诊断超声及低声压治疗超声对微泡的作用效果研究

目的 探讨低机械指数诊断超声及低声压治疗超声对造影剂微泡的作用效果.方法 低机械指数的诊断超声及不同声压下低能量治疗超声体外辐照造影剂微泡,通过获得的超声造影图像间接分析微泡总浓度的变化,探讨不同机械指数诊断超声及不同声压治疗超声下微泡发生稳定空化及惯性空化情况.结果 低机械指数(<0.2)诊断超声及低声压(<0.15...     

治疗性超声介导微泡破裂促进大鼠体内肌肉基因转染的参数优化实验研究

【摘要】 目的 探讨治疗性超声介导微泡破裂促进基因体内转染大鼠肌肉的最适合的转染条件。方法 在大鼠双侧胫前肌内局部注射微泡与增强型绿色荧光蛋白（EGFP）质粒混合物,应用不同输出功率、不同占空比及不同超声辐照时间的治疗性超声辐照胫前肌,分别用肌肉局部注射及静脉注射微泡和质粒联合超声辐照,根据荧光染色及免疫组化染色下EGFP表达结果判断转染效果,根据转染效果最好且HE染色下肌肉损伤最小选出最适合的超声辐照条件和最适合的注射方式。将大鼠分为4组:①超声辐照+微泡+质粒组,②微泡+质粒组,③超声辐照+质粒组,④单纯质粒组。选取最适辐照及注射方式后,将大鼠在超声辐照后5 d处死,荧光染色及免疫组化染色下观察肌肉EGFP表达情况,HE染色观察肌肉损伤情况。结果 在1 Mz治疗性超声辐照下,输出功率2 W/cm2,占空比20%,超声辐照3 min的最适合的辐照条件下,肌肉EGFP表达明显,且无明显损伤。肌肉局部注射较静脉注射更适合。荧光染色和免疫组化染色示4组中超声辐照+微泡+质粒组肌肉EGFP表达高于其余3组,微泡+质粒组高于其余2组（P<0.05）。HE染色下未见超声辐照及微泡造成的肌肉损伤。结论 在适合转染条件下,治疗性超声联合微泡能明显增强基因体内转染大鼠肌肉的效率,且不损伤肌肉细胞,可作为基因治疗的一种安全有效的非病毒性基因转染方法。     

超声辐照超声微泡介导siRNA 转染膀胱癌T24细胞的实验研究

 目的探讨超声辐照超声微泡介导siRNA 转染人膀胱癌T24 细胞的有效性。方法使用超声辐照超声微泡的方法介导FITC-siRNA转染人膀胱癌T24 细胞,通过荧光显微镜及流式细胞学方法观察siRNA的转染效率,使用MTT方法观察细胞活力。结果与各阴性对照组相比,超声辐照超声微泡的方法可以明显增加siRNA向T24 细胞中的转染效率。随着超声辐照强度的增加,siRNA的转染效率在一定范围内有所提高,同时也增加了对T24细胞毒性。与脂质体介导的siRNA体外转染相比,超声辐照超声微泡介导的siRNA 转染效率仍然较低。结论超声辐照超声微泡能够有效提高siRNA 转染膀胱癌细胞的效率,有可能为膀胱癌的基因靶向治疗提供新的转染手段。     

