超声联合微泡作用下即时产生非酶化一氧化氮的实验

背景:一氧化氮是体内重要的气体信号分子,近年来研究发现一氧化氮可以在无一氧化氮合成酶存在的条件下合成,即非酶化一氧化氮合成,而非酶化途径合成一氧化氮,可能是许多生物学效应的机制之一.目的:观察超声联合微泡是否可以实现或者增效一氧化氮非酶途径的产生.方法:将L-精氨酸和H2O2按照1:1,10:1,10:0.1,1:10的比例混合,应用频率为1 MHz,输出功率为0.5,1.0,1.5 W/cm2的超声分别持续辐射60 s,论证最优的L-精氨酸和H2O2的浓度比例以及最适的超声输出功率.确定最适浓度比和超声输出功率后,实验设立4组,分别进行超声联合微泡、单纯超声、微泡进行干预,空白对照组不进行干预,比较各组一氧化氮生成量.结果与结论:体外试验中,L-精氨酸和H2O2的浓度比例以10:1为最佳,选择1.5 W/cm2声强作为超声最优值.以此最适浓度比和超声输出功率干预,超声联合微泡组的一氧化氮生成量多于超声组(P < 0.01);超声组一氧化氮生成量优于空白对照组(P < 0.01);而微泡组的一氧化氮生成量与空白对照组没有差异.结果表明超声联合微泡可以增效一氧化氮的非酶合成.     

超声联合微泡增加内皮细胞中内皮型一氧化氮合酶和一氧化氮生成的体外实验

目的 评估诊断性超声联合微泡对内皮细胞中内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)及一氧化氮(nitric oxide,NO)生成的增强效应.方法 将正常培养的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)分为空白对照组(A组)、纯微泡组(B组)、单纯超声组(C组)和超声联合微泡组(D组).其中,D组按条件不同又分为:辐照时间不同组(1 min、5 min、10 min)、机械指数不同组(0.09、0.4、1.0)、微泡浓度不同组(5×10~8/ml、2.5×10~8/ml、1.25×10~8/ml).干预后即刻和干预后24 h分别在光镜下观察细胞形态结构,RT-PCR测细胞eNOS相对表达量,NO试剂盒测培养液中NO水平,并用统计学方法对上述指标进行组间比较.结果 D组中eNOS及NO含量明显较其他三组高;当参数选择为机械指数1.0、微泡浓度2.5×10~8/ml、辐照时间10 min时,D组中eNOS和NO的增加程度最明显;并且干预后即刻和干预后24 h各组细胞及细胞膜形态结构在光镜下均无显著变化.结论 超声联合微泡辐照能增加内皮细胞中eNOS和NO生成;给予的超声及微泡条件不同,其增强效果也不同.     

微泡增强超声空化联合血凝酶对兔肝局部微血管阻断作用的实验研究

目的探讨微泡增强超声空化联合血凝酶对兔肝局部微血管的损毁及阻断作用。方法 40只健康新西兰大白兔,随机分为生理盐水组、血凝酶组、微泡组和微泡联合血凝酶组(联合组)。各组均分别在实验前后行超声造影检查,使用专用图像处理系统分析各组实验前后造影的视觉图像及峰值灰阶值(GSV)的变化,并与病理改变进行对照。结果实验前后,生理盐水组和血凝酶组超声图像造影视觉改变及定量分析峰值GSV均无明显变化(P>0.05)。微泡组和联合组视觉观察有造影灌注缺损,联合组更为明显;定量分析微泡组峰值GSV从(62.94±6.19)降至(36.36±7.30)(P<0.05),联合组峰值GSV从(63.52±8.53)降至(22.91±5.39)(P<0.05)。实验后生理盐水组和血凝酶组病理无明显改变;微泡组偶见局灶性小片状出血;联合组可见明显的细胞变性和坏死,伴有微血栓形成。结论微泡增强超声空化联合血凝酶可以造成更明显的兔肝局部微血管的损毁及血流阻断。     

