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.     

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。     

Experimental Study on Heterograft of Glomus Cells of Carotid Body for Hemiparkinsonian Rats

Parkinson disease(PD) is a neuro- degenerativedisorder commonly found in middle- aged or olderpeople,which ischaracterized by the loss ofmelanin-containing neurons in the substantia nigra pars com-pacta(SNpc) and a reduction in dopamine in stria-tum.After several years of treatment by dopamineprecursor,L- dihydroxyphenylalanine (L - DOPA ) ,and due to the reduced effectof L- DOPA and dyski-nesia,some PD patients need to be switched to othertherapeutic approaches such as neural transplant…     

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。     

胚胎神经上皮干细胞PD大鼠模型脑内移植的比较

目的观察胚胎神经上皮干细胞分别植入PD大鼠模型两侧纹状体内的存活、分化及对疾病的治疗情况。方法将GFP转基因鼠的胚胎神经上皮干细胞体外克隆增殖后,分别立体定向移植到PD模型鼠的毁损侧纹状体和健康侧纹状体内,术后观察动物的行为改变,分期取材后检测移植细胞的存活与分化状况,比较两种移植方式的治疗作用。结果移植后实验动物的旋转行为明显改善,移植细胞在宿主脑内存活良好,并分化出TH阳性神经元,损伤侧移植组效果更好。结论神经上皮干细胞植入损伤侧纹状体内的治疗效果更好。     

Transplantation of VEGF165-gene-transfected endothelial progenitor cells in the treatment of chronic venous thrombosis in rats

Background The organization and recanalization of thrombi is a dynamic and complex process. The aim of this research was to study the cotherapeutic effect of stem cell transplantation and gene transfection on chronic venous thrombosis. Methods We constructed a recombinant adenoviral vector carrying the vascular endothelial growth factor 165 （VEGF165） gene by using the pAdEasy system, which was subsequently identified and amplified. Simultaneously, endothelial progenitor cells （EPCs） were isolated from rat bone marrow using Ficoll, cultured in EBM-2MV medium, and identified. Then, the cells were transfected with the recombinant Ad-VEGF165. The EPCs were labeled with 1 ,1＇-dioctadecyl-3,3,3＇,3＇-tetramethylindocarbocyanine （Dil） before transplantation. A rat model of chronic vein thrombosis was developed by partial ligation of the inferior vena cava. The rats were randomly divided into 4 groups （n=25, each）： A, Ad-VEGF165/EPC-transplantation group received 1 ml （10^6） of Ad-VEGF165/EPCs; B, EPC-transplantation group received 1 ml （10^6） of EPCs; C, Ad/EPC-transplantation group received 1 ml （10^6） of Ad/EPCs; D, control group received 1 ml of the transplantation medium. The thrombi and adjacent caval walls were harvested 28 days after transplantation; real-time quantitative polymerase chain reaction was used to detect the expression level of vascular endothelial growth factor （VEGF） mRNA; and western blotting was used to measure changes in VEGF protein expression. Hematoxylin-eosin staining and immunohistochemical staining were performed to detect recanalization. Neovascularization was detected by immunohistochemical staining using the antibody for von Willebrand factor （vWF）, which is a component of endothelial cells.The capillary density was quantitatively determined by counting the capillaries under a high-power microscope. Results The Ad-VEGF165 was constructed, and bone-marrow-derived EPCs were cultivated and successfully identified. We determined the optimum transfection ratio that promoted the growth of EPCs. After transfection, the EPCs secreted the VEGF protein. After transplantation, the in vivo survival of EPCs and their differentiation into endothelial cells were determined by detecting the fluorescence associated with the Dil stain. VEGF mRNA was expressed in groups A, B, C and D after transplantation, and the VEGF mRNA level in group A was significantly higher than those in groups B, C and D （P〈0.05）; the VEGF mRNA levels in groups B and C were significantly higher than those in group D （P〈0.05）, and there was no statistical significance between the VEGF mRNA levels in groups B and C. The recanalization capillary density in group A was significantly higher than those in groups B, C （P 〈0.05） and D （P 〈0.01）; the recanalization capillary densities in groups B and C were significantly higher than that in group D （P 〈0.05）. Moreover, there was no statistical significant difference between the values for groups B and C. Conclusions The EPCs were successfully transfected by Ad-VEGF165. A suitable transfection ratio can improve the efficiency of EPCs and the possibility of promotion of angiogenesis after transplantation. Transfected EPCs caused accelerated organization and recanalization of vein thrombi.     

