IL—6转基因表达诱导大肠癌细胞凋亡

通过分子克隆技术将ＩＬ－６基因导入大肠癌细胞ＨＴ－２９，建立了目的基因转导株ＨＴ－２９ＩＬ－６细胞，该转导株接种子裸鼠皮下后，与非转导株相比，长出肿瘤的时间延后，最终瘤径明显较小，而且移植瘤标本镜下可见大量的核固缩，核染色质加深，核断裂等凋亡细胞，用携带ＩＬ－６基因的重组逆转病毒液感染大肠癌细胞Ｌｏｖｏ后，仅以１．２５μｍｏｌ／Ｌ的ＶＵＭＯＮ－２６（简称ＶＭ－２６）即可诱导该转染株发生明显凋亡，而     

Polymorphisms in the vitamin D receptor gene, ultraviolet radiation, and susceptibility to prostate cancer

Ultraviolet radiation (UVR) exposure may protect against prostate cancer development via a mechanism involving vitamin D. The vitamin D receptor (VDR) gene is therefore a candidate susceptibility factor for prostate cancer. This possibility has been previously investigated with conflicting results. We examined the association of VDR genotypes (variants at the CDX-2, Fok1, and Taq1 sites), haplotypes, and genotype combinations with risk by studying 368 prostate cancer and 243 benign prostatic hypertrophy (BPH) patients. CDX-2, Fok1, and Taq1 genotype and haplotype frequencies were not significantly different in cancer and BPH patients. As the impact of VDR polymorphisms may depend on UVR exposure, we studied associations of variants with risk in men stratified into low (below median) and high (above median) cumulative exposure/year groups. In men with UVR exposure above the median (1,100 hr/year), CDX-2 GA and AA (odds ratios [OR] = 2.11 and 2.02, respectively) and Fok1 ff (OR = 2.91) were associated with increased prostate cancer risk. No associations were observed for Taq1 genotypes. Of the genotype combinations, relative to all other CDX-2 and Taq1 and combinations, GGTT (P = 0.022, OR = 0.30), and relative to all other Fok1 and Taq1 combinations, FFTT (P = 0.026, OR = 0.35) were associated with reduced prostate cancer risk in the presence of the main effects. None of the other two- or three-genotype combinations was associated with risk. These data indicate that VDR variants influence prostate cancer risk and that this association is dependent on the extent of UVR exposure.     

DPC4基因转染对结肠癌细胞生长的抑制作用及其作用机制

目的 研究DPCA基因转染对结肠癌细胞生长及p21^WAFI mRNA表达的影响。方法 构建pcDNA3．1-DPCA真核表达质粒．利用脂质体转染技术将pcDNA3．1质粒和pcDNA3．1-DPCA质粒分别导入结肠癌细胞系SW620;采用免疫细胞化学和Western蛋白印迹检测细胞中DPCA基因的表达;通过检测细胞生长曲线、克隆形成实验研究DPCA基因转染对SW620细胞生长的抑制作用。通过逆转录．聚合酶链反应(RT-PCR)检测各组细胞内p21^WAFI mRNA的表达。结果 转染DPC4基因后,在SW620细胞中可检测到高表达的DPCA蛋白;细胞生长倍增时间(74h)明显延长;细胞克隆形成率(21％)明显降低。DPCA基因转染后SW620细胞p21^WAFI mRNA含量增加。结论 DPC4基因的高表达能够抑制结肠癌细胞的生长,DPCA基因可能通过诱导p21^WAFI基因的表达而抑制结肠癌细胞的生长。     

