不同方法转染人前列腺癌PC-3细胞pEGFP-N1基因的体外实验研究

目的:探讨转染人前列腺癌PC-3细胞pEGFP-N1基因的最佳转染方法。方法:以超声微泡造影剂、超声辐照、脂质体转染及其相互结合的方法,将质粒pEGFP-N1基因转染人前列腺癌PC-3细胞,24h后以荧光显微镜观察前列腺癌PC-3细胞中的绿色荧光蛋白表达情况,并用流式细胞仪测定转染率。结果:以超声+微泡+脂质体组基因转染效率最高,与其他组比较,差异均有统计学意义(P<0.05)。结论:超声联合微泡与脂质体结合能明显提高pEGFP-N1基因在人前列腺癌细胞中的转染率,是一种较理想的基因转染方法。     

超声靶向微泡破裂介导EGFP质粒转染肝癌细胞的研究

目的探讨超声靶向微泡破裂(Ultrasound Targeted Microbubble Destruction,UTMD)介导EGFP质粒转染肝癌细胞株HepG2的有效性、安全性并优化超声辐照参数。方法体外培养HepG2细胞,在不同治疗超声的声强、占空比和辐照时间作用下,观察pEGFP-N3质粒在HepG2细胞中的转染。荧光显微镜下观察绿色荧光蛋白在HepG2细胞中的表达,流式细胞仪检测细胞的转染率,MTT法检测细胞活性。结果在超声声强为2 w/cm2、占空比为20%、照射时间为60 s时,HepG2细胞的7转染率最高,达到11.53%±2.15%,且细胞生存率大于85%。结论 UTMD是一种有效的基因转染方法,不同的超声转染参数对细胞活力和基因传输效率有较大影响,对其进行优化后可减少细胞损伤,增强基因转染。     

Myocardium-targeted delivery of endothelial progenitor cells by ultrasound-mediated microbubble destruction improves cardiac function via an angiogenic response

Application of ultrasound-mediated destruction of microbubbles (US + Bubble) to skeletal muscle creates capillary ruptures leading to leakage of the cell components. We studied whether US + Bubble combined with bone-marrow-derived mononuclear cells (BM-MNCs) infusion enables the targeted delivery of endothelial-lineage cells into the myocardium and improves cardiac function of the cardiomyopathy model due to the paucity of neocapillary formation. Pulsed US was applied to the anterior chest of BIOTO2 cardiomyopathy hamsters for 90 s after the intravenous injection of microbubble (Optison) followed by infusion of BM-MNCs. Cardiac samples from US + microbubble + BM-MNCs (US + Bubble + BM), US + Bubble, US + BM without Bubble, and saline infusion control groups were analyzed 12 weeks after treatment. Labeled BM-MNCs transplanted by US + Bubble were found to be mainly localized in the microvessels, but not by US stimulation without microbubble (121.2 +/- 24.5 vs. 2.80 +/- 1.30 cells/mm2, P < 0.001). Capillary densities in US + Bubble + BM group were increased 1.7-fold (P < 0.05) over the control, and neither US + Bubble nor US + BM enhanced neocapillary formation. 99mTc-Tetrofosmin scintigraphy revealed that blood perfusion area in the US + Bubble + BM group was 48% greater than the control (P < 0.01). US + Bubble stimulation induces the expression of adhesion molecules (VCAM-1 and ICAM-1) in capillaries, and the US + Bubble-mediated supply of BM-MNCs increased the myocardial content of VEGF and bFGF. The left ventricular wt/body wt, area of cardiac fibrosis, and apoptotic cell numbers in the US + Bubble + BM group significantly (P < 0.05) decreased by 82%, 73%, and 64% relative to the control, respectively. The cardiac function in myopathic hamsters (assessed by fractional shortening) was markedly improved 36% (P < 0.05) by US + Bubble + BM treatment. Targeted delivery of BM-MNCs by US + Bubble to the myocardium of the cardiomyopathic hamster increased the capillary densities and regional blood flow and inhibited cardiac remodeling, resulting in the prevention of heart failure. This non-invasive cell delivery system may be useful as a novel efficient approach for angiogenic cell therapy to the myocardium.     

Apoptosis Induced by Microbubble-Assisted Acoustic Cavitation in K562 Cells: The Predominant Role of the Cyclosporin A-Dependent Mitochondrial Permeability Transition Pore

Acoustic cavitation of microbubbles has been described as inducing tumor cell apoptosis that is partly associated with mitochondrial dysfunction; however, the exact mechanisms have not been fully characterized. Here, low-intensity pulsed ultrasound (1 MHz, 0.3-MPa peak negative pressure, 10% duty cycle and 1-kHz pulse repetition frequency) was applied to K562 chronic myelogenous leukemia cells for 1 min with 10% (v/v) SonoVue microbubbles. After ultrasound exposure, the apoptotic index was determined by flow cytometry with annexin V–fluorescein isothiocyanate/propidium iodide. In addition, mitochondrial membrane potential (ΔΨ_m) was determined with the JC-1 assay. Translocation of apoptosis-associated protein cytochrome c was evaluated by Western blotting. We found that microbubble-assisted acoustic cavitation can increase the cellular apoptotic index, mitochondrial depolarization and cytochrome c release in K562 cells, compared with ultrasound treatment alone. Furthermore, mitochondrial dysfunction and apoptosis were significantly inhibited by cyclosporin A, a classic inhibitor of the mitochondrial permeability transition pore; however, the inhibitor of Bax protein, Bax-inhibiting peptide, could not suppress these effects. Our results suggest that mitochondrial permeability transition pore opening is involved in mitochondrial dysfunction after exposure to microbubble-assisted acoustic cavitation. Moreover, the release of cytochrome c from the mitochondria is dependent on cyclosporin A–sensitive mitochondrial permeability transition pore opening, but not formation of the Bax-voltage dependent anion channel complex or Bax oligomeric pores. These data provide more insight into the mechanisms underlying mitochondrial dysfunction induced by acoustic cavitation and can be used as a basis for therapy.     

