粒细胞集落刺激因子对卵巢癌细胞株生长曲线的影响

目的 :探讨粒细胞集落刺激因子 (G- CSF)对卵巢癌细胞株生长曲线的影响。方法 :采用 XTT比色法检测不同浓度 G-CSF对卵巢癌细胞株 3AO及 CAOV3细胞生长曲线的影响。结果 :不同浓度 G- CSF作用 2 4～ 1 2 0 h范围内 ,对 3AO及 CAOV3细胞的生长曲线均无明显性的影响 (P >0 .0 5 )。结论 :G- CSF作用较长时间对卵巢癌细胞株 3AO及

鼠抗人多聚体蛋白聚糖单克隆抗体对骨髓瘤细胞株生长的抑制作用

目的研究多聚体蛋白聚糖(syndecan 1,CD138)单克隆抗体(mAb)4B3 对天然表达syndecan 1的人多发性骨髓瘤(MM)细胞XG 1 和XG 2 的生物学效应。方法采用间接免疫荧光标记和流式细胞术分析,通过4B3mAb观察XG 1和XG 2细胞膜上syndecan 1荧光标记阳性率;按常规培养方式对XG 1、XG 2进行细胞培养和台盼蓝染色记数,观察不同浓度的4B3mAb对XG 1和XG 2细胞体外生长作用。结果4B3mAb能识别XG 1 和XG 2 细胞表达的syndecan 1 分子,表达率与商品化的鼠抗人syndecan 1 mAb(BB4)相似;同时,4B3mAb与XG 1 和XG 2 细胞膜上synde can 1荧光标记阳性率基本一致,反应均呈强阳性;加入不同浓度的4B3mAb,XG 1 细胞数量2 d后开始下降,而XG 2细胞3 d后数量出现减少。结论4B3mAb抗体对XG 1 和XG 2 均具有明显的生长抑制作用,但XG 2细胞的生长抑制作用早于XG 1,可为MM的生物治疗提供新途径。

塞来昔布对胶质瘤U251细胞株生长的影响

目的:探讨COX-2抑制剂塞来昔布(celecoxib)对胶质瘤的抑制作用及可能的作用机制。方法:采用胶质瘤细胞株U251移植裸鼠后,服用塞来昔布,测量肿瘤重量;流式细胞仪检测药物对肿瘤细胞凋亡的影响;电镜观察药物作用前后肿瘤细胞超微结构的变化;免疫组织化学观察肿瘤微血管密度(MVD)。结果:药物治疗组和对照组肿瘤的重量分别为(51.3±3.85)mg和(125.2±7.34)mg;治疗组细胞凋亡增多;超微结构可见有凋亡特征的细胞。免疫组织化学可见MVD分别为(25.7±3.25)/HP和(76.4±9.41)/HP。结论:塞来昔布能够减少肿瘤血管生成,促进肿瘤细胞凋亡,从而对肿瘤具有抑制作用,对胶质瘤的治疗具有重要意义。

阿维A对人表皮鳞状细胞癌A431细胞株生长及凋亡的影响

目的研究阿维A对上皮鳞状细胞癌(鳞癌)细胞生长的影响及其诱导凋亡作用。方法以体外培养的人表皮鳞癌细胞A431细胞和角质形成细胞HaCaT细胞为研究对象,用MTT法观察阿维A对A431细胞和HaCaT细胞的生长抑制作用,用光镜和电镜观察细胞形态,流式细胞仪检测凋亡峰的出现,Annexin-V染色证实凋亡的发生。结果随着阿维A作用时间的延长及剂量的增加,对A431细胞增殖的抑制作用增加。同样浓度和同样作用时间的阿维A对A431细胞增殖的抑制率高于对HaCaT细胞的抑制率。光镜和电镜观察显示随阿维A作用时间延长,A431凋亡细胞增多。流式细胞仪检测显示随阿维A作用时间的延长,亚G1期凋亡峰明显增加,Annexin-V染色阳性细胞数增多。结论阿维A具有抗上皮鳞癌细胞生长及诱导上皮鳞癌细胞凋亡作用。

