CD95抑制大肠癌细胞生长的实验研究

目的建立表达外源性CD95基因的大肠癌细胞株,观察CD95表达细胞株在CD95抗体作用下对体外培养的大肠癌细胞的抑制效应。方法采用分子克隆技术将CD95基因插入真核表达载体pBK-CMV的多克隆位点之间。以脂质体介导法将目的基因导入受体细胞的HT-29,用G418筛选克隆细胞。以Northern blot,Western blot检测转导细胞CD95基因的表达。MTT法和直接记数法以及软琼脂集落形成实验检测转导株在CD95抗体作用下的细胞增殖水平、生长曲线及细胞克隆形成率。结果成功地构建了真核表达载体pBK-CMV/CD95 cDNA。转导细胞并经筛选后,获得了2株稳定的抗性细胞,从而建立了CD95基因表达株(HT-29 CD95 cells)。杂交结果表明、转导株CD95mRNA及其蛋白水平的表达均明显高于非转导株,转导细胞增殖速度、倍增时间、对数生长期等均比非转导株更为缓慢和处于抑制状态,集落形成能力低下,但差异无显著性意义,而在CD95抗体作用下效果更为显著,差异有非常显著性意义。结论 CD95基因在大肠癌细胞中处于低表达状态;通过真核表达载体的介导,CD95基因导入大肠癌细胞后,能有效地表达CD95mRNA及其蛋白。CD95表达细胞株在CD95抗体的作用下可明显抑制体外培养的大肠癌细胞的生长增殖,其作用机制与CD95诱导细胞凋亡有关。     

CD95基因抑制胃癌细胞生长的体外抑瘤效应

目的　将CD95基因导入胃癌细胞 ,建立CD95基因表达株 ,并比较转导前后mRNA与蛋白的表达水平。观察CD95蛋白对体外培养胃癌细胞的抑制作用。方法　采用分子克隆技术将CD95基因插入真核表达载体pBK CMV的多克隆克隆位点之间 ,以脂质体介导法将目的基因导入受体细胞SGC790 1,用G418筛选克隆细胞 ;以Northernblot,Westernblot检测CD95基因的表达。MTT法检测转导株对化疗药物的敏感性 ;直接记数法描述转导株的细胞生长曲线 ;软琼脂集落形成实验观察基因转导前后细胞的克隆形成力。结果　成功地构建了真核表达载体pBK CD95cDNA。转导细胞后 ,从 1× 10 5细胞中筛选出 10 0个抗性克隆以上 ,转导率大于 0 1% ,随机挑选 2个克隆扩增培养 ,获得了 1株稳定的抗性细胞 ,从而有效地建立了CD95基因表达株 (SGC790 1CD95cells)。杂交结果表明 ,转导株在mRNA及蛋白水平的表达均明显高于非转导株。转导细胞的细胞倍增时间、对数生长期等均体现了比非转导株更为缓慢和处于抑制状态 ,集落形成能力低下 ,而对VCR、5 FU等化疗药物的敏感性明显增强。结论　CD95基因在胃癌细胞中处于低表达状态 ;通过真核表达载体的介导 ,CD95基因导入胃癌细胞后 ,能有效地表达CD95mRNA及其蛋白。CD95基因转导株的表达蛋白能有效地抑制体外培     

