超声介导击破微泡增强型绿色荧光蛋白转染正常大鼠关节滑膜组织

目的 探讨超声击破微泡介导增强型绿色荧光蛋白(EGFP)转染大鼠关节滑膜组织的可行性。 方法 将20只正常清洁级Wister大鼠分为4组,每组5只(10个膝关节)。单纯质粒组:10 μg EGFP注射入大鼠关节腔内;超声+质粒组:EGFP注射入大鼠关节腔内,超声持续照射10 min;微泡+质粒组:300 μl SonoVue与10 μg EGFP混合注射入大鼠关节腔内;超声+微泡+质粒组:将300 μl SonoVue与10 μg EGFP混合后注射入大鼠关节腔内,超声持续照射10 min。3天后取出大鼠膝关节滑膜组织,直接贴在载玻片上,在荧光显微镜480 nm波长激发状态下观察EGFP转染效果。 结果 单纯质粒组、超声+质粒组和微泡+质粒组的大鼠膝关节滑膜组织EGFP的荧光强度稍有增强;超声+微泡+质粒组EGFP的荧光强度明显增强。 结论 超声介导微泡可以实现EGFP质粒对正常大鼠关节滑膜组织的转染。     

低频超声联合SonoVue微泡促进裸鼠基因体内转染的可行性

目的 探讨利用低频超声联合SonoVue微泡造影剂促进基因转染人前列腺癌裸鼠皮下移植瘤的可行性。方法 采用人前列腺癌PC-3细胞建立裸鼠皮下移植瘤模型,成瘤后随机分为空白组（仅注射生理盐水）、单纯质粒组（注射生理盐水和质粒混悬液）、低频超声组（注射生理盐水和质粒混悬液后超声辐照）和低频超声+微泡组（注射超声微泡造影剂和质粒混悬液后超声辐照）。超声辐照条件：功率3 W/cm²,占空比5：5,频率80 kHz,辐照10 min。基因转染3天后在激光共聚焦显微镜下观察各组绿色荧光蛋白表达情况,并分析其平均荧光强度;同时行常规病理检查,观察低频超声联合微泡辐照对组织的损伤程度。结果 4组中,仅低频超声+微泡组可见明显绿色荧光,与其他各组差异均有统计学意义（P均<0.05）;空白组、单纯质粒组和低频超声组均未见明显绿色荧光。各组均未见明显组织损伤。结论 SonoVue微泡造影剂在低频超声辐照下能够安全有效地促进pEGFP基因转染进入人前列腺癌裸鼠皮下移植瘤组织内。     

诊断超声联合微泡对大鼠移植瘤血管通透性影响的实验研究

目的 研究诊断超声联合微泡对大鼠移植瘤血管通透性的影响.方法 将48只已建立Walker-256皮下移植瘤模型的SD大鼠随机分为4组:空白对照(A)组、单纯微泡剂(B)组、单纯超声(C)组和超声联合微泡(D)组.以伊文思蓝(Evens blue,EB)为指示剂,选择频率为1.75 MHz、机械指数为1.4的诊断超声持续照射肿瘤区5 min,经大鼠尾静脉注射/不注射微泡造影剂.使用分光光度计和标准曲线法测量大鼠肿瘤组织内EB含量变化,激光共聚焦显微镜观察EB外溢情况.结果 超声联合微泡可增加肿瘤组织微血管通透性.D组瘤内EB含量较A、B、C组明显增加(P＜0.05),血管周围EB外溢明显.A、B、C组EB含量比较差异无统计学意义(P＞0.05),血管周围均未见明显EB渗出.结论 在一定条件下,诊断超声联合微泡可明显提高辐照肿瘤组织血管通透性,这对临床肿瘤化疗患者具有潜在的应用意义.     

