全反式维甲酸对结肠癌不同增殖潜能细胞株VEGF表达的影响

目的:探讨全反式维甲酸对结肠癌不同增殖潜能细胞株VEGF表达的作用；研究VEGF在结肠癌侵袭和转移中的作用。 方法:采用细胞培养观察、ATRA干预、MTT和FACS方法确定结肠癌细胞株CW-2和LS174T的生长增殖状况，用Northern blotting方法检测结肠癌中VEGF mRNA的表达量，用免疫细胞化学观察细胞VEGF蛋白的表达。 结果:MTT生长曲线显示结肠癌细胞株LS174T的生长增殖比CW-2快;FACS结果显示LS174T细胞的S期细胞较CW-2细胞数多；Northern blotting和免疫细胞化学检测在CW-2中有明显的VEGF表达，但在高增殖细胞株LS174T中VEGF的表达更明显。 结论:VEGF在结肠癌细胞株中有较高的表达。在高增殖结肠癌细胞株VEGF表达更明显。ATRA可能通过抑制VEGF表达，而抑制结肠癌细胞的增生。     

白藜芦醇对小鼠胚胎成纤维细胞增殖和细胞周期的影响

目的:探讨白藜芦醇(Res)对小鼠胚胎成纤维细胞(MEF)增殖和细胞周期的影响及变化规律.方法:通过不同浓度(1～150 μmol/L)Res作用于增殖期的MEF,用MTT法检测72 h后各组细胞增殖变化,用流式细胞术检测并分析其细胞周期.结果:Res 1 μmo/L组A值明显高于对照组(P<0.05),Res 50 μmol/L和150 μmol/L组A值分别为0.196±0.055和0.185±0.034,均明显低于对照组(分别P<0.05,P<0.01),其细胞生长抑制率分别为14.0%和18.9%.细胞周期检测结果表明,Res 1 μmol/L组中G2/M期细胞占(14.82±0.49)%显著高于对照组(P<0.01).50 μmol/L和150 μmol/L组G0/G1期细胞所占比例分别为(59.61±6.03)%和(65.31±0.27)%均显著高于对照组(P<0.01).结论:Res对MEF增殖的影响,具有双向浓度依赖性,增殖和抑制增殖均与细胞周期调控有关,其中阻断G1期细胞进入S期是其抑制MEF增殖主要因素.     

不同增殖能力结肠癌细胞株iNOSmRNA表达的比较研究

目的:探讨iNOSmRNA在结肠癌不同增殖能力细胞株中的表达和作用,研究ATRA对于结肠癌不同增殖能力细胞株iNOSmRNA表达的影响。方法:采用MTT方法确定结肠癌细胞株CW-2和LS174T的生长增殖状况,用RT-PCR和Northernblot方法检测结肠癌中iNOSmRNA的表达量。结果:MTT生长曲线显示结肠癌细胞株LS174T的生长增殖比CW-2快;RT-PCR显示CW-2细胞株有较强的iNOSmRNA表达,而LS174T细胞株iNOSmRNA的表达较弱;Northernblot检测在CW-2中有明显的iNOSmRNA表达,但在细胞株LS174T中表达相对较弱;ATRA对结肠癌CW-2和LS174T细胞株iNOSmRNA的表达量无明显影响。结论:iNOSmRNA对结肠癌细胞株生长有双重作用,即在低增殖结肠癌CW-2呈高表达,可以通过细胞毒或诱导细胞凋亡等作用发挥抗肿瘤效应;在高增殖结肠癌LS174T呈低表达,产生NO作为信号转导的重要分子,增加血供和血管生成,促进肿瘤生长、侵袭和转移。ATRA可以抑制结肠癌细胞株的生长。     