逆转录病毒载体介导IL-6基因修饰大肠癌细胞的抗瘤效应

目的:将外源性 IL-6基因导入大肠癌细胞,建立 IL-6基因表达株,观察 IL-6转基因表达对大肠癌细胞的体内外抑制作用.方法:参照分子克隆技术将构建成功的重组逆转录病毒载体 pZIPIL-6cDNA 转染 PA317包装细胞,以 G418筛选抗性细胞,常规制备重组病毒液并感染大肠癌 HT-29细胞,采用 Northern Blot 分析基因转录水平,ELISA 法和 MTT 显色法检测蛋白表达的量与活性,以细胞生长曲线和集落形成实验以及裸鼠移植瘤实验观察转导株的体内外抑瘤作用。结果:制备了高滴度的重组病毒液(5.1×10~5cfu/ml),建立了稳定表达 IL-6的转导细胞(HT-29IL-6),表达的量与活性分别为1132.5pg/ml/10~5cells 24h 与150U/ml,转导株的细胞群体倍增时间为2.5天,对数生长期在4～7天之间,集落形成率和抑制率分别是2.21%和50%,接种裸鼠皮下的出瘤时间为13.5天,最终瘤体直径在6.5～8.5mm之间,移植瘤标本镜下可见大量凋亡细胞,以上结果与非转导株 HT-29细胞相比,均有显著差异。结论:通过逆转录病毒载体的介导,IL-6基因能稳定整合在靶细胞染色体并进行有效的转录表达,IL-6转基因表达可明显抑制大肠癌细胞的体外增殖和体内移植瘤的形成与发展。     

IL—6基因转导大肠癌细胞的表达

目的:IL-6基因导入大肠癌细胞,建立能有效表达IL-6的转导株HT-29IL-6.方法:采用酶切与连接技术构建重组IL-6基因逆转录病毒载体,基因转染包装细胞,C418筛选克隆,常规制备重组病毒液并感染HT-29细胞,筛选抗性细胞,Southern blot和Northem blot分析基因的整合与mRNA转录水平,MTT显色法及ELISA法检测表达产物的量与活性.结果:成功地构建了重组载体pZIPIL-6cDNA,制备了高滴度的重组病毒液(5.1×10~5cft/ml),其感染率达80%以上,建立了HT-29IL-6表达株,杂交结果证实具有目的基因的稳定整合和相应mRNA的有效转录,表达IL-6的量与活性分别为1132.5pg/ml和15OU/ml.结论:逆转录病毒载体介导的IL-6基因通过转染及筛选能稳定整合在大肠癌细胞染色体,并进行有效的转录与表达,为IL-6转基因治疗大肠癌的研究奠定了基础.     

逆转录病毒载体介导IL-6基因修饰大肠癌细胞的抗瘤效应

目的：将外源性IL-6基因导入大肠癌细胞，建立IL-6基因表达株，观察IL-6转基因表达对大肠癌细胞的体内外抑制作用。方法：参照分子克隆技术将构建成功的重组逆转录病毒载体pZIPIL-6cDNA转染PA317包装细胞，以G418筛选抗性细胞，常规制备重组病毒液并感染大肠癌HT-29细胞，采用Northern Blot分析基因转录水平，ELISA法和MTT显色法检测蛋白表达的量与活性，以细胞生长曲线和集落形成实验以及棵鼠移植瘤实验观察转导株的体内外抑瘤作用。结果：制备了高滴度的重组病毒液(5.1×10^5cfu／ml)，建立了稳定表达IL-6的转导细胞(HT-29IL-6)，表达的量与活性分别为1132.5pg/ml/10^6cells 24h与150U／ml，转导株的细胞群体倍增时间为2．5天，对数生长期在4～7天之间,集落形成率和抑制率分别是2．21%和50％，接种裸鼠皮下的出瘤时间为13．5天,最终瘤体直径在6．5～8.5mm之间，移植瘤标本镜下可见大量凋亡细胞，以上结果与非转导株HT-29细胞相比．均有显差异。结论:通过逆转录病毒载体的介导，IL-6基因能稳定整合在靶细胞染色体并进行有效的转录表达，IL-6转基因表达可明显抑制大肠癌细胞的体外增殖和体内移植瘤的形成与发展。     

