Expression and role of ubiqutin carboxyl-terminal hydrolase L1 in lens with age-related cataract under oxidative stress / 中华实验眼科杂志

Background Oxidative damage is a major cause of age-related cataracts,and the ubiquitinproteasome system is involved in lens differentiation and development.Ubiquitin carboxy-terminal hydrolase L1 (UCHL1),one of key enzymes of ubiquitin-proteasome system,was discovered to participate in the age related diseases and oxidative stress damage. Objective This study was to investigate the effects of UCHL1 on the formation and development of age-related cataract. Methods Lens capsule were collected from 24 patients with age-related cataract(including 12 cases of cortical cataract and 12 cases of nuclear cataract) during the surgery.Five normal lens capsule membranes were obtained from eye bank of Tongji University.Human lens epithelial cells (LECs) line (SRA01/04) was also collected in this study.Expression of UCHL1 in the lens epithelial layer of different samples was assayed using immunofluorescence technology.UCHL1 eukaryotic expressing vector was constructed and transfected into cultured SRA01/04 by liposome,and green fluorescent protein (GFP) eukaryotic expressing vector was transfected at the same method as the control group.UCHL1 over-expressing cells were then exposed to different concentrations (0.2,0.3,0.4 and 0.5 mol/L) of tert-butyl hydroperoxide (TBHP) for 24 hours and subsequently monitored for cell viability evaluation by MTT assay. Results Immunofluorescence showed that UCHL1 was expressed in human lens epithelial layer,but significantly different expressing levels were seen among normal lens capsular membrane,cortical cataract and nuclear cataract ( F =13.411,P =0.000),and UCHL1 expressing levels were lower in cortical cataract and nuclear cataract than the normal lens (P =0.000,P =0.000).No significant difference was found in UCHL1 expressing level between cortical cataract and nuclear cataract ( P =0.164).Western blot analysis verified that UCHL1 exhibited a stranger expression in the UCHL1 transfected group compared with the GFP transfected group,illuminating a successful transfection of UCHL1 in SRA01/04 cells.MMT assay revealed that the A570/630 value in UCHL1 transfected cells was significantly elevated in comparison with GFP transfected cells following the treatment of 0.3 mol/L TBHP. Conclusions UCHL1 has an antioxidative ability,and it might plays an important role in the progress of age-related cataract.

Construction of Sense and Antisense of Human Heparanase and Green Fluorescent Protein Eukaryotic Co-expressing Vectors and Their Expression in Pancreatic Cancer Cell Lines SW1990 / 昆明医科大学学报

Objective To construct sense and antisense of human heparanase and green fluorescent protein eukaryotic expressing vectors and then transfect these vectors into Pancreatic Cancer Cell Lines SW1990.Methods The human Heparanase cDNA fragment contained in the pcDNA3 Hpa vector was cloned into the enhanced green fluorescent protein eukaryotic expressing vector pIRES2-EGFP in cis-direction or trans-direction.The recombinant vectors were identified by digestion of BamH I and further identified by DNA sequencing.The recombinant were transfected to pancreatic cancer cells SW1990 by liposome method.After 24h,the transfected cells were observed under fluorescent inverted microscope.Results After digested by BamH I,two fragments with the length of 5.3 kb and 1.7 kb were formed in sense fluorescent eukaryotic expressing vector,while two other fragments with the length of 6.5 kb and 0.5 kb were formed in antisense fluorescent vector.The DNA sequencing was also confirmed the linking direction of sense and antisense recombinant.Green fluorescence of the transfected cells could be observed under fluorescent inverted microscope after 48h of transfection.Conclusion Human heparanase sense and antisense fluorescent eukaryotic expressing vectors are successfully constructed and transfected to human pancreatic cancer cell lines.

Establishment of HEK293 cell line expressing sense and antisense Alu-Sx stably and efficiently / 第三军医大学学报

Objective To establish a HEK293 cell line stably and highly expressing sense and antisense Alu-Sx.Methods According to Alu subfamily Sx sequence,a pair of primers containing the sites for given restrictive endonuclease at both ends were designed and synthesized.PCR of the total DNA extracted from HEK293 cell line was performed,the products of which were cloned into a highly efficient eukaryotic expression vector pcDNA3.1/myc-His A.The recombinants were sequenced and identified by restrictive endonuclease digestion and then transfected into the HEK293 cell line by lipofectamine2000.The stable transfectants were screened by G418.Cell subclones were isolated by gradient dilution.The highly expressing clones were identified by Northern blotting.Results Eukaryotic expressing vectors stably and efficiently expressing sense and antisense Alu-Sx were constructed and cell subclones stably and efficiently expressing sense and antisense Alu-Sx were established.Conclusion Cell subclones stably and efficiently expressing sense and antisense Alu-Sx can used for our further study.