C-erbB2基因shRNA表达质粒对结肠癌细胞生物学行为的影响

目的:观察C-erbB2基因shRNA表达质粒对结肠癌HT-29细胞生长抑制、细胞周期和凋亡的影响。方法:用MTT法及流式细胞仪分别检测pGenesil-erbB2实验组(PEG)、转染试剂对照组(TRCG)、阴性质粒对照组(NPCG)细胞对结肠癌细胞生长曲线、细胞周期及细胞凋亡率的影响。结果:pGenesil-erbB2实验组、转染试剂对照组、阴性质粒对照组的生长抑制率分别为39.65%、7.23%、8.05%,实验组明显高于其他两组(P<0.01);pGenesil-erbB2实验组、转染试剂对照组、阴性质粒对照组的G0/G1期细胞分别占74.93%、67.19%、68.05%,实验组明显高于其他两组(P<0.05);S期细胞分别占7.81%、14.02%、13.70%,实验组明显低于其他两组(P<0.05);pGenesil-erbB2实验组、转染试剂对照组、阴性质粒对照组的凋亡率分别为19.21%、3.13%、4.08%,实验组明显高于其他两组(P<0.01)。结论:C-erbB2基因siRNA重组质粒可以明显抑制结肠癌细胞的生长;可以将细胞周期阻滞于G0/G1期;可以明显增加结肠癌细胞的发生凋亡;说明C-erbB2基因在结肠癌的发生和发展中也起到非常重要的作用。     

蛋白酶体抑制剂MG132对NK/T淋巴瘤细胞增殖、凋亡和细胞周期的影响

目的： 探讨蛋白酶体抑制剂MG132（Z-leu-leu-leu-CHO,三肽基乙醛）对NK/T淋巴瘤细胞的增殖和细胞周期分布的影响,研究蛋白酶体抑制剂MG132治疗NK/T细胞淋巴瘤的潜在应用价值。方法: 用蛋白酶体抑制剂MG132处理细胞,噻唑蓝［ 3（4,5二甲基噻唑2）2,5二苯基四氮唑溴盐,3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide,MTT］法检测细胞增殖抑制率,倒置显微镜观察细胞形态改变,流式细胞仪检测细胞周期分布和凋亡。结果: 1 μmol/L MG132作用24 h,HANK1细胞增殖抑制率为（57.72±7.44）%,而0.1 μmol/L MG132作用24 h,增殖抑制率仅为（3.98±0.07）%;MG132剂量大,增殖抑制率高;1 μmol/L MG132作用24 h后,HANK1细胞G1和G2期细胞分别为（72.33±3.44）%和（12.86±1.29）%,对照组为（63.63±2.29）%和（7.94±1.91）%,用药组G1和G2期细胞明显高于对照组（P<0.01,P<0.05）; 早期凋亡细胞和晚期凋亡细胞分别为（33.57±2.10）%和（16.66±0.47）%,而对照组仅为（7.18±0.82）%和（3.81±1.06）%,与对照组相比,凋亡率明显增高 (P<0.01,P<0.01)。结论: MG132抑制NK/T细胞淋巴瘤细胞增殖,使细胞周期G1和G2期阻滞,促进细胞凋亡,并存在剂量和时间依赖关系,蛋白酶体抑制剂MG132很可能成为治疗进展期NK/T细胞淋巴瘤的药物。     

miR-124对C6胶质瘤细胞增殖和迁移能力的影响

目的:研究miR-124对C6胶质瘤细胞增殖和迁移能力的影响.方法:体外培养C6胶质瘤细胞,依据转染不同分为空白对照组、阴性对照组和miR-124拟似物组.实时荧光PCR检测转染效率,绘制生长曲线,MTT实验检测miR-124对C6细胞增殖能力影响,计算抑制率.划痕实验检测miR-124对C6细胞迁移能力影响,计算迁移率.结果:生长曲线显示miR-124拟似物组C6细胞生长能力受到明显抑制,转染后第3天,miR-124拟似物组C6细胞数目显著低于阴性对照组(5.410±0.463Vs.6.917±0.385;P＜0.01);miR-124拟似物组mRNA表达量显著高于阴性对照组和空白对照组(5.92±0.56 vs.0.93±0.13和1.00±0.12;P＜0.01);MTT增殖实验显示miR-124抑制C6细胞增殖能力,miR-124拟似物组抑制率显著高于阴性对照物组(42.90±5.169 vs.10.24±3.351;P＜0.01);划痕实验显示miR-124明显抑制C6细胞迁移能力,阴性对照组迁移率显著高于miR-124拟似物组(98.79±1.210vs.81.72±5.972;P＜0.05).结论:miR-124能够显著抑制C6胶质瘤细胞的增殖和迁移能力.     

