缓激肽对大鼠缺血区血脑屏障的影响

目的研究缓激肽对局部脑缺血区超微结构、血脑屏障的通透性及继发性脑水肿的影响。方法制备大鼠大脑中动脉缺血模型,在大脑中动脉缺血2hr再灌注1hr末,颈内动脉灌注小剂量缓激肽(10μg/kg/min),应用电镜观察血脑屏障超微结构的改变;测定脑组织伊文思蓝含量来判断血脑屏障的通透性;应用干湿法测定脑含水量的变化。结果缓激肽灌注大鼠脑毛细血管紧密连接开放,对照组大鼠紧密连接完整;与对照组相比,缓激肽灌注组缺血侧脑组织伊文思蓝含量明显高于缺血对照组(P<0.01),但再灌注24hr后脑含水量并没有增加(P>0.05)。结论小剂量缓激肽通过开放紧密连接来增加缺血区血脑屏障的通透性,但并不会增加继发性脑水肿的程度。     

胶质瘤的白细胞介素-4免疫基因治疗实验研究

胶质瘤是颅内最常见的恶性肿瘤。本研究通过逆转录病毒载体将白细胞介素 4 (IL 4 )基因导入逆转录病毒包装细胞 ,注入到脑内荷瘤大鼠肿瘤组织中 ,观察其对胶质瘤的治疗作用。首先构建和鉴定携带IL 4基因的逆转录病毒载体Pbabe IL 4 ,将其导入逆转录病毒包装细胞PA317,通过G4 18抗性筛选 ,得到携带目的基因的包装细胞 ;应用NIH3T3细胞测定抗性细胞克隆上清的病毒滴度 ,扩增培养高滴度产毒细胞克隆 ,命名为PA317IL 4 ;常规提取PA317IL 4细胞基因组总RNA ,通过RTPCR鉴定IL 4基因的整合 ;ELISA法检测PA317IL 4细胞培养上清的I…     

单纯疱疹病毒胸苷激酶基因／Ganciclovir系统对脑胶质瘤动物模型的治疗

目的　探讨单纯疱疹病毒Ⅰ型胸苷激酶基因 (HSV1 tk)结合Ganciclovir(GCV)治疗方案 ,在脑胶质瘤动物模型中基因治疗的疗效以及杀伤肿瘤细胞的机制。　方法　采用Southern杂交法 ,证实逆转录病毒载体生产细胞系pN2 A tk VPC(VPC)带有tk基因并稳定整合到基因组DNA上 ;再将VPC与人脑多形性胶质母细胞瘤细胞系DBTRG 0 5MG(0 5MG)分别以 1∶1、1∶4比例混合接种于BALB c裸鼠皮下 (以只接种 0 5MG细胞作为对照 ) ,建立裸鼠皮下胶质瘤动物模型 ,然后腹腔注射GCV治疗 [30mg (kg·d) ],连续 14d ,治疗后 1周 ,取出瘤块作病理组织学分析。　结果　 1∶1组瘤块较 1∶4组和对照组明显减小 (P <0 0 1) ,且有 30 %的瘤块完全消失 ,提示HSV1 tk GCV对肿瘤细胞有显著杀伤效应。光镜观察可见 ,1∶1组细胞核固缩、溶解甚至破裂等坏死特征 ,说明HSV1 tk GCV在invivo水平杀伤肿瘤细胞是细胞毒杀伤效应。　结论　HSV1 tk GCV系统能有效杀伤肿瘤细胞 ,并可能通过旁观者效应扩大肿瘤杀伤效应     

雷公藤对致敏小鼠T细胞IL-5 mRNA表达及转录因子GATA-3活性的抑制作用

探讨雷公藤内酯醇 (TP )对致敏小鼠T淋巴细胞IL 5mRNA表达的影响及其机制。采用卵蛋白 (OVA )致敏的方法建立模型 ;运用原位杂交染色法 (ISH )观察TP对T淋巴细胞IL 5mRNA表达的影响 ;通过凝胶电泳迁移率实验 (EMSA )对CD4+T淋巴细胞核转录因子GATA 3的DNA结合活性进行检测 ,同时就TP的作用与地塞米松 (DM )相比较。结果表明致敏小鼠T淋巴细胞IL 5mRNA表达显著高于正常对照组 (P <0 0 1) ,经TP、DM处理后 ,其IL 5mRNA表达显著低于致敏组(P <0 0 1)。致敏小鼠CD4+ T淋巴细胞体外经伴刀豆蛋白A (ConA )刺激后 ,GATA 3的DNA结合活性与正常对照组比较显著增强 ,并呈时间依赖关系 ,经TP、DM处理后 ,GATA 3的DNA结合活性显著减弱。TP抑制IL 5基因转录的分子机制可能与其抑制GATA 3的DNA结合活性有关。     