Fas基因修饰胃癌耐药细胞的表达

目的将 Fas 基因导入胃癌耐药细胞,建立 Fas 基因表达耐药株,并比较转导前后 mRNA 与蛋白的表达水平。方法:采用分子克隆技术将 Fas 基因插入真核表达载体 pBK-CMV 的多克隆克隆位点之间,以脂质体介导法将重组表达载体转染受体细胞 SGC7901/VCR,G418筛选克隆细胞,Southern blot、Northern blot、Weston blot 检测Fas 基因和 Bcl-2基因的表达。结果:成功地构建了真核表达载体 pBK-Fas cDNA;转导细胞后,从2×10~5细胞中筛选出大约120个抗性克隆,转导率大于0.5‰,随机挑选2个克隆继续筛选与扩增培养,获得了1株稳定的抗生细胞,从而建立了 Fas 基因表达耐药株,我们命名为 SGC7901/VCR Fas cells;杂交结果表明,转导株与非转导株均有Fas cDNA 的表达,但转导株 Fas mRNA 及其蛋白水平的表达则显著高于非转导株。结论:在胃癌耐药细胞中 Fas基因处于低表达状态;通过脂质体介导的基因转染,Fas 基因可成功地导入胃癌耐药细胞,并能有效地增强 FasmRNA 及其蛋白的表达,为诱导细胞凋亡逆转胃癌耐药细胞的研究奠定了重要基础。     

Fas基因修饰胃癌耐药细胞的表达

目的 将Fas基图导入胃癌耐药细胞，建立Fas基因表达耐药株，并比较转导前后mRNA与蛋白的表达水平。方法：采用分子克隆技术将Fas基因插入真核表达载体pBK-CMV的多克隆克隆位点之间，以脂质体介导法将重组表述载体转染受体细胞SGC7901／VCR，G418筛选克隆细胞，Southern blot、Northern blot、Weston blot检测Fas基因和Bel-2基因的表达．结果：成功地构建了真核表述载体pBK-Fas eDNA-转导细胞后．从2×10^5细胞中筛选出大约120个抗性克隆，转导率大于0．5‰，随机挑选2十克隆继续筛选与扩增培养-获得了l株稳定的抗生细胞，从而建立了Fas基困表达耐药株，我们命名为SCXC7901／VCRFascells杂交结果表明,转导株与非转导株均有FaseDNA的表达，但转导株Fas mRNA及其蛋白水平的表过则显高于非转导株。结论：在胃癌耐药细胞中Fas基镯处于低表达状态;通过脂质体介导的基因转染．Fas基因可成功地导入胃癌耐药细胞，并能有效地增强FasmRNA及其蛋白的表达，为诱导细胞凋亡逆转胃癌耐药细胞的研究奠定了重要基础。     

Fas转导大肠癌细胞中凋亡相关基因表达的变化

目的观察Fas转导大肠癌细胞株在诱导细胞凋亡中凋亡相关基因的变化,进一步探讨凋亡信号传导途径。方法运用流式细胞直接免疫荧光术(FICM)检测凋亡相关基因的表达。细胞分组包括:Fas转导细胞组(对照组)、Fas转导细胞+Fas抗体组(12 h)、Fas转导细胞+Fas抗体组(24 h),Fas抗体浓度1∶100。结果在Fas抗体诱导的Fas转导株大肠癌细胞凋亡中,bcl-2蛋白表达水平明显下降(P0.01);bax蛋白表达水平有所增加(P0.05)。初步验证了Fas途径诱导的细胞凋亡中部分凋亡相关基因变化。结论Fas表达大肠癌细胞株在Fas抗体作用下能够有效通过下调bcl-2及上调bax表达诱导细胞凋亡。     

CD95基因转导食管癌细胞的体外抑瘤效应

目的 观察CD95基因表达株表达的CD95蛋白对体外培养食管癌细胞的抑制作用。方法 Western blot检测CD95基因转导前后CD95蛋白的表达水平。直接记数法描述转导株的细胞生长曲线;MTT显色法比较转导前后细胞的体外增殖能力;药敏实验观察转导细胞对VCR、5-FU等化疗药物的敏感性;软琼脂集落形成实验观察基因转导前后细胞的克隆形成力。结果 杂交结果表明．转导株CD95蛋白水平的表达明显高于非转导株。转导细胞的细胞倍增时间(2d)、对数生长期(3d～7d)等均体现了比非转导株(0．8d,2d～8d)更为缓慢和处于抑制状态,体外生长速度的增长指数下降,集落形成能力低下(0．9％),而对VCR、5-FU等化疗药物的敏感性明显增加。结论 CD95基因在食管癌细胞中处于低表达状态;通过真核表达载体的介导,CD95基因导入食管癌细胞后．能有效地表达CD95蛋白。CD95基因转导株的表达蛋白能有效地抑制体外培养的食管癌细胞的生长。     

