yu JY, Xu HL, et al. Concanavalin A:a potential anti-neoplastic agent targeting apoptosis, autophagy and anti-angiogenesis for cancer therapeutics. [J]. Biochem Biophys Res Commun.2011,22,414(2):282-6
[213]Li CY, Xu HL, Liu B, Concanavalin A, from an old protein to novel candidate anti-neoplastic drug[J]. Curr Mol Pharmacol.2010,3(3):123-8.
[214]Chih-Peng Chang, Ming-Cheng Yang, Hsiao-Sheng Liu, et al. Concanavalin A induces autophagy in hepatoma cells and has a therapeutic effect in a murine in situ hepatoma model[J]. Hepatology,2007,45(2):286-296.
[215]Caserta TM, Smith AN, Gultice AD. Q-VD-oph, a broad spectrum caspase inhibitor with potent antiapoptotic properties [J]. Apoptosis.2003,8(4):345-52
[216]Sass DA, Shakil AO. Fulminant hepatic failure[J]. Liver Transpl,2005,11:594-605.
[217]Agrawal N, Dasaradhi PV, Mohmmed A, et al. RNA interference:biology, mechanism, and applications[J]. Microbiol Mol Biol Rev,2003,67:657-685.
[218]Kawakami S, Hashida M. Targeted delivery systems of small interfering RNA by systemic administration[J]. Drug Metab Pharmacokinet,2007,22:142-151.
[219]Akhtar S, Benter I. Toxicogenomics of non-viral drug delivery systems for rnai potential impact on sirna-mediated gene silencing activity and specificity [J]. Adv Drug Deliv Rev,2007,59:164-182.
[220]Ryther RC, Flynt AS, Phillips JA 3rd, et al. Sirna therapeutics:big potential from small rnas[J]. Gene Ther,2005,12:5-11.
[221]Scherr M, Battmer K, Dallmann I, et al. Inhibition of GM-CSF receptor function by stable RNA interference in a NOD/SCID mouse hematopoietic stem cell transplantation model[J]. Oligonucleotides,2003,13:353-363.
[222]Song E, Lee SK, Wang J, et al. RNA interference targeting Fas protects mice from fulminant hepatitis[J]. Nat Med,2003,9:347-351.
[223]Yano J, Hirabayashi K, Nakagawa S, et al. Antitumor activity of small interfering RNA/cationic liposome complex in mouse models of cancer[J]. Clin Cancer Res, 2004,10:7721-7726.
[224]Grzelinski M, Urban-Klein B, Martens T, et al. RNA interference-mediated gene silencing of pleiotrophin through polyethyle-nimine-complexed small interfering rnas in vivo exerts antitumoral effects in glioblastoma xenografts[J]. Hum Gene Ther,2006,17:751-766.
[225]Ying RS, Zhu C, Fan XG, et al. (2007)Hepatitis B virus is inhibited by RNA interference in cell culture and in mice[J]. Antiviral Res,2007,73:24-30.
[226]Kittiwongwattana C, Lutz K, Clark M, et al. Caspase 3 activation is essential for effcient influenza virus propagation[J].The EMBO Journal Vol.2003,22:2717-2728.
[1]Munger J, Hagglund R, Roizman B. Infected cell protein no.22 is subject to proteolytic cleavage by caspases activated by a mutant that induces apoptosis. Virology 2003,305:364-370.
[2]Barkett M, Dooher JE, Lemonnier L, et al. Three mutations in v-Rel render it resistant to cleavage by cell-death protease caspase-3. Biochim Biophys Acta 2001, 1526:25-36.
[3]Wang L, Zhi B, WuW, et al. Requirement for shrimp caspase in apoptosis against virus infection. Dev Comp Immunol 2008,32:706-715.
[4]Satoh S, Hirota M, Noguchi T, et al. Cleavage of hepatitis C virus nonstructural protein 5A by a caspase-like protease(s)in mammalian cells. Virology,2000;270: 476-487.
[5]Eleouet JF, Chilmonczyk S, Besnardeau L, et al. Transmissible gastroenteritis coronavirus induces programmed cell death in infected cells through a caspase-dependent pathway. J Virol 1998;72:4918-4924.
[6]Eleouet JF, Slee EA, Saurini F, et al. The viral nucleocapsid protein of transmissible gastroenteritis coronavirus(TGEV)is cleaved by caspase-6 and-7 during TGEV-induced apoptosis. J Virol 2000;74:3975-3983.
[7]Al-Molawi N, Beardmore VA, Carter MJ, et al. Caspase-mediated cleavage of the feline calicivirus capsid protein. J Gen Virol 2003;84:1237-1244.
[8]Roberts LO, Al-Molawi N, Carter MJ, et al. Apoptosis in cultured cells infected with feline calicivirus. Ann NY Acad Sci 2003;1010:587-590.
[9]Grand RJ, Schmeiser K, Gordon EM, et al. Caspase-mediated cleavage of adenovirus early region 1A proteins. Virology 2002;301:255-271.
[10]Majerciak V, Kruhlak M, Dagur PK, et al. Caspase-7 cleavage of Kaposi sarcoma-associated herpesvirus ORF57 confers a cellular function against viral lytic gene expression. J Biol Chem 2010;285:11297-11307.
[11]Cheng F, Chen AY, Best SM, et al. The capsid proteins of Aleutianmink disease virus activate caspases and are specifically cleaved during infection. J Virol 2010;84:2687-2696.
[12]Karlberg H, Tan YJ, Mirazimi A. Induction of caspase activation and cleavage of the viral nucleocapsid protein in different cell types during Crimean-Congo hemorrhagic fever virus infection. J Biol Chem 2011;286:3227-3234
[13]Moody CA, Fradet-Turcotte A, Archambault J, et al. Human papillomaviruses activate caspases upon epithelial differentiation to induce viral genome amplification. Proc Natl Acad Sci USA 2007; 104:19541-19546.
[14]Fei JW, de Villiers EM. Degradation of HPV20E6 by p53:delta Np63 alpha and mutant p53R248W protect the wild type p53 mediated caspase-degradation. Int J Cancer 2008; 123:108-116.
[15]Leu JH, Chen LL, Lin YR, Kou GH, Lo CF. Molecular mechanism of the interactions between white spot syndrome virus anti-apoptosis protein AAP-1 (WSSV449) and shrimp effector caspase. Dev Comp Immunol 2010;34:1068-1074.
[16]Best SM, Wolfinbarger JB, Bloom ME. Caspase activation is required for permissive replication of Aleutianmink disease parvovirus in vitro. Virology 2002;292:224-234.
[17]Zhou Q, Krebs JF, Snipas SJ, et al. Interaction of the baculovirus anti-apoptotic protein p35 with caspases. Specificity, kinetics, and characterization of the caspase/p35 complex. Biochemistry 1998;37:10757-10765.
[18]Fisher AJ, Cruz W, Zoog SJ, et al. Crystal structure of baculovirus P35:role of a novel reactive site loop in apoptotic caspase inhibition. EMBO J 1999; 18:2031-2039.
[19]Xu G, Cirilli M, Huang Y, et al. Covalent inhibition revealed by the crystal structure of the caspase-8/p35 complex. Nature 2001;410:494-497.
[20]Zoog SJ, Schiller JJ, Wetter JA, et al. Baculovirus apoptotic suppressor P49 is a substrate inhibitor of initiator caspases resistant to P35 in vivo. EMBO J 2002;21:5130-5140.