白藜芦醇对帕金森病大鼠黑质多巴胺能神经元损伤的保护作用

 目的 观察白藜芦醇(Res)对脂多糖（LPS）所致大鼠黑质多巴胺能（DA）神经元损伤的保护作用。方法 黑质内注射LPS制作帕金森病（PD）大鼠模型。应用Res对实验动物进行处理。通过行为学、酪氨酸羟化酶（TH）、核因子κB（NF-κB）等免疫组化及免疫印迹技术观察Res对DA能神经元损伤的作用。结果 对照组大鼠无行为学变化,PD组大鼠平均旋转圈数为（196.90±9.52）圈,Res组为（106.57±7.89）圈,差异有统计学意义（P<0.01）。TH免疫组化结果表明,对照组大鼠黑质TH阳性神经元数量较多,胞体较大,突起明显;PD组神经元数量明显减少（P<0.01）,甚至消失,神经元胞体萎缩,突起亦不清晰。Res组TH阳性神经元数量与PD组相比明显增加（P<0.01）,神经元形态变化亦不明显。NF-κB免疫组化结果表明,对照组黑质仅见少数NF-κB阳性细胞,无明显核转位现象;PD组黑质NF-κB阳性细胞较多,胞质呈黄褐色,部分细胞核也着色,有明显核转位现象;与PD组相比,Res组NF-κB阳性细胞数明显减少（P<0.01）。小胶质细胞特异性抗体（OX-42）免疫组化结果表明,对照组大鼠黑质小胶质细胞多呈静止的“分枝样”状态,PD组多为激活状态,呈典型的“阿米巴样”;Res组激活状态的小胶质细胞与PD组相比明显减少,形态介于静止和激活状态之间。Western blot分析结果显示,与对照组相比,PD组TH蛋白表达减少,NF-κB P65蛋白表达明显增高（P<0.01）;与PD组相比,Res组TH蛋白表达增加,NF-κB P65蛋白表达显著下降（P<0.01）。结论 Res可对LPS所致黑质DA能神经元损伤起防护作用,具有抗炎及神经保护作用。     

胚胎中脑细胞植入帕金森病模型大鼠脑内的实验研究

目的：观察胚胎中脑细胞脑内移植治疗帕金森病(PD)模型大鼠的长期疗效。方法：将取自11．5天胚胎(E11．5)大鼠的中脑细胞植入PD模型大鼠的绞状体，观察大鼠旋转行为的改变，并于移植手术1年后用免疫组织化学的方法检测移植细胞的存活及其分化为多巴胺能神经元的状况。结果：E11．5大鼠中脑细胞在植入脑内后能够明显改善PD大鼠的旋转行为，但只有不到0．1％的移植细胞能够分化成为多巴胺能神经元，这些分化良好的多巴胺能神经元可以在脑内长期存活。结论：E11．5大鼠的中脑细胞植入PD模型大鼠的绞状体后，虽然有一定的疗效，但其最终分化成为多巴胺能神经元的比例却较低，提示E11．5大鼠胚胎中脑细胞不如E13—E15大鼠胚胎中脑细胞脑内移植治疗PD的效果好。     

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.     

神经干细胞移植对帕金森病鼠旋转行为的影响

目的 :观察神经干细胞定向移植后对帕金森病 (Parkinsondisease,PD)鼠的旋转行为的影响。方法 :建立PD模型大鼠以及体外培养神经干细胞 ,然后将神经干细胞悬液立体定向移植到PD模型鼠黑质纹状体区 ,测定PD模型鼠旋转行为的变化 ,并与对照组相比。结果 :神经干细胞移植后PD模型鼠旋转行为比对照组明显减少。结论 :神经干细胞移植能减轻 6 羟基多巴胺对黑质纹状体多巴胺能神经元的损伤。     

