经心包腔转染腺病毒血管内皮生长因子165基因对猪冠脉血管形成的影响

目的:探讨以腺病毒为载体的血管内皮生长因子165(Ad-VEGF165)基因经心包腔转染的表达规律、合适的剂量及对缺血心肌血管生成的作用.方法:随机将20头中华小型猪分为实验组和对照组,每组10头.采用球囊堵塞前降支第一对角支远端建立心肌梗死模型.构建后即刻,实验组采用经皮剑突下穿刺,中心静脉导管插入心包腔内,以含胶原酶1 200 u及透明质酸酶3 000 u的生理盐水2 ml预处理心包后,在心包腔内注入含Ad-VEGF165基因2.0×109pfu的生理盐水1 ml;对照组同样方法预处理心包后,在心包腔内注射生理盐水1 ml.注射后3 d(n=2)、7 d(n=2)及28 d(n=6)分别用免疫组化、超声心动图检测缺血心肌血管新生情况及心功能,并以酶联免疫吸附试验(ELISA法)检测血浆及心肌组织中Ad-VEGF165的表达.结果:Ad-VEGF165基因经心包腔转染缺血心肌组织后,在心肌组织中呈高表达,于7 d达到高峰,28 d降至基线水平,血浆中无目的基因的表达;28 d时,实验组缺血心肌微血管密度(MVD)、心功能均明显高于对照组(MVD(517.00±75.70)mm-2vs(226.50±54.10)mm-2,P=0.009;LVEF(%)(72.11±5.20)w(55.14±4.37),P=0.005).结论:用胶原酶及透明质酸酶预处理心包后,经其转染Ad-VEGF165可以诱导急性心肌梗死模型局部VEGF蛋白表达,促进缺血心肌组织血管新生并能改善心功能.     

重组腺病毒血管内皮生长因子165基因心包腔与冠状动脉转染猪心肌效果比较

目的:比较经心包腔与冠状动脉转染重组腺病毒血管内皮生长因子165(Ad-VEGF165)基因的效果.方法:20头小型猪随机等分为心包转染组和冠状动脉转染组,2组均采用球囊堵塞前降支第一对角支远端,心包组心肌梗死模型建立后即刻,采用经皮剑突下穿刺中心静脉导管心包腔内转染,胶原酶和透明质酸酶预先处理心包,然后注射Ad-VEGF165 1.0 ml(2.0×109pfu);冠状动脉组心肌梗死模型建立后即刻,经冠状动脉注射Ad-VEGF1651.0 ml(2.0×109pfu),于注射后3 d、7 d、28 d分别测定2组心肌组织内VEGF水平、微血管密度、心功能.结果:心包转染组和冠状动脉转染组的心肌组织均表达有VEGF165基因,组织内VEGF水平在7 d时达高峰,28 d时降至基线水平,心包转染组高于冠状动脉转染组((702±85)ng/Lvs(592±59)ng/L,P=0.026).2组微血管密度随转染时间增加,心包转染组心功能优于冠状动脉转染组(FS/%:32.9±2.2vs 30.6±2.1,P=0.049;EF/%:72.11±5.20vs65.87±2.16,P=0.034).结论:导管介导的心包腔与冠状动脉转染Ad-VEGF165基因治疗心肌缺血是有效、切实可行的,而前者可能是更有前途的新方法.     

心包穿刺简易装置在基因转染中的应用

目的探讨自制心包穿刺装置转染心脏的安全性、可行性。方法应用磷酸钙沉淀方法制备携带大肠杆菌LacZ基因复制缺陷的重组腺病毒(Ad-LacZ),将12头中国小型猪分为实验组和对照组,采用球囊堵塞前降支第一对角支远端,心肌梗死模型建立后即刻,采用自制简易心包腔穿刺装置经皮剑突下穿刺,成功后置中心静脉导管于心包腔内并行转染,28 d后处死。实验组:胶原酶1200 U及透明质酸酶3000 U预处理心包后,在心包腔内注射Ad-LacZ基因2.0×109p.f.u;对照组:同样方法预处理心包后,在心包腔内注射生理盐水1 mL。于注射后3、7及28 d分别对缺血心肌进行染色及病理观察。结果冠状动脉造影证实前降支远端完全闭塞,病理显示心肌有缺血和梗死;实验组注射Ad-LacZ基因后第3、第7天及28d后X-gal染色有阳性细胞,以第7天最明显,对照组无阳性细胞。结论自制的心包腔简易穿刺装置将腺病毒载体转染至缺血心肌是安全的,可行的,并且腺病毒可持续表达4周。     

