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Tumor necrosis factor-α (TNF-α) inhibitors have been widely used and proven to be effective in the treatment of various inflammatory disorders in recent years, but there is a noteworthy and paradoxical adverse drug reaction that cannot be ignored, that is, TNF-α inhibitor-induced psoriasis. Its pathological manifestations could be psoriasiform or spongiotic changes, and its pathogenesis may be related to the imbalance between TNF-α and type Ⅰinterferon, the involvement of interleukin-23/T helper 17 axis, or infection. This review elaborates the epidemiological, histopathological features and possible pathogenesis of TNF-α inhibitor-induced psoriasis, and provides a basis for recognition and treatment of TNF-α inhibitor-induced psoriasis.
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Objective To evaluate the effect of mefformin on the human keratinocyte line HaCaT,and to explore its molecular mechanism.Methods HaCaT cells were divided into several groups to be treated with mefformin at different concentrations of 1,2,5,10,20,50 mmol/L for 24,48 and 72 hours.Cell counting kit-8 (CCK-8) assay was performed to evaluate the effect of metformin on the survival rate of HaCaT cells.After 48-hour treatment with metformin at concentrations of 0 (control group),0.5,1,2,5,10 mmol/L,flow cytometry was conducted to evaluate the effect of metformin on cell cycle and apoptosis.Western blot analysis was performed to determine the expression of cell proliferation-and differentiation-related proteins (keratin-16 [K16],K17,K1,involucrin),apoptosis-related proteins (Bax,Bcl-2) and AKT/mTOR/STAT3 pathway proteins.Enzyme-linked immunosorbent assay (ELISA) was conducted to detect the levels of interleukin (IL)-8,tumor necrosis factor (TNF)-α and IL-23 (inflammatory factors) in the culture supernatant of HaCaT cells.Statistical analysis was carried out with SPSS19.0 software using one-way analysis of variance for comparison of the above indices among the 0.5-,1-,2-,5-,10-mmol/L metformin groups and control group,repeated measures analysis of variance for comparisons among different time points or different metformin groups,least significant difference (LSD)-t test for multiple comparisons.Results CCK-8 assay showed that mefformin had inhibitory effects on the proliferation of HaCaT cells (F =116.87,P ＜ 0.05),and the cell survival rates gradually decreased along with the increase in the concentrations of mefformin.After 48-hour treatment with mefformin at concentrations of 0,0.5,1,2,5,10mmol/L,the proportion of HaCaT cells in G2/M phase gradually increased (5.55％ ± 1.03％,6.37％ ±0.93％,8.57％ ± 1.18％,10.05％ ± 0.60％,10.76％ ± 0.87％,13.63％ ± 1.41％,respectively,F =24.98,P ＜0.05),and the early apoptosis rate also gradually increased (0.78％ ± 0.71％,19.18％ ± 1.41％,25.67％ ±1.34％,28.45％ ± 0.92％,34.97％ ± 2.12％,40.41％ ± 1.49％,respectively,F =296.08,P ＜ 0.05).Along with the increase in the concentrations of metformin,there were increasing trends in the expression of K1 and the pro-apoptotic protein Bax (F =8.86,5.38 respectively,both P ＜ 0.05),while there were decreasing trends in the expression of K16,K17 and the apoptotic protein Bcl-2 (F =8.02,4.82,12.10 respectively,all P ＜ 0.05).There was no significant difference in the expression of involucrin among different metformin groups (F =0.57,P ＞ 0.05).After 48-hour treatment with mefformin at different concentrations,the levels of IL-8 and TNF-α in HaCaT cells significantly decreased (F =33.89,14.99 respectively,both P ＜0.05),while there was no significant change in the IL-23 level (F =2.12,P ＞ 0.05).Along with the increase in the concentrations of metformin,the expression of p-AKT,p-mTOR and p-STAT3 significantly decreased (F =11.38,0.35,4.38 respectively,all P ＜ 0.05),but there were no significant changes in the protein expression of AKT,mTOR and STAT3 (F =0.66,0.35,4.24 respectively,all P ＞ 0.05).Conclusion Metformin can inhibit the proliferation,promote the differentiation and apoptosis of HaCaT cells,and inhibit the secretion of inflammatory factors by regulating the AKT/mTOR/STAT3 signaling pathway.