莪术醇对鼻咽癌细胞CEN-2增殖与凋亡的影响

目的:观察莪术醇对鼻咽癌CEN-2的增殖抑制与凋亡的影响,探讨其抗鼻咽癌的分子机制。方法:以不同浓度的莪术醇(12.5、25、50、100 mg/L)及阴性对照组处理鼻咽癌CNE-2细胞,应用MTT法检测不同浓度的莪术醇对CNE-2细胞生长增殖抑制作用;Hochest33342荧光染色法观察凋亡细胞的发生及形态学变化,流式细胞术检测其凋亡率,Western blot法检测核转录因子-κB(NF-κB)蛋白的表达。结果:莪术醇处理组能明显抑制CNE-2的增殖(P<0.01),流式细胞术检测药物作用48 h后,CNE-2细胞的凋亡率也随莪术醇浓度的增加而增加,100 mg/L的莪术醇处理组其凋亡率可达45.5%,与阴性组比差异极显著(P<0.01);NF-κB的蛋白相对表达量随莪术醇剂量的增大而下降,与对照组相比有显著性意义(P<0.01)。结论:莪术醇能对体外鼻咽癌CNE-2细胞具有明显增殖抑制和诱导凋亡的作用,其分子机制可能与NF-κB的表达下调有关。     

抗人DR5单克隆抗体诱导HL-60细胞凋亡机制

探讨抗人DR5单克隆抗体(mDRA-6)诱导白血病细胞系HL-60细胞凋亡的可能机制。MTT法检测mDRA-6对HL-60细胞的生长增殖的影响,以及Caspase 8、10、3抑制剂对mDRA-6抑制HL-60细胞生长增殖的影响;琼脂糖凝胶电泳检测HL-60细胞DNA片断化降解;Western blot检测mDRA-6对HL-60细胞Caspase 8、10、3的激活改变。结果发现,mDRA-6呈时间、浓度依赖性地抑制HL-60细胞的生长增殖,10 mg/L的mDRA-6作用HL-60细胞6 h、8 h和10h,细胞增殖抑制率分别为23.96%、44.10%和50.28%;琼脂糖凝胶电泳显示,10 mg/L的mDRA-6作用6 h,HL-60细胞呈现凋亡细胞特有的DNA梯形条带;Western blot检测结果发现,mDRA-6作用HL 60细胞不同时间,Caspase 8均只有酶原条带出现,无激活片段产生,而Caspase 10和Caspase 3显示明显裂解片段产生,并随mDRA-6作用时间延长而增多;预先使用15μmol/L的Caspase 10及Caspase 3抑制剂孵育细胞1 h,能够使mDRA-6对HL-60细胞的生长抑制率分别降低64.15%(t=10.13、P<0.01)和53.69(t=8.93、P<0.01)%,而预先使用15μmol/L的Caspase 8抑制剂孵育细胞1 h,mDRA-6对HL-60细胞的生长抑制率仅降低4.40%(t=0.52、P>0.05)。以上实验结果提示,mDRA-6通过激活Caspase 10启动凋亡信号分子,诱导白血病HL-60细胞凋亡。     

mTOR在5637细胞中的表达与增殖及侵袭能力的关系

目的探讨mTOR在膀胱癌5637细胞中的表达及西罗莫司对5637细胞增殖和侵袭能力的影响。方法将5637细胞贴壁培养于10%胎牛血清、100 U/mL青霉素、100μg/mL链霉素的RPMI-1640培养液中,37℃、5%CO_2饱和湿度培养箱中培养。取对数生长期细胞,处理组加入浓度分别为10nmol/L,100nmol/L,1000nmol/L西罗莫司;对照组不加药物,每天更换1640培养液。MTT法检测各组细胞生长抑制率,Transwell法检测5637细胞的体外侵袭能力,Real-time PCR法检测mTOR在5637细胞中的表达。结果西罗莫司对5637细胞有增殖抑制作用,呈剂量依赖关系。西罗莫司处理后的5637体外侵袭能力明显下降,呈剂量依赖性。西罗莫司处理组的m TOR表达量明显下降,与增殖和侵袭能力下降相关。结论西罗莫司抑制膀胱癌5637细胞增殖和侵袭能力与抑制mTOR表达相关。     