复制缺陷的重组腺病毒经心包腔转染急性心肌梗死猪观察

目的:评价重组腺病毒经心包腔转染的效率及持续时间.方法:应用磷酸钙沉淀方法制备携带大肠杆菌LacZ基因复制缺陷的重组腺病毒(Ad-LacZ).12头中国小型猪随机等分为实验组和对照组,采用球囊堵塞前降支第一对角支远端,心肌梗死模型建立后即刻采用经皮剑突下穿刺中心静脉导管心包腔内注射转染,28 d后处死.实验组胶原酶1 200 u及透明质酸酶3 000 u预处理心包后,在心包腔内注射Ad-LacZ基因2.0×109噬斑集落形成单位;对照组:同样方法预处理心包后,在心包腔内注射生理盐水1 ml.于注射后3 d、7 d及28 d分别对缺血心肌进行HE、HBFP及X-gal染色.结果:冠状动脉造影证实前降支远端完全闭塞,病理学检查显示心肌有缺血和梗死.实验组注射Ad-LacZ基因后第3 d、7 d及28 d后X-gal染色有阳性细胞,以7 d最明显,对照组28 d内均未发现阳性细胞.结论:应用球囊堵塞法可成功建立猪急性心肌梗死模型,胶原酶及透明质酸酶预处理心包后,腺病毒载体可转染缺血心肌,并持续表达4周.     

腺病毒介导的血管内皮生长因子基因转移对帕金森病大鼠多巴胺能神经元的保护作用

目的探讨血管内皮生长因子(VEGF)基因转移对帕金森病(PD)大鼠的保护作用。方法携带VEGF165基因的腺病毒(Ad-VEGF165)感染PD模型大鼠纹状体后,采用大鼠旋转行为观察、免疫组织化学方法和高效液相色谱技术检测VEGF165基因转移的PD大鼠行为学变化、黑质纹状体酪氨酸羟化酶(TH)、层黏蛋白(laminin)和胶质纤维原性酸性蛋白(GFAP)的表达以及纹状体区多巴胺(DA)及代谢产物含量变化。并与重组腺病毒载体(Ad-Lacz)组及磷酸盐缓冲液(PBS)组进行比较。结果Ad-VEGF165成功感染PD大鼠并高表达目的基因和蛋白。感染Ad-VEGF165的大鼠获得行为学改善,其黑质和纹状体TH阳性细胞数和纤维密度与对侧半球的比值(0．42±0．11和0．56±0．10)高于Ad-LacZ组(0．20±0．10和0．28±0．09)和PBS组(0．22±0．13和0．24±0．08),P<0．01;纹状体区血管和胶质细胞数量均明显增多;Ad-VEGF165感染组大鼠纹状体区DA及代谢产物含量与对侧比值也高于对照组。结论Ad-VEGF基因转移能保护PD大鼠多巴胺能神经元,血管和胶质细胞的增生可能参与了VEGF对在体多巴胺能神经细胞的保护机制。     