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Primary cilium has attracted increasing attention in the biomedical field in recent years.It exists in the surface of various cells and is micro-sized,but its complex structures have not been clear.The primary cilium has an important role in sensory perception.Cells can receive extracellular mechanical and chemical signals through primary cilia,and primary cilia can assist in transferring signals into cells,followed by cellular responses.Recent studies have shown that the primary cilium also plays an important role in embryonic development and malignant transformation of cells.The investigation into primary cilia will facilitate the understanding of malignant transformation of cells and development of tumors.According to the related literature in recent years,this review summarizes the relationship between the primary cilium and common skin tumors,as well as potential targets for these tumors,so as to provide references and new sights for further researches.
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Objective To establish a rapid and accurate method for quantitative determination of erlotinib hydrochloride by proton nuclear magnetic resonance(1H-NMR). Methods Maleic acid was used as an internal standard,and DMSO-d6 was employed as solvent.The pulse width was 9.54 μs,the delay time was 20 s,the scanning frequency was 64 and the testing temperature was set as 300℃.Under this condition,the 1H-NMR data of the mixture of erlotinib hydrochloride and maleic acid were obtained.The content of erlotinib hydrochloride was calculated by comparing the response signal area of sample (As) and internal standard (Ar). Results Proton signal peaked at δ 8.84 and δ 8.47 for erlotinib hydrochloride and at δ6.26 for maleic acid served as quantitation peaks.The content of erlotinib hydrochloride was obtained by calculating the average of the test results of the two quantitative peaks.The concentration of erlotinib hydrochloride (2.88~19.08 mg·mL-1) and the peak area of quantification peaks showed a good linear correlation.The precision RSD was 0.36% (n=6),the repeatability RSD was 0.83%(n=6) and the sample recovery was 100.91% (n=6).The content of 3 batches of erlotinib hydrochloride was 92.26%,91.94%and 92.09%,the average was 92.10% and its RSD was 0.17%.The obtained results were generally consistent with those obtained from mass balance method. Conclusion This established method is simple to handle as compared to traditional methods,and the analysis results are accurate.1H-NMR provides a novel method for the determination of erlotinib hydrochloride and can be used for the quality control of erlotinib hydrochloride.
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ObjectiveTo observe the efficacy of electroacupuncture in treating post-operative intractable hiccup.Method Sixty-seven patients with post-operative intractable hiccup were divided into a treatment group of 34 cases and a control group of 33 cases. The treatment group was intervened by electroacupunctureplus diaphragmatic training, while the control group was by Metoclopramide and Baclofen tablets. The effective rate, recovery time, recovery rate of the first treatment week, and relapse rate within 2 weeks were statistically analyzed.ResultThe total effective rate was 88.2% in the treatment group versus 63.6% in the control group; the mean recovery time was (5.75±3.14)d in the treatment group versus (6.11±3.40)d in the control group. The recovery rate of the first treatment week was 41.2% in the treatment group versus 18.2% in the control group, and the difference was statistically significant (P<0.05). The relapse rate within 2 weeks was 20.0% in the treatment group versus 76.2% in the control group, and the difference was statistically significant (P<0.01).ConclusionElectroacupuncture plus diaphragmatic training is an effective approach in treating post-operative intractable hiccup.