Ultrasound with Microbubble Contrast Agent and Urokinase for Thrombosis

This study was aimed at assessing the effects of urokinase (UK) in combination with ultrasound and microbubbles in in vitro and in vivo thrombolytic therapy for the treatment of deep vein thrombosis (DVT). Thrombi with formation times of 1, 3, 7, 14 and 21 d were used for thrombolysis. Forty-five adult mongrel dogs were used to evaluate thrombosis in vivo. Both in vitro and in vivo analyses revealed that UK?+?microbubbles had the best effect among the combinations. Thrombolysis <7 d was more effective at a thrombolysis rate of about 50%, but the thrombolytic effect of thrombi >7 d was poor at thrombolysis rates <30%. Ultrasound?+?UK significantly increased the thrombolysis rate of thrombi <7 d. These results suggest that the combination of ultrasound with microbubble contrast agents and UK may have a synergistic effect on thrombolysis.     

诊断超声激励微泡对增强临床乳腺癌血流灌注的研究

目的 观察并探讨诊断超声激励超声造影剂微泡对浸润性乳腺癌病灶血流灌注的增强效应。方法 5例经病理证实的浸润性乳腺癌患者,在每次新辅助化疗结束后1h内对病灶进行诊断超声激励造影剂微泡治疗。治疗前后实施常规超声造影动态观察其血流灌注情况,应用时间-强度曲线分析软件分析并获取峰值强度(Peak Intensity, PI)、曲线下面积(Area Under Curve, AUC)及曲线上升斜率(Ascending Slope, AS)。结果 诊断超声激励微泡治疗后乳腺癌病灶的PI、AS及AUC均有所提高,治疗前后PI及AS比较差异均有统计学意义(Z=-2.13,-2.09；P=0.03,0.03),但治疗前后AUC比较差异无统计学意义(t=-1.15；P=0.28)。结论 诊断超声激励造影剂微泡能够增加新辅助化疗乳腺癌病灶的血流灌注量,加快血流灌注速度。     

超声联合微泡在肿瘤化疗增敏的基础研究与临床应用进展

恶性肿瘤是一类死亡率很高的疾病，化疗是其重要的治疗方法之一。应用微泡的声孔效应提高细胞对化疗药物的摄取以增加肿瘤组织局部药物浓度达到增敏化疗的目的，是目前肿瘤治疗研究的新方向之一。超声联合微泡可增加肿瘤组织局部药物浓度、增强细胞毒作用，促进肿瘤细胞凋亡，缩小肿瘤体积，改善肿瘤对化疗药物的耐药性，具有良好的临床应用价值及前景。本文拟从超声联合微泡增敏肿瘤化疗的基础研究及临床运用方面进行综述。     

小鼠颊癌颈部淋巴结超声造影的实验研究

目的 探讨局部注射造影剂对移植颊癌原发灶及颈部转移淋巴结增强显像效果.方法 建立小鼠颊癌颈部转移淋巴结动物模型后,经原发灶局部注入自制表面活性剂类超声微泡,以谐波显像方式观察原发灶及颈部淋巴结增强显像效果,同时进行回声强度的比较.结果 造影前,转移淋巴结检出率为61%（17/28）,炎性淋巴结检出率为50%（6/12）;造影后转移淋巴结检出率为82%（23/28）,炎性淋巴结检出率为75%（9/12）.与造影前比较,造影后原发灶和淋巴结的回声强度增大,差异有统计学意义（P〈0.01）结论自制表面活性剂类超声微泡能够经原发灶注入,进入淋巴道,实现显著的淋巴结超声显像增强效果.     

载多西紫杉醇脂质微泡联合超声靶向微泡破裂对兔VX2肝癌微血管的作用

目的探讨载多西紫杉醇脂质微泡（DLLM）联合超声靶向微泡破裂（UTMD）对兔VX2肝癌微血管的抑制作用。方法建立60只兔VX2肝癌动物模型,将其随机分为6组（n=10）：单纯药物组（Doc组）、单纯载药微泡组（DLLM组）、药物＋超声组（Doc＋US组）、单纯微泡＋超声组（PLM＋US组）、载药微泡＋超声组（DLLM＋US组）及对照组,比较各组动物肿瘤组织的血管密度（MVD）、CD34及VEGF的表达。结果处理后DLLM＋US组的CD34表达水平低于其他各组,MVD明显降低,与其他各组相比差异有统计学意义（P〈0.01）;DLLM＋US组的VEGF表达水平明显低于其他各组（P〈0.01）。结论 DLLM联合UTMD能抑制兔VX2肝癌的微血管生成,从而抑制兔VX2肝癌的生长。