3种常用抗癌药物对宫颈癌Hela细胞和肿瘤干细胞抑制作用研究

目的:探讨3种常用抗肿瘤药物对宫颈癌Hela细胞和Hela肿瘤干细胞的抑制率。方法:采用无血清悬浮培养法从宫颈癌Hela细胞中分离出Hela肿瘤干细胞,鉴定后研究丝裂霉素、顺铂和紫杉醇等3种抗癌药物对Hela细胞和Hela肿瘤干细胞的抑制率并进行比较。结果:不同质量浓度的3种抗肿瘤药物对肿瘤细胞和肿瘤干细胞均有抑制作用,且呈剂量依赖性(r=0.944,P<0.05)。在最高终浓度40 mg/L时,丝裂霉素对肿瘤细胞和肿瘤干细胞的抑制率分别为(88.75±2.59)%和(38.95±5.37)%;顺铂的抑制率分别为(92.28±1.57)%和(52.68±3.55)%;紫杉醇的抑制率分别为(75.22±0.99)%和(36.71±2.42)%。结论:3种药物对肿瘤干细胞的抑制率均明显低于肿瘤细胞的抑制率(P<0.05);比较发现对于肿瘤细胞和肿瘤干细胞,顺铂抑制率均最高,其次是丝裂霉素,紫杉醇的抑制率最低(P<0.05),说明宫颈癌肿瘤干细胞对目前常用的3种治疗药物有不同程度的耐受。     

X线照射对大肠癌细胞株Lovo细胞TNFR-p55表达及释放的影响

目的 研究X线照射在体外培养人大肠癌细胞株LoVo后．其膜TNFR-p55表达及释放sTNFR-p55情况。方法免疫细胞化学检测细胞膜TNFR-p55蛋白的表达．EUSA法检测培养上清中sTNFR-p55水平,流式细胞仪分析细胞凋亡率,光镜观察细胞形态变化。结果x线照射后的细胞膜TNFR-p55蛋白表达显著增强（P〈0．01）;照射后大肠癌Lovo细胞上清sTNFRR-p55水平显著低于照射前sTNFRR-p55水平（P〈0．01）;光镜发现细胞体积缩小、变圆、胞浆浓缩、核固缩深染;流式细胞仪分析细胞凋亡率显著增高（P〈0．05）。结论x线可通过上调细胞膜TNFR-p55蛋白的表达,抑制其脱落释放sTNFRR-p55到上清中,诱导细胞凋亡增强照射肿瘤的杀伤作用。     

转录因子NF-kB在肿瘤坏死因子α介导的胰岛β细胞凋亡中的作用

目的 探讨肿瘤坏死因子(17NFα)是否可引起胰岛B细胞凋亡以及转录因子核因子kB(NF—kB)在TNFα诱导的β细胞凋亡中所起的作用。方法培养INS-1胰岛β细胞,采用DNA重组、转染和再感染技术获得可表达NF—kB的特异性抑制物：IkBα的突变体IkBα△N的INS-1／IkB△N细胞。应用DNA片段分析技术和kB—luc报告基因荧光分析技术观察NF—kB活性和β细胞的凋亡情况。结果IL—Iβ可诱导INS-1细胞的NF—kB激活,但在INS-1／IkB△N细胞则无此作用。将细胞与IL-1β(10μg/L)、TNFα(100μg/L)以及IFNγ(100000U／L)一道培养48h后显示,TFNFα与IFNγ合用可诱导INS-1细胞发生凋亡,而在INS-1／IkB△N细胞未见此现象。单用IL—Iβ或IFN-γ或IL—IB加IFNγ均未能诱导INS-1细胞凋亡。结论凋亡是TNFα导致B细胞死亡的方式之一,NF—kB在TNFα介导的β细胞凋亡中具有重要作用,抑制NF—kB激活可能保护β细胞免于TNFα诱导的凋亡。     

热休克蛋白CpkB对nrhTNF体外复性的促进作用

目的;探讨嗜热菌热休克蛋白ＣｐｋＢ在体外与新型重组人肿瘤坏死因子蛋白质复性的关系。方法：在原核细胞中表达并纯化ＯｐｋＢ蛋白;将ｎｒｈＴＮＦ在８ｍｏｌ／Ｌ脲中变民生,然后在不同浓度的ＣｐｋＢ存在下进行体外复性,以复性后ｎｒｈＴＮＦ的和活性检测指标进行分析。     

