Association of UCP3,APN,and TNF-α Gene Polymorphisms with Type 2 Diabetes in a Population of Northern Chinese Han Patients

We observed the polymorphism distribution and coaction of uncoupling protein 3（UCP3）-55C/T,adiponectin（APN）＋45T/G and tumor necrosis factor（TNF）-α-308G/A on the onset and development of T2DM in a Northern Chinese Han population of 213[100 type 2 diabete（T2DM） patients and 113 health control subjects] by polymerase chain reaction-restriction fragment length polymorphisum（PCR-RFLP） method.Results demonstrate the polymorphism of UCP3-55C/T,APN＋45T/G,and TNF-α-308G/A related to T2DM onset and developement.And the individuals carrying UCP3-55T,APN＋45G and TNF-α-308A allele had higher T2DM risk.Those results are the first report to evaluate the association of the coaction of UCP3,APN,TNF-α genes polymorphism on T2DM risk and the susceptibility of T2DM in the Northern Chinese Han population.

DNA Extraction from Formalin-fixed and Paraffin-embedded Tissues by Triton X-100 for Effective Amplification of EGFR Gene by Polymerase Chain Reaction

For first-line non-small-cell lung cancer（NSCLC） therapy,detecting mutation status of the epidermal growth factor receptor（EGFR） gene constitutes a prudent test to identify patients who are most likely to benefit from EGFR-tyrosine kinase inhibitor（TKI） therapy.Now,the material for detecting EGFR gene mutation status mainly comes from formalin-fixed and paraffin-embedded（FFPE） tissues.DNA extraction from FFPE and the amplification of EGFR gene by polymerase chain reaction（PCR） are two key steps for detecting EGFR gene mutation.We showed a simple method of DNA extraction from FFPE tissues for the effective amplification of EGFR gene.Extracting DNA from the FFPE tissues of NSCLC patients with 1% Triton X-100（pH=10.0） was performed by heating at 95 °C for 30 min.Meanwhile,a commercial kit was used to extract DNA from the same FFPE tissues of NSCLC patients for comparison.DNA extracted products were used as template for amplifying the exons 18,19,20 and 21 of EGFR by PCR for different amplified fragments.Results show that DNA fragment size extracted from FFPE tissues with 1% Triton X was about 250―500 base pairs（bp）.However,DNA fragment size extracted from FFPE tissues via commercial kit was about from several hundreds to several thousands bp.The DNA yield extracted from FFPE tissues with 1% Triton X was larger than that via commercial kit.For about 500 bp fragment,four exons of EGFR could not be amplified more efficiently from extracted DNA with 1% Triton X than with commercial kit.However,for about 200 bp fragment.This simple and non-laborious protocol could successfully be used to extract DNA from FFPE tissue for the amplification of EGFR gene by PCR,further screening of EGFR gene mutation and facilitating the molecular analysis of a large number of FFPE tissues from NSCLC patients.

Immunofluorescent Labeling of Human HepG2 Cells with CdTe Quantum Dot Probe Conjugated with Anti-pan CK MAb

A relatively sensitive, specific, and photostable method for the detection of cytokeratin of cancer cells via conjugation with cadmium telluride quantum dots（CdTe QDs） was described. Water soluble CdTe QDs were conjugated to anti-pan-cytokeratin（CK） monoclonal antibody（MAb） through coupling reagent [1-ethyl-3-（3-dimethyla- mino propyl）carbodiimide, EDC] and the conjugates were purified by dialysis. The expression of pan CK protein in HepG2 cells was observed by immunocytochemistry and direct immunofluorescence via QDs-Ab conjugates respectively. Fluorescence intensity and photostability of QDs were compared with those of FITC（fluorescein isothiocyanate）. The results show that the QDs-Ab conjugates recognized specifically pan CK protein in HepG2 cells. Compared with FITC, CdTe QDs had higher brightness and photostability without obvious photobleaching under continuous exciting light illumination for 30 min and after the placement at room temperature for 3 d. The results indicate that conjugates of CdTe quantum dot with anti-pan CK MAb can be used for labeling cancer cells derived from epithelial tissues, which provides the basis for the detection of circulating tumor cells（CTCs）.

Effect of Regulation of HSV-tk Gene Expression and Tumor Killed Activity with a Single Tetracycline-regulatable Plasmid Vector on HeLa Cells

To construct a single tetracycline-regulatable plasmid vector based on the double tetracycline-regulatable plasmid vector system for regulating HSV-tk gene expression so as to effectively kill HeLa cells. Two tetracycline operator（TetO2） was cloned into pcDNA3.1 and a cassette was made for a cytomegalovirus-type 2 tetracycline operator（CMV-TetO2） promoter, and the obtained vector was named pcDNA3.1-CMV-TetO2. Herpes simplex virus thymidine kinase（HSV-tk） gene and tetracycline repressor（TR） gene were cloned into pcDNA3.1-CMV-TetO2 and the two genes were linked with internal ribosome entry site（IRES） to gain a vector named pcDNA3.1-CMV-TetO2- HSV-tk-IRES-TR. The HeLa cells were stablly transfected with pcDNA3.1-CMV-TetO2-HSV-tk-IRES-TR plasmid. The expression of HSV-tk and TR were detected by RToPCR, the tumorcidal activity of HSV-tk/GCV was determined by MTT assay. In Hela cells transfected with the above plasmid vector, HSV-tk gene and TR gene can be expressed lowly and the concentration of GCV producing a 50% decrease in cell viability was about 50 lag/mL without adding deoxycycline; in contrast, the expessions of HSV-tk gene and TR gene increased significantly and the concentration of GCV producing a 50% decrease in cell viability was about 5 μg/mL with adding deoxycycline. Therefore tetracycline can regulate the expression and tumorcidal activity of HSV-tk gene in HeLa cells with this single plasmid vector.

Fluorosis Caused Cellular Apoptosis and Oxidative Stress of Rat Kidneys

As the strongest electronegative element, fluorine can stimulate the production of superoxide radicals in cells. In view of the important roles of kidneys in bone metabolism, the authors analyzed the quantitative pathomor- phological characteristics of renal damage and the potential cellular apoptosis and oxidative stress mechanisms in rats treated with excessive fluoride. Wistar rats were exposed to 50 mg F （110.5 mg NaF）/L, 100 mg F-（221.0 mg NaF）/L and 150 mg F-（331.5 mg NaF）/L in drinking water for 70 and 140 d, respectively. Microscope with image analysis was used to quantitate pathomorphological changes in renal tissues of the rats. Reactive oxygen species（ROS）, the cell cycle and apoptosis of renal cells were measured by flow cytometry and TUNEL technique（terminal deoxynuc- leotidyl transferase dUTP nick end labeling）, respectively. The ion concentrations in serum and renal functional pa- rameters were detected by automatic biochemical analyzer. Quantitative analysis results demonstrate the expanded Bowman＇s space of glomerulus and obvious dilatation of renal tubule. TUNEL technique revealed that NBT/BCIP （nitro blue tetrazolium/5-bromo-4-chloro-3＇-indolylphosphate, p-toluidine salt）-staining positive apoptotic cells se- lectively located in medullocortical junction areas. The data suggest that renal damage in chronic fluorostic rats is associated with the cellular apoptosis and oxidative stress.