P-glycoprotein (P-gp) is encoded by the multidrug resistance (MDR1) gene and is well studied as a multi-drug resistance transporter. Peritoneal adhesion formation following abdominal surgery remains an important clinical problem. Here, we found that P-gp was highly expressed in human adhesion fibroblasts and promoted peritoneal adhesion formation in a rodent model. Knockdown of P-gp expression by intraperitoneal injection of MDR1-targeted siRNA significantly reduced both the peritoneal adhesion development rate and adhesion grades. Additionally, we found that operative injury up-regulated P-gp expression in peritoneal fibroblasts through the TGF-β1/Smad signaling pathway and histone H3 acetylation. The overexpression of P-gp accelerated migration and proliferation of fibroblasts via volume-activated Cl(-) current and cell volume regulation by enhancing phosphorylation of the chloride channel-3. Therefore, P-gp plays a critical role in postoperative peritoneal adhesion formation and may be a valuable therapeutic target for preventing the formation of peritoneal adhesions.

The chloride channel-3 (ClC-3) protein is known to be a component of Cl- channels involved in cell volume regulation or acidification of intracellular vesicles. Here, we report that ClC-3 was highly expressed in the cytoplasm of metastatic carcinomatous cells and accelerated cell migration in vitro and tumor metastasis in vivo. High-grade expression of cytoplasmic ClC-3 predicted poor survival in cancer patients. We found that independent of its volume-activated Cl- channel properties, ClC-3 was able to promote cell membrane ruffling, required for tumor metastasis. ClC-3 mediated membrane ruffling by regulating keratin 18 phosphorylation to control β1 Integrin recycling. Therefore, cytoplasmic ClC-3 plays an active and key role in tumor metastasis and may be a valuable prognostic biomarker and a therapeutic target to prevent tumor spread.

P-糖蛋白(P-gp)是多药耐药基因MDR1编码的蛋白,其介导肿瘤产生多药耐药性的药泵功能已明确。但它也广泛的分布于人体的肠、肝、肾、等部位的管腔上皮细胞和脑毛细血管内皮细胞以及免疫细胞,其在这些正常细胞中的生理功能却尚未完全明确。本文综述了正常组织中P-gp的表达分布及其在正常组织中非耐药功能的研究进展。P-gp非多药耐药功能的研究将拓展P-gp的功能研究,揭示其与疾病发生的潜在关系,为探索和开发新的药物提供新的研究思路。

Chloride channel-3 (ClC-3) is suggested to be a component and/or a regulator of the volume-activated Cl− channel in the plasma membrane. However, ClC-3 is predominantly located inside cells and the role of intracellular ClC-3 in tumor growth is unknown. In this study, we found that the subcellular distribution of endogenous ClC-3 varied in a cell cycle-dependent manner in HeLa cells. During interphase, ClC-3 was distributed throughout the cell and it accumulated at various positions in different stages. In early G1, ClC-3 was mainly located in the nucleus. In middle G1, ClC-3 gathered around the nuclear periphery as a ring. In late G1, ClC-3 moved back into the nucleus, where it remained throughout S phase. In G2, ClC-3 was concentrated in the cytoplasm. When cells progressed from G2 to the prophase of mitosis, ClC-3 from the cytoplasm translocated into the nucleus. During metaphase and anaphase, ClC-3 was distributed throughout the cell except for around the chromosomes and was aggregated at the spindle poles and in between two chromosomes, respectively. ClC-3 was then again concentrated in the nucleus upon the progression from telophase to cytokinesis. These results reveal a cell cycle-dependent change of the subcellular distribution of ClC-3 and strongly suggest that ClC-3 has nucleocytoplasmic shuttling dynamics that may play key regulatory roles during different stages of the cell cycle in tumor cells.

