目的：探讨沉默 G 蛋白偶联受体激酶 3（G protein-coupled receptor kinase 3，GRK3）对口腔鳞状细胞癌（oral squamous cell carcinoma，OSCC）细胞增殖、迁移和侵袭的影响及其可能的机制。方法：利用 Oncomine 数据库分析 GRK3 在正 常口腔组织及 OSCC 组织中的表达水平。用 RNA 干扰技术敲降 GRK3 在 OSCC 细胞 WSU-HN6 和 CAL27 中的表达，用 qPCR 法验证干扰效率后，采用 CCK-8 法和流式细胞术分别检测敲降 GRK3 对 OSCC 细胞增殖和凋亡的影响，Transwell 小室 法检测对 OSCC 细胞迁移、侵袭能力的影响，qPCR 法检测对 OSCC 细胞周期、上皮间质转化（epithelial to mesenchymal transition，EMT）和基质金属蛋白酶（matrix metallopeptidase，MMP）相关分子 mRNA 水平表达的影响，WB 法检测 EMT 及 MMP 相关分子的蛋白表达水平变化。结果：OSCC 组织中 GRK3 的表达水平显著高于正常口腔组织（P<0.01）。转染 si-GRK3 后，OSCC 细胞中 GRK3 mRNA 表达水平均下调 70% 以上。敲降 GRK3 可显著抑制 OSCC 细胞的增殖、迁移和侵袭能力（均 P<0.01），对细胞凋亡无显著影响（P>0.05）。敲降 GRK3 表达后，OSCC 细胞的 G0/G1 期比例显著增高（t=5.799，P<0.01），细胞 周期蛋白 D1（Cyclin D1）、Cyclin D3、周期蛋白依赖性激酶 2（cyclin-dependent kinases 2，CDK2）和 CDK4 基因的 mRNA 表达降 低（均 P<0.05）；EMT 相关分子波形蛋白（Vimentin）、Zeb1 和 Slug 表达降低，E-钙黏蛋白（E-Cadherin）表达升高（均 P<0.05）； MMP3 和 MMP9 表达降低（均 P<0.05），MMP2 和 MMP7 表达无明显变化（均 P>0.05）。结论：GRK3 可通过调节细胞周期促 进 OSCC 细胞的增殖能力，并通过调控 EMT 和 MMP 增强细胞的迁移和侵袭能力。

Objective To explore the efficacy of alprostadil combined with Bailing capsule in the treatment of early chronic kidney disease. Methods A total of 94 early stage chronic kidney disease patients were selected and divided into treatment group(n=46) and control group(n=48).The patients in control group were treated with Bailing capsule,5 capsules, tid,po.The patients in treatment group were treated with Bailing capsule combined with 2 mL alprostadil in 20 mL 0.9% sodium chloride injection,intravenous injection,qd.The patients were treated for 4 weeks as a course of treatment in both groups.After 2 courses of treatment,the improvement of renal function,the changes in cytokine levels including NK cells and T cell subsets CD+3, CD+4,CD+8,adverse reactions of two groups were observed. Results The effective rates of the control group and the treatment group were 60.42%,91.30%,respectively(P<0.05).The renal function index 24 h urine protein were(1.15± 0.35) g,serum creatinine were(78.52±10.63) μmol·L-1,urea nitrogen were(8.23±1.65) mmol·L-1,all of which were decreased significantly (P<0.05).The levels of NK cells were(21.89±2.73)%,T cell subsets CD+3were(71.02±5.61)%,CD+4were(38.84±3.52)%, CD+4/CD+8were(1.28 ± 0.14),which were increased significantly,while the level of CD+8were(30.21± 3.03)% was decreased significantly(P<0.05).There was no significant difference between two groups in the adverse reactions(P>0.05). Conclusion The combination of alprostadil and Bailing capsule is effective to early stage chronic kidney disease by improving the renal function and regulating the level of cytokines.