rAAV2VEGF165与rAAV2ANG-1联合转染促猪缺血心肌血管生成的研究

目的:探讨联合应用血管内皮细胞生长因子(VEGF165) 及血管生成素(ANG-1),对猪慢性缺血心肌中血管生成效应及心功能改善的影响.方法:完全腔镜下放置血管缩窄环于小型猪左冠状动脉回旋支(LCX),建立慢性心肌缺血模型.6周后行心电图、冠状动脉造影和心脏超声检查,确认LCX闭塞或相应心肌的缺血.16只动物随机均分为:单纯注射转染VEGF165组(组Ⅰ,n=4) 、单纯注射转染ANG-1组(组Ⅱ,n=4)、联合注射转染VEGF165与ANG-1组(组Ⅲ,n=4)和注射磷酸盐缓冲液PBS对照组(组Ⅳ,n=4).各组在胸腔镜下,心肌内直接注射rAAV2 ANG-1、rAAV2 VEGF165,剂量为5×1011 vg(virus/genome).治疗后,不同时间点ELISA检测VEGF165和ANG-1蛋白的分泌,冠状动脉造影观察侧枝循环形成的情况,行心脏超声检查观察左心室功能变化.3个月后取注射部位心肌,用Western blot检测VEGF165和ANG-1蛋白的表达情况,免疫组织化学观察心肌毛细血管密度与微小动脉生成情况.结果:放置血管缩窄环后6周,所有动物均出现LCX完全/次全闭塞,或LCX支配区域的心肌缺血.Ⅰ、Ⅲ两组猪体内的VEGF165蛋白分泌水平从基因转染后第7天开始上升,并于第14天达到高峰,随后逐渐下降,尤以Ⅰ组分泌水平为高;其它两组各时间点及组间的VEGF165蛋白分泌水平差异无统计学意义(P>0.05).同样,Ⅱ、Ⅲ两组猪体内的ANG-1蛋白分泌水平的变化与VEGF165蛋白分泌水平类似,尤以Ⅱ组分泌水平为高(P<0.01);其它两组各时间点及组间的ANG-1蛋白分泌水平差异无统计学意义;组Ⅲ中VEGF165与ANG-1蛋白水平变化同步.Western blot检测显示,基因治疗后3个月,组Ⅲ VEGF165、ANG-1蛋白水平都显著高于组Ⅳ(P<0.01),组Ⅲ与组Ⅰ间VEGF165蛋白表达无显著差异(P ＞0.05),组Ⅲ与组Ⅱ间ANG-1蛋白表达差异无统计学意义(P＞0.05).基因治疗3个月后,组Ⅲ左心室室腔缩小,心肌收缩力增强,LCX及其分支较基因转染前增粗、迂回,左回旋支发出侧支,左回旋支远端重新有造影剂充填.组Ⅰ、Ⅱ则有不同程度心肌收缩力增强,充盈改善,但均较组Ⅲ改善幅度小,回旋支见少许造影剂充填,但较组Ⅲ明显少,组Ⅳ处理前后无明显改变.超声检查显示,模型建立术后6周,左心室下壁及后壁变薄,局部运动减弱、不协调,收缩异常;基因转染后1个月、3个月,组Ⅰ、组Ⅱ、组Ⅲ心功能都较转染前有明显改善,组Ⅳ无明显变化;组Ⅲ 的EF与FS改善程度要明显好于其他各组.CD34免疫组化染色评价血管密度,组Ⅲ缺血区血管计数高于组Ⅰ、组Ⅱ,差异有统计学意义(P<0.05),对照组Ⅳ的心肌内毛细血管数目很少.应用α-SMA免疫组化染色评价小动脉形成,结果提示,基因转染3个月后组Ⅳ心肌中小动脉密度最低,其余各组心肌中均有较多的小动脉新生,尤以组Ⅲ最为显著.结论:完全腔镜下放置血管缩窄环及完成心肌内注射切实可行,在胸腔镜下建立的小型猪慢性心肌缺血模型中,心肌内注射rAAV2 VEGF165 、rAAV2 ANG-1可以有效地转染小型猪心肌组织,外源基因长期稳定表达,同时ANG-1能协同VEGF165作用,从而显著地提高其促进缺血心肌毛细血管和小动脉生成,促进有效侧枝循环形成并改善心功能.     