[21]Guy MP, Friesen PD. Reactive-site cleavage residues confer target specificity to baculovirus P49, a dimeric member of the P35 family of caspase inhibitors. J Virol 2008;82:7504-7514.
[2]Taylor RC, Cullen SP, Martin SJ. Apoptosis:controlled demolition at the cellular level[J]. Nat Rev Mol Cell Biol.2008,9(3):231-241.
[3]Mukherjee B, Kessinger C, Kobayashi J, et al. DNA-PK phosphorylates histone H2AX during apoptotic DNA fragmentation in mammalian cells[J]. DNA Repair(Amst)2006,5:575-590.
[4]Oberst A, Pop C, Tremblay AG, et al. Inducible dimerization and inducible cleavage reveal a requirement for both processes in caspase-8 activation[J]. J Biol Chem. 2010,285(22):16632-16642.
[5]Lamkanfi M, Festjens N, Declercq W, et al. Vandenabeele P. Caspases in cell survival, proliferation and differentiation[J]. Cell Death Differ.2007,14(1):44-55.
[6]Festjens N, Cornelis S, Lamkanfi M, et al. Caspase-containing complexes in the regulation of cell death and inflammation[J]. Biol Chem.2006,387(8):1005-1016.
[7]Oberst A, Pop C, Tremblay AG, et al. Inducible dimerization and inducible cleavage reveal a requirement for both processes in caspase-8 activation[J]. J Biol Chem. 2010,285(22):16632-16642.
[8]Pop C, Salvesen GS. Human caspases:activation, specificity, and regulation[J]. J Biol Chem.2009,284(33):21777-21781.
[9]Choi BH, Kim W, Wang QC, et al. Kinetin riboside preferentially induces apoptosis by modulating Bcl-2 family proteins and caspase-3 in cancer cells[J]. Cancer Lett., 2008,261 (1):37-45.
[10]mackenzie SH, Clark AC. Death by caspase imerization. [J]. Adv Exp Med Biol. 2012,747:55-73.
[11]Keller N, Mares J, Zerbe O, Gruttermg. Structural and Biochemical Studies on Procaspase-8:New Insights on Initiator Caspase Activation[J]. Structure,2009, 17(3):438-448.
[12]Mrudula Donepudi, Markus G Grutter. Structure and zymogen activation of caspases[J]. Biophysical Chemistry.2002,101-102:145-53.
[13]Walters J, Schipper JL, Swartz P, et al. Allosteric modulation of caspase 3 through mutagenesis[J]. Biosci Rep.2012,32(Pt 4):401-411.
[14]Virag L. Curr Vasc Pharmacol. Structure and Function of Poly (ADP-ribose) Polymerase-1:Role in Oxidative Stress-Related Pathologies[J]. Current Vascular Pharmacology.2005,3(3):209-14.
[15]Dell'Acquaml, Smith KE, Gorski JA, Regulation of neuronal PKA signaling through AKAP targeting dynamics[J]. Eur J Cell Biol.2006,85(7):627-33.
[16]Wang CZ, Johnson KM. The Role of Caspase-3 Activation in Phencyclidine-Induced Neuronal Death in Postnatal Rats[J]. Neuropsychopharmacology,2007, 32(5):1178-1194
[17]Abdul-Ghani M, Megeney LA. Rehabilitation of a Contract Killer:Caspase-3 Directs Stem Cell Differentiation[J]. Cell Stem Cell.2008,2(6):515-516.
[18]Mazumdar B, Meyer K, Ray R. N-Terminal Region of Gelsolin Induces Apoptosis of Activated Hepatic Stellate Cells by a Caspase-Dependent Mechanism [J].Plos One.2012;7(8):e44461.
[19]Woo DH, Han IS, Jung G. Mefenamic acid-induced apoptosis in human liver cancer cell-lines through caspase-3 pathway[J]. Life Sci.2004,75(20):2439-49.
[20]Zender L, Hutker S, Liedtke C, et al. Caspase 8 small interfering RNA prevents acute liver failure in mice[J]. Proc Natl Acad Sci,2003,100(13):7797-802.
[21]Piekarska A, Kubiak R, Omulecka A, et al. Expression of tumour necrosis factor-related apoptosis inducing ligand and caspase-3 in relation to grade of inflammation and stage of fibrosis in chronic hepatitis C. Histopathology 2007;51 (5),597-604.
[22]Malene Hvid, Claus Johansen, Bent Deleuran, et al. Regulation of caspase 14 expression in keratinocytes by inflammatory cytokines-a possible link between reduced skin barrier function and inflammation?[J]. Experimental Dermatology, 2011,20(8):633-636.
[23]Howard C. Masuoka, Maria Eugenia Guicciardi. Caspase Inhibitors for the Treatment of Hepatitis C[J]. Clin Liver Dis.2009,13(3):467-475.
[24]Kalamvoki M, Georgopoulou U, Mavromara P. The NS5 A protein of the hepatitis C virus genotype 1a is cleaved by caspases to produce C-terminal-truncated forms of the protein that reside mainly in the cytosol[J]. J Biol Chem 2006,281:3449-13462.
[25]Kalamvoki M, Mavromara P. Calcium-dependent calpain proteases are mplicated in processing of the hepatitis C virus NS5A protein[J]. J Virol,2004,78:11865-11878.
[26]Rust C, Gores GJ. Apoptosis and liver disease[J]. Am J Med.2000,108(7):567-574.
[27]Canbay A, Friedman S, Gores GJ. Apoptosis:the nexus of liver injury and fibrosis[J]. Hepatology,2004,39(2):273-278.
[28]Jaeschke M, Gujral JS, Bajtml. Apoptosis and necrosis in liver disease. Liver Int. 2004,24(2):85-89.
[29]Grassi A, Susca M, Ferri S, et al. Detection of the M30 neoepitope as a new tool to quantify liver apoptosis:timing and patterns of positivity on frozen and paraffin-embeddedsections[J]. Am J Clin Pathol.2004,121(2):211-219.
[30]Wieckowska A, Zein NN, Yerian LM, et al. In vivo assessment of liver cell apoptosis as a novel biomarker of disease severity in nonalcoholic fatty liver disease. Hepatology[J].2006,44(1):27-33.
[31]Maria Eugenia Guicciardi and Gregory J. Gores. Apoptosis as a Mechanism for Liver Disease Progression[J]. Semin Liver Dis,2010,30(4):402-410.
[32]Canbay A, Feldstein A, Baskin-Bey E, et al. The caspase inhibitor IDN-6556 attenuates hepatic injury and fibrosis in the bile duct ligated mouse[J]. J Pharmacol Exp Ther.2004,308(3):1191-1196.
[33]Wieckowska A, Zein NN, Yerian LM, et al. In vivo assessment of liver cell apoptosis as a novel biomarker of disease severity in nonalcoholic fatty liver disease[J]. Hepatology.2006,44(1):27-33.
[34]Taylor RC, Cullen SP, Martin SJ. Apoptosis:controlled demolition at the cellular level[J]. Nat Rev Mol Cell Biol.2008,9(3):231-241.
[35]Malhi H, Guicciardi ME, Gores GJ. Hepatocyte death:a clear and present danger. Physiol Rev[J].2010,90(3):1165-1194.