Objective To explore the evaluation value of dynamic monitoring of neutrophil/lymphocyte ratio(NLR)in severity of illness and prognosis of bacterial sepsis.Methods Clinical data and laboratory index of 72 cases of patients with bacterial sepsis in ICU of Yangpu Hospital Affiliated to Tongji University were retrospectively analyzed.According to the severity of illness,patients were divided into sepsis group(n=20),severe sepsis group(n=30)and septic shock group(n=22).According to the mortality within 28 d,patients were divided into survival group(n=47),death group(n=25).The 0 h NLR,48 h NLR and change rate of NLR between two groups were compared,and the influence factors of prognosis were analyzed.Results With the increase of severity of illness,the time of mechanical ventilation was shortened((8.8±1.9)d,(4.6±0.6)d,(3.9±0.4)d),and PLT((146.4±45.8)×109/L,(110.6±41.3)×109/L,(102.5±38.6)×109/L),NLR rate(0.61±0.26,0.26±0.11,0.22±0.09)were decreased significantly,APEACHE Ⅱ score,CRP,PCT,0 h NLR,48 h NLR were increased obviously((18.5±2.3)points,(20.4±3.6)points,(23.1±3.9)points;(72.6±10.4)mg/L,(78.2±11.6)mg/L,(85.2±12.5)mg/L;(1.5±0.4)μg/L,(2.3±0.6)μg/L,(2.7±0.9)μg/L;11.3±2.6,14.2±3.4,15.7±3.5;3.4±0.9,9.7±2.4,11.2±2.6),the differences were statistically significant(P＜0.05).Compared with death group,the time of mechanical ventilation in survival group was prolonged((4.1±0.3)d vs.(8.7±1.4)d),APEACHE Ⅱ score,CRP,PCT,0 h NLR,48 h NLR were decreased obviously((21.4±3.5)points vs.(18.3±2.6)points,(78.2±11.6)mg/L vs.(71.5±10.8)mg/L,(2.5±0.7)μg/L vs.(1.4±0.6)μg/L,(15.0±3.3)vs.(11.6±2.4),(10.5±2.8)vs.(3.2±0.8)),and PLT,NLR rate were increased significantly((106.5±41.5)×109/L vs.(148.4±50.8)×109/L,0.24±0.10 vs.0.65±0.24),the differences were statistically significant(t=16.18,4.26,2.44,6.99,5.01,16.73,3.54,8.15,P＜0.05).Multivariate logistic regression analysis showed that APEACHE Ⅱ score,0 h NLR were the independent risk factors of death in patients with bacterial sepsis(OR=3.99,3.01,95％CI:1.65-2.38,1.99-4.54,P＜0.05),and NLR rate was independent protection factor(OR=0.95,95％CI:0.91-0.97,P＜0.05).Conclusion Dynamic monitoring of peripheral blood NLR can help to judge the prognosis and severity of illness of patients with bacterial sepsis,and NLR before treatment and change rate of NLR are an independent predictors of death.

Objective Explore the spine fractures treated by percutaneous one-way pedicle screws surgery compared with curative effect of traditional open surgery.Methods From October 2012 to October 2015,to collect 61 patients with single segmental thoracolumbar fractures were retrospectively analyzed,respectively for percutaneous minimally invasive one-way long tail pedicle screw(Observation group,n =32) of internal fixation were compared with traditional open surgery (control group,n =29).Compare two groups of paticnts in the differences of operation time,amount of operative bleeding,operative wound,postoperative VAS scores,the ratio of postoperative injured vertebral front height,and hospitalization expenses.Results All patients were followed up for an average of 9.6 months (from 7 to 14months),neither of the groups showed internal fixation of related complications.Time of operation(min):observation group(87.4 ± 13.6) min,control group (92.3 ± 10.3) min,(t =-1.648,P ＞ 0.05);Mount of operative bleeding:observation group (73 ± 8.8) mL,control group (352 ± 63.7) mL,(t =-23,385,P ＜ 0.05);Wound of operative (cm2):observation group (12.3 ± 2.30) cm2,control group (81.5 ± 14.2) cm2,(t =-25.937,P ＜ 0.05);Expenses of hospitalization (RMB ten thousand):observation group (3.5 ± 0.3),control group(2.3± 0.5),(t =-11.223,P ＜ 0.05);VAS score 2 days and 6 months after surgery:observation group (3.0±0.4) and (1.3±0.6),controlgroup(4.2±0.5) and (2.7±0.7),(t=-10.396 and-8.409,P＜ 0.05),and comparcd with the preoperative also statistically significant (P ＜ 0.05);2 days and 6 months after surgery in the ratio of injured vertebral front height:observation group(89.6 ±7.2)％ and (84.2 ±5.7)％,control group (91.3 ± 5.8) ％ and (86.3 ± 4.6) ％,(t =-1.009 and-1.573,P ＞ 0.05),but in the same group recovery of injured vertebral leading edge height ratio compared with preoperative postoperative were significantly (P ＜ 0.05).Conclusion For a single section of thoracolumbar spine fracture,Compared with the control group,Percutaneous one-way long tail pedicle screw internal fixation technology have the advantage of little trauma,less sequel,rapid postoperative recovery,also in the recovery of vertebral body height and loss of late has achieved the same cur-ative effect,but patients with costly.