外源性骨桥蛋白对人大肠癌LS-174T细胞增殖和侵袭能力的影响

目的观察人重组骨桥蛋白(recombinated human osteopontin,rhOPN)对大肠癌LS-174T细胞增殖、侵袭及其相关基因表达的影响,探讨骨桥蛋白在大肠癌演进中的作用。方法添加不同浓度rhOPN于人大肠癌LS-174T细胞培养液中,采用四甲基偶氮唑蓝(MTT)试验检测细胞的增殖能力,重组人工基底膜实验评价细胞侵袭能力,并运用实时荧光定量RT-PCR检测大肠癌细胞中尿激酶型纤溶酶原激活物(uPA)、基质金属蛋白酶-2(MMP-2)和血管内皮生长因子(VEGF)等mRNA表达水平。结果LS-174T细胞经不同浓度rhOPN作用24h后,实验组的MTT值显著高于对照组(P<0.01),并呈剂量依赖关系。Transwell上室加入5nmol/L、30nmol/L的rhOPN后,实验组LS-174T细胞穿膜数量明显多于对照组(P<0.05)。经5nmol/L、30nmol/L的rhOPN处理24h后,实验组LS-174T细胞的uPA mRNA及VEGF mRNA的表达水平明显高于对照组(P<0.05),而MMP-2 mRNA表达水平与对照组无显著性差别(P>0.05)。结论rhOPN促进人大肠癌LS-174T细胞的增殖,并提高其侵袭能力,可能与uPA和VEGF基因表达上调有关。     

阿托伐他汀对人NK 细胞杀伤结肠癌细胞的影响及其机制研究

目的:探讨阿托伐他汀对人NK 细胞杀伤结肠癌细胞的影响及其机制。方法:不同浓度的阿托伐他汀作用于3 株结肠癌细胞(HCT-116、SW-480、Caco-2),CCK-8 法测定阿托伐他汀对结肠癌细胞生长抑制率的影响。SCGM 培养基体外扩增人NK 细胞,自动生化分析仪测定NK 细胞对3 株结肠癌细胞的杀伤活性;流式细胞仪检测结肠癌细胞MICA/ B 的表达率。结果:(1)NK 细胞的培养:培养前CD3- CD56+的NK 细胞占外周血单个核细胞的比例为4.5%,培养10 d 时NK 细胞的比例增至93.1%。(2)阿托伐他汀对3 株结肠癌细胞生长抑制率的影响:阿托伐他汀作用48 h 后,在浓度5 ~40 μmol/ L 的4 个实验组中,HCT-116 细胞的生长抑制率较对照组升高(P<0.05)。作用96 h 后,在1.25 ~ 40 μmol/ L 的所有浓度组(6 个)中,HCT-116 细胞的生长抑制率均显著高于对照组(P<0.05)。另外, HCT-116 细胞的生长抑制率随着阿托伐他汀浓度的升高而逐渐上升,相关分析显示,阿托伐他汀的浓度与HCT-116 细胞的生长抑制率呈正相关(r[48 h] = 0.13,r[96 h] = 0.22,P<0.05)。(3)NK 细胞经阿托伐他汀作用96 h 后,除浓度为20 μmol/ L 和40 μmol/ L 时抑制率高于对照组,其他各组对NK 细胞生长无明显影响。(4)阿托伐他汀对NK 细胞杀伤结肠癌细胞活性的影响:NK 细胞对HCT-116 细胞的杀伤活性在阿托伐他汀浓度2.5 ~10 μmol/ L 组均显著高于对照组,对SW-480 的杀伤活性在5 ~20 μmol/ L 组较对照组明显升高,对Caco-2 的杀伤活性在2.5 ~20 μmol/ L 组显著高于对照组(P<0.05),其中同一浓度时对HCT-116 细胞杀伤作用最强。(5)阿托伐他汀对结肠癌细胞MICA/ B 表达的影响:在阿托伐他汀浓度2.5 μmol/ L 及5 μmol/ L 组,HCT-116 细胞MICA/ B 的表达率与对照组比较显著升高(P <0.05),在10 μmol/ L 及20 μmol/ L 组,SW-480 细胞MICA/ B 表达率显著高于对照组(P <0.05),在2.5 ~40 μmol/ L 组,Caco-2 细胞MICA/ B 的表达率均较对照组显著升高(P<0.05)。结论:(1)阿托伐他汀能够呈剂量依赖性抑制结肠癌细胞HCT-116、SW-480 及Caco-2 的生长;(2)阿托伐他汀能够增强NK 细胞对结肠癌细胞的杀伤活性,可能与阿托伐他汀上调结肠癌细胞MICA/ B 的表达有关;(3)阿托伐他汀可以上调3 株结肠癌细胞MICA/ B 的表达率,提高结肠癌细胞的免疫原性。     

