摘要
目的:本研究旨在探讨超声造影剂介导下增强标记有GFP报告基因的腺相关病毒载体(rAAV2-GFP)心肌内靶向转染的转染效率。本研究探讨经系统输入法,超声介导下rAAV2-GFP心肌转染的重组腺相关病毒的最佳滴度;探讨超声造影剂介导下增强rAAV2-GFP心肌靶向转染前后心功能的变化;探讨超声造影剂介导下rAAV2-GFP在心肌内的靶向转染效率。方法:将同种属同窝别同年龄雌雄不限SD大鼠,体重为250～350克,随机分为7组,每组3只,共21只。包括6个实验组和1个对照组。根据rAAV2-GFP滴度不同将实验组分为以下6组:1组滴度为1.5×109vg/ml;2组滴度为3.0×109vg/ml;3组滴度为1.5×1010vg/ml;4组滴度为3.0×1010vg/ml;5组滴度为1.5×1011vg/ml;6组滴度为3.0×1011vg/ml。苯巴比妥钠50mg/kg腹腔麻醉。各实验组通过尾静脉注入携带上述不同滴度rAAV2-GFP的SonoVue微泡造影剂1.2ml。对照组通过尾静脉注入1.2ml的SonoVue。首先使用GE Vivid 7 Dimension超声诊断仪所配备的i13L线阵探头,探头频率为10～14MHz,经左胸壁进行心脏超声心动图检查。仪器设置为低机械指数MI﹦0.4(mechanical index,MI)。当心肌组织内见微泡造影剂充盈时立即换成M3S探头,探头频率为2.0～3.5MHz,启用二次谐波功能,发射频率为1.3MHz,接收频率为2.6MHz并进入反向编码实时心肌造影模式,使机械指数调至1.02,利用FLASH(fast low-angle shot,FLASH)功能破坏心腔及心肌组织中的造影剂微泡,并进行心电触发,每三到六个心动周期触发一次,以达到定点爆破造影剂微泡的目的。每触发一次间隔2～5秒,以利于下一心动周期有足够的超声造影剂进入心肌组织内。14天后,麻药过量处死大鼠。取心脏组织做冰冻切片,切片厚度为5μm以下。荧光显微镜下观察心肌内荧光的表达量代表腺相关病毒载体的转染量;将同种属同窝别同年龄雌雄不限SD大鼠,体重250～350克,随机分为2组,每组10只,共20只。采用苯巴比妥钠50mg/kg腹腔麻醉。对照组给予单纯尾静脉注射超声造影剂SonoVue并于体表心脏部位进行超声照射破坏造影剂微泡(不携带rAAV2-GFP);实验组给予尾静脉注射携带有rAAV2-GFP的微泡造影剂SonoVue并于体表心脏部位进行超声照射。使用i13L线阵变频探头,探头频率为10～14MHz,仪器设置为低机械指数MI=0.4,行超声心动图实时监测,当超声微泡进入心肌组织内时,立即换成M3S探头,参数设置同前,爆破微泡至微泡完全消失,从而达到定点爆破造影剂微泡、定点释放rAAV2-GFP、促进腺相关病毒载体定点转染至心肌组织内的目的。分别于实验前及实验后14天由同一名有经验的超声心动图医师完成心功能数据的采集。分别采集大鼠二尖瓣水平短轴、乳头肌水平短轴及心尖短轴二维灰阶动态图(帧频为92～123frames/s)各3个心动周期,保持心率一致,储存于EchoPAC工作站。使用EchoPAC工作站做后处理分析。应用2DS分析软件,对每一动态图像分别于收缩末期手动勾画左室心内膜边界,软件自动生成感兴趣区(ROI),调整ROI宽度使其包纳心肌全层。运行程序后软件自动逐帧追踪ROI内心肌运动,并将室壁划分为6个节段,得出各节段及左室整体的应变、应变率、旋转角度、扭转角度、达峰时间等参数:乳头肌水平短轴切面测量左室周向应变(SC)、收缩期周向应变率(SrcSYS)、舒张早期周向应变率(SrcE)舒张晚期周向应变率(SrcA)。二尖瓣水平短轴及心尖水平短轴测量心底整体最大收缩期旋转角度(RotMV)、心尖整体最大收缩期旋转角度(RotAP)。逆时针方向旋转角度定义为正值,顺时针方向旋转角度定义为负值。左室扭转角度(LVtor)定义为:LVtor=RotMV-RotAP。对不同大鼠间心率差异进行时间校标,将主动脉瓣关闭时间点(AVC)设定为收缩期末,下一个心动周期的R波顶点设定为舒张期末,测量扭转峰值角度、达峰时间。采用二维及M型超声心动图分别记录心率(HR)、左室舒张末期容积(EDV)、左室收缩末期容积(ESV)、左室射血分数(EF)、短轴缩短率(FS);将同种属同窝别同年龄体重相近雌雄不限的SD大鼠随机分为6组,每组10只,共60只,体质量为250～350克。给予苯巴比妥钠50mg/kg腹腔麻醉。分别采用以下方法给予干预:第一组给予单纯经尾静脉注射超声造影剂SonoVue并同时进行体表心脏部位超声照射爆破微泡造影剂(不携带rAAV2-GFP);第二组给予单纯经鼠尾静脉注射rAAV2-GFP,但不进行超声照射;第三组给予尾静脉注射rAAV2-GFP并进行体表心脏部位超声照射爆破微泡造影剂;第四组先给予尾静脉注射超声造影剂后进行体表心脏部位超声照射爆破造影剂微泡,再给予尾静脉注射rAAV2-GFP;第五组只给予尾静脉注射携带rAAV2-GFP超声造影剂但不进行超声照射;第六组给予造影剂携带rAAV2-GFP尾静脉注射,当心肌内见微泡造影剂充盈时于体表心脏部位进行超声照射,爆破微泡造影剂。进行超声照射的所有实验组,首先用i13L线阵探头,探头频率为10～14MHz,仪器设置为低机械指数MI=0.4进行超声心动图实时观察。当心肌组织内可见造影剂微泡充盈时立即更换探头为M3S,参数设定同前,爆破心脏内的微泡造影剂,至心肌内微泡完全消失。14天后麻药过量处死动物,取心脏、肝脏、脑组织做冰冻切片荧光显微镜下进行荧光表达情况的观察。结果:各实验组中均见不同程度的荧光表达,而对照组中心肌内未见荧光表达。