ObjectiveTo develop a new nested PCR to detect human papillomavirus (HPV) DNA in lesions of extragenital Bowen's disease.Methods DNA was extracted from the lesions of 41 patients with extragenital Bowen's disease and suhjected to the amplification of HPV by a nested PCR.Five primers,including CN1FR,CN2FR,CN3FR,CN4FR and CN5FR,were designed and used in the second round PCR.ResultsBased on the ClustalX analysis,69 HPV subtypes,including mucosal types,cutaneous types and epidermodysplasia verruciformis-associated types,could be amplified by using the 5 designed primers.The detection limit varied from 10-3 to 10-2 copies of HPV DNA for this PCR.Of the 41 lesional specimens,5 were positive for HPV DNA,including 3 cases of high-risk HPV types(2 cases of HPV 16,1 case of HPV 33) and 2 cases of cutaneous HPV types(1 case of HPV 27 and 1 case of HPV 76).ConclusionsThe improved nested PCR is highly sensitive and specific for the detection of HPV DNA in lesions of extragenital Bowen's disease.The development of extragenital Bowen's disease may be associated with the infection with high-risk mucosal HPV types in some patients.

Objective To assess the relationship between human papillomavirus (HPV) and extrageni-tal Bowen's disease. Methods Regular PCR with consensus primers for LI region as well as mix primers and nested PCR were performed to detect the DNA of a broad range of cutaneous and mucosal HPV types in tissue samples from lesions of 41 patients with extragenital Bowen's disease and from normal skin of 48 human controls. Semiquantitative PCR and tyramide-based in situ hybridization (ISH) were also conducted to determine the load and localization of HPV DNA in HPV-positive samples. Results HPV DNA was detected in lesions from 5 (12%) of the 41 patients with extragenital Bowen's disease. Of the 5 HPV-positive patients, 3 carried mucosal HPV types (HPV16 in 2 cases, HPV 33 in 1 case) with a viral load of 101 to 103 copies, 2 cutaneous HPV types (HPV27 in 1 case and HPV76 in 1 case). As ISH showed, there was a generalized expression of mucosal HPV DNA in most tumor cell nuclei but not in peritumoral normal tissue, and no expression of cutaneous HPV DNA was observed in lesions. HPV DNA was detected in 1 (2.1%) control tissue sample, which proved to be epidermodysplasia verruciformis-associated HPV23. There was no significant difference in the detection rate of HPV DNA between the patients and controls. The viral load of cutaneous HPV types amounted to 10-2 to 10-3 copies in the 2 patients, which was similar to that of HPV 23 in the normal control. Conclusion Mucosal HPV types may be closely associated with the development of extragenital Bowen's disease.

BACKGROUND:Bone marrow mesenchymal stem cellcontent is less under normal conditions, and easily confounded with other cells. Therefore, to establish a simple feasible in vitro cultured amplification method and to obtain a large number of stable bone marrow mesenchymal stem cells are of important theoretical significance and application value. OBJECTIVE:To establish a simple feasible in vitro culture and amplification method and to obtain numerous stable bone marrow mesenchymal stem cells. METHODS:Bone marrow mesenchymal stem cells were isolated from Sprague-Dawley rats and cultured in vitro using density gradient centrifugation and adherence separation screening. cellmorphological changes were observed under an inverted light microscope. Submicroscopic structure of cells was observed under a transmission electron microscope. Living cellnumber was counted using Trypan blue exclusion method. cellgrowth curve was drawn. cellcycle was analyzed using flow cytometry. The expression of c-kit and CD45 was measured using immunocytochemistry. CD45 expression was analyzed using flow cytometry. RESULTS AND CONCLUSION:(1) celladhesion and pseudopodia could be seen under an inverted light microscope at 24 hours after inoculation;cellcolony formed at 4 days;90%cellconfluence was found at 14 days. After passage, the cells were tended to homogenous and became fibroblast-like cells, which showed whirl-like growing or flamboyancy growing. (2) The size of passage 3 bone marrow mesenchymal stem cells was smal and nucleolus was large, with clear nucleoli under a transmission electron microscope, with the presence of sparse chromatin and low electron density. There was microvil us on the surface of cells, abundant ribosomes could be seen in the cytoplasm with few other cellular organs such as endoplasmic reticulum, mitochondria and Golgi complex. These showed that ultrastructural structure had undifferentiated features. (3) The number of passage 3 bone marrow mesenchymal stem cells was reduced at 1 day after inoculation. The cells began to grow at 2 days, and entered the period of exponential growth at 3 days, entered the flat period at 7 days and the number of cells began to decrease at 9 days. Growth curve exhibited“S”shape. (4) The percentage of S phase cells was 21.1%as detected by flow cytometry after passage 3 bone marrow mesenchymal stem cells were stained by DNA. (5) Immunocytochemistry and flow cytometry had proved that the c-kit positive rate of cells was 53.3%and CD45 positive rate was 1.68%. (6) After osteogenic induction of mesenchymal stem cells for 16 days, cells became oval, and short processes connected each other. The cytoplasm was dark, suggesting that it may be rich in rough endoplasmic reticulum and Golgi complex. They can secrete osteoid. Results verified that obtained bone marrow mesenchymal stem cells stably grew, and actively proliferated.