腺病毒载体介导的VEGF165基因治疗大鼠缺血皮瓣的实验研究

目的 探讨局部应用以腺病毒为载体的血管内皮生长因子(Ad-VEGF165)基因治疗对大鼠缺血皮瓣生存的影响.方法 选用SD大鼠30只,体重350～400 g,随机分成3组,每组10只,在其背部形成8 cm×2 cm的全厚随意型皮瓣.于皮瓣远端皮下分别注射携带血管内皮生长因子基因的腺病毒(Ad-VEGF165目的基因组,简称VEGF165组),携带绿色荧光蛋白基因的腺病毒(Ad-GFP基因组对照,简称GFP组),磷酸盐缓冲液(PBS空白对照组,简称PBS组),48 h后按原设计掀起皮瓣并原位缝合,术后7天,测量皮瓣的成活面积,计算成活面积百分比,并采集成活皮瓣组织行免疫组化、组织学检查等.结果 皮瓣成活面积百分比VEGF165组为(69.4±1.3)%,GFP组为(52.2±1.7)%,PBS组为(52.9±2.9)%,VEGF165组皮瓣成活面积百分比明显高于GFP组和PBS组(P＜0.01).免疫组化显示VEGF165组毛细血管周围VEGF沉积,组织切片微血管密度明显高于GFP组和PBS组(P＜0.01).结论 局部皮下注射Ad-VEGF165可诱导局部VEGF蛋白表达,促进新生血管的形成,提高缺血皮瓣的成活面积,是一种简单有效的基因治疗方法.     

纳米粒子介导血管内皮生长因子基因治疗兔心肌缺血的疗效

目的观察纳米粒子介导血管内皮生长因子(VEGF)基因转染的可行性,了解心肌缺血动物模型经心肌直接注射VEGF基因纳米粒子后新生血管以及心功能改善情况。方法应用聚乳酸聚乙醇酸共聚物(PLGA)和聚乙烯醇(PVA)包载VEGF165基因质粒,制备纳米级粒子混合物,检测其载药量、体外释放情况及粒径;培养乳鼠心肌细胞,转染VEGF165基因纳米粒子(VEGF纳米粒子),应用RT-PCR法检测VEGF mRNA水平,ELISA法检测VEGF蛋白表达水平;将VEGF纳米粒子悬浮液注射至活体家兔心肌内,96h后心肌注射部位取材,电镜观察其向心肌组织内传递基因的可行性;建造兔心肌缺血模型,分别将VEGF纳米粒子(12只)、VEGF165裸质粒(12只)或生理盐水(对照组,8只)注射至缺血部位心肌,1个月后心脏超声监测,然后处死,组织学切片观察心肌组织中毛细血管生成情况。结果制备的纳米粒子载药量为1·87%,粒径为50~300nm;RT-PCR和ELISA结果提示纳米粒子可将目的基因转移至培养的心肌细胞中;体内实验显示心肌细胞胞质内及胞核内可见大量被吞噬的纳米粒子;术后1个月超声检查显示,VEGF纳米粒子组室壁运动幅度(1·87mm±0·32mm)、左室射血分数(60%±10%)较裸质粒组(1·59mm±0·24mm,50%±6%)及对照组(0·93mm±0·40mm,40%±8%)改善更加明显,各组之间差异均有统计学意义(均P<0·05);组织学检查显示,在100倍光镜视野下心肌内毛细血管数VEGF纳米粒子组(57个±12个)明显高于裸质粒组(41个±14个)及对照组(24个±8个),各组之间差异有统计学意义(均P<0·05)。结论纳米粒子可作为VEGF基因向心肌组织转运的载体,在兔心肌缺血模型经心肌直接注射VEGF165基因纳米粒子,能够促进心肌内毛细血管新生,达到改善心功能的目的。     

