ObjectiveTo explore the clinical characters and histopathologic differences between patients with culture-positive and culture-negative prosthetic joint infection (PJI). MethodsBetween January 2012 and July 2013, 66 PJI patients in accord with diagnostic criteria were enrolled. According to the results of preoperative aspiration and intraoperative cultures, the patients were divided into culture-negative group (CN group, n=21) and culture-positive group (CP group, n=45). There was no significant difference in gender, age, height, weight, and body mass index between 2 groups (P>0.05). Preoperative C reactive protein (CRP), erythrocyte sedimentation rate (ESR), and prosthesis survival time were compared between 2 groups. Intraoperative frozen sections and paraffin sections were both performed to identify infections, and histological typing was performed according to Morawietz's methods. ResultsThe preoperative CRP was (1.29±1.84) mg/ dL in CN group and (5.08±9.57) mg/dL in CP group, showing significant difference (t=2.094, P=0.038). The preoperative ESR was (22.86±28.42) mm/1 h in CN group and (36.74±31.26) mm/1 h in CP group, showing significant difference (t=7.761, P=0.000). The median survival time of prosthesis was 72 months (range, 8-504 months) in CN group and 25 months (range, 15 days-300 months) in CP group, showing significant difference (U=2.231, P=0.026). Morawietz's histological typing results showed that 2 cases were rated as type I, 7 cases as type II, and 12 cases as type III in CN group; 6 cases were rated as type I, 25 cases as type II, 13 cases as type III, and 1 case as type IV in CP group. The positive culture rate was 68.18% (45/66), and pathogenic bacteria was dominated by Staphylococcus, accounting for 68.89%. ConclusionThe patients with culture-negative PJI have slow onset and mild inflammatory response, so comprehensive diagnosis should be made based on pathological detection, laboratory examination, and intraoperative cultures.
ObjectiveTo investigate regulation mechanism of glypican-3 (GPC3) on Hippo signaling pathway and its effects on biological behavior of hepatocellular carcinoma Huh7 cells. MethodsShort hairpin RNAs (shRNA) targeting GPC3 and Yes-associated protein 1 (YAP1) genes were constructed. All of the shRNAs were transfected into Huh7 cells by liposome transfection in order to screen out the stable expression cell lines. The expressions of GPC3 and YAP1 in Huh7 cells were detected by PCR and Western blot in order to screen out the effective shRNAs. The proliferation, invasion, and apoptosis of Huh7 cells were detected by Edu cell proliferation assay, transwell, and flow cytometry. GPC3 shRNA transfection experiments were divided into 6 groups:non-transfection group, empty vector group, GPC3-714-shRNA group, GPC3-647-shRNA group, GPC3-1718-shRNA group, and GPC3-2134-shRNA group. YAP1 shRNA transfection experiments were divided into 6 groups:non-transfection group, empty vector group, YAP1-906-shRNA group, YAP1-1363-shRNA group, YAP1-1666-shRNA group, and YAP1-2895-shRNA group. GPC3 regulation experiments were divided into 5 groups:non-transfection group, empty vector group, GPC3-1718-shRNA group, GPC3-1718-shRNA+ rhYAP1 group, and YAP1-1666-shRNA group. Results① GPC3-1718-shRNA and YAP1-1666-shRNA plasmids were successfully constructed to silence the expressions of GPC3 and YAP1. ② The expressions of GPC3 mRNA and protein in each transfection group were significantly lower than those in the non-transfection group (P<0.05) and the empty vector group (P<0.05), while which in the GPC3-1718-shRNA group was significantly lower than those in all the other transfection groups (P<0.05). The expressions of YAP1 mRNA and protein in each transfection group were significantly lower than those in the non-transfection group and empty vector group (P<0.05), while which in the YAP1-1666-shRNA group was significantly lower than those in all the other transfection groups (P<0.05). ③ The expressions of YAP1 mRNA and protein in the GPC3-1718-shRNA group and the YAP1-1666-shRNA group were significantly lower than those in the non-transfection group (P<0.05) and the empty vector group (P<0.05), while which in the GPC3-1718-shRNA+rhYAP1 group were significantly higher than those in the GPC3-1718-shRNA group (P<0.05) and the YAP1-1666-shRNA group (P<0.05). ④ Compared with the non-transfection group and the empty vector group, the abilities of cell proliferation and invasion in the GPC3-1718-shRNA group and the YAP1-1666-shRNA group were significantly decreased, and the cell apoptosis was significantly increased (P<0.05); The cell proliferation, invasion, and apoptosis in the GPC3-1718-shRNA+rhYAP1 group were significantly improved (P<0.05). ConclusionGPC3 is likely to affect biological behavior of hepatocellular carcinoma Huh7 cells through regulation of YAP1 in Hippo signaling pathway.
ObjectiveTo investigate the anti-apoptotic ability of synovium-derived mesenchymal stem cells (SMSCs) by comparing the apoptosis induced by tumor necrosis factor α (TNF-α) between SMSCs and bone marrow mesenchymal stem cells (BMSCs). MethodSMSCs and BMSCs were isolated with tissue adhering and density gradient centrifugation respectively, and cells at passages 3-5 were used in further experiments. After immunophenotype identification and differentiation induction, cells were divided into 4 groups. In the experimental groups, apoptosis of SMSCs and BMSCs were induced by 20 ng/mL TNF-α and 10 μg/mL cycloheximide, and cells were cultured in normal culture medium in the control groups. Cellular morphology were observed by inverted phase contrast microscope. After apoptosis induction for 24 hours, cell viability was determined by cell counting kit 8 assay and apoptotic index was detected by flow cytometer. Moreover, the level of Cleaved Caspase-8, 3 were determined by Western blot. ResultsBoth SMSCs and BMSCs accorded with the definition criteria of MSCs according to results of immunophenotype identification and differentiation induction. After apoptosis induction, cells became shrinking and partially floated and cellular morphologies became worse than those in the control groups. After apoptosis induction for 24 hours, cell viabilities of SMSCs and BMSCs in the control groups were both 100%, and no apoptotic cells were observed. However, cell viabilities of SMSCs and BMSCs in the experimental groups were 60.13%±8.63% and 46.55%±10.54% respectively, which were both significantly lower than those in the control groups (P<0.05) , and cell viability in the SMSCs experimental group was significantly higher than that in the BMSCs experimental group (t=3.152, P=0.006) . The apoptotic index was 36.54%±8.63% in the SMSCs experimental group and was 53.77%±11.52% in the BMSCs experimental group, both were significantly higher than the control groups (1.12%±0.24% and 1.35%±0.31%) (P<0.05) . What's more, it was significantly lower in SMSCs experimental group than that in BMSCs experimental group (t=3.785, P=0.001) . Moreover, no expression of Cleaved Caspase-8, 3 was detected in the control groups. But the levels of Cleaved Caspase-8, 3 were significantly enhanced in the experimental groups and they were lower in SMSCs than in BMSCs (t=13.870, P=0.000; t=7.309, P=0.000) . ConclusionsTNF-α induced apoptosis is lower in SMSCs than in BMSCs, which means that SMSCs may have stronger anti-apoptosis ability than BMSCs.