结肠癌干细胞样细胞的体外培养、鉴定及n-3多不饱和脂肪酸对结肠癌干细胞样细胞的抗增殖作用

 目的:采取无血清培养法培养出SW620细胞球,并对细胞球细胞进行干细胞鉴定;在细胞水平研究n-3多不饱和脂肪酸（n-3 PUFAs）对结肠癌干细胞样细胞的作用。 方法: 正常培养人结肠癌细胞株SW620并使其逐步适应无血清培养条件,经无血清培养1周后收集SW620细胞球。用免疫荧光法检测胚胎干细胞标志物SSEA-1和TRA-1-81;采用real-time PCR的方法检测干细胞相关基因Sox-2和Oct-4的表达情况;对比SW620贴壁细胞和干细胞样细胞（CSCLC）在软琼脂上的克隆形成能力;采用裸鼠移植瘤模型比较2种细胞的成瘤能力;用MTS法对比2种细胞在递增浓度的5-氟尿嘧啶（5-FU）或mitomycin C处理下的生长抑制情况;用MTS法、Annexin V/PI染色和台盼蓝染色分别观察递增浓度二十二碳六烯酸（DHA）和二十碳五烯酸（EPA）作用于SW620 CSCLC后细胞生长抑制情况、凋亡情况和死亡情况;MTS法检测5-FU或mitomycin C联合n-3 PUFAs对结肠癌CSCLC增殖的影响。 结果: 无血清培养法成功从SW620中培养出细胞球。细胞球细胞高表达SSEA-1和TRA-1-81并一过性表达Sox-2和Oct-4基因;对5-FU及mitomycin C相对抵抗;在软琼脂上克隆形成率及在裸鼠皮下的成瘤率均显著高于贴壁细胞,表明这些细胞具有干细胞特性,即为来自于SW620的CSCLC。DHA和（或）EPA作用于SW620 CSCLC能抑制细胞生长、诱导细胞凋亡,并能增强5-FU及mitomycin C对其抑制作用。 结论: 无血清培养法能够从SW620细胞中培养出具有干细胞特性的细胞,它们具有高克隆形成能力及高致瘤性,对化疗药物相对抗拒;DHA和EPA能够诱导SW620来源CSCLC发生凋亡并增强化疗药物的抗肿瘤活性。     

羊栖菜多糖体外诱导人大肠癌细胞凋亡

目的研究羊栖菜多糖(SFPS)对人大肠癌lovo细胞和RKO细胞增殖的作用及其意义。方法MTT法检测SFPS在体外抑制大肠癌细胞增殖的抑制率;扫描电镜、透射电镜和流式细胞术鉴定细胞凋亡。结果SFPS对lovo细胞和RKO细胞具有显著的生长抑制作用,并在一定范围内呈量效关系。电镜下可见细胞膜表面微绒毛减少、染色质固缩、边集,凋亡小体形成。流式细胞术结果显示,G0/G1期的细胞比例有不同程度的增高,相应的S期细胞比例显著下降(P<0.01);而lovo细胞的细胞周期时相比例无明显改变。结论SFPS在体外能够显著诱导lovo和RKO细胞凋亡,这可能是SFPS抑制人大肠癌细胞增殖的机制之一。     

MicroRNA-451 is involved in the self-renewal, tumorigenicity, and chemoresistance of colorectal cancer stem cells

Many antitumor therapies affect rapidly dividing cells. However, tumor proliferation may be driven by cancer stem cells (CSCs), which divide slowly and are relatively resistant to cytotoxic drugs. Thus, many tumors may progress because CSCs are not sensitive to the treatment. In this work, we searched for target genes whose expression is involved in proliferation and chemoresistance of CSCs. Both of these processes could be controlled simultaneously by cell regulators such as microRNAs (miRNAs). Therefore, colonospheres with properties of CSCs were obtained from different colon carcinoma cells, and miRNA profiling was performed. The results showed that miR-451 was downregulated in colonspheres versus parental cells. Surprisingly, expression of miR-451 caused a decrease in self-renewal, tumorigenicity, and chemoresistance to irinotecan of colonspheres. We identified cyclooxygenase-2 (COX-2) as an indirect miR-451 target gene involved in sphere growth. Our results indicate that miR-451 downregulation allows the expression of the direct target gene macrophage migration inhibitory factor, involved in the expression of COX-2. In turn, COX-2 allows Wnt activation, which is essential for CSC growth. Furthermore, miR-451 restoration decreases expression of the ATP-binding cassette drug transporter ABCB1 and results in irinotecan sensitization. These findings correlate well with the lower expression of miR-451 observed in patients who did not respond to irinotecan-based first-line therapy compared with patients who did. Our data suggest that miR-451 is a novel candidate to circumvent recurrence and drug resistance in colorectal cancer and could be used as a marker to predict response to irinotecan in patients with colon carcinoma.     

维生素D3诱导的U937细胞中CD14表达的实验研究

目的: 探讨维生素D3诱导U937细胞上CD14蛋白的表达及其对内毒素 (LPS)刺激的反应性。方法: 用0. 1μmol/LVitD3与U937细胞共同培养 24h诱导CD14基因的表达, 并观察U937细胞对不同浓度的LPS刺激不同时间的反应性。结果: VitD3能稳定诱导U937细胞表达CD14mRNA和CD14蛋白。经VitD3诱导的U937细胞对LPS刺激的敏感性显著增强, 表现为低浓度LPS刺激即能诱导该细胞核中NF -κB激活, 促进TNF- αmRNA的转录和表达, 并将表达的TNF α释放入培养上清中。结论: VitD3能诱导U937细胞中CD14基因和蛋白的表达, 并增加其对LPS刺激的反应性。     

Soluble CD44 secretion contributes to the acquisition of aggressive tumor phenotype in human colon cancer cells