腺病毒介导的血管内皮生长因子165对扩张型心肌病大鼠心功能的影响

目的:观察经心包腔注射腺病毒介导的血管内皮生长因子165基因(Ad-VEGF 165)对扩张型心肌病(DCM)大鼠心功能的影响并探讨其作用机制。方法:35只雌性SD大鼠腹腔注射多柔比星建立扩张型心肌病模型,随机分为2组:VEGF组(18只)心包腔内注射0.1 mL Ad-VEGF 165 1.0×109pfu;DCM组(17只)注射生理盐水。比较4周后大鼠左室收缩末期容积(LVES)、舒张末期容积(LVED)、左室收缩末期内径(SD)、左室舒张末期内径(DD)、左室内径缩短率(FS)、左室射血分数(LVEF)和体重(BW)、心率(HR)的变化,观察大鼠心肌组织改变情况,ELISA法测定血清APO-1/Fas含量。结果:VEGF组BW、LVEF、HR、FS较DCM组明显增加(P<0.01);LVES、LVED、SD、DD均明显降低(P<0.05),APO-1/Fas水平较DCM组下降(P<0.01);注射VEGF后大鼠BW、LVEF、HR、FS较注射前明显增加(P<0.01);而LVES、LVED、SD、DD则明显降低(P<0.05),大鼠的心肌病变减轻。结论:心包注射Ad-VEGF165基因可改善DCM鼠心功能,降低其死亡率,有利于心肌细胞的存活,其机制可能与减少心肌细胞凋亡有关。     

血管内皮细胞生长因子基因转染血管内皮祖细胞移植促进缺血皮瓣存活的实验研究

目的探讨VEGF165基因转染血管内皮祖细胞(EPCs)移植促进缺血皮瓣的血管新生,提高皮瓣存活率。方法体外分离、培养人脐血中EPCs,利用脂质体介导血管内皮细胞生长因子165(VEGF165)基因体外转染EPCs,然后移植于裸鼠随意皮瓣,皮瓣早期断蒂。结果脐血中分离培养的EPCs表达CD34、KDR及CD133,VEGF165基因转染EPCs体外及体内检测均有VEGF165蛋白的表达。转染VEGF165基因的EPCs组和EPCs组移植裸鼠皮瓣后,EPCs整合到缺血部位新生血管中,与对照组的皮瓣存活率分别为97 2% ±2 8%、60 3% ±2 1%、34 2% ±1 8% (P<0 05 ),而且前二组毛细血管密度、血流灌注差异均有统计学意义(P<0 05),较对照组均有明显改善(P<0 05)。术后第7d时三组皮瓣中的EPCs密度分别为136个/mm2 ±10个/mm2、75个/mm2 ±6个/mm2、0个/mm2 (P<0 05 ); 第11天时EPCs密度分别为305个/mm2 ±26个/mm2、199个/mm2 ±18个/mm2、0个/mm2 (P<0 05)。结论脐血中的EPCs体外培养后移植体内可促进缺血皮瓣的血管新生,提高存活率,而转染VEGF165基因的EPCs具有更强大的促血管新生的作用。     

Efficacy and safety of therapeutic angiogenesis from direct myocardial administr ation of an adenoviral vector expressing vascular endothelial growth factor 165

Objective To investigate whether direct administration of adenoviral vectors (Ad) containing the complementary deoxyribonucleic acid (cDNA) of vascular endothelial growth factor 165 (Ad- VEGF165) induces porcine coronary collateral vessel formation, improves regional myocardial perfusion and function and is safe. Methods Three weeks after miniature swine underwent left thoracotomy and placement of an Ameroid constrictor on the left circumflex coronary artery (LCX), Ad- VEGF165 (n=6) or the control, Ad expressing β- galactosidase cDNA (Ad- Gal, n=6), was directly administered into the ischemic myocardium in the circumflex distribution. All animals were sacrificed 4 wk after the second surgery. Myocardial perfusion and function were assessed by electrocardiogram- gated single photon emission computed tomography (GSPECT) imaging. Ex vivo coronary angiography was performed to examine collateral vessels. Toxicity was assessed by blood analyses on the day just before (day 0) and on day 1, 3, 7, 28 after vector delivery and by vascular, myocardial and liver histology after sacrifice. Results GSPECT imaging 4 wk after administration of Ad- VEGF165 demonstrated significant reduction in ischemic area (P＜0.01) and rest ischemic severity (P＜0.01) and significant improvement in the left ventricular ejection fraction (P＜0.01) and regional wall motion (P＜0.05) compared with that of Ad- Gal and before administration of Ad- VEGF165. Collateral vessel development assessed by coronary angiography was significantly greater in the Ad- VEGF165 group than in the Ad- Gal group (P＜0.05). General safety parameters, including routine blood parameters, liver and kidney function and cardiac specific parameters demonstrated no difference between Ad- VEGF165 and Ad- Gal animals except for the red blood cell count on day 28 (P＜0.05) and blood urea nitrogen on day 7 (P＜0.05).Only transient elevations in creatine phosphokinase (P＜0.05) and aspartate transaminase (P＜0.05) on day 1 were revealed compared with that before vector administration in both groups. Histologically, no atherosclerotic lesion in the circumflex and no inflammation in liver were revealed and only a small myocardial necrosis was observed in one Ad- VEGF165 animal (area≤20%) and one Ad- Gal animal (area＜10%). Conclusions Ad- VEGF165 can induce coronary collateral vessel formation, improve regional myocardial perfusion and function and is safe by means of direct injection, which suggesting that this strategy may be useful in treating human ischemic heart disease.     

