CDglyTK双自杀基因杀伤C6胶质瘤细胞的体外实验研究

目的观察CDglyTK双自杀基因对C6胶质瘤细胞的杀伤作用及旁观者效应。方法利用逆转录病毒介导的胞嘧啶脱氨酶(CD)、单纯疱疹病毒胸苷激酶(HSV-tk)融合基因转染C6胶质瘤细胞,通过细胞集落形成试验、细胞生长抑制率(GIR)测定(MTT法),检测和分析CD/5-氟胞嘧啶(5-FC)、HSV-tk/更昔洛韦(GCV)双自杀基因系统对肿瘤细胞的杀伤作用和旁观者效应。结果RT-PCR检测分析融合基因的表达,显示逆转录病毒介导的双自杀基因在C6胶质瘤细胞中表达。不加5-FC和GCV时,转染组和对照组肿瘤细胞集落形成和倍增时间分别为94h、96h和25.7h、26.6h,组间差异比较无显著意义(P>0.05)。在5-FC(80.0mg/L)和GCV(10-1mg/L)浓度下,GIR分别为83.36%、7.08%,差异比较有显著意义(P<0.01)。转染C6胶质瘤细胞在混育细胞中比例占5%时,即可获得明显的旁观者效应,细胞生长抑制率可达38.48%。结论CDglyTK融合基因联合双前药治疗能取得显著的抗胶质瘤作用。     

CDglyTK双自杀基因杀伤C6胶质瘤细胞的体内实验研究

目的 观察CDgtyTK双自杀基因对C6实体胶质瘤生长的抑制作用. 方法 利用逆转录病毒介导的大肠杆菌胞嘧啶脱氨酶(CD)和单纯疱疹病毒胸苷激酶(HSV-TK)融合基因转染小鼠C6实体胶质瘤,RT-PCR检测分析融合基因的表达,并观察对照组和治疗组肿瘤体积、重量、抑瘤牢及生存期的变化,同时观察其对C6胶质瘤细胞凋亡的影响,分析CDglyTK双自杀基因系统对肿瘤的抑制作用. 结果 RT-PCR检测显示逆转录病毒介导的COgtyTK双自杀基因在C6胶质瘤中有表达.对照组第4周肿瘤已长至20 mmx30 mm大小.并出现小鼠死亡现象,至第6周末全部死亡;治疗组肿瘤体积未增长,约5 mm大小.部分肿瘤消失.肉眼观察可见对照组肿瘤体积大,色红,血供丰富,治疗组肿瘤体积减小或消失.21 d后处死取瘤称重,对照组[(2.51±0.58)g]与治疗组肿瘤质量[(0.35±0.26)g]比较差异有统计学意义(P<0.05),治疗组抑瘤牢为86.1%.流式细胞仪检测可见治疗组细胞凋亡率(34.41%±5.20%)明显高于对照组(2.92%±1.30%),差异有统计学意义(P<0.05).电镜观察可见治疗组肿瘤细胞凋亡小体形成. 结论 效 CDglyTK双自杀基因联合双前药治疗能取得显著的抗胶质瘤作用.     

人原代胶质瘤细胞自杀基因治疗体外研究

目的：应用原代培养的胶质瘤细胞作不研究对象,观察ＡＣＶ对转染了ＨＳＶ－ｔｋ基因的肿瘤细胞的杀伤作用。方法：表达单纯疱疹病毒胸苷激酶（ＨＳＶ－ｔｋ）重组逆转录病毒载体ＳＴＫ,经包装细胞ＰＡ３１７包装成重组逆转录病毒,ＮＩＨ３Ｔ３细胞测定病毒滴度,用此病毒感染原代培养的人胶质瘤细胞,以Ｇ４１８选择后获得ＴＫ＋肿瘤细胞。ＭＴＴ法及活细胞动态观察ＡＣＶ对转基因细胞的毒性作用。结果：ＡＣＶ对ｔｋ＋胶质瘤细胞     