超声联合一氧化氮微泡介导间充质干细胞移植对心肌梗死大鼠心功能的影响

目的 探讨超声联合一氧化氮(NO)微泡介导间充质干细胞(MSCs)移植对心肌梗死大鼠心功能的影响,并探讨其可能作用机制.方法 选用冠状动脉左前降支结扎法制作心肌梗死模型大鼠28只,采用随机数字表法将其分为对照组(经尾静脉注入磷酸盐缓冲液)、MSCs组(经尾静脉注入MSCs)、普通微泡组(经尾静脉注入普通微泡,同时给予超声干预,然后经尾静脉注入MSCs)及NO微泡组(经尾静脉注入NO微泡,同时给予超声干预,然后经尾静脉注入MSCs),每组7只.各组大鼠分别经治疗4周后行M型心功能彩超检查,计数比较各组大鼠缺血心肌局部平均毛细血管密度,采用蛋白免疫印迹法及实时PCR检测各组大鼠心肌局部血管内皮生长因子(VEGF)表达.结果 治疗4周后发现NO微泡组射血分数[(56.27±3.66)％]较普通微泡组[(51.31±3.22)％]、MSCs组[(45.81±3.37)％]及对照组[(43.66±4.79)％]均显著提高(P＜0.05);NO微泡组心肌缺血区域平均毛细血管密度[(45.96±9.01)个/每高倍镜视野]较普通微泡组[(28.07±4.93)个/每高倍镜视野]、MSCs组[(21.41 ±5.27)个/每高倍镜视野]及对照组[(18.04±4.82)个/每高倍镜视野]均显著增加(P＜0.05).NO微泡组VEGF相对表达量(0.67 ±0.024)较普通微泡组(0.54 ±0.011)、MSCs组(0.44±0.020)及对照组(0.12±0.009)均显著增强(P＜0.05).结论 超声联合NO微泡介导MSCs移植治疗心肌梗死大鼠,能进一步提高模型大鼠心功能,其治疗机制可能与促进局部VEGF表达、加速梗死区域血管生成有关.     

超声微泡促进肿瘤细胞报告基因转染

目的探讨超声破坏微泡造影剂后对β-半乳糖苷酶报告基因在人肝癌细胞(QGY)中转染的影响。方法将培养QGY细胞转于24孔培养板后,分为5组,分别为单纯加入质粒组(D);质粒 微泡组(D M);质粒 超声组(D U);质粒 超声 微泡组(D U M);脂质体 质粒组(L D);进行β-半乳糖苷酶质粒DNA的转染。2d后,进行β-半乳糖苷酶染色,测定各组细胞转染率。结果单纯质粒组细胞转染率为4．92％;质粒 微泡组转染率为6．742％;质粒 超声组转染率为29．73％;质粒 超声 微泡组转染率为50．88％;脂质体 质粒组转染率为12．15％;结论超声破坏微泡造影剂可促进报告基因β-半乳糖苷酶质粒在QGY细胞的转染,为肿瘤基因治疗提供一种新型基因转移系统。     

长脉冲超声联合脂质体微泡溶解微血栓的体外实验研究

目的评价长脉冲超声联合脂质体微泡造影剂溶解微血栓的疗效。方法构建评价溶栓效能的体外循环系统,通过造成200~300μm的微血栓阻塞于滤网来模仿体内的微循环栓塞。然后治疗组给予脂质体微泡加超声治疗15min,超声频率1MHz、声压1.5MPa、脉冲间隔3s,包括100、1 000、5 000 3种周期,对照组给予超声照射但无声学微泡。结果治疗组溶栓的效果随着脉冲的延长而增加,且对应的惯性空化效应强度也随之增加。1 000和5 000周期组的溶栓率显著高于单纯超声组(P均<0.01)。结论长脉冲超声联合脂质体微泡能实现良好的溶栓效果,且在一定范围内溶栓的效果随着脉冲的延长而增加。惯性空化可能是长脉冲超声介导微泡溶栓的重要机制之一。     

聚焦超声联合微泡开放血脑屏障的研究进展

血脑屏障可以阻止有害物质进入脑内,同时阻碍了药物治疗颅内疾病。如何安全、可逆地开放血脑屏障成为研究重点。聚焦超声联合微泡技术开放血脑屏障可能是基于超声的空化效应和声辐射力作用,其开放程度可在MRI的监控下准确评估,且微泡作为载体可运载辅助药物治疗颅内疾病,已在大量动物实验中获得初步成效。该技术具有靶向、无辐射和无毒性、不损害大脑组织及可重复性等优点,具有很大的发展前景。     

