Objective To establish a method to quantitatively assess melanosome transfer with incorporation of 14C-thiouracil (TU) into nascent melanin. Methods To characterize whether 14C-TU was exclusively incorporated into melanin-producing cells, the same number of mouse melan-a or SP-1 keratinocytes were labeled with 14C-TU for 12 hours and 48 hours, respectively, followed by the measurement of radioactivity. Mouse melan-a melanocytes were pre-labeled with 1 Ci/mL 14C-TU, and cocultured with mouse SP1 keratinocytes to develop an assay system for melanosome transfer to keratinocytes. Following co-culture, the keratinocytes with transferred radioactivity were separated from melanocytes at different time points via two times of differential trypsinization. Transferred radioactivity in keratinocytes, denoting the amount of melanosome transfer, was measured with liquid scintillation counting. Meanwhile, the effects of forskolin, a PKA activator, and nicotinamide on melanosome transfer were also investigated with this assay system.Results The incorporated radioactivity in melan-a cells was 66- or 80-fold as high as that in SP-1 cells,indicating that 14C-TU would be a suitable tracer for melanosome transfer in co-culture with keratinocytes. A purity of 84.5% was achieved for keratinocytes with transferred radioactivity by twice differential trypsinization.As shown by this assay, there was an approximately 0.67-fold decrease in melanosome transfer with the treatment of 1 g/L nicotinamide and 2.3-fold increase with 20μmol/L forskolin treatment. After coculture with SP1 cells for 8-12 hours, melan-a cells developed well-extending dendrites with detectable melanosome transfer, while no proliferation of melan-a cells induced by forskolin was seen. Conclusion An optimized protocol for selective incorporation of 14C-TU into nascent melanin has been successfully applied to the quantitative measurement of melanosome transfer from melanocytes to keratinocytes induced by forskolin or nicotinamide.

Objective　To explore the therapeutic effect of treatment of uterine leiomyoma with different dosages of mifepristone and the effects of mifepristone on sex hormones.Methods　The patients of uterine leiomyoma were divided into two groups,group A included 45 patients who were mifepristone orally given neoplasma,25mg per day for 3～6 months,group B included 30 patients who took mifepristone 12.5mg per day for 3～6 months.The size of uterus and uterine leicomyoma were detected with type B sonarography and serum sex hormones level were tested with radioimmunoassay before the treatment.Results　Amenorrhea was found during the treatment of two groups and after tree months' the symptoms were improved,the size of uterine leiomyoma in group A was meanly decreased by 52.56% and 61.69% respectively,after six months' treatment;The size of uterine leiomyoma in group B was averagely decreased by 48.61% after three months' treatment and by 52.12% after six months' treatment.There was no significant difference between A and B group after three months' treatment(P＞0.05),But there was significant difference between the two groups after six months' treatment and it dependent on the time of the treatment.Contents of serum estradiol and progestagen of the two groups were droped dwon significantly after treatment(P＜0.01).Conclusions　Mifepristone applied to treat the uterine leiomyoma have definitve therapeutic effect,and less side effects.Therefore,mifepristone provides a new avenue to treat uterine leiomyoma and suggesting its best dosage is 25mg every day.

Objective To explore the effects of wound healing and histological changes by utilizing ultrapulse CO2 fractional laser for the treatment of superficial scar in dermal-epidermal junction (DEJ) in rabbit ears.Methods We adopted traditional iodophor treatment and moist exposed burn ointment treatment repairing the wound and utilizing ultra pulse CO2 fractional laser for the treatment of rabbit ears superficial scar.The superficial scars in rabbit ear were induced with ultra pulse CO2 fractional laser.In 1,2,4,8 weeks tissue samples would be taken.Using electron microscopy we observed DEJ ultrastructure and light microscopy to observe the histological changes of the dermal papilla and PAS staining for epidermis basement membrane.Results The wound healing time of moist exposed burn ointment group was shorter than that of iodophor group (P＜0.05).The number of hemidesmosomes and anchoring fibers in the moist exposed burn ointment group on the 8th week was more than the 1th week,The number of hemidesmosomes and anchoring fibers in the iodophor group on 8th week was more than 1th week (P ＜0.05).The number of hemidesmosomes and anchoring fibers in the iodophor group was less than the moist exposed burn ointment group (P＜0.05).Conclusions U1trapulse CO2 fractional laser can cause ultra structural changes in scarring dermal-epidermal junction area,wet wound healing environment promotes the reconstruction of DEJ district organization,and rebuilding the function of the scar skin has the positive significance.

0 05),But there was significant difference between the two groups after six months' treatment and it dependent on the time of the treatment.Contents of serum estradiol and progestagen of the two groups were droped dwon significantly after treatment(P

Objective To study the relationship of HPV pseudo-neutralizing titers detected by two different reporter genes: Zoanthus sp. green fluorescent protein (ZsGreen) and secreted alkaline phosphatase (SEAP) , and the relationship between HPV the pseudovirus-neutralizing antibody titer and the antibody titer determined by ELISA method. Methods The plasmids with expression cassettes of the HPV capsid protein L1 and L2 genes after codon optimization and the plasmid with reporter gene (ZsGreen or SEAP) were co-transfected into 293FT cells. The cell lysate supernatants were collected after 48 h culture, then the pseudovirus was purified through POROS column chromatography from the supernatants. After the titer of pseudovirus bulk were measured, HPV-16 and HPV-18 pseudovirus-neutralization assays were carried out for determining the titer of sera collected from immunized mice with HPV candidate vaccine and Gardasil HPV vaccine. Results In statistical analysis, the two reporter gene systems for the detection of the pseudovirus neutralizing antibody titer are highly relevant to each other (Spearman coefficient; r = 0. 760). And their neutralizing antibody titers bear a high degree of correlation with the antibody titer (Spearman coefficient: r= 0.577 and r =0. 741). Conclusion ZsGreen and SEAP pseudovirus neutralizing antibody titers are highly relevant to each other. The neutralizing antibody and the antibody titer are also relevant. These results reveal some mechanism of HPV vaccines to prevent the virus from invading the host cells, and are absolutely useful in the protection efficiency evaluation of the HPV-16 and HPV-18 candidate vaccines.

