眼镜蛇毒神经生长因子诱导人鼻咽癌CNE-2细胞凋亡作用的研究

目的：研究广西眼镜蛇毒神经生长因子（Nerve growth factor，NGF）对人鼻咽癌CNE2细胞增殖的抑制作用和诱导细胞凋亡。方法：应用三磷酸腺苷-生物荧光肿瘤化疗药物敏感试验法（ATP—TCA法）测定不同质量浓度（0．0125，0．025，0．05，0．1，0．2g·L^-1）NGF处理24，48，72，96h对人鼻咽癌CNP2生长的抑制率；Hoechst 33342荧光染色观察凋亡细胞的形态；琼脂糖凝胶电泳测定DNA断裂状况；流式细胞术检测细胞周期变化和细胞凋亡率。结果：不同浓度的NGF能抑制CNE-2细胞增殖，并呈浓度时间效应关系，作用24，48，72，96h的半数抑制浓度（IC50）分别为0．213，0．095，0．048，0．033g·L^-1；经NGF（0．1g·L^-1）处理细胞48h后，荧光染色呈现典型的细胞凋亡形态学特征；DNA凝胶电泳可见凋亡细胞特有的DNA片段；0．（15，0．1，0．2g·L^-1 NGF作用48h后，CNB2细胞被阻滞于G0／G1期，细胞凋亡率分别为（28．2±2．0）％、（39．9±1．9）％、（50．3±1．3）％。结论：NGF通过诱导人鼻咽癌CNE-2细胞凋亡而产生抗鼻咽癌活性。     

眼镜蛇毒因子的研究进展

目的：介绍眼镜蛇毒因子的性质,应用现状及发展前景,方法：参考国内外的文献,介绍眼镜蛇毒因子的理化性质,分子生物学特性,免疫学作用机制及其临床应用。结果与结论：眼镜毒因子的一种强效的补体抑制剂,具有突出的优点和特异的临床应用范围,有广阔的开发前景。     

马蔺子甲素诱导鼻咽癌细胞凋亡的研究

目的:初步探讨马蔺子甲素体外抗鼻咽癌作用和诱导鼻咽癌细胞凋亡作用.方法:细胞毒测定以MTT法,细胞凋亡的测定以流式细胞仪法.结果:马蔺子甲素对多种鼻咽癌细胞株均具有细胞毒作用,对CNE_2、SUNE_1、Fadu细胞的IC50分别为18.8±3.1、22.4±12.0、19.3±6.8μmol/L;马蔺子甲素也能诱导鼻咽癌细胞株CNE_2细胞凋亡.Fadu细胞与58.8、29.4μmol/L浓度马蔺子甲素共同培养48h,其诱导细胞的凋亡率分别为59.3%±4.5%、33.2%±2.3%,半数凋亡浓度为56.3μmol/L,且具有剂量依赖性.结论:马蔺子甲素本身也具有抗鼻咽癌作用及诱导鼻咽癌细胞凋亡的作用.     

马蔺子甲素诱导鼻咽癌细胞凋亡的研究

目的:初步探讨马蔺子甲素体外抗鼻咽癌作用和诱导鼻咽癌细胞凋亡作用.方法:细胞毒测定以MTT法,细胞凋亡的测定以流式细胞仪法.结果:马蔺子甲素对多种鼻咽癌细胞株均具有细胞毒作用,对CNE_2、SUNE_1、Fadu细胞的IC50分别为18.8±3.1、22.4±12.0、19.3±6.8μmol/L;马蔺子甲素也能诱导鼻咽癌细胞株CNE_2细胞凋亡.Fadu细胞与58.8、29.4μmol/L浓度马蔺子甲素共同培养48h,其诱导细胞的凋亡率分别为59.3%±4.5%、33.2%±2.3%,半数凋亡浓度为56.3μmol/L,且具有剂量依赖性.结论:马蔺子甲素本身也具有抗鼻咽癌作用及诱导鼻咽癌细胞凋亡的作用.     