4-1BB(CD137)配体在几种肿瘤细胞株上的表达

目的：研究4—1BBL在几种人肿瘤细胞株上的表达。方法：用RT—PCR及法检测肿瘤细胞内4—lBBL mRNA水平的表达；用流式细胞仪检测肿瘤细胞膜表面4—1BBL的表达。结果：4—1BBL在Jurkat细胞株上高表达(98．97％)，在HL-60和K562上表达较低(分别为24．77％和19．23％)，而在HL-29细胞株上基本不表达(2．13％)。结论：4—1BBL在不同肿瘤细胞株表达水平不同。     

洛拉曲克在肿瘤细胞中的药代动力学研究

目的：探讨新型药物洛拉曲克在肿瘤细胞中的转运特点与其敏感性之间的关系。方法：采用MTT法测定洛拉曲克对三株肿瘤细胞C6、SRS－82、LoVo的生长抑制作用。通过高效液相色谱法测定经洛拉曲克处理后的细胞内外药物浓度。结果：C6是三株细胞中对洛拉曲克最敏感的，SRS－82和LoVo细胞对洛拉曲克的IC50值分别是C6细胞的6．8和13．8倍。洛拉克克在这三株细胞中胞内与胞外浓度存在线性关系。C6的细胞内稳态浓度显著高于其他两株细胞。结论：本研究的结果表明，洛拉曲克可快速进入细胞，不同细胞株对洛拉曲克有不同的吸收能力，这种现象可部分解释肿瘤细胞对洛拉曲克不同的敏感性。     

干扰素对肿瘤细胞Fas表达的上调作用及与细胞凋亡的关系

OBJECTIVE: To investigate effect of interferon-gamma (IFN-gamma) on Fas expression and Fas-mediated apoptosis in tumor cell lines. METHODS: Fas expression was detected by flow cytometry, and the tumor cell apoptosis evaluated by enzyme-linked immunosorbent assay and AnnexinV staining assay. RESULTS: All the 4 tumor cell lines used in this study, e.g. gastric cancer cell lines Kato-III AGS, lung cancer cell line A549 and laryngeal cancer cell line Hep-2, expressed Fas protein to varied degrees. Fas expression was up-regulated by IFN-gamma (P<0.05), which alone was enough to induce apoptosis in tumor cells and increase the susceptibility of them to anti-Fas antibodies. IFN-gamma induced Fas expression up-regulation and tumor cell apoptosis in a time-dependent manner. CONCLUSION: Fas-mediated apoptosis is one of the mechanisms for IFN-gamma to exercise its anti-tumor effect.     

人增殖抑制基因(HSG)对肿瘤细胞系化疗敏感性的作用

目的:将人增殖抑制基因(hHSG)用电子穿孔的转基因方式转染到体外培养的人肿瘤细胞系中,观察hHSG 基因对内源性hHSG表达水平不同的肿瘤细胞系的化疗敏感性的影响.方法:首先用免疫组化方法检测不同组织来源的肿瘤细胞系中hHSG的表达水平,然后选择内源性hHSG表达水平较低的肺腺癌(A549)和内源性hHSG表达水平较高的宫颈癌(HeLa S3)细胞系,用电穿孔方法转染含有hHSG的真核表达载体(pEGFP-hHSG)24 h后,加入放线菌酮(CHX),采用细胞计数、MTT法观察hHSG对肿瘤细胞增殖抑制作用及对CHX的化疗敏感性的影响.结果:hHSG在不同组织来源的肿瘤细胞系都有不同程度的表达, pEGFP-hHSG转染到两种内源性hHSG表达水平不同的肿瘤细胞系后,这两种肿瘤细胞的生长增殖都明显受到抑制,同时外源性的hHSG也增强了这些肿瘤细胞系对CHX的敏感性.结论:外源性hHSG可不依赖其内源性表达水平而抑制肿瘤细胞的增殖,与CHX并用可增强肿瘤细胞对CHX的敏感性,具有协同作用.     

