In order to investigate the effect of sequence-specific small interfering RNA on suppressing cyclin D1 expression and proliferation and cell cycle and expression of G1 phase regulators of fibroblasts derived from keloid, the plasmid expression vector of siRNA targeted against cyclin D1 was constructed and transfected into fibroblasts with LipofectamineTM 2000. The changes of cyclin D1 expression were detected by fluorescent quantitative PCR(FQ-PCR), semi-quantitative RT-PCR. The effect of sequence- specific small interfering RNA in suppressing the proliferation of fibroblasts was detected by MTT assay. Flow cytometry were used for evaluation ofceU cycle. The expression of cyclin D1, CDK4, pRb and P16 was detected by immunohistochemical method. The results showed that: (1) The sequence- specific siRNA effectively suppressed cyclin D1 expression at both mRNA levels with inhibition rate of 63.68% and 92.83% (P<0.01). (2) Significantly inhibited the proliferation of fibroblasts, and changed cell cycle in percentage of G0/G1 phase cells was increased compared with the controls groups in fibroblasts(P < 0.05). (3) 72 h after transfection, the expression of cyclin D1, CDK4 and pRb decreased, and the expression of P16 increased. It can be concluded that the plasmid expression vector for the RNAi against cyclin D1 constructed in the study can effectively and specifically suppress cyclin D1 expression, and progression of G1/S is effected by G1 phase related regulatory protein, and suppresses proliferation of fibroblasts derived fiom keloid.

Objective To evaluated the role of wt-P53 protein in telomerase regulation in keloid fibroblasts(KFBs). Methods The fibroblasts were derived from human keloid tissue which was proved by pathological diagnosis. KFBs were divided into 2 groups, the transfection group and the untransfection group. wt-p53 gene was transfected into the fibroblasts by adenovirus vectors in the transfection group. The KFBs untransfected with wt-p53 gene served as control (untransfection group). After 48 hours of transfection, the expression of wt-P53 protein was analyzed by both Western blotting and immunofluorescence method, respectively. The telomerase activity was evaluated by TRAP-ELISA after 1-7 days of transfection.Results All the KFBs from 2 groups expressed wt-P53 protein. But the expression level of wt-P53 protein in the transfection group was significantly higher than that in the untransfection group. At the same time of high expression of wt-P53 protein, the telomerase activity of KFBs in transfection group was significantly lower than that in the untransfection group( P＜0.05). Conclusion High level expression of wt-P53 protein can transiently inhibit the telomerase activity of KFBs.

Objective To evaluate catheter-directed thrombolysis via the popliteal vein in the treatment of the lower limb acute deep venous thrombosis.Methods From July 2009 to October 2010,32patients of the lower limb acute deep venous thrombosis including 3 patients with concurrent pulmonary embolism underwent uhrasound-guided catheter-directed thrombolysis via the popliteal vein.The thrombolytic catheter was inserted into thrombus,through which urokinase was infused at a dosage of 100 × 104U/d.The venous patency score and the rate of patency improvement were observed by venograms before and after therapy.Results In every patient,the lower limb swell and pulomonary symptoms relieved.The circumferences between affected and normal thigh before and after the thrombolysis were(5.4 ± 1.4)cm and (1.7 ± 1.3)cm(t =9.92,P ＜0.01).The circumferences between affected and normal leg before and after the thrombolysis were(4.1 ± 1.5)cm and(1.5 ±0.7)cm(t =7.65,P ＜0.01).The venous patency score before and after the thrombolysis were(15 ± 4)and(4 ± 3)(t =7.12,P ＜ 0.01).The mean rate of venous patency was 88.21％.In the 3 patients with pulmonary embolism,thrombus was complete dissolved in 1 case and partial dissolved in 2 cases.No major complications occurred in all these patients.29 patients were followed up for 3-12 months.There were no thrombosis relapsed.Conclusions Catheter-directed thrombolysis via the popliteal vein with urokinase for acute lower limb deep venous thrombosis is safe and effective.

Objective To investigate the relationship between aggrecan and YKL-40 in knee articular cartilage of Sprague-Daw-lay(SD) rats with osteoarthritis (OA) .Methods Fifty-six healthy SD rats were randomly divided into 7 groups ,8 cases per group . The one side of knee joint was randomly selected for performing the anterior cruciate ligment transection (ACLT) and establishing the OA model .The rats in one group were randomly killed on the day of operation and at postoperative 0 ,2 ,4 ,8 ,12 ,16 ,20 weeks . The femoral condyle cartilage samples at different time periods in the operated side were collected for conducting safranin O /fast green staining and HE staining .Meanwhile ,the OA pathological grade was made out according to the modified Mankin scale .The expression of aggrecan and YKL-40 in the cartilage with different stages of OA were analyzed by the immunohistochemistry meth-od ,and the status of expression were measured by average optical density (AOD) .The correlation between aggrecan and YKL-40 was analyzed .Results With the aggravation of OA ,the expression of aggrecan was gradually reduced and the expression of YKL-40 was gradually increased .The differences during the early ,middle and late phases of OA had statistical significance (P<0 .05) . The expression of aggrecan was negatively correlated with the expression of YKL-40(P<0 .05) .Conclusion The level of aggrecan is gradually reduced with the aggravation of OA .Aggrecan is negatively correlated with the YKL-40 level ,which may reflect the dedifferentiation degree of joint chondrocyte to some extent .