[36]Malhi H, Gores GJ. Cellular and molecular mechanisms of liver injury [J]. Gastroenterology.2008,134(6):1641-1654.
[37]Maria Eugenia Guicciardiand Gregory J. Gores. Apoptosis as a Mechanism for Liver Disease Progression[J]. Semin Liver Dis.2010,30(4):402-410.
[38]Po w er CP, W ang J H, M anni ng B. Bacterial li poprotein delays apoptosis in human neutrophils through inhibition of caspase-3 activity:regulatory roles for CD14 and TLR-2[J]. J. Immuno 1.,2008,180(l):664.
[39]Cheung ZH, Leung M CP, Yip HK. A neuroprotective herbal mixture inhibits caspase-3-independent apoptosis in retinal ganglion cells[J]. Cell Mol. Neurobiol., 2008,28(1):137-155.
[40]de Oca J, Azuara D, Sanchez-Santos R, et al. Caspase-3 activity, response to chemotherapy and clinical outcome in patients with colon cancer[J]. Int J Colorectal Dis.2008,23(1):21-27.
[41]James KE, Asgian JL, Li ZZ, et al. Design, synthesis, and evaluation of aza-peptide epoxides as selective and potent inhibitors of caspases-1,-3,-6, and-8[J]. J Med Chem.2004;47(6):1553-1574.
[42]Motchkiss RS, Nicholson DW. Apoptosis and caspases regulate death and inflammation in sepsis[J]. Nature Rev Immunol.2006;6(11):813-822.
[43]Fleischer A, Ghadiri A, Dessauge F, et al. Modulating apoptosis as a target for effective therapy[J]. Mol Immunol.2006;43(8):1065-1079.
[44]Alkhalaf M, El-Mowafy A, Renno W, Resveratrol induced apoptosis in human breast cancer cells is mediated primarily through the caspase-3-dependent path w ay[J]. Arch Med Res.2008,39(2):162-168.
[45]Johansen C, Moeller K, Kragballe K, et al. The activity of caspase-1 is increased in lesional psoriatic epidermis [J]. J Invest Dermatol.2007,127(12):2857-2864.
[46]Joo JH, Yoon SY, Kim JH, et al. S100A6(calcyclin)enhances the sensitivity to apoptosis via the upregulation of caspase-3 activity in Hep3B cells[J]. J Cell Biochem,2008,103(4):1183-1197.
[47]Degterev A, Boyce M, Yuan J. A decade of caspases[J]. Oncogene,2003;22(53): 8543-8567.
[48]Ekici OD, Gotzmg, James KE, et al. Aza-peptide Michael acceptors:A new class of inhibitors specific for caspases and other clan CD cysteine proteases[J]. J Med Chem.2004,47(8):1889-1892.
[49]Ullman BR, Aja T, Deckwerth TL, et al. Structure-activity relationships within a series of caspase inhibitors:Effect of leaving group modifications [J]. Bioorg Med Chem Lett.2003;13(20):3623-3626.
[50]Rudolphi K, Gerwin N, Verzijl N, et. al, an inhibitor of interleukin-1β converting enzyme, reduces joint damage in two murine models of osteoarthritis[J]. Osteoarthritis Cartilage.2003,11(10):738-746.
[51]Yunxia Tao, Iram Zafar, Jun Kim, et al.Caspase-3 Gene Deletion Prolongs Survival in Polycystic Kidney Disease[J]. J Am Soc Nephrol,2008,19(4):749-755.
[52]Graczyk PP. Caspase inhibitors as anti-inflammatory and anti apoptotic agents[J]. Prog Med Chem.2002;39:1-72.
[53]Meier M, Nitschke M, Hocke C, et al. Insulin Inhibits Caspase-3 Activity in Human Renal Tubular Epithelial Cells via the PI3-Kinase/Akt Pathway [J]. Cell Physiol Biochem,2008,21(4):279-286.
[54]Toulmond S, Tang K, Bureau Y, et al. Neuroprotective effects of M826, a reversible caspase-3 inhibitor, in the rat malonate model of Huntington's disease[J]. Br J Pharmacol.2004;141(4):689-697.
[55]Akdemir O, Berksoy I, Karaoglan A, et al. Therapeutic efficacy of Ac-DMQD-CHO, a caspase 3 inhibitor, for rat spinal cord injury[J]. J Clin Neurosci,2008, 15(6):672-678.
[56]Han BH, Xu D, Choi J, et al. Selective, reversible caspase-3 inhibitor is neuroprotective and reveals distinct pathways of cell death after neonatal hypoxic-ischemic brain injury. J Biol Chem.2002;277(33):30128-30136.
[57]Janzen V, Fleming HE, Riedt T, et al. Hematopoietic stem cell responsiveness to exogenous signals limited by Caspase-3 [J]. Cell Stem Cell,2008,2(6):584-594.
[58]Cheng YJ, Lee CH, Lin YP, et al. Caspase-3 enhances lung metastasis and cell migration in a protease-independent mechanism through the ERK pathway [J]. Int J Cancer.,2008,123(6):1278-1285.
[59]Wannamaker W, Davies R, Namchuk M, et al. (S)-1-((S)-2-{[1-(4-Amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide(VX-765), an orally available selective interleukin(IL)-converting enzyme/caspase-1 inhibitor, exhibits potent anti-inflammatory activities by inhibiting release of IL-1β andil-18[J]. J Pharmacol Exp Ther.2007,321(2):509-516.
[60]Puga I, Rao A, Macian F. Targeted cleavage of signaling proteins by caspase 3 inhibits T cell receptor signaling in anergic T cells[J]. Immunit y.2008, 29(2):193-204.
[61]Yao K, Wang K, Xu W, et al. Caspase-3 and its inhibitor Ac-DEVD-CHO in rat lens epithelial cell apoptosis induced by hydrogen in vitro [J]. Chin Med J.2003, 116(7):1034-1038.
[62]Knoblach SM, Alroy DA, Nikolaeva M, et al. Caspase Inhibitor z-DEVD-fink Attenuates Calpain and Necrotic Cell Death in Vitro and After Traumatic Brain Injury[J]. J Cereb Blood Flow Metab.2004,24(10):1119-1132.
[63]Graessmann M, Berg B, Fuchs B, et a. Chemotherapy resistance of mouse WAP-SVT/t breast cancer cells is mediated by osteopontin, inhibiting apoptosis downstream of caspase-3 [J]. Oncogene,2007,26(20):2840-2850.
[64]Grassi A, Susca M, Ferri S, et al. Detection of the M30 neoepitope as a new tool to quantify liver apoptosis:timing and patterns of positivity on frozen and paraffin-embeddedsections[J]. Am J Clin Pathol.2004;121(2):211-219.
[65]D'Amelio M, Cavallucci V, Cecconi F. Neuronal caspase-3 signaling:not only cell death[J]. Cell Death and Differentiation.2010,17:1104-1114.
[66]Fischer U, Schulze-Osthoff K. Apoptosis-based therapies and drug targets[J]. Cell Death Differ.2005,12(Suppl 1):942-961.
[67]Linton SD, Aja T, Armstrong RA, et al. First-in-class pan caspase inhibitor developed for the treatment of liver disease. J Med Chem.2005,48(22):6779-6782.
[68]Pockros P, Schiff E, Shiffman M, et al. Oral IDN-6556, an antiapoptotic caspase inhibitor, may lower aminotransferase activity in patients with chronic hepatitis C[J]. Hepatology.2007,46(2):324-329.