实验组中注入rAAV2-GFP的滴度为1.5×109vg/ml和3.0×109vg/ml组心肌内荧光表达量极少;注入rAAV2-GFP滴度为1.5×1010vg/ml和3.0×1010vg/ml组心肌内有荧光表达,但表达量亦较少;注入滴度为1.5×1011vg/ml组荧光的表达量较低滴度组有了大幅度的增加,近12倍,差异有统计学意义(P<0.01),且荧光亮度清晰可见,完全可以满足研究中对rAAV2-GFP转染情况的观察。虽然随着注入滴度增加心肌内荧光表达量亦有增加,注入rAAV2-GFP滴度为3.0×1011vg/ml组心肌内荧光的表达量更多,然而只比注入1.5×1011vg/ml滴度组增加了一倍,且增加了经济成本。因而认为经尾静脉注入1.5×1011vg/ml的rAAV2-GFP为超声介导下增强rAAV2-GFP心肌转染的最佳滴度;对照组与实验组大鼠的心率、左室舒张末期容积、左室收缩末期容积、左室射血分数、短轴缩短率、左室周向应变、收缩期周向应变率、舒张早期周向应变率、舒张晚期周向应变率、左室扭转角度,各项心功能指标处理前后比较均无明显变化,差异无统计学意义(P>0.05)。并且处理后对照组与实验组心功能的上述指标亦均无明显变化,差异无统计学意义(P>0.05);给予尾静脉注射携带rAAV2-GFP的超声微泡造影剂并进行体表心脏部位超声爆破微泡造影剂组,心肌内荧光的表达量较其它组明显增加,差异有统计学意义(P<0.01)。先给予尾静脉注射造影剂微泡再给予体外心脏部位超声照射,而后再给予rAAV2-GFP尾静脉注射组,心肌内荧光表达较单纯尾静脉注射rAAV2而不给予超声照射组明显增加,差异有统计学意义(P<0.01)。尾静脉注射rAAV2-GFP并同时进行超声照射组心肌内荧光的表达量较给予造影剂携带rAAV2-GFP注射而不给予超声照射组明显增加,差异有统计学意义(P<0.05)。只给予rAAV2-GFP不行超声照射组与给予携带rAAV2-GFP的微泡造影剂注射而不予超声照射组相比心肌内荧光表达量差异无统计学意义(P>0.05)。单纯给予尾静脉注射超声造影剂并超声爆破造影剂微泡组心肌内未见荧光表达。除只给予尾静脉注射超声造影剂并进行超声照射组,肝脏内未见荧光表达外,其他各组肝脏内均见荧光表达。但肝脏内荧光表达量各组间相比差异均无统计学意义(P>0.05)。而给予尾静脉注入携带rAAV2-GFP超声造影剂并进行超声照射组心肌内荧光表达量较肝脏中增多,差异有统计学意义(P<0.01)。其它各组中心肌内荧光的表达与肝脏中荧光的表达差异无统计学意义。脑组织冰冻切片内未见荧光表达。结论:超声及超声造影剂介导下同时在体表心脏部位进行定点爆破可以高效安全地促进腺相关病毒载体在心肌内的靶向转染。经尾静脉输入法超声介导下增强腺病毒相关病毒心肌靶向转染的腺病毒相关病毒的最佳滴度为1.5×1011vg/ml;超声介导下增强腺病毒相关病毒心肌靶向转染对大鼠左心功能无影响。该方法安全可靠;超声介导下微泡破裂法可以实现腺病毒相关病毒心肌靶向转染且可提高其在心肌内的转染效率。
Objective: The achievement of organ-specific delivery of gene/drug represents a major problem limiting the clinical application of retroviral vectors for gene therapy, whilst non-viral techniques have the disadvantage of lack of efficiency and longevity of gene expression. Ultrasound-targeted microbubble destruction (UTMD) has been newly developed for destructing the bubbles carrying drugs or genes, and thus for achieving the local release of these target molecules. The aim of this study was to evaluate the potential for achieving to the effective, local delivery of recombinant adeno-associated virus serotypes 2 (rAAV2) transgene into rat myocardium by UTMD. This study involves three parts. The aim of the first part is to study the best titer of rAAV2 delivered to myocardium by targeted ultrasound microbubbles and being infused into tail vein. The aim of the second part is to study the changes of left ventricular function before and after rAAV2-GFP targeted to myocardium by the ultrasound mediated microbubbles. The aim of the third part is to evaluate the potential for achieving to the effective, local delivery of recombinant adeno-associated virus serotype-2 (rAAV2) transgene into rat myocardium by UTMD. Methods: The methods of the fist part: Twenty one adult SD rats were divided