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Objective To evaluate the effect of Rab23 on the proliferation of a squamous cell carcinoma cell line Sa3,and to investigate its mechanisms.Methods Cultured Sa3 cells were classified into four groups:normal control group transfected with green fluorescent protein (GFP),Rab23-overexpressing group transfected with a GFP-labelled Rab23-overexpressing plasmid,Rab23-silencing group transfected with a plasmid carrying a Rab23-targeting shRNA,empty vector group transfected with an empty vector.After additional culture for different durations,plate colony formation assay and flow cytometry were performed to evaluate the proliferative activity of Sa3 cells,and Western blot was conducted to detect the expression of Erl/phosphorylated-Erk in Sa3 cells.Statistical analysis was carried out by t test,one-way analysis of variance and Bonferroni's multiple comparison test.Results Stable Sa3 cell lines with overexpression or silencing of Rab23 were established by plasmid construction and lentivirus-mediated transfection.The plate colony formation assay showed that the colony formation rate was significantly lower in the Rab23-overexpressing group than in the normal control group (2.3％ ± 0.2％ vs.3.6％ ± 0.3％,P ＜ 0.05),but higher in the Rab23-silencing group than in the empty vector group (4.1％ ± 0.2％ vs.1.8％ ± 0.03％,P ＜ 0.01).Rab23 overexpression induced G1 phase arrest in Sa3 cells.The proliferation index was significantly decreased in the Rab23-overexpressing group compared with the normal control group (0.581 ± 0.035 vs.0.698 ± 0.018,P ＜ 0.05),but increased in the Rab23-silencing group compared with the empty vector group (0.567 ± 0.015 vs.0.444 ± 0.014,P ＜ 0.01).As Western blot showed,there were no significant changes in the expression of Erk in the Rab23-silencing or-overexpressing group compared with the normal control group,whereas the expression of p-Erk was attenuated in the Rab23-overexpressing group compared with the normal control group,but enhanced in the Rab23-silencing group compared with the empty vector group.Conclusions Rab23 could inhibit the proliferation of Sa3 cells,which may be associated with the Erk pathway.
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Objective To evaluate the effect of leptin on K17 expression in HaCaT human keratinocytes.Methods Some cultured HaCaT cells were treated with leptin (100 ng/ml) or remained untreated for 24 hours followed by the quantification of K17 mRNA expression by real-time PCR and detection of K17 protein expression by Western blot and immunofluorescence staining.To investigate the action mechanism of leptin,some cultured HaCaT cells were divided into several groups to be treated with leptin (100 ng/ml) alone,Piceatannol (an inhibitor of the STAT3 pathway) + leptin (100 ng/ml),PD-98059 (an inhibitor of the Erk1/2 pathway) + leptin (100 ng/ml),respectively for 24 hours,with the cells receiving no treatment as the negative control.Subsequently,the mRNA and protein expressions of K17 were measured by the above methods.Statistical analysis was done by the two-sample ttest.Results The mRNA expression of K17 was significantly higher in HaCaT cells treated with leptin alone than in those remaining untreated (3.086 7 ± 0.186 1 vs.1.000 0 ± 0.000 0,P ＜ 0.01),but significantly downregulated in HaCaT cells treated with Piceatannol + leptin and those with PD-98059 + leptin compared with those treated leptin alone (0.674 1 ± 0.060 0 and 0.855 0 ± 0.390 3 vs.2.242 7 ± 0.188 7,both P ＜ 0.01).The results of Western blot and immunofluorescence staining were in agreement with those of real-time PCR.Conclusions Leptin can induce K17 expression in HaCaT cells,likely by activating the STAT3 and Erk1/2 signaling pathways.
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Objective To estimate the biological effects of leptin on human HaCaT keratinocytes and explore their molecular mechanisms.Methods Cell counting kit-8 (CCK-8) was used to evaluate the proliferation of cultured HaCaT cells treated with different concentrations of leptin for 24 and 48 hours.Some HaCaT cells were classified into four groups to remain untreated,be treated with leptin (100 μg/L) and piceatannol (a specific inhibitor of STAT3 phosphorylation) alone or in combination for 24 hours,respectively,followed by the evaluation of cell proliferation using CCK-8 kit.Flow cytometry was performed to assess cell cycle of HaCaT cells treated with leptin of 100 μg/L,Western blot to determine the phosphorylation level of Erk1/2 and STAT3 in HaCaT cells treated with leptin of 100 μg/L for different durations.Statistical analysis was done by Student's t-test for unpaired data using GraphPad Prism 5 software.Results The proliferation of HaCaT cells was accelerated to different degrees after treatment with leptin of 50 and 100 μg/L for 24 and 48 hours,and the accelerating effect was in a dose-dependent manner within 24 hours (r =0.9989,P ＜ 0.05).Piceatannol apparently inhibited the promotive effect of leptin on the proliferation of HaCaT cells.There was an obvious elevation in the percentage of cells at S phase ((57.70 ± 5.88)％ vs.(42.50 ± 7.55)％,P ＞ 0.05),but a significant decrease in that at G0/G1 phase ((39.70 ± 1.57)％ vs.(45.20 ± 1.44)％,P ＜ 0.05),with a significant increase in proliferation index (0.603 ±0.0157 vs.0.564 ± 0.0144,P ＜ 0.05) in HaCaT cells treated with leptin of 100 μg/L for 24 hours compared with the untreated controls.Western blot showed that leptin of 100 μg/L markedly enhanced the phosphorylation level of STAT3 in HaCaT cells.Conclusion Leptin may upregulate the proliferation of HaCaT cells through activation of STAT3 pathway.
