转染连接蛋白基因对胶质瘤细胞细胞间隙连接通讯及其生长变化的研究

目的　研究连接蛋白 (Cx)基因对C6胶质瘤细胞的增殖抑制及细胞间隙连接通讯(GJIC)的作用 ,探讨以Cx43基因治疗胶质瘤的可行性。方法　将含Cx43cDNA的质粒以Lipofec tamine介导转染Cx43表达缺失的C6胶质瘤细胞 ,通过Northern印迹杂交、原位杂交及免疫组织化学染色检测Cx43mRNA及蛋白表达 ,划痕标记荧光染料示踪技术 (SLDT)检测GJIC ,MTT法测定细胞增殖率 ,核仁组成区嗜银蛋白 (AgNOR)染色检测细胞增殖活性 ,TUNEL法检测细胞凋亡。结果　转染后C6细胞有不同程度的Cx43mRNA及蛋白表达和GJIC恢复。表达Cx43的克隆细胞增殖明显下降 ,培养 2～ 6d时 ,每天除C6组与空载组差异无显著性外 ,其余各组差异均有非常显著性 (P <0 .0 1) ,但细胞凋亡并未增加。结论　Cx43基因及GJIC在恶性胶质瘤的发生发展过程中起重要作用 ,可能成为恶性胶质瘤基因治疗的优选靶的之一。     

人脑胶质瘤白介素13受体基因表达与肿瘤增殖活性的关系研究

目的探讨人脑胶质瘤白介素13受体（IL-13R）基因表达与肿瘤增殖活性的关系。方法对6例正常脑组织，50例人脑胶质瘤和2个脑瘤体外细胞系采用RT—PCR法和免疫组织化学法检测IL-13R。结果人脑胶质瘤组织IL-13RαmRNA总阳性表达率70％，正常脑组织中仅1例有极弱的表达；2例恶性胶质瘤体外细胞系均高表达。IL-13RαmRNA表达率和表达丰度与胶质瘤分级（前者rs=0．87，P〈0．01；后者rs=0．69，P〈0．01）、肿瘤增殖活性Ki-67LI（r=0．64，P〈0．01）呈正相关，即胶质瘤恶性程度越高，IL-13RαmRNA表达率和表达水平越高。结论IL-13Rα基因在人脑胶质瘤中表达上升，与肿瘤的分级和肿瘤增殖活性呈正相关，可作为预测某些肿瘤治疗效果及监测复发的指标之一。     

人脑胶质瘤血管生成素-2表达及其与管生成和脑水肿的关系

目的 研究血管生成素.2(Ang-2)基因在人脑胶质瘤表达及其与胶质瘤血管生成及瘤周水肿的关系。方法 用半定量逆转录-聚合酶链反应(RT-PCR)、免疫组织化学方法测定42例人脑胶质瘤和8例正常脑组织中Ang-2 mRNA及其蛋白表达情况。用免疫组织化学方法检测肿瘤微血管密度(MVD)。结果 正常脑组织中无或弱表达Ang-2。42例胶质瘤组织中均有Ang-2 mRNA表达,不同级别间Ang-2 mRNA的表达差异有显著性(P<0.05)。随着脑胶质瘤恶性程度的增加,Ang-2 mRNA的表达增高(r=0.894,P<0.01)。免疫组织化学结果显示,胶质瘤细胞及肿瘤血管内皮细胞中均有Ang-2蛋白表达。Ang-2 mRNA表达与MVD、脑水肿指数(EI)显著相关(分别为r=0.853,P<0.01;r=0.784,P<0.01)。结论 Ang-2可能参与胶质瘤血管生成,对胶质瘤瘤周脑水肿及恶性进展有促进作用。     

