一种更有效的肝癌靶向性条件复制型腺病毒的构建及体外抑瘤作用

目的:分别构建以甲胎蛋白(alpha fetoprotein,AFP)300bp启动子和800bp增强子-300bp启动子调控复制的条件复制型腺病毒(CRAd),对两种病毒的务件复制性以及溶瘤作用进行比较,为肝癌靶向性治疗提供更优良的载体.方法:以HepG2基因组DNA为模板,PCR扩增AFP基因启动子(AFPp)和增强子(AFPe),构建表达质粒pAFPp-EGFPluc和pAFPep-EGFPluc,通过检测报告基因EGFP和luciferase的表达鉴定启动子和增强子的活性后,构建穿梭质粒pDC311-AFPp-E1A,pDC311-AFPep-E1A并包装出腺病毒Ad.AFPp-E1A和Ad.AFPep-E1A.利用Western blot、病毒增殖、细胞病变、细胞活力等鉴定并比较两种病毒的复制能力和溶瘤作用.结果:Ad.AFep-E1A和Ad.AFPep-E1A均可在AFP阳性细胞中选择性复制并且具有一定的溶瘤作用,以后者的选择复制性和溶瘤性更为明显.感染病毒Ad.AFPp-E1A后的HepG2、Hep3B细胞的存活率分别为(54.23±7.13)%、(61.18±12.63)%;感染病毒Ad.AFPep-E1A后的HepG2、Hep3B细胞存活率分别为(26.65±5.43)%、(24.49+3.31)%.结论:被截短的800bp增强子片断,在对AFP启动子起到增强作用的同时,可以为腺病毒包装进更多的治疗基因提供空间,从而为肝癌靶向治疗提供更为良好的条件复制型病毒载体.     

Limitation in use of heterologous reporter genes for gene promoter analysis. Silencer activity associated with the cloramphenicol acetyltransferase reporter gene

Various heterologous reporter genes have been widely used for the functional characterization of gene promoters. Many such studies often found weak to very strong silencer activities to be associated with specific parts of the basal promoter or further upstream regions. In this study, we carried out a systematic study on human blood coagulation factor IX (hFIX) and anti-coagulant protein C (hPC) genes, previously shown to have silencer activities associated with their 5'-flanking regions containing promoter sequences. With newly constructed chloramphenicol acetyltransferase (CAT) reporter vectors carrying hFIX or hPC gene promoter sequences, we confirmed the strong silencer activities associated with the regions nt -1895 through nt -416 of the hFIX gene or with the region nt -802 through nt -82 of the hPC gene. However, no such silencer activities associated with the specific regions were found when autologous hFIX cDNA, hFIX minigenes, or hPC minigenes were used as reporters in the expression vector system. Relative levels of CAT, hFIX, and hPC proteins produced in the transient assays correlated well with their mRNA levels. Human FIX minigene constructs containing a simian virus 40 (SV40) 3'-untranslated region (UTR) taken from the CAT reporter gene showed no silencer activity, indicating that SV40 3'-UTR sequence of the CAT reporter gene does not contribute to the silencer activity. Expression vectors constructed with the beta-galactosidase gene under the control of hFIX gene promoter sequences also showed no silencer activity associated with the region nt -1895 through nt -416. These findings indicate that silencer activities associated with specific regions of promoter sequences as analyzed with CAT reporter genes may represent artifacts specific to the CAT reporter genes. Our findings strongly suggest a need for re-examination of promoter characterizations of many eukaryotic genes, which have been studied to date with CAT reporter genes.     

人IL-2/IFNα2b融合基因在肝癌细胞中靶向表达

 根据细胞因子之间协同作用的特点,采用重组 Ｄ Ｎ Ａ 技术设计并构建了人 Ｉ Ｌ ２ 与 Ｉ Ｆ Ｎα融合基因,并用肝癌组织特异的 Ａ Ｆ Ｐ增强子／ Ａ Ｌ Ｂ启动子调控融合基因在肝癌细胞中的靶向表达．实验结果表明,克隆的 Ｅ Ａ Ｆ Ｐ Ｐ Ａ Ｌ Ｂ联合转录调控序列能调控细胞因子基因在 Ａ Ｆ Ｐ阳性人肝癌细胞中靶向表达, Ｉ Ｌ ２／ Ｉ Ｆ Ｎα２ｂ 融合基因的表达水平与感染肝癌细胞的 Ａ Ｆ Ｐ表达水平呈正相关性．实验证明表达的融合蛋白具有 Ｉ Ｌ ２ 和 Ｉ Ｆ Ｎ 两种生物学活性的细胞因子．这可能为肝癌基因治疗开辟新途径．     