诊断超声联合微泡对肿瘤微循环的作用

目的 探讨高机械指数诊断超声联合微泡对大鼠Walker-256肿瘤微循环的作用。 方法 将29只皮下荷Walker-256肿瘤SD大鼠随机分为超声微泡组(n=15)、单纯超声组(n=7)和假照组(n=7):对超声微泡组采用声辐射力脉冲(ARFI)成像模式下诊断超声连续激励20次辐照肿瘤,同时经尾静脉推注微泡0.04 ml;对单纯超声组在行超声辐照的同时以等量生理盐水代替微泡;对假照组则采用假照方式,仅推注等量微泡溶液,但不发射超声能量。对所有动物于辐照前、辐照后即刻、10、20 min行CEUS检查。最后每组随机选取3只动物获取肿瘤组织标本,行病理学检查。 结果 辐照后即刻,超声微泡组辐照区几乎无造影剂充填,呈负性显影,肿瘤区平均造影峰值强度(PI)由25.17%减低到12.01%(P<0.01);单纯超声组及假照组辐照后即刻可见造影剂快速充填,灌注良好(P>0.05)。10 min后,超声微泡组造影可见血流逐渐恢复,但PI仍降低;20 min后肿瘤血流基本完全恢复,呈高灌注(P>0.05)。 结论 高机械指数诊断超声联合微泡能特异性地暂时降低大鼠Walker-256皮下移植瘤的微循环。     

靶向和非靶向微泡联合尿激酶超声溶栓的电镜表现

目的 比较靶向和非靶向微泡联合尿激酶超声溶栓的电镜表现。方法 将精氨酸-甘氨酸-天冬氨酸-丝氨酸片段（RGDS）与尿激酶（UK）以及超声微泡（SonoVue）通过机械振荡法,制备成靶向微泡。于30只新西兰大白兔单侧股动脉制备在体混合性血栓,并分为单纯超声辐照组（US组）、超声辐照+非靶向微泡造影剂+UK组（US+M+UK组）、超声辐照+靶向微泡造影剂+UK组（US+RGDS+UK组）。通过超声及多普勒血流仪观察溶栓效果,然后对股动脉离体标本行HE染色,并观察电镜表现。结果 溶栓20 min后,与US组和US+M+UK组比较,US+RGDS+UK组血流量明显恢复（P均<0.05）,US组与US+M+UK组比较差异无统计学意义（P均>0.05）。US+M+UK组HE染色显示管腔内充满血栓,血小板梁呈颗粒状、不致密,扫描电镜示粗大束状的胶原纤维上疏松附着少量细小纤维蛋白丝,大部分断裂;透射电镜示血栓大部分溶解为空泡状,可见白细胞或血小板降解的碎片。US+RGDS+UK组HE染色显示血栓完全溶解;扫描电镜示血栓的纤维网状结构被破坏,纤维蛋白完全的溶解;透射电镜示血栓降解为高电子密度的颗粒。结论 血栓结构的空泡化、纤维蛋白网架结构完全崩解和纤维蛋白的完全溶解是靶向微泡和UK联合溶栓的主要电镜改变。     

超声靶向微泡破碎联合半乳糖聚乙烯亚胺介导凋亡素基因诱导肝癌细胞凋亡的研究

目的:探讨超声靶向微泡破碎(ultrasound targeted microbubble destruction,UTMD)联合半乳糖聚乙烯亚胺(PEI-Gal)介导凋亡素基因(VP3)诱导肝癌细胞HepG2凋亡的影响。方法:体外培养HepG2细胞,随机分为4组:(1)对照组;(2)PEI-Gal组;(3)PEI-Gal+超声组;(4)PEI-Gal+超声+微泡组。转染24h后荧光显微镜观察pEGFP-VP3在HepG2细胞中的表达,流式细胞仪检测转染效率,RT-PCR和Western blot分别检测VP3 mRNA和蛋白表达水平,AnnexinV-FITC/PI检测细胞凋亡率。结果:流式细胞仪、RT-PCR和Western blot分析表明,PEI-Gal+超声+微泡组的转染效率为(28.83±2.07)%、VP3 mRNA水平为0.92±0.02,蛋白水平为1.65±0.06,HepG2凋亡率为(37.40±2.12)%,均显著高于其他各组(均P<0.01)。结论:UTMD联合PEI-Gal可明显提高VP3基因转染HepG2细胞的效率及在HepG2细胞内的表达,增加HepG2细胞的凋亡率。     

