心血管疾病基因治

2020 4 6 1 心血管疾病基因治疗 新世纪的挑战CardiovascularGeneTherapy CenturyChallenge 齐向前上海第二医科大学仁济医院 2020 4 6 2 一 Introductions 20年来分子生物学取得了飞速的发展 基因 Gene 可作为一种 药物 直接或间接对基因损伤及其表达产物进行修复和补充 基因移植 Transfer 已经作为一种新的潜在的方法用来治疗和纠正某些心血管疾病 2020 4 6 3 心血管疾病的研究的微观化过程 基因治疗在人类疾病治疗上的地位和作用以及作为一种新兴的治疗手段和方法已被广大研究人员和临床医生所接受并实践 目前世界上有关心血管疾病基因治疗的实验和临床研究多达1200项 3000多例患者已经进行了基因治疗临床试验 2020 4 6 4 现就基因治疗某些心血管疾病的动物实验和初步临床观察结果做一简单介绍 2020 4 6 5 二 ChronicMyocardialIschemia 正常成人毛细血管细胞翻转率 心脏新生血管生成可以有两种形式 1 真性血管生成 Angiogenesis 即新生毛细血管形成 一种涉及细胞基质分解 细胞迁移 粘连 增生和管腔形成过程 2020 4 6 6 2 可利用性血管生成 RecapitulatedVasculogenesis 即原有的小动脉转变形成肌小动脉 或原有的侧枝循环血管进一步扩展 2020 4 6 7 血管内皮生长因子 VEGF 是特异性血管内皮细胞促分裂物 一种在线粒体上第7号外显子基因编码的蛋白质 糖蛋白 以同源二聚体的形式存在 在正常心脏发展中VEGF是主要血管生成蛋白 具有促进微静脉 小静脉通透性增加 血管内皮细胞分裂 增殖 细胞质钙聚集以及诱导血管生成的强大的促新生血管生成作用 2020 4 6 8 根据氨基酸数量的不同 VEGF A可有不同的分离形式 VEGF 121 145 165 189和206 其中VEGF121和165十分重要 VEGF蛋白的功能是与内皮细胞的特殊受体相互作用 产生格状物增生 积聚 形成管状结构的动静脉网络 2020 4 6 9 冠心病心肌缺血的基因治疗是将促血管生长因子表达基因导入心脏组织 在局部产生蛋白多肽而发挥作用 局部移植有直接器官组织内注射 或将载体溶液选择性地注射或灌注在靶器官的动脉内 缺血性冠心病的基因运载方式包括直接冠脉内注射 直接心肌内 经心外膜下或心内膜下 注射和心包腔内注射 灌注 2020 4 6 10 2020 4 6 11 2020 4 6 12 1998年 Sellke等报告8例严重冠心病患者 在冠脉搭桥术中无法搭桥的缺血心肌区域中埋植含有bFGF蛋白的藻酸肝素颗粒 在随访3个月中 其中7例无心绞痛发作 3例同位素心肌灌注显像在药物治疗区域心肌灌注增加 8例中无一例发生不良反应 2020 4 6 13 Losordo等报告5例药物治疗无效以及又不适合PTCA或冠脉搭桥的冠心病患者 经左前胸微切口将编码VEGF165的裸质粒 phVEGF165 DNA直接注入心肌内 DouglasW Losordo MDSt Elizabeth sMedicalCenter Boston MACirculation 1998 98 2800 2804 2020 4 6 14 手术10天后5例患者心绞痛症状明显减轻 其中2例在30天后症状完全消失 3例术后左室射血分数无改变 2例明显改善 术后60天多巴胺同位素心肌灌注显像显示所有病人心肌缺血明显改善 冠脉造影Rentrop分级明显增加 全部5例患者未发现任何毒付作用 2020 4 6 15 Figure3 SPECT sestamibiperfusionimaging Top Exampleofimprovementina fixed defect perfusionabnormalityonrestingimage Inpatient Pt 4 amoderateareaofdecreasedperfusionisseenintheinfero lateralwall arrow beforegenetherapy Aftergenetherapy perfusionisimproved Bottom Exampleofimprovementinanareaofischemia InPt 2 asmallzoneofdecreasedperfusionisseenintheinferiorwall arrow beforetreatment Aftertreatment thematchingscanshowsnoevidenceofthisperfusiondefectwhileazoneofischemiaontheanterior opposite wallpersists GeneTherapyforMyocardialAngiogenesis DouglasW Losordo MDSt Elizabeth sMedicalCenter Boston MACirculation 1998 98 2800 2804 2020 4 6 16 他们在随后的随机双盲安慰剂对照试验中 证实100 gbFGF对心绞痛有较好的治疗效果 Rosengant等报告21例在冠脉搭桥术中心肌内注射Ad GV VEGF121 10的30天随访I期临床观察结果 Circulation 1999 100 468 RosengartTK MD WeillMedicalCollegeofCornellUniversity NewYorkPresbyterianHospital NewYork NY GenVec Inc T M G Rockville Md andDivisionofBiostatistics M L L NorthShoreUniversityHospital Manhasset NY 2020 4 6 17 他们证实所有患者心绞痛症状明显改善 注射病毒载体区域的节段运动异常减轻 单纯 非冠脉搭桥 基因治疗的患者 6例 运动平板试验提示心肌缺血明显好转 在随后的6个月随访中 未发现任何与VEGF载体移植有关的全身或心血管系统的毒付作用 2020 4 6 18 Vale等报告另外一组13例VEGF临床治疗观察 经左前胸切口直接心肌内注射phVEGF165后 同位素心肌显像和经皮左室导管电机械标测均证实心肌灌注改善 PeterR Vale MD Circulation 2000 102 965 St Elizabeth sMedicalCenterandTuftsUniversitySchoolofMedicine Boston Mass 2020 4 6 19 Figure4 A NOGALVEMMperformedin48 year oldwoman NOGAimagesinrightanteriorobliqueprojectionbeforeGTxshowUpVmap topleft withnormalvoltagessuggestiveofviablemyocardium purple pink blue green andLLSmap topright