Effect of glutathion on cadmiuminduced cytotoxicity in nasopharyngeal carcinoma cell

To study the possible role of glutathion (GSH) in cadmium-induced anti-tumor effects on human nasopharyngeal carcinoma(NPC).CNE1 cell line was treated with CdCl2,and together with N-acetyl cysteine(NAC),the predecessor of GSH,or with L-buthionine-[S,R]-sulfoximine (BSO),a selective inhibitor of GSH synthesis.The intracellular GSH level,the LDH leakage and MTT assay were measured.Results showed that there was remarkable proliferation in NAC CdCl2 treated CNE1 cells,with high activity of GSH.However,in BSO CdCl2 exposed cells,with low level GSH,the proliferation was significantly inhibited.It was indicated that GSH depletion would sensitize the inducible cytotoxicity in NPC cell,which correlated with cadmium-induced anticancer effects.     

去甲斑蝥素诱导人鼻咽癌细胞CNE1凋亡的实验研究

目的检测去甲斑蝥素对人鼻咽癌细胞CNE1增殖、凋亡的影响,为临床应用NCTD治疗鼻咽癌细胞提供实验基础。方法体外常规培养CNE1细胞用于实验,经NCTD作用后,采用MTT比色法检测细胞增殖情况,流式细胞仪观察鼻咽癌细胞凋亡、细胞周期。结果 NCTD对鼻咽癌细胞的生长有明显的抑制作用,呈剂量依赖性,与对照组比较差异有统计学意义(P<0.01);NCTD可剂量和时间依赖性地诱导肿瘤细胞凋亡;NCTD可剂量依赖性地诱导肿瘤细胞周期发生改变,将鼻咽癌细胞阻滞在G2期。结论 NCTD对体外培养的CNE1细胞的生长有明显抑制作用和诱导凋亡的作用。     

Direct cytotoxicity of Lentinula edodes mycelia extract on human hepatocellular carcinoma cell line

Lentinula edodes mycelia (L.E.M.) is a dried powder extracted from shiitake mushrooms (Lentinula edodes). We previously demonstrated that it has immunomodulatory effects. In this paper, the direct cytotoxic effects of the polysaccharide-rich fraction of L.E.M. (L.E.M. ethanol precipitate; LEP) on HepG2 human hepatocellular carcinoma (HCC) cells were investigated. LEP directly killed the HepG2 cells efficaciously, but had only minor effects on normal rat hepatocytes and normal mouse dermal cells under the same conditions. Characteristic morphological changes associated with apoptosis such as shrinkage, rounding, and floating as well as chromatin condensation were confirmed; terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was positive as determined by fluorescence microscopy analyses. The caspase-3 and -8 death receptor pathway was found largely responsible for the apoptotic death of HepG2 cells treated with LEP. In conclusion, LEP can directly induce apoptosis of HepG2 cells, and thus may have potential chemotherapeutic applications for the treatment of HCC.     

细胞角蛋白13基因联合放疗对人鼻咽癌细胞株的作用

目的观察CK13基因对人鼻咽癌HNE1细胞株放疗敏感性的影响。方法将HNE1细胞株分为HNE1-CK13A、HNE1-CK13B、HNE1-pEGFP-N1、HNE1四组,经放疗后进行Western blotting及凋亡坏死率的检测。结果转染了CK13基因的HNE1细胞株在放疗后比对照组的凋亡坏死率多,且随着放射剂量的增大,凋亡率也随之提高。结论 CK13基因对人鼻咽癌HNE1细胞株的放疗起协同作用,为HNE1细胞株的放射增敏剂。     

中华眼镜蛇毒组分抑制肺癌细胞H1299的血管生长因子VEGF和Bfgf

目的:了解中华眼镜蛇毒活性组分(CCVAF)对人非小细胞肺癌细胞株H1299VEGF、bFGF表达的抑制作用。方法:采用RTPCR方法检测H1299细胞中VEGF、bFGFmRNA表达的变化;利用ELISA检测培养上清中VEGF、bFGF蛋白的含量。结果:不同浓度的(2.0、4.0μg·ml-1)CCVAF对H1299bFGFmRNA水平有降低作用,其中4.0μg·ml-1CCVAF作用7h后bFGFmRNA的表达明显降低,但对VEGFmRNA的表达无明显影响。CCVAF作用H1299细胞24h后,其细胞上清中VEGF、bFGF的含量较对照组明显减少(P<0.05),且呈剂量依赖性。结论:CCVAF可抑制H1299细胞bFGFmRNA和蛋白的表达及VEGF蛋白的表达。     