[69]Hardy JA, Lam J, Nguyen JT, et al. Discovery of an allosteric site in the caspases[J]. Proc Natl Acad Sci USA.2004,101(34):12461-12466.
[70]Scheer JM, Romanowski MJ, Wells JA. A common allosteric site and mechanism in caspases[J]. Proc Natl Acad Sci USA.2006,103(20):7595-7600.
[71]Laxman B, Hall DE, Bhojani MS, et al. A Noninvasive real-time imaging of apoptosis[J]. PNAS,2002,99(26):16551-16555.
[72]Coppola JM, Ross BD, Rehemtulla A, et al. Non-invasive imaging of apoptosis and its application in cancer therapeutics[J]. Clin Cancer Res.2008,14(8):2492-2501.
[73]Li S, Tang Z, Yu H, et al. Administration of Naked Plasmid Encoding Hepatic Stimulator Substance by Hydrodynamic Tail Vein Injection Protects Mice from Hepatic Failure by Suppressing the Mitochondrial Permeability Transition[J]. J Pharmacol Exp Ther.2011,338(3):750-7.
[74]Barbara Bonamassa, Li Hai, and Dexi Liu. Hydrodynamic Gene Delivery and Its Applications in Pharmaceutical Research[J]. Pharm Res.2011,28(4):694-701.
[75]Gao X, Kim KS, Liu D D. Nonviral gene delivery:what we know and what is next[J]. AAPS J.2007,9:E92-E104.
[76]Herweijer H, Wolff JA. Gene therapy progress and prospects:hydrodynamic gene delivery[J]. Gene Ther.2007,14:99-107.
[77]Kobayashi N, Nishikawa M, Takakura Y, et al. The hydrodynamics-based procedure for controlling the pharmacokinetics of gene medicines at whole body, organ and cellular levels[J]. Drug. Adv. Deliv. Rev.2005,57:713-731
[78]Hodges BL, Scheule RK. Hydrodynamic delivery of DNA[J]. Expert Opin. Biol. Ther.2003,3:911-918.
[79]Zhang G, Gao X, Song YK, et al. Hydroporation as the mechanism of hydrodynamic delivery[J]. Gene Ther.2004,11:675-682.
[80]Takehara T, Uemura A, Tatsumi T, et al. Natural killer cell-mediated ablation of metastatic liver tumors by hydrodynamic injection of ifnalpha gene to mice[J]. Int. J. Cancer.2007,120:1252-1260.
[81]Suda T, Gao X, Stolz DB, et al. Structural impact of hydrodynamic injection on mouse liver[J]. Gene Ther.2007,14:129-137.
[82]Kobayashi N, Nishikawa M, Hirata K, et al. Hydrodynamics-based procedure involves transient hyperpermeability in the hepatic cellular membrane:implication of a nonspecific process in efficient intracellular gene delivery [J]. J. Gene Med. 2004,6:584-592.
[83]Hagstrom JE, Hegge J, Zhang G, et al. A facile nonviral method for delivering genes and sirnas to skeletal muscle of mammalian limbs[J]. Mol. Ther.2004,10:386-398.
[84]Alino SF, Crespo F, Dasi A. Long-term therapeutic levels of human alpha-1 antitrypsin in plasma after hydrodynamic injection of nonviral DNA[J]. Gene Ther. 2003,10:1672-1679.
[85]Suda T, Liu D. Hydrodynamic gene delivery:its principles and applications [J]. Mol. Ther.2007,15:2063-2069.
[86]Neal ZC, Bates MK, Albertini MR, et al. Hydrodynamic limb vein delivery of a xenogeneic DNA cancer vaccine effectively induces antitumor immunity [J]. Mol. Ther.2007,15:422-430.
[87]Ortaldo JR, Winkler-Pickett RT, Bere EW, et al. In vivo hydrodynamic delivery of cdna encoding IL-2:rapid, sustained redistribution, activation of mouse NK cells, and therapeutic potential in the absence of NKT cells[J]. J. Immunol.2005, 175:693-699.
[88]Yazawa H, Murakami T, Li HM, et al. Hydrodynamics-based gene delivery of naked DNA encoding fetal liver kinase-1 gene effectively suppresses the growth of pre-existing tumors [J]. Cancer Gene Ther.2006,13:993-1001.
[89]Tada M, Hatano E, Taura K, et al. High volume hydrodynamic injection of plasmid DNA via the hepatic artery results in a high level of gene expression in rat hepatocellular carcinoma induced by diethylnitrosamine[J]. J. Gene Med.2006, 8:1018-1026.
[90].詹林盛,饶林,彭剑淳,等.HCV IRES介导荧光素酶小鼠体内表达模型的建立[J].中华微生物和免疫学杂志.2004,24(3)：40-241.
[91]Thyagarajan B, Olivares EC, Hollis RP, et al. Site-specific genomic integration in mammalian cells mediated by phage phic31 integrase[J]. Mol Cell Biol.2001, 21(12):3926-3934.
[92]Groth AC, Calos MP.Phage integrases:biology and applications[J]. J Mol Biol.2004, 335(3):667-678.
[93]Olivares EC, Hollis RP, Chalberg TW, et al. Site-specific genomic integration produces therapeutic Factor IX levels in mice[J]. Nat Biotechnol.2002,20(11): 1124-1128.
[94]Chen L, Woo SL. Complete and persistent phenotypic correction of phenylketonuria in mice by site-specific genome integration of murine phenylalanine hydroxylase cdna[J]. PNAS.2005,102(43):15581-15586.
[95]Bertoni C, Jarrahian S, Wheeler TM, et al. Enhancement of plasmid-mediated gene therapy for muscular dystrophy by directed plasmid integration[J]. PNAS.2006, 103(2):419-424.
[96]Welsh DK, Kay SA. Bioluminescence imaging in living organisms[J]. Curr Opin Biotechnol.2005,16(1):73-78.
[97]Negrin RS, Contag CH. In vivo imaging using bioluminescence:a tool for probing graft-versus-host disease[J]. Nat Rev.2006,6:484-489.
[98]Keravala A, Chavez CL, Hu G, et al. Long-term phenotypic correction in factor IX knockout mice by using ΦC31 integrase-mediated gene therapy. [J]. Gene Ther. 2011,18(8):842-848
[99]Woodard LE, Hillman RT, Keravala A, Effect of nuclear localization and hydrodynamic delivery-induced cell division on phic31 integrase activity. [J]. Gene Ther.2010,17(2):217-226.
[100]Sun Z, Wang Y, Fu Q, et al. Long-term hepatitis C internal ribosome entry site-dependent gene expression mediated by phage phic31 integrase in mouse model. [J]. Antivir Ther.2009,14(3):393-400.
[101]Fu Q, Jia S, Sun Z, et al. Phic31 integrase and liver-specific regulatory elements confer high-level, long-term expression of firefly luciferase in mouse liver. [J]. Biotechnol Lett.2009,31(8):1151-1157
[102]Lee S, Hong SW, Choi HS, et al. Experimental parathyroid hormone gene therapy using (?)C31 integrase. [J]. Endocr J.2008 Dec;55(6):1033-41
[103]Chen L, Woo SL. Complete and persistent phenotypic correction of phenylketonuria in mice by site-specific genome integration of murine phenylalanine hydroxylase cdna. [J]. PNAS,2005 Oct 25;102(43):15581-6.
