Objective To explore if keratinocytes that stably maintain HPV11 genome can be obtained by transfection and selection methods. Methods Escherichia coil containing pBR322.HPV11 plasmid was cultured and amplified. Then the plasmid was extracted, purified and digested with BamH Ⅰ enzyme to release viral genome from the bacterial vector. After recovering from the low-melting point agarose gel by electrophoresis, the genome was self-circulated with T4 DNA ligase. The religated DNA was cotransfected with pTK-neo DNA into HaCaT keratinocytes using Lipofectamine reagent. After selection with G418 for 2 to 3 weeks, clonal and pooled cultures were expanded and analyzed. Fluorescent quantitative PCR (FQ-PCR) and nested reverse transcriptase PCR (nRT-PCR) were applied to detect HPV11 DNA and spliced HPV11 E1^E4 mRNA expression in the transfected cells. Results After the cotransfection of HPV11 genome into HaCaT keratinocytes and two-week selection,G418-resistant cell colonies were obtained with morphological features indistinguishable from normal HaCaT keratinocytes. As shown by FQ-PCR, HPV11 DNA was present in G418-selected HaCaT keratinocytes. The average viral DNA load capacity was 15.9±16.8 copies/cell in the primary culture of G418-selected HaCaT cells and 23.9±1.1 copies/cell in the third passage of the cells; there was no statistical difference between the two passages of cells (t=-0.822, P>0.05). nRT-PCR targeting HPV11 E1^E4 mRNA transcript produced a specific 628-bp fragment, which was shown by agarose gel electrophoresis. Conclusions Our data indicate that HPV11 genome can be successfully introduced into HaCaT keratinocytes by transfection and HPV11 DNA-positive cells can be obtained by G418 selection. Moreover, HPV11 DNA is still present in the third passage of transfected cells.

Isocitrate dehydrogenase 1 (IDH1), a key rate-limiting enzyme in tricarboxylic acid cycle,is found within the cytoplasm and peroxisomes.In recent years, IDH1 mutation is discovered in most gliomaand its large scale metabolites, 2-hydroxyglutarate, may be involved in tumor as potential carcinogens, and isshown to be closely related to better patients' survival as well.IDH1 mutation is anticipated to be a novel genet-ic biomarker which will guide the treatments of clinical glioma.

Objective To investigate the effects of the compound T0 extracted from Tripterygium wilfordii, a traditional Chinese herb, in comparison with those of cyclosporin A(CyA), on down－ regulating the expression and activities of IL－ 1 and IL－ 2. Methods The cellular reactive systems of peritoneal macrophage (rat)－ thymocytes (rat), and spleen cells (rat)－ CTLL－ 2 (mouse) were set up. The technique of 3H－ TdR incorporation was applied. Results The production of IL－ 1 and IL－ 2 were significantly inhibited by T0 and CyA. The activities of IL－ 1 and IL－ 2 were down－ regulated by T0,rather than by CyA. Conclusion T0 and CyA are potent inhibitors of the production of IL－ 1 and IL－ 2. Effects of T0 on the activities of IL－ 1 and IL－ 2 are different from those of CyA.

Objectives To establish an experimental model for Th1 typ e shifting and meet the requirements of studying on the mechanisms of some immun omodulators. Methods The levels of cytokines, IL-12, IFN-ice were detected by using ELISA. Sple en cells of the BALB/c mice were incubated under the following conditions: with different concentrations of T cell mitogen ConA (1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL), mononuclear phagocyte system activator LPS (50 mg/mL, 5 mg/mL, 0. 5 mg/mL) or LPS (50 mg/mL, 5 mg/mL, 0.5 mg/mL) combined with 0.25 mg/mL ConA. Re sults LPS could induce the production of IL-12 from spleen cells. The lowest concentration that ConA could induce the measurable production of IFN-rom sp leen cells was 0.25 mg/mL. When different concentrations of LPS were combined wi th 0.25 mg/mL ConA, LPS could accelerate the production of IFN- and positively with that of IL-12. Conclusion LPS combined with ConA can induce the activation of spleen cells from mice towards Th1 type response.