单纯疱疹病毒载体转基因治疗神经疾病的潜力

基因治疗人类神经系统疾病是最近才提出来的并且已取得成功〔1,2〕。目前,国外已建立了许多病毒载体,如单纯疱疹病毒(HSV)、腺病毒、反转病毒、腺病毒相关病毒等载体,但主要是用于体外试验和动物。现已成功地把外源基因插入到上述病毒载体。而这种外源基因可以激活神经系统的一些特殊区域。从病毒在神经元细胞建立潜在感染的能力来看,HSV特别适合于神经元产生基因。病毒载体用于治疗某些疾病,如恶性脑胶质瘤、帕金森氏病及已知的单基因疾病和脑缺血等具有很大的潜力。本文着重综述病毒载体的以下几个方面。1　病毒载体途径的总则〔3〕基…     

雷公藤抑制致敏小鼠T细胞IL-5 mRNA的表达及核因子NFAT活性

目的：探讨雷公藤内酯醇（TP）对致敏小鼠T淋巴细胞IL－5 mRNA表达的影响及其机制。方法：采用卵蛋白（OVA）致敏的方法建立模型;运用原位杂交染色法（ISH）观察TP对T淋巴细胞IL－5 mRNA表达的影响;通过凝胶电泳迁移率实验（EMSA）对CD4^ T淋巴细胞核转录因子NFAT的DNA结合活性进行检测,同时就TP的作用与地塞米松（DM）相比较。结果：致敏小鼠T淋巴细胞IL－5 mRNA表达显著高于正常对照组（P＜0．01）,经TP、DM处理后,其IL－5 mRNA表达显著低于致敏组（P＜0．01）。致敏小鼠CD4^ T淋巴细胞体外经刀豆蛋白A（ConA）刺激后,NFAT的DNA结合活性与正常对照组比较显著增强,并呈时间依赖关系,经TP、DM处理后,NFAT的DNA结合活性显著减弱。结论：TP抑制IL－5基因转录的分子机制可能与其抑制DFAT的DNA结合活性有关。     

Cyclin D1和p16、p27在脑胶质瘤中的表达及其与PCNA的相关性

目的 :检测CyclinD1 和p16、p2 7以及PCNA在脑胶质瘤中的表达状况 ,以探讨不同病理类型脑胶质瘤中各自的表达及其相关性。方法 :应用免疫组化S P法 ,检测 12例正常脑组织、5 8例脑胶质瘤组织中CyclinD1 、p16、p2 7及PCNA表达及其特征。结果 :CyclinD1 在由低度恶性胶质瘤向高度恶性胶质瘤转化中阳性表达逐渐增强 (χ2 检验 ,P <0 0 0 5 ,χ2 =5 1 6 7) ;而p16、p2 7阳性表达却是随着胶质瘤恶性程度的升高而降低 (χ2 检验 ,P值均 <0 0 0 5 ,χ2 分别为 15 4 1和 12 81)。CyclinD1 与PCNA呈正相关 ,rs =0 74 5 ;p16和p2 7与PCNA呈负相关 ,rs分别为 - 0 5 6 6和 - 0 6 12。结论 :脑胶质瘤中CyclinD1的表达程度对胶质瘤的细胞增殖活性起促进作用 ,而p16、p2 7的表达则起抑制作用。     

缓激肽选择性增加局部脑缺血大鼠血脑屏障的通透性

目的　研究颈动脉灌注小剂量缓激肽对缺血后血脑屏障通透性的影响及机制。方法　免疫组化分析正常脑组织的缓激肽B2受体所在。大鼠大脑中动脉结扎 1h或 2h再灌流 1h。用放射自显影方法检测缓激肽对血脑屏障通透性的变化。WesternBlot方法检测bNOS ,iNOS和B2受体。NOS检测盒检测NOS的活性。结果　正常脑组织毛细血管内皮未见B2受体表达 ,在神经细胞上发现B2受体的表达。缺血 2h再灌流1h缓激肽灌注缺血区血脑屏障通透性显著增加。WesternBlot结果提示 ,在缓激肽灌注组和对照组间 ,缺血皮质区bNOS和B2受体没有明显变化 ,各组中均未检测出iNOS。缓激肽灌注组的NOS活性显著高于对照组。结论　正常脑组织毛细血管内皮未表达B2受体 ,神经细胞上可见B2受体的表达。灌注小剂量缓激肽能选择性增加局部脑缺血大鼠血脑屏障的通透性     