Up-regulated expression of ADAM17 in human colon carcinoma: co-expression with EGFR in neoplastic and endothelial cells

The ADAM17 metalloproteinase (a disintegrin and metalloprotease 17) controls epidermal growth factor receptor (EGFR) activation through regulated shedding of EGFR ligands. With the advent of new therapeutic options targeting EGFR signalling in colon carcinoma, it was decided to determine ADAM17 status in relation to clinico-pathological parameters and EGFR status. To this end, a series of 39 colon carcinomas were analysed. Immunohistochemistry and immunofluorescence were used to localize ADAM17, EGFR, and the activated forms of EGFR. The activated form of ADAM17 was assessed in primary cancers and colon cell lines by immunoblotting. ADAM17 and EGFR mRNA levels were assessed by quantitative RT-PCR. Chromogenic in situ hybridization (CISH) was used to quantify the HER1 gene. ADAM17 was strongly expressed in all tumours, by both neoplastic and endothelial cells. It was expressed both as a pro- and as an active form in tumours and colonic cancer cell lines. ADAM17 mRNA was up-regulated in 90% of colon carcinomas relative to the paired normal mucosa, whatever the tumour grade or stage. When present, activated EGFR was co-expressed with ADAM17 by colon carcinomas, although at a variable level among tumour cells, and by endothelial cells. EGFR mRNA was overexpressed in 77% of colon carcinomas compared with the paired normal mucosa. One case showed high-level amplification of HER1. In conclusion, this study is the first demonstration that ADAM17 is overexpressed in human primary colon carcinoma, whatever the tumour stage and differentiation and whatever the level of EGFR expression. Its co-expression with EGFR, in both neoplastic and endothelial cells, suggests a role for ADAM17 in tumour growth and angiogenesis.     

Constitutive expression of growth regulated oncogene (gro) in human colon carcinoma cells with different metastatic potential and its role in regulating their metastatic phenotype

The purpose of this study was to examine the expression and functional significance of the growth-regulated oncogene (gro) family in human colon carcinoma growth and metastasis. We examined constitutive expression of CXCL1 (gro-alpha), CXCL2 (gro-beta), CXCL3 (gro-gamma) and their receptor, CXCR2 in human colon carcinoma cells with different metastatic potentials. Non-metastatic and low metastatic cells expressed lower levels of CXCL1 and CXCR2 mRNA and protein as compared to high metastatic colon carcinoma cells. No difference in CXCL2 and CXCL3 mRNA expression levels was observed. Colon carcinoma cells expressing higher levels of CXCL1 exhibit increased proliferation and invasive potential. Furthermore, exogenous addition of recombinant human CXCL1 significantly enhanced the proliferation and invasiveness of colon carcinoma cells. Furthermore, treatment of KM12C cells with exogenous CXCL1 enhanced their invasiveness. Neutralizing antibody to CXCL1 in combination with antibody to CXCR2 inhibited highly metastatic KM12L4 (high CXCL1 expressor) cell proliferation. These data demonstrate that the constitutive expression of CXCL1 and its receptor CXCR2 is associated with metastatic potential and modulates colon cancer cell proliferation and an invasive phenotype.     

