Objective To introduce comet assay and to analyze the DNA damage and repair ability of human fetal lung diploid fibroblasts (2BS) as well as lymphocytes. Methods The mixture of 2BS cells (or lymphocytes) and 1% LMA is put on the gel of 0. 5% NMA. After breakage, electrophoresis and stain, the comet tail distance and comet tail area of 100 cells (per sample) are analyzed with Leica image analysis system. Results There are no obvious comet tail for most of young and senescent 2BS cells as well as lymphocytes from young and senescent donors. The comet tails of 28PD 2BS cells treated with H2O2 are obvious. Moreover, the comet tail distances and tail areas of the cells increase as the concentration of H2O2 increasing. The stastics data of the 28PD 2BS cells exposed to H2O2 are as following. Comet tail distance: s = 1.7 ~3. 5 (?m)、CV = 1. 6% ~ 2. 8% . Comet tail area: s = 1. 5% ~ 2. 3%、CV = 1. 8% ~ 2. 5% . Conclusions The DNA breakage level is low both for young and senescent cell. H2O2 can damage DNA obviously. Comet assay is one of important methods used to comment on DNA damage and DNA repair ability.

Objective The differentially expressed genes in the progress of cellular senescence were studied. Methods Suppression subtractive hybridization was used to young and senescent human diploid fibroblast 2BS cells, then screening and analyzing the subtractive libraries. Results Two kinds of subtraction libraries were obtained, respectively representing the differential genes with higher expression in young cells or senescent cells. Screened by dot blot, thirty of genes with differential ratio larger than 2 were sequenced, showing changes in much of cell functions. Some of them also had expression change between blood samples from newborn or old people. Conclusions The differential expression of RBM4?FBXO7?TOM1 is reported during 2BS cell senescence for the first time. There are the similarities during the senescence of culture cell and organism.

Objective　The relationship between DNA methylation and the overexpression of cell cycle negative regulator p16MTS1/INK4a in senescent cells was studied. 　Methods　PCR amplification of p16 exon I following digestion with Sma I , a methylation sensitive DNA endonuclease, was adapted to determine the methylation status at specific site. 　Results　 T-he increased expression of p16 in the aging process of human fetal lung diploid fibroblasts (2BS) was observed. In middle-aged and old cells, the p16 level was about 3 folds and 10 folds respectively as that in young cells. The methylation level of the Sma I site in p16 exon I tended to decline with aging, being about 64% and 41% in young and middle-aged cells respectively, but still maintain relatively as high as about 24% in senescent cells. 　Conclusions　 The overexpression of p16 in senescent human fibroblasts might be related to the alteration of methylation level of exon I, its mechanisms need to be defined further.

Objective　To investigate the involvement of cell cycle negative regulator p16 in the replicative senescence of normal human diploid fibroblasts. 　Methods　 p16 cDNA and retroviral vector was introduced into normal human fibroblast 2BS cells by transfection technique. Then the effects of p16 on replicative cellular senescence of 2BS cells were examined. 　Results　 Compared with the control cells, 2BS cells transfected with p16 cDNA showed significant suppression of growth rate (decrease 50%) with cell cycle arrested at G1 phase. This phenomenon similar to that of the senescent cells, with the cellular response to stimulation by growth factor decreased 79.4%, showing the characteristics in morphology of senescent fibroblasts. 　Conclusions　 The over expression of p16 gene contributes to the process of cellular senescence of normal human diploid fibroblasts.

55 population doubling, PD) were compared with those of the young cells(

Objective:To explore the preparation procedure of pediatric Kechuan black plasters. Methods:A single factor method was used to observe and study the preparation process of pediatric Kechuan black plaster from the following aspects: the selection of matrix raw material and refined equipment, and the temperature of refinery, “Xiadan”, pouring white wax and adding fine material powder in production processing. Results:The best temperature of“Xiadan” for pure sesame oil and mineral orange with the purity a-bove 95% was 313 ℃. The “Xianzha” temperature was 264 ℃, the temperature of “Houxia” was between 180 ℃ and 200 ℃, and white wax instead of the traditional method was used to eliminate fire toxin. Conclusion:The research is helpful to improving the quali-ty of the black plasters. The preparation process is stable and feasible, and suitable for the industrial production.

Objective To investigate the effects of ?-2-macroglobulin on the aging process of human diploid fibroblasts. Methods pIRES-A2M sense and antisense vectors were constructed and transferred into 2BS cells mediated by lipofectamine.2BS/A2Ms and 2BS/A2Ma cell lines were verified by Southern and Northern blot analysis respectively.Cell growth curve,the population doublings,cell cycle analysis,staining of senescent-associated-?-galactosidase and expression of p16 and p21 in transfected cells were measured. Results Southern and Northern blot analysis verified that the exogenous cDNAs were integrated into genomic DNA in the transfected cells.The ultmost population doublings of 2BS/A2Ms cells were slightly higher than normal 2BS cells.Cell growth curve,cell cycle analysis,staining of senescent-associated-?-galactosidase and the population doublings all revealed that 2BS/A2Ms cells demonstrated obvious difference compared with 2BS/A2Ma cells((P0.05). Conclusions The aging process of 2BS cells is influenced slightly by expression of A2M.

AIM: To investigate the volume percentage of infarct and expression level of glucose transporter-3 (GLUT3) transcription and protein at different ischemic time points and different reperfusion time points in rat focal cerebral ischemic penumbra. METHODS: Focal ischemic models of middle cerebral artery occlusion (MCAO) in rats were made by inserting nylon thread. Brain samples were harvested from ischemic penumbra. Infarct volume was analyzed quantitatively by Kontron IBAS 2.5 image auto-analyses system. The change of GLUT3 mRNA was assessed by RT-PCR, and the expression of GLUT3 protein was assessed by immunohistochemistry. RESULTS: The infarction volume in MCAO 1 h/R group was obviously smaller than that in MCAO 3 h/R group. GLUT3 began to ascend at 3 h in MCAO 1 h/R group, reached to climax at 24 h and remained higher than normal at 1 week. In contrast, in the MCAO 3 h/R group, GLUT3 had a descent at 3 h. Later on, it ascended rapidly, and reached climax at 24 h. At 1 week, it approached to normal. The expression level of GLUT3 protein corresponds with that of mRNA. CONCLUSION: GLUT3 expression is up-regulated in the penumbra region after focal cerebral ischemia, it may be a protective reaction against ischemia/reperfusion injury. [

Dental Implantation, Endosseous ; Dental Implants ; Humans ; Maxilla ; Tissue Engineering

Objective:To observe the distribution of vascular endothelial growth factor (VEGF) and microvessel density (MVD) in different brain regions in aged rats and determine the role of VEGF and MVD in the aging process of the nervous system. Methods:We observed the expression of VEGF and MVD in different parts of rat brain in the 3- month group and 30-month group with immunohistochemical technique. Results:Compared with the 3-month group, the 30-month group showed fewer VEGF-positive cells and MVD in the brain (P<0.01), and the number varied signiifcantly in different brain regions(P<0.01). The motor cortex region contained more VEGF-positive cells and MVD than the hippocampus and cerebellum. Conclusion:VEGF-positive cells and MVD are decreased in every brain region of aged rats, and the motor cortex region contains more positive cells, suggesting exogenous VEGF may enhance the formation of microvessels and delay the aging of the nervous system.