Objective To observe the expression of retinol-binding protein 4 (RBP4) and phosphatidylinositol 3 kinase (PI3K) in insulin resistant (IR) rats and the role of pioglitazone intervention. MethodsThirty-five male Wistar rats in SPF level were randomly divided into control fed with normal diet (n= 11 ) and IR model fed with high fat sucrose diet (HFSD) (n= 24). The IR rats were then randomly divided into two subgroups, namely IR fed with HFSD(n = 12) and pioglitazone intervention (n= 12) given a daily dose of 20 mg · kg 1 · d-1 pioglitazone and HFSD (IR +Pio group )for 8 weeks.All rats were killed after 16 weeks and the levels of serum TG, high density lipoprotein (HDL-C), low density lipoprotein (LDL-C), fasting blood-glucose (FBG), fasting serum insulin (FIns) and insulin resistance index (HOME-1R) were measured.Enzyme-linked immunosorbent assay (ELISA) and RT-PCR were employed to detect the level of serum RBP4 and the mRNA expression of RBP4 in the epididymis adipose tissues, respectively. Immunohistochemistry was used to detect the protein expression of PI3K in skeletal muscles while UV spectrophotometer was employed to measure level of serum AOPP. The ratio of visceral fat weight of mesentery, epididymis and peritoneum in abdominal cavity to body weight (BW) was calculated. Results(1) the level of BW, TG, LDL-C, FINS and the ratio of visceral fat weight to BW were higher in IR group than in control group, but HDL-C decreased significantly. After the intervention of pioglitazone, the level of BW,TG, LDL-C, FINS, and the ratio of visceral fat weight to BW in IR+Pio group were lower and HDL-C increased significantly than those in IR group.(2) The level of RBP4 from serum and epididymis adipose and serum AOPP were higher in IR group than in control and lower significantly in IR+Pio group than in IR group (all P＜0.05). (3) The expression of PI3K in skeletal muscles were lower in IR group than in NC group, and increased after the invention of pioglitazone. (4) FIns, the ratio of visceral fat weight to BW and LDL-C were positively correlated with RBP4 while HDL-C and PI3K negatively correlated with RBP4. Conclusions(1) The increased RBP4 can lead to metabolic disturbances and oxidative stress in IR rats.(2) RBP4 may decrease the insulin sensibility by weakening insulin signal transduction. (3) Pioglitazone can ameliorate insulin resistance by decreasing the level of RBP4 and serum AOPP and increasing the level of PI3K in skeletal muscles of IR rats.

Objective To study wt-p53 gene's influence on the proliferation of keloid fibroblasts in vitro. Methods wt-p53 gene was transfected into keloid fibroblasts by adenovirus vector. wt-p53 mRNA was analyzed by RT-PCR; wt-p53 protein was evaluated by indirectiy immunofluorescence; The ability of proliferation of keloid fibroblasts was analyzed by cell growing curves; The cell cycle of KFB was checked by FCAS. Results The expression of wt-p53 mRNA and protein was obviously higher in the fibroblasts of the experimental group than those of control group; the rate of G_0~G_1 in cell cycle was higher in the fibroblasts of the experimental group than those of control group; at the same time, the rate of G_2~M was lower in fibroblasts of the experimental group than those of control group (P

Objective To investigate the risk factors of acute kidney injury(AKI) after intracoronary stent implantation in order to provide the basis for clinical prophylaxis and treatment.Methods Retrospectively analyzed 626 consecutive patients who underwent isolated intracoronary stent implantation in our institution from January 2007 to July 2011.Multivariate logistic regression model was constructed to identify the risk factors for the development of AKI defined as a serum creatinine (SCr) 130 to 199 μ mol/L or estimated creatinine clearance(Ccr) 30 to 60 ml/min per 1.73 m2.Results Ninety-three patients of 626 (14.9％) underwent isolated intracoronary stent implantation developed AKI.The results of the multivariate forward stepwise logistic regression analysis found that risk factors for the development of AKI following isolated intra-coronary stent implantation was associated with age (OR =1.570,95％ CI 1.308-1.885),ejection fraction (EF) ≤ 30％(OR =11.526,95％ CI 2.452-54.177),hypotension during perioperative and postoperation (OR =11.074,95％ CI 2.439-50.282),operation duration(OR =1.032,95％ CI 1.012-1.051),sex (OR =0.010,95％ CI 0.001-0.086),NYHA class Ⅲ & Ⅳ (OR =0.209,95％ CI 0.059-0.737),peripheral vascular disease (OR =0.528,95％ CI 0.286-0.973),chronic obstructive pulmonary diseases (OR =0.546,95％ CI 0.304-0.982),preoperation Cr (OR=1.418,95％CI 1.216-1.654) (and all P＜0.05).Conclusion AKI is the common complications after intracoronary stent implantation,especially age,EF ≤ 30％,hypotension during perioperative and postoperation,operation duration are independent risk factors.