fas基因与bcl-2反义RNA转导胃癌耐药细胞的药敏上调效应

目的比较fas基因和bcl-2基因在胃癌耐药细胞与非耐药细胞表达的差异,将fas基因和bcl-2反义核酸导入胃癌耐药细胞,并分析转导前后的细胞在mRNA与蛋白的表达水平,研究转导株与非转导株对化疗药物敏感性的区别.方法采用分子克隆技术将fas 基因和反义bcl-2片段分别插入真核表达载体pBK-CMV和pDOR-SV40的多克隆克隆位点之间,以脂质体介导法将两个重组表达载体分别转染受体细胞SGC7901/ VCR,G418筛选克隆细胞,Northern blot, Western blot检测耐药细胞与非耐药细胞以及转导细胞中fas基因和bcl-2基因mRNA及其蛋白的表达,MTT法药敏实验检测转导细胞与非转导细胞对VCR、顺铂、5-FU 的敏感性.结果真核表达载体pBK-fas cDNA和pDOR-bcl-2 cDNA转导胃癌耐药细胞后,分别从2×105细胞中筛选出大约80和120个抗性克隆,转导率各为0.4‰和0.6‰,随机各挑选2个克隆继续筛选与扩增培养,均获得了稳定的抗性细胞,我们将此命名为SGC7901 fas/ VCR cell和SGC7901 anti bcl-2/ VCR cell. 杂交结果表明,胃癌耐药细胞SGC7901/ VCR与非耐药细胞相比,bcl-2基因的表达明显较高,而fas基因表达极其微弱. 转导株SGC7901 fas/ VCR cell的fas mRNA及其蛋白水平的表达显著高于非转导株,SGC7901 anti bcl-2/ VCR cell中bcl-2蛋白的表达明显低于非转导株. 2株转导细胞对VCR、顺铂、5-FU的敏感性明显高于非转导株.结论胃癌耐药细胞与非耐药细胞相比,bcl-2基因处于高表达状态,而fas基因处于极其低弱的表达状态. 通过脂质体介导的基因转染,有效地阻断了bcl-2蛋白在SGC7901 anti bcl-2/ VCR cell的表达,明显增强了fas mRNA及其蛋白在SGC7901 fas/ VCR cell的表达. bcl-2反义核酸和fas基因转导胃癌耐药细胞后,对化疗药物的敏感性明显增加.     

APO-1-induced apoptosis of leukemia cells from patients with adult T- cell leukemia

The 48-Kd cell-surface protein APO-1 is a new member of the nerve growth factor (NGF)/tumor necrosis factor (TNF) receptor superfamily. APO-1 is expressed on various cells, including activated T and B cells and some lymphoid and nonlymphoid cell lines. Triggering of APO-1 by the monoclonal antibody anti-APO-1 induces programmed cell death (apoptosis) in APO-1-expressing cells. APO-1 is also present on T-cell lines derived from patients with adult T-cell leukemia (ATL). Therefore, we investigated APO-1 expression and APO-1-mediated induction of apoptosis ex vivo in cells from patients with ATL. Fresh leukemic cells from nine patients with ATL were assayed for APO-1 expression by two-color immunofluorescence. The leukemic cells from all patients strongly expressed APO-1. Incubation of ATL cells with anti- APO-1 in vitro inhibited spontaneous and cytokine-mediated DNA synthesis. Furthermore, DNA isolated from cells treated with anti-APO-1 exhibited polynucleosomal DNA fragmentation (DNA ladder) characteristic for apoptotic cell death. The analysis of APO-1-mediated apoptosis may represent a new approach to the study of growth control in lymphoid malignancies. In addition, induction of apoptosis by administration of anti-APO-1 may represent a new therapeutic approach for aggressive T- cell malignancies such as ATL.     