苯乙酸对胶质瘤U-251细胞中RNA编辑酶表达的影响

目的观察正常人脑胶质细胞和胶质瘤U-251细胞中RNA编辑酶RED1,RED2 mR- NA水平的表达,并观察诱导分化剂苯乙酸对U-251细胞中RED1 mRNA表达的影响。方法对原代培养的正常人脑胶质细胞和胶质瘤U-251细胞,应用RED1,RED2全长序列合成引物及合成的特异引物,分别通过逆转录-聚合酶链反应(RT-PCR)检测RED1,RED2 mRNA水平的表达。用RT- PCR及图像分析法,检测胶质瘤细胞U-251在不同浓度的苯乙酸处理前后,RED1 mRNA表达水平的变化。RED1基因表达水平用基因/β-肌动蛋白(β-actin)灰度比值表示。结果RED1在正常人脑胶质细胞中表达极弱,在高恶性度的胶质瘤U-251细胞中明显表达。诱导分化剂苯乙酸作用后, U-251细胞中RED1表达水平降低。RED2在正常人脑胶质细胞及苯乙酸处理前后的胶质瘤细胞中,均未见表达。结论RED1 mRNA水平高表达,可能与高恶性度胶质瘤的发生有关。诱导分化剂苯乙酸可能通过降低RED1mRNA水平的表达,作用于胶质瘤细胞的RNA编辑过程。     

脑胶质瘤中p27和Cyclin D1的表达及意义

目的探讨人脑胶质瘤组织中Cyclin D1和p27蛋白的表达及其与细胞凋亡的关系。方法采用免疫荧光法检测脑胶质瘤和非肿瘤组织中p27和Cyclin D1蛋白的表达,并用图像分析系统作定量分析。原位末端标记（TUNEL）法检测细胞凋亡情况,计算其凋亡细胞密度。结果p27蛋白的表达强度随肿瘤恶性程度增高而下降;Cyclin D1蛋白的表达强度随肿瘤恶性程度增加而升高;并且p27基因表达强度与细胞凋亡密度呈显著正相关。结论p27表达下降或缺失和Cyclin D1的过表达与脑胶质瘤的发生、发展密切相关。p27蛋白表达的下降可能导致肿瘤细胞凋亡减少。p27kip1有望成为胶质瘤基因治疗的新策略。     

间隙连接蛋白家族中Cx26、Cx32、Cx43在前列腺癌组织中的表达

目的:研究间隙连接蛋白家族中Cx26、Cx32、Cx43在前列腺癌(PCa)及良性前列腺增生(BPH)组织中的表达情况,分析三者与PCa生物学行为关系,探索其在PCa发生、发展中的作用机制,为PCa的诊断及治疗提供新的实验依据。方法:随机收集我院近4年存档的PCa石蜡标本31例、BPH 23例,采用免疫组化染色SABC法回顾性研究Cx26、Cx32、Cx43在PCa和BPH组织中的表达情况,半定量研究Cx26、Cx32、Cx43的表达与PCa、BPH的临床和病理参数的关系。结果:①Cx26、Cx32、Cx43在BPH、PCa组织中的阳性表达率分别为82.6%与74.2%(χ2=0.541,P0.05)、78.3%与61.3%(χ2=1.763,P0.05)和87.0%与38.7%(χ2=12.730,P0.01),Cx43在PCa组织中的阳性表达率较BPH中显著降低。②Cx26、Cx43在PCa组织中的阳性表达强度与肿瘤的恶性程度呈负相关(r Cx26=-0.476,P0.01;r Cx43=-0.484,P0.01);Cx32的表达与肿瘤恶性程度无相关性(r=-0.242,P0.05)。③3种Cx表达与年龄、血清PSA浓度及组织中PSA表达强度无相关性,且3种Cx间的表达亦无相关性。结论:Cx26、Cx32、Cx43在BPH和PCa组织中均有不同程度表达,其中Cx43在PCa的发生、发展中可能起到一定的作用,其也许可作为PCa除PSA外的另一标志物以及PCa生物治疗的新靶点,Cx26在PCa进展过程中可能起一定的作用,但其机制尚需进一步研究。     

