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expression and purification of fusion protein HSP70-EGFP and its application in the study of dendritic cells internalization

QU Ping, SUI Yanfang, MA Jiahai, CHEN Guangsheng, LIU Libing, CHEN Jiankang, LIU Fang'e

Frontiers of Medicine 2008, Volume 2, Issue 1,   Pages 82-86 doi: 10.1007/s11684-008-0015-0

Abstract: To study the endocytic activity of dendritic cells (DCs) by obtaining fusion protein HSP70-EGFP as exogenous antigen and loading it with DCs derived from human peripheral blood. Fusion protein HSP70-EGFP was prokaryotically expressed, isolated and purified. DCs were isolated and cultured from human peripheral blood. The DCs were divided into 3 groups in the endocytic experiment. There were 10 DCs in each group. Group 1 and 2 were respectively incubated for 30 min. with HSP70-EGFP and EGFP. Group 3 was incubated with HSP70 for 30 min, and then incubated for 30 min. with HSP70-EGFP. Subsequently, 3 groups were placed in an incubator at 37°C for 0.5, 1, 2 and 24 h. Flow cytometry (FCM) was adopted to detect the amount of DCs with EGFP inside. IL-12 Eli-spot was adopted to detect the amount of DCs which secreted IL-12. There were 5 types in the experiment: LPS, inactive LPS, HSP70-EGFP, EGFP and no antigen. Fusion protein HSP70-EGFP was successfully obtained and its molecular weight was 97 000. It accounted for 35.32% of the total protein. Under irradiation of an ultraviolet lamp, the protein solution sent out viridescent fluorescence. The result detected by FCM indicated that after incubation for 0.5 h at 37°C, the positive rate in group 1 was 63%, while the other 2 groups were negative. After incubation for 1, 2 and 24 h at 37°C, the positive rates in the 3 groups were above 80%. The IL-12 Eli-spot examination shows that with HSP70-EGFP being loaded, the amount of DCs secreting IL-12 was 134.09 ± 31.78/10 cells, a little lower than that of DCs with LPS loaded (with the average point of 156.36 ± 15.73). There was no significant difference between the 2 groups ( < 0.01). By contrast, both of them were significantly higher than inactive LPS-(33.78 ± 1.40)/10 cells and EGFP-loaded (23.13 ± 4.57)/10 cells DC groups in the amount of DCs secreting IL-12 ( < 0.01). The results suggest that receptor-mediated phagocytosis plays a main role in the preliminary stage of DCs internalizing HSP70-EGFP. With increasing incubation time, pinocytosis begins to dominate. HSP70-EGFP may promote DCs to secret cell factor IL-12.

Keywords： significant difference     irradiation     peripheral     preliminary     EGFP    

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Endostatin specifically targets both tumor blood vessels and lymphatic vessels

Wei Zhuo, Yang Chen, Xiaomin Song, Yongzhang Luo

Frontiers of Medicine 2011, Volume 5, Issue 4,   Pages 336-340 doi: 10.1007/s11684-011-0163-5

Abstract: not only angiogenesis but also lymphangiogenesis, and the underlying mechanism by which endostatin internalization

Keywords： endostatin     angiogenesis     lymphangiogenesis     nystatin     internalization     tumor    

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Assessing combined toxic effects of tetracycline and P25 titanium dioxide nanoparticles using

Barsha Roy, Khushboo Kadam, Suresh Palamadai Krishnan, Chandrasekaran Natarajan, Amitava Mukherjee

Frontiers of Environmental Science & Engineering 2021, Volume 15, Issue 1, doi: 10.1007/s11783-020-1298-x

Abstract: Abstract • UVA pre-irradiation to TiO2 NPs enhanced its toxicity toward plant A. cepa. • UVA TiO2 NPs increased intracellular ROS, resulting in more cell damage. • Cell death enhanced cell permeability and increased uptake of NPs. • Being highly toxic (EC50 = 0.097 µmol/L), TC did not increase ROS generation. • Even at a low dose, TC enhanced the toxic potential of TiO2 NPs significantly. Usage of titanium dioxide nanoparticles (TiO2 NPs) and tetracycline (TC) has increased significantly in the present era. This leads to their release and accumulation in the environment. Both the compounds, individually, can have adverse toxic effects on the plants. Their binary mixtures can increase this degree of damage. The present study aimed to evaluate the toxicity of both the contaminants in individual and binary mixtures in Allium cepa. Further, the toxicity of TiO2 NPs upon UVA pre-irradiation was also measured. Results showed that UVA pre-irradiated NPs (UVA-TiO2 NPs) had a significant decrease in cell viability than their non-irradiated counterparts (NI-TiO2), denoting an increase in photocatalytic activity upon UVA pre-irradiation. Very low concentrations of TC (EC10 = 0.016 µmol/L) mixed with TiO2 NPs significantly increased the toxicity for both UVA-TiO2 and NI-TiO2 NPs. Intracellular ROS generation was significantly high for UVA-TiO2 NPs. However, TC did not have any effects on ROS production. Both the compounds exhibited genotoxic potential in A. cepa. Different chromosomal abnormalities like anaphase bridges, telophase bridges, laggard chromosomes, binucleate cells, etc. were observed. The binary mixture of UVA-TiO2 NPs and TC showed the highest chromosomal aberrations (64.0%±1.26%) than the mixture with NI-TiO2 or the individual contaminants. This decreased significantly after recovery (46.8%±1.92%), denoting the self-repair processes. This study proved that UVA-TiO2 NPs were more toxic and could be enhanced further when mixed with a sub-lethal concentration of TC. This work will help to assess the risk of both compounds in the environment.

Keywords： toxicity     Cell viability     Mitotic index     Chromosomal aberration     Reactive oxygen species     Nanoparticles internalization    

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Title Author Date Type Operation

expression and purification of fusion protein HSP70-EGFP and its application in the study of dendritic cells internalization

QU Ping, SUI Yanfang, MA Jiahai, CHEN Guangsheng, LIU Libing, CHEN Jiankang, LIU Fang'e

Journal Article

Endostatin specifically targets both tumor blood vessels and lymphatic vessels

Wei Zhuo, Yang Chen, Xiaomin Song, Yongzhang Luo

Journal Article

Assessing combined toxic effects of tetracycline and P25 titanium dioxide nanoparticles using

Barsha Roy, Khushboo Kadam, Suresh Palamadai Krishnan, Chandrasekaran Natarajan, Amitava Mukherjee

Journal Article