中华眼镜蛇毒活性组分抑制微血管形成研究

目的探讨中华眼镜蛇毒活性组分(CCVAF)抗血管生成的作用。方法采用原代培养牛肺主动脉内皮细胞克隆形成实验、Transwell小室趋化实验及大鼠胸主动脉环体外无血清培养微血管样结构形成实验,分别观察CCVAF对内皮细胞克隆形成、细胞迁移及大鼠动脉环微血管生成的影响。结果 CCVAF浓度为1.25,2.5,5.0μg/mL时,其对内皮细胞克隆形成抑制率分别为17.7%,40.3%,62.9%,呈浓度依赖性(r=0.982,P<0.05),而对内皮细胞迁移抑制率分别为20.5%,59.0%,73.5%,呈浓度依赖性(r=0.902,P<0.05);大鼠动脉环培养至第13天,CCVAF组较溶剂对照组微血管生成率下降了86.5%,CCVAF+VEGF组较VEGF对照组微血管生成率下降了88.1%。结论 CCVAF能够抑制内皮细胞克隆的形成、Hela细胞诱导的内皮细胞迁移和大鼠动脉环微血管的生成。     

Cantharidin-induced cytotoxicity and cyclooxygenase 2 expression in human bladder carcinoma cell line

Mylabris is used in clinical therapy, but is always accompanied by cystitis. The toxic effects of mylabris on bladder are attributed to its active principle: cantharidin. In the present study, we explored how cantharidin induces cytotoxicity in the bladder. Human bladder carcinoma cell line T 24 cells were used as target cells, and human colon carcinoma HT 29 cells as native cells. Cantharidin exhibited acute cytotoxicity in the T 24 cells, and IC(50) was 21.8, 11.2 and 4.6 microM after treatment for 6, 24 and 48 h, respectively. The cytotoxicity of cantharidin was not significantly enhanced when T 24 cells were treated for a longer time. Moreover, PARP proteins and pro-caspase 3, Bcl-2 were significantly inhibited after cantharidin treatment in T 24 cells. Pretreatment with the caspase 3 inhibitor markedly inhibited cantharidin-induced cell death. Therefore, we suggested that cantharidin could induce apoptosis via active caspase 3 in T 24 cells. When T 24 cells were treated with cantharidin at a low dose, the cell cycle was arrested in the G(2)/M phase. Furthermore, p21(Cip1/Waf1) was enhanced, and cyclin A, B1 and cdk1 decreased. At a high dose (more 12.5 microM), cantharidin could stimulate T 24 cells to deplete a large number of ATP and induce secondary necrosis. In addition, cantharidin also stimulated COX 2 over-expression and PGE(2) production in T 24 cells, in a dose-dependent manner. However, cantharidin also induced apoptosis and G(2)/M phase arrest in HT 29 cells, but did not induce COX 2 over-expression. Therefore, we suggest that cantharidin may induce cystitis through secondary necrosis and COX 2 over-expression.     

Bioactivities of nerve growth factor from Chinese cobra venom

The nerve growth factor, NGF, from Chinese cobra Naja naja atra venom was isolated by gel-filtration and ion-exchange chromatography. Cobra NGF was characterized by analytical HPLC techniques as well as SDS-PAGE, and was proven to be a glycoprotein with a mol. wt. of 23 (+/- 2) kD and a pI of 9.2. The amino acid analysis and N-terminal sequencing were performed using conventional methods. Bioassays with cultured chick embryos ganglia and rat pheochromocytoma PC-12 cells revealed a promotion of fiber outgrowth, which is typical of NGF activity. Absence of enzymatic, toxicological, and teratogenic activities were shown by quality inspection. Since 1994, many clinical cases about volunteers receiving NGF treatment have been reported in mainland China. Bioactivities of NGF deal with a wide range of disciplines and technologies. In this paper we will discuss neuronal and non-neuronal effects of NGF treatment. Does the NGF cross the blood-brain barrier by transcytosis into the brain tissue? How is NGF important in wound healing, especially in peripheral nerve injury and diabetic neuritis? NGF may also be useful for male volunteers suffering from sterility, because it is possible that the sexual cells of testis can be promoted to maturity.     