小鼠IL-12单链融合基因真核表达质粒构建及其肿瘤基因治疗初探

目的　构建小鼠IL 12单链融合基因 (msIL 12 ) ,进行基因治疗初探。方法　用RT PCR法从小鼠腹腔巨噬细胞总RNA分别获取p4 0cDNA和p35cDNA ,2次PCR引入linker后 ,依次克隆入pcDNA3质粒 ,构建成msIL 12真核表达质粒 (pmsIL 12 ) ;用DEAE dextran法将PEG纯化的pmsIL 12转染COS 7细胞 ,用ELISA法检测其培养上清 (转染上清 )IL 12蛋白含量。皮内注射PEG纯化的pmsIL 12 ,用MTT法检测其对正常小鼠脾细胞NK活性的影响 ,观察其对荷H2 2腹水型肝癌细胞瘤小鼠生存期的影响。结果　所得p35cDNA序列与GenBank登录号M86 6 72完全一致 ;所得IL 12p4 0cDNA序列与GenBank登录号AH0 0 4 85 9报道完全相同 ,除第 10 0 0位碱基是G而不是A(但所编码氨基酸相同 ,都是丝氨酸 )外也与GenBank登录号M86 6 71报道相同。成功构建了小鼠IL 12单链融合基因真核表达质粒pmsIL 12 ,其转染COS 7细胞后的转染上清IL 12蛋白含量达 (2 .5 5± 0 .6 0 )ng ml。皮内注射pmsIL 12后 ,使正常小鼠脾细胞NK活性显著增强 (P <0 .0 1) ,使荷H2 2肝癌细胞瘤小鼠的生存期明显延长(P <0 .0 5 )。结论　构建了小鼠IL 12单链融合基因的真核表达质粒 ,可用于基因治疗研究。     

胶质瘤的单纯疱疹病毒胸腺激酶基因治疗

胶质瘤是颅内最常见的原发恶性肿瘤,约占成人原发脑肿瘤的30%,年发病率约为14.7/10万人.虽然在神经外科技术、放射与化学治疗等方面近年已有很大的进展,但胶质瘤的预后并无明显的改善,5年生存率仍<5.5%.近几年,许多学者提出胶质瘤的基因治疗,已有几种基因转移系统利用于局部治疗肿瘤,这些系统利用体外转移基因和细胞因子或大的组织相容性基因来诱导免疫介导的肿瘤消退,而另一种基因治疗方法是用药物敏感基因(“自杀”基因,如HSV—tK基因)来选择性地破坏肿瘤.这些分子生物学方面的进展,给胶质瘤治疗带来新的希望.本文就胶质瘤的单纯疱疹病毒胸腺激酶(HSV—tK)基因治疗综述如下:     

Herpetic infection of the oral cavity in guinea pigs]

A convenient experimental model for serial studies of herpes simplex of the buccal mucosa has been developed in guinea pigs by application of fresh isolates of herpes simplex virus type 1 from a patient with primary herpetic gingivo-stomatitis on prescarified buccal mucosa. The herpetic nature of the eruptions in the oral cavity was confirmed by virus isolations and serologically. The possibility of exogenous reinfection of the buccal mucosa in convalescent animals against the background of humoral virus-neutralizing antibody has been demonstrated. No virus was isolated from the blood, brain, regional lymph nodes, oesophagus, trachea, lungs or liver of the animals with primary herpes simplex of the buccal mucosa. The model is recommended for experimental investigations of herpetic infections of the buccal mucosa.     

