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。     

超声微泡介导pGPU6/Neo质粒转染神经干细胞的实验研究

目的 探讨超声微泡介导pGPU6/Neo 质粒转染神经干细胞(NSCs)的效率及可行性.方法 以一定能量的超声介导质粒转染大鼠NSCs,分为:空白组、质粒组、质粒+微泡组、质粒+超声组、质粒+微泡+超声组,并按不同转染条件分为亚组.转染48 h后荧光显微镜观察转染效率,台盼蓝染色法检测细胞活性.结果 最优参数为声强1....     

超声靶向微泡介导PEDF质粒转染小鼠肝癌的实验研究

目的探讨超声靶向微泡介导色素上皮源性因子(PEDF)质粒转染HepG2荷瘤裸鼠的治疗价值。方法建立裸鼠HepG2肝细胞肝癌模型,随机分为5组。Ⅰ组:对照组;Ⅱ:PEDF质粒组;Ⅲ组:PEDF质粒+微泡组;Ⅳ组:微泡+超声辐照组;Ⅴ组:PEDF质粒-微泡+超声辐照组。疗程结束后观察瘤结节的超声造影特征,计算抑瘤率,并检测瘤组织PEDF蛋白表达。应用SPSS软件分析各组间各参数的差异。结果疗程结束后,Ⅴ组瘤结节超声造影增强明显弱于Ⅰ组;Ⅳ组及Ⅴ组抑瘤率明显高于Ⅱ、Ⅲ组;Ⅴ组瘤组织PEDF蛋白表达高于其他各组。结论超声靶向微泡介导PEDF质粒有助于提高质粒转染率,抑制肿瘤血管生成及肿瘤生长,为肝细胞肝癌的治疗提供了一个新的途径。     

超声微泡介导EGFP转染正常大鼠关节滑膜组织的研究

目的 探讨超声微泡介导EGFP转染大鼠关节滑膜组织的可能性.方法 24只正常清洁级Wister大鼠;4只为单纯对照组,20只为实验组.实验组根据质粒的剂量又分4组,每组5只(10个膝关节),1组:超声+微泡+1μg EGFP;2组:超声+微泡+5μgEGFP;3组:超声+微泡+10μg EGFP;4组:超声+微泡+15μg EGFP.将300μl SonoVue与EGFP混合,注射入大鼠关节腔内,超声持续照射10 min.3 d后取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 1μg和5μg EGFP组与单纯对照组比较,荧光强度稍有增强,但是不明显;而10μg和15μg组荧光强度与单纯对照组比较均有明显增强.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,10μg EGFP是本实验的最佳剂量.     

携抗ICAM-1靶向微泡定向转染Ang-1基因治疗急性心肌梗死

研究背景 基因治疗为难治性心肌缺血提供了新方法,但通过静脉途径导入体循环的外源性基因缺乏组织特异性,难以在靶组织局部停留,疗效较低。目的 利用缺血时心肌内皮细胞黏附分子-1(ICAM-1)表达增高的特点,探讨心肌梗死时利用抗ICAM-1靶向微泡能否提高血管生成素-1基因在体内的转染效率。方法 体外实验检测抗ICAM-1靶向微泡与经人白细胞介素-1β刺激后的ECV304细胞结合的程度。体内实验分三组:①对照组:hAng-1基因+超声照射;②非靶向微泡组:携hAng-1基因微泡+超声照射;③靶向微泡组:携hAng-1基因的抗ICAM-1靶向微泡+超声照射。制作兔急性心肌梗死模型,分别用上述三种方法从静脉导入hAng-1基因,各组动物均于梗死模型建立后当天和基因转染2周后进行常规超声检查和心肌造影;2周后心肌造影检测转染区心肌灌注状况,RT-PCR检测hAng-1基因的mRNA 表达以评价转染疗效。结果 携抗ICAM-1抗体的微泡在体外能大量靶向结合到产生炎性反应的ECV304内皮细胞周边。应用靶向微泡转染后缺血心肌2周后,左心室内径减小,左心室射血分数升高,但与非靶向微泡组无显著性差异,而心肌造影显示心肌内造影剂填充量较非靶向微泡组增多,RT-PCR定量灰度分析hAng-1mRNA高于非靶向微泡组(1.04±0.08vs0.53±0.04,P<0.01)。结论 微泡与抗ICAM-1抗体连接后形成靶向微泡载体,可明显提高hAng-1基因在体内的转染效率,增加心肌梗死后局部的血管新生效应。     

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.     