withlargezoneofabnormalwallmotion red arrow representingelectromechanicaluncouplingthatsuggestsischemicorhibernatingmyocardiumpredominantlyinvolvingtheseptum UpVandLLSmapsinrightanteriorobliqueprojection60daysafterGTx bottomleftandright respectively demonstratealmostcompleteresolutionofischemiczone ischemicarea9 57cm2beforeGTxvs0 39cm2afterGTx ChangesinLLScorrespondtochangesobservedonSPECTscan panelB Redlinerepresentslongaxisthroughapex B PersantineSPECT sestamibimyocardialperfusionscanning Selectedshort axisandhorizontal axisstressandrestingimagestakenbeforeandafterphVEGF165GTxinsamepatientasshowninpanelA White yellowcolordepictsmaximaluptakeofradionuclide andreddepictsimpaireduptake Pre GTxscans top showfixedanteroapicaldefect arrowhead andpartiallyreversibleinferoseptaldefect arrow Post GTxscans bottom showcompletenormalizationofrestingperfusionwithsmallpersistentreversibleanteroapicaldefectafterpharmacologicalstress PeterR Vale MD Circulation 2000 102 965 2020 4 6 20 Figure5 A NOGALVEMMperformedin58 year oldman NOGAimagesinleftanteriorobliqueprojectionbeforeGTxshowUpVandLLSmapsusingsamecolorscaleasinFigure1A redzoneonLLSmap topright andpreservedviability purple pink blue green onUpVmap topleft constituteafocusofelectromechanicaluncouplingthatsuggestsischemicorhibernatingmyocardium arrow inlateralLVwall UpVandLLSmapsinsameprojectionconstructed60daysafterGTx bottomleftandright respectively disclosenear completeresolutionoflateralwallischemiczone 8 08cm2beforeGTxvs0 16cm2afterGTx correspondingtochangesobservedonSPECTscan panelB Verticalandhorizontalaxes x y andz arepresentedaswhitelines B PersantineSPECT sestamibimyocardialperfusionscanning Selectedshort axisandhorizontal axisstressandrestingimagesthatusedsamecolorscaleasinFigure1BweretakenbeforeandafterphVEGF165GTxinsamepatientasshowninpanelA Pre GTxscans top showlargepartiallyreversiblelateralwalldefect arrows andmoderatereversibleseptalwalldefect arrowheads Post GTxscans bottom showalmostcompletenormalizationofrestingperfusionandmarkedimprovementinstressimages 2020 4 6 21 Figure6 A NOGALVEMM UpVandLLSNOGAimages samecolorscaleasinFigure1A inanteroposteriorprojection topleftandright respectively of55 year oldmanbeforephVEGF165GTxshowinganareaofelectromechanicaluncouplingsuggestiveofischemicorhibernatingmyocardiumthatinvolvestheanteriorregion arrow SixtydaysafterGTx UpVandLLSimages bottomleftandright respectively showcompleteresolutionofischemia ischemicarea5 29cm2beforeGTxvs0 00cm2afterGTx thatcorrespondstochangesobservedonSPECTscan panelB Redlinerepresentslongaxisthroughapex Verticalandhorizontalaxes x y andz arepresentedaswhitelines B PersantineSPECT sestamibimyocardialperfusionscanning Selectedshort axisandvertical axisstressandrestingimages samecolorscaleasinFigure1B weretakenbeforeandafterphVEGF165GTxinsamepatientasshowninpanelA Pre GTxstressscans top showfixedanteriordefect arrowheads atrestandreversibleanteroseptaldefect arrow thatappearswithstress Post GTxscans bottom shownormalizationofrestingperfusion 2020 4 6 22 Figure7 NOGALVEMMperformedin61 yearoldman