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ObjectiveTo investigate the effects of imatinib mesylate as a tyrosine kinase inhibitor on the biological activity of and Wnt/β-catenin pathway in Hs294T melanoma cells.MethodsAfter Hs294T cells were incubated with imatinib mesylate at various concentrations(4,8,10,16,20 and 24 μmol/L) for 24 hours or imatinib mesylate at 10 μmol/L for 24,48 and 72 hours,methyl thiazolyl tetrazolium (MTT) assay was performed to estimate the proliferation of cells and to determine the effects of imatinib mesylate on the proliferation of Hs294T cells.Then,Hs294T cells were treated with imatinib mesylate at 10 μmol/L or dimethyl sulfoxide (DMSO) for different durations,followed by the detection of cell apoptosis with flow cytometry,localization of β-catenin with annexin V/propidium iodide-double staining and laser confocal microscopy,quantification of β-catenin and cyclin D1 protein with Western blot,and measurement of LEF1 and C-myc mRNA expression with real time fluorescence-based quantitative PCR.Matrigel invasion assay was performed to evaluate the invasiveness of Hs294T cells after treatment with imatinib mesylate at 5 μmol/L or DMSO for 24 hours.ResultsImatinib mesylate at 4-10 μmol/L elicited a dose-dependent decline in the proliferation of Hs294T cells (F =125.3,P ＜ 0.05),and imatinib mesylate at 10 μmol/L induced a time-dependent decrease from 24 to 72 hours(F =714.6,P ＜ 0.01 ).The percentage of early and late apoptotic cells was markedly increased,while the invasiveness was decreased by about 48％(P ＜ 0.01 ),together with a downregulation in the expression of LEF1,C-myc and Cyclin D1 in imatinib mesylate-treated Hs294T cells compared with the DMSO-treated cells.No obvious changes were observed in the protein expression of β-catenin,but a decline in the nuclear localization of β-catenin was noted in Hs294T cells after being treated with imatinib mesylate.ConclusionImatinib mesylate may suppress the proliferation and invasion of,but promote the apoptosis in,melanoma cells,by downregulating the Wnt/β-catenin pathway.
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Objective To retrospectively review the efficacy and side effects of intense pulsed light (IPL) and red light emitting diode (LED) in the treatment of steroid-dependent dermatitis.Methods Seventy patients with steroid-dependent dermatitis mainly manifesting as facial telangiectasis were treated with IPL for an average of 3.49 sessions with a 4-week interval.The energy density of IPL varied from 20 to 23 J/cm2,pulse width from 2.6 to 5.0 ms,and delay from 15 to 20 ms.Meantime,197 patients with steroid-dependent dermatitis,who mainly presented with facial skin sensitivity,were treated with red LED (633 ± 3 nm wave length) twice a week for an average of 4.23 sessions.The energy density of red LED was 128 J/cm2,and the irradiation lasted 20 minutes at each treatment.The efficacy and adverse reactions were assessed and recorded for each treatment.Results The total response rate was 88.57％ for IPL,and 83.76％ for red LED.There was a significant difference in the clinical efficacy between triple-pulse and double-pulse IPL (x2 =8.14,P ＜ 0.05).No severe adverse reaction was observed in any of the patients.Conclusion IPL and red LED are both effective in treating steroid-dependent dermatitis.