Expression of the APO-1 antigen in Burkitt lymphoma cell lines correlates with a shift towards a lymphoblastoid phenotype.

APO-1 is a cell surface molecule that induces apoptosis when ligated with the monoclonal antibody anti-APO-1. Expression of APO-1 and response to anti-APO-1 was investigated in a number of Epstein-Barr virus (EBV)-positive and -negative Burkitt lymphoma (BL) cell lines, in EBV-immortalized lymphoblastoid cell lines, and in cells from fresh BL biopsies. APO-1 was not expressed in EBV-negative cell lines and in EBV-positive BL cell lines with a phenotype corresponding to BL tumor biopsy cells (CD10+, CD21-, CD23-, CD30-, CD39-, CDw70-, CD77+). Accordingly, fresh BL cells obtained from three BL biopsies were APO-1 negative. EBV-positive BL cell lines that had acquired a lymphoblastoid phenotype (CD10-, CD21+, CD23+, CD30+, CD39+, CDw70+, CD77-) upon prolonged in vitro cultivation, as well as normal B-lymphoblastoid cell lines, expressed a high density of APO-1. APO-1 may, therefore, be regarded as a B-cell activation marker. APO-1 expression is not the only prerequisite for anti-APO-1-induced apoptosis because 6 of 7 APO-1-expressing EBV-positive BL cell lines were not sensitive to anti-APO-1, whereas all lymphoblastoid cell lines were killed by anti-APO-1. The sensitivity of lymphoblastoid cell lines to anti-APO-1-mediated apoptosis may open a new therapeutic approach for the treatment of EBV-induced lymphoproliferative lesions in immunocompromised individuals, because these are composed of cells with a lymphoblastoid phenotype.     

Endothelial leukocyte adhesion molecule 1: direct expression cloning and functional interactions.

A cDNA for endothelial leukocyte adhesion molecule 1 (ELAM-1) was isolated by transient expression in COS-7 cells of a subtracted cDNA library from cytokine-treated human umbilical vein endothelial cells (HUVECs), with selection of ELAM-1-expressing clones by adhesion of transfected cells to the human promyelocytic cell line HL-60. This cloning method requires neither antibody nor purified ligand. ELAM-1-expressing COS cells bind the promyelocytic cell line HL-60 by a Ca2(+)-dependent but temperature-independent mechanism. Although ELAM-1 is homologous to mammalian lectins, its interaction with HL-60 cells is not inhibited by simple carbohydrate structures. ELAM-1-expressing COS cells also bind human neutrophils and the human colon carcinoma cell line HT-29, but not the B-cell line Ramos. However, Ramos cells adhere to cytokine-treated HUVECs but not control HUVECs, confirming the existence of other inducible adhesion molecules. In addition, the binding of HL-60 cells or neutrophils to ELAM-1-expressing COS cells is not inhibited by a monoclonal antibody (60.3) directed to an inhibitory epitope on CD18, indicating that the ELAM-1 ligand, although uncharacterized, is not a member of the CD11/CD18 family.     

丁酸钠对结肠癌细胞株HT-29血管内皮生长因子表达水平的影响

目的 观察丁酸钠对结肠癌细胞株HT-29的生长抑制情况以及对血管内皮生长因子(VEGF)表达水平的影响。方法 运用细胞增生抑制实验(MTT法)，免疫细胞化学技术观察丁酸钠对HT-29细胞株的生长和对、VEGF表达水平的影响。结果 丁酸钠能抑制HT-29细胞株增生，并降低VEGF表达水平。结论 丁酸钠能够降低VEGF的表达水平，提示可用作抗血管生成物质降低肿瘤细胞侵袭力。     