土贝母皂苷诱导人鼻咽癌细胞株CNE-2Z细胞凋亡的研究

目的　研究土贝母皂苷 (下称皂苷 )对人鼻咽癌细胞株CNE 2Z细胞凋亡的影响。方法　MTT法检测皂苷对CNE 2Z细胞生长的影响 ;形态学方法 (荧光显微镜和透射电镜 )、流式细胞仪和DNA琼脂糖凝胶电泳观察和分析在皂苷作用下CNE 2Z细胞形态、DNA含量的变化和DNA断裂的情况 ;蛋白免疫印迹法检测凋亡相关基因bcl 2、bax和cas pase 3表达的变化。 结果　皂苷抑制CNE 2Z细胞的生长 ,其效果与皂苷的浓度和作用时间相关 ;诱导CNE 2Z细胞发生典型程序性死亡 ;凋亡抑制基因bcl 2表达逐渐减少 ;而凋亡诱导基因bax和caspase 3在 1、3、5h表达增高。 结论　诱导细胞凋亡是皂苷发挥抗瘤效果的一种重要机制 ;皂苷诱导的CNE 2Z细胞凋亡与bcl 2、bax和caspase 3基因有关     

眼镜蛇毒因子抑制小鼠疼痛反应

<正>疼痛是诸多病征共有的症状,是动物的神经系统对伤害刺激的感觉。疼痛研究的焦点在于疼痛发生的机制以及干预治疗的策略和药物研究~([1-2])。补体旁路激活会导致炎症和组织损伤~([3])。有研究表明,补体与某些神经病理性疼痛关系密切~([4-5]),但其与非神经性疼痛的关系尚不清楚。本研究用眼镜蛇毒因子(cobra venom factor,CVF)特异抑制补体旁路途径,观察小鼠对疼痛的反应。1材料与方法     

中华眼镜蛇毒活性组分选择性抑制内皮细胞增殖

目的观察中华眼镜蛇毒活性组分(Ch ina cobra venomactive factor,CCVAF)对内皮细胞增殖的选择性抑制作用。方法实验分对照组和不同浓度的CCVAF组,应用MTT法分析CCVAF对牛肺主动脉内皮细胞(bovine arteria pu lmona-lis vascu lar endothelial cells,BAVEC)、SD大鼠主动脉血管平滑肌细胞(smooth musc le cells,SMC)、人胚肺成纤维细胞HLF以及人支气管上皮细胞HBE生长增殖的影响;并运用直线回归分析统计对比CCVAF在不同时间点对BAVEC的IC50及上述各细胞的24 h IC50。结果CCVAF呈剂量及时间依赖性抑制内皮细胞的增殖:6、12、24、48、72 h其IC50分别为4.955、4.932、2.443、3.048、3.133 mg.L-1,24 h抑制作用最强;观察此时浓度与抑制作用的关系,0.625、1.25、2.5、5、10、20 mg.L-1,抑制率分别是4.41%、30.94%、61.81%、86.17%、96.05%、98.68%,10 mg.L-1抑制率即达高峰,且各浓度组抑制作用与对照组相比差异有显著性(P<0.05)。CCVAF抑制内皮细胞增殖具有一定的选择性,作用24 h后,抑制BAVEC生长的作用明显高于其它3种细胞,对BAVEC、HLF、HBE、SMC的24 h IC50分别为2.443、22.233、35.318、32.810 mg.L-1。结论CCVAF能够选择性抑制内皮细胞增殖,这在抗血管生成及其靶向治疗研究中可能具有重要意义。     

Monofunctional platinum complexes showing potent cytotoxicity against human liver carcinoma cell line BEL-7402