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To assess and grade facial nerve dysfunction according to the extent of facial paralysis in the clinical course of acupuncture treatment for Bell's palsy, and to observe the interrelationship between the grade, the efficacy and the period of treatment, as well as the effect on prognosis.
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Objective To evaluate the efficiency and safety of hair removal with Lightsheer 800 nm diode laser. Methods Lightsheer, 800 nm diode laser, was used to remove hairs from different areas, including hairlines, lips, whiskers, armpit, chest, back, limbs and bikinis area, in 432 patients. Results At least twice treatments, the maximum 29 times, were needed to achieve satisfactory results. Total effective rate increased with the treatment times but depended on the different area:63.4 % of hairlines, 67.7 % of lips, 71.9 % of whiskers, 100 % of armpit, 100 % of chest, 100 %of back, 82.6 % of limbs and 100 % of bikinis area, respectively. The adverse effect was observed with temporal hypopigmentation in 4 patients and hyperpigmentation in 9 patients, without any scar formation. Conclusions Lightsheer 800 nm diode laser is a safe and efficient method of laser hair removal.
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Objective To detect the proliferation of and production of interferon-γ by drug-specific peripheral T cells from patients with severe drug eruption.Methods Peripheral blood mononuclear cells (PBMCs) were isolated from 10 patients with severe drug eruption,10 patients with mild or moderate drug eruption and 10 normal human controls,stimulated with causative drugs to obtain drug-specific T cells.Then,both PBMCs and drug-specific T cells were stimulated with causative drugs or unrelated drugs followed by the detection of secretion levels of IFN-γ with ex vivo enzyme-linked immunodotting (ELISpot) assay and cultured ELlSpot assav respectively.Results After stimulation with causative drugs,a higher level of IFN-γ was secreted by PBMCs and drug-specific T cells from patients with severe drug eruption compared with those from normal human controls (both P<0.01).and by drug-specific T cells than by PBMCs (P<0.01).The culture with unrelated drugs could neither induce the generation of drug-specific T cells nor promote the secretion of IFN-γ by PBMCs from the patients.Drug-specific T cells still existed in the peripheral blood of 3 patients within 1 to 3 years after recovery of drug eruption.Conclusions There are drug-specific T cells in peripheral blood of patients with severe drug eruption,and they may persist for a certain period of time after recovery of drug eruption.Ex vivo ELISpot combined with cultured ELISpot may be applied to the identification of causative drugs in vivo.
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Objective To study to the effect of 1064-nm Q-switched Nd:YAG laser irradiation on the melanogenesis in a human epidermal melanocyte line PIG. Methods Cultured PIG cells were irradiated with 1064-nm Q-switched Nd:YAG laser (Medlite C6) at different energy densities for 10 times. After additional culture for various durations, cell viability was detected by MT assay, tyrosinase activity by dopa oxidation assay, mRNA and protein expressions of tyrosinase, tyrosinase related protein (TRP)-1 and TRP-2 by real-time quantitative fluorescent RT-PCR and Westen blotting respectively, Results The irradiation with Q-switched Nd:YAG laser at energy densities from 1 to 3 J/cm2 had no obvious effect on the viability of PIG cells. After irradiation with Nd:YAG laser at 1 J/cm2, PIG cells showed a significant increase in the tyrosinase activity,mRNA expressions of tyrosinase and TRP-1 compared with unirradiated cells (0.563 ± 0.014 vs 0.501 ±0.019, 1.40±0.11 vs 1.0, 1.28 ± 0.03 vs 1.0, all P＜ 0.05), but both the mRNA (0.91 ± 0.17 vs 1.0, P＞0.05) and protein expressions of TRP-2 experienced no significant changes before and after the irradiation.However, a significant decrease was noted in PIG cells irradiated with Nd:YAG laser at 3 J/cm2 in tyrosinase activity, mRNA and protein expressions of tyrosinase (0.70 ± 0.02 vs 1.0, 0.64 ± 0.05 vs 1.0, both P ＜ 0.05),TRP-1 (0.73±0.04 vs l.0, 0.86±0.17 vs l.0, both P＜0.05) andTRP-2 (0.68±0.04 vs l.0,0.69±0.11vs 1.0, both P ＜0.05) in comparison with unirradiated PIG cells. Conclusions The 1064-nm Q-switched Nd:YAG laser irradiation may affect the melanogenesis in PIG cells. With no influence on cell viability, the 1064-nm Q-switched Nd:YAG laser at 1 J/cm2 could enhance melanogenesis, while that at 3 J/cm2 could suppress melanogenesis, in PIG cells.
