Objective To design a pair of allele-specific diagnostic primer for authenticating Dendrobium huoshanense from other species of Dendrobium Sw.only by PCR.Methods Based on rDNA ITS sequences database of D.huoshanense and other species of Dendrobium Sw.,an allele-specific diagnostic primer pairs was designed.Diagnostic PCRs were performed using the primer with the total DNAs of 19(original) plants from Dendrobium Sw.as templates.The positive was the genuine.Results When the(annealing) temperature was raised to 60 ℃, the template DNA of D.huoshanense could be amplified whereas the diagnostic PCR of the rest species were all negative.Conclusion The diagnostic PCRs can authenticate D.huoshanense from other species of Dendrobium Sw.efficiently.Compared to the authentication method by sequencing DNA fragments and morphological traits etc.,the allele-specific diagnostic PCR is not only simple with time-saving but also practical and effective.

Colorectal cancer is a common malignant tumor,and invasion and metastasis are the major causes of death. Epithelial-mesenchymal transition(EMT)occurs along with transition from epithelial phenotype to mesenchymal phenotype. EMT is involved in the genesis,development,invasion and metastasis of various tumors including colorectal cancer. EMT is closely associated with chemotherapy resistance of tumors. Various signaling pathways are involved in the pathogenesis of EMT and its mechanism is not fully clear. This article reviewed the molecular mechanism and role of EMT in colorectal cancer.

Objective To investigate effects of ACUMED self-orientated elbow plate system in treatment of comminuted distal humeral fractures (type C) in adults.Methods Thirty-two adult cases of comminuted distal humeral fractures treated by open reduction and internal fixation with parallel dual plates from May 2009 to October 2010 were enrolled in the study.There were 17 males and 15 females,at average age of 60 years (range,21-85 years).All cases were subjected to closed fractures,involving left side in 15 cases and right side in 17.Besides,two cases had associated ulnar nerve injury.According to AO classification,there were 12 cases of type C1,12 of type C2 and eight of type C3 fractures.The time from injury to operation averaged 5.6 days.Distal humeri were exposed through posterior median incision of elbow and V shape osteotomy of olecroanon.Muscular contractile and relaxant functional exercise was taken within 24 hours postoperatively.Clinical results were assessed based on Mayo elbow performance score (MEPS) and X-ray films.Results X-ray films showed that all cases obtained primary bone healing in the follow-up (mean 12 months).Three cases of type C3 fractures had heterotopic ossification.All cases had no complications like infections or implant loosening postoperatively.Range of motion in elbow flexion and extension averaged 85° (range,8°-140°).MEPS was average 90 points (range,60-100 points),including excellent outcomes in 13 cases,good in 15,fair in one and poor in three,with excellence rate of 88％.Conclusion ACUMED parallel dual plate fixation technique based on stability theory of arch structure achieves solid fixation of adult comminuted distal humeral fractures,successfully reconstructs bone structure of distal humeri,and effectively restores elbow joint function in response to early rehabilitation exercise.

Objective To investigate the expression of stathmin and evaluate its influence to anti-microtubule adjuvant chemotherapy in non-small-cell lung cancer(NSCLC).Methods The clinical data and survival status of 78 NSCLC patients were collected,and their paraffin-embedded tissue were detected immunohistochemically with a rabbit anti-human stathmin polyclonal antibody.The clinical significance of stathmin expression and its influence to overall survival rate were analyzed statistically between patients who received paclitaxel or vinblastine adjuvant chemotherapy.Results The positive expression of stathmin could only be observed in the cytoplasm of cancer cells.Among 78 patients,40 (51.3 ％ ) patients were stained stathmin-positive.The positive rate of stathmin was significantly higher in male than female,in central type than peripheral type,in pleura-involved than non-involved,and in dead patients than survival patients ( P ＜ 0.05 ),but showed no significant differences in patients with different age,differentiated grade,pathological type,clinical stage,or lymph-node metastasis status.The expression of stathmin had a significant influence to overall survival rate(x2 =4.348,P ＜0.05 ),and those stathmin-negative patients showed a longer survival time.In stathmin-negative patients,those who received adjuvant chemotherapy with vinblastine exhibited a shorter survival time than those with paclitaxel,but the P =0.06.In stathmin-positive patients,the survival rate or time showed no difference between groups with paclitaxel and vinblastine.The differentiated grade,metastasis to lymph node and expression of stathmin were independent risk factors influencing survival rate.The positive expression of stathmin could only be observed in the cytoplasm of cancer cells.Among 78 patients,40 (51.3 ％ )patients were stained stathminpositive.The positive rate of stathmin was significantly higher in male than female,in central type than peripheral type,in pleura-involved than non-involved,and in dead patients than survival patients ( P ＜ 0.05 ),but showed no significant differences in patients with different age,differentiated grade,pathological type,clinical stage,or lymph-node metastasis status.The expression of stathmin had a significant influence to overall survival rate(x2 =4.348,P ＜ 0.05 ),and those stathmin-negative patients showed a longer survival time.In stathmin-negative patients,those who received adjuvant chemotherapy with vinblastine exhibited a shorter survival time than those with paclitaxel,but the P =0