血管内皮生长因子和间隙连接蛋白43在胰腺癌组织中的表达及其临床意义

目的探讨血管内皮生长因子（VEGF）和间隙连接蛋白43（Cx43）在胰腺癌组织中的表达特征及其临床意义。方法采用免疫组织化学链霉菌抗生物素蛋白-过氧化物酶连结法观察70例胰腺癌组织和癌周正常组织中VEGF和Cx43的表达情况，分析其表达与胰腺癌临床病理特点之间的关系。结果VEGF在胰腺癌组织中的阳性表达率为77．1％（54／70），在癌旁正常组织中的阳性表达率为18．6％（13／70），两者差异有统计学意义（P〈0．01）；Cx43在胰腺癌组织中的阳性表达率为30．0％（21／70），在癌旁正常组织中的阳性表达率为72．9％（51／70），两者差异有统计学意义（P〈0．01）。VEGF表达水平与胰腺癌肿瘤大小、TNM分期、淋巴结转移有关（P〈0．05），Cx43表达水平与胰腺癌组织学分化程度、TNM分期、淋巴结转移有关（P〈0．05）。结论同时检测VEGF与Cx43表达水平有助于判断胰腺癌恶性程度和预后。     

长链非编码RNA在神经胶质瘤中的相关作用机制及代表分子

胶质瘤是最常见的原发性颅内肿瘤之一,按恶性程度为Ⅰ～Ⅳ,其组织学亚型包括星形细胞瘤,多形性胶质母细胞瘤,少突胶质细胞瘤和混合型肿瘤.胶质瘤具有细胞快速增殖和血管生成的特征,其中胶质母细胞瘤恶性程度最高,预后极差,复发及死亡率高.长链非编码RN A在神经胶质瘤组织和细胞系中显著差异表达,其对神经胶质瘤的发生,发展和其他恶...     

细胞周期因子4抑制因子a和星形细胞上调基因-1与少突胶质细胞瘤的预后关系

目的 探讨细胞周期因子4抑制因子a(inhibitor of CDKda,p16INK4a)和星形细胞上调基因-1(astrocyte elevated gene-1,AEG-1)在少突胶质细胞瘤中的表达,以及其与少突胶质瘤预后的关系.方法 回顾性分析经病理证实的42例少支胶质瘤资料,采用EnVision免疫组化法检测p16INK4a和AEG-1表达,随访患者生存情况.结果 42例中6例失访,p16INK4a总阳性表达率为66.7%,不同级别肿瘤p16INK4a表达阳性率随肿瘤恶性程度增加而降低,而AEG-1阳性率随肿瘤恶性程度的增加而增高,其总阳性表达率为45.2%.p16INK4a表达与生存时间呈正相关,而AEG-1表达则呈负相关.AEG-1与p16INK4a表达之间呈负相关.结论 p16INK4a和AEG-1的表达强度对判断少支胶质瘤恶性程度和预后具有参考价值,可作为临床评估预后的生物学指标之一.     

间隙连接蛋白43的表达与黑色素瘤侵袭能力和预后转归的关联

目的探讨间隙连接蛋白43(connexin 43,Cx43)的表达与黑色素瘤侵袭能力和预后转归的关联。方法筛选自2015年2月至2017年4月沧州市中心医院的70例恶性黑色素瘤患者为研究对象。通过询问以及查阅病例等方式收集患者的一般信息与临床病理信息;采用RT-PCR法测定患者癌组织、癌旁组织中Cx43 mRNA相对含量,并根据癌组织中Cx43 mRNA相对含量将患者分为两组,其中Cx43 mRNA相对含量0.700为高表达组(n=39);≥0.700为低表达组(n=31)。比较两组患者的预后转归情况。结果黑色素瘤患者癌组织中Cx43 mRNA相对含量为0.671±0.052,明显低于癌旁组织的0.748±0.063,两种组织中Cx43 mRNA相对含量差异具有统计学意义(P0.05)。TNM分期为Ⅰ、Ⅱ期的患者,癌组织中Cx43 mRNA相对含量明显高于Ⅲ、Ⅳ期的患者(0.749±0.031∶0.662±0.044);肿瘤直径4 cm的患者癌组织中Cx43 mRNA相对含量明显高于肿瘤直径≥4 cm患者(0.752±0.073∶0.684±0.070);浸润深度为浅层的患者癌组织中Cx43 mRNA相对含量明显高于深层患者(0.744±0.051∶0.677±0.050);发生淋巴结转移患者的Cx43 mRNA相对含量明显低于未发生淋巴结转移的患者(0.692±0.064∶0.733±0.065);不同TNM分期、肿瘤直径、浸润深度、发生与未发生淋巴结转移患者间Cx43 mRNA相对含量存在明显差异(P0.05)。Cx43 mRNA低表达组患者中位生存期为(25.43±5.29)个月,1年生存率为51.61%;高表达组患者中位生存期为(17.18±3.46)个月,1年生存率为23.08%;低表达组患者中位生存期以及1年生存率均显著优于高表达组(P0.05)。结论 Cx43的表达与黑色素瘤侵袭能力以及预后相关,Cx43表达水平越低,表明患者肿瘤侵袭能力越强、病情越严重,且患者往往预后较差。     