烷基-去-血根碱-N^5-甲基衍生物的合成及肿瘤细胞株生长抑制活性评价

采用汇聚合成方式,分别以6-溴-2,3-二羟基苯甲醛、5-硝基-2,3-萘二酚和二溴甲烷为起始原料,经二氧-去-二溴亲核取代反应、还原反应、席夫碱生成反应以及三丁基锡烷和AIBN诱导的自由基关环反应等反应合成2:3,7:8-双亚甲二氧基苯并[c]菲啶;以2:3,7:8-双亚甲二氧基苯并[c]菲啶为中间体,以NaBH4和不同的脂肪酸为烷基化试剂,合成5,6-二氢-2:3,7:8-双亚甲二氧基-N^5-烷基苯并[c]菲啶;以DDQ为氧化剂,在碱性条件下将2:3,7:8-双亚甲二氧基-5,6-二氢-N^5-烷基苯并[c]菲啶氧化芳构化,并经盐酸盐化反应,得到具有系列性特征的目标化合物2,3:7,8-双亚甲二氧基-N^5-烷基苯并[c]菲啶-5-阳离子季铵盐。与阳性对照药物以及天然血根碱对比,本文合成的系列烷基-去-血根碱-N^5-甲基型血根碱类似物在体外显示出明显获得改善的肿瘤细胞株生长抑制活性;在对5种肿瘤细胞株进行的药理实验中,与血根碱比较,目标化合物的活性提高5倍;表明了对血根碱-N^5-甲基进行长链烷基的替换修饰,可以通过增加脂溶性和空间位阻、提高5,6-位亚胺结构的稳定性而改善其肿瘤细胞株生长抑制活性。

PH Ⅱ-7作用于慢性粒细胞白血病细胞k562的机制研究

目的 PH Ⅱ-7是以靛玉红为模板合成的一种抗肿瘤药物,并有显著逆转耐药作用。实验室前期研究发现在人慢性粒细胞白血病k562细胞中,PH Ⅱ-7可与烯醇化酶ENO1相结合。本研究旨在探究PH Ⅱ-7通过ENO1对k562细胞体外抑癌作用机制。方法构建ENO1表达稳定干扰细胞株k562-shENO1和对照细胞株k562-shCON。通过MTT法检测PH Ⅱ-7对k562-shCON和k562-shENO1细胞株的生长抑制作用;通过细胞计数法检测k562-shCON和k562-shENO1细胞株生长差异;采用流式细胞仪测定细胞凋亡水平。采用逆转录PCR,实时荧光定量PCR和免疫印迹技术检测相关基因表达水平。结果通过real-time-PCR和免疫印迹实验验证k562-shCON和k562-shENO1干扰细胞株成功建立。k562-shENO1和k562-shCON细胞株体外生长无显著差异,PH Ⅱ-7对k562-shENO1和k562-shCON细胞株IC50无显著性差异。PH Ⅱ-7可诱导k562-shENO1和k562-shCON细胞株凋亡,并可激活caspase3、caspase9和PARP等凋亡相关蛋白。与k562-shCON相比较,k562-shENO1细胞对高浓度PH Ⅱ-7诱导凋亡作用更敏感。结论烯醇化酶ENO1与PH Ⅱ-7在人慢性粒白血病k562细胞中作用密切相关,干扰ENO1表达能在一定程度上增强PH Ⅱ-7的抑癌作用。

东亚钳蝎蝎毒多肽组分提取物的抗癌作用

目的 探讨东亚钳蝎蝎毒抗癌多肽组分对不同癌细胞株生长的抑制作用及凋亡的影响。方法 采用离子交换层析法分离纯化获得蝎毒多肽组分精提取物，然后体外直接作用培养的各组癌细胞，通过MTT法观察各组分对不同癌细胞株增殖的影响；采用Annexin V／PI染色的流式细胞技术检测癌细胞早期凋亡率。结果 与阴性对照组比较，蝎毒多肽组分IV对癌细胞株HeLa-3、HepG2、Bewo、QGY、MGC-803增殖的抑制作用显著(P<0．001)，组分Ⅲ针对Hep G2、BeWo、HeLa-3、MGC-803有抑制作用。BeWo流式细胞学检测表明，组分Ⅱ、Ⅲ、Ⅳ、Ⅴ可使癌株的早期细胞凋亡率增高(P<0．01)。结论 蝎毒多肽多种组分提取物Ⅰ-Ⅴ等具有抗癌作用，组分Ⅳ、Ⅲ抗癌谱广，活性强．能促进癌细胞凋亡，有进一步开发为抗癌新药的可能。