超声微泡介导内皮抑素基因转染人脐静脉内皮细胞的实验研究

目的：探讨超声微泡介导内皮抑素（endostatin,ES）基因对人脐静脉内皮细胞（human umbilical vein endothelial cells ,HUVECs）转染的效率及可行性。方法 HUVECs种于24孔板内,培养24 h后加入5μg pIRES2-EGFP-ES质粒及超声造影剂SonoVue微泡,启动超声照射进行转染,设定MI分别为0.7、1.0、1.5,辐照时间分别为10 s、30 s、60 s、120 s,微泡浓度分别为10％、20％、50％。转染24 h后观察绿色荧光蛋白表达,计数活细胞数,MTT检测细胞增殖抑制率。结果超声爆破微泡介导了内皮抑素基因转染HUVECs,并具有强度、时间及微泡浓度依赖关系,但随着强度、微泡浓度增加及辐照时间延长,细胞凋亡率增加、细胞活力下降,转染率随之下降,当超声强度MI1.5、辐照时间60 s、微泡浓度20％转染率相对最高。结论超声微泡可介导内皮抑素基因转染脐静脉内皮细胞,但转染受多因素影响,在相对最佳转染条件下可实现内皮抑素的高表达。     

低频脉冲超声联合微泡对在体微小血管作用的实验研究

目的 探讨低频脉冲超声联合微泡对微血管的渗出作用.方法 20只新西兰大白兔分为4组:空白组、 单纯微泡组、 单纯超声组、 超声微泡组,进行实验观察.用频率为1 MHz,声压2000 MPa的脉冲超声辐照兔肠系膜及肠壁血管,在荧光显微镜下观察辐照前后肠系膜及肠壁上微血管的损伤,并静脉注入伊文思蓝溶液,观察超声辐照后对伊文思蓝溶液的渗出.结果 空白组、 单纯微泡组、 单纯超声组在超声辐照后肠系膜及肠壁上微血管内血流通畅,注入伊文思蓝溶液后,微血管内呈蓝色染色,血管周围未见渗出;超声微泡组在超声辐照后微血管周围可见渗出,部分形成血肿.结论 低频脉冲超声联合微泡对微小血管管壁产生损伤作用,血管周围可见渗出,部分形成血肿.     

超声及超声微泡介导基因转染的安全性研究

目的:探讨超声及超声微泡介导基因转染的安全性。方法:将人脐静脉内皮细胞分别按超声辐照时长及所加入的微泡剂量分组,超声辐照后分别培养24h及48h,应用台盼蓝拒染法进行细胞计数,与对照组进行比较。超声辐照含有不同微泡剂量的细胞后,行扫描电镜观察细胞膜形态。结果:不同辐照时长组经超声辐照后培养24h,10min、15min、30min组细胞计数比对照组降低,差异有统计学差异(P<0.05);继续培养48h,各组与对照组比较无统计学差异。不同微泡剂量组经超声辐照5min后培养24h,200μl、500μl组细胞计数比对照组降低,差异有统计学差异(P<0.05);继续培养48h,500μl组细胞计数比对照组降低,差异有统计学差异(P<0.05)。扫描电镜可见超声辐照后细胞膜完整性变差,继续培养24h后有一定程度改善。结论:超声辐照+微泡对细胞增殖有一定的抑制作用,对细胞膜有一定程度的损害,但这些作用在超声辐照时长不超过5min及微泡剂量不超过100μl时是轻微、可逆的。超声及超声微泡介导基因转染是相对安全的。     

高频诊断超声联合微泡对肝血管通透性影响的实验研究

目的探讨高频诊断级超声联合微泡对肝血管通透性的影响。方法选择机械指数(mechanical index,MI)为1.2的高频诊断超声,并经实验大鼠尾静脉注射0.2ml/kg微泡造影剂,超声辐照大鼠肝脏时间分为1、3、5、7min。用伊文氏蓝(Evens Blue,EB)观察超声辐照后肝血管通透性的改变,透射电镜观察肝血管内皮细胞及肝细胞结构改变。结果高频诊断超声联合微泡辐照肝脏后,肝组织EB含量与对照组相比差异有统计学意义,透射电镜证实肝血管内皮细胞肿胀,连接中断明显。结论高频诊断超声联合微泡辐照肝脏时可增加肝脏血管通透性。     