超声破坏SonoVue微泡介导Ang-1基因的体外体内转染

目的 探讨超声破坏微泡造影剂在体外及体内介导Ang-1基因转染的效率及其促血管新生的作用.方法 293T细胞悬液分为3组:A组(微泡+超声+质粒组),B组(单纯超声照射+质粒组)及C组(对照组,每孔仅加入目的基因质粒);转染后48 h用荧光显微镜和流式细胞仪分别定性、定量检测基因的转染效率,PCR及Western-blot分别检测细胞转染后的目的基因表达与蛋白翻译.27只健康成年中国家兔行急性前壁心肌梗死造模后随机分为3组:组Ⅰ(超声辐照+微泡+质粒组)、组Ⅱ(超声辐照+质粒组)、组Ⅲ(对照组,心肌梗死后不做任何处理);于基因转染前后分别行心肌超声造影(MCE)检查,并于2周后处死取心肌组织行PCR及Western blot检测Ang-1 mRNA及其蛋白的表达,心肌组织Ⅷ因子染色检测其新生毛细血管密度.结果 48 h后可在荧光显微镜下观察到A组及B组转染成功的细胞胞浆内发出绿色荧光,其中C组未见明显绿色荧光表达,A组的绿色荧光表达量明显高于B组;流式细胞仪检测A组转染效率较B组显著提高(P＜0.01).体内转染中,心肌超声造影可见组Ⅰ转染前充盈缺损处出现片状造影剂回声,PCR可检测出Ang-1 mRNA表达,Western blot可检测出目的基因的蛋白产物,余各组则无明显造影剂充填,PCR、Western blot均无法检测出Ang-1基因及其蛋白的表达;心肌毛细血管计数显示组Ⅰ新生毛细血管数量显著提高,余各组仅见少量新生毛细血管.结论 超声破坏微泡造影剂可明显提高Ang-1基因的体外及体内转染效率,具有良好的促血管新生作用.     

超声辐照声诺维与重组腺相关病毒经不同途径转染大鼠视网膜的实验研究

目的 探讨超声辐照微泡造影剂声诺维(SonoVue)能否增强rAAV2-EGFP转染视网膜的效率.方法 62只Wistar大鼠进行玻璃体腔(10只)及视网膜下(52只)注射.实验分组:病毒+生理盐水、病毒+微泡、病毒+生理盐水+超声辐照和病毒+微泡+超卢辐照.第4、7、35、49、120 d分别用荧光体视镜观察,利用软件进行定量分析,并进行组织切片光镜检查.结果 玻璃体腔注射组持续观察2个月未见荧光;视网膜下注射组的超声辐照微泡组在第4、7、35 d较其他组转染效率高,超声辐照微泡造影剂对眼底组织无明显损伤.结论 超声辐照SonoVue可在体内促进rAAV2-EGFP转染大鼠视网膜.     

超声波与微泡声学造影剂增强基因定位转染动物实验研究

目的观察经静脉注射基因与微泡声学造影剂,同时经皮超声辐照肝脏方式能否增强小鼠肝脏基因定位转染及其转染效率.方法昆明种小白鼠24只,随机分为4组,每组6只.第1组为单纯质粒转染组:经尾静脉快速注入2 ml含20 μg乙型肝炎核心抗原(pcDNA3.1/HBV)质粒的生理盐水溶液.第2组为单纯微泡转染组:经尾静脉快速注入2 ml含20 μg质粒的微泡声学造影剂.第3组为单纯超声转染组:经尾静脉快速注入2 ml含20 μg质粒的生理盐水溶液,同时采用频率为1 MHz、声强为0.5 W/cm2的超声波经小白鼠体表肝区辐照1 min.第4组为超声与微泡转染组:经尾静脉快速注入2 ml含20 μg质粒的微泡声学造影剂,同时经小白鼠体表肝区局部采用相同剂量超声辐照1 min.7天后处死小鼠,分别取肝、肾、肺、心、脾、骨骼肌组织进行pcDNA3.1/HBV免疫组化检测,记录不同组织每高倍视野(×400)pcDNA3.1/HBV表达阳性细胞数.结果第1、2组肝、肾组织内偶见pcDNA3.1/HBV表达弱阳性细胞,肺、心、脾、骨骼肌组织未见表达阳性细胞.第3组肝组织内可见少量pcDNA3.1/HBV阳性细胞,每高倍视野表达阳性细胞(26.5±3.9)个.肾、肺、心、脾、骨骼肌组织未见表达.第4组肝组织内pcDNA3.1/HBV表达最强,每高倍视野表达阳性细胞(84.2±4.4)个,为第3组的约3.2倍.肾、肺、心、脾、骨骼肌组织未见表达.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,能显著增强辐照部位局部组织的基因转染与表达,有望成为一种安全、高效的体内基因定位转染新技术.     