超声爆破微泡介导内皮抑素基因抑制兔颈动脉粥样硬化的实验研究

目的 探讨利用超声爆破微泡介导内皮抑素(endostatin,ES)基因转染对兔颈动脉粥样硬化斑块内新生血管及斑块生长的抑制作用.方法 20只颈动脉粥样硬化模型兔随机分为三组:A组,微泡+超声;B组,对照质粒+微泡+超声;C组,ES质粒+微泡+超声.建模2周行超声爆破微泡介导基因转染,间隔3周时重复转染1次.建模14周时行超声及数字减影血管造影(DSA)检查,病理检测病变血管新生内膜、斑块内新生血管及ES表达情况.结果 超声显示A组和B组内膜明显增厚,斑块较大,管腔明显狭窄,收缩期峰值流速(PSV)增加,C组上述指标明显较A组和B组低(P＜0.05).病理检测C组内中膜厚度(IMT)、内膜厚度(IT)、内中膜厚度比(IT/MT)、内膜面积(IA)、内中膜面积比(IA/MA)及新生内膜狭窄率与A组和B组比较差异均有统计学意义(P＜0.05).C组颈动脉新生血管及血管内皮生长因子(VEGF)表达均较A组和B组低,内膜及胫前肌可见较多ES表达,而A组和B组无明显ES表达.结论 一定超声辐照条件下,超声爆破微泡介导ES基因转染可抑制兔颈动脉粥样硬化病变的发展,有可能为动脉粥样硬化性疾病的基因治疗提供一种安全有效的方法 .

Abstract：

Objective To explore the inhibition effect on angiogenesis and plaque growth of carotid atherosclerosis by transfection of endostatin gene using microbubbles combined with ultrasound exposure.Methods Twenty rabbit models of carotid atherosclerosis were randomly divided into 3 groups:group A,microbubble+ ultrasound; group B, control plasmid + microbubble + ultrsound; group C, ES plasmid +microbubble+ ultrasound. Two weeks after surgery, ultrasound/microbubble mediated gene transfer was performed,and it was performed once again three weeks after the first transfection. Ultrasonography and digital subtraction angiography(DSA) were performed at the time of 14 weeks. The carotid arteries were taken to detect the neointima and angiogenesis, and the expression of endostatin was detected using pathological means. Results The imagings of ultrasound showed that the intima in group A and B were thick significantly with larger plaques, and the lumen became stenosis with the peak systolic velocity increasing,however,in group C,the parameters mentioned above were significantly less than those of group A and B ( P＜0.05). Pathological results displayed that intima-media thickness (IMT), intima thickness (IT), intima thickness/media thickness (IT/MT), intima area (IA), intima area/media area (IA/MA) and neointimal stenosis rates were greater in group A and B, however, they were less in group C ( P＜0.05).The number of neovascularization and vascular endothelial growth factor(VEGF) expression in group A and B were more than those of group C. There was more endostatin positive expression in carotid arteries and anterior tibial muscles of group C, while there was nearly no expression in group A and B. Conclusions Under the conditioned ultrasonic irradiation, ultrasound/microbubble mediated endostatin gene transfection can inhibit the development of carotid atherosclerosis in rabbits, which might provide a safe and effective strategy for gene therapy of atherosclerotic disease in future.     