携自杀基因微泡转染卵巢癌细胞株SKOV_3的超声参数及转染效率

目的:探究超声介导携Ⅰ型单纯疱疹病毒胸苷激酶(hsv1tk)自杀基因微泡转染SKOV3细胞的超声参数及转染效率.方法:超声在不同微泡浓度与辐照时间(8,15,30,60s间隔1s)组合下辐照SKOV3细胞,MTT法筛选最适辐照时间和微泡浓度.将实验分成6组分别进行以下处理:超声辐照组,脂质体+超声辐照组,脂质体+微泡+超声辐照组,微泡+超声辐照组,裸质粒组(阴性对照),脂质体组(阳性对照).用裸质粒及脂质体处理后分别转染pcDNA3.1-EGFP/hsv1tk质粒;采用荧光显微镜、流式细胞技术分别定性和定量检测各组转染效率;采用逆转录聚合酶链反应(RT-PCR)方法检测hsv1tk基因的表达.结果:MTT检测在超声强度0.5W/cm2,频率1MHz,微泡浓度0.56×1011个/L,辐照时间8s间隔1s条件下,超声辐照微泡转染对细胞活性无明显抑制.经此条件转染后荧光显微镜下可观察到脂质体+微泡+超声辐照组的绿色荧光强度最强;流式细胞技术检测表明,微泡+超声辐照组转染效率为(11.74±0.19)%,比超声辐照组(2.19±0.22)%高,而脂质体+微泡+超声辐照组(25.62±0.08)%均高于其他各组(P<...     

低声压超声调控微泡空化对大鼠Walker-256肿瘤血管效应的初步研究

目的 探讨低声压超声调控的微泡空化对大鼠Walker-256肿瘤产生的血管效应.方法 健康雄性SD大鼠26只,双侧大腿内侧细胞接种法移植Walker-256肿瘤52个,利用随机数字表法进行分组后,分别给予不同强度的治疗声压,即假照组(n=6)、200 kPa组(n=12)、400 kPa组(n=10)、600 kPa组(n=12)、800 kPa组(n=12).超声治疗占空比1％,治疗时间5 min.各不同声压治疗组中半数肿瘤在超声辐照同时经尾静脉注射0.1 mL脂质微泡.治疗前后分别进行超声造影,利用Adobe Photoshop软件对图像进行灰阶定量分析,得到肿瘤造影峰值平均灰阶值.治疗结束后获取肿瘤标本行病理检查.结果 假照组和各实验组在治疗前后的超声造影分析结果显示,肿瘤造影峰值平均灰阶值差异无统计学意义(P＞0.05),但病理检查发现600 kPa组肿瘤组织血浆外渗和轻度水肿,800 kPa组肿瘤组织明显出血、水肿.结论 低声压超声联合微泡对大鼠Walker-256肿瘤的血流灌注影响不大,但600 ～ 800 kPa声压可引起血管通透性的增加.     

新型纳米微泡超声造影剂的制备及超声显像研究

【目的】 制备可稳定显像的新型小粒径纳米级超声微泡造影剂,并评价其体内外超声显像效果。【方法】 通过薄膜-水化、声振-通气及多级分离等多步骤方法制备纳米级脂质微泡,并针对制备过程中的主要影响因素设计了正交试验,筛选出粒径小、重复性好的实验条件。通过光学显微镜及Zeta粒径仪检测纳米微泡大小形态、粒径分布,体外及动物体内超声显像检测其增强显像效果。【结果】 纳米脂质微泡呈圆形,分布均匀,无明显聚集,粒径范围238.6 ~ 340.7 nm,均值为 (276.0 ± 24.1)nm,Zata电位:(-14.0 ± 0.6)mV。体外超声检测在超声造影状态下能持续稳定显像,进入到二维灰阶状态时,瞬间破坏完全,显像效果消失,粒径变小。体内超声显像在造影状态下纳米微泡可明显增强大鼠肝脏、肾脏回声信号。【结论】 通过本研究方法成功制备了粒径小、可稳定显像的新型纳米级超声造影剂,在二维灰阶高机械指数状态下该纳米微泡造影剂可被瞬间破坏,这为进一步肿瘤组织血管外靶向显像及肿瘤局部药物释放研究奠定基础。     

