AIM:Long bone growth plate injury induced by wound and infection may cause limb reduction or angular deformity. Tissue engineering provides a promising treatment of growth plate injuries. In this study,we investigated the feasibility of establishing tissue-engineered growth-plate by allograft demineralized bone matrix(DBM) co-cultured with rabbit iliac growth-plate cells. METHODS:The experiment was performed at Department of Orthopaedics,Fourth Military Medical University of Chinese PLA from June 2005 to June 2006. ①Two 3-week-old New Zealand rabbits irrespective of gender(clean grade,2.0-2.5 kg) were selected. Growth plate cells were harvested from iliac crest epiphyseal cartilage of the rabbits by dissection and digestion with type Ⅱ collagenase. The third passage cells cultured were collected and incubated on allograft demineralized bone matrix. ② Histology,immunohistochemical staining and electronic scanning microscope(SEM) examinations were performed to observe cell growth on DBM 24 hours,7,14 and 21 days after culture. RESULTS:①Growth plate chondrocytes exhibited polygonal in monolayer culture,and immunohistochemical staining for type Ⅱ collagen was positive. ②SEM examination showed that twenty-four hours after coculture,the cells adhered to DBM scaffolds;Seven days after culture,the growth-plate chondrocytes rapidly proliferated and began to secret extracellular matrix;cells covered the whole scaffold and became overlapped on the 21st day. ③HE staining showed after 14 days of culture,growth plate cells adhered DBM scaffold in spherical shape with abundant cytoplasm. CONCLUSION:Tissue-engineered growth-plate is successfully constructed by allograft mineralized bone matrix co-cultured with rabbit iliac growth-plate cells.

Objective To investigate the accuracy and promptness of thromboelastography (TEG) to assess the blood transfusion requirements after abdominal operation in comparison with conventional assessments including vital signs (MAP,heart rate,breathing rate),urine output,hemoglobin and hematocrit.Methods From June to December in 2010,there were 57 patients were suspected bleeding in abdominal cavity after operation in SICU.Recorded data including vital signs (MAP,heart rate,breathing rate,oxygen saturation),urine volume per hour,the coagulation tests (Fib,PT,aPTT,INR),TEG parameters (R,K,Angle,MA,CI),the results of blood routine (Hb,Hct,Ph) and whether bleeding or not,blood product requirements within 24 h.Results Vital signs (MAP,heart rate,breathing rate,oxygen saturation),urine output per hour and the coagulation tests (Fib,PT,INR) showed no significant correlations (P ＞ 0.05) with blood transfusion requirements,but aPTT (R =0.513,P =0.000) and MA (R =0.578,P =0.000) correlated with the blood transfusion requirement.Patients with reduced MA needed more blood transfusion requirements.Patients were divided into active bleeding group and insidious bleeding group.MA had significant difference between two groups (P =0.025),but aPTT had not.Conclusions Thrombelastography is a more accurate indicator of blood transfusion requirements,especially in active bleeding patients.

Objective To investigate the effects of intravenous fluid restriction on complications after biliary surgery.Methods The clinical data of 168 patients who received biliary surgery at the Nanjing General Hospital of Nanjing Military Command from October 2006 to March 2008 were prospectively analyzed.All patients were randomly divided into test group(85 patients received fluid restriction treatment)and control group(83 patients received conventional treatment)by the sealed envelope method.The difference in the fluid volume between the 2groups was observed.Differences in systemic complication rate,local complication rate,general complication rate,time to bowl movement,length of hospital stay and mortality between the 2 groups were compared.All data were analyzed using the chi-square test,t test,Fisher exact test,Results The median total volumes of fluid in test group and control group were 1450 ml and 2420 ml,respectively,with significant difference between the 2 groups (t=-5.067,P<0.05).The median volumes of erystalloid solution in the test group was 850 ml,which was significantly lower than 1500 ml of the control group(t=-15.190,P<0.05).The postoperative systemic complication rate and general complication rate of the test group were 9%(8/85)and 19%(16/85),which were lower than 22%(18/83)and 30%(25/83)of the control group.There was a significant difference in the postoperative systemic complication rate between the test group and the control group(x2=4.837,P<0.05).The time to bowl movement and length of hospital stay were 2 days and 9 days in the restriction fluid group,which were significantly shorter than4 days and 12 days in the control group(t=-8.102,-2.003,P<0.05).The mortalities of test group and control group were 2%(2/85)and 4%(3/83),respectively,with no significant difference between the 2 groups(P>0.05).Conclusion Fluid restriction reduces the complication rate,shortens the length of hospital stay and accelerates recovery after biliary operation.