胸苷激酶基因治疗恶性胶质瘤Ⅰ期临床研究

目的用插入单纯疱疹病毒胸苷激酶基因的逆转录病毒包装细胞(pLTKcSN/VPC)和更昔洛韦(Ganciclovir,GCV)治疗恶性胶质瘤,探讨其毒副作用和疗效。方法14例恶性胶质瘤尽可能切除肿瘤,瘤床多点注射pLTKcSN/VPC,注射点每人平均50点,接种量依据瘤床面积而定,每人注射量8～16ml不等,平均13.92ml/人,于术后7～10d开始,静脉点滴GCV,10mg·kg-1·d-1,分2次,12h1次,持续2周。结果本组毒性比较轻,均能耐受。胶质母细胞瘤组平均存活23.9个月,存活时间超过12个月病人占75%,存活时间超过23个月病人占50%,最长存活时间50个月的病人死于其他疾病。恶性胶质瘤组平均存活29.2个月,存活时间超过24个月病人占67%,存活时间超过36个月病人占33%,最长存活时间54个月,病人至今存活,无肿瘤复发迹象。结论pLTKcSN/VPC和GCV治疗恶性胶质瘤毒副作用低,对部分胶质瘤有比较明确的治疗作用。     

逆转录病毒载体介导中国单疱病毒TK基因转导脑胶质瘤细胞

目的：研究载中国单疱病毒胸苷激酶基因逆转录病毒重组体（ＲＶ－ＨＳＶ－ＴＫｃ）和羟甲基无环鸟苷（ＧＣＶ）体外转染和杀伤脑胶质瘤效果，探索应用该系统基因治疗脑胶质瘤。方法：ＲＶ－ＨＳＶ－ＴＫｃ重组体病毒感染胶质瘤细胞，Ｇ４１８筛选转染阳性胶质瘤细胞克隆。Ｎｏｒｔｈｅｒｎ杂交检测抗Ｇ４１８胶质瘤细胞中的ＴＫｃ基因表达。药物敏感实验观察ＴＫｃ胶质瘤细胞对ＧＣＶ的毒性反应。结果：１．０ｍｇ／ｍｌＧ４１８筛选１０～１４天获抗性胶质瘤细胞克隆；Ｎｏｒｔｈｅｒｎ杂交显示抗Ｇ４１８细胞ＴＫｃ基因明显表达；ＴＫｃ胶质瘤细胞对ＧＣＶ高度敏感，其ＥＤ５０为：Ｃ６ＴＫｃ０．０２μｇ／ｍｌ，Ｕ２５１ＴＫｃ０．００６μｇ／ｍｌ，Ｕ８７ＴＫｃ０．００４μｇ／ｍｌ，ＴＫｃ胶质瘤细胞对ＧＣＶ的敏感性是非转基因胶质瘤细胞的５００倍以上。结论：载中国ＴＫｃ基因逆转录病毒重组体可有效转染脑胶质瘤细胞并使其对ＧＣＶ高度敏感，该系统可望用于脑胶质瘤的基因治疗。     

胸苷激酶基因治疗人脑胶质瘤SHG44的研究

构建了含有ｐｇｋ启动子驱动的ＨＳＶ－ｔｋ基因的反转录病毒载体ｐＬＮＴＫ，并成功地转移至人脑胶质瘤ＳＨＧ４４细胞。体内实验证实，ＡＣＶ可抑制ＳＨＧＬＮＴＫ的肿瘤形成，并可抑制ＳＨＧＬＮＴＫ肿瘤的生长。原位基因转移治疗ＳＨＧ４４肿瘤效果明显。提示其可作为脑肿瘤治疗的一种新方法。     

姜黄素对胶质瘤C6细胞垂体瘤转化基因下调作用的体外研究

目的 探讨姜黄素对体外培养胶质瘤C6细胞垂体瘤转化基因(PTTG)表达的影响.方法 体外培养胶质瘤C6细胞,给予不同浓度的姜黄素(10μmol/L、20 μmol/L、30μmol/L)作用24h后应用半定量逆转录聚合酶链式反应(RT-PCR)检测PTTG mKNA表达情况,Western blot方法 检测PTTG蛋白水平的变化. 结果 姜黄素作用后,C6细胞PTTG mRNA表达与PTTG蛋白水平均下降,差异有统计学意义(P<0.01),且随着浓度的增加,其下降越明显. 结论 姜黄素具有下调胶质瘤C6细胞PTTG表达的作用,并呈剂量依赖性.     