CD44, a widely distributed cell surface glycoprotein and a receptor for hyaluronan (HA), has been implicated in facilitating tumor growth and metastasis, antiapoptosis and directional motility of cancer cells. In order to investigate the role of soluble CD44 (CD44(sol)) in colon cancer cell growth, SW620, a human colon cancer cell line deficient in CD44 expression was stably transfected with human CD44 cDNA containing exons 1-5, 15 and 16 of the human CD44. Western blot analyses demonstrated the presence of 78 kDa soluble CD44 protein in the culture supernatant of stably transfected cell lines (CD44(sol) clones) and were not detected in the empty vector control line (clone m). The CD44(sol) transfected cells showed higher cell proliferation and clonal growth in vitro, confirmed by MTT and clonogenic assays respectively, when compared to the control cells. Cell adhesion to hyaluronan was significantly lower with CD44(sol) cells compared to the control cells. Western blot analyses were negative for cleaved PARP in lysates from CD44(sol) cells, suggesting resistance to apoptosis. These findings indicate that the secretion of soluble CD44 contributes to colon cancer growth in vitro, possibly as a decoy receptor.     

TRAIL联合氟尿嘧啶体外诱导结肠癌细胞凋亡

目的:探讨氟尿嘧啶(Fu)对重组人肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-relatedapoptosis-inducing ligand,TRAIL)诱导结肠癌细胞凋亡的影响及其作用机制。方法:采用四甲基偶氮唑蓝比色法检测Fu及联合rmhTRAIL对人结肠癌SW480的IC50值;流式细胞术检测不同浓度Fu与rmhTRAIL联合处理SW480 24 h后细胞的凋亡率;实时荧光定量PCR和Western印迹法检测不同浓度Fu与rmhTRAIL联合处理SW480 24 h后细胞survivin基因mRNA和蛋白表达变化。结果:细胞经药物处理24 h后,单用组IC50值为29.5μmol/L,Fu联合rmhTRAIL组IC50值为6.4μmol/L,高质量浓度联合组药物相互作用系数为0.92;Fu与rmhTRAIL联合作用24 h后,随Fu质量浓度增高survivin mRNA和蛋白的表达显著下调。结论:Fu能增强TRAIL诱导的结肠癌细胞凋亡,其机制可能与下调survivin表达有关。     

Nonlinear responses for chromosome and gene level effects induced by vinyl acetate monomer and its metabolite,acetaldehyde in TK6 cells

Vinyl acetate monomer (VAM) produced rat nasal tumors at concentrations in the hundreds of parts per million. However, VAM is weakly genotoxic in vitro and shows no genotoxicity in vivo. A European Union Risk Assessment concluded that VAM's hydrolysis to acetaldehyde (AA), via carboxylesterase, is a critical key event in VAM's carcinogenic potential. In the following study, we observed increases in micronuclei (MN) and thymidine kinase (Tk) mutants that were dependent on the ability of TK6 cell culture conditions to rapidly hydrolyze VAM to AA. Heat‐inactivated horse serum demonstrated a high capacity to hydrolyze VAM to AA; this activity was highly correlated with a concomitant increase in MN. In contrast, heat‐inactivated fetal bovine serum (FBS) did not hydrolyze VAM and no increase in MN was observed. AA's ability to induce MN was not impacted by either serum since it directly forms Schiff bases with DNA and proteins. Increased mutant frequency at the Tk locus was similarly mitigated when AA formation was not sufficiently rapid, such as incubating VAM in the presence of FBS for 4 hr. Interestingly, neither VAM nor AA induced mutations at the HPRT locus. Finally, cytotoxicity paralleled genotoxicity demonstrating that a small degree of cytotoxicity occurred prior to increases in MN. These results established 0.25 mM as a consistent concentration where genotoxicity first occurred for both VAM and AA provided VAM is hydrolyzed to AA. This information further informs significant key events related to the mode of action of VAM‐induced nasal mucosal tumors in rats. Environ. Mol. Mutagen. 54:755–768, 2013. © 2013 Wiley Periodicals, Inc.     