Objective To investigate the effect of capsaicin on hepatic stellate cells (HSCs) and liver fibrogenesis.Methods HSCs were cultured.The reactive oxygen in HSCs under capsaicin at different concentrations was tested by DCFH-DA kit.The proliferation of HSCs was detected by CCK-8 test kit.Smoothmuscle α-actin (α-SMA) expression of HSCs was evaluated by Western blot.The fibrosisrelated genes were tested by RT-PCR.The apoptosis of HSCs was measured by flow cytometer.Bcl-2,bax and cyt-c was detected by Western blot.A murine model of liver fibrogenes was established.Capsaicin of different concentration was injected intraperitoneally.Liver pathology was observed using HE staining.Hydroxyproline content of liver and levels of collagen Ⅲ and hyaluronic acid in serum were tested.Results In dose dependent manner capsaicin inhibited the generation of the reactive oxygen species.Proliferation and activation of HSCs was inhibited by capsaicin (respectively F =13.267,57.392,all P ＜ 0.05) and the apoptosis of HSCs was promoted by capsaicin (F =235.571,P ＜ 0.05).Bax,cyt-c and caspase-3 was increased obviously (respectively F =29.334,38.274,138.329,all P ＜ 0.05).Capsaicin changed the expression of fibrosis-related genes (TGF-β1,TIMP-1) in HSCs (respectively F =376.534,253.751,all P ＜0.05).Capsaicin downregulated the level of hydroxyproline,collagen Ⅲ and hyaluronic acid in the rat model (respectively F =153.397,27.149,38.392,all P ＜ 0.05).Conclusions Capsaicin inhibits the proliferation and activation of hepatic stellate cells.Capsaicin promotes the apoptosis of hepatic stellate cells,and inhibits liver fibrogenesis.

Objective To investigate the Fsp27 gene's influence on the regulation of hepatic stellate cells (HSCs) in vitro.Methods HSCs were isolated from rat liver,the Fsp27 gene was detected in primary HSCs,and activated HSCs were detected by RT-qPCR.After 72 h of Fsp27 transduction through a lentivirus expressing Fsp27 (pLV-Fsp27),the proliferation of HSCs was tested by the CCK-8 test kit,smooth muscle α-actin (α-SMA) expression of HSCs was tested by Western blot,and the fibrosis-related genes were tested by RT-qPCR.Results The HSCs were isolated and cultured successfully,and the Fsp27 genetic difference between primary and activated HSCs was significant (P＜0.01).After coculture for 72 h,Fsp27 inhibited the proliferation and activation of HSCs (P＜0.05).Fsp27 can enhance expression of the MMP-2 gene and down-regulate expression of the TIMP-1 and TGF-β1 gene in activated HSCs (P＜0.05).Conclusion The Fsp27 gene can inhibit the proliferation and activation of HSCs,regulate the expression of fibrosis-related genes,and may play an important role in maintaining the quiescent phenotype of HSCs.

Objective To study the protective effects of resveratrol against hepatic stellate cells (HSCs) and liver fibrogensis.Methods HSCs were isolated from liver of SD rats.The reactive oxygen output in HSCs under resveratrol in different concentrations was tested by DCFH-DA kit.The proliferation of HSCs was tested by CCK-8 test kit.Smoothmuscle α-actin (α-SMA) expression of HSCs was evaluated by Western blotting.The activity-related genes were measured by PCR.The models of liver fibrogenes were established.Resveratrol in different concentrations was administrated intraperitoneally.Liver was studied by pathology and SMA staining.Hydroxyproline content of liver and levels of collagen Ⅲ and hyaluronic acid in serum were tested.Results HSCs were isolated from liver and cultured successfully.Resveratrol inhibited the generation of the reactive oxygen.Proliferation and activation of HSCs was inhibited by resveratrol (0.536 ±0.052,0.411 ±0.047,0.327 ±0.063,0.312 ±0.032,F =12.776,P ＜0.05) (103 ±7,90 ±7,63 ± 4,53 ± 3,F =62.179,P ＜ 0.05).Resveratrol inhibited the expression of genes (myogenic determination gene MyoD,collagen 11 and collagen Ⅰ) in HSCs(122 ± 5,96 ± 3,68 ± 3,60 ± 3,F =180.600,P＜0.05) (100±8,82 ±3,53 ±3,51 ±2,F=77.451,P ＜0.05) (170 ±3,147 ±4,92 ±3,90 ±2,F =462.878,P ＜ 0.05).Resveratrol downregulated the level of hydroxyproline,collagen Ⅲ and hyaluronic acid (358.3 ± 20.2,320.5 ± 15.3,290.3 ± 24.5,F =23.929,P ＜ 0.05) (32.8 ± 3.1,28.9 ±1.3,25.3±1.8,F=20.050,P＜0.05)(276.3 ±17.8,225.3 ±28.3,195.4 ±11.2,F=18.585,P＜0.05).Conclusions Resveratrol can inhibit the proliferation and activation of HSCs and downregulate the fibrogensis level of the liver of rats.

