Objective To estimate the effects ot rosiglitazone on cultured HaCaT human keratinocytes and their possible mechanism.Methods HaCaT cells were cultured and treated with different concentrations ( 10,20,40,80 μ mol/L) of rosiglitazone or solvent for 24,48,72 and 96 hours,respectively.Cell proliferation was detected with methyl thiazolyl tetrazolium (MTT) assay.Western blot was performed to measure the protein expression of β-catenin and cyclin D1.Results Compared with the solvent-treated cells,the proliferation of HaCaT cells was significantly inhibited by 18.9％,23.7％,35.1％ and 44.6％ (all P＜ 0.05) after treatment with rosiglitazone of 10,20,40 and 80 μmol/L,respectively,for 48 hours.The expressions of β-catenin and cyclin D 1 were significantly lower in rosiglitazone-treated HaCaT cells than in solvent-treated cells (all P ＜ 0.05).Conclusion Rosiglitazone could inhibit the proliferation of HaCaT cells,likely by downregulating the expressions of β-catenin and cyclin D 1.

BACKGROUND:Traditional total knee arthroplasty adopts medial parapatel ar approach, which induces severe trauma, requires long-term rehabilitation, and interferes the end point of quadriceps femoral tendon on superior patel ar pole. Total knee arthroplasty by subvastus approach has no impact on the knee-extension equipment, but it cannot provide sufficient exposure of surgical field and can induce damage to femoral muscle, so its application and safety need further exploration.
OBJECTIVE:To explore clinical effect of total knee arthroplasty by subvastus approach and medial parapatel ar approach.
METHODS:A total of 78 patients with 82 knees who were candidates for total knee arthroplasty were randomly divided into two groups. Treatment group (39 cases;41 knees) was given subvastus approach and control group (39 cases;41 knees) was given median parapatel ar approach. The knee function, range-of-motion of knee joint and complications after total knee arthroplasty were observed and compared.
RESULTS AND CONCLUSION:Compared with control group, the operation time in treatment group was significantly increased, while the postoperative wound drainage, straight leg raising time and walking time were obviously reduced (P<0.05). The scores of HSS before surgery and 12 weeks after surgery were not significantly different between the two groups (P>0.05), while at 1 and 6 weeks after surgery the scores in treatment group were significantly higher than those in control group (P<0.05). The range-of-motion of knee joint and MMT rank for quadriceps muscle were significantly improved in two groups after treatment (P<0.05), and those indicators in treatment group were more significant than the control group (P<0.05). The incidences of complications in treatment group and control group were 2.6%and 15.4%, respectively, with statistical y significant difference (P<0.05). The total knee arthroplasty by subvastus approach has less impact on the extensor mechanism, improves the recovery of knee function and range-of-motion of knee joint, and reduces the incidence of complications.

Objective To investigate the effects of recombinant human pigment epithelium derived factor (rhPEDF)on in vitro proliferation of and expressions of interleukin 6(IL-6), IL-8 and vascular endothelial growth factor (VEGF)in cultured human HaCaT keratinocytes. Methods Some cultured HaCaT cells were treated with rhPEDF at various concentrations(25, 50, 100 μg/L)for different durations, and some treated with RPMI 1640 medium only served as the control group. Cell counting kit-8(CCK8)assay was performed to evaluate cell proliferation after 24-, 48- and 72-hour treatment, reverse transcription (RT)-PCR to measure the mRNA expressions of IL-6, IL-8 and VEGF in HaCaT cells after 24-hour treatment, and Western blot to detect the protein expressions of IL-6, IL-8 and VEGF in HaCaT cells after 48-hour treatment. Statistical analysis was carried out by two- and one-way analysis of variance, Student-Newman-Keuls(SNK)-q test and Pearson correlation analysis. Results After treatment with rhPEDF of 25-100 μg/L for 24 - 72 hours, the proliferation of HaCaT cells was significantly inhibited to different extents compared with the control group(all P < 0.05), and the inhibition rate significantly increased with the increase in treatment duration and concentrations of rhPEDF(F = 1115, 329.9, respectively, both P < 0.001). Moreover, there was a significant decrease in the expressions of IL-6, IL-8 and VEGF mRNAs(at 24 hours)and proteins(at 48 hours)in HaCaT cells after treatment with rhPEDF of 25 - 100 μg/L compared with the control group(all P < 0.05). The expression levels of VEGF mRNA as well as IL-6 and IL-8 proteins all significantly decreased with the increase of rhPEDF concentrations (all P < 0.05). The mRNA expressions of IL-6 and IL-8 were significantly lower in the 100-μg/L rhPEDF group than in the 25-μg/L rhPEDF group (both P < 0.05), and the protein expression of VEGF was significantly weaker in the 100-μg/L rhPEDF group than in 25-and 50-μg/L rhPEDF groups (both P < 0.05), but similar between the 25- and 50-μg/L rhPEDF groups (P > 0.05). Conclusions rhPEDF can inhibit the proliferation of HaCaT cells, and down-regulate the mRNA and protein expressions of IL-6, IL-8 and VEGF.