人CD137L在肿瘤细胞的表达及其对肿瘤细胞分泌IL-8的影响

目的 :检测人CD137L在肿瘤细胞的表达及其功能。方法 :采用RT PCR、FACS方法检测人CD137L在肿瘤细胞的表达 ,用ELISA方法检测CD137L对肿瘤细胞分泌IL 8的影响。结果 :9种不同来源的人肿瘤细胞系均不同程度表达CD137L ,其中 ,BEL 74 0 2、HL 6 0、A2细胞系呈高水平表达 ;HepG2 .2 .15、L 78、HT 2 9细胞系中度表达 ;U937、H6、HLE细胞系低表达。胚胎细胞系ECV 30 4中度表达 ,而新分离的正常外周血单个核细胞不表达。CD137L的表达水平与肿瘤细胞系的来源无关 ,同一组织来源的细胞系 ,有的高表达 ,有的低表达。肿瘤细胞上的CD137L对肿瘤细胞本身IL 8的分泌可产生不同的影响 :可使HepG2 .2 .15分泌IL 8的水平增加 ,但对HLE、A2 ,只能微弱地增强肿瘤细胞释放IL 8。结论 :CD137L在肿瘤细胞上的表达是普遍现象 ,其表达可能与细胞的快速生长有关 ;肿瘤细胞的CD137L可增强某些细胞系分泌IL 8。     

Transportation characteristics of nolatrexed in three tumor cell lines

Objective：To investigate the association of the transportation characteristics of nolatrexed in tu-mor cells with its drug sensitivity. Methods: The sensitivity of 3 tumor cell lines, C6, SRS82 and LoVo, to nolatrexed were determined by growth inhibition study. After exposure to 20μmol/L nolatrexed at different time intervals ranging from 0 to 30 rain, or to nolatrexed at different concentrations ranging from 0 to 40μmol/L for 10 min, the intracellular drug concentration was measured using high-performance liquid chro-matography. Results: C6 was the most sensitive cell line among the three, with sensitivity 6.8-fold and 13.8-fold those of SRS-82 and LoVo cells respectively. Transportation of nolatrexed in the 3 cell lines were qualitatively similar, which rapidly achieved steady-state within 5 min, and linear relationship between the in-tracellular and extracellular drug concentration was observed. The intracellular steady-state level achieved in C6 was significantly higher than those in the other two cell lines, the latter having comparable levels. Con-clusion: Nolatrexed enters the cell very quickly and different transport capacities are involved in the genera-tion of varied sensitivity to nolatrexed in tumor cells.     

胶质瘤细胞株U251,SHG-44,BT325中β-catenin的表达及其成瘤性分析

目的：探讨β-catenin在人神经胶质细胞株（HA）、人神经胶质瘤细胞株U251,SHG-44,BT325中的表达及其与恶性程度的相关性.方法：运用实时荧光定量PCR检测β-catenin在HA,U251,SHG-44,BT325细胞内的表达水平,并通过免疫荧光辅助分析β-catenin的表达及细胞内定位情况.建立裸鼠胶质瘤模型,观察肿瘤的生长.结果：β-catenin基因在人神经胶质瘤U251,SHG-44,BT325细胞株内的表达均高于HA细胞株,其中U251细胞表达最高,SHG-44细胞次之,而BT325细胞表达最低.在HA细胞株中,β-catenin蛋白主要在细胞膜表达,而在人神经胶质瘤U251,SHG-44,BT325细胞株中β-catenin蛋白表达出现了明显的胞质和胞核的易位表达.通过对荷瘤鼠模型的观察发现U251细胞所造肿瘤生长速度最快,SHG-44细胞次之,而BT325细胞的肿瘤生长速度最慢.结论：β-catenin基因表达与人神经胶质瘤U251,SHG-44,BT325细胞株恶性程度呈正相关.U251细胞表达β-catenin最高,更适合神经胶质细胞瘤Wnt/β-catenin信号传导通路相关研究.     