ObjectiveTo investigate the protective effect of sevoflurane postconditioning (Sevo-Postcon)on the hearts isolated from rats with diabetes mellitus (DM) of different duration against ischemia-reperfusion (I/R)injury.MethodsSeventy-two pathogen-free male SD rats weighing 200-240 g were randomly divided into 3 groups ( n =24 each):group Ⅰ rats without DM (C) ; group Ⅱ rats with 2 week DM (2w DM) and group Ⅲ rats with 6 week DM (6w DM).DM was produced by intraperitoneal (IP) 1％ streptozocin (STZ) 60 mg/kg and confirmed by fasting blood glucose concentration ＞ 16.7 mmol/L in groups Ⅱ and Ⅲ.Hearts were isolated from rats and perfused with Krebs-Henseleit buffer (KHB) in a Langendorff apparatus.After a 15 min stabilization period,the isolated hearts were subjected to 30 min of global no-flow ischemia followed by 75 min of reperfusion.Twelve hearts in each group were perfused after ischemia with KHB saturated with 3％ Sevo for 15 min followed by perfusion with regular KHB for 60 min.LVEDP,LVDP, ± dp/dt and HR were measured and recorded after 15 min stabilization (T0,baseline) and at 15 and 75 min of reperfusion (T1,2 ).Myocardial specimens were obtained at 15 min of reperfusion (T1) for detection of p-Akt expression (by Western blot analysis).Infarct size was determined at 75 min of reperfusion (T2).ResultsSevo-Postcon significantly improved cardiac function,reduced infarct size and up-regulated p-Akt expression in groups Ⅰ (C) and Ⅱ (2w DM),while in group Ⅲ (6w DM) Sevo-Postcon did not cause any change in cardiac function,infarct size and p-Akt expression as compared with the isolated hearts without Sevo-Postcon.ConclusionThe cardioprotective effect of Sevo-Postcon can be attenuated with increasing duration of DM by impairing PI3K/Akt signaling pathway.

Objective To study the roles of micheliolide on ovarian cancer cells. Methods Firstly, human ovarian cancer cell lines HeyA8, SKOV3 and A2780/DDP were treated with different concentration of micheliolide (0.25, 0.5, 1, 2.5, 5, 10, 20, 50 μmol/L) for 72 hours, then methyl thiazolyl tetrazolium (MTT) assay was used wo detect the growth of the human ovarian cancer cell lines and the stongest inhibited cell line were selected for the following test. Secondly, after HeyA8 cell line was treated with different concentration (5, 10, 20μmol/L) of micheliolide for 24 hours, the HeyA8 cell apoptosis was measured byflow cytometry. Thirdly, the expression of RelA mRNA in HeyA8 cell was detected through real-time PCR, the expressions of nuclear factor κB(NF-κB)signal pathway related protein RelA and the activited cysteinyl aspartate specific proteinase (caspase-9) were detected by western blot analysis. Results (1) The growth of HeyA8, SKOV3 and A2780/DDP cells were all significantly inhibited after being treated with different concentration of micheliolide for 72 hours and the roles of inhibition were all concentration dependant (P<0.05). The half maximal inhibitory concentration (IC50) of HeyA8, SKOV3 and A2780/DDP were (9.8±2.2), (12.0±2.1) and (12.8±1.8)μmol/L, respectively. We chose HeyA8 cell to do the following expreriments because of its best inhibited effect. (2) After HeyA8 cell was treated with micheliolide of different concentrations, as the concentration increased (20 and 0μmol/L, for example), the apoptosis rate of HeyA8 cell raised from (7.2±1.0)%to (17.4±1.1)%, the percentage of survived cells reduce from (92.8 ± 1.3)% to (82.6 ± 1.4)%,and the relative mRNA level of RelA decreased from 1.00 ± 0.13 to 0.18 ± 0.00 (P<0.01); furthermore, the expression of RelA protein was weaken and the activited caspase-9 protein expression was increased gradually. Conclusions Micheliolide plays a significantly inhibited role in HeyA8, SKOV3 and A2780/DDP cells. The inhibited role of micheliolide inovarian cancer cells might through inhibiting nuclear factor-kappa B (NF-кB) signaling pathway, and inducing the expression of activited caspase-9 protein to promoting apoptosis of HeyA8 cell.