心包腔内注入腺病毒-血管内皮生长因子165基因治疗心肌缺血的实验研究

目的:探讨经心包腔途径转染血管新生基因对缺血心肌的血管生成及舒缩功能的影响。方法:第一部分:随机将12头中国小型猪分为实验组和对照组,每组6头。两组猪均采用球囊堵塞前降支第一对角支远端以建立心肌梗死模型,心肌梗死模型建立后即刻,采用经皮剑突下穿刺方法,将中心静脉导管插入心包腔内转染Ad-LacZ。以胶原酶1200 u及透明质酸酶3000u预处理心包后,在心包腔内注射含Ad-LacZ基因2.0×109pfu。对照组心包腔内注射生理盐水。分别于注射后3天、7天及28天处死动物,对缺血心肌进行染色及病理观察。第二部分:随机将20头中国小型猪分为实验组和对照组,每组10头,每组又分3天(n=2)、7天(n=6)及28天(n=6)三个亚组。注射后3天、7天及28天分别用免疫组化、超声心动图对缺血心肌血管新生情况进行检测,并以酶联免疫吸附试验(ELISA)检测血浆、心包及心肌组织中Ad-VEGF165的表达。第三部分:20头小型猪随机分为心包转染组(心包组)和冠脉转染组(冠脉组)。心包组和冠脉组均注射Ad-VEGF1651.0 ml(2×109pfu),于注射前及其后3、7、28天分别测定组织内VEGF水平、微血管密度(MVD)、心功能。结果:①实验组注射Ad-LacZ基因后第3天、第7天及28天后X-gal染色有阳性细胞,以第7天最明显,对照组无阳性细胞。②Ad-VEGF165基因经心包腔转染缺血心肌组织后,在心包及组织中成高表达,于7天达到高峰,28天降至基线水平,血浆中无目的基因的表达;28天时,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,517.0±75.7/mm2vs 226.5±54.1/mm2,P=0.009;LVEF72.11±5.2%vs 55.14±4.37%,P=0.005]。③心包组和冠脉组的心脏均表达有VEGF165基因,组织内VEGF水平在7天时达高峰,28天时降至基线水平,前组高于后组(702±85pg/ml vs 592±59 pg/ml,P=0.026)。而两组的MVD、心功能随转染时间延长均明显增加,但心包组优于冠脉组(28d,MVD,517.0±75.7/mm2vs 326.4±24.1/mm2,P=0.001;FS,32.9±2.2%vs 30.6±2.1%,P=0.049;LVEF,72.11±5.2%vs 65.87±2.16%,P=0.034)。结论:①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。②用胶原酶及透明质酸酶预处理心包腔后,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。③导管介导的心包腔与冠脉转染Ad-VEGF165基因治疗心肌缺血是有效的、切实可行的,而前者可能是更有前途的新方法。     

早期帕金森病大鼠模型的建立

目的 探索建立早期帕金森病（Parkinson disease,PD）大鼠模型.方法 成年大鼠单侧纹状体内注射6-羟多巴胺（6-hydroxydopamine,6-OHDA）.注射后第7天,通过姿势不对称性、前肢始动不能、前肢使用不对称性、阿朴吗啡诱发旋转实验进行行为学评估.观察组织学变化,取鼠脑黑质节段（n=6）,固定、石蜡包埋、连续冠状切片.焦油紫（Nissl）染色显示黑质神经细胞;抗酪氨酸羟化酶（tyrosine hydroxylase,TH）抗体免疫组织化学染色显示黑质多巴胺（DA）能神经元,光镜下观察并进行细胞计数,统计学分析处理数据.而且,使另一部分模型动物（n=6）继续存活到第28天,鉴定所选动物模型的可靠性.结果 纹状体内注射6-OHDA后第7天,动物行为学出现有意义改变,黑质内神经细胞出现肿胀等变性坏死的早期形态变化,DA能神经元出现有意义减少;注射后的第28天,行为学改变更加显著,神经细胞大量减少,DA能神经元数量减少到92%.结论 纹状体内注射6-OHDA后第7天,通过行为学方法能确定早期PD动物模型.     

帕金森病大鼠中脑腹侧被盖区多巴胺能神经元的改北

目的：探讨帕金森病（PD）大鼠中脑腹侧被盖区（VTA）多巴胺（DA）能神经元的改变.方法：20只3月龄Wistar雄性大鼠,随机分为模型组和对照组,每组10只.6-羟基多巴胺（6-OHDA）注射右侧黑质致密区（SNC）制作PD大鼠模型,腹腔注射阿朴吗啡后进行行为学观察;尼氏染色观察左侧中脑VTA神经元、酪氨酸羟化酶（TH）的改变;免疫组织化学ABC法观察其DA能神经元的改变并进行图像分析.结果：阿朴吗啡诱发PD大鼠模型异常旋转行为,尼氏染色见PD大鼠左侧中脑VTA有神经细胞肿胀、坏死等变化;VTA TH阳性神经元形态学改变,数量减少,与对照组相比,差异有统计学意义.结论：中脑VTA DA能神经参与PD模型大鼠的改变.     