[104]Cook S, Griffin D. Luciferase imaging of a neurotropic viral infection in intact animals[J]. J Virol,2003,77:5333-5338.
[105]Gross S, Moss B, Piwnica-Worms D. Veni, vidi, vici:in vivo molecular imaging of immune response[J]. Immunity 2007,27:533-538.
[106]Hutchens M, Luker GD. Applications of bioluminescence imaging to the study of infectious diseases[J]. Cell Microbiol 2007,9:2315-2322.
[107]Hwang S, Wu T-T, Tong L, Kim K, Martinez-Guzman D, Colantonio A, Uittenbogaart C, Sun R. Persistent gammaherpesvirus replication and dynamic interaction with the host in vivo[J]. J Virol,2008,82:12498-12509.
[108]Luker G, Bardill J, Prior J, et al. Noninvasive bioluminescence imaging of herpes simplex virus type 1 infection and therapy in living mice[J]. J Virol 2002, 76:12149-12161.
[109]Luker G, Pica C, Song J, et al. Imaging 26S proteasome activity and inhibition in living mice[J]. Nat Med 2003,9:969-973.
[110]Luker K, Hutchens M, Schultz T, et al. Bioluminescence imaging of vaccinia virus:effects of interferon on viral replication and spread[J]. Virology 2005, 341:284-300.
[111]Luker K, Luker G. Applications of bioluminescence imaging to antiviral research and therapy.multiple luciferase enzymes and quantitation[J]. Antiviral Res 2008, 78:179-187.
[112]Luker K, Schultz T, Romine J, et al. Transgenic reporter mouse for bioluminescence imaging of herpes simplex virus 1 infection in living mice[J]. Virology 2006, 347:286-295.
[113]Mandl S, Schimmelpfennig C, Edinger M, et al. Understanding immune cell trafficking patterns via in vivo bioluminescence imaging[J]. J Cell Biochem 2002, 39:239-248.
[114]Mandl S, Schimmelpfennig C, Edinger M, et al. Understanding immune cell trafficking patterns via in vivo bioluminescence imaging[J]. J Cell Biochem 2002, 39:239-248.
[115]Yuan J, Shaham S, Ledoux S, et al. The C. Elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1β-converting enzyme[J]. Cell.1993, 75(4):641-652.
[116]kuidak, zhengts, nas, et al. Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice[J]. Nature,1996,384:368-372
[117]Fernander-Alnemri T, Litwack G, Alnemrt ES, et al. Cpp32, a novel human apoptotic protein with homology to caenorhabait is elegans cell death protein ced-e and mammalian ICE[J]. J Biol Chem,1994,269(49):30761-30764.
[118]Tis o N, Pall avicini A, Muraro T, et al. Chromosomal localization of the human genes, Cpp32, Mch2, Mch3, and ichi, in volved in cellular apoptosis[J]. Bioch em Biophys Res Commun,1996,225:983-989.
[119]Nicholson DW, Ali A, Thornberry NA, e t a. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis[J]. Nature,1995,376 (6535):37-43.
[120]Matteo Scabini, Fabio Stellari, Enrico Pesenti et al. In vivo imaging of early stage apoptosis by measuring real-time caspase-3/7 activaiton[J]. Apoptosis,2011,16: 198-207.
[121]Michitaka Ozaki, Sanae Haga and Takeaki Ozawa. In vivo monitoring of liver damage using caspase-3 probe[J]. Theranostics,2012 2(2):207-214.
[122]Leu JH, Chen LL, Lin YR, et al. Molecular mechanism of the interactions between white spot syndrome virus anti-apoptosis protein AAP-1(WSSV449)and shrimp effector caspase[J]. Dev Comp Immunol 2010,34:1068-1074.
[123]Karanewsky DS, Bai X, Linton SD, et al. Conformationally constrained inhibitors of caspase-1(interleukin-1β converting enzyme)and of the human ced-3 homologue caspase-3(cpp32, apopain). Bioorg Med Chem Lett.1998;8(19):2757-2762.
[124 Ullman BR, Aja T, Deckwerth TL, etal, et al. Structure-activity relationships within a series of caspase inhibitors:Effect of leaving group modifications. Bioorg Med Chem Lett.2003;13(20):3623-3626.
[125]付秋霞.靶向HCV IRES和NS3蛋白抑制剂筛选及评价小鼠模型的建立.[D][博士学位论文].北京：中国人民解放军军事医学科学院,2006：15
[126]Joseph Sambrook, David W. Russell.分子克隆试验指南,下册,黄培堂等译,第三版,科学出版社,2002,1571.
[127]Joseph Sambrook, David W. Russell.分子克隆试验指南,下册,黄培堂等译,第三版,科学出版社,2002,1570.
[128]Joseph Sambrook, David W. Russell.分子克隆试验指南,下册,黄培堂等译,第三版,科学出版社,2002,1595.
[129]Joseph Sambrook, David W. Russell.分子克隆试验指南,上册,黄培堂等译,第三版,科学出版社,2002,1599.
[130]Putt KS,Chen GW, Pearson JM, et al. Small-molecule activation of procaspase-3 to caspase-3 as a personalized anticancer strategy. Nat Chem Biol.2006;2:543-550.
[131]Kwo PY, Vinayek R. The therapeutic approaches for hepatitis C virus:protease inhibitors and polymerase inhibitors[J]. Gut Liver,2011,5(4):406-17.
[132]Shiryaev SA, Thomsen ER, Strongin AY, et al. New details of HCV NS3/4A proteinase functionality revealed by a high-throughput cleavage assay[J]. Plos One, 2012,7(4):e35759.
[133]Shiryaev SA, Cheltsov AV, Strongin AY. Probing of Exosites Leads to Novel Inhibitor Scaffolds of HCV NS3/4A Proteinase[J]. Plos One,2012,7(7):e40029.
[134]Iro M, Witteveldt J, Patel AH, et al. A reporter cell line for rapid and sensitive evaluation of hepatitis C virus infectivity and replication[J]. Antiviral Res,2009, 83(2):148-55.
[135]Chou CF, Shen S, Tan YJ, et al. The use of hepatitis C virus NS3/4A andsecreted alkaline phosphatase to quantitate cell-cell membrane fusion mediated by severe acute respiratory syndrome coronavirus S protein and the receptor angiotensin-converting enzyme[J]. Anal Biochem,2007,366(2):190-6.
[136]Cole MJ, Pirity M, Hadjantonakis AK, et al. Shedding light on bioscience[J]. EMBO reports,2003,4(9):838
[137]Jeon YH, Choi Y, Kang JH, et al. In vivo monitoring of DNA vaccine gene expression using firefly luciferase as a naked DNA[J]. Vaccine,2006,24:3057
[138]Pianko S, Patella S, Ostapowicz G, et al. Fas-mediated hepatocyte apoptosis is increased by hepatitis C virus infection and alcohol consumption, and may be associated with hepatic fibrosis:mechanisms of liver cell injury in chronic hepatitis C virus infection[J]. J Viral Hepat 2001,8:406-413.
[139]Harmeet Malhi, Gregory J. Gores and John J. Lemasters. Apoptosis and Necrosis in the Liver:A Tale of Two Deaths?[J]. HEPATOLOGY 2006,43(2):31-44.