Objective To investigate the anti-proliferation mechanisms of Phenethyl isothiocyanate (PEITC)in human lung cancer cells line NCI-H446. Methods Human lung cancer cell line NCI-H446 was cultured in vitro,and treated with 10,30,50μmol/L PEITC for 24 h respectively. Cell viability and apoptosis were analyzed by methylthiazolyldiphenyl-tetrazolium bromide (MTT)assay and flow cytometry,respectively. Phosphorylation of Akt,activation of caspase-3 and caspase-9 in NCI-H446 cells,and production of cytochrome C in cytoplasm were detected by Western blot. Expression of phosphatase and tensin homologue (PTEN)was detected by real-time PCR. Results PEITC significantly reduced NCI-H446 cells proliferation in dose-dependent manner (P<0.05 ),and apoptosis was also inducted after PEITC administration. PEITC also markedly induced expression of caspase-3 and caspase-9,as compared with control group. And the level of cytochrome C in the cytoplasm was also increased after treatment with PEITC.Furthermore,PEITC treatment significantly increased the mRNA level of PTEN in NCI-H446 cells. Conclusion PEITC may be used as a potential anti-lung cancer agent by regulationg PI3K/AKT pathway and increasing PETN expression.

Objective:To investigate the protective effects and mechanisms of targeted inhibition of type 3 deiodinase (Dio3) on skeletal muscle mitochondria in sepsis.Methods:① In vivo experiments: adeno-associated virus (AAV) was employed to specifically target Dio3 expression in the anterior tibial muscle of rats, and a septic rat model was generated using cecal ligation and puncture (CLP). The male Sprague-Dawley (SD) rats were divided into shNC+Sham group, shD3+Sham group, shNC+CLP group, and shD3+CLP group by random number table method, with 8 rats in each group. After CLP modeling, tibial samples were collected and Western blotting analysis was conducted to assess the protein levels of Dio3, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), and silence-regulatory protein 1 (SIRT1). Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was utilized to examine mRNA expression of genes including thyroid hormone receptors (THRα, THRβ), monocarboxylate transporter 10 (MCT10), mitochondrial DNA (mtDNA), and PGC1α. Transmission electron microscopy was employed to investigate mitochondrial morphology. ② In vitro experiments: involved culturing C2C12 myoblasts, interfering with Dio3 expression using lentivirus, and constructing an endotoxin cell model by treating cells with lipopolysaccharide (LPS). C2C12 cells were divided into shNC group, shD3 group, shNC+LPS group, and shD3+LPS group. Immunofluorescence colocalization analysis was performed to determine the intracellular distribution of PGC1α. Co-immunoprecipitation assay coupled with Western blotting was carried out to evaluate the acetylation level of PGC1α. Results:① In vivo experiments: compared with the shNC+Sham group, the expression of Dio3 protein in skeletal muscle of the shNC+CLP group was significantly increased (Dio3/β-Tubulin: 3.32±0.70 vs. 1.00±0.49, P < 0.05), however, there was no significant difference in the shD3+Sham group. Dio3 expression in the shD3+CLP group was markedly reduced relative to the shNC+CLP group (Dio3/β-Tubulin: 1.42±0.54 vs. 3.32±0.70, P < 0.05). Compared with the shNC+CLP group, the expression of T3-regulated genes in the shD3+CLP group were restored [THRα mRNA (2 -ΔΔCt): 0.67±0.05 vs. 0.33±0.01, THRβ mRNA (2 -ΔΔCt): 0.94±0.05 vs. 0.67±0.02, MCT10 mRNA (2 -ΔΔCt): 0.65±0.03 vs. 0.57±0.02, all P < 0.05]. Morphology analysis by electron microscopy suggested prominent mitochondrial damage in the skeletal muscle of the shNC+CLP group, while the shD3+CLP group exhibited a marked improvement. Compared with the shNC+Sham group, the shNC+CLP group significantly reduced the number of mitochondria (cells/HP: 10.375±1.375 vs. 13.750±2.063, P < 0.05), while the shD3+CLP group significantly increased the number of mitochondria compared to the shNC+CLP group (cells/HP: 11.250±2.063 vs. 10.375±1.375, P < 0.05). The expression of mtDNA in shNC+CLP group was markedly reduced compared with shNC+Sham group (copies: 0.842±0.035 vs. 1.002±0.064, P < 0.05). Although no difference was detected in the mtDNA expression between shD3+CLP group and shNC+CLP group, but significant increase was found when compared with the shD3+Sham group (copies: 0.758±0.035 vs. 0.474±0.050, P < 0.05). In the shD3+CLP group, PGC1α expression was significantly improved at both transcriptional and protein levels relative to the shNC+CLP group [PGC1α mRNA (2 -ΔΔCt): 1.49±0.13 vs. 0.68±0.06, PGC1α/β-Tubulin: 0.76±0.02 vs. 0.62±0.04, both P < 0.05]. ② In vitro experiments: post-24-hour LPS treatment of C2C12 cells, the cellular localization of PGC1α became diffuse; interference with Dio3 expression promoted PGC1α translocation to the perinuclear region and nucleus. Moreover, the acetylated PGC1α level in the shD3+LPS group was significantly lower than that in the shNC+LPS group (acetylated PGC1α/β-Tubulin: 0.59±0.01 vs. 1.24±0.01, P < 0.05), while the expression of the deacetylating agent SIRT1 was substantially elevated following Dio3 inhibition (SIRT1/β-Tubulin: 1.04±0.04 vs. 0.58±0.03, P < 0.05). When SIRT1 activity was inhibited by using EX527, PGC1α protein expression was notably decreased compared to the shD3+LPS group (PGC1α/β-Tubulin: 0.92±0.03 vs. 1.58±0.03, P < 0.05). Conclusion:Inhibition of Dio3 in skeletal muscle reduced the acetylation of PGC1α through activating SIRT1, facilitating nuclear translocation of PGC1α, thereby offering protection against sepsis-induced skeletal muscle mitochondrial damage.