超声微泡造影剂介导EGFP质粒转染大鼠骨骼肌

目的 探讨增强型绿色荧光蛋白质粒(EGFP)在大鼠骨骼肌微血管通透性增加的条件下表达的可行性.方法 20只清洁级Sprague-Dawley(SD)大鼠,随机均分为裸质粒组(P)、质粒 超声组(P U)、质粒 微泡组(P M)和质粒 超声 微泡组(P U M)共4组进行实验.选择增强绿色荧光蛋白质粒与白蛋白微泡相混合,以超声介导白蛋白微泡破裂的方法 对大鼠骨骼肌行基因转染,转染7 d后,荧光显微镜下观察质粒在大鼠脊斜肌组织中的表达情况.结果 P、P M组大鼠脊斜肌组织中均未见增强型绿色荧光蛋白表达;P U组可见少量微弱绿色荧光,荧光强度较P和P M组明显增强(P<0.05),P U M组可见明显特异性增强型绿色荧光蛋白表达,荧光强度约为P、P M组的10倍,P U组的3倍(P<0.05);P U M组转染率为(42.72±10.07)%较之P U组(13.62±6.17)%明显增高(P<0.05).结论 超声介导微泡造影剂破裂造成的脊斜肌组织毛细血管通透性的增加,是血管内基因成功跨越内膜屏障的主要途径之一.     

超声激励荧光微泡对兔乳腺癌转移淋巴结的释放作用

目的 观察超声激励荧光微泡空化对兔乳腺癌转移淋巴结的荧光释放作用。 方法 使用二甲基亚砜(DMSO)溶解绿色细胞膜荧光分子探针(DiO),抽取少量溶解液与脂质微泡混和,通过高速机械振荡制成荧光微泡。选取16只荷VX2乳腺癌的新西兰大白兔随机分成荧光微泡联合超声空化组和单纯荧光微泡组。荧光微泡联合超声空化组经瘤周皮下注射1 ml荧光微泡并按摩,待瘤周引流淋巴结超声显影后,采用脉冲式治疗超声间歇辐照淋巴结5次,共30 min;单纯荧光微泡组同样经瘤周皮下注射荧光微泡并按摩,但给予治疗超声假照。剖取淋巴结标本行冰冻组织切片,于激光共聚焦显微镜下观察淋巴结内的荧光分布情况,并定量分析荧光面积、累积光密度(IOD)及平均光密度(AOD)。 结果 荧光微泡联合超声空化组淋巴结的荧光面积、IOD和AOD均高于单纯荧光微泡组(P均<0.05)。 结论 荧光脂质微泡经瘤周注射不仅可进入兔乳腺癌模型的淋巴管使淋巴结显影,且可在治疗超声的激励作用下实现荧光物质在局部淋巴结高浓度释放。     

超声声致孔隙效应增强兔前列腺组织通透性的研究

目的 探讨超声联合造影剂微泡的超声声致孔隙效应开放血-前列腺屏障,提高前列腺组织通透性的可行性.方法 15只健康8月龄性成熟新西兰兔,随机分为单纯微泡组、单纯超声组、超声联合微泡辐照组,超声直接辐照前列腺,荧光显微镜、体视显微镜、伊文思蓝(EB)示踪观察前列腺组织通透性的变化.结果 体视显微镜下超声联合微泡辐照组前列腺组织可见蓝色EB分布于前列腺包膜,部分EB渗透到腺体实质,腺体呈均匀蓝染,单纯超声组及单纯微泡组EB均分布于包膜,腺体实质部分渗透不明显.单纯超声组、单纯微泡组、超声联合微泡辐照组前列腺冰冻切片均可见红色EB荧光分布;经苏木素复染后可见超声联合微泡辐照组腺体管腔内有EB荧光,而单纯超声组、单纯微泡组均未见管腔内红色EB荧光;超声联合微泡辐照组前列腺EB平均浓度较单纯超声组、单纯微泡组高,差异有统计学意义(P<0.01),单纯微泡组及单纯超声组EB浓度均值未见显著差异(P>0.05).结论 超声联合造影剂微泡的超声声致孔隙效应可以有效开放血-前列腺屏障,明显提高兔前列腺组织通透性.     

评价超声联合白蛋白微泡造影剂实现基因转染靶向性的研究

目的 评价超声联合白蛋白微泡造影剂实现基因转染的靶向性.方法 选择增强型绿色荧光蛋白质粒为报告基因,经股静脉输入黏附质粒的白蛋白微泡的同时在大鼠背部脊斜肌区域经皮辅予一定强度的超声照射对大鼠脊斜肌行基因转染,转染7 d后,取脊斜肌、肝、肾、心肌组织快速冷冻切片,荧光显微镜下观察各组织内的荧光表达.结果 脊斜肌组织中可见较多特异性绿色荧光,多位于微血管周围,荧光强度较其余组织明显增强(P<0.05),肝脏、肾脏组织中见少量特异性绿色荧光,肝脏荧光强度约为肾脏的4倍(P<0.05),心肌组织中未见特异性绿色荧光.结论 静脉注射黏附质粒的微泡声学造影剂同时经体表给予一定强度的超声辐照,可以较好地实现基因的靶向转染.     