BACKGROUND:It has been verified that exogenous basic fibroblast growth factor(bFGF) has obvious inhibitory effect on apoptosis in vascular endothelial cells.OBJECTIVE:To construct a fluorescent eukaryotic cell expression vector carrying the gene of human bFGF,and to investigate its effect on apoptosis induced by hydrogen peroxide(H2O2) and related gene expression in vascular endothelial cells.METHODS:bFGF and GFP gene was subcloned into eukaryotic expression plasmid pcDNA3.1 by means of gene cloning to obtain pcDNA3.1-bFGF-GFP.pcDNA3.1-bFGF-GFP was transfected into umbilical vein endothelial cells by liposome mediating RT-PCR and observing green fluorescence by fluorescence microscope were used to determine the expression of bFGF and GFP.Umbilical vein endothelial cells were divided into three groups:control group(transfected pcDNA3.1),H2O2 group(transfected pcDNA3.1+H2O2) and bFGF-transfected+H2O2 group(transfected pcDNA3.1-bFGF-GFP+H2O2),the apoptosis of umbilical vein endothelial cells was detected by flow cytometry,the expression of caspase-3 P17 subunit and bax protein were determined by Western blot.RESULTS AND CONCLUSION:pcDNA3.1-bFGF-GFP was successfully constructed,bFGF mRNA was increased significantly and specific green fluorescence was observed by fluorescence microscope after pcDNA3.1-bFGF-GFP transfected.Compared with the control group,apoptosis rate,the expression level of caspase-3 P17 subunit and bax protein significantly increased in H2O2 group(P < 0.01).Compared with H2O2 group,bFGF gene transferring significantly decreased apoptosis rate,down-regulated the expression of caspase-3 P17 subunit and bax protein(P < 0.01).bFGF gene transferring can inhibit the apoptosis induced by H2O2 in vascular endothelial cells,the underlying mechanism might be associated with regulation on the expression of bax protein and activity of caspase-3.

Objective To construct a targeting ribozyme(M1GS) which is specific to HCV genome.MethodsAccording to the sequence of conservative region(5′UTR) of HCV genome,a guide sequence was designed and synthesized.And then by the PCR method,a kind of targeting ribozyme can be constructed by covalently linking the guide sequence to the 3′terminus of M1 RNA,the catalytic subunit of RNase P from Escherichia coli.ResultsThe engineered ribozyme(M1GS-HCV/C67) is targeted to the 5′UTR of HCV genome,and can effectively cleave the substrate RNA segment in vitro.The cleavage is specific and the cleavage site is between 67 nt and 68 nt of the target region.Conclusion The M1GS-HCV/C67 would be a useful experimental material to further study its cleavage activity in vivo,and can be even used for evaluating its anti-viral effect in the animal model.It was believed that this study would markedly facilitate the research of a general gene targeting agent for anti-HCV applications,and layed the foundation for developing a new nucleic acid drug of anti-HCV therapy.

Objective To investigate the effects and its posssible mechanisms of basic fibroblast growth factor(bFGF)gene transfection on the migratory activity of vascular endothelial cells(HUVECs).Methods Eukaryotic expression plasmid pcDNA3.1-bFGF was constructed by gene subclone,and then was transfected into HUVECs by liposome mediating,RT-PCR and ELISA were used to determine the expression of bFGF mRNA.Transwell Chamber assay was performed to detect the cell migratory capacity changes of HUVECs after bFGF gene transferred.The expression level of Raf-1,extracellular regulated protein kinases 2(ERK2) and focal adhesion kinase(FAK) protein were assessed by Western blot.Results pcDNA3.1-bFGF was successfully constructed,bFGF mRNA and protein both increased significantly after the transfection.bFGF gene transfection promoted the migratory capacity of HUVECs in vitro,and up-regulated the expression of ERK2 and FAK protein ERK2 and FAK protein,but not affected the expression of Raf-1 protein.Conclusions The transfection of bFGF gene can promote the migration of vascular endothelial cell,its mechanisms might be associated with up-regulation of the expression of ERK2 and FAK protein.