[Abstract] Objective: To investigate the effect of RG108 on the proliferation and apoptosis of human non-small cell lung cancer (NSCLC) cell lines (A549, H1299) and explore its molecular mechanism. Methods: A549 and H1299 cells were cultured in vitro and treated with different concentrations of RG108. The cell proliferation, cell cycle and apoptosis were detected by MTT assay and Flow cytometry, respectively. qPCR and Western blotting (WB) were used to detect the TFPI-2 mRNA and protein expressions as well as the expression of TMPRSS4 in cells. Meanwhile, the methylation status and degree of TFPI-2 promoter in cells were detected with Methylation-specific PCR (MSP) and colorimetry. Finally, siRNA-TFPI-2 and pcDNA3.0-TMPRSS4 plasmids were used to silence TFPI-2 or overexpress TMPRSS4, and then the changes in cell proliferation and apoptosis were detected. Results: After treatment with RG108, the proliferation rate of A549 and H1299 cells were significantly decreased (all P<0.05), while the apoptosis rate were significantly increased(P<0.05), the cell cycle were arrested in G1/S phase (P<0.05), and the intracellular mRNA and protein expressions of TFPI-2 were significantly increased (P<0.01 or P<0.05). Meanwhile, the methylase degree in TFPI-2 promoter region and the expression of TMPRSS4 in cells were all significanly decreased ( all P<0.05). After TFPI-2 silence, the proliferation levels of A549 and H1299 cells were significantly increased(all P<0.05); however, the apoptosis rate of A549 and H1299 cells were significantly reduced after transfection with pcDNA3.0-TMPRSS4(all P<0.05). Conclusion: RG108 can inhibit proliferation of A549 and H1299 cells and promote apoptosis by inhibiting the methylation of TFPI-2 and negatively regulates the expression of TMPRSS4.