微泡增强的超声空化阻断肿瘤微循环的病理观察

目的 探讨微泡增强的超声空化阻断肿瘤微循环的病理机制。方法 建立兔肝VX2转移瘤模型18只,随机分为超声微泡组(n=6)、单纯超声组(n=6)及假照组(n=6)。对超声微泡组经耳缘静脉注射微泡,并进行超声辐照;对单纯超声组以生理盐水代替微泡,并进行超声辐照;对假照组注射生理盐水,并进行超声假照。观察辐照后各组病理变化。结果 大体解剖观察,超声微泡组辐照面可见较多出血点;单纯超声组及假照组辐照后肿瘤组织均未见明显改变。光镜下观察,超声微泡组肿瘤组织可见大片出血,血管内皮细胞损伤、连续性中断,红细胞外溢;单纯超声组可见局灶性小片状出血,血管内皮尚完整;假照组肿瘤组织未见明显出血,血管内皮完整。电镜下观察,超声微泡组肿瘤血管内皮破损严重,可见大量红细胞渗出,线粒体肿胀及吞饮小泡;单纯超声组及假照组仅见内质网略水肿。结论 微泡增强的超声空化阻断兔VX2肝脏肿瘤微循环的机制可能为血管机械性损伤。     

超声破坏微泡法介导增强型绿色荧光蛋白质粒转染兔股骨头组织的安全性及可行性(英文)

背景:超声微泡转染系统已尝试于体内多部位的基因转染,但尚未见有用于骨部位基因转染的报告。目的:观察超声破坏微泡法介导增强型绿色荧光蛋白质粒转染兔股骨头组织的效率及可行性。方法:将日本大耳白兔按随机均分为裸转染组,预照射+裸转染组,超声定位转染组,预照射+超声定位转染,重复定位转染组。其中前2组不给予超声定向转染照射,后3组利用超声微泡破裂法介导增强型绿色荧光蛋白质粒,定向基因转染兔股骨头。各组转染1周后,于荧光显微镜下观察增强型绿色荧光蛋白在股骨头组织中的表达情况。结果与结论:超声定位转染组,预照射+超声定位转染,重复定位转染组均有增强型绿色荧光蛋白表达,且复定位转染组增强型绿色荧光蛋白质粒在兔股骨头内的转染效率明显高于其他组(P<0.01)。超声定位转染组,预照射+超声定位转染,重复定位转染组兔超声照射部位软组织和骨组织切片未观察到明显损伤病灶。结果证实,超声微泡破裂法能安全、有效实现增强型绿色荧光蛋白质粒在兔股骨头组织的转染。     

超声介导载sPD-1和miR-206基因纳米微泡协同抑制小鼠H22肝癌皮下移植瘤

目的 探讨超声介导载可溶性程序性死亡因子1（sPD-1）联合miR-206纳米微泡对小鼠H22肝癌皮下移植瘤的干预效果。方法 自制载sPD-1和miR-206基因的纳米微泡,构建H22肝细胞癌皮下移植瘤小鼠模型,并将模型小鼠随机分为模型组、空微泡组、miR-206微泡组、sPD-1微泡组、联合组（给予miR-206微泡组及sPD-1微泡）,分别每2天经尾静脉给予生理盐水及相应的微泡,每次注射后给予超声辐照瘤体治疗1次。5次治疗后取小鼠肿瘤组织,测量肿瘤质量及体积,计算肿瘤体积及质量抑瘤率,HE染色观察肿瘤组织病理变化,免疫组织化学法检测小鼠肿瘤组织Bax、Bcl-2蛋白表达,半定量PCR检测Bax、Bcl-2、c-met、γ干扰素（IFN-γ）、程序性死亡因子-1配体（PD-L1）mRNA表达,实时荧光定量PCR检测miR-206表达。结果 制备的纳米微泡呈球形,分布均一。与模型组比较,各组肿瘤体积、肿瘤质量均降低,体积抑瘤率及质量抑瘤率均升高（P均<0.05）;上述变化以联合组为著（P均<0.05）。与模型组比较,其余各组Bax蛋白和mRNA均高表达、Bcl-2蛋白和mRNA均低表达,以联合组为著（P均<0.01）。各组小鼠肿瘤组织Bax、Bcl-2、c-met、PD-L1、IFN-γ、miR-206 mRNA表达差异均有统计学意义（P均<0.01）。结论 超声介导载sPD-1和miR-206纳米微泡可协同抑制小鼠H22肝癌皮下移植瘤生长。     