肿瘤坏死因子相关诱导凋亡配体与肿瘤治疗的关系进展

 肿瘤坏死因子相关诱导凋亡配体（tumor necrosis factor-related apoptosis-inducing ligand, TRAIL）能选择性诱导肿瘤细胞和转化细胞凋亡,对机体正常组织细胞无毒副作用,有望成为新型的抗肿瘤制剂。探讨TRAIL的生物学特点及其对肿瘤的治疗策略,能够为其临床应用于肿瘤治疗提供理论基础。     

原位杂交测肿瘤坏死因子mRNA在宫颈细胞及子宫内膜组织的定位表达

目的 观察肿瘤坏死因子（mRNA）在宫颈细胞及子宫内膜组织的表达及其在不同病变组织中的分布特点。方法 采用非同位素原位杂交法对3个传代的宫颈细胞系（HiSa、W12、NCx），石蜡包埋的宫颈组织96例、子宫内膜组织75例进行了TNFmRNA写位表达的研究。结果 TNFαmRNA和W12细胞中有阳性表达，NCx细胞未见阳性表达；在宫颈组织中阳性表达见于上皮、间质、血管壁及平滑肌；子宫内膜癌组织中阳性表达见于间质单个核细胞和平滑肌细胞，对照组未见阳性表达。结果 宫颈尖脱湿疣及宫颈鳞癌来源的上皮为TNF生成细胞之一;TNFmRNA表达强度与细胞增生程度有关；宫颈HPV感染对细胞内TNFmRNA表达有促进作用。     

RNA干扰沉默survivin基因在抑制卵巢癌裸鼠移植瘤中的作用

目的 观察pshRNA-survivin在体内是否能抑制卵巢癌移植瘤体的生长.方法 建立人卵巢癌裸鼠移植瘤模型,瘤内注射pshRNA-survivin,观察肿瘤生长情况.半定量RT-PCR、免疫组织化学检测移植瘤的survivin表达情况,TUNEL检测移植瘤的细胞凋亡情况.结果 pshRNA-survivin能明显抑制肿瘤组织中survivin的表达,促进肿瘤细胞的凋亡,减缓肿瘤生长速度.结论 pshRNA-survivin明显抑制了卵巢癌裸鼠移植瘤的增殖.     

Differential thymosin β10 expression levels and actin filament organization in tumor cell lines with different metastatic potential

Background To investigate the differential expression levels of thymosin [β10 (Tβ10) and the corresponding changes of actin filament organization in human tumor cell lines with different metastatic potential.Methods Four groups of nine human tumor cell lines with different metastatic potential were analyzed for the amount of Tβ10 mRNAs by Northern blot and for their peptide expression levels by immunohistochemistry. The filamentous actin (F-actin) was observed by staining of TRITC-phalloidin to detect changes in actin organization.Results In comparison with non-/weakly metastatic counterparts, Tβ10 was upregulated in highly metastatic human lung cancer, malignant melanoma and breast cancer cell lines. Staining of TRITC-phalloidin revealed less actin bundles, a fuzzy network of shorter filaments and some F-actin aggregates in the highly metastatic tumor cells. Meanwhile, the actin filaments were robust and orderly arranged in the non-/weakly metastatic cancer cell lines.Conclusion Tβ10 levels correlate positively with the metastatic capacity in human tumors currently examined. The increasing metastatic potential of tumor cells is accompanied by a loss of F-actin,poorly arranged actin skeleton organizations and presence of F-actin aggregates. There is a consistent correlation between the elevated Tβ10 expression and the disrupted actin skeleton.     

大鼠急性胰腺炎中核因子NF-κB抗凋亡机制的研究

目的探讨核因子NF—kB激活在急性胰腺炎抗凋亡机制中的作用。方法利用原位细胞凋亡检测法和免疫组织化学方法检测大鼠胰腺细胞凋亡及Bcl-2和Bcl—XL在胰腺组织中的表达。结果预防或治疗性给予N-乙酰半胱氨酸可不同程度地降低NF—kB激活，减轻胰腺坏死程度，增加胰腺腺泡细胞凋亡程度，降低Bcl-2和Bcl—XL蛋白的表达。结论细胞凋亡对腺泡细胞具有保护作用，与急性胰腺炎的严重程度呈负相关。Bcl-2和Bcl—XL基因表达可能参与NF—kB抗胰腺腺泡细胞凋亡过程。     