[140]Fu QX, Wang LC, Zhan LS, et al. Screening compounds against HCV based on MAVS/IFN-β pathway in a replicon model[J]. World J Gastroenterol,2010, 16(44):5582-7.
[141]田粉梅.持续表达HCV全基因组小鼠模型的建立[D][硕士学位论文].北京：中国人民解放军军事医学科学院,2008.
[142]Picazo F., Domingo B., Franco S., et al. Fluorescence resonance energy transfer-based assay for characterization of Hepatitis C Virus NS3-4A protease activity in live cells[J]. Antimicrob Agents Chemother.2009,53:728-734.
[143]王志强.报告基因标记的HCV复制/感染模型的建立[D][硕士学位论文].北京：中国人民解放军军事医学科学院,2009.
[144]樊炜.报告基因标记的HCV长期表达小鼠模型的优化及在疫苗评价中的应用 [D][硕士学位论文].北京：中国人民解放军军事医学科学院,2011.
[145]高博.荧光素酶标记的HCV细胞模型的建立及其在药物评价等方面的应用[D][博士学位论文].北京：中国人民解放军军事医学科学院,2011.
[146]孙志东.HCV IRES小鼠体内长期表达模型的建立及其在药物评价中的应用[D][博士学位论文].北京：中国人民解放军军事医学科学院,2007.
[147]闫虎.树突状细胞的标记及活体荧光成像示踪其在体内的分布归巢[D][硕士学位论文].北京：中国人民解放军军事医学科学院,2011.
[148]Laxman B, Hall DE, Rehemtulla A, et al. Noninvasive real-time imaging of apoptosis[J]. Proc Natl Acad Sci USA 2002 99(26):16551-16555.
[149]Fan F., Wood K., Engineered luciferases for molecular sensing in living cells[J]. Curr Opion Biotechnol.2009,20:14-18.
[150]Zhang N, Weber A, Li B, et al. An inducible nitricoxide synthase-luciferase reporter system for in vivo testing of anti-inflammatory compounds in transgenic mice[J]. J Immunol,2003,170:6307
[151]Juan Du, Fang Zhao, Lin-sheng Zhan, et al. Bioluminescence Imaging allows monitoring hepatitis C virus core protein inhibitors in mice. Plos ONE[J].,2010, 5(11).
[152]Licui Wang, Qiuxia Fu, Linsheng Zhan, et al. Bioluminescence imaging of Hepatitis C virus NS3/4A serine protease activity in cells and living animals[J]. Antiviral Research 2010 87:50-56.
[153]Deroose CM, Reumers V, Gijsbers R, et al. Noninvasive monitoring of long-term lentiviral vector-mediated gene expression in rodent brain with bioluminescence imaging[J]. Mol Ther,2006,14(3):423
[154]Du J, Zhao F, Zhan LS, et al. Bioluminescence Imaging allows monitoring hepatitis C virus core protein inhibitors in mice[J]. Plos One,2010,5(11):e14043.
[155]Tseng J C, Levin B, Hurtado A, et al. Systemic tumor targeting and killing by sindbisviral vectors[J]. Nat Biotechnol,2004,22:70
[156]Sato M, Johnson M, Zhang L Q, et al. Optimization of adenoviral vectors to direct highly amplified prostate-specific expression for imaging and gene therapy[J]. Mol Ther,2003,8(5):726
[157]Gao X, Cui Y, Levenson RM, et al. In vivo cancer targeting and imaging with semiconductor quantum dots[J]. Nat Biotechnol,2004,22(8):969
[158]Wang LC, Fu QX, Zhan LS, et al. Bioluminescence imaging of Hepatitis C virus NS3/4 serine protease activity in cells and living animals[J]. Antiviral Research, 2010,8(7):50-56.
[159]Khalid Shah, Yi Tang, Xandra Breakefield, et al. Real-time imaging of TRAIL-induced apoptosis of glioma tumors in vivo[J].Oncogene 2003,22:6865-6872.
[160]Steinmann E., Wedemeyer H., Manns M. P., et al. Cyclosporine A inhibits Hepatitis C Virus nonstructural protein 2 through cyclophilin A[J]. Hepatology.2009,50: 1638-1645.
[161]Hosseinzadeh L, Behravan J, Mosaffa F, et al. Curcumin potentiates doxorubicin-induced apoptosis in H9c2 cardiac muscle cells through generation of reactive oxygen species[J]. Food Chem Toxicol.2011,49(5):1102-9
[162]Halestrap AP, Clarke SJ, Javadov SA. Mitochondrial permeability transition pore opening during myocardial reperfusion-a target for cardioprotection[J]. Cardiovasc Res 2004,61:372-385.
[163]Baines CP, Kaiser RA, Purcell NH, et al. Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death[J]. Nature 2005, 434:658-662.
[164]Reinehr R, Becker S, Keitel V, et al. Bile salt-induced apoptosis involves NADPH oxidase isoform activation[J]. Gastroenterology 2005,129:2009-2031.
[165]Deng T, Zhang Y. Possible involvement of activation of P53/P21 and demethylation of RUNX 3 in the cytotoxicity against Lovo cells induced by 5-Aza-2'-deoxycytidine[J]. Life Sci.2009,27;84(9-10):311-20.
[166]Deng T, Zhang Y.5-Aza-2'-deoxycytidine reactivates expression of RUNX3 by deletion of DNA methyltransferases leading to caspase independent apoptosis in colorectal cancer Lovo cells[J]. Biomed Pharmacother.2009,63(7):492-500.
[167]Zender L, Hutker S, Liedtke C, et al. Caspase-8 small interfering RNA prvents acute liver failure in mice[J]. PNAS,2003,100(13):7797-7802
[168]Walter J. Wurzer, Oliver Planz, Christina Ehrhardt. Caspase 3 activation is essential for efficient influenza virus propagation[J]. The EMBO Journal,2003,22(11):2717-2728
[169]W ang J, liw, M i n J, et a 1. Fas sirna reduces apoptotic cell death of al logeneic transplanted hepatocytes in mouse spleenl[J]. Transplant Proc,2003,35(4):1594-51
[170]Song E, Lee SK, Wang J, et al. RNA interferencet argeting Fas protects mice from fulminant hepatitis[J]. Nat Med,2003,9:347-511
[171]Eszter Kapusi, Katja Kempe, Myroslava Rubtsova, et al. Phic31 Integrase-Mediated Site-Specific Recombination in Barley[J]. plos One.2012,7(9):e45353.
[172]Kittiwongwattana C, Lutz K, Clark M, et al. Plastid marker gene excision by the phic31 phage site-specific recombinase[J]. Plant Mol Biol,2007,64:137-143.
[173]Thomason LC, Calendar R, Ow DW. Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage phic31 site-specific recombination system[J]. Mol Genet Genomics,2001,265:1031-1038.
[174]Allen BG, Weeks DL. Transgenic Xenopus laevis embryos can be generated using phic31 integrase[J]. Nat Methods,2005,2:975-979.
[175]Liu F, Song Y, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA[J]. Gene Ther.1999,6:1258-1266.
[176]Zhang G, Gao X, Song YK, et al. Hydroporation as the mechanism of hydrodynamic delivery[J]. Gene Ther.2004,11:675-682.
[177]Suda T, Gao X, Stolz DB, et al. Structural impact of hydrodynamic injection on mouse liver[J]. Gene Ther.2007,14:129-137.