Comparative Phenotypic Studies of Duct Epithelial Cell Lines Derived from Normal Human Pancreas and Pancreatic Carcinoma

We have investigated the mRNA/protein expression of several tyrosine kinase receptors, growth factors, and p16^INK4A cyclin inhibitor in cell lines derived from normal human pancreatic duct epithelium (HPDE) and compared them with those of five pancreatic ductal carcinoma cell lines. Cultured HPDE cells express low levels of epidermal growth factor receptor (EGFR), erbB2, transforming growth factor (TGF)-α, Met/hepatocyte growth factor receptor (HGFR), vascular endothelial growth factor (VEGF), and keratinocyte growth factor (KGF). They also expressed high levels of amphiregulin but did not express EGF and cripto. The expression levels were similar in primary normal HPDE cells and those expressing transfected E6E7 genes of human papilloma virus-16, but their immortalization appeared to enhance the expression of EGFR and Met/HGFR. In comparison, pancreatic carcinoma cell lines commonly demonstrated overexpression of EGFR, erbB2, TGF-α, Met/HGFR, VEGF, and KGF, but they consistently showed marked down-regulation of amphiregulin mRNA expression. In contrast to all carcinoma cell lines that showed deletions of the p16 gene, HPDE cells consistently demonstrated normal p16 genotype and its mRNA expression. This is the first report that compares the phenotypic expression of cultured pancreatic ductal carcinoma cells with epithelial cell lines derived from normal human pancreatic ducts. The findings confirm that malignant transformation of human pancreatic duct cells commonly results in a deregulation of expression of various growth factors and receptors.     

不同浓度人羊膜匀浆上清液培养大鼠许旺细胞的增殖

背景：许旺细胞是周围神经修复过程的重要细胞,而研究发现人羊膜细胞分泌的多种细胞因子能够促进许旺细胞增殖。 目的：观察不同浓度人羊膜匀浆上清液对鼠许旺细胞(RSC96)生长的影响。 方法：使用含体积分数20%胎牛血清的高糖DMEM培养基原代培养RSC96细胞株,传代至第2代用于实验研究。根据人羊膜匀浆上清液在培养基中的不同体积分数(0,10,15,20,25%)分组。 结果与结论：人羊膜匀浆上清液的总蛋白浓度为675 mg/L,表皮生长因子、碱性成纤维生长因子和血管内皮生长因子浓度分别为(470.625±2.546),(4.121±0.026)和(0.172±0.002) ng/L。在培养第1-7天,10%和15%人羊膜匀浆上清液组的增殖率大于20%和25%人羊膜匀浆上清液组(P < 0.05);10%、15%人羊膜匀浆上清液组显示出促进细胞增殖的作用,而20%、25%人羊膜匀浆上清液组显示出抑制细胞增殖的作用;各实验组的细胞活力与对照组接近(P > 0.05)。提示人羊膜匀浆上清液低浓度时(10%和15%)具有促进RSC96增殖作用,高浓度时(20%和25%)抑制RSC96增殖。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Cellular distribution of a human I-A-like (DS/DC) antigen on normal and neoplastic lymphoid cells

Previous biochemical studies have shown that B cell specific, monoclonal antibody, McAb 33.1, reacts with a class II antigen that represents a human analogue of murine I-A (DS/DC) antigens (J. Exp. Med. 158:1924, 1983). McAb 33.1 recognizes a polymorphic human B lymphocyte specific antigen present on mu+, B1+ peripheral B cells, B lymphoid cell lines, activated B cells, and neoplastic B lymphoid cells. Of 100 HLA-D/DR typed EBV-transformed lymphoblastoid cell lines tested, only those from DR3,3 and DR7,7 individuals failed to react with McAb 33.1. The 33.1 antigen is present at lower concentrations on B cells from blood, tonsil, spleen, and lymph node when compared to B cell lines. By contrast, the antigen is not detectable on blood T lymphocytes, T cell lines, or mitogen activated T cells and it is absent on monocytes of some individuals or is present only on a minor subpopulation (approximately 20%). McAb 33.1 should facilitate the functional, structure, and molecular dissection of the human Ia system.     