into seven groups. SonoVue attached with different titers (1.5×109vg/mL ;3.0×109vg/mL;1.5×1010 vg/mL;3.0×1010vg/mL; 1.5×1011vg/mL; 3.0×1011vg/mL) of rAAV2-GFP was infused into the tail vein of rats, following ultrasound mediated microbubbles destruction, as experiment groups. Normal saline was infused into the tail vein of rats as the control group (without rAAV2). Rats were killed after 14 days and hearts were harvested. GFP protein expression which showed rAAV2 transfer was observed under fluorescence microscope in frozen section. The methods of second part: The rats were divided into 2 groups of 10 rats. Control group: Echocardiographic destruction of microbubbles without rAAV2-GFP. Experimental group: Echocardiographic destruction of microbubbles containing rAAV2-GFP. The mean value of Left ventricular rotation of rats was obtain in apical and base plane using speckle tracking imaging (STI). LV twist was defined as apical rotation relative to the base. Circumferential strain; systolic and diastolic circumferential strain rate E and A; torsion and time to peak were measured using STI, before and after 14 days of experiments. Rats were killed after 14 days and hearts were harvested. GFP protein expression which showed rAAV2 transfer was observed under fluorescence microscope in frozen section. The methods of the third part: Recombinant adenovirus vector marked with Green Fluorescent Protein (GFP) was attached to the surface of the SonoVue, sulphur hexafluoride-filled microbubbles. These bubbles were infused into the tail vein of rats with or without simultaneous echocardiography. Rats were divided into six groups of 10: Group 1 echocardiographic destruction of microbubbles without rAAV2-GFP; Group 2 infusion of rAAV2-GFP (no microbubbles) without echocardiography; Group 3 echocardiography during infusion of rAAV2-GFP (no microbubbles); Group 4: echocardiographic destruction of microbubbles followed by rAAV2-GFP infusion; Group 5: microbubbles containing rAAV2-GFP without echocardiography; Group 6: echocardiographic destruction of microbubbles containing rAAV2-GFP. Group 6 was the experimental target; the remaining groups were controls. We administered infusions (1.2 mL) over 5～8 minutes because of the risk of volume overload. Results: The results of the first part: When the titer of rAAV2 was 1.5×1011 vg/ml infused into the tail vein of rats there was much more GFP expression in myocardium than lower titers (P<0.01) However the 3.0×1011 vg/ml group