DPC4基因对人胰腺癌细胞JF305增殖的影响

目的研究肿瘤抑制基因DPC4(deleted in pancreatic carcinoma)对人胰腺癌细胞系JF305增殖能力的影响。方法将携带DPC4基因的真核表达载体转入JF305细胞内,经G418筛选获得DPC4稳定表达细胞株,免疫细胞化学和RT-PCR法检测转染前后细胞内DPC4的表达。用MTT法、细胞计数法和流式细胞仪测定细胞生长曲线、细胞周期、细胞贴壁率和细胞克隆形成率。比较转染前后细胞增殖能力的变化。结果未转染及转染空质粒的JF305细胞无DPC4的表达,转染pBK-CMV-DPC4的JF305细胞可检测到DPC4的表达,且其细胞增殖能力较前明显下降(P<0．001),细胞倍增时间显著延长(由11．8d延长至18d),细胞贴壁率和克隆形成率均明显下降(P<0．0001),G_1期细胞所占比例明显增加(P<0．0001),G_2／M期细胞所占比例明显下降(P<0．0001)。结论无DPC4表达的胰腺癌细胞株JF305可转基因获得DPC4稳定表达,DPC4转基因后可抑制其增殖能力,细胞G_1期延长,G_2／M期缩短。DPC4有望成为胰腺癌基因治疗新的候选基因。     

bax基因对大肠癌细胞株HT-29作用的实验研究

目的 探索用双重腺病毒载体介导bax基因大肠癌进行基因治疗。方法 双重腺病毒载体介导bax表达，相关显微镜、MTT比色法、流式细胞仪观察bax基因对HT－29的作用，Western Blot检测Bax蛋白的表达。结果 Ad/Gt-bax能有效地引起HT－29形态学的改变，当加入Ad/PGK-GV16后，该作用更为显著，呈典型的凋亡形态学特征 ，其生长抑制率及凋亡诱导率分别为57.4%和28.2%，与Ad/GT-bax组的29.2%和12.6%相比，差异有显著性（P＜0．05）。Ad/GT-bax与Ad/PGK-GV16同时应用能起Bax蛋白大量表达,而单用Ad/GT-Bax，则Bax蛋白表达量较低。结论 bax基因具有有效诱导人大肠癌细胞株HT－29凋亡的作用。     

Effects of inositol hexaphosphate on proliferation of HT-29 human colon carcinoma cell line

AIM: To investigate the effects of inositol hexaphosphate (IP6) on proliferation of HT-29 human colon carcinoma cell line. METHODS: Cells were exposed to various concentrations (0, 1.8, 3.3, 5.0, 8.0, 13.0 mmol/L) of IP6 for a certain period of time. Its effect on growth of HT-29 cells was measured by MTT assay. The expressions of cell cycle regulators treated with IP6 for 2 d were detected by immunocytochemistry. RESULTS: IPe inhibited the HT-29 cell growth in a dose- and time-dependent manner. Analysis of cell cycle regulator expression revealed that IPe reduced the abnormal expression of P53 and PCNA and induced the expression of P21. CONCLUSION: IPe has potent inhibitory effect on proliferation of HT-29 cells by modulating the expression of special cell cycle regulators.     

Significance of Bcl-xL in human colon carcinoma

AIM：To investigate the clinical significance of Bcl-xL gene in the pathogenesis of human colon carcinoma. METHODS：Fifty-six pair tissue samples from patients with colon cancer were collected, and protein level of the Bcl-xL gene was measured by immunohistochemistry method. The correlation of Bcl-xL expression with clinical index was evaluated. After human colon cancer cell line HT29 was transfected with Bcl-xL small interfering RNA （siRNA）, the anchorage-independent growth of cancer cells was detected by colony formation in soft agar and invasion ability of cancer cells was determined by a transwell model.
RESULTS：The Bcl-xL expression was higher in cancerous tissue samples than in normal tissue samples （38.78 ± 11.36 vs 0.89 ± 0.35, P 〈 0.001）, and was associated with the pathological grade, lymphnode metastasis and Duke＇s stage of colorectal carcinoma. Transfection with Bcl-xL siRNA inhibited the colony formation and invasion ability of human colon cancer cell line HT29 in vitro.
CONCLUSION：Bcl-xL gene plays an important role in carcinogenesis of human colorectal carcinoma and is associated with malignant biological behaviors of human colorectal carcinoma.