OBJECTIVE:To provide reference for rational use of leucocyte increasing drugs in patients with lung cancer. METHODS:The application of drugs in lung cancer patients in 40 d each year were collected from 11 Zhejiang hospitals during 2009-2014,and then analyzed retrospectively in consumption sum,DDDs,DDC and department distribution,etc. RESULTS:The proportion of consumption sum of leucocyte increasing drugs in total consumption sum decreased generally in 11 Zhejiang hospital during 2009-2014,decreasing from 781 995.50 yuan in 2009(3.28%)to 626 792.80 yuan in 2014(1.53%). Top 3 departments in the list of consumption sum of leucocyte increasing drugs were oncology department(29.00%),radiotherapy department(27.08%) and respiratory medicine department(9.93%). Top 3 drugs in the list of consumption sum during 2009-2014 were recombinant hu-man granulocyte colony-stimulating factor,Coenzyme complex for injection and Leucogen tablets;top 3 drugs in the list of DDDs were Leucogen tablets,Berbamine tablet and recombinant human granulocyte colony-stimulating factor;Coenzyme complex for in-jection,recombinant human granulocyte colony-stimulating factor and human GM-CSF took the first three place in the list of DDC during 2009-2013,and Leucogen tablets were the top one in 2014. CONCLUSIONS:The application of leucocyte increasing drugs in lung cancer patients is decreasing year by year in 11 Zhejiang hospitals,and those with definite therapeutic efficacy and moder-ate price predominate clinical application predominate in the clinical practive.

Objective: To investigate the utilization status of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) in 11 hospitals of Zhejiang province from 2009 to 2015, and to analyze the use rationality.Methods: The doctor's advice in 40 days annually was collected in 11 hospitals of Zhejiang province from 2009 to 2015, and the drug consumption, frequency of utilization (DDDs), defined daily cost (DDC) and drug utilization index (DUI) were analyzed for the patients with lung cancer treated with EGFR-TKI.Results: Icotinib, erlotinib and gefitinib were the three prevalent EGFR-TKIs used in Zhejiang province, and icotinib started to be used in clinics in 2013.The overall cost of EGFR-TKIs increased year by year during 2009 and 2015, and the total amount of sales increased by 4.67 times in 2015 when compared with that in 2009.Generally, the DDDs value of erlotinib showed a decreasing trend, however, that of icotinib and gefitinib rose year by year during 2009 and 2015.Erlotinib had the highest DDC followed by gefitinib and icotinib.The mean value of DUI of the three targeted drugs was about 1.Conclusion: The utilization of EGFR-TKI is reasonable in 11 hospitals of Zhejiang province with increasing comsuption.

Humans ; Jaw, Edentulous ; therapy ; Maxilla ; Prostheses and Implants

OBJECTIVETo investigate the influences of recasting on the mechanical properties of Au-Pt ceramic alloy.

METHODSAu-Pt ceramic alloy samples were prepared and recast for 3 times without adding any new Au-Pt ceramic alloy. The tensile strength, 0.2% yield strength, percentage of elongation, flexural strength, flexural modulus and Vickers hardness of each specimen were measured.

RESULTSBeing cast for different times, the Au-Pt ceramic alloy showed no significant differences on their tensile strength, 0.2% yield strength, percentage of elongation, flexural strength or Vickers hardness. The flexural modulus of the Au-Pt alloys being cast for 2 or 3 times was significantly higher than that of the alloys being cast for 1 time (P < 0.05). CONCLUSION Au-Pt ceramic alloy can be recast for 3 times at least, without any decrease in the mechanical properties.

Objective To observe c-Fos expression in visual cortex of infant rhesus monkeys with myopia induced by hyperopic defocus and preliminarily investigate the possibility of visual cortex participating in myopia. Methods Eight SPF grade healthy infant rhesus monkeys aged 20 to 30 days were randomly divided into hyperopic defocused group and control group,4 monkeys for each group. The monkeys in hyperopic defocused group wore -3 D spectacle lenses. The monkeys in control group wore 0 D lenses. The monkeys' refractive error,corneal topography, vitreous chamber depth were measured at the start of lens wear and at 2,4,6,8,12 weeks post-treatment. At 12 weeks post-treatment,the visual cortex tissues were removed for c-Fos protein measurement by immunohistochemistry and Western blot assays. The results were analyzed semiquantitatively to compare the differences of c-Fos expression between hyperopic defocused group and control group. The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. This study protocol was approved by Ethic Committee of Zhongshan Ophthalmic Center ( No. 2013-014). Results After 12 weeks'lens wear,the vitreous chamber elongation amplitude of hyperopic defocused group monkeys was more obvious than that of the control group ([0.93±0.24]mm vs. [0.72±0.09]mm;t=2.292,P=0.047). The decrease of hyperopic degrees of hyperopic defocused group monkeys was more obvious than that of the control group ([-3.23± 1.36]D vs. [-1.55±0.52]D;t=-3.273,P=0.006). The eyes of hyperopic defocused group monkeys appeared a remarkable myopic shift after treatment. The number of c-Fos immunoreactive neurons was less in the hyperopic defocused group than that in the control group,with a statistically significant difference between them ([1 843±191]/mm2vs. [2 296±503]/mm2;t=2.381,P=0.041). Western blot assay showed that the optical density of c-Fos protein in the hyperopic defocused group was significantly less than that in the control group (0.50±0.17 vs. 0.99± 0.22;t=-4.982,P<0.01). Conclusions Hyperopic defocus,as an abnormal visual stimulus,can induce the onset of myopia in infant rhesus monkeys and inhibit c-Fos expression in visual cortex. Visual cortex may participate in myopia induced by hyperopic defocus.