Granulocyte colony-stimulating factor facilitates the angiogenesis induced by ultrasonic microbubble destruction

Ultrasonic destruction of microbubbles (US/MB) in the microcirculation causes local inflammatory cell infiltration, which has been shown to induce angiogenesis. Granulocyte colony-stimulating factor (G-CSF), which mobilizes myelomonocytic cells from the bone marrow and enhances vascular endothelial growth factor (VEGF) release from these cells, has also been applied to therapeutic angiogenesis induction. In the present study, we sought to examine the potential of G-CSF pretreatment to enhance the angiogenic effect of US/MB. Ischemic hindlimbs in mice were treated with either a predetermined minimal effective dose (300 mug/kg) of G-CSF, US/MB alone or G-CSF pretreatment followed by US/MB at seven days after removal of the femoral artery. Ultrasonic destruction of microbubbles was performed as intermittent pulsed local insonation using a diagnostic ultrasound scanner at a peak negative pressure of 1.4 MPa after intravenous injection of perfluorocarbon microbubbles. At 21 days after the treatment, we quantified the surface vascularity using a grid method and the capillary density using an alkaline phosphatase stain. Relative to the capillary density in normal muscle, the capillary density in the treated limbs was restored to 74 +/- 13% by G-CSF alone and 90 +/- 20% by US/MB alone (p < 0.05 vs. both untreated and G-CSF alone), and further increased to 101 +/- 21% by G-CSF pretreatment. The collateral growth induced by the combination of G-CSF pretreatment and US/MB was 2.8- and 1.4-fold greater than the growth induced by G-CSF alone and US/MB alone, respectively (p < 0.05 for both). Thus, pretreatment with a single minimal effective dose of G-CSF can augment the angiogenic effect of US/MB.     

超声微泡造影剂实现肿瘤靶向性治疗研究进展

低功率超声辐照微泡栓塞肿瘤血管是治疗肿瘤的一种新方法,特异性抗体偶联微泡介导的药物或基因靶向运输在肿瘤治疗方面也显示出巨大潜力,具有广阔的应用前景。现对超声微泡造影剂介导肿瘤靶向性治疗的研究进展做一综述。     

Augmentation of cardiac protein delivery using ultrasound targeted microbubble destruction

Gas-filled microbubbles have become an important tool as ultrasonic contrast agents. We have previously shown that ultrasound-targeted microbubble destruction (UTMD) can direct plasmids to the heart. The aim of this study was to evaluate UTMD for protein delivery. Six different groups of rats received 1 microg of luciferase protein with varying protocols: (1) luciferase-loaded microbubbles and ultrasound; (2) luciferase only; (3) luciferase and ultrasound; (4) luciferase-loaded microbubbles; (5) unloaded microbubbles incubated with luciferase and ultrasound; (6) unloaded microbubbles with ultrasound followed by luciferase. Relative luminescence units per mg protein per s were determined in hearts and control organs. The rats that received ultrasound and luciferase-loaded bubbles showed a six-fold higher cardiac luciferase uptake compared with control groups that did not include bubbles. None of the other groups significantly augmented cardiac luciferase activity. We conclude that ultrasound-targeted microbubble destruction can substantially and noninvasively augment organ-specific delivery of proteins.     

微泡浓度对于超声辐照诱导生物效应的影响

目的 探讨全氟显微泡浓度对于超声辐照诱导体外培养的正常肝细胞"声致孔隙"效应、凋亡和坏死的影响.方法 应用超声辐照含不同量微泡的HL-7702细胞悬液.实验设空白对照组(无造影剂,无辐照)、超声辐照组(无造影剂,超声辐照)及不同微泡浓度组[微泡数/细胞数(B/C)值为1～200,辐照10 min].辐照后观察大分子荧光物质进入细胞情况以检测"声致孔隙"效应,检测细胞活力及凋亡.结果 与空白对照组及超声辐照组比较,B/C为5～200组荧光染色阳性率明显增加,差异有统计学意义(P＜0.05).与空白对照组及超声辐照组比较.分别为B/C 50、100及200组的细胞死亡率明显增加,差异有统计学意义(P＜0.05);超声辐照组与空白对照组比较,各项结果差异无统计学意义(P＞0.05).结论 一定浓度下诊断超声辐照国产造影剂全氟显可诱导正常肝细胞发生"声致孔隙"效应及细胞死亡.应用于诊断目的 时,B/C值尽可能在10以下;应用于基因转染时则选择B/C值50较为适宜,必要时可达100.     