GENE TRANSFER INTO PORCINE MYOCARDIUM VIA PERICARDIAL CAVITY BY HOMEMADE EASY PUNCTURE DEVICE

Objective To explore the feasibility and safety of gene transfer into porcine myocardium via the pericardial cavity by a homemade easy device. Methods Replication-deficient recombinant adenoviral vector carrying LacZ report gene (Ad-LacZ) was constructed by the calcium phosphate precipitation method. Twelve healthy Chinese mini-swine were randomly divided into experimental group (n=6) and control group (n=6). Acute myocardial infarction (AMI) model was established by balloon occlusion of the distal part of D1 branch of left anterior descending (LAD) artery, at the same time the intra-pericardial cavity injections were performed through the small incision of the abdominal wall below the xyphoid appendix using a homemade device. Then gene transfer was performed using a central venous catheter. The pericardium was pretreated with injection of a mixture of collagenase (1 200 U) and hyaluronidase (3 000 U) in both groups. Then 2.0×109 plaque formation unit (PFU) Ad-LacZ was injected into the pericardial cavity in experimental group, while 1 mL of normal saline was injected in the control group. The β-galactosidase activity detection and X-gal staining of the ischemic myocardium were performed on the 3rd, 7th, and 28th day after injection. Results The LAD artery was occluded completely and infarction and ischemia were detected by histological assessment. In experimental group, the X-gal staining positive cells and β-galactosidase activity quantification were detectable on the 3rd day after injection, increased markedly on the 7th day, and then declined on the 28th day. The transfer efficiencies indicated by the positive myocardial cells were 16.7%, 45.6%, 22.8% on the 3rd, 7th, 28th day, respectively. In control group, no positive cells and β-galactosidase activity were observed. Conclusion Adenovirus can be transferred into ischemic myocardium and express target gene in the AMI model for four weeks with the homemade easy device via pericardial cavity pretreated by collagenase and hyaluronidase.     

GENE TRANSFER INTO PORCINE MYOCARDIUM VIA PERICARDIAL CAVITY BY HOMEMADE EASY PUNCTURE DEVICEA

Objective To explore the feasibility and safety of gene transfer into porcine myocardium via the pericardial cavity by a homemade easy device.Methods Replication-deficient recombinant adenoviral vector carrying LacZ report gene (Ad-LacZ) was constructed by the calcium phosphate precipitation method. Twelve healthy Chinese mini-swine were randomly divided into experimental group (n = 6) and control group (n =6). Acute myocardial infarction (AMI) model was established by balloon occlusion of the distal part of D[ branch of left anterior descending (LAD) artery, at the same time the intra-pericardial cavity injections were performed through the small incision of the abdominal wall below the xyphoid appendix using a homemade device. Then gene transfer was performed using a central venous catheter. The pericardium was pre-treated with injection of a mixture of collagenase (1 200 U) and hyaluronidase (3 000 U) in both groups. Then 2. 0 × 109 plaque formation unit (PFU) Ad-LacZ was injected into the pericardial cavity in experimental group, while 1 mL of normal saline was injected in the control group. The p-galactosidase activity detection and X-gal staining of the ischemic myocardium were performed on the 3rd, 7th, and 28th day after injection.Results The LAD artery was occluded completely and infarction and ischemia were detected by histological assessment In experimental group, the X-gal staining positive cells and (3-galactosidase activity quantification were detectable on the 3rd day after injection, increased markedly on the 7th day, and then declined on the 28th day. The transfer efficiencies indicated by the positive myocardial cells were 16. 7% , 45. 6% , 22. 8% on the 3rd, 7th, 28th day, respectively. In control group, no positive cells and (3-galactosidase activity were observed.Conclusion Adenovirus can be transferred into ischemic myocardium and express target gene in the AMI model for four weeks with the homemade easy device via pericardial cavity pretreated by collagenase and hyaluronidase.     