Highly efficient gene transfer into murine liver achieved by intravenous administration of naked Epstein-Barr virus (EBV)-based plasmid vectors

Naked plasmid DNA (pDNA) injection could become an alternative procedure to viral and nonviral gene delivery systems. We have previously shown that Epstein-Barr virus (EBV)-based plasmid vectors containing the EBV nuclear antigen 1 (EBNA1) gene and the oriP sequence enable quite high and long-lasting expression in various in vitro and in vivo transfection systems. The EBV-based plasmids were intravenously injected into mice via their tail vein under high pressure. A large amount of the marker gene product was expressed in the liver; as much as 320 microg of luciferase was demonstrated per gram of liver at 8 to 24 h after a single injection with 10 microg of DNA. More than 70% of liver cells stained with X-gal when beta-gal gene was transferred. The expression level was significantly higher than that obtained by conventional pDNA lacking the EBNA1 gene and oriP. On day 35 after the transfection, the expression from the EBV-based plasmid was approximately 100-fold stronger than the conventional pDNA gene expression. Both the EBNA1 gene and oriP are a prerequisite for the augmentation of the transfection efficiency. These results suggest that the intravascular transfection with naked EBV-based plasmid may provide a quite efficient, simple and convenient means to transduce therapeutic genes in vivo into the liver.     

Intrapericardial delivery of fibroblast growth factor-2 induces neovascularization in a porcine model of chronic myocardial ischemia

Therapeutic angiogenesis is a novel approach to the treatment of myocardial ischemia based on the use of proangiogenic growth factors to induce the growth of new blood vessels to supply the myocardium at risk. This study was designed to assess the safety and efficacy of a single intrapericardial injection of basic fibroblast growth factor (FGF-2) in a porcine model of chronic myocardial ischemia. Yorkshire pigs underwent ameroid placement around the left circumflex coronary artery. At 3 weeks, animals were randomized to receive a single intrapericardial injection of either saline (n = 10), 3 mg of heparin (n = 9), 3 mg of heparin + 30 microgram of FGF-2 (n = 10), 200 microgram of FGF-2 (n = 10), or 2 mg of FGF-2 (n = 10). Coronary angiography, microsphere flow, magnetic resonance functional, and perfusion imaging were performed before and 4 weeks after treatment, at which time histologic analysis was also performed on 3 animals in each group. In ischemic pigs, FGF-2 treatment resulted in significant increases in left-to-left angiographic collaterals and left circumflex coronary artery blood flow. These benefits were accompanied by improvements in myocardial perfusion and function in the ischemic territory, as well as histologic evidence of increased myocardial vascularity without any adverse effects. Not one of these benefits was seen in saline- or heparin-treated ischemic animals. A single intrapericardial injection of FGF-2 in a porcine model of chronic myocardial ischemia results in functionally significant myocardial angiogenesis, without any adverse outcomes. This mode of FGF-2 administration may prove to be a useful therapeutic strategy for the treatment of patients with ischemic heart disease.     