建立类似于绝经期妇女帕金森病大鼠模型的实验研究

目的探讨利用6-羟基多巴(6-OHDA)制备模拟绝经期妇女帕金森病的大鼠模型。方法应用6-OHDA制备OVX PD模型大鼠,应用免疫组织化学染色、高效液相色谱法等技术对大鼠黑质(SN)的酪氨酸羟化酶(TH)阳性神经元数目、纹状体多巴胺(DA)及其代谢物二羟基苯乙酸(DOPAC)和高香草酸(HVA)含量进行考察并评价。结果阿朴吗啡可诱导出明显的PD大鼠旋转行为;大鼠损伤侧黑质TH阳性神经元数量较健侧显著减少(P<0.01),纹状体DA及其代谢物DOPAC和HVA含量也较健侧明显减少(P<0.01)。结论本模型具有绝经期妇女帕金森病的基本的病理特点。     

稳定表达GDNF的MN9D/GDNF工程细胞可明显改善帕金森病模型大鼠的旋转行为

目的：观察稳定表达ＧＤＮＦ的ＭＮ９Ｄ／ＧＤＮＦ工程细胞对６羟基多巴胺损毁所致帕金森病（Ｐａｒｋｉｎｓｏｎｄｉｓｅａｓｅ，ＰＤ）模型大鼠旋转行为有无治疗作用。方法：用６羟基多巴胺损毁大鼠单侧中脑黑质多巴胺能神经元、２周后腹腔注射阿朴吗啡诱导大鼠出现偏侧旋转行为的方法制备ＰＤ模型大鼠。通过脑立体定位技术，将稳定表达ＧＤＮＦ的ＭＮ９Ｄ／ＧＤＮＦ工程细胞注入模型鼠损毁侧纹状体，１周后观察阿朴吗啡诱导的旋转行为有无改善。结果：稳定表达ＧＤＮＦ的ＭＮ９Ｄ／ＧＤＮＦ工程细胞可明显降低ＰＤ模型大鼠的异常旋转行为，治疗作用达１０周之久。结论：运用ｅｘｖｉｖｏ方法和防治兼顾的策略对于ＰＤ基因治疗的基础研究及临床应用具有重要意义。     

腺病毒载体介导血管内皮生长因子165基因治疗新生大鼠缺氧缺血性脑损伤

目的 研究腺病毒介导血管内皮生长因子(VEGF)165基因转移对新生大鼠缺氧缺血性脑损伤(HIBD)的治疗作用.方法 采用细菌内同源重组技术构建腺病毒重组载体Ad-VEGF.7日龄SD大鼠随机分成3组:假手术组(20只)、HIBD(20只)、Ad-VEGF移植组(Ad-VEGF组,20只),Ad-VEGF移植组在HIBD后3 d于大鼠左侧感觉运动皮层区(坐标AP:+0.3mm,ML:-2 mm,DV:-1.5 mm)立体定位注射2μl重组体腺病毒悬液.移植后7 d采用免疫组织化学法检测鼠脑VEGF蛋白;采用原位缺口末端标记法(TUNEL法)检测鼠脑神经元凋亡情况;大鼠3～4周龄时采用T迷宫觅食实验及足错误、姿势反射、肢体放置实验对其学习记忆、空间能力和感觉运动功能等行为学进行评估;采用尼氏染色法检测鼠脑海马CAl区和皮层单位面积内神经元数目.结果 免疫组织化学法结果 显示皮层与海马Ad-VEGF组VEGF阳性细胞数均明显多于HIBD组(68.09±3.37比24.65±3.37,68.37±3.17比25.14±1.86,均P<0.05).TUNEL结果 显示Ad-VEGF组鼠脑神经元凋亡数目明显低于HIBD组(151.4±21.7比264.4±16.3,P<0.05);行为学检测结果 显示Ad-VEGF组的各项行为学测试成绩均较HIBD组有明显改善(均P<0.05),尼氏染色结果 显示Ad-VEGF组鼠脑海马CAl区和皮层单位面积内神经元数日明显多于HIBD组(70.6±2.3比55.3±2.1,95.1±2.8比70.1±2.7,均P<0.05).结论 腺病毒载体介导的VEGF165基因治疗可增加VEGF蛋白的表达,减少脑细胞凋亡、改善脑损伤后的远期行为学功能,减轻缺氧缺血后的脑损伤,具有神经保护作用.     