Comparative study of catheter-mediated gene transfer into heart

ObjectiveTo investigate the feasibility and features of 3 catheter-mediated approaches of gene transfer into heart, including direct myocardial injection (DMI), coronary artery perfusion (CAP), and intrapericardial cavity injection (ICI).MethodsFifteen dogs were used, and 03ml (1×10［9］ pfu) of an adenovirus (Adex1SR LacZ) was injected into the heart by 3 methods.The dogs were killed 5 days following injection, and gene expressions in heart and liver were evaluated by histochemical analysis.ResultsThe results showed that ① the CAP method was relatively less damaging and induced sparse LacZ expression in the myocardium, and the gene expression was also foundin both vessels within the myocardium and liver; ② gene transfer by DMI resulted in intense LacZ expression around the injection accompanied by a local inflammatory response; ③ LacZ expression elicited by ICI was detected in either the inner surface of the parietal pericardium or epicardial surface of the heart,and also in the myocardium underlying the visceral pericardium.ConclusionThree catheter-mediated methods of gene transfer into the heart may be used and a reasonable approach should be chosen according to purpose     

人VEGF_(165)和bFGF基因联合治疗大鼠急性心肌梗死

目的观察直接心肌内注射人血管内皮细胞生长因子(VEGF165)和碱性成纤维细胞生长因子(bFGF)基因对大鼠急性心肌梗死模型血管及梗死面积的治疗效果,评估比较联合应用2种基因与单纯应用一种基因的疗效。方法建立大鼠急性心肌梗死模型,将生理盐水(对照组)、空载体(A组)、VEGF165(B组)、bFGF(C组)、VEGF165和bFGF混合质粒(D组)分3点注射于梗死交界处心肌内,4周后取材做常规HE染色和Masson染色,分别测量各组微血管数量和梗死面积;用免疫组织化学染色鉴定VEGF、bFGF的表达。结果B组、C组和D组心肌毛细血管总数明显大于对照组和A组(P<0.01),D组毛细血管总数大于B组和C组(P<0.01)。D组和C组的梗死面积百分比小于其他3组(P<0.01),D组和C组之间差异无统计学意义(P>0.05)。VEGF和bFGF免疫组化染色显示有相应蛋白表达。结论联合应用VEGF和bFGF基因治疗急性心肌梗死,具有明显促进血管生成、缩小梗死面积的作用。     

Intrastriatal Gene Transfer of Vascular Endothelial Growth Factor Rescues Dopaminergic Neurons in a Rat Parkinson＇s Disease Model

To examine the ability of intrastriatal gene transfer of vascular endothelial growth factor 165 mediated by adenoviral vector to rescue dopaminergic neurons in a rat model of Parkinson＇s disease （PD）, we constructed recombinant replication-deficent adenoviral vectors carrying the gene of VEGF165 （Ad-VEGF）, and injected Ad-VEGF （or Ad-LacZ and PBS as controls） into the striatum of rats 7 days after the lesion by 6-hydroxydopamine. The rat rotational behavior analysis and tyrosine hydroxylase （TH） immunohistochemistry were performed to assess the change of dopaminergic neurons. Our results showed that the rats receiving Ad-VEGF injection displayed a significant improvement in apomorphine-induced rotational behavior and a significant preservation of TH-positive neurons and fibers compared with control animals, It is concluded that intrastriatal gene transfer by Ad-VEGF may rescue the dopaminergic neurons from degeneration in a rat model of PD.     