犬脐血干细胞肌源性诱导分化移植对细胞间连接的影响

背景:脐血间充质干细胞诱导分化为心肌细胞后移植入心肌缺血组织,可以通过与宿主细胞建立联系来修复取代缺血心肌组织.目的:对犬脐血干细胞进行肌源性诱导,观察其植入后与宿主细胞间的连接情况.设计、时间及地点:随机对照动物实验,于2006-07/2007-10在上海交通大学医学院附属新华医院动物实验中心完成.材料:接近足月的妊娠杂种犬2只,用于分离培养脐血间充质干细胞.成年杂种犬36只,随机数字表法分为细胞移植组、模型对照组,18只/组.方法:取传至第4代的犬脐血间充质干细胞,加入含lacZ基因的重组腺相关病毒载体进行基因转染,继续培养3 d后,行10 μmol/L5-氮杂胞苷肌源性诱导,常规培养3周.两组犬均建立心肌梗死模型,造模后细胞移植组经冠状动脉和局部注射将2 mL细胞悬液(约107个脐血间充质干细胞)移植到梗死区,模型对照组同法注射等量生理盐水.分别于移植后2,4,8周取材制备心肌组织切片,免疫组化检测细胞间连接情况.主要观察指标:脐血间充质干细胞的基因转染、肌源性诱导分化情况,移植细胞与宿主心肌细胞的细胞间连接情况.结果:转染72 h后大部分细胞表达LacZ基因,并合成半乳糖苷酶,X-gal染色呈蓝色.5-aza诱导3周后,脐血干细胞α-Actin(+),Desmin(+),Connexin43(+),而诱导前均呈阴性.移植后第8周,心肌切片中移植细胞和宿主心肌相连处可形成连接,连接处可见cadherin和connexin43绿色荧光阳性表达;模型对照组仅表达cadherin和connexin43,未见带红色荧光的移植细胞.结论:脐血间充质干细胞可在体外诱导为心肌样细胞,移植后能与宿主心肌可能形成有效连接,从而具有通讯联系.     

Efficiency of eight different AAV serotypes in transducing rat myocardium in vivo

Recombinant adeno-associated (AAV) viruses have unique properties, which make them ideal vectors for gene transfer targeting the myocardium. Numerous serotypes of AAV have been identified with variable tropisms towards cardiac tissue. In the present study, we investigated the time course of expression of eight different AAV serotypes in rat myocardium and the nature of the immunity against these serotypes. We first assessed whether neutralizing antibodies (NAb) were present for any of the serotype in the rats. We injected 100 microl of each AAV 1-8 serotype (10(12) DNAse resistant particles/ml), encoding LacZ gene, into the apical wall of rat myocardium. At 1, 4, 12 and 24 weeks after gene delivery, the animals were killed and beta-galactosidase (beta-gal) activity was assessed by luminometry. Additionally, LacZ genomic copies and AAV capsids copies were measured through standard polymerase chain reaction analysis and cryo-sections from the area of viral injection were stained for X-gal detection at the same time points. No NAbs were detected against any of AAV serotypes. At all the time points studied, AAV1, 6 and 8 demonstrated the highest efficiency in transducing rat hearts in vivo. Parallel to the results with beta-gal activity, the highest levels LacZ and AAV DNA genomic copies were with AAV1, 6 and 8. The positive X-gal staining depicted by these serotypes confirmed these results. These results indicate that among the various AAV serotypes, AAV1, 6 and 8 have differential tropism for the heart unaffected by pre-existing NAb in the rat. Although AAV 1 and 6 vectors induced rapid and robust expression and reach a plateau at 4 weeks, AAV 8 continued increasing until the end of the study. AAV 2, 5 and 7 vectors were slower to induce expression of the reporter gene, but did reach levels of expression comparable to AAV1 and AAV6 vectors after 3 months.     

携抗ICAM-1靶向微泡定向转染Ang-1基因治疗急性心肌梗死的实验研究

目的 探讨携抗ICAM-1的靶向微泡定向转染基因至心肌细胞的能力及其作为新型载体治疗急性心肌梗死的可行性.方法 37只家兔制备急性心肌梗死模型.3只经耳缘静脉注射携ICAM-1抗体靶向微泡,心肌组织冷冻切片后观察其对受损血管内皮的吸附.余34只兔随机分为3组:IM组(心肌注射Ang-1基因组)、ICAM-1组(注射携抗ICAM-1靶向微泡+Ang-1基因组)、对照组(空白对照),于基因转染前后分别行常规超声心动图检查,处死后取心肌组织及ICAM-1组家兔肝、肾组织行RT-PCR及Western-Blot分别检测目的 基因及蛋白表达;免疫组化Ⅷ因子染色检测其新生毛细血管密度.结果 IM组、ICAM-1组基因转染后2周左室射血分数(LVEF)均较转染前显著提高(P＜0.05),其中IM组及ICAM-1组之间差异无统计学意义;IM组及ICAM-1组行RT-PCR及Western-Blot均可检测出目的 基因及蛋白表达,且两组之间表达量差异无统计学意义;对照组及ICAM-1组肝肾组织未见明显Ang-1 mRNA及蛋白表达.心肌组织Ⅷ因子染色后高倍镜下显示IM组及ICAM-1组均可见大量新生毛细血管,其中相对于ICAM-1组,IM组新生毛细血管计数更高.结论 携抗ICAM-1的靶向微泡可定向转染Ang-1基因至心肌细胞,其转染效率与目前认为最高效的心肌内注射基因的转染效率近似,可作为新型靶向载体定向转染血管新生基因,对急性心肌梗死具有治疗作用.

