【作者】 段相国；

【导师】 丁剑冰； 詹林盛； 付秋霞；

【作者基本信息】 新疆医科大学 ， 劳动卫生与环境卫生学， 2012， 博士

【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。更多还原

【Abstract】 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. ConclusionOur 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.更多还原