血管内皮细胞生长因子和血管紧张素Ⅱ对蛙心包间皮,淋巴？…

目的 观察血管内皮细胞生长因子（ＶＥＧＦ）和血管紧张素Ⅱ（ＡＮＧⅡ）对蛙心包间皮、淋巴孔和心肌生血管的作用,探讨ＶＥＧＦ和ＡＮＧⅡ对心包间皮的通透性、淋 的调控作用和心肌肥厚的影响。方法 应用ＶＥＧＦ和ＡＮＧⅡ蛙腹膜腔注扫描电镜和光镜观察,计算机图象处理。结果 正常蛙心包壁层有一些散在２的心包淋巴孔秒量血窦是皮。蛙心肌无血管,其血供仅由心腔内血液直接进入心肌小梁间隙。ＶＥＧＦ组和ＡＮＧⅡ组的心包淋     

过表达HGF促慢性缺血心肌处血管生成的实验研究

目的研究过表达HGF对促进缺血心肌处血管生成作用及对心功能的影响。方法经左胸冠状动脉回旋支放置Ameroid环的方法建立稳定有效的慢性心肌缺血猪模型。随机分为3组(n=6)于缺血心肌处分别注射4×109pfu 200μL AdHGF、4×109pfu 200μL AdNull、200μL Ns。继续饲养4周,检测心脏功能及心肌HGF蛋白表达等。结果全部实验动物成功构建慢性缺血性心脏病模型;治疗后心脏彩超检查示AdHGF组左室前壁缺血区室壁增厚,灌注明显改善,运动增强,LVEDV、LEEF、FS明显增加(P<0.05);AdHGF组心肌组织HGF蛋白较对照组获得较高表达(P<0.05);AdHGF组心肌血管数量及密度均明显高于对照组(P<0.05);Ad-HGF组VIII因子阳性的血管数量明显高于AdNull组(P<0.05)。结论过表达HGF促进慢性缺血心肌处血管生成,增加心肌血流灌注,改善缺血心肌局部微环境,从而改善和保护心功能的作用。     

REGULATING EFFECTS OF VASCULAR ENDOTHELIAL GROWTH FACTOR AND ANGⅡ ON FROG'S PERICARDIAL STOMATA, MESOTHELIUM AND ANGIOGENESIS

Objective. To observe the regulating effects of vascular endothelial growth factor (VEGF) and angiotensinⅡ (ANG II) on the frog's pericardium, lymphatic stomata and angiogenesis so as to reveal their effects and mechanism on the mesothelial permeability, lymphatic stoma regulation and myocardial hypertrophy. Methods. VEGF and ANGⅡ were injected into the frog's peritoneal cavity so as to examine the changes of the pericardial stromata by using transmission electron microscopy, scanning electron microscopy and computerized imaging analysis. Results. Scattered distributed pericardial stomata were found on the parietal pericardium of the frog with a few sinusoid mesothelial cells, whose blood supply was directly from the cardiac chambers flowing into the trabecular spaces of the myocardium (because there are no blood vessels in the myocardium of the frog). The average diameters of the pericardial stomata in VEGF and ANGⅡ groups were 1.50 μ m and 1.79 μ m respectively, which were much larger than those in the control group (0.72 μ m, P< 0.01); the average distribution densities of the stomata were 8.25/0.1 mm2 and 12.80/0.1 mm2 in VEGF and ANGⅡ groups, which were also much higher than those in the control group (3.57/0.1 mm2, P< 0.01); the sinusoid areas in VEGF and ANGⅡ groups were 2442.95 μ m2/0.1 mm2 and 2121.79 μ m2/0.1 mm2, which were larger than that in the control group (995.08 μ m2 /0.1 mm2 , P< 0.01); no angiogenesis was found in the frogs of the experimental groups. Conclusions. VEGF and ANGⅡ could strongly regulate the pericardial stomata by increasing their numbers and openings with larger diameters and higher distribution density. They could also increase the sinusoid areas with the result of the higher permeability of the pericardium, which clearly indicated that VEGF and ANGⅡ could speed up the material transfer of the pericardial cavity and play an important role in preventing myocardial interstitial edema. Yet there was no strong evidence to show the angiogenesis in the myocardium.