Abstract：

Objective To explore the capability of Ang-1 gene delivery to acute myocardial infarction using targeted microbubbles carrying ICAM-1 antibody.Methods Thirty-seven rabbits' left circumfles branch coronary arteries were ligated for models.Three rabbits were injected with microbubbles carrying ICAM-1 to detect the ability of targeting.Thirty-four rabbit models were divided into 3 groups randomly as follow:IM group (n=12,accept direct intramuscular injection),ICAM-1 group (n=12,accept intravenous injection of targeted microbubbles and Ang-1) and control group (n=10,without any treatment).Ultrasonography were executed on all animals before and 2 weeks after the treatment.All rabbits were killed after 2 weeks and examined for Ang-1 mRNA and protein by RT-PCR and Western-Blot respectively.Microvessel density (MVD) counting of infracted myocardium,observed by factor Ⅷ immunochemical staining,was performed to value the proangiogenesis effect of Ang-1 delivered by targeted microbubbles carrying ICAM-1 antibody.The liver and the kidney in ICAM-1 group were taken to assess the systemic delivery.Results IM and ICAM-1 group showed significantly improvement in the ejection fraction (P＜0.05) while control group did not.Ang-1 mRNA and protein could be detected in IM and ICAM-1 group;however,the expression between the two groups showed no siginificant difference.None of the control animals showed Ang-1 expression.compared with ICAM-1 group,the MVD was greater in IM group.Ang-1 was not detected either in liver or in kidney in ICAM-1 group.Conclusions Targeted microbubbles carrying ICAM-1 antibody can deliver Ang-1 gene to ischemic myocardium directly.Meanwhile,it's as effective as IM injections besides the greater angiogenesis effect.This strategy improves the perfusion of acute myocardial infarction and the function of heart.     

Transthoracic direct current shock facilitates intramyocardial transfection of naked plasmid DNA infused via coronary vessels in canines

Catheter-mediated, percutaneous, transluminal delivery of naked plasmid DNA (pDNA) into myocardium may offer a valuable strategy to heart diseases. Here, we examined whether clinically available transthoracic direct current (DC) shock improves intracoronary naked DNA transfection into myocardium. Plasmid vector encoding the GL3 luciferase was infused retrogradely into the coronary veins of beagle dogs, whereas another pDNA solution was infused into the left coronary artery. During and after these procedures, the coronary venous sinus was occluded by balloon, and transthoracic DC shock of 200 J was applied immediately after the infusions. Without DC shock, no remarkable increase in luciferase activity was demonstrated in any part of the left ventricular myocardium. In the presence of DC pulsation, significant luciferase expression was detected in the regions that were supplied by left anterior descending coronary artery (LAD), whereas the gene expression in the right coronary artery (RCA) regions was much less drastic. X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) staining of cardiac cross-sections also revealed regional expression of beta-galactosidase. Immunohistochemical examinations of heart cryosections revealed that cardiomyocytes in LAD regions successfully expressed transgene product. The present system may enable a new strategy for myocardial gene therapy, without any special device or technique other than cardiac catheterization and DC cardioversion that are generally performed in ordinary hospitals.     

超声心动图对经心内膜注射VEGF165质粒治疗慢性心肌缺血的疗效评价

目的运用超声心动图评价经心内膜注射血管生长因子165（VEGF165）质粒治疗慢性心肌缺血的疗效。方法20只实验用猪建立心肌梗死模型,随机分成两组,即VEGF治疗组（7只）和空质粒对照组（8只）。梗死模型建立3个月后,采用左心室心内膜电机械标测系统进行经心内膜心肌内质粒注射实验。分别于质粒注射前、注射后3个月进行超声心动图指标检测。同时进行心内膜电机械标测、冠状动脉造影随访及组织病理分析。结果质粒注射治疗后3个月,与对照组相比,治疗组左室前壁局部室壁运动幅度、背向散射积分的心动周期变异和左室射血分数等均显著升高,测值差异有统计学意义（P〈0.05）。冠状动脉造影显示心肌缺血区侧支循环形成明显,闭塞部位远端血管充盈良好;心内膜电机械标测表明心肌缺血状况改善;病理结果证实新生血管数目多于对照组。结论经心内膜注射VEGF质粒治疗慢性心肌缺血可促进缺血区血管生成,改善左室功能。超声心动图为质粒注射治疗心肌缺血疗效评价提供了有效的方法。     