Acute Morphine Administration Reduces Cell-Mediated Immunity and Induces Reactivation of Latent Herpes Simplex Virus Type 1 in BALB/c Mice

Acute morphine administration is known to alter the course of herpes simplex virus infection. In this study, the effect of acute morphine administration on the reactivation of latent herpes was investigated in a mouse model. Because of the important role of cytolytic T lymphocyte （CTL） activity in the inhibition of herpes simplex virus type 1 （HSV-1） reactivation, the effect of acute morphine administration on CTL responses was also evaluated. Furthermore, lymphocyte proliferation and IFN-γ production were evaluated for their roles in the induction of the CTL response. The findings showed that acute morphine administration significantly reduced CTL responses, lymphocyte proliferation, and IFN-γ production. Furthermore, acute morphine administration has been shown to reactivate latent HSV-γ. Previous studies have shown that cellular immune responses have important roles in the inhibition of HSV reactivation. These findings suggest that suppression of a portion of the cellular immune response after acute morphine administration may constitute one part of the mechanism that induces HSV reactivation.     

缓激肽及其类似物RMP-7对血脑屏障通透性的影响

缓激肽及其类似物RMP-7能选择性增加血肿瘤屏障的通透性,而对正常脑组织的血脑屏障几乎无影响。研究表明,缓激肽选择性开放血脑屏障是由于缓激肽与大脑毛细血管内皮细胞膜上的B2受体结合,引起细胞内Ca2+水平升高,从而引发包括NO和cGMP在内的一系列的信号传递反应,使胞饮小泡数量增加或紧密连接开放。本文就缓激肽及其类似物RMP-7选择性开放血脑屏障的新进展做一综述。     

Experimental herpetic infection of the CNS with predominant involvement of the spinal cord

Clinical, morphological, and virological characteristics of a model of herpes infection of the CNS with predominant involvement of the spinal cord, an acute ascending myelitis with primary demyelinization, are presented. The development of spinal cord lesions after HSV inoculation into the spleen was observed in different species of laboratory animals: mice, hamsters, guinea pigs, and rabbits; some animals (30%-70%) developed generalized infection with the involvement of the brain. The frequency of the spinal cord involvement was found to depend on the age of the animal and the dose of the virus-containing inoculum. Differences in the clinical and morphological manifestations of herpes infection were due to different mechanisms of herpes simplex virus spread in the body: either centripetally on the axons of the visceral nerves innervating the spleen into the thoracal cord, or hematogenic disseminations with virus penetration across the hematoencephalic barrier. The total duration of the disease was 3-12 days depending on the animal species.     

Herpes simplex virus type 1 DNA is located within Alzheimer's disease amyloid plaques

The brains of Alzheimer's disease sufferers are characterized by amyloid plaques and neurofibrillary tangles. However, the cause(s) of these features and those of the disease are unknown, in sporadic cases. We previously showed that herpes simplex virus type 1 is a strong risk factor for Alzheimer's disease when in the brains of possessors of the type 4 allele of the apolipoprotein E gene (APOE-epsilon4), and that beta-amyloid, the main component of plaques, accumulates in herpes simplex virus type 1-infected cell cultures and mouse brain. The present study aimed to elucidate the relationship of the virus to plaques by determining their proximity in human brain sections. We used in situ polymerase chain reaction to detect herpes simplex virus type 1 DNA, and immunohistochemistry or thioflavin S staining to detect amyloid plaques. We discovered a striking localization of herpes simplex virus type 1 DNA within plaques: in Alzheimer's disease brains, 90% of the plaques contained the viral DNA and 72% of the DNA was associated with plaques; in aged normal brains, which contain amyloid plaques at a lower frequency, 80% of plaques contained herpes simplex virus type 1 DNA but only 24% of the viral DNA was plaque-associated (p < 0.001). We suggest that this is because in aged normal individuals, there is a lesser production and/or greater removal of beta-amyloid (Abeta), so that less of the viral DNA is seen to be associated with Abeta in the brain. Our present data, together with our finding of Abeta accumulation in herpes simplex virus type 1-infected cells and mouse brain, suggest that this virus is a major cause of amyloid plaques and hence probably a significant aetiological factor in Alzheimer's disease. They point to the usage of antiviral agents to treat the disease and possibly of vaccination to prevent it.     