我国发现致皮肤恶性黑素瘤恶化关键因子

中南大学湘雅医院皮肤科研究人员从2003年起。对皮肤恶性黑素瘤的发病机理和有效治疗途径进行了系统研究。研究人员采用先进的核糖核酸（RNA）干扰技术，来抑制恶性黑素瘤细胞株生长扩散的诱导因子（CD147）；继而。将经过带有抑制加工诱导因子（CD147）和未加工诱导因子的肿瘤细胞分别注入两组无免疫能力的实验鼠体内进行观察。6～8周后，前者肿瘤恶化程度轻微，后者则明显加重，并出现肿瘤细胞转移现象。

Partial Characteristics of Hydrogen Production by Fermentative Hydrogen-producing Bacterial Strain B49

To investigate the characteristics of hydrogen production by a novel fermentative hydrogen-producing bacterial strain B49 (AF481148 in EMBL), batch experiments are conducted under different conditions. Hydrogen production has a correlation with cell growth and the consumption of glucose and soluble protein. The optimum pH for cell growth is 4.5±0.15. At acidic pH 4.0±0.15, the bacteria has the maximum accumulated hydrogen volume of 2382 ml/L culture and the maximum hydrogen evolution rate of 339.9 ml/L culture·h with 1% glucose. The optimum temperature for cell growth and hydrogen production is 35℃. In addition, fermentative hydrogen-producing bacterial strain B49 can generate hydrogen from the decomposition of other organic substrates such as wheat, soybean, corn, and potato. Moreover, it can also produce hydrogen from molasses wastewater and brewage wastewater, and hydrogen yields are 137.9 ml H 2/g COD and 49.9 ml H 2/g COD, respectively.

Growth-inhibitory effects of MOB2 on human hepatic carcinoma cell line SMMC-7721

AIM:To investigate the growth-inhibiting and apoptosis-inducing effects of the gene MOB2 on human hepatic carcinoma cell line SMMC-7721.METHODS:The full-length cDNA of the MOB2 gene was amplified from human umbilical vein endothelial cells.The correct full-length MOB2 cDNA was subcloned into the eukaryotic expression vector pEGFP-C1.After lipofection of the MOB2 gene into cancer cells,the levels of MOB2 protein in the cancer cells were detected by immunoblotting.To transfect the recombined plasmid vector pEGFP-CI-MOB2 into SMMC-7721 cells,the cells were cultured in Dulbecco's Modified Eagle'sMedium with 10% fetal calf serum and glutamine,and then mixed with liposomes,Lipofectamine 2000 and the plasmid vector pEGFP-CI-MOB2.RESULTS:We observed the growth and proliferation of SMMC-7721 cells containing pEGFP-CI-MOB2 and analyzed their apoptosis and growth cycle phases by flow cytometry.We successfully transfected the recombined plasmid vector pEGFP-CI-MOB2 into SMMC-7721 cells and screened for a single clone cell containing MOB2.After transfection,MOB2 enhanced growth suppression,induced apoptosis,increased the ratio of G0/G1,significantly inhibited the advance of cell cycle phase,and arrested cells in G0/G1 phase.CONCLUSION:MOB2 overexpression induces apoptosis and inhibits the growth of human hepatic cancer cells,which may be useful in gene therapy for hepatic carcinoma.

Effects of transfecting Bcl-2 shRNA and celecoxb on the SGC-7901 cell line

Objective:The aim of this study was to study the changes of SGC-7901 cells transfecting small hairpin RNA(shRNA) targeted Bcl-2 and celecoxib in vitro.Methods:To use the effective siRNA targeted to Bcl-2 by Lipofectamine 2000,the rate of cell growth inhibition of all groups was detected by multiply-table tournament(MTT) method,apoptosis rate was examined by flow cytometry and the expression of Bcl-2 was assayed by Elisa.Results:After siRNA was transfected to SGC-7901 cells,the rate of cell growth inhibition was increased,the joint group was higher by flow cytometry and the expression of Bcl-2 was lower by Elisa.Conclusion:The growth of SGC-7901 cells which was transfected siRNA has been inhibited,and the sensitivity to celecoxib has been increased.