Objective To investigate the effects of rottlerin on in vitro proliferation of and expressions of interleukin (IL)-17C,CCL20 chemokine,and nuclear factor (NF)-κB in cultured human HaCaT keratinocytes.Methods Some HaCaT cells were divided into several test groups treated with rottlerin at concentrations of 0.5,1.0,2.0 and 4.0 μmol/L,a solvent group treated with RPMI 1640 culture solution containing the same volume of dimethyl sulfoxide (DMSO) as that of 4.0 μmol/L rottlerin,and a control group treated with RPMI 1640 culture solution.Cell counting kit-8 (CCK8) assay was conducted to estimate the proliferative activity of HaCaT cells after 24-,48-and 72-hour culture,RT-PCR to determine the mRNA expressions of IL-17C and CCL20 after 48-hour culture,and Western blot to measure the protein expressions of IL-17C,CCL20 and NF-κB after 48-hour culture.Statistical analysis was carried out by using repeated-measures analysis of variance,one-way analysis of variance and Pearson correlation analysis with the SPSS16.0 software.Results Rottlerin showed an inhibitory effect on the proliferation of HaCaT cells,and the inhibitory effect increased over time (F =126.936,P ＜ 0.05) and with the increase of rottlerin concentrations (F =838.308,P ＜ 0.05),with a significant interaction effect between rottlerin concentrations and treatment duration (F =15.961,P ＜ 0.05).After 48-hour treatment,a significant decrease was observed in the mRNA and protein expressions of IL-17C (F =206.041,233.887,respectively,both P ＜ 0.05) and CCL20 (F =143.883,162.431,respectively,both P ＜ 0.05),as well as in the protein expression of NF-κB (F =577.915,P ＜ 0.05) in the test groups with the increase in rottlerin concentrations.Conclusions Rottlerin can inhibit the proliferation of HaCaT cells in vitro,and decrease the mRNA and protein expressions of IL-17C and CCL20 likely by downregulating the protein expression of NF-κB.

Objective To investigate the in vitro effect of sirolimus on the apoptosis of a cutaneous squamous cell carcinoma cell line, Colo-16 cells. Methods Cultured Colo-16 cells were treated with different concentrations (50, 100, 150, 200 nmol/L) of sirolimus for various durations ( 12, 24, 48, 72 hours). Subse-quently, cell proliferation was detected by MTT assay, and cell apoptosis by Annexin V-FITC and PI double staining. Morphological changes of the cells were observed with Hoechst 33258 fluorescent staining. Total RNA was extracted from Colo-16 cells treated with sirolimus for 48 hours, and subjected to reverse tran-scription (RT)-PCR for the detection of mRNA expression of B cell lymphoma/leukmia-2 (Bcl-2) and Bcl-2-associated X Protein (Bax). Results Sirolimus inhibited the proliferation of Colo-16 cells in a time-and dose-dependent fashion. The early apoptosis rate was 7.26%±0.26%, 8.34% ±0.19%, 9.86%±0.14%, 11.92% ±0.15% in Colo-16 cells treated with sirolimus of 50, 100, 150, and 200 nmol/L, respectively, signifi-candy higher than that in untreated cells (1.53%±0.09%, P < 0.05); a positive correlation was observed between the apoptosis rate and concentrations of sirolimus (r = 0.955, P = 0.000). Typical morphological changes of apoptosis, such as chromatin condensation and margination as well as nuclear fragmentation were observed by fluorescence staining. After treatment with sirolimus for 48 hours, a significant decrease was observed in the mRNA expression of Bcl-2, while an increase in that of Bax was noticed. Conclusion Sirolimus could induce Colo-16 cells apoptosis in vitro, which may be associated with the modulation of expression of apoptosis-regnlating genes, such as Bcl-2 and Bax.

Objective To investigate the effects of a short hairpin RNA targeting epidermal growth factor recereceptor (EGFR-shRNA) on Colo-16 cell apoptosis and sensitivity to rapamycin. Methods The expression vector of EGFR-specific shRNA was constructed. Colo-16 cells were classified into 4 groups, normal control group remaining untreated, liposome group transfected with lipofectamine 2000, negative control group transfected with shRNA-NC/Iipofectamine 2000 and positive interference group transfected with the expression vector of shRNA-EGFR/Lipofectamine 2000. After additional culture, immunocytochemistry and Western blot were conducted to detect the protein expression of EGFR, and flow cytometry to measure the apoptosis in Colo-16cells. MTT assay was performed to measure the sensitivity of Colo-16 cells to rapamycin. Results Compared with the normal control group, the expression of EGFR was down-regulated by 43.3% in positive interference group (F= 44.6, P< 0.05), and the sensitivity to rapamycin was increased by 2.44 folds. The apoptosis rate in positive interference group was (12.65±0.091)%, significantly different from that in the normal control group (F = 2042.9, P < 0.05). Conclusion The plasmid expression vector containing shRNA targeting EGFR can effectively suppress the expression of EGFR by Colo-16 cells, enhance the sensitivity of Colo-16 cells to rapamycin and induce the apoptosis in Colo-16 cells.