Investigation Into the Impact of Diagnostic Ultrasound with Microbubbles on the Capillary Permeability of Rat Hepatomas

Ultrasound-targeted microbubble destruction (UTMD) takes advantage of transiently increased capillary permeability to enhance the release of tumor-specific drugs from blood vessels into sonicated tumor tissues. However, the application of focused ultrasound is limited because of the lack of an appropriate image-monitoring system. In this study, hepatoma-bearing Sprague-Dawley rats were insonicated with low-frequency diagnostic ultrasound and injected with Evans Blue (EB) dye and microbubbles through their tail veins to test changes in capillary permeability. We studied how the mechanical index, sonication duration and the injected microbubble (MB) concentration affect the hepatoma vascular permeability by quantitatively evaluating the EB delivery efficiency. Confocal laser scanning microscopy was used to observe the deposition of red fluorescence–dyed EB in tumor tissues. In addition, P-selectin, a type of biochemical marker that reflects vascular endothelial cell activation, was identified using an immunoblotting analysis. The experimental results reveal that EB delivery efficiency in tumor tissues was greater in groups with the diagnostic ultrasound–mediated UTMD (8.40 ± 0.71 %ID/g) than in groups without UTMD (1.73 ± 0.19 %ID/g) and EB delivery efficiency could be affected by MI, sonication duration and MB dose. The immunoblotting analysis indicates that diagnostic ultrasound–induced UTMD results in the vascular endothelial cell activation to increase capillary permeability, justifying the high quantity of EB deposited in tumor tissues.     

Spatial Distribution of Ultrasound Targeted Microbubble Destruction Increases Cardiac Transgene Expression But Not Capillary Permeability

Ultrasound targeted microbubble destruction (UTMD) has evolved as a promising tool for organ specific gene and drug delivery. Using DNA-loaded microbubbles, cardiac transfection has been shown to be feasible. However, two-dimensional properties of the ultrasound beam limit cardiac transgene expression to the focal zone, thus, reducing its potential therapeutic effect. The aim of this study was to test if spatial distribution of ultrasound targeted microbubble destruction in the heart could lead to augmented transgene expression or increased capillary permeability. Lipid microbubbles containing plasmids with a luciferase transgene were used to target rat hearts. The diagnostic ultrasound probe was fixed in a mid-short axis view with a gel stand-off between the chest and probe. Ultrasound (1.3 MHz) with a mechanical index of 1.6 was intermittently applied to rats during microbubble infusion. Rats were randomized to either stay in that position or move horizontally in a cranio-caudal direction (3 mm sweep) relative to the ultrasound probe during UTMD. After 4 days, organs were harvested and analyzed for reporter gene expression. Another group of rats received Evans Blue, followed by UTMD with unloaded microbubbles. Again, rats were randomized into a static or moving group. Hearts were harvested to evaluate extravasation of Evans Blue. Moving rats in a cranio-caudal direction significantly increased transgene expression by 19-fold in the anterior heart, by sixfold in the posterior heart and by 32-fold in the apex. Interestingly, Evans Blue extravasation was not augmented in the moving group. Spatial distribution of UTMD may increase transgene expression due to sonication of larger areas in the heart. In contrast, capillary permeability does not increase, indicating less capillary damage. (E-mail: raffi.bekeredjian@med.uni-heidelberg.de)     

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。     

Ultrasound-Mediated Microbubble Destruction Increases Renal Interstitial Capillary Permeability in Early Diabetic Nephropathy Rats