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Objective: To observe the clinical effect of puncturing Back-Shu points in treating chronic fatigue syndrome. Methods: Twenty-two subjects were recruited and treated by puncturing corresponding Back-Shu points based on syndrome differentiation. The short-form General Health Survey (MOSSF GHS) and the Chalder Questionnaires for Fatigue were adopted for evaluating the therapeutic effects. Results: Of the 22 patients, 4 cases showed a marked effect, 11 got effect, 7 got failure, and the total effective rate was 68.2%. Conclusion: Puncturing Back-Shu points is effective in relieving the symptoms of chronic fatigue syndrome and enhancing the patients' health standard.
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Objective To investigate the expression of Ptch-1 and Gli-1, hedgehog pathway-related genes in squamous cell carcinoma (SCC), and the effect of cyclopamine, a specific inhibitor of hedgehog signaling pathway, on the proliferation of a SCC cell line Tca. Methods Skin samples were resected from 42 patients with SCC and 10 normal human controls. Immunohistochemistry and in situ hybridization were employed to study the expression and distribution of Ptch-1 and Gli-1 in these specimens. Tca cells were incubated with cyclopamine (1, 2, 5, 10 μmol/mL) for 48 hours, or cyclopamine (5 μmol/mL) for 1-8 days. The same concentrations of lycopersicin served as the control treatment. Then, MTT assay was performed to detect the proliferation of Tca cells. A fraction of Tca cells were cultured in the presence of 5 μmol/mL cyclopamine for 72 hours followed by BrdU assay for the evaluation of cell growth and proliferation. Results A significant increment was shown in the expression of both Patch-1 and Gli-1 by immunohistochemistry (χ2= 5.656, 6.732, P<0.05, 0.01, respectively) and in situ hybridization (χ2=6.787, 9.600, respectively, both P<0.01) in SCC tissue compared with the control specimens. And both of them were predominantly distributed in the cytoplasm of SCC cells. As MTT assay revealed, cyclopamine notably inhibited the proliferation of Tea cells, and the effect increased with the concentration and action time of cyclopamine. Further more, the percentage of BrdU-positive cells was 26% in cyclopamine-treated Tca cells, significantly higher than that in the blank control cells (77%) and lycopersicin-treated cellls (72%). Conclusions Hedgehog signaling pathway is activated in the lesions of SCC, and inhibition of the pathway may facilitate the treatment of SCC.
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Objective To evaluate the feasibility and safety of vaginal enlarged amputation of cervix to treat patients with cervical cancer of stage Ⅰ a1 and cervical intraepithelial neoplasia grade Ⅲ(CIN Ⅲ)who were unfit for conization surgery.Methods From July 2002 to May 2007,patients with cervical cancer at stage Ⅰ a1,diagnosed by pathology after loop electrosurgical excision procedure(LEEP),large area CIN Ⅲ(the area of lesion≥3/4 on colposcopy),CIN Ⅲ coexisted with vaginal intraepithelial neoplasia (VAIN)in the superior segment of vagina,CIN Ⅱ-Ⅲ recurrence or with residual lesion,positive margin after conization of cervix,who wanted to preserve fertility and(or)corpus uteri were selected to receive vaginal enlarged amputation of cervix.Results Forty-eight eases including 5 with cervical cancer in stage Ⅰ a1,38 with large area CIN Ⅲ(9 with gland involvement),2 with residual lesion and 2 with positive margin after LEEP,1 recurrence after cold knife conization,received the procedure successfully.The median age was 34 years(range 27-40),median operation time was 60 minutes(range 30-100),median blood loss was 40 ml(range 5-300),and median hospital stay was 10 days(range 7-17).After follow-up 1-39 months,no patient had postoperative complications and recurrence,and all patients resumed normal menstrual cycle and sexual life.Condusion Vaginal enlarged amputation of cervix appears to be a safe and feasible procedure for patients with cervical cancer at stage Ⅰ a1 and CIN Ⅲ who are unfit for conization surgery.