Growth inhibitory effects of Phyllanthus niruri extracts in combination with cisplatin on cancer cell lines

AIM：To investigate the cytotoxic effects of spray-dried extracts of Phyllanthus niruri in combination with cis- platin on two cancer cell lines. METHODS： Colorectal carcinoma （HT29） and human hepatocellular carcinoma （HepG2） cells were treated with spray-dried extracts of Phyllanthus niruri （SDEPN） either alone or in combination with cisplatin at differ- ent concentrations （0.5 mg/mL and 1 mg/mL） for 4 h and 24 h. To verify and quantify cancer cells treated with these products as well as identify the cell cycle stage and cell viability, we stained the cells with prop- idium iodide and assessed them by flow cytometry. The percentage of cells in different cell cycle phases was quantified and data were expressed as histo- grams. Significant differences between groups were determined using analysis of variance and Bonferroni＇s test, as indicated. A value of P 〈 0.05 was considered to be statistically significant. RESULTS： SDEPN had significantly different cyto- toxic effects on HT29 （2.81 4- 0.11 vs 3.51 4- 1.13, P 〉 0.05） and HepG2 （5.07± 0.3 vs 15.9 ± 1.04, P 〈 0.001） cells when compared to control cells for 4 h. SDEPN also had significantly different cytotoxic effects on HT29 （1.91 ± 0.57 vs 4.53± 1.22, P 〉 0.05） and HepG2 （14.56 ± 1.6 vs 35.67 ± 3.94, P 〈 0.001） cells when compared to control cells for 24 h. Both cell lines were killed by cisplatin in a dose-dependent manner compared to control cells （HepG2 cells for 4 h： 10.78 ± 1.58 vs 53.89 ± 1.53, P 〈 0.001; 24 h： 8.9 ± 1.43 vs 62.78 ± 1.87, P 〈 0.001 and HT29 cells for 4 h： 9.52 ±0.913 vs 49.86 ± 2.89, P 〈 0.001; 24 h： 11.78 ± 1.05 vs 53.34 ± 2.65, P 〈 0.001）. In HT29 cells, pretreat- ment with SDEPN and subsequent treatment with cis-platin resulted in a greater number of cells being killed （12.78 ± 1.01 vs 93.76 ± 1.6, P 〈 0.001）. HepG2 cells showed significant cell killing with treatment with SDEPN when combined with cisplatin （12.87 ± 2.78 vs 78.8 ± 3.02, P 〈 0.001）. CONCLUSION： SDEPN is selectively toxic against two cancer cell lines. Moreover, SDEPN in combination with cisplatin induces a synergistic increase in the cell death of both HT29 and HepG2 cells.

The difference between multi-drug resistant cell line A549/Gem and its parental cell A549

Objective:To discuss the difference between multi-drug resistant cell line A549/Gem and its parental cell A549 on the basis of establishment of human gemcitabine-resistant cell line A549/Gem so as to elaborate the possible mechanisms of gemcitabine resistance.Methods:Human gemcitabine-resistant non-small cell lung cancer cell line A549/Gem was estab-lished by the method of repeated clinical serous peak concentration plus gradually increasing concentration of gemcitabine from its parental cell human lung adenocarcinoma cell line A549 which was sensitive to gemcitabine.During the course of inducement,we had monitored their morphology,checked their resistance indexes and resistant pedigree by MTT method,gathered their growth curves and calculated their doubling time,examined their DNA contents and cell cycles by FCM;at the same time,we had measured their expressions of P53,EGFR,Cerb-B-2,PTEN,PCNA,c-myc,VEGF,MDR-1,Bcl-2,nm23,MMP-9,TIMP-1,and CD44v6 proteins via immunocytochemistry staining,RRM1 and ERCC1 mRNA by real-time fluorescent quantitative-PCR.Results:The resistance index of A549/Gem' cells(the deputy of cells in the process of inducement) to gemcitabine was 163.228,and the cell line also exhibited cross-resistance to vinorelbine,taxotere,fluorouraci,etoposide and cisplatin,but kept sensitivity to paclitaxol and oxaliplatin.The doubling time of A549/Gem' was shorter and figures in G0-G1 phases were increased than A549 cells.Compared with A549 cells,A549/Gem' cells achieved EGFR and c-myc proteins expressions,nm23 protein expression enhanced,P53,Cerb-B-2 and Bcl-2 proteins expressions reduced,PTEN,PCNA and MDR-1 proteins expressions vanished,but those of MMP-9,VEGF,CD44v6 and TIMP-1 proteins changed trivially.Meanwhile,expressions of RRM1 and ERCC1 mRNA were augmented markedly.The resistance index of A549/Gem cells to gemcitabine was 129.783,and the cell line also held cross-resistance to vinorelbine,taxotere,etoposide,cisplatin and sensitivity to paclitaxol.But the resistance to fluorouracil and sensitivity to oxaliplatin vanished.And the expression of RRM1 and ERCC1 mRNA decreased visibly.The doubling time of A549/Gem cells was longer and figures in G0-G1 phases were decreased than A549/Gem' cells.In A549/Gem cells,expressions of P53,EGFR,PCNA and MDR-1 proteins was same to those of A549/Gem' cells.A549/Gem cells achieved TIMP-1 and PTEN proteins expressions,Cerb-B-2,MMP-9,c-myc and Bcl-2 proteins expressions enhanced,nm23 protein expressions vanished,but the expressions of VEGF and CD44v6 proteins changed trivially.Furthermore,Compared with its parental cell A549,A549/Gem cell was mixed with giant cells of different sizes and was larger and more irregular.Conclusion:The human gemcitabine-resistant non-small cell lung cancer cell line A549/Gem had achieved multi-drug resistance and great changes of biological characters compared with its parental cells A549.And these changes possibly participated in the formation of multidrug resistance.