EphB4在人胶质瘤过表达及意义

目的探讨人脑胶质瘤中EphB4表达情况及临床病理学意义。方法收集正常脑组织和胶质瘤组织，行逆转录-聚合酶链式反应（RT—PCR）和免疫组织化学染色（IHC），分析与病理级别、年龄、性别的相关性。结果EphB4在胶质瘤中呈过表达，表达于肿瘤细胞和血管上，与病理级别正相关，与年龄、性别无明显相关。结论EphB4／ephnnB2信号可能通过直接影响肿瘤细胞和间接影响瘤性血管的双重作用促进肿瘤发生发展。     

Neural stem cell markers, nestin and musashi proteins, in the progression of human glioma: correlation of nestin with prognosis of patient survival

BACKGROUND: The IF protein nestin and the RNA-binding protein musashi are expressed by neural progenitor cells during CNS development. Their expression in glial tumors was evaluated by immunohistochemistry, and the histopathological scores correlated with levels of cysteine cathepsins that are known prognostic markers in several tumors. METHODS: The levels of nestin, musashi, and cathepsins B and L were assessed by immunohistochemical analysis of biopsies from 87 patients with primary CNS tumors. To confirm the immunohistochemical data, nestin expression was analyzed by real-time PCR in 12 brain tumor biopsies. The exact location of nestin-positive cells was determined by mapping the distribution of nestin in a highly invasive human glioma xenograft model. RESULTS: Immunostaining revealed nestin to be expressed in 95.8% and musashi in 80% of the patient biopsies. The total IHC score for nestin was significantly higher in high- than in low-grade tumors (P < .0001). No difference was observed for musashi (P = .11). Real-time PCR of nestin expression confirmed the immunohistochemical data. Nestin expression was shown to be a strong prognostic marker for decreased overall survival (P = .0001), whereas musashi expression has no prognostic significance. Moreover, nestin was shown by Cox regression analysis to be a stronger prognostic marker than cathepsins B and L. IHC staining of nestin in a xenograft model showed that its expression is localized mainly in the invasive tumor cells at the tumor periphery. CONCLUSIONS: Nestin is shown to be a strong prognostic marker for glioma malignancy. The presented data links the invasive glioma cells to CNS precursor cells, indicating that the most malignant cells in the gliomas may well be closely related to the glioma stem cells.     