胰岛素受体在糖尿病大鼠心肌组织中的表达

目的 通过研究胰岛素受体（insulin receptor，IR）在链脲佐菌素诱导的糖尿病大鼠模型中心肌组织中的表达，探讨IR与糖尿病性心肌病发病机制的关系。方法 成年Sprague—Dawley雄性大鼠20只，成实验组和对照组。实验组用链脲佐菌素诱导产生糖尿病模型，4周后对各组大鼠心肌进行IR免疫组织化学染色，观察IR在糖尿病大鼠和正常大鼠左心室心肌细胞中的表达差异。结果 发现糖尿病大鼠中心肌间质纤维增生，心肌间微血管壁增厚，IR表达阳性心肌细胞的数目为（9．42±2．32）个／mm^2及其光密度均为（66．58±6．32）较正常对照组减少（t分别：17．24、8．75，P均〈0．05）。结论 糖尿病心肌病可作为一种特异性的病变发展为心肌自主神经系统的紊乱和冠脉血流储备的障碍。IR表达阳性心肌细胞减少可能会造成心肌糖代谢的损害与心力衰竭的发生。     

高原环境对移居大鼠子代主要脏器指数及心脏形态学的影响

目的探讨移居高原大鼠子代在高原环境中主要脏器发育状况,观察移居高原大鼠子代心脏组织形态学改变。方法将24只(18只雌鼠,6只雄鼠)8周龄wistar大鼠平均分为高原组和平原组,每组12只(9只雌鼠,3只雄鼠)按3:1雌雄比例合笼受孕,所有孕鼠均自然分娩。比较高原组和平原组子代大鼠出生后的第1天、第30天、第60天心、肝、脾、肺、肾、脑的脏器指数,取心脏组织做HE染色检测病理学变化。结果高原组子代大鼠出生第1天体重低于平原组,分别为6.20±0.77g,6.60±0.90g(P<0.05)。第60天高原组子代大鼠心脏指数高于平原组,分别为0.76±0.11,0.61±0.01(P<0.05)。高原组子代大鼠心肌组织HE染色可见心肌间质内有炎性细胞浸润,心肌细胞结构破坏,胞核消失,胞质溶解,均质红染,部分心肌纤维呈不规则断裂,出现大小不等的空泡,心肌间隙扩大,小动脉玻璃样变。结论高原环境对移居孕鼠子代心脏的发育有影响,心肌组织病理改变明显,可能与高原低压、低氧有关。     

超声造影剂及超声辐照对TIMP-1 siRNA在肝纤维化大鼠肝组织中表达的影响

目的观测超声造影剂及超声辐照对TIMP-1 siRNA质粒转染肝纤维化大鼠肝组织内TIMP-1基因表达的影响。 方法采用二甲基亚硝胺（DMN）方法建立大鼠肝纤维化模型，72只造模大鼠在第6周随机均分成3组，质粒组（P组）、质粒＋造影剂及超声照射组（P＋U组）、对照组。2d、6d及12d后各组分别随机取8只处死、取肝、肾、肺、脑、心肌组织快速冷冻切片，荧光显微镜下观察各组织内TIMP-1 siRNA荧光表达；FQ—PCR测定肝组织TIMP-1 mRNA表达。 结果2d后，P组肝、肾、肺、脑、心肌组织内均有少量TIMP-1 siRNA绿色荧光表达，组间无显著差异，P＞0．05；P＋U组肝组织内TIMP-1 siRNA荧光表达明显增强，较其它组织差异显著，P＜0．001。P组及P＋U组肝组织TIMP-1 mRNA表达量均较对照组明显降低，P＜0．001；P＋U组降低较P组显著，P＜0．001；P＋U组6d时TIMP-1 mRNA表达量最低，后依次为48h、12d。 结论TIMP-1 siRNA对肝纤维化大鼠TIMP-1基因表达抑制作用明显；超声造影剂及超声辐照能够明显增高基因转染效率，并有将基因载体靶向定位于肝组织释放的作用。