also showed a large quantity of GFP expression, the GFP expression in the 1.5×1011 vg/ml group is clear and enough to observe the rAAV2 transfection rate. The results of the second part:There is no significant difference of left ventricular myocardial function of rats between before and after rAAV2-GFP targeted to myocardium by the ultrasound mediated microbubbles. The results of the third part: The hearts of all 10 rats in the experimental groups showed luciferase activity, indicating rAAV2-GFP expression. All of the treatment groups that received the virus showed less luciferase activity in the myocardium than the group of echocardiographic destruction of microbubbles containing rAAV2-GFP. This confirmed that destruction of the microbubbles containing the virus was responsible for the observed GFP expression in the myocardium of rats. The livers of all rats that received the virus showed extensive GFP activity, whereas none of the brains showed GFP activity. GFP expression was 2.2-fold higher in the group treated with echocardiographic destruction of microbubbles (group 6) containing rAAV2-GFP than in the controls (P, 0.001). The group that underwent microbubble destruction followed by an infusion of rAAV-GFP had a 1.6-fold increase in GFP expression, suggesting that disruption of the endothelial barrier is an important factor in viral transduction. There is significant difference of GFP activity only in group of echocardiographic destruction of microbubbles containing rAAV2-GFP, between liver and heart (P<0.01), however other groups showed no significant difference (P>0.05). Conclusion: Ultrasound-mediated destruction of SonoVue is a promising method for the delivery of rAAV2 to the heart in vivo. And it is safe invasive and convenient and efficient way for gene transfection. 1) The first part of conclusion is that 1.5×1011 vg/ml is the best titer of rAAV2-GFP delivered to myocardium by being infused into tail vein and ultrasound mediated microbubbles destruction. The second part of conclusion is that the left ventricular function between before and after the experiment had not significant difference. It means the method of enhancement of rAAV2 delivered to the myocardium by ultrasound mediated microbubbles destruction is safe and efficient. The third part of conclusion is that ultrasound-mediated destruction of microbubbles is a promising method for the delivery of rAAV2 to the heart in vivo.
引文
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