Cardiomyocyte-specific gene expression following recombinant adeno-associated viral vector transduction

Recombinant adeno-associated viral (rAAV) vectors hold promise for delivering genes for heart diseases, but cardiac-specific expression by the use of rAAV has not been demonstrated. To achieve this goal rAAV vectors were generated expressing marker or potentially therapeutic genes under the control of the cardiac muscle-specific alpha myosin heavy chain (MHC) gene promoter. The rAAV-MHC vectors expressed in primary cardiomyocytes with similar kinetics to rAAV-CMV; however, expression by the rAAV-MHC vectors was restricted to cardiomyocytes. rAAV vectors have low cytotoxicity, and it is demonstrated here that rAAV fails to induce apoptosis in cardiomyocytes compared with a recombinant adenoviral vector. rAAV-MHC or rAAV-CMV vectors were administered to mice to determine the specificity of expression in vivo. The rAAV-MHC vectors expressed specifically in cardiomyocytes, whereas the control rAAV-CMV vector expressed in heart, skeletal muscle, and brain. rAAV-MHC transduction resulted in long term (16 weeks) expression of human growth hormone following intracardiac, yet not intramuscular, injection. Finally, we defined the minimal MHC enhancer/promoter sequences required for specific and robust in vivo expression in the context of a rAAV vector. For the first time we describe a panel of rAAV vectors capable of long term cardiac specific expression of intracellular and secreted proteins.     

Promoter interactions in retrovirus vectors introduced into fibroblasts and embryonic stem cells.

The activity of the Moloney murine leukemia virus promoter is restricted in mouse embryonic stem cells. Gene expression with retrovirus vectors can be achieved in these cells if internal promoters are used. To address the possible influence of the viral enhancer sequences on expression from the internal promoter, we have constructed high-titer, self-inactivating retrovirus vectors which delete viral regulatory sequences upon integration in the host genome. We show that deleting most of the viral enhancer sequences has no significant effect on viral titer. This enhancer deletion leads to either an increase or a decrease in the amount of RNA transcribed from the internal promoter, but no consistent change can be found with any type of vector. The same changes in expression from the internal promoter observed in embryonic stem cells are also observed in 3T3 fibroblast cells, in which the viral promoter is active. These results indicate that viral regulatory elements influence expression from an internal promoter independently of expression from the virus promoter.     

hMAM启动子／增强子调控表达载体构建和调控作用

目的构建人乳腺珠蛋白（human mammaglobin,hMAM）启动子／增强子调控报告基因表达载体,探讨hMAM启动子／增强子序列在乳腺癌细胞中的特异性调控作用。方法应用PCR技术,从基因组DNA中扩增出hMAM启动子／增强子DNA序列,构建于PGL3报告基因上游,分别转染体外培养的乳腺癌细胞MDA—MB-415、T47D及胃癌细胞7901,分析启动子和增强子序列对乳腺癌细胞的基因表达调控作用。结果酶切图谱分析、DNA序列测定表明成功构建hMAM启动子／增强子调控的表达载体;荧光素酶报告基因检测结果分析表明,hMAM启动子／增强子能够调控报告基因的表达。结论hMAM启动子／增强子,在MDA—MB-415乳腺癌细胞具有调控基因表达的作用;     

Identification and Functional Validation of a 5′ Upstream Regulatory Sequence in the Human Tyrosinase Gene Homologous to the Locus Control Region of the Mouse Tyrosinase Gene

Comparison analysis of the sequences of the mouse and human genomes has proven a powerful approach in identifying functional regulatory elements within the non‐coding regions that are conserved through evolution between homologous mammalian loci. Here, we applied computational analysis to identify regions of homology in the 5′ upstream sequences of the human tyrosinase gene, similar to the locus control region (LCR) of the mouse tyrosinase gene, located at ?15 kb. We detected several stretches of homology within the first 30 kb 5′ tyrosinase gene upstream sequences of both species that include the proximal promoter sequences, the genomic region surrounding the mouse LCR, and further upstream segments. We cloned and sequenced a 5′ upstream regulatory sequence found between ?8 and ?10 kb of the human tyrosinase locus (termed h5′URS) homologous to the mouse LCR sequences, and confirmed the presence of putative binding sites at ?9 kb, homologous to those described in the mouse tyrosinase LCR core. Finally, we functionally validated the presence of a tissue‐specific enhancer in the h5′URS by transient transfection analysis in human and mouse cells, as compared with homologous DNA sequences from the mouse tyrosinase locus. Future experiments in cells and transgenic animals will help us to understand the in vivo relevance of this newly described h5′URS sequence as a potentially important regulatory element for the correct expression of the human tyrosinase gene.