抗人肝癌单克隆抗体HL2的制备及鉴定

用肝癌细胞株QGY-7703、BEL-7402、SMMC-7721以贯序法免疫BALB/c小鼠,获得一株分泌肝癌单抗的杂交窟株HL_2,其染色体数目为97—105。单抗HL_2系IgG_2b亚类。吸收实验及阻断实验证明HL_2抗原与CEA、AFP、HBsAg、HBcAg及HBeAg无免疫同源性。ABC法和ELISA法说明单抗HL_2与四株肝癌细胞、六例肝癌组织均呈明确阳性反应,除与SGC-7901、H_(128)、K_(562)、Hela细胞株及部分胚胎肝脏,胚胎结肠有较弱性反应外,与肝癌旁组织、正常肝脏、其它肿瘤组织和细胞株、二种正常人细胞及其它胚胎组织无反应。显示了单抗HL_2的特异性较好、阳性率较高,是一个有应用前景的单抗。     

Up-regulation of vascular endothelial growth factor-C by nicotine in cervical cancer cell lines

PROBLEM: Smoking and infection with human papilloma virus (HPV) are major risk factors for cervical cancer. Our earlier work shows that nicotine enhances cellular proliferation of cervical cancer cell lines by up-regulating epidermal growth factor (EGF) and its receptor EGF-R, which leads to increased insulin-like growth factor II in vitro. We found that the vascular endothelial growth factor (VEGF)-C, one of the five isoforms of VEGF, may be specifically involved in lymphogenic metastasis of cervical cancer. This has prompted us to study if in vitro nicotine treatment will up-regulate VEGF-C alongside EGF-R levels, while down regulating the anti-proliferative transforming growth factor (TGF)-beta levels in HPV positive cervical cancer cell lines. METHOD OF STUDY: Cervical cancer cell lines CaSki, HeLa and ME-180, were cultured in serum free DMEM medium for 24-hr, and treated with 10 ng/mL nicotine in the medium supplemented with 10% fetal bovine serum. A group of untreated cells served as controls. The cells were cultured in chamber slides (for immunofluorescent antibody assay) as well as microtiter plate wells (for BrdU cell proliferation assay). The cellular levels of VEGF-C, TGF-beta, EGF-R and HPV-E6 (early protein 6) were measured by a semi-quantitative immunofluorescent antibody assay. The cell proliferation and immunofluorescent assay data were analyzed by a Student's t-test. RESULTS: Cell proliferation was significantly increased after nicotine treatment in all the cell lines. The VEGF-C levels were significantly increased, while TGF-beta levels were decreased by nicotine in all the cell lines (P < 0.00001). EGF-R levels were also significantly increased after nicotine treatment in HeLa and ME-180, while HPV-E6 levels remained unchanged in all three. CONCLUSIONS: Nicotine up regulates expression of cell proliferative VEGF-C and EGF-R, while down-regulating anti-proliferative TGF-beta. Our data suggest that nicotine in circulation and in cervical squamous epithelial cells may promote not only rapid tumor growth but its lympho-angiogenic spread (VEGF-C) as well. It appears that nicotine does not promote HPV spread in the cervical cancer cells.     

利用基因重组抗原研制人CD20单克隆抗体及其功能的研究

目的　利用基因重组抗原免疫小鼠 ,制备人CD2 0单克隆抗体 ,研究其特异性和功能。方法　用表达重组人CD2 0基因的细胞NIH 3T3免疫BALB c小鼠 ,取其脾细胞与Sp2 0细胞融合 ,以间接免疫荧光法筛选杂交瘤上清。免疫沉淀法及流式细胞术鉴定其识别抗原的相对分子质量 (Mr)和特异性 ;以MTT法、流式细胞术及形态学方法检测诱导细胞凋亡和抑制细胞增殖的功能。结果　利用杂交瘤技术获得了 1株抗人CD2 0的单克隆抗体 1 2 8,它具有CD2 0抗体特异的细胞反应谱 ,其识别的抗原Mr 为 33× 10 3 。MTT实验证实 1 2 8能显著抑制Daudi和Raji细胞生长。结论　利用基因重组抗原制备了 1株能够稳定分泌抗人CD2 0的单克隆抗体的杂交瘤细胞株 1 2 8,此单抗具有抑制CD2 0阳性细胞增殖和诱导其凋亡的功能。