Diabetic nephropathy (DN) is defined as persistent proteinuria corresponding to a urinary albumin excretion rate >300 μg/mg in the absence of other non-diabetic renal diseases. The aim of this study was to determine if ultrasound (US)-mediated microbubble (MB) destruction could increase renal interstitial capillary permeability in early DN rats. Diabetes was induced with streptozotocin. DN rats presented with mild micro-albuminuria 30 d after onset of diabetes. DN rats (N = 120) were divided into four groups that received Evans blue (EB) followed by: (i) no treatment (control group); (ii) continuous ultrasonic irradiation for 5 min (frequency = 7.00 MHz, mechanical index = 0.9, peak rarefactional pressure = 2.38 MPa: US group); (iii) microbubble injection (0.05 mL/kg: MB group); and (iv) both ultrasound and microbubble injection (US + MB group). Another 8 DN rats were subjected to ultrasound and microbubbles and then injected with EB after 24 h (recovery group). EB content, EB extravasation and E-selectin mRNA and protein expression significantly increased, and interstitial capillary walls became discontinuous in the US + MB group. Neither hemorrhage nor necrosis was observed on renal histology. Urine samples were collected 24 h post-treatment. There was no hematuria, and the urinary albumin excretion rate did not increase after ultrasound-microbubble interaction detected by urinalysis. EB content returned to the control group level after 24 h, as assessed for the recovery group. In conclusion, ultrasound-mediated microbubble destruction locally increased renal interstitial capillary permeability in DN rats, and should be considered a therapy for enhancing drug and gene delivery to the kidney in the future.     

超声作用下CTGF-siRNA对大鼠肝纤维化基因治疗的实验研究

目的探讨利用超声微泡造影剂介导携带结缔组织生长因子-小干扰RNA(CTGF-siRNA)真核表达质粒转染大鼠肝细胞的有效性。方法 (1)构建CTGF-siRNA真核表达质粒;(2)将40只实验大鼠随机分为7组:正常组,实验对照组,CTGF-siRNA质粒组,超声联合微泡组,超声联合微泡介导基因低、中、高剂量组;(3)建立肝纤维化模型,基因治疗4周后处死大鼠,超声及病理切片HE染色评价肝纤维化程度,masson染色观察胶原纤维含量,反转录-聚合酶链反应(RT-PCR)检测肝组织中CTGF、Ⅰ型和Ⅲ型胶原的mRNA表达。结果 (1)CTGF-siRNA基因治疗后超声及病理切片显示干预组纤维化程度降低;(2)masson染色显示超声介导微泡基因组胶原纤维含量表达量随着剂量增高而降低(P<0.05);(3)RT-PCR显示超声介导微泡基因组CTGF、Ⅰ型和Ⅲ型胶原的mRNA表达明显低于其他组,且随着质粒剂量增大而减少(P<0.05)。结论超声微泡作用下CTGF-siRNA基因能特异地作用于靶位点,有效抑制肝纤维化进程,提高对肝纤维化干预的特异度。     

体外实验观察微泡-阳离子纳米脂质体复合物促进HGF基因转染

目的通过生物素-亲和素的特异结合,制备一种高效载肝细胞生长因子(HGF)基因的脂质微泡(LMB)-阳离子纳米脂质体复合物(LMB-CNLP),观察转染情况,探讨其对对数生长期的肝星状细胞株HSC-T6活性的影响。方法将HSC-T6细胞分为载HGF基因的LMB组、LMB-CNLP组和对照组(单纯质粒),超声辐照各组进行HGF转染。以荧光显微镜及流式细胞仪观测转染情况,ELISA试剂盒检测HGF蛋白表达情况,流式细胞仪检测HSC-T6细胞凋亡。结果 LMB-CNLP组HGF基因的转染率为41.29%,明显高于其他2组。转染后的细胞能持续表达HGF,且随着HGF蛋白增多,HSC-T6细胞凋亡率增高。结论超声辐照LMB-CNLP复合物能提高HGF基因在体外培养的HSC-T6细胞中的转染情况,促进HSC-T6凋亡,为基因治疗肝纤维化提供实验依据。     

Insonation of the eye in the presence of microbubbles: preliminary study of the duration and degree of vascular bioeffects--work in progress.