Establishment, Growth kinetics, and Susceptibility to AcMNPV of Heat Tolerant Lepidopteran Cell Lines

Lepidopteran heat-tolerant (ht) cell lines have been obtained with sf-9, sf-21 and several Bombyx cells. They have a distinct karyotype, membrane lipid composition, morphology and growth kinetics from the parental cell lines. In this paper, we report the development of ht cell lines from other insect species and examination of their growth characteristics and virus susceptibility. Adaptation of cell lines sf-9, BTI-TN-5B1-4 (High5) and BTI-TN-MG1 (MG1) to 33℃ and 35℃ was carried out by shifting the culture temperature between 28℃ and higher temperatures by a gradual stepwise increase in temperature. The process of adaption to a higher culture temperature was accomplished over a period of 2 months. The cell lines with the temperature adaption were designated as sf9-ht33, sf9-ht35, High5-ht33, High5-ht35, MG1-ht33, MG1-ht35. These cell lines have been subcultured over 70 passages. Adaption to high temperatures was confirmed by a constant population doubling time with individual cell lines. The population doubling time of heat adapted cell lines were 1-4 h less than these of parental cell lines. Cell shapes did not show obvious change, however, the cell size of sf9-ht cells was enlarged and those of High5 and MG1 ht cells were reduced after heat adaption. When the cell lines were infected with Autographa californica nuclear polyhedrosis virus (AcMNPV) at 28℃, 33℃, 35℃ and 37℃, production of budded virus and occlusion bodies in each cell line was optimum at its own adapted temperature.

水飞蓟宾对荷人肝癌BALB／c鼠NK细胞的免疫活性的调节作用

水飞蓟宾是菊类植物水飞蓟的提取物。近来，体外研究发现水飞蓟宾可以直接抑制肝癌细胞株生长。我们观察水飞蓟宾对荷人肝癌BALB／c鼠自然杀伤细胞（natural killer cell，

Establishment of Kinetics Models for Batch Fermentation Process of β-mannase with Bacillus licheniformis HDYM-04

In order to improve the yield of β-mannase and to investigate the rules of fermentation production, a high-yield β-mannase producing strain, Bacillus licheniformis HDYM-04, was used to investigate the kinetics models based on the optimal fermentation conditions： HDYM-04 strain was fermented at 37℃ for 30 h with agitation speed at 300 r/min and aeration rate at 3 L/min in a 5 L fermenter, the initial addition amount of konjac flour was 2%（w/v）, the initial pH of medium was 8.0, and the inoculum concentration was 6.7%（v/v）. Three batch fermentation kinetic models were established （cell growth kinetic model, substrate consumption kinetic model, product formation kinetic model） bases on Logistic and Luedeking-Piret equations. To be specific, cell growth kinetic model was dX/dt =0.431X （1- X/ 15.522 ）, substrate consumption kinetic model was -ds/dt =1.11 dX/dt ＋0.000 2 dP/dt ＋0.000 8X, and product formation kinetic model was dP/dt=133.1 dX ＋222.87X. The correlation coefficients R^2 of the three equations were 0.990 21, 0.989 08 and 0.988 12, respectively, which indicated a good correlation between experimental values and models. Therefore, the three equations could be used to describe the processes of cell growth, enzyme synthesis and substrate consumption during batch fermentation using B. licheniformis strain HDYM-04. The establishment of batch fermentation kinetic models （cell growth kinetic model, substrate depletion kinetic model, product formation kinetic model） could lay the theoretical foundation and provide practical reference for the applica- tion of HDYM-04 in fermentation industry.