蒿甲醚对C6胶质瘤细胞的抑制作用

目的 研究蒿甲醚对大鼠C6胶质瘤细胞的抑制作用.方法 C6胶质瘤细胞经培养后,按是否加入蒿甲醚分为实验组和对照组,均采用四甲基偶氮唑盐(MTT)法、流式细胞技术(FCM)及Hoechst33258荧光染色法检测细胞凋亡情况.结果 MTT法检测显示:24、48、72h3个时间组蒿甲醚对C6胶质瘤细胞的半数抑制浓度(IC50)分别为(195.08±3.27) μmol/L、(119.64±4.06) μmol/L、(87.84±0.93) μmol/L.FCM法检测显示:24、48、72 h3个时间组C6胶质瘤细胞凋亡率分别为:7.95％、22.01％、31.22％;且G0-G1期细胞比例增加,S期和G2-M期细胞比例降低.荧光染色显示:实验组细胞内出现凋亡小体;而对照组细胞呈弥散均匀荧光.结论 蒿甲醚能抑制C6胶质瘤细胞生长,且其抑制作用呈现时间依赖性和浓度依赖性;蒿甲醚能干扰C6胶质瘤细胞的细胞周期,可将其阻滞在G0-G1期并诱导其凋亡.     

wt-p53基因联合HSV-TK/GCV对C6鼠胶质瘤杀伤作用的实验研究

目的研究wtp53基因联合自杀基因治疗恶性胶质瘤的可行性。方法体外试验以腺病毒为载体转导wtp53和HSVTK入C6鼠胶质瘤细胞,给以不同浓度的GCV,以MTT法检测细胞生存率。体内实验以C6鼠胶质瘤细胞和SD大鼠建立动物模型,原位注射腺病毒为载体的wtp53和HSVTK基因,腹腔给以GCV(15mg.kg-1day-1×14)。观察荷瘤鼠生存期;强化MRI动态监测荷瘤鼠肿瘤大小;TUNEL法检测凋亡。结果wtp53基因明显提高鼠胶质瘤细胞对HSVTK/GCV的敏感性,p53联合HSVTK基因转染C6的GCVID100=0.01μg.ml-1,而单独HSVTK转染C6的GCVID100=1μg.ml-1,约提高100倍;p53基因还可使C6细胞凋亡增加。体内p53基因联合HSVTK/GCV治疗使荷瘤鼠生存期明显延长,凋亡明显增加(P<0.001);强化MRI示4周肿瘤消失。结论p53基因和HSVTK基因的联合基因治疗可作为临床上优化自杀基因治疗方案的一种可能选择。     

温热疗法对人脑胶质瘤细胞杀伤作用研究

目的温热疗法对人胶质瘤细胞的杀伤作用.方法用人胶质瘤细胞NP2, U251,行37℃(对照组),42℃,43℃,44℃(治疗组)加温1 h,用WST-1方法测定细胞生存率,分析肿瘤细胞杀伤效果.结果 43℃,44℃加温1 h,均取得了显著的肿瘤细胞杀伤效果.结论温热疗法作为脑胶质瘤新的临床治疗方法是可行的,可靠的.     

Herpes simplex encephalitis: an autopsy case with isolation of type 1 herpes simplex virus

Summary An adult case of herpes simplex encephalitis was studied after autopsy. Postmortem examination revealed necrotizing encephalitis associated with Cowdry type A intranuclear inclusion bodies in glial cells. Herper simplex virus type 1 was isolated from the removed brain. Herpes simplex virus antigens were detected diffusely in wide areas of the brain by immunofluorescent test and viral particles characteristic to herpes simplex virus were demonstrated by electron microscopy. There was an apparent discrepancy between severity of histological changes and distribution of virus antigen.     

Seroprevalence of herpes simplex virus type 2 in multiple sclerosis

BACKGROUND: It has been proposed that multiple sclerosis (MS) might be a sexually transmitted disorder. There is evidence that seropositivity to herpes simplex virus type 2 (HSV-2) correlates well with the number of sexual partners. Accordingly, a raised overall HSV-2 seroprevalence in MS would lend support to this theory. MATERIALS AND METHODS: Serum from 497 UK subjects with clinically definite MS was tested for antibodies to HSV-2 and compared with matched historical controls from within and outside London, blood donors and genito-urinary medicine (GUM) clinics. RESULTS: The unadjusted MS seropositivity rate was 14%. HSV-2 seroprevalence in MS patients aged 35-64 years was significantly higher overall compared with a non-London general population in an unadjusted comparison. HSV-2 seroprevalence in London MS patients compared with London blood donors was significantly greater irrespective of age, but the MS seropositive rate was lower than GUM clinic attenders. In a logistic regression analysis, increased age, female sex and MS diagnosis all independently increased the odds of seropositivity after adjustment for each other. CONCLUSION: It is concluded that there is increased likelihood of HSV-2 exposure in patients with MS and this may indicate a higher than average number of partners.     