评价超声破坏微泡联合骨髓间充质干细胞移植的靶向性

目的 评价超声破坏微泡联合骨髓间充质干细胞移植的靶向性。方法 Wister大鼠10只,离断右侧股动脉后7d,经颈静脉输入白蛋白微泡和骨髓间充质干细胞(MSCs,6×106/ml～7×106/ml)的混悬液2 ml的同时用参数为1.9 W/cm2的超声间断作用大鼠右侧缺血下肢骨骼肌180s.处理后第7d,取右侧缺血下肢骨骼肌及各重要脏器,用免疫组化的方法检测移植的MSCs。结果 免疫组化结果显示大鼠下肢缺血骨骼肌及脾脏均可见较多移植的MSCs,而在心脏、肝脏、肾脏组织未检测到移植的MSCs。结论 利用超声破坏微泡技术联合MSCs移植具有较好的靶向性,有希望成为一种介导干细胞移植的新方法。     

Ultrasound microbubble contrast agents for diagnostic and therapeutic applications: current status and future design

Ultrasound contrast agents are highly echogenic microbubbles with many unique properties. Microbubbles can basically improve the sensitivity of conventional ultrasound imaging to the microcirculation. The resonance of microbubbles in response to an incident ultrasound pulse results in nonlinear harmonic emission that serves as the signature of microbubbles in microbubble-specific imaging. Inertial cavitation and destruction of microbubbles can produce a strong mechanical stress enhancing the permeability of the surrounding tissues, and can further increase the extravasation of drugs from the blood into the cytoplasm or interstitium. Stable cavitation by high-frequency ultrasound can also mildly increase tissue permeability without causing any damage even at a high acoustic pressure. Microbubbles can carry drugs, release them upon ultrasound-mediated microbubble destruction, and simultaneously enhance vascular permeability to increase drug deposition in tissues. Various targeting ligands can be conjugated to the surface of microbubbles to attain ligand-directed and site-specific accumulation for targeted imaging. In addition to current developments in microbubble technology, this review introduces our studies of the applications of microbubble- specific imaging, ultrasound-aided drug delivery, and targeted imaging. These applications are promising but may require further improvement for clinical use.     

超声靶向破坏微泡技术介导小鼠肝癌细胞株JNK1基因表达、细胞迁移和侵袭抑制

目的: 探讨应用超声靶向破坏微泡 (UTMD) 技术介导小鼠肝癌细胞株JNK1基因的表达、细胞迁移和侵袭抑制的作用,阐明其作用机制。方法: 构建并筛选RNA干扰效果最好的短发夹RNA(shRNA)。将小鼠肝癌细胞株Hca-F分为正常Hca-F细胞组、shRNA质粒组、脂质体组、超声微泡结合超声辐照组及脂质体结合超声微泡加超声辐照组。采用倒置荧光显微镜观察各组细胞转染率,荧光定量PCR和Western blotting 法检测JNK1基因mRNA和蛋白表达水平,CCK-8法检测各组细胞的细胞活性,应用Transwell 实验检测各组细胞的体外迁移能力。结果: 脂质体结合超声微泡加超声辐照组细胞转染率高于shRNA质粒组、脂质体组和超声微泡结合超声辐照组(均P<0.05),脂质体组和超声微泡结合超声辐照组比较差异无统计学意义(P>0.05)。脂质体结合超声微泡加超声辐照组JNK1 mRNA和蛋白表达水平低于其他各组(P<0.05);脂质体结合超声微泡加超声辐照组细胞活性和平均穿膜细胞数均低于其他各组(P<0.05)。结论: UTMD技术结合脂质体转染法可以提高小鼠肝癌细胞株JNK1 shRNA的转染效率,增强其对基因表达、细胞活力、迁移和侵袭能力的抑制。     