Evaluation of tumor formation of three bladder cancer cell lines in nude mice

This study examined the differences in tumor formation of three bladder tumor cell lines (BIU-87, T24 and EJ) after subcutaneously transplanted into nude mice, in order to find the best technique for establishing in vivo bladder tumor model. BIU-87, T24 and EJ cells at logarithmic phase were re-suspended in serum-free medium. The cells suspensions of the identical concentration were subcutaneously transplanted into nude mice and then the success rate and tumor growth were compared among the three cell groups. The results of tumor formation were pathologically evaluated. Lung, liver and kidney tissues were also pathologically examined for distant metastasis. The proliferation of the three cells were determined by immunohistochemically detecting the PCNA expression in the tumors. The results showed that the success rates of EJ and T24 cells were significantly higher than that of BIU-87 cells and no distant metastasis was noted among the three groups. The proliferation levels of EJ and T24 cells was significantly higher than that of BIU-87. But at the later stage of tumor formation, as compared with T24 cells, EJ grew more vigorously, soon resulting in the central necrosis of tumor, which affected the measurement of the actual size of the tumors. Moreover, PCNA staining exhibited that the proliferation of EJ and T24 was significantly higher than that of BIU-87 cells. It is concluded that as compared with BIU-87 cells, EJ and T24 cells had higher success rates, with not significant differences in death rate and distant metastasis found among them. There existed no significant difference in tumor formation between EJ and T24 cells and T24 cells do not rupture easily, which makes it a better cell line for the establishment of in vivo bladder tumor model.     

洛拉曲克在肿瘤细胞中的药代动力学研究

目的探讨新型药物洛拉曲克在肿瘤细胞中的转运特点与其敏感性之间的关系。方法采用MTT法测定洛拉曲克对三株肿瘤细胞C6、SRS-82、LoVo的生长抑制作用。通过高效液相色谱法测定经洛拉曲克处理后的细胞内外药物浓度。结果C6是三株细胞中对洛拉曲克最敏感的,SRS-82和LoVo细胞对洛拉曲克的IC50值分别是C6细胞的6.8和13.8倍。洛拉曲克在这三株细胞中胞内与胞外浓度存在线性关系。C6的细胞内稳态浓度显著高于其他两株细胞。结论本研究的结果表明,洛拉曲克可快速进入细胞,不同细胞株对洛拉曲克有不同的吸收能力,这种现象可部分解释肿瘤细胞对洛拉曲克不同的敏感性。     

核因子κB在大鼠多器官功能障碍综合征中的表达及意义

目的:探讨多器官功能障碍综合征(MODS)大鼠肝、肺、肾和小肠组织中核因子xB(NF-kB)的DNA结合蛋白和NF-kB P65蛋白的表达及意义。方法:32只雄性SD大鼠随机分成MODS组(n=16)和正常对照组(n=16),采用二次打击建立MODS大鼠模型,检测大鼠肝功能(ALT、AST)、肾功能(Cr、BUN)、动脉血氧分压(PaO2);用电泳迁移率改变试验(EMSA)检测大鼠肝、肺、肾和小肠组织中NF-kB的DNA结合活性;用Western杂交检测NF-kB P65蛋白表达。结果:衰竭组大鼠肝、肺、肾和小肠组织中NF—kB的DNA结合活性及NF—kB P65蛋白表达明显高于正常组(P<0.01);血清中ALT、BUN分别与肝、肾组织NF—kB的活性呈正相关(P<0.01)。结论:NF—kB活化在MODS的发生发展中可能起重要作用。     

Cotransfection of TrkA and p75~(NTR) in neuroblastoma cell line (IMR-32) promotes differentiation and apoptosis of tumor cells

Objective To assess the effects of both TrkA and p75NTR on nerve growth factor (NGF)-induced differentiation of neuroblastoma cells.Methods Retroviral vectors were constructed to express the high affinity NGF receptor (TrkA) and low affinity NGF receptor (p75NTR). Neuroblastoma cell line IMR-32 was transfected by the vectors expressing either TrkA or p75NTR or both by using lipofectmine?reagent separately or cotransfected at the same time. Southern blot, Northern blot, RT-PCR and flow cytometry were used to determine the success of the transfection. MTT technique was to monitor the cell proliferation. Colony formation in soft agar and tumor forming assay in nude mice were used to test the biological characteristics of the tumor cells. Terminal-deoxynucleotidytransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay was used to test the apoptosis of the tumor cells.Results Stable transformant cell lines expressing TrkA, p75NTR or both genes were established. Studies on these transformant cell line