Resistance of rat CNS to brain stem infection with herpes simplex virus type 1

Infection of the CNS by herpes simplex type 1 (HSV-1) via the trigeminal route to the brain stem was elucidated in a rat model. In contrast to the earlier described cortical and hippocampal infection after intracranial injection, the CNS showed a profound resistance to HSV-1 infection when the virus was administred by nose inoculation, as judged by histopathology and immunohistochemistry. In contrast, when the distribution of HSV-1 in the brain was investigated after nose inoculation by polymerase chain reaction, viral DNA was detected at all levels from the ganglia to the cortex. When replication of HSV-1 was assayed in primary cell cultures of rat astrocytes derived from brain stem, striatum, hippocampus and cerebral cortex, significantly lower virus yields were obtained in brain stem-derived astrocytes cultures as compared with in cortex-derived astrocytes. This finding was independent of whether HSV-1 strains used originated from brains of patients suffering from herpes simplex encephalitis or from patients with oral cutaneous lesions and lacking neurological symptoms. Also, by immunocytochemistry of cultures after HSV-1 infection, a lower number of plaques were seen in brain stem-derived astrocytes as compared with cortex-derived astrocytes. The observed relative resistance of brain stem-derived astrocytes to replicate HSV-1 might contribute to the ability of the brain stem to withstand infection during reactivation of this virus in the trigeminal neurons.Financial support was received from the Swedish Medical Research Council, the Medical Society of Göteborg, the Swedish Medical Society, and the Faculty of Medicine, University of Göteborg     

Gene delivery to rat enteric neurons using herpes simplex virus-based vectors

Neurons of the enteric (gut) nervous system can be cultured in vitro and readily survive transplantation into the brain making close connections with host neurons. As such, they could potentially be used to deliver therapeutic gene products to the brain after transduction with appropriate genes in culture. Here the authors report the first example of gene delivery to such cultured neurons using herpes simplex virus based vectors. They show that viruses lacking the immediate early gene encoding ICP27 (which are unable to replicate lytically) can efficiently deliver a marker gene to enteric neurons without producing extensive cellular damage. In contrast, viruses lacking only the viral neurovirulence factor encoded by ICP34.5 are inefficient in gene delivery, and produce extensive cellular damage, although they cannot replicate lytically in enteric neurons. A virus lacking both ICP27 and ICP34.5, however, produces less cellular damage than one lacking only ICP27, and is as efficient in gene transfer, whereas inactivation of VMW65 reduces toxicity further. The identification of this virus as a safe and efficient gene delivery vector for enteric neurons paves the way for the eventual delivery of therapeutic genes and subsequent transplantation of engineered neurons into the CNS.     

Infection with herpes simplex virus type 1 is associated with cognitive deficits in bipolar disorder.

BACKGROUND: In a previous investigation, we found an association between reduced cognitive functioning and the prevalence of antibodies to herpes simplex virus type 1 in individuals with schizophrenia. The current study was undertaken to determine if this association also occurs in individuals with bipolar disorder. METHODS: Cognitive functioning and serologic evidence of infection with potentially neurotropic herpesviruses were measured in 117 individuals with bipolar disorder and in 100 individuals without a history of psychiatric disorder. Cognitive functioning was evaluated with the Repeatable Battery for the Assessment of Neuropsychological Status. For each patient, serologic evidence of infection was ascertained by the measurement of serum immunoglobulin G class antibodies with specificities for six potentially neurotropic human herpesviruses. The association between serologic evidence of herpesvirus infection and cognitive functioning was analyzed by univariate analyses, as well as multivariate analyses that included demographic and clinical factors associated with cognitive functioning. RESULTS: Serologic evidence of infection with herpes simplex virus type 1 was an independent predictor of decreased cognitive functioning in the individuals with bipolar disorder (F = 20.5, p <.0001). Discriminant function analysis indicated that most of the difference in cognitive functioning between individuals who were antibody positive and antibody negative for herpes simplex virus type 1 could be attributed to immediate verbal memory (F = 12.07, p <.001). There was no significant association between cognitive functioning and the other human herpesviruses. No association between antibodies to herpesviruses and cognitive functioning was found in the control individuals without a history of psychiatric disorder. CONCLUSIONS: Serologic evidence of herpes simplex virus type 1 infection is associated with cognitive impairment in individuals with bipolar disorder.     