缓激肽B2受体在大鼠C6脑胶质瘤细胞上的表达

目的　研究缓激肽选择性开放脑肿瘤的血肿瘤屏障的机制。方法　通过双重免疫组化染色 ,确定缓激肽B2受体是存在于血管内皮细胞上还是存在于肿瘤细胞上。结果　在正常脑组织和肿瘤的血管内皮细胞上未见缓激肽B2受体的表达 ,而在肿瘤细胞上发现了高水平表达的缓激肽B2受体。结论　肿瘤细胞上B2受体的高水平表达可能是小剂量缓激肽灌注能够选择性开放恶性脑肿瘤的血 -肿瘤屏障而未影响正常血脑屏障通透性的重要原因之一。     

Potential treatment of herpes simplex virus encephalitis by brain-specific delivery of trifluorothymidine using a dihydropyridine in equilibrium pyridinium salt type redox delivery system

A newly described, drug-carrier delivery system in which a lipophilic derivative is enzymatically converted to a hydrophilic compound was used to treat experimental herpes simplex virus (HSV) encephalitis. Because trifluorothymidine (TFT) does not cross the blood brain barrier, the lipophilic dihydropyridine derivative 3'-(N-methyl-1, 4-dihydronicotinoyl)-5-'pivaloyltrifluorothymidine (DHTFT) was synthesized and characterized by HPLC. After intravenous administration of 20 mg/kg of DHTFT to rats, the quaternary, intermediate compound 3'-N-methyl-1,4-nicotinoyltrifluorothymidine was measured at levels of 7-8 micrograms/g brain at 1 hour and 13.5 +/- 0.8 micrograms/g brain at 4 hours. This compound had antiviral activity equivalent to that of TFT against HSV-1 in a plaque reduction assay (ID 50 = 0.5-1.0 microgram/ml), either directly or by conversion to TFT. Although survival was not prolonged in a rat model of HSV encephalitis, a statistically significant reduction in titer of HSV/g brain was achieved with daily intravenous treatment with DHTFT. TFT was not detected in brains of rats at 1 and 4 hours after intravenous DHTFT, but a low level was observed at 18 hours, 0.3 +/- 0.05 microgram/g brain. These data suggest that the lipophilic compound DHTFT or a lipophilic metabolite crossed the blood brain barrier and was converted to a quaternary compound, which accumulated in the brain and which was either active directly or was converted to TFT. The drug-carrier delivery system described here can potentially be used in the treatment of HSV or other viral encephalitides.     

Immunogenicity of subviral herpes simplex virus preparations: protection of mice against intraperitoneal infection with live virus

The capability of crude antigen extracts from herpes simplex virus type 1-infected human diploid cells (AM) to induce protection against intraperitoneal challenge with homotypic but heterologous virus in mice was investigated. The administration of repeated doses of AM without adjuvant failed to confer significant protection. But one relatively small dose of AM with complete Freund's adjuvant resulted in a protective effect. A good correlation was observed between the protective activity of AM preparations and the content of neutralizing antigens as determined by the chromium release-inhibition test. Protection was observed even in animals which were free of neutralizing antibody at the time of challenge. The administration of one dose of cyclophosphamide (120 mg/kg body weight) on day 7 or day 14 after immunization depressed antibody formation but had no effect on the rate of protection. These findings seem to corroborate previous observations on the important role of cell-mediated immunity in protection against herpes simplex.     

Specific neuro-radiological diagnosis of Herpes encephalitis in an animal model

Summary The potential of utilizing a radio-labelled derivative of the antiviral drug (E)-5-(2-iodovinyl)-2-deoxyuridine (IVDU) for the specific, non-invasive, in vivo diagnosis of Herpes simplex virus encephalitis (HSVE) was investigated in a rat model of the disease. Following pharmacological disruption of the blood brain barrier radiolabelled IVDU was administered by intra-carotid injection. Brain radioactivity was compared between control and infected animals via gamma camera scintigraphy. After clearance of non-metabolized drug, markedly higher levels of activity were found in infected brain. Post-mortem studies of cryostat sections of brain examined by autoradiography and immunochemical staining showed the radioactivity selectively accumulated in areas of virus infection. These results indicate that radio-labelled derivatives of antiviral drugs may allow the specific neuro-radiological diagnosis of HSVE.     

Antiherpesviral activity of acycloguanosine H-phosphonate in experiments using laboratory animals

A study of the antiherpesviral activity of acycloguanosine (ACG) H-phosphonate (ACG-P) on a model of fatal herpesvirus infection in inbred BALB/c albino mice has established that ACG-P reduces death rates in the animals, considerably increases their average lifespan, and significantly decreases brain virus titers with both 60% mortality in the control and 92% mortality in the control group. There was also a significant inhibition of herpes simplex virus type 1 (HSV-1) replication in the brain tissue of animals receiving ACG-P on a model of ACG-resistant HSV-1/L2/RACG(TK-).