灯盏花素松弛家兔离体回肠的作用

【目的】研究灯盏花素对家兔离体回肠管收缩反应的影响。【方法】在离体家兔回肠管上,观察不同浓度灯盏花素对各致痉剂引起肠平滑肌收缩的影响。【结果】灯盏花素对乙酰胆碱、氯化钾和氯化钡引起家兔离体回肠管收缩的抑制作用呈一定的剂量依赖性。【结论】灯盏花素对回肠管的抑制作用可能由胆碱受体介导,或直接作用于肠道平滑肌,具有钙拮抗作用。     

丘脑出血80例分析

目的探讨丘脑出血的临床特点及预后。方法回顾总结80例丘脑出血患者的临床表现与预后。结果本组80例中,既往有高血压病史68例,入院时高血压者70例,意识障碍38例,瞳孔变化34例,双眼垂直运动障碍22例,感觉障碍66例,运动障碍68例,合并上消化道出血30例,死亡26例,病死率32.5%。结论高血压病和动脉硬化为本病的主要病因,意识障碍主要与脑脊液循环受阻或血肿压迫导致颅内压增高有关。双眼垂直运动障碍、瞳孔缩小是丘脑出血的特征性体征,运动障碍发生率高,其预后与出血量多少有关,合并上消化道出血预后差。     

Ultrasound-mediated microbubble delivery of pigment epithelium-derived factor gene into retina inhibits choroidal neovascularization

Background Many studies have suggested that the imbalance of angiogenic factor and anti-angiogenic factor expression contributes significantly to the development of choroidal neovascularization （CNV）, and ultrasound microbubble combination system can increase the gene transfection efficiency successfully. This study was designed to investigate whether ultrasound-mediated microbubble destruction could effectively deliver therapeutic plasmid into the retina of rat, and whether gene transfer of pigment epithelium-derived factor （PEDF） could inhibit CNV.
Methods Human retinal pigment epithelial cells were isolated and treated either with ultrasound or plasmid alone, or with a combination of plasmid, ultrasound and microbubbles to approach feasibility of microbubble-enhanced ultrasound enhance PEDFgene expression; For in vivo animal studies, CNV was induced by argon lasgon laser in rats. These rats were randomly assigned to five groups and were treated by infusing microbubbles attached with the naked plasmid DNA of PEDF into the vitreous of rats followed by immediate ultrasound exposure （intravitreal injection）; infusing liposomes with the naked plasmid DNA of PEDF into the vitreous （lipofectamine ＋ PEDF）; infusing microbubbles attached with PEDF into the orbit of rats with ultrasound irradiation immediately （retrobular injection）; infusing microbubbles attached with PEDF into the femoral vein of rats with exposed to ultrasound immediately （vein injection）. The CNV rats without any treatment served as control. Rats were sacrificed and eyes were enucleated at 7, 14, and 28 days after treatment. Gene and protein expression of PEDF was detected by quantitative real-time RT-PCR, Western blotting and immunofluorescence staining, respectively. The effect of PEDF gene transfer on CNV was examined by fluorescein fundus angiography.
Results In vitro cell experiments showed that microbubbles with ultrasound irradiation could significantly enhance PEDF delivery as compared with microbubbles or ultrasound alone. In the rat CNV model, transfection efficiency mediated by ultrasound/microbubbles was significantly higher than that by lipofectamine-mediated gene transfer at 28 days after treatment. The study also showed that with the administration of ultrasound-mediated microbubbles destruction, the CNV of rats was inhibited effectively.
Conclusions Ultrasound-microbubble technique could increase PEDF gene transfer into rats＇ retina and chorioid, in association with a significant inhibition of the development of CNV, suggesting that this noninvasive gene transfer method may provide a useful tool for clinical gene therapy.     

