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　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.

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 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.

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.

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

Objective To observe the treatment response and potential issues related to antiretroviral therapy (ART) in pediatric patients with acquired immunodeficiency syndrome (AIDS) and to provide a basis for revising the treatment guideline and improving the clinical practice. Methods Children eligible for ART according to the current treatment guideline were recruited from Dehong area. Enrolled patients were provided with ART and followed up for regular clinical check and laboratory tests. Results By the end of March 2009, a total of 70 children had received ART. Among them, 60 patients were treated with regimen including zidovudine (AZT)+ lamivudine (3TC)+nevirapine (NVP). Twelve, eighteen, twenty-three and nineteen patients have tested for HIV viral load after 3 month, 6 month, 12 month and 18 month treatment, respectively. Among them, 7, 12,14 and 14 patients respectively achieved HIV viral load lower than 1000 copy/mL. Average CD4+ Tlymphocyte count increased from (317.8±288.8) × 106/L at baseline to (1037.2±1086. 1) × 106/Lafter 18 month treatment. Side effects mainly occurred within the first 3 months of treatment. Nearly 50% of children had gastrointestinal symptoms. Resistance to 3TC, NVP and efavirenz (EFV) were found in five patients who have completed 12 months of treatment. Conclusions Pediatric AIDS patients show good compliance and treatment response to ART. Most side effects happen in the first 3months of treatment and the most common side effects are gastrointestinal symptoms.

BACKGROUND: Recent researches indicate that ischemia and hypoxia can lead to abnormal brain metabolism and even energy failure, which is an important reason for brain damage and necrosis and identifies energy metabolism disorder as the key event in brain ischemia-reperfusion (IR)injury. Glucose transporter-3 plays the vital role in brain energy metabolism.OBJECTIVE: To observe the changes of cerebral infarct volume and glucose transporter-3 mRNA and protein expressions in cerebral cortical penumbra at different stages of focal cerebral ischemia and reperfusion in rats.DESIGN: Randomized controlled experiment.SETTING: Department of Neurosurgery, Second Hospital Affiliated to Sun Yat-sen University.MATERIALS: This experiment was conducted in the Animal Laboratory of Medical Research Center, Second Hospital Affiliated to Sun Yat-sen University between August and October 2002.Totally 56 SD rats were randomized into 3 groups which were subjected to ① ischemia for 1 hour followed by reperfusion (n=28), ② ischemia for 3 hours followed by reperfusion (n=24), and ③ sham operation (n=4). The rats in the first group were subdivided into 7 subgroups for examination at 1, 3, 6, 12, 24, and 72hours and 1 week after ischemia, with 7 rats in each subgroup; the rats in the second ischemia group were also subdivided in similar manner but without a 1 hour postischemic subgroup. The rats in the sham operation group only received the operation but without arterial occlusion.METHODS: Focal cerebral ischemia-reperfusion (IR) injury model was induced in the rats in the two ischemic groups by means of insertion of suture for arterial occlusion, and the ratio of central ischemic area to cerebral infarct volume in the ischemic penumbra was examined at the specified time points. Reverse transcription-PCR (RT-PCR) was used to detect the expression of glucose transporter-3 mRNA in the cerebral cortex in ischemic penumbra region, and semi-quantitative immunohistochemistry (IHC) employed to detect the level of glucose transporter-3 protein.MAIN OUTCOME MEASURES: Cerebral infarct volume after IR injury, changes of transporter-3 mRNA and protein expressions after IR injury.RESULTS: Totally 56 rats were used in this experiment and all entered result analysis. The post-IR cerebral infarct volume was obviously smaller in 1-hour ischemia group than in 3-hour ischemia group. Glucose transporter-3 mRNA expression began to increase 3 hours after ischemia in 1-hour ischemia group, reaching the peak level at 24 hours and still mainrained higher level than that of the sham operation 1 week; in 3-hour ischemia group, the mRNA expression was slightly decreased at 3 hours but began to increase afterwards till reaching the peak level at 24 hours, followed then by recovery of normal level at 1 week. The changes in glucose transporter-3 protein and mRNA expressions followed almost the same pattern.CONCLUSION: Glucose transporter-3 expression is up-regulated in the ischemic penumbra region, possibly as a protective response to cerebral IR injury.