Cerebrovascular connexin expression: effects of traumatic brain injury

Traumatic brain injury (TBI) results in dysfunction of the cerebrovasculature. Gap junctions coordinate vasomotor responses and evidence suggests that they are involved in cerebrovascular dysfunction after TBI. Gap junctions are comprised of connexin proteins (Cxs), of which Cx37, Cx40, Cx43, and Cx45 are expressed in vascular tissue. This study tests the hypothesis that TBI alters Cx mRNA and protein expression in cerebral vascular smooth muscle and endothelial cells. Anesthetized (1.5% isoflurane) male Sprague-Dawley rats received sham or fluid-percussion TBI. Two, 6, and 24?h after, cerebral arteries were harvested, fresh-frozen for RNA isolation, or homogenized for Western blot analysis. Cerebral vascular endothelial and smooth muscle cells were selected from frozen sections using laser capture microdissection. RNA was quantified by ribonuclease protection assay. The mRNA for all four Cx genes showed greater expression in the smooth muscle layer compared to the endothelial layer. Smooth muscle Cx43 mRNA expression was reduced 2 h and endothelial Cx45 mRNA expression was reduced 24 h after injury. Western blot analysis revealed that Cx40 protein expression increased, while Cx45 protein expression decreased 24 h after injury. These studies revealed significant changes in the mRNA and protein expression of specific vascular Cxs after TBI. This is the first demonstration of cell type-related differential expression of Cx mRNA in cerebral arteries, and is a first step in evaluating the effects of TBI on gap junction communication in the cerebrovasculature.     

Multidrug resistance gene (MDR1) expression in human brain tumors.

Multidrug resistance for many types of cancer outside the central nervous system (CNS) has been found to be due to the overexpression of the multidrug resistance gene MDR1, of which the gene-product P-glycoprotein acts as a membrane-bound efflux pump for many anticancer drugs. To examine whether brain tumors overexpress the MDR1 gene, 25 brain-tumor specimens were subjected to Northern blot analysis: 10 gliomas, eight meningiomas, three schwannomas, one malignant lymphoma, and three tumors metastatic to the brain. Ten fresh-frozen autopsy specimens of various parts of normal brain were also analyzed. Blots were hybridized with 32P-labeled Chinese hamster complementary deoxyribonucleic acid (cDNA) and 32P-labeled human MDR1 cDNA. The MDR1 gene messenger ribonucleic acid (mRNA) was detected in two tumors using the Chinese hamster probe (one sphenoid wing meningioma and one metastatic prostate tumor) and in one CNS lymphoma using the human probe. Intact mRNA could not be extracted from the fresh-frozen autopsy specimens of normal brain. Seventeen tumors were examined for P-glycoprotein by immunohistochemical staining using murine monoclonal antibody C219: eight gliomas, eight meningiomas, and one craniopharyngioma. The neoplastic cells from two gliomas and three meningiomas and the blood vessels within six gliomas and two meningiomas stained positively for P-glycoprotein. Seven of 10 normal brain specimens stained positively for P-glycoprotein in blood vessels but no specimen demonstrated staining of parenchymal cells. This study demonstrates that the MDR1 gene can be detected in normal brain, and in malignant, benign, and metastatic lesions. P-glycoprotein can be present in tumor blood vessels even when it is not seen in neoplastic cells. Although the role of P-glycoprotein in tumor blood vessels needs to be further examined and more clearly defined, drug resistance in malignant primary brain tumors may result from characteristics not solely of neoplastic cells but also tumor vasculature.     

脑胶质细胞瘤低密度脂蛋白受体活性研究

目的 观察人脑胶质瘤组织的低密度脂蛋白受体(LDLR)活性和其mRNA水平,探讨其与胶质瘤肿瘤级别的关系.方法 以125I-LDL为配体,采用受体-放射性配体结合分析法测定63例肿瘤及其周边脑组织的LDLR活性;逆转录-聚合酶链反应(RT-PCR)技术检测LDLR蛋白mRNA在瘤组织的表达.结果 胶质瘤组织LDLR活性为(129.43±48.04)μmol/g组织,其周边正常脑组织LDLR为(34.06±27.62)μmol/g组织,两者比较差异有统计学意义(P＜0.01);高级别胶质瘤组织的LDLR为(154.59±31.68)μmol/g组织,低级别胶质瘤组织LDLR活性为(92.32±24.37)μmol/g组织,高级别胶质瘤LDLR活性高于低级别(P＜0.05);胶质瘤组织LDLR蛋白mRNA水平均增高,但组间差异无统计学意义(P＞0.05).结论 脑胶质瘤组织LDLR活性明显增高,肿瘤级别越高,LDLR活性越高;因此,LDLR有可能成为脑胶质瘤治疗的潜在靶点.