OBJECTIVE: The purpose of this study was to investigate the presence and duration of vascular permeability changes induced by the combination of ultrasound and an intravascular microbubble contrast agent in the rabbit eye. METHODS: Five eyes were studied in 8 anaesthetized rabbits. Insonation was performed with a diagnostic B-mode system (center frequency = 2 MHz; mechanical index [MI] = 0.2 and 1.7) for 5 minutes after administration of perflutren microbubbles (0.07 mL/kg). Fluorescein fundus angiography was performed before and 3 minutes after insonation; at 6 minutes, color fundus photography was used to assess the dye leakage, bleeding, and alteration of the diameter of fundus vessels. RESULTS: Alteration of fundus vessel diameters was observed in 1 of 5 cases at a low MI and in 4 of 5 cases at a higher MI. In 1 case, leakage of fluorescein indicated increased permeability at the higher MI. No bleeding was detected in any case. CONCLUSIONS: The permeability change induced by insonation and this dose of an ultrasound contrast agent appears to be transient under the conditions studied, although the time delay between insonation and optical assessment limits the completeness of the findings. This preliminary study may be relevant to drug delivery strategies using ultrasound and microbubbles.     

Effects of transcranial ultrasound and intravenous microbubbles on blood brain barrier permeability in a large animal model

We sought to determine whether transtemporal-applied 1-MHz ultrasound-induced microbubble destruction may be a safe method of transiently altering blood brain barrier (BBB) permeability for drug delivery in a large animal model. Endothelial cells are an integral component of the BBB but also prevent passage of potentially therapeutic drugs. Ultrasound-mediated destruction (UMD) of microbubbles has been shown to disrupt this barrier in small animals when ultrasound is delivered through bone windows. However, the effects of temporal bone attenuation and scattering in a large animal may limit the clinical application of such a technique. Twenty-four pigs were studied. One-MHz pulsed-wave ultrasound at 2.0 W/cm(2) (20% duty cycle) across the temporal bone was applied for 30 min after intravenous injections of either albumin-coated perfluorocarbon microbubble (PESDA, 8 pigs), lipid-encapsulated perfluorocarbon microbubbles (LEMB, 8 pigs) or ultrasound alone (8 pigs). BBB leak was quantified at 30 and 120 min after insonation using Evans blue. Serial magnetic resonance imaging (MRI) was performed in nine of the pigs (3 for each group) to quantify Gadolinium leak within the parenchyma. Peak negative pressures decreased ten-fold when ultrasound was transmitted across the pig temporal bone. Despite this, spectrophotometric analysis showed that both IV LEMB and PESDA combined with transtemporal ultrasound resulted in a significant increase in Evans blue extravasation across BBB of the treated side at 30 min after insonation (p < 0.001; compared with ultrasound alone) but not at 120 min. There was significant retention of Gadolinium within the insonified parenchyma at 60 and 90 min after insonation, but not at 120 min. Oxygen saturation and arterial pressures were not changed after any microbubble injection. Intravenous microbubbles, combined with transtemporal ultrasound, can transiently increase BBB permeability in a large animal. This induced opening of BBB is reversible and may be a safe noninvasive method of achieving drug or gene delivery across the BBB.     

超声微泡造影剂介导EGFP质粒转染大鼠视网膜的实验研究

目的探讨超声破坏微泡介导EGFP质粒转染大鼠视网膜的效率及可行性，为实现外源基因高效、定向的转移目的奠定基础。方法将30只Long-evans大鼠分为6组，第1组仅以0．5w／cm。的超声波辐照大鼠眼球，第2组于尾静脉输入适当剂量的微泡造影剂，并立即以相同能量的超声波辐照大鼠眼球，第3组于尾静脉输入质粒，第4组于尾静脉输入质粒，并以超声辐照大鼠眼球，第5组于尾静脉输入质粒与微泡，第6组尾静脉输入质粒、微泡，并用超声辐照眼球。转染2周后，在激光共聚焦显微镜下观察EGFP表达情况。结果超声微泡介导的EGFP质粒对大鼠视网膜的转染效率，明显高于其他实验组。一定能量和时间的超声波辐照，及适当浓度的微泡，对大鼠视网膜脉络膜无明显损伤。结论利用低频率和一定能量的超声击碎携带EGFP质粒的超声微泡造影剂，能够有效地提高EGFP质粒在大鼠视网膜的转染效率。