[178]Kobayashi N, Nishikawa M, Hirata K, et al. Hydrodynamics-based procedure involves transient hyperpermeability in the hepatic cellular membrane.implication of a nonspecific process in efficient intracellular gene delivery[J]. J. Gene Med. 2004,6:584-592.
[179]Maruyama H, Higuchi N, Nishikawa Y, et al. High-level expression of naked DNA delivered to rat liver via tail vein injection[J]. J. Gene Med.2002,4:333-341.
[180]Eastman SJ, Baskin KM, Hodges BL, et al. Development of catheter-based procedures for transducing the isolated rabbit liver with plasmid DNA[J]. Hum. Gene Ther.2002;13:2065-2077.
[181]Dooley CT, Dore TM, Hanson GT et al. Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators. J Biol Chem.2004, 279:22284-93.
[182]Kanno A, Yamanaka Y, Hirano H et al. Cyclic luciferase for real-time sensing of caspase-3 activities in living mammals[J]. Angew Chem Int Ed.2007,46:7595-9.
[183]Zhu L, Xie J, Swierczewska M et al. Real-time video imaging of protease expression in vivo[J]. Theranostics.2011,1:18-27.
[184]Lexman B, Hall DE, Bhojani MS et al. Noninvasive real-time imaging of apoptosis[J]. PNAS.2002,99:16551-5.
[185]Jiequn Wu, Qinglin Zhang, Wei Deng, et al.Toward Improvement of Erythromycin A Production in an Industrial Saccharopolyspora erythraea Strain via Facilitation of Genetic Manipulation with an Artificial attb Site for Specific Recombination[J]. Appl Environ Microbiol.2011,77(21):7508-7516.
[186]Oliynyk M. Complete genome sequence of the erythromycin-producing bacterium Saccharopolyspora erythraea NRRL23338[J]. Nat. Biotechnol.2007,25:447-453.
[187]Rodriguez E. Rapid engineering of polyketide overproduction by gene transfer to industrially optimized strains[J]. J. Ind. Microbiol. Biotechnol.2003 30:480-488.
[188]Lister JA. Chapter 11-Use of Phage phic31 Integrase as a Tool for Zebrafish Genome Manipulation J]. Methods Cell Biol.2011,104:195-208.
[189]Karow M, Calos MP. The therapeutic potential of phic31 integrase as a gene therapy system[J]. Expert Opin Biol Ther.2011,11(10):1287-96. Epub 2011 Jul 8.
[190]Ginsburg DS, Calos MP. Site-specific integration with phic31 integrase for prolonged expression of therapeutic genes. Adv Genet.2005,54:179-87
[191]Sung CM, Yeh CT, Shiau SS, Hydrodynamics-Based Transfection of the Combination of Betacellulin and Neurogenic Differentiation 1 DNA Ameliorates Hyperglycemia in Mice with Streptozotocin-Induced Diabetes[J]. Diabetes Technol Ther.2011,3(5):519-25
[192]Tsung-Hsing chen, chau-Ting YEH, Yu-Pin HO, et al. Hydrodynamics-based Transfection of Pancreatic Duodenal Homeobox 1 DNA Improves Hyperglycemia and is Associated with Limited Complications in Diabetic Mice[J]. Endocrine Journal 2009,56(6),783-790
[193]Changml, Chen JC, Yeh CT, et al. Gene gun Bombardment with dna-Coated gold Particles is a Potential alternative to Hydrodynamics-Based Transfection for delivering genes into superficial Hepatocytes[J]. Hum Gene Ther,2008,19:391-5.
[194]Huang LR, Wu HL, Chen PJ, at al. An immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection[J]. PNAS,2006,103: 17862-7.
[195]Allen BG, Weeks DL. Using phic31 integrase to make transgenic Xenopus laevis embryos[J]. Nat Protoc,2006,1:1248-1257.
[196]Bateman JR, Lee AM, Wu CT. Site-specific transformation of Drosophila via phic31 integrase-mediated cassette exchange[J]. Genetics,2006,173:769-777.
[197]Venken KJ, He Y, Hoskins RA, et al. P[acman]:a BAC transgenic platform for targeted insertion of large DNA fragments in D. Melanogaster[J]. Science,2006, 314:1747-1751.
[198]Zhang G, Budker V, Wolff JA. High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA[J]. Hum. Gene Ther. 1999,10:1735-1737.
[199]Hodges BL, Scheule RK. Hydrodynamic delivery of DNA[J]. Expert Opin. Biol. Ther.2003,3:911-918.
[200]. Hagstrom JE, Hegge J, Zhang G, et al. A facile nonviral method for delivering genes and sirnas to skeletal muscle of mammalian limbs[J]. Mol. Ther.2004, 10:386-398.
[201]Zhang G, Budker V, Williams P, et al. Efficient expression of naked DNA delivered intraarterially to limb muscles of nonhuman primates [J]. Hum. Gene Ther.2001, 12:427-438.
[202]mccaffrey AP, Ohashi K, Meuse L, et al. Determinants of hepatitis C translational initiation in vitro, in cultured cells and mice[J]. Mol. Ther.2002,5:676-684.
[203]Zhang G, Song YK, Liu D. Long-term expression of human alphal-antitrypsin gene in mouse liver achieved by intravenous administration of plasmid DNA using a hydrodynamics-based procedure[J]. Gene Ther.2000,7:1344-1349.
[204]Ozaki M, Haga S, Ozawa T. Theranostics. In Vivo Monitoring of Liver Damage Using Caspase-3 Probe [J]. Theranostics,2012,2(2):207-14.
[205]Ray P, De A, Patel M, Gambhir SS. Monitoring caspase-3 activation with a multimodality imaging sensor in living subjects[J]. Clin Cancer Res.2008,14: 5801-9.
[206]Yhee JY, Kim SA, Koo H et al. Optical imaging of cancer-related proteases using near-infrared fluorescence matrix metallopro-teinase-sensitive and cathepsin B-sensitive probes[J]. Theranostics.2012,2:179-89.
[207]Kim GB, Kim Y.-P. Analysis of protease activity using quantum dots[J]. Theranostics.2012;2:127-38.
[208]Jiang N, Zhang X, Zheng X, A Novel In Vivo sirna Delivery System Specifically Targeting Liver Cells for Protection of cona-Induced Fulminant Hepatitis[J]. plos One.2012,7(9):e44138.
[209]Zheng X, Lian D, Wong A, Bygrave M, Ichim TE, et al. (2009)Novel small interfering RNA-containing solution protecting donor organs in heart transplanta-tion[J]. Circulation 120:1099-1107
[210]Gabizon A, Horowitz AT, Goren D, et al. Targeting folate receptor with folate linked to extremities of poly(ethylene glycol)-grafted liposomes:in vitro studies[J]. Bioconjugate Chem,2009,10:289-298.
[211]Takeda K, Hayakawa Y, Van Kaer L, et al. OCritical contribution of liver natural killer T cells to a murine model of hepatitis[J].2000,97:5498-5503
[212]Li WW, Yu JY, Xu HL, et al. Concanavalin A:a potential anti-neoplastic agent targeting apoptosis, autophagy and anti-angiogenesis for cancer therapeutics. [J]. Biochem Biophys Res Commun.2011,22,414(2):282-6
[213]Li CY, Xu HL, Liu B, Concanavalin A, from an old protein to novel candidate anti-neoplastic drug[J]. Curr Mol Pharmacol.2010,3(3):123-8.