超声击破微泡效应抑制兔VX_2肿瘤生长的实验研究

目的 探讨超声击破微泡效应抑制兔VX_2肿瘤生长的可行性.方法 21只移植有皮下VX_2肿瘤的新西兰大白兔,随机分为超声微泡治疗组、单纯超声组和假照组.经静脉注射脂质微泡的同时,脉冲式聚焦超声直接辐照肿瘤区域.单纯超声组和假照组分别用5 ml生理盐水和超声假照替代,每次10 min,每隔72 h治疗一次,采集各时间点肿瘤二维声像图和超声造影影像,测量肿瘤切面最大直径.结果 在30 d的实验周期内,微泡超声治疗组、单纯超声组和假照组的肿瘤平均直径分别从(1.1±0.1)cm、(1.2±0.1)cm、(1.2±0.1)cm生长至(2.1±0.5)cm、(3.0±0.9)cm、(3.4±0.7)cm,微泡超声治疗组肿瘤直径明显小于另两组.结论 超声击破微泡效应可阻断肿瘤微循环,有一定的抑制肿瘤生长作用.     

超声破坏微泡对大鼠皮肤创面愈合影响的实验研究

目的 探讨超声破坏微泡(声诺维)对大鼠皮肤创面愈合的影响.方法 96只SD大鼠建立皮肤创面模型后随机分成4组:超声破坏微泡组、单纯微泡组、单纯超声组和对照组.超声破坏微泡组经鼠尾静脉注射微泡造影剂0.5 ml,同时用声强度1 MHz、2.0 W/cm2超声辐照3 min单纯微泡组经鼠尾静脉注射微泡造影剂0.5 ml;单纯超声组经鼠尾静脉注射生理盐水0.5 ml,同样条件超声辐照3 min对照组经鼠尾静脉注射生理盐水0.5 ml.于创伤后第1、3、5、7、14、21 d利用HE染色和免疫组化方法 观察各组创面肉芽组织生长及血管内皮生长因子(VEGF)表达的情况.结果 HE染色:伤后第7 d,超声破坏微泡组肉芽组织明显比其他3组厚,新生毛细血管均垂直于创面生长,其他3组毛细血管排列紊乱.免疫组化观察VEGF表达变化:超声破坏微泡组在第3 d形成表达高峰,其他3组表达高峰在第5～7 d.结论 超声破坏微泡可以提高大鼠背部皮肤的创面愈合质量;新生肉芽组织成熟早,创面愈合加速.超声破坏微泡时产生的高温、高压及某些化学效应可以刺激内源性VEGF分泌,促进血管生成,从而促进创面愈合.

Abstract：

Objective To investigate the effect of microbubble(Sono Vue) destruction with ultrasound on wound healing in rats. Methods Total 96 SD rats were accepted one rounded whole-layer skin incision on back each other and randomly divided into four groups:microbubble destruction with ultrasound(US + MB),microbubble(MB), ultrasound(US) and control group. Rats in US + MB group were injected with 0.5 ml microbubble contrast agent via tail vein,and then ultrasound irradiated for 3 minutes immediately. MB group were injected with 0.5 ml microbubble contrast agent. US group were injected with 0.5 ml physiological saline,and then ultrasound irradiated for 3 minutes immediately under the same condition. Control group were injected with 0.5 ml physiological saline. Feed each rat in single cage. On day 1,3,5,7,14 and 21 after wound creation,the excised wound tissues were analyzed by histology and VEGF expression in wounds by immunohistochemistry. Results HE staining: On day 7, wounds of US + MB group displayed the most accumulation of granulation tissue and all new capillaries were perpendicular to the wound surface, but the new capillaries of other 3 groups were disordered. Immunohistochemical examination of VEGF expression:the peak expression appeared on day 3 in US + MB group, other 3 groups were on day 5 to day 7.Conclusions US + MB treatment could improve the quality of wound healing and granulation tissues were maturated earlier than MB, US treatment and control group, which could accelerate wound healing. High temperature,high pressure and some kind of chemistry effecs induced by microbubble destruction with ultrasound can stimulate the secretion of endogenous VEGF, which may be the mechanism of promoting angiogenesis and wound healing.