Objective To investigate the protective effect of the first window(FW)of liver ischemic preconditioning(IPC),the second window(SW) of remote(leg) ischemic preconditioning(RPC) and conbined applications of liver and lges IPC to against liver ischemia/reperfusion(I/R) injury in the rat,and to investigate the mechanism of the protection.Methods Rats were randomly divided into five groups(n=8 each):(1) Sham group(S group),rats without IPC,(2) Rats with 5 min IPC(IPC group);(3) Rat wiht both liver and lower limbs IPC and repeated three times(RPC group);(4) IPC 24 h after RPC group;(5) IR without IP(I/R group);except S group,the rats were subjected to 60 min sustained liver ischemia followed by 180 min reperfusion.All ischemia rats were only subjected to 70% liver ischemia.Finally,blood and liver samples were obtained to determine the activity of ALT and AST,the expressions of TNF-? and HSP70 protein,and liver wet/dry weight(W/D) and pathology.Results All IPC group and RPC group and IPC+RPC group had obviously lower levels of ALT,AST,W/D,TNF-? than that of the I/R group(P0.05).Conclusions The FW of the IPC,the SW of the RPC and combined applications can lessen hepatic I/R injury.There is no significant difference in the protective intensity of the 3 motheds.The protective effects possibly are due to suppression of TNF-? production,induction of protein HSP70 expression and improvement of liver microcirculation.

A matrix solid phase dispersion extraction_dispersed liquid phase microextraction_gas chromatography_mass spectrometric method has been developed for the determination of three pyrethroids ( tetramethrin, permethrin and deltamethrin) in soil. The optimal conditions for the analysis were as follows. About 0. 5 g soil and 1. 5 g C18 solid phase extraction powder were grinding for 5 min. The mixture was eluted with 10 mL of acetone. The eluent was concentrated to 0. 4 mL, and then mixed with 20 μL of tetrachloromethane and 5. 0 mL of ultrapure water to form a homogeneous cloudy solution. The emulsion was broken by centrifugation. About 1 μL of sediment was injected and analyzed directly by GC_MS. Good linearities for three pyrethroids were ranged from 5 to 200 μg/kg (r2≥0. 9989), and recoveries at three spiked levels were ranged from 86 . 5% to 108 . 0% with RSDs less than 7 . 8%. The LODs of three pyrethroids were 1. 00-1. 48 μg/kg. This method can meet the determination of trace pyrethroids in soil.

Objective To study the expression of GRHL-3 in diffuse large B-cell lymphoma (DLBCL) tissues and its clinical significance.Methods One hundred and sixty-eight pathology paraffin-embedded diffuse large B-cell lymphomas tissues were collected from January 2006 to September 2011.Immunohistochemistry was used to detect the expression of GRHL-3 protein in the above tissues.Results The positive expression rate of GRHL-3 protein in the GCB type tissues was higher than that in the non-GCB type tissues [84.87 ％(101/119) vs 14.29 ％ (7/49), P ＜ 0.01].Further analysis indicated that in the non-GCB type tissues,the positive expression rate of GRHL-3 in the latter stage group was significantly higher than that in the early stage group [90.00 ％ (63/70) vs 77.56 ％ (38/49), P ＜ 0.01].The positive expression rate of GRHL-3 in the lactatede hydrogenase increased group was significantly higher than that in the normal lactated hydrogenase [91.67 ％ (77/84) vs 68.57 ％ (24/35), P ＜ 0.01].The positive expression rate of GRHL-3 in the extranodal involvement status ≥ 2 group was significantly higher than that in the extranodal involvement status 0-1 group [96.29 ％ (26/27) vs 81.52 ％ (75/92), P ＜ 0.05].The positive expression rate of GRHL-3 in the IPI score 4-5 group was significantly higher than that in the IPI score 0-1 group [91.30 ％ (65/69) vs 66.67 ％ (18/27), P ＜ 0.01] and IPI score 2-3 group [91.30 ％ (65/69) vs 79.96 ％ (18/23), P ＜ 0.05].However, the expression of GRHL-3 had no correlation with the gender, age, and performance status of DLBCL.Conclusion The positive expression rate of GRHL-3 protein in the GCB type tissues is higher than that in the non-GCB type tissues.The positive expression rate of GRHL-3 in the DLBCL is correlated with the Ann Arbor stage, lactate dehydrogenase, extranodal involvement status and IPI score.