下调TSG101基因对人急性髓性白血病细胞的调节作用

构建肿瘤易感基因101(TSG101)基因的小干扰RNA载体并将其转导入HL-60细胞,获得稳定转染的阳性克隆后,应用RT-PCR和Western印迹进行鉴定;MTT法和流式细胞仪检测细胞转染前后生长速度和细胞周期的变化;DNA梯带(ladder)法和流式细胞仪检测细胞对顺铂诱导的凋亡的变化;Western印迹检测耐药相关蛋白P-gp和MRP的表达变化。结果表明,成功构建了TSG101的小干扰RNA载体;经蛋白质水平检测证实成功建立了稳定的TSG101低表达的白血病细胞模型;转染TSG101小干扰RNA后的细胞生长速度显著减慢,出现G1期阻滞;对顺铂的敏感性明显增强,形成DNA条带,且低表达P-gp。因此,下调TSG101基因能抑制HL-60细胞生长,增加细胞对化疗药物的敏感性,并提示该基因具有进行白血病基因治疗的可行性。     

Citrate Synthase Expression Affects Tumor Phenotype and Drug Resistance in Human Ovarian Carcinoma

Citrate synthase (CS), one of the key enzymes in the tricarboxylic acid (TCA) cycle, catalyzes the reaction between oxaloacetic acid and acetyl coenzyme A to generate citrate. Increased CS has been observed in pancreatic cancer. In this study, we found higher CS expression in malignant ovarian tumors and ovarian cancer cell lines compared to benign ovarian tumors and normal human ovarian surface epithelium, respectively. CS knockdown by RNAi could result in the reduction of cell proliferation, and inhibition of invasion and migration of ovarian cancer cells in vitro. The drug resistance was also inhibited possibly through an excision repair cross complementing 1 (ERCC1)-dependent mechanism. Finally, upon CS knockdown we observed significant increase expression of multiple genes, including ISG15, IRF7, CASP7, and DDX58 in SKOV3 and A2780 cells by microarray analysis and real-time PCR. Taken together, these results suggested that CS might represent a potential therapeutic target for ovarian carcinoma.     

Production and Purification of a Bioactive Substance Inhibiting Multiple Drug Resistant Bacteria and Human Leukemia Cells from a Salt-Tolerant Marine Actinobacterium sp. Isolated from the Bay of Bengal

Four marine actinobacteria tolerant to 200 g NaCl l⁻¹ were screened for antibacterial activity against eight patient-derived multiple drug resistant (MDR) bacteria. The active compound (MW 300.2, predicted molecular formula C₂₀H₂₈O₂) from an actinobacterium, was inhibitory to three Gram-positive and three Gram-negative MDR bacteria, seven non-clinical Gram-positive, four Gram-negative bacteria and five fungi (MIC: 3.5–4.0 µg ml⁻¹). Also, 54% of human leukemia (HL-60) cells were killed by the compound at 0.05 µg ml⁻¹. Bioreactor production demonstrated unusual primary metabolite kinetics. Molecular phylogenetic analysis showed this typical intertidal inhabitant to be a member of the Streptomyces genus and distinct from other salt-tolerant actinobacteria. As no compound was found to match the properties in several electronic databases, our screening strategy should increase the possibility of discovering bioactive molecules from rare actinobacteria.     

HIV-1的表型及其感染的细胞嗜性

HIV-1的表型分为合胞体诱导型(syncytium-inducing,SI)和非合胞体诱导型(non-syncytium-inducing,NSI)。依据所用辅助受体和感染靶细胞的不同,HIV-1又被分为R5、X4和R5X4型。R5和X4型病毒分别利用CCR5和CXCR4作为辅助受体,而R5X4型病毒可利用这两种辅助受体。在病毒的复制力、细胞嗜性以及合胞体诱导能力上,SI型与X4型病毒一致,NSI型与R5型病毒一致。在HIV-1感染过程中,疾病的发展伴随着病毒从NSI型向SI型、及R5型向X4型的转变。HIV-1的表型影响和决定着HIV-1的感染、传播及AIDS的疾病进程。HIV-1的表型和细胞嗜性主要由病毒gp120的V3区(特别是第11和25位的氨基酸)决定。V3区的氨基酸序列信息,将为预测HIV-1的表型,以及病毒感染后的疾病进程提供生物信息学的依据。     

Toward a CRISPR Picture: Use of CRISPR/Cas9 to Model Diseases in Human Stem Cells In Vitro

Human induced pluripotent stem cells (iPSCs) can be differentiated into any cell in the body unlocking enormous research potential. Combined with the recent discovery of CRISPR/Cas9 endonucleases in bacteria and their modification for use in biomedical research, these methods have the potential to revolutionize the field of genetic engineering and open the door to generating in vitro models that more closely resemble the in vivo system than ever before. Use of CRISPR/Cas9 has created a whirlwind within the scientific community in the last few years, as the race to move beyond just disease analysis and toward the goal of gene and cell therapy moves further. This review will detail the CRISPR/Cas9 method and its use in stem cells as well as highlight recent studies that demonstrate its use in creating robust disease models. Finally, recent results and current controversies in the field are reviewed and lingering challenges to further development are explored. J. Cell. Biochem. 119: 62–68, 2018. © 2017 Wiley Periodicals, Inc.