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Objective To investigate the in vitro effects of KAAD-cyclopamine, a specific inhibitor of hedgehog signaling pathway, on the growth and apoptosis of human squamous cell carcinoma cell line A431. Methods A431 cells were cultured and treated with KAAD-cyclopamine(0.5, 1, 2, 5 μmol/L).Then, MTT assay was used to detect the proliferation of A431 cells, and light microscopy to observe cell morphology at different time points with a 24-hour interval. Flow cytometry was used to assess cell cycle,and annexin-V/propidium iodide double staining to evaluate the apoptosis in these cells after 48 hours of treatment with KAAD-cyclopamine. Results KAAD-cyclopamine of 0.5, 1, 2 and 5 μmol/L inhibited the proliferation of A431 cells by (7.0±2.3)%, (20.6±2.8)%, (48.3±3.4)% and (61.6±3.3)%, respectively (F = 49.92, P＜0.01 ). Furthermore, in the presence of KAAD-cyclopamine of 5 μmol/L, on day 1, 2, 3, 4,and 5 the proliferation of A431 cells was suppressed by (18.5±2.6)%, (56.1±3.7)%, (65.4±2.8)%,(71.2±1.9)% and (75.9±3.0)%, respectively, the difference was significant among these time points(F =16.32, P＜0.01 ). Statistical analysis showed that KAAD-cyclopamine downregulated the growth of A431 cells in a dose-and time-dependent manner (r = 0.91, 0.86, P＜0.01 and 0.05, respectively). Light microscopy revealed typical morphological changes of cell damage in A431 cells. KAAD-cyclopamine in creased the percentage of cells in G1 phase from (51.8±2.9)% to(76.2±1.8)% (F = 26.34, P＜0.01 ), the proportion of hypoploid cells from (1.7±0.3 )% to (8.7±0.2)% (F = 6.32, P＜0.05 ), which suggested that KAAD-cyclopamine could arrest A431 cells in G1 phase of the cell cycle. The apoptosis ratio in KAAD-cyclopamine-treated cells was significantly higher than that in the untreated control [ (46.2±2.8)% vs (18.5±3.1 )%, F = 32.01, P＜0.01 ]. Tomatidine treatment did not affect the proliferation or apoptosis of A431 cells (both P＞0.05).Conclusion KAAD-cyclopamine can markedly suppress the proliferation and induce apoptosis of A431 cells.
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Objective To construct an anti-keratin autoantibody (AK auto Ab) transgenic mouse model. Methods Linearized transgene plasmid was microinjected into the zygotes of CBA?C57BL/6 mice, which were transplanted into the oviducts of pseudo-pregnant mice. PCR was used to identify the genotype of the offsprings, and ELISA was applied to measure the serum levels of AK auto Ab. Results Twelve transgene positive founder mice were obtained, and 9 of them produced offsprings as the third generation. The serum level of AK auto Ab was increased in 3 of the transgenic mice. Conclusions AK auto Ab transgenic mice were successfully established; these mice could serve as an animal model with increased serum levels of AK auto Ab.
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Objectives To investigate the binding of natural IgM to Staphylococcus aureus and its role in the phagocytosis of S. aureus by phagocytes, and to pave way for further study on the role and mechanism of natural IgM in defense of bacteria. Methods The binding of natural antikeratin IgM 3B4 to S. aureus was analyzed by ELISA and indirect immunoiluorescence. The role of 3B4 in the phagocytosis was analyzed by colony forming assay and flow cytometry (FCM). Results Both ELISA and indirect immunofluorescence proved the binding of natural IgM 3B4 to S. aureus. Colony forming assay found that the amount of colony forming units decreased significantly when 3B4 was added. The analysis of FCM showed that 3B4 augmented phagocytosis of 5. aureus by phagocytes. Conclusions Natural antikeratin IgM 3B4 can bind to S. aureus and regulate the phagocytosis of it, indicating that natural IgM may play some role in the defense against bacterial infection.