In order to reinforce the development and sharing of elaborate educational resources and improve the educational quality, the course team completed the transformation and upgrading of the original fine course for the construction of national high-quality resource sharing class. In the con-struction of courses the organic combination of course content, method, skills, quality was focused on, and the transformation and upgrading of the course achieved further optimization of the excellent resources and full sharing.

Objective To explore the efficacy of multiplex ligation-dependent probe amplification (MLPA)combined with fluorescence in situ hybridization(FISH)and comparative genomie hybridization (CGH)combined with FISH in genetic analysis of chorionic villi specimen(CVS)of spontaneous abortion.Methods CGH+FISH and MLPA+FISH were used for genetic analysis of 29 CVS from spontaneous abortion and 6 normal CVS from selective abortion,in the mean time,those results were compared with conventional eytogenetic karyotyping.Results The report time were 40 hours in MLPA+FISH and 120 houm in CGH+FISH.The mean time of chorionic villi culture was(240±72)hours.The successful rate of specimen analysis were 97%(34/35)in CGH,100%(35/35)in MLPA,100%(35/35)in FISH and 91%(32/35)in conventional cytogenetic karyotyping.Apart from 1 case failed in CGH analysis,the results from MLPA+FISH were almost similar to that from CGH+FISH,however,that 1 specimen failed in CGH were detected successfully by MLPA+FISH.The discrepancy rate were 13%(4/31)in CGH+FISH and 12%(4/32)in MLPA+FISH respectively when compared with conventional cytogenetic analysis.Conclusions MLPA+FISH analysis present shorter detecting time and achieve 100%tale of successful report.This combined method was an important adjuvant approach to conventional cytogenetic karyotyping in CVS from spontaneous abortion.

The herpes simplex virus type 1 (HSV-1) infected-cell protein 27 (ICP27) is an essential,highly conserved protein involved in various steps of HSV-1 gene regulation as well as in the shut-off of host gene expression during infection.It functions primarily at the post-transcriptional level in inhibiting precursor mRNA splicing and in promoting nuclear export of viral transcripts.Recently,many novel functions performed by the HSV-1 ICP27 protein were shown,including leptomycin B resistance,inhibition of the type I interferon signaling,regulation of the viral mRNA translation and determining the composition of HSV-1 virions.

Objective To investigate the role of autophagy in radiation-induced death process of human esophageal squamous carcinoma Eca-109 cells.Methods Esophageal carcinoma cell line Eca-109 was divided into 6 groups of control,5 mmol/L 3-Methyladenine treatment,10 mmol/L treatment,6 Gy irradiation,irradiation + 5 mmol/L drug,and irradiation + 10 mmol/L drug.Some cells were transferred with GFP-LC3 plasmid and the changes of autophagosome were obserred.After each treatment,the expression of autophagy marker LC3B was measured by Western Blot,cell viability was detected by MTT,morphological characteristics of apoptosis cells were stained with a fluorescein of Hoechst 33342 and the percentage of apoptotic cells and cell cycle distribution were measured by flow cytometry.Clonogenic survival were used to evaluate the cell radiosensitivity.Results Autophagy level was increased after radiation,and the LC3B Ⅱ expression and LC3B Ⅱ/LC3B Ⅰ ratio were significantly decreased by autophagy inhibitor 3-Methyladenine (F =25.64,P ＜ 0.05).The number of autophagosome fluorescent foci were significantly increased in the GFP-LC3 transfected cells after radiation,but reduced by 3-Methyladenine (F =127.36,P ＜ 0.05).Compared with radiation alone group,autophagy inhibition combined with radiation significantly decreased cell viability (F =129.54,P ＜ 0.05) and colony formation,increased apoptosis and the percentage of G2/M-phase cells.Conclusions 3-Methyladenine enhances the radiosensitivity of esophageal squamous carcinoma Eca-109 cells,suggesting that inhibition of autophagy could be used as an adjuvant treatment of radiotherapy in esophageal squamous carcinoma.

Objective: To explore the effect of high glucose on NADPH oxidase (NOX) expression and intracellular reactive oxygen species (ROS) generation in human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were divided into a control group, a mannite group, a glucose group and aglucose plus diphenylene iodonium (DPI) group. Intracellular ROS was detected by lfow cytometry. RNA and protein expression of NOX in HUVECs was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot, respectively. Results: 1) Compared with the control group, the intracellular ROS were signiifcantly increased in the glucose group (P<0.05,n=3), but there were no signiifcant change in the glucose plus DPI group (P>0.05,n=3); 2) Compared with the control group, the mRNA and protein expression of NOX4 in the glucose group were signiifcantly increased (P<0.05), but there were no signiifcant change in the mRNA and protein expression of NOX2, p22phox, p67phox and rac (allP>0.05); 3) there were no signiifcant difference in the mRNA and protein expression of NOX2, NOX4, p22phox, p67phox and rac between the glucose plus DPI group and the control group (allP>0.05). Conclusion: High glucose may increases intracellular ROS generation by increasing the expression of NOX4 in HUVECs, which might mediate the oxidative stress.