Experimental Study on Heterograft of Glomus Ccl ls of Carotid Body for Hemioarkinsonian Rats

Summary: To observe the effects of heterograft of glomus cells of carotid body on hemiparkinsonian rat models, rats with unilateral 6-hydroxydopamine (6-OHDA)-induced lesions of the right dopamin ergic neurons of substantia nigra received intrastriatal glomus cells heterograft. Apomorphine-induced rotation was monitored for 30 rmin at various time points after grafting. The striata were cut and ex-amined for dopamine content by HPLC and for immunohistochemical staining of tyrosine hydroxylase positive neurons (TH ) at the end of the experiments. The results showed that apomorphine-induced rotational behavior was significantly reduced for 12 weeks and the dopamine contents were signifi cantly elevated after grafting (P＜0.01), and TH cells survived better. The present study demon strates that intrastriatal heterograft of glomus cells within carotid body in rats with 6-OHDA-elicited lesions could reduce apomorphine-induced rotational behavior and elevate the dopamine contents and numbers of TH cell surviving within striatum, and can serve as a new and effective alternative for Parkinson disease.     

腺病毒介导的血管内皮生长因子体外转染心肌细胞的研究

目的:构建携带人血管内皮生长因子(VEGF)基因的重组腺病毒载体,并转染体外培养的心肌细胞,检测VEGF的表达.方法:将人源性的VEGF165cDNA正向插入到腺病毒载体PDC315,构建重组质粒,通过脂质体共转染293细胞,经同源重组获得携带人VEGF165基因的重组腺病毒,通过PCR扩增法鉴定所构建的腺病毒,扩增并测定滴度后,体外转染培养的心肌细胞,利用ELISA、Western印迹分析等方法检测VEGF在心肌细胞中的表达.结果:人VEGF165cDNA成功地正向插入到PDC315载体中,以重组病毒基因组DNA为模板,同时扩增出了610bp的VEGF165cDNA基因片段,证实了所构建病毒的正确性,病毒滴度为2.8×108 pfu/ml,Ad VEGF165体外转染心肌细胞3 d后,在培养细胞的上清液及细胞内检测到了VEGF的表达.结论:成功构建了表达人VEGF 165基因的腺病毒载体,体外转染心肌细胞后能够满意表达VEGF,为基因治疗心肌缺血奠定基础.     

VEGF165基因转染的内皮祖细胞移植对大鼠肾脏缺血再灌注后的保护作用

评估VEGF165基因转染的内源性内皮祖细胞(EPCs)移植治疗肾脏缺血再灌注损伤(IRI)的作用,进一步完善EPCs对肾脏IRI产生保护作用的旁分泌机制。利用重组腺病毒载体Ad-VEGF165感染EPCs,并进行转染效率鉴定。并进一步移植治疗大鼠肾脏IRI,术后24h和72h分别评估血清肌酐及尿素氮水平;组织病理学检查评估各组大鼠术后肾损伤程度;Western blot检测大鼠肾脏IRI中血管内皮生长因子 (VEGF)、血管生成素-1 (Ang-1)及血管生成素-2 (Ang-2) 表达情况。结果发现利用重组腺病毒载体Ad-VEGF165转染的EPCs移植治疗后,大鼠肾脏功能明显改善,术后72h检测大鼠患肾,其内VEGF、Ang-1以及Ang-2表达水平均明显提升。因此,VEGF165基因转染的EPCs移植治疗可以有效的治疗大鼠肾脏IRI,其作用机制可能与EPCs归巢后旁分泌过量VEGF并进一步促进Ang-1、Ang-2等血管新生因子表达,从而促使肾脏血管新生有关。     

GAP-43基因及TH基因治疗帕金森病的实验研究

目的：利用基因工程化细胞及双基因表达的方式对生长相关蛋白质（Growth associated protein,GAP-43)基因及酪氨酸羟化酶（Tyrosine hydroxylase,TH)基因在治疗帕金森病（Parkinson's disease,PD)中的作用进行探讨。方法：将GAP－43及TH基因分别构建入逆转录病毒载体中，再将含重组DNA的病毒上清感染培养的星形胶质细胞后移植入PD大鼠脑内，了解PD鼠旋转行为的改善情况。结果：TH基因治疗组及TH＋GAP－43基因治疗组PD鼠旋转行为改善明显，GAP－43基因治疗组无明显治疗作用，结论：TH对PD有明显的治疗作用，GAP－43可促进TH的疗效。