阿昔洛韦对单纯疱疹病毒脑炎小鼠脑细胞的保护作用

目的 了解单纯疱疹病毒脑炎(HSE)脑细胞结构的改变及药物的影响。方法 采用光学显微镜及透射电子显微镜观察HSE小鼠脑细胞结构的变化，并给予阿昔洛韦(ACV)及地塞米松(DEX)治疗，观察治疗后脑细胞结构的变化。结果 HSE小鼠脑神经细胞明显肿胀，核仁固缩，核内结构破坏，线粒体及高尔基体可见空泡样变性，核仁内可见病毒颗粒。用药物干预的小鼠脑神经细胞改变较轻微，未找到病毒颗粒；与单用ACV干预的小鼠相比，用ACV DEX干预的小鼠脑神经细胞及毛细血管周围水肿明显减轻。结论 HSE早期给予ACV DEX治疗，对HSE脑细胞结构有明显保护作用。     

Morphological and physiological studies on cultured nerve cells from guinea pigs infected with herpes simplex virus in vivo

Adult guinea pigs were inoculated with type 2 herpes simplex virus (HSV) on the whole back skin, and nerve cells from the dorsal root ganglia (DRG) 6 days after infection were grown in tissue culture. Morphological and physiological properties of the cultured nerve cells from HSV-infected animals (HSV-NC) were compared to those of nerve cells of DRG from the control, non-virus infected animals (CON-NC). During the early period of the culture (0-4 days) growth of nerve cells and non-neuronal cells from the HSV infected animals was essentially the same as that from the control animals. HSV-antigen was present in only a small percentage of the HSV-NC by immunofluorescence (IF). Electrophysiological examination revealed that most of the HSV-NC exhibited a reduced capability of generating spikes, which was quantitatively described as a reduction in the Vmax of the Na spikes. This was interpreted as a reduction in the number of Na channel molecules in the plasma membrane of the HSV-NC, while the resting membrane properties of the same cells, such as the resting membrane potential, input resistance and capacitance, were essentially the same as those of the CON-NC. On 4-5 days in culture, some HSV-NC regained a full capability to generate Na spikes. We considered these nerve cells to have overcome the HSV infection and were now entering a latent period of HSV infection. About 1/3 of the HSV-NC still remained incapable of generating Na spikes. Viral antigen was detected in only 10% of the nerve cells. In the late stage of the culture, HSV infection in vitro was first observed as lysis of non-neuronal cells growing close to some HSV-NC. Nerve cells then started to lose their neurites and became spherical. Finally on 8-9 days most of the cells, including the nerve cells, were lost from the dishes as a result of a generalized infection of supporting cells, i.e. fibroblasts and Schwann cells. This study confirms our previous finding that the electrophysiological technique is much more sensitive than the IF method for the detection of HSV-infection in nerve cells. The results indicate that some nerve cells infected with HSV overcome the infection in vitro. This is interpreted as the entering of these nerve cells into a latent period of HSV infection in vitro.     

Targets of herpes simplex virus type 1 infection in a mouse corneal model

Summary In animal models, spread of herpes simplex virus type 1 (HSV-1) from epithelial replication sites to the peripheral and central nervous system is known from analysis of individually dissected tissues. To examine virus spread in undissociated tissues, corneas of adult mice were inoculated with HSV-1. After 1 to 13 days groups of mice were perfused with formalin, and decalcified blocks of head and neck were embedded in paraffin. At intervals, serial sections were screened for HSV antigen. On days 1 and 2, viral antigen was restricted to cornea and conjunctiva but by days 3 and 4 was also seen in autonomic ganglia and the trigeminal system. On day 6, HSV antigen reached its maximum extent; infected sites included the trigeminal complex (ganglion, root, peripheral ophthalmic and maxillary branches and spinal nucleus and tract), ehtmoid sinus and olfactory buld, visual system, and autonomic ganglia (ciliary, pterygopalatine and superior cervical). Antigen progressively diminished on days 8 and 10, and was not detected on day 13. This method demonstrates a broader range of infected tissues and suggests a more complex pattern of HSV spread than has been previously recognized. Virus appears to reach the intracranial compartment by four different neural routes. When effects of higher and lower corneal inoculation doses were compared, a lower dose resulted in lower peak HSV titers in trigeminal ganglion and brain stem and later virus appearance in these tissues. Thus, dose may influence the kinetics of HSV spread from the peripheral inoculation site to the CNS.Supported in part by U.S.U.H.S. grant, R07396. the opinions or assertions contained herein are the private views of the authors and should not be construed as official or necessarily reflecting the views of the Uniformed Services University of the Health Sciences or Department of Defense. There is no objection to its presentation and/or publication