NOGAimagesinleftlateralprojectionbeforeGTxshowUpVandLLSmaps redzone whichdepictsabnormalwallmotion onLLSmap topright togetherwithpreservedviability purple pink blue green onUpVmap topleft constitutefocusofelectromechanicaluncouplingthatsuggestsischemicorhibernatingmyocardium arrowheads inposterolateralwall UpVandLLSmapsinsameprojection90daysafterGTx bottom disclosealmostcompleteresolutionofposterolateralischemiczone 11 8cm2beforeGTxversus1 1cm2afterGTx correspondingtochangesobservedonSPECTscan Figure4 CircularbrowniconsinlowerpanelsrepresentsitesofphVEGF 2injection Verticalandhorizontalaxes x y andz arepresentedbywhitelines Redlinerepresentslongaxisthroughtheapex Circulation 2000 102 965 2020 4 6 23 尽管上述冠心病基因治疗初步临床试验结果令人鼓舞 不过开胸手术创伤性大 限制了VEGF治疗的常规使用 2020 4 6 24 1999年 Henry等根据动物实验在冠脉内注射生长因子对慢性心肌缺血有良好的新生血管形成作用 采用静脉或冠脉内直接注射VEGF和安慰剂的方法治疗178例顽固性心绞痛患者 HenryTDJAmCollCardiol1999 33 348A 2020 4 6 25 术后随访60天无一例发现严重的毒付作用和不良反应 但患者的运动耐力 心绞痛分级和生活质量在试验组和对照组之间无差别 2020 4 6 26 晚近 Vale等报告经皮导管介入直接左室心内膜下心肌注射VEGF治疗6例慢性心肌缺血性冠心病获得成功 为基因移植在方法学上带来新的曙光 PeterR Vale MDCirculation 2001 103 2138CardiovascularResearch andCardiothoracicSurgery StElizabeth sMedicalCenter TuftsMedicalSchool Boston Mass 2020 4 6 27 他们在左室导管电机械标测系统 NOGAleftventricularelectromechanicalmapping 导引下用导管在缺血区域注射200 gphVEGF 2 治疗后最长随访者360天 心绞痛每周发作频率以及心肌耗氧量均显著下降 2020 4 6 28 心室电机械标测和同位素心肌显像心肌缺血均改善 该研究采用随机单盲安慰剂对照的方法 同时导管介入经心内膜下心肌注射共36例均未引起心率 血压改变 亦无持续性室性心律失常 心肌梗塞或心肌穿孔的证据 2020 4 6 29 Figure9 NOGALVEMMperformedina64 yearoldmanwhowasinitiallyrandomizedtocontrol NOGAimagesinleftanteriorobliqueprojectionbeforeGTxshowUpVandLLSmaps redzoneonLLSmap topright togetherwithpreservedviability blue green onUpVmap topleft constitutefocusofelectromechanicaluncouplingthatsuggestsischemicmyocardium arrows locatedpredominantlyposterolateralbutalsoanteroseptal arrowheads UpVandLLSmapsinsameprojection constructed90daysaftermockprocedure center disclosenochangeinposterolateralischemiczonewithextensiontoinferoapicalsurface UpVandLLSmapsinsameprojection 90daysaftercrossingovertoactiveagent bottom disclosealmostcompleteresolutionofischemia 22 9cm2beforeGTxversus2 3cm2afterGTx Browniconsinupperpanelsrepresentdatapointsinischemiczonewherecatheterwasplacedformockprocedure BrowniconsincenterpanelsrepresentsitesofphVEGF 2injection Verticalandhorizontalaxes x y andz arepresentedbywhitelines PeterR Vale MDCirculation 2001 103 2138 2020 4 6 30 Figure10 SelectedstressandrestimagesusingsamecolorscaleasFigure4 takenbeforeandaftercontrolassignmentandagainafterVEGF 2GTxinpatientshowninFigure3 Scanstakenbeforecontrol top showlargefixedlateralwalldefect arrow andpartiallyreversibleinferoseptalandanteroapicaldefects arrowheads Scanstakenaftermockprocedure center shownosignificantchangeinmyocardialperfusion By90daysaftercrossovertoVEGF 2GTx bottom lateralwallispartiallyreversibleandanteroapicalrestingperfusionhasnormalized Largeposterolateralscarpersists PeterR Vale MDCirculation 2001 103 2138 2020 4 6 31 Figure11 PersantineSPECT sestamibiscansrecordedbeforeandafterphVEGF 2GTxinsamepatientshowninFigure2 White yellowcolorde