选择性环氧合酶-2抑制剂对结肠癌细胞的生长抑制作用及机制探讨

目的：研究NS-398（一种选择性环氧合酶-2抑制剂）对结肠癌HT-29细胞的生长抑制作用并探讨其机制。 方法： 通过MTT法检测细胞的增殖情况，通过流式细胞术检测细胞凋亡率和细胞周期，通过RT-PCR检测bcl-2 mRNA和bax mRNA表达，通过共聚焦激光扫描显微镜观察细胞骨架成分F-actin的变化。 结果： NS-398对结肠癌HT-29细胞生长的抑制具有时间和剂量依赖性。流式细胞术结果显示NS-398可剂量依赖地诱导HT-29细胞凋亡，使其停滞于G0/G1期。经不同浓度的NS-398处理72 h后，bcl-2 mRNA 在HT-29细胞的表达降低，bcl-2／bax比值降低。细胞骨架成分F-actin主要分布在HT-29细胞核周围，呈环状结构，NS-398作用后细胞核周围的环状结构消失。 结论： NS-398可显著抑制结肠癌细胞的体外生长并诱导其凋亡，这与下调bcl-2／bax比值以及细胞骨架的破坏有关。     

Prolonged survival effects induced by immature dendritic cells and regulatory T cells in a rat liver transplantation model

BackgroundDendritic cells (DCs) and regulatory T (Treg) cells are crucial for inducing immune tolerance. However, the suppressive function of infused Treg cells and immature DCs (imDCs) following solid organ transplantation remains unclear.MethodsImDCs derived from DA-donor rats and Treg cells isolated from spleens of Lewis rats were prepared. A heterotopic liver transplantation model was established to examine the immune tolerance effects of infusion of Treg-imDCs, imDCs and Treg cells individually. Th1/Th2 cytokines and TRAL were detected by ELISA. The overall rejection grade was assessed and the rejection activity index (RAI) was calculated. TUNEL-positive lymphocytes were detected in the portal area in liver sections.ResultsThe infusion of Treg-imDCs was more effective than imDCs or Treg cells individually. Moreover, the expression of IL-10 and TGF-β1 was significantly up-regulated, and IL-12 expression was significantly down-regulated, especially in the Treg-imDCs group. The percentage of TUNEL-positive cells was significantly higher in the Treg cells and imDCs groups. The RAI values in Treg-imDCs group on days 3 and 7 were lower than control, imDCs and Treg cells groups individually (p < 0.05). Both TBIL and ALT levels in the Treg-imDCs and imDCs groups were significantly lower than those of the control and Treg cells groups, and serum TRAL levels increased significantly 10 days after transplantation in the imDC and Treg-imDC groups compared with the control and Treg cells groups (P < 0.001).ConclusionThese data demonstrated that infusion of Treg cells and/or imDCs induces alloantigen tolerance and prolongs liver allograft survival. The infusion of Treg-imDCs was more effective than imDCs or Treg cells individually. ImDCs synergize with Treg cells in inducing and maintaining the feedback loop between imDCs and Treg cells in vivo.     

胰腺癌患者树突状细胞诱导I型调节性T细胞的特征及其意义

为了检测胰腺癌患者树突状细胞(dendritic cells,DC)诱导I型调节性T细胞(typeⅠregulatory T cells,Tr1)的特征、功能及其临床意义。采用外周血单核细胞来源的未成熟DC,诱导同种异体初始T细胞分化为Tr1,ELISA、流式细胞仪检测Tr1细胞因子表达水平。用混合淋巴细胞反应(mixed lymphocyte reaction,MLR)检测Tr1的免疫抑制功能。结果:经胰腺癌患者DC诱导分化的Tr1分泌IL-10(P<0.05)和TGF-β(P<0.01)水平高于正常对照组。IL-10胞内染色结果也表明,分泌IL-10的Tr1在胰腺癌组明显增加(P<0.01)。胰腺癌患者DC诱导的Tr1抑制MLR增殖的能力也明显增强(P<0.01)。胰腺癌患者DC诱导同种异体Tr1分化的能力明显增强,提示过度Tr1活化可能与胰腺癌的病理形成有关。     

Shp2 expression is upregulated in cervical cancer,and Shp2 contributes to cell growth and migration and reduces sensitivity to cisplatin in cervical cancer cells

Src homology phosphotyrosine phosphatase 2 (Shp2) has been found to be overexpressed in cervical cancer tissues. However, the influence of Shp2 on the biological behavior and sensitivity to cisplatin of cervical cancer cells remains unclear. We aimed to assess Shp2 expression in cervical tissues and cell lines and to detect the influence of Shp2 knockdown and overexpression on the biological behavior and sensitivity to cisplatin in cervical cancer cells. We found that Shp2 expression was significantly upregulated in cervical cancer tissues and cell lines, and Shp2 overexpression was associated with lymph node metastasis and a high human papillomavirus (HPV) DNA load. Shp2 knockdown inhibited cell growth and migration and enhanced sensitivity to cisplatin in the HeLa and SiHa cervical cancer cell lines. In contrast, Shp2 overexpression had the opposite effects. These tumor-promoting effects of Shp2 may be partly related to Akt signaling. In conclusion, Shp2 is involved in the occurrence and development of cervical cancer and may confer cisplatin resistance in cervical cancer. Shp2 blockade may be a new strategy for cervical cancer treatment.