Objective To investigate the influence of fat-specific protein 27 (Fsp27) gene on the regulation of liver fibrogenesis in vivo.Methods Hepatic stellate cells (HSCs) were isolated from rat liver.Fsp27 gene was detected in primary HSCs and activated HSCs by real-time quantitative PCR (RTqPCR).Lentiviral vector carrying Fsp27 gene was constructed.The model of liver fibrosis was established by infusing carbon tetrachloride (CC14).The rats with liver fibrogenesis were infected by the virus.Liver sections were made to observe the structure and form of liver histocytes.The content of fibrous protein in liver and serum was detected by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay.Resukts HSCs were isolated and cultured successfully.The difference of Fsp27 gene between primary HSCs and activated HSCs was significant(P ＜ 0.01).The model of liver fibrosis was achieved.After infecting the model rats,we found the fibrosis level in treatment group was lower compared with control group.Conclusions Fsp27 treatment can decrease collagen deposition in the liver and inhibit the formation of fibrosis.

In our recent study by exploring an intein-based dual-vector to deliver a B-domain-deleted FVIII (BDD-FVIII) gene, it showed that covalently ligated intact BDD-FVIII molecules with a specific coagulant activity could be produced from expressed heavy and light chains by protein trans-splicing. Here, we assessed the hypothesis that the efficiency of trans-splicing may be increased by adding to the intein sequences a pair of leucine zippers that are known to bring about specific and strong protein binding. The intein-fused heavy and light chain genes were co-transferred into cultured COS-7 cells using a dual-vector system. After transient expression, the intracellular BDD-FVIII splicing was observed and the spliced BDD-FVIII and bioactivity secreted to culture media were quantitatively analyzed. An enhanced splicing of BDD-FVIII with decreased protein precursors from gene co-transfected cells was observed by Western blotting. The amount of spliced BDD-FVIII and bioactivity secreted to the culture media were 106 +/- 12 ng x mL(-1) and 0.89 +/- 0.11 U x mL(-1) analyzed by ELISA and Coatest method respectively, which was greater than leucine zipper free intein-fused heavy and light chain genes co-transfected cells (72 +/- 10 ng x mL(-1) and 0.62 +/- 0.07 U x mL(-1)). The activity of cellular mechanism-independent protein splicing was also improved, as showed by the increasing of spliced BDD-FVIII and bioactivity in culture media from combined cells separately transfected with heavy and light chain genes which was 36 +/- 11 ng x mL(-1) and 0.28 +/- 0.09 U x mL(-1). It demonstrated that the leucine zippers could be used to increase the efficiency of protein trans-splicing to improve the efficacy of a dual-vector mediated BDD-FVIII gene delivery by strengthening the interaction between the two intein-pieces fused to heavy and light chains. It provided evidence for further study in animal model using a dual-adeno-associated virus vector to deliver FVIII gene in vivo.

To investigate the improving effect of inter-chain disulfide formation on protein trans-splicing, we introduce a Cys point mutation at Tyr(664) in heavy chain and at Thr(1826) in light chain of B-domain-deleted FVIII (BDD-FVIII). By co-transfection of COS-7 cell with the two Cys mutated chain genes, the intracellular protein splicing, inter-chain disulfide formation, secreted BDD-FVIII and bioactivity in culture supernatant were observed. The data showed that a strengthened spliced BDD-FVIII with an inter-chain disulfide detected by Western blotting and an elevated secretion of spliced BDD-FVIII (128 +/- 24 ng mL(-1)) compared to control (89 +/- 15 ng mL(-1)), assayed by a sandwich ELISA. A Coatest was performed to assay the secretion of bioactivity in culture supernatant and shown a much higher value (0.94 +/- 0.08 u mL(-1)) compared to that of control (0.62 +/- 0.15 u mL(-1)). It suggests that inter-chain disulfide formation could improve protein trans-splicing based dual-vector delivery of BDD-FVIII gene providing experimental evidence for ongoing in vivo study.