胶质瘤中LGI 1的突变及微卫星不稳定性研究

目的 为了检测人脑胶质瘤组织及胶质瘤细胞系LGI 1基因编码区中有无碱基突变及微卫星不稳定性(MSI)和杂合性缺失(LOH).方法 收集30例胶质瘤标本,2例脑膜瘤、2例瘤旁及2例颅脑损伤内减压脑组织标本及体外培养5个脑胶质瘤细胞系.提取组织标本及培养细胞基因组DNA.设计特异性引物分别扩增LGI 1各外显子序列,采用SSCP银染分析;扩增LGI 1微卫星序列,凝胶电泳银染分析;发现异常泳动条带进行DNA序列分析.结果 ①PCR-SSCP分析琼脂糖凝胶电泳检测有1例Ⅱ级胶质瘤标本未检测到第1 a外显子处扩增产物以及Ⅱ、Ⅲ级各1例胶质瘤标本均未检测到第8 c外显子处扩增产物.未在30例胶质瘤标本和4个脑胶质瘤细胞系中检测到电泳条带异常,在细胞系TJ905第5外显子处检测到电泳条带异常,经DNA测序分析证实确有突变.②在30例胶质瘤标本及5个细胞系中均未检测到MSI和LOH.结论 基因突变及MSI和LOH可能不是LGI 1在胶质瘤恶性进展过程中失活的主要原因.     

bFGF小分子干扰RNA诱导胶质瘤U251细胞凋亡作用的研究

目的:初步探讨以小分子干扰RNA沉默碱性成纤维细胞生长因子(bFGF)诱导胶质瘤细胞系U251凋亡的机制.方法:将U251细胞分为正常对照组、空载体组和实验组,按4×105个细胞每孔接种于6孔细胞培养板,培养24h后,空载体组和实验组按MOI=50分别进行空载体腺病毒(rAd5-null)和含有bFGF小分子干扰RNA(siRNA)的重组腺病毒(rAd5-bFGF)转染,转染72h后检测各组细胞的凋亡及细胞周期变化情况及其相关蛋白的表达.结果:与正常对照组、空载体组比较,实验组U251细胞经转染bFGF-siRNA后出现了明显的细胞凋亡(P<0.05),但细胞周期未见变化(P>0.05);Western blot结果显示,实验组U251胶质瘤细胞经转染bFGF-siRNA 72 h后,bFGF蛋白表达明显降低,同时STAT3及Bcl-2蛋白表达降低,Bax及Caspase-3蛋白表达增强.结论:bFGF小分子干扰RNA能够诱导U251细胞凋亡,其作用可能是通过调节STAT3信号转导通路实现的.     

一次性造口袋用于肠外瘘患者引流肠液的临床观察

目的：观察一次性造口袋用于肠外瘘病人引流肠液的临床效果。方法：对18例肠外瘘病人使用一次性造口袋引流肠液（实验组）与2008年前18例采用单纯放置引流管引流肠液（对照组）进行分组对比。结果：对照组出现红肿糜烂18例,伤口敷料污染14例,衣物污染14例,病人下床活动受限,护理工作强度大。实验组出现皮肤红肿糜烂3例,伤口敷料污染2例,衣物污染2例,病人可下床活动,降低了护理工作强度。两组对比有明显差异性,P〈0.05。结论：一次性造口袋应用于肠外瘘病人,在引流肠液的同时起到保护皮肤作用,增加病人舒适度,提高病人满意度,减轻护理工作量,收到良好临床效果。     

Adenovirus-mediated transfer of siRNA against basic fibroblast growth factor mRNA enhances the sensitivity of glioblastoma cells to chemotherapy

Basic fibroblast growth factor (bFGF) is an important growth factor for glioma cell proliferation and invasion. BFGF is overexpressed in malignant gliomas and its level is associated with malignant grades and clinical outcome of patients with gliomas. Small interfering RNAs (siRNA) are synthetic forms of microRNA made of short double stranded RNA, and they effectively catalyze the degradation of their target mRNA. The use of siRNA has become a key method in the suppression of gene expression and the development of therapeutic agents. In this study, we used an adenovirus(Ad)-mediated transfer of siRNA against bFGF mRNA (Ad-bFGF-siRNA) to study the effect of down-regulating bFGF expression on the sensitivity of glioma cells to chemotherapeutics. An optimal siRNA sequence specific for bFGF mRNA was cloned into an adenoviral vector and transfected into three glioma cell lines: U251, A172, and LN229. Methyl thiazolyl tetrazolium (MTT) assays were used to examine changes in cell proliferation, and changes in bFGF mRNA and protein levels in U251 cells were detected using quantitative RT-PCR and Western blot, respectively. Apoptosis of U251 cells was detected using Hoechst staining and flow cytometry, with expression of apoptosis-related proteins evaluated by Western blot. Following the transfection of a bFGF-specific siRNA, mRNA and protein levels of bFGF decreased significantly. Lower rates of proliferation and increased levels of apoptosis also were associated with the Ad-bFGF-siRNA-transfected group compared to control group. Decreased expression of Bcl-2, Bcl-xL, Jak-1, and STAT-3 and increased expression of Bax also were detected in the Ad-bFGF-siRNA-transfected group. For cells treated with both Ad-bFGF-siRNA and chemotherapeutics, a significant reduction in cell survival was observed compared to treatment with Ad-bFGF-siRNA or chemotherapeutics alone. Overall, we found that targeting bFGF mRNA with a siRNA resulted in lower rates of proliferation, increased apoptosis, and enhanced sensitivity of glioma cells to chemotherapy drugs. This suggests that specific targeting of bFGF mRNA may provide a novel approach for the treatment of glioblastoma multiforme (GBM).     

HIV-1 viral protein R (Vpr) induction of apoptosis and cell cycle arrest in multidrug-resistant colorectal cancer cells

Colorectal cancer is a significant health problem, and the advanced stages of the disease have a low response rate to chemotherapy and easily acquire chemoresistance. HIV-1 viral protein R (Vpr) has been shown to possess inhibitory effects on various malignant cells in vivo and in vitro. In this study, an Ad-Vpr construct was used to infect the multidrug-resistant human colorectal cancer HCT-8/5-FU(MDR) cell line in vitro for cell viability, apoptosis, gene expression and gene activity using the MTT, flow cytometry, immunoblotting and gel shift assays, respectively. The data showed that Ad-Vpr significantly reduced HCT-8/5-FU(MDR) cell viability in a dose- and time-dependent manner. Ad-Vpr infection promoted HCT-8/5-FU(MDR) cells to undergo apoptosis and to arrest at the G2 phase of the cell cycle. The G2 cell cycle protein Cyclin B1 accumulated in the cells after Ad-Vpr infection. Furthermore, Ad-Vpr induced activation of caspase-3 and -9, but not caspase-8, in HCT-8/5-FU(MDR) cells. Ad-Vpr suppressed expression of the Bcl-xl protein, but upregulated Bax expression and cytochrome c release from the mitochondria in HCT-8/5-FU(MDR) cells. Ad-Vpr infection also resulted in a time-dependent decrease in nuclear translocation of NF-κB/p65 protein and p65 DNA-binding activity in HCT-8/5-FU(MDR) cells. The data from the current study provide mechanistic insights into understanding the molecular basis and utility of Ad-Vpr as a novel anticancer agent for multidrug resistance in human colorectal cancer.