RNA Interference-Mediated Gene Silencing of Vascular Endothelial Growth Factor

OBJECTIVE To inhibit the expression of the vascular endothelial growth factor (VEGF) by RNA interference, and to observe the effect in different cells line. METHODS Using the services of E-RNAi, we designed and constructed two kinds of shRNAs expression vectors which were aimed at the VEGF gene. These vectors were then transfected into HEK293, colon cancer HT29, Hela and HepG2 cells by LipofectamineTM 2000. The level of VEGF mRNA was determined by RT-PCR and Northern blotting and the VEGF expression was examined by immunofluoresence staining. RESULTS The two kinds of VEGF specific shRNAs expression vectors were found to efficiently inhibit the expression of VEGF in HEK293 and HT29 cells by RT-PCR analysis, with inhibition rates of 72% and 42%, respectively; but the inhibition rates were reduced to 28% in Hela cells and 13% in HepG2 cells. Northern blotting showed that the inhibition rates of VEGF mRNA expression were 88% and 89% in HEK293 and HT29 cells, respectively. The inhibition rate of VEGF protein expression in HT29 cells was 73% based on immunofluoresence staining. CONCLUSION The expression of VEGF was inhibited by RNA interference, but differed with various cells lines, showing that RNA interference was cell-line dependent.

Syrosingopine,an anti-hypertensive drug and lactate transporter(MCT1/4)inhibitor,activates hepatic stellate cells and exacerbates liver fibrosis in a mouse model

Syrosingopine is an anti-hypertensive drug and can cause high intracellular lactate levels and end-product inhibition of lactate dehydrogenase by inhibiting the lactate transporters MCT1 and MCT4.Previous studies have shown that syrosingopine plays an essential role in the process of glycolytic blockade,ATP depletion,and cell death in cancer due to high intracellular levels of lactate.

A simplified noncryogenic strategy to transport mesenchymal stem cells: Potential applications in cell therapy and regenerative medicine

With the rapid advances in stem cell research and po-tential cell-based therapies,there is an urgent need to develop safe and reliable cell transport strategies.Except for autologous stem cell-based therapies,allogeneic stem cell therapies and ex vivo genetically engineered cell therapies would require safe,efficient,and reliable cell preservation and transport methods.

Twenty years of Gendicine?rAd-p53 cancer gene therapy:The first-in-class human cancer gene therapy in the era of personalized oncology

Genetic mutations in TP53 contribute to human malignancies through various means.To date,there have been a variety of therapeutic strategies targeting p53,including gene therapy to restore normal p53 function,mutant p53 rescue,inhibiting the MDM2-p53 interaction,p53-based vaccines,and a number of other approaches.This review focuses on the functions of TP53 and discusses the aberrant roles of mutant p53 in various types of cancer.Recombinant human p53 adenovirus,trademarked as Gendicine,which is the first anti-tumor gene therapy drug,has made tremendous progress in cancer gene therapy.We herein discuss the biological mechanisms by which Gendicine exerts its effects and describe the clinical re-sponses reported in clinical trials.Notably,the clinical studies suggest that the combination of Gendicine with chemotherapy and/or radiotherapy may produce more pronounced efficacy in slowing tumor growth and progression than gene therapy/chemotherapy alone.Finally,we summarize the methods of administration of recombinant human p53 adenovirus for different cancer types to provide a reference for future clinical trials.

All-trans retinoic acid inhibits the malignant behaviors of hepatocarcinoma cells by regulating ferroptosis

All-trans retinoic acid(ATRA)can reverse the malignant behaviors of hepatocellular carcinoma(HCC)cells,thereby exerting anti-HCC effect;however,the underlying mechanism is yet to be understood.This study aimed to demonstrate that ATRA is vital to ferroptosis in HCC.Ferroptosis-related genes exhibit different expression in patients with HCC compared to that in healthy individuals.A total of 20 amino acid products were detected in HepG2 cells,the expression level of 5 was decreased after ATRA treatment.ATRA improved the levels of lipid ROS,MDA,and NAPDt/NADPH,and reduced the mt-DNA copy number and changed the structure of mitochondria,in HepG2 and Hep3B cells.We found the expression of genes positively correlated with ferroptosis to increase and those negatively correlated to decrease with ATRA treatment.Inhibition of ferroptosis by Ferrostatin-1 reversed ATRA-inhibited proliferation of HCC cells,along with cell migration and invasion.GSH synthesis was blocked by ATRA,accompanied by decreased cystine content and increased glutamate content,and downregulation of the expression of GSH synthesis-related genes.Our findings suggested that ATRA inhibited the malignancy of HCC cells by improving ferroptosis,and that inhibition of GSH synthesis contributed to ATRA-induced ferroptosis.

关节软骨修复细胞基因治疗中软骨细胞支架载体特点

目的探讨四种不同类型三维支架中培养的关节软骨细胞活性状况及其转基因特性。方法原代培养兔膝关节软骨细胞，并且以表达绿色荧光蛋白（GFP）和萤火虫荧光素酶（GL3）的腺病毒载体AdGFP和AdGL3进行感染。在Ⅰ型胶原海绵、纤维蛋白胶、透明质酸和聚乳酸四种类型的支架材料中，以荧光显微镜和荧光素酶分析法检测细胞的活性状况及其转基因特性；以阿辛蓝染色评价软骨基质的产生。结果表达绿色荧光蛋白的腺病毒（AdGFP）成功感染兔关节软骨细胞，并且持续表达绿色荧光蛋白。被感染的软骨细胞在所有被测试的细胞支架上均能存活，并能表达GFP和荧光素酶报告基因。与其余三种支架比较，聚乳酸支架中转基因表达率最高（P〈0．01）。而且聚乳酸培养体系中，4周时可以检测到阿辛蓝染色阳性的基质材料。结论在关节软骨修复的细胞基因治疗领域，聚乳酸可能是一种合适的支架材料。