Objective To determine whether leflunomide could control the proinflammatory cytokine expression induced by anthralin via inhibiting the activation of nuclear factor ?B (NF-?B). Methods The expression of NF-?B inhibitory protein ? (I?B?), was analyzed by using Western blot method. MTT assay and RT-PCR were used to assess the proliferating activity and mRNA expression of intercellular adhesion molecule-1 (ICAM-1) of HaCaT keratinocytes, respectively. Results Leflunomide inhibited the degradation of I?B? by anthralin, i.e. the activation of NF-?B signaling pathway, in a dose-dependent manner. The inhibition of keratinocyte growth by anthralin did not correlate with the activation of NF-?B. Under the experimental conditions used, leflunomide was shown to be able to significantly inhibit the over-expression of ICAM-1 on keratinocytes induced by anthralin; this inhibition occurred in a dose dependent manner. Conclusions Growth inhibition by topical anti-psoriatic medication anhtralin is unrelated to the NF-?B-dependent signaling pathway, and leflunomide can control ICAM-1 expression induced by anthralin via inhibiting the activation of NF-?B.

In mammals,there are two RNA-binding proteins,Musashi (Msi)1 and Msi2,constituting the Msi family.Msi2 is mainly distributed among neural,hematopoietic,gastrointestinal,pancreatic and epithelial stein cells.It is of great importance to maintain the balance between proliferation and differentiation of stem cells and regulate their growth and development.The changed expression of this protein will induce genesis and progression of malignant tumor through many kinds of signal pathways.Thus,Msi2 is trusted to provide a predictive mark and a therapeutic target for related tumors.

Objective To investigate the effects of some compounds,bestatin,tripdiolide,etc,on leukotriene A4hydrolase activity.Methods LTB4,product of leukotriene A4hydrolase,was determined by reverse phase high performance liquid chromatography(PR-HPLC).Bestatin,an inhibitor of LTA4hydrolase was used as a positive control.Results Tripdiolide could inhibit LTA4hydrolase activity in a dose-depen-dent pattern.The50%inhibitory concentration values(IC 50 )was2.58?10 -5 ?g/mL.The other compounds,an-thralin,cyclosporin A,tretinoin,calcipotriene,clobetasol propionate,methotrexate,and erythromycin,had no effects.Conclusions Tripdiolide possibly has anti-inflammatory effect through down-regulation of leukotriene A4hydrolase in psoriasis.

Objective To investigate the expression of chemokine receptors CXCR1and CXCR2in progressive plaque psoriasis and their clinical significance.Methods Reverse transcription-polymerase chain reaction(RT-PCR)technique was applied to semi-quantatatively analyze CXCR1and CXCR2mRNA expression in peripheral blood polymorphonuclears(PMNs)from30cases of psoriatic patients,14of the30patients which were treated until the70%of skin lesions resolved,and30healthy controls.The correlation between CXCR1/2and psoriatic area and severity index(PASI)was evaluated.Results CXCR1and CX-CR2mRNA levels in PMNs from psoriatic patients were1.30?1.18和1.62?0.97,respectively,which were markedly higher than those in normal controls(0.56?0.36,0.74?0.58,respectively)and treated patients(0.49?0.34,0.51?0.51,respectively.)(P

Objective To investigate the effects of keratinocyte growth factor (KGF) and KGF receptor (KGFR) antisense oligonucleotide (ASODN) on cell cycle and apoptosis of HaCat cells. Methods HaCaT cell, an immortalized keratinocyte cell strain, was cultured in vitro. Flow cytometry was used to measure the cell cycle and apoptosis mediated by KGF and ASODN. Results The rates of S phase and apoptosis in the group treated with KGF increased significantly than those in the control group (both P

According to the characteristics of pain and disease process， postherpetic neuralgia （PHN） can be classified into the category of "collaterals disease" in traditional Chinese medicine. The key pathogenesis of PHN includes toxin， stasis and deficiency. The main syndromes of collaterals disease are pathogenic toxin burning collaterals， static blood blocking collaterals， and collaterals depletion. Ashi points are mainly selected for treatment of PHN. For the syndrome of pathogenic toxin burning collaterals， dense fire needling is suitable for removing toxin and relieving pain. For the syndrome of static blood blocking collaterals， collateral bloodletting is suitable to remove stasis and relieve pain. For the syndrome of collaterals depletion， the combination of encircling fire needling， hair needling and transverse insertion with filiform needles， and Jiaji acupoint （EX-B2） needling is suitable for nourishing collaterals and relieving pain. In clinical practice， the most appropriate acupuncture regimen can be selected according to the differentiated syndrome of "collaterals disease"， and with adoption of the holistic pattern differentiation and treatment， the clinical efficacy can be strengthened.