Three novel Pt(II) complexes [PtL(1)'Cl] I (L(1)' = glycine-N'-8-quinolylamide), [PtL(2)'Cl] II (L(2)' = l-alanine-N'-8-quinolylamide), and [PtL(3)Cl] III [L(3) = N-(tert-butoxycarbonyl)-l-methionine-N'-8-quinolylamide] have been synthesized and characterized. The crystal structure of complexes II and III showed that the ligands are three-coordinated with only one Cl(-) as the leaving group. Complex II crystallized in the monoclinic system with space group P2(1), a = 9.502(2) A, b = 4.724(1) A, c = 14.800(3) A, while complex III crystallized in the orthorhombic system with space group P2(1)2(1)2(1), a = 5.441(1) A, b = 12.978(3) A, c = 29.438(6) A. These complexes have been tested against a wide range of tumor cell lines including BEL-7402, HCT-116, SPC-A4, MOLT-4, P388, HL-60, A-549, SGC-7901, MKN-28, and HO-8910. Complex III is highly cytotoxic against the HCT-116 (IC(50) = 0.38 microM), SPC-A4 (IC(50) = 0.43 microM), BEL-7402 (IC(50) = 0.43 microM), and MOLT-4 (IC(50) = 0.61 microM) cell lines. The cell line most sensitive to III is human liver carcinoma cell line BEL-7402, which has a response rate of 75.1% at 6.6 x 10(-7) M, nearly 6 times higher than that of cisplatin.     

siRNA沉默Twist基因对鼻咽癌细胞CNE-2增殖的影响

目的探讨RNA干扰沉默Twist基因对人鼻咽癌细胞株CNE-2的迁移、侵袭和增殖等生物学活性的影响。方法构建Twist-shRNA表达载体,脂质体法将重组质粒分别转染入鼻咽癌细胞株CNE-2中,RT-PCR法检测Twist mRNA、Western blot法检测Twist蛋白表达量的变化。MTT法检测细胞的增殖,细胞创伤愈合试验观察细胞迁移能力,Tran-swell小室试验检测细胞侵袭能力的变化。结果沉默Twist基因可减慢CNE-2细胞生长速度(与对照组比较P<0.05),降低CNE-2细胞的迁移速度,抑制CNE-2的体外侵袭能力。结论靶向封闭Twist基因的表达,可明显抑制CNE-2细胞的增殖、迁移及侵袭能力,提示Twist可作为鼻咽癌防治的一个新的靶点。     

左旋棉酚对人鼻咽癌细胞凋亡的影响

目的探讨左旋棉酚对人鼻咽癌细胞株CNE2凋亡的影响及其机制。方法采用MTT实验检测左旋棉酚对CNE2细胞的增殖抑制作用,流式细胞术分析左旋棉酚诱导细胞凋亡及其对Bcl-2、Bax蛋白表达的影响,caspase-3分光光度法检测试剂盒测定caspase-3活性。结果≥10μmol／L左旋棉酚可显著抑制CNE2细胞增殖,并表现出剂量、时间依赖效应。左旋棉酚可调节CNE2细胞的细胞周期,使其主要被阻滞于G0／G1期,在诱导细胞凋亡过程中,凋亡相关基因Bcl-2表达下降,而Bax表达则相对上调,caspase-3活性在24h达到高峰。结论左旋棉酚在体外可诱导鼻咽癌细胞CNE2凋亡,其机制可能与Bcl-2基因下调、Bax基因上调、caspase-3的激活有关。     

中华眼镜蛇毒活性组分诱导内皮细胞凋亡

目的观察中华眼镜蛇毒活性组分(China cobra venom active factor,CCVAF)诱导内皮细胞凋亡的作用。方法采用荧光显微镜、流式细胞仪等方法检测CCVAF对牛肺主动脉内皮细胞(bovine arteria pulm onalis vascular endothelialcells,BAVEC)凋亡形态学、DNA、细胞周期以及凋亡指数的影响。结果经AO染色、流式细胞仪分析均证实CCVAF能够诱导内皮细胞出现凋亡。荧光显微镜下观察到CCVAF各浓度(0.15625、0.3125、0.625、1.25μmol.L-1)组处理的内皮细胞,核染色质固缩或断裂成片段状,染色不均匀,出现凋亡小体,凋亡细胞的比例随浓度升高而增加,0.625μmol.L-1组视野内凋亡细胞最多;流式细胞仪分析结果表明CCVAF在浓度低于0.625μmol.L-1,细胞生长被阻滞在S期,1.25μmol.L-1时则被阻滞在G0/G1期,且细胞凋亡率随浓度升高而逐渐增加。结论CCVAF能够诱导内皮细胞出现凋亡,通过此途径抑制内皮细胞的生长增殖,这可能是其对抗血管生成的机制。