[214]Chih-Peng Chang, Ming-Cheng Yang, Hsiao-Sheng Liu, et al. Concanavalin A induces autophagy in hepatoma cells and has a therapeutic effect in a murine in situ hepatoma model[J]. Hepatology,2007,45(2):286-296.
[215]Caserta TM, Smith AN, Gultice AD. Q-VD-oph, a broad spectrum caspase inhibitor with potent antiapoptotic properties [J]. Apoptosis.2003,8(4):345-52
[216]Sass DA, Shakil AO. Fulminant hepatic failure[J]. Liver Transpl,2005,11:594-605.
[217]Agrawal N, Dasaradhi PV, Mohmmed A, et al. RNA interference:biology, mechanism, and applications[J]. Microbiol Mol Biol Rev,2003,67:657-685.
[218]Kawakami S, Hashida M. Targeted delivery systems of small interfering RNA by systemic administration[J]. Drug Metab Pharmacokinet,2007,22:142-151.
[219]Akhtar S, Benter I. Toxicogenomics of non-viral drug delivery systems for rnai potential impact on sirna-mediated gene silencing activity and specificity [J]. Adv Drug Deliv Rev,2007,59:164-182.
[220]Ryther RC, Flynt AS, Phillips JA 3rd, et al. Sirna therapeutics:big potential from small rnas[J]. Gene Ther,2005,12:5-11.
[221]Scherr M, Battmer K, Dallmann I, et al. Inhibition of GM-CSF receptor function by stable RNA interference in a NOD/SCID mouse hematopoietic stem cell transplantation model[J]. Oligonucleotides,2003,13:353-363.
[222]Song E, Lee SK, Wang J, et al. RNA interference targeting Fas protects mice from fulminant hepatitis[J]. Nat Med,2003,9:347-351.
[223]Yano J, Hirabayashi K, Nakagawa S, et al. Antitumor activity of small interfering RNA/cationic liposome complex in mouse models of cancer[J]. Clin Cancer Res, 2004,10:7721-7726.
[224]Grzelinski M, Urban-Klein B, Martens T, et al. RNA interference-mediated gene silencing of pleiotrophin through polyethyle-nimine-complexed small interfering rnas in vivo exerts antitumoral effects in glioblastoma xenografts[J]. Hum Gene Ther,2006,17:751-766.
[225]Ying RS, Zhu C, Fan XG, et al. (2007)Hepatitis B virus is inhibited by RNA interference in cell culture and in mice[J]. Antiviral Res,2007,73:24-30.
[226]Kittiwongwattana C, Lutz K, Clark M, et al. Caspase 3 activation is essential for effcient influenza virus propagation[J].The EMBO Journal Vol.2003,22:2717-2728.
[1]Munger J, Hagglund R, Roizman B. Infected cell protein no.22 is subject to proteolytic cleavage by caspases activated by a mutant that induces apoptosis. Virology 2003,305:364-370.
[2]Barkett M, Dooher JE, Lemonnier L, et al. Three mutations in v-Rel render it resistant to cleavage by cell-death protease caspase-3. Biochim Biophys Acta 2001, 1526:25-36.
[3]Wang L, Zhi B, WuW, et al. Requirement for shrimp caspase in apoptosis against virus infection. Dev Comp Immunol 2008,32:706-715.
[4]Satoh S, Hirota M, Noguchi T, et al. Cleavage of hepatitis C virus nonstructural protein 5A by a caspase-like protease(s)in mammalian cells. Virology,2000;270: 476-487.
[5]Eleouet JF, Chilmonczyk S, Besnardeau L, et al. Transmissible gastroenteritis coronavirus induces programmed cell death in infected cells through a caspase-dependent pathway. J Virol 1998;72:4918-4924.
[6]Eleouet JF, Slee EA, Saurini F, et al. The viral nucleocapsid protein of transmissible gastroenteritis coronavirus(TGEV)is cleaved by caspase-6 and-7 during TGEV-induced apoptosis. J Virol 2000;74:3975-3983.
[7]Al-Molawi N, Beardmore VA, Carter MJ, et al. Caspase-mediated cleavage of the feline calicivirus capsid protein. J Gen Virol 2003;84:1237-1244.
[8]Roberts LO, Al-Molawi N, Carter MJ, et al. Apoptosis in cultured cells infected with feline calicivirus. Ann NY Acad Sci 2003;1010:587-590.
[9]Grand RJ, Schmeiser K, Gordon EM, et al. Caspase-mediated cleavage of adenovirus early region 1A proteins. Virology 2002;301:255-271.
[10]Majerciak V, Kruhlak M, Dagur PK, et al. Caspase-7 cleavage of Kaposi sarcoma-associated herpesvirus ORF57 confers a cellular function against viral lytic gene expression. J Biol Chem 2010;285:11297-11307.
[11]Cheng F, Chen AY, Best SM, et al. The capsid proteins of Aleutianmink disease virus activate caspases and are specifically cleaved during infection. J Virol 2010;84:2687-2696.
[12]Karlberg H, Tan YJ, Mirazimi A. Induction of caspase activation and cleavage of the viral nucleocapsid protein in different cell types during Crimean-Congo hemorrhagic fever virus infection. J Biol Chem 2011;286:3227-3234
[13]Moody CA, Fradet-Turcotte A, Archambault J, et al. Human papillomaviruses activate caspases upon epithelial differentiation to induce viral genome amplification. Proc Natl Acad Sci USA 2007; 104:19541-19546.
[14]Fei JW, de Villiers EM. Degradation of HPV20E6 by p53:delta Np63 alpha and mutant p53R248W protect the wild type p53 mediated caspase-degradation. Int J Cancer 2008; 123:108-116.
[15]Leu JH, Chen LL, Lin YR, Kou GH, Lo CF. Molecular mechanism of the interactions between white spot syndrome virus anti-apoptosis protein AAP-1 (WSSV449) and shrimp effector caspase. Dev Comp Immunol 2010;34:1068-1074.
[16]Best SM, Wolfinbarger JB, Bloom ME. Caspase activation is required for permissive replication of Aleutianmink disease parvovirus in vitro. Virology 2002;292:224-234.
[17]Zhou Q, Krebs JF, Snipas SJ, et al. Interaction of the baculovirus anti-apoptotic protein p35 with caspases. Specificity, kinetics, and characterization of the caspase/p35 complex. Biochemistry 1998;37:10757-10765.
[18]Fisher AJ, Cruz W, Zoog SJ, et al. Crystal structure of baculovirus P35:role of a novel reactive site loop in apoptotic caspase inhibition. EMBO J 1999; 18:2031-2039.
[19]Xu G, Cirilli M, Huang Y, et al. Covalent inhibition revealed by the crystal structure of the caspase-8/p35 complex. Nature 2001;410:494-497.
[20]Zoog SJ, Schiller JJ, Wetter JA, et al. Baculovirus apoptotic suppressor P49 is a substrate inhibitor of initiator caspases resistant to P35 in vivo. EMBO J 2002;21:5130-5140.
[21]Guy MP, Friesen PD. Reactive-site cleavage residues confer target specificity to baculovirus P49, a dimeric member of the P35 family of caspase inhibitors. J Virol 2008;82:7504-7514.