Objective To investigate the effects of COX-2 antisense oligonucleotide (AsODN) on the proliferation and expression of COX-2 in human skin squamous cell carcinoma cell line Coio-16. Methods The COX-2 AsODN was synthesized artificially, and various concentrations (50, 100, 200, 400 nmol/L) of the AsODN were transfected into Colo-16 cells with lipofectin followed by additional culture for different durations. The transfection results were observed with fluorescence microscopy. Subsequently, MTT assay,Western blotting and reverse transcription PCR were used to detect the cell proliferation, protein and mRNA expression of COX-2 in Coio-16 cells, respectively. Restults Compared with untreated cells, the proliferation of Colo-16 cells was inhibited significantly at 24, 48, 72 and 96 hours after transfection with different concentrations of COX-2 AsODN (all P < 0.05), and the COX-2 AsODN of 400 nmol/L exerted the highest inhibition rate of 60.3% at 48 hour. The average gray scale was 0.763±0.070, 0.600±0.065, 0.430±0.074 and 0.251±0.045 for COX-2 protein, 0.778±0.025, 0.602±0.041, 0.417±0.031 and 0.297±0.051 for COX-2 mRNA in Colo-16 cells transfected with COX-2 AsODN of 50, 100, 200, and 400 nmol/L respectively,significantly lower than that in untreated Colo-16 cells (all P < 0.05); there was a significant difference in the expression of COX-2 protein and mRNA among the cells transfected with the four concentrations of COX-2 AsODN and untreated cells (F = 83.54, 132.48, respectively, both P < 0.05). Conehtsions COX-2 AsODN can inhibit the proliferation, as well as the expression of COX-2 protein and mRNA in Colo-16 cells, which suggests that COX-2 AsODN has a potential therapeutic effect on skin squamous cell carcinoma.

Objective To evaluate effects of cucurbitacin Ⅰ on in vitro proliferation of HaCaT cells and the expression of keratin 17 (K17),signal transducer and activator of transcription 3 (STAT3) and vascular endothelial growth factor (VEGF) in cultured HaCaT cells.Methods In vitro cultured HaCaT cells were divided into 6 groups to be treated with cucurbitacin Ⅰ at different concentrations of 0.0125,0.025,0.05 and 0.1 μmol/L (0.0125,0.025,0.05 and 0.1 μmol/L cucurbitacin Ⅰ groups),DMEM containing the same volume of DMSO as 0.1 pmol/L cucurbitacin Ⅰ (DMSO group),DMEM (negative control group) and 10 nmol/L calcipotriol (positive control group),respectively.Cell counting kit-8 (CCK8) assay was performed to assess in vitro cellular proliferative activity after 12-,24-,36-hour treatment,reverse transcription (RT)-PCR to measure the mRNA expression of K17 and VEGF in HaCaT cells after 24-hour treatment,and Western blot analysis to determine the protein expression of K17,STAT3,phosphorylated-STAT3 (p-STAT3) and VEGF after 24-hour treatment.Statistical analysis was carried out by one-way analysis of variance (ANOVA),repeated measures ANOVA,Student-Newman-Keuls (SNK)-q test and Pearson correlation analysis.Results The proliferative activity of HaCaT cells started to decrease after 12-hour treatment with cucurbitacin Ⅰ at the concentration of 0.0125 μmol/L.When the concentration of cucurbitacin Ⅰ increased up to 0.1 μmol/L,the cell proliferation rates were inhibited by 43.00％ ± 2.11％ and 48.98％ ± 2.27％ after 24-and 36-hour treatment respectively.Compared with the negative control group,cucurbitacin Ⅰ at different concentrations all could inhibit in vitro proliferation of HaCaT cells (all P ＜ 0.05),and the inhibitory effects increased gradually with the increase of cucurbitacin Ⅰ concentration and treatment duration.After 24-hour treatment,the mRNA expression of K17 and VEGF and the protein expression of K17,VEGF and P-STAT3 were significantly decreased along with the increase of concentration of cucurbitacin Ⅰ (all P ＜ 0.05).Conclusion Cucurbitacin Ⅰ can inhibit in vitro proliferation of HaCaT cells,and down-regulate the mRNA expression of K17 and VEGF and protein expression of K17,VEGF and P-STAT3.

10.3969/j.issn.2095-4344.2013.23.006

Objective:To increase the critical relative humidity(CRH) of Qubi granule,thus to improve its humidity resistance. Methods:The best prescription of Qubi granule was optimized by comparative method,and to determine its critical relative humidity. Results:The old prescription of the Qubi granule's CRH was 50%.With new prescription,the Qubi granule’ s CRH was 61%. the latter has increased by 11% as compared with the former . Conclusion:Considering above result,there is a hope to improve the hygroscopicity of Qubi granule.