常规超声检查难以定性的复杂盆腔包块超声造影增强特点

 摘要：【目的】探讨常规超声检查难以定性的盆腔肿块超声造影增强特点。【方法】对常规超声检查难以定性的137例盆腔肿块进行超声造影检查。分析病灶增强开始时间、增强水平、形态和增强模式的特点。【结果】良恶性病灶超声造影表现各不相同。增强时间早于子宫肌层呈高增强的恶性、良性病灶分别为85.3%（29/34）和13.7%（14/103）;增强形态不均匀的恶性、良性病灶分别为 97.1%（33/34）和31.0 % (32/103);增强模式I型的恶性、良性病灶分别为85.3%（29/34）和9.7%（10/103）,上述两组结果比较,差异均有统计学意义(p＜0.01)。【结论】复杂的盆腔良恶性肿块造影增强模式具有特征性,可为病变的定性诊断提供信息。     

超声e-Flow检测脑梗塞患者颈动脉斑块内新生血管效果评价

【目的】应用超声e-Flow方法,检测颈动脉斑块内新生血管,揭示斑块内新生血管的特点,间接反映斑块的稳定性。【方法】选取脑梗塞患者共69例进行颈动脉超声检查,检出颈动脉内斑块做为超声e-Flow观察对象,并进行对比。【结果】梗塞侧颈动脉检出斑块100个,非梗塞侧检出斑块87个,梗塞侧斑块在检出数量及新生血管的比例上,明显高于非梗塞侧,差异有统计学意义(P<0.05)。显示有新生血管的斑块在软斑比例及斑块的厚度上,明显高于未显示新生血管的斑块,差异有统计学意义(P<0.05)。【结论】超声不仅能够显示斑块的大小和形态,同时能够敏感探测斑块内新生血管血流,从而间接判定斑块的稳定性,有助于预防脑梗死的发生。     

Ultrasonic destruction of albumin microbubbles enhances gene transfection and expression in cardiac myocytes

Background  It has been proven that ultrasonic destruction of microbubbles can enhance gene transfection efficiency into the noncardiac cells, but there are few reports about cardiac myocytes. Moreover, the exact mechanisms are not yet clear; whether the characteristic of microbubbles can affect the gene transfection efficiency or not is still controversial. This study was designed to investigate whether the ultrasound destruction of gene-loaded microbubbles could enhance the plasmids carried reporter gene transfection in primary cultured myocardial cell, and evaluate the effects of microbubbles characteristics on the transgene expression in cardiac myocytes.

Methods  The β-galactosidase plasmids attached to the two types of microbubbles, air-contained sonicated dextrose albumin (ASDA) and perfluoropropane-exposed sonicated dextrose albumin (PESDA) were prepared. The gene transfection into cardiac myocytes was performed in vitro by naked plasmids, ultrasound exposure, ultrasonic destruction of gene-loaded microbubbles and calcium phosphate precipitation, and then the gene expression and cell viability were analyzed.

Results The ultrasonic destruction of gene-loaded microbubbles enhanced gene expression in cardiac myocytes compared with naked plasmid transfection ((51.95±2.41) U/g or (29.28±3.65) U/g vs. (0.84±0.21) U/g, P <0.01), and ultrasonic destruction PESDA resulted in more significant gene expression than ASDA ((51.95±2.41) U/g vs. (29.28±3.65) U/g, P <0.05). Ultrasonic destruction of microbubbles during calcium phosphate precipitation gene transfection enhanced β-galactosidase activity nearly 8-fold compared with calcium phosphate precipitation gene transfection alone ((111.35±11.21) U/g protein vs. (14.13±2.58) U/g protein, P <0.01). Even 6 hours after calcium phosphate precipitation gene transfection, ultrasound-mediated microbubbles destruction resulted in more intense gene expression ((35.63±7.65) U/g vs. (14.13±2.58) U/g, P <0.05 ) .

Conclusions  Ultrasonic destruction of microbubbles might be a promising method for the delivery of non-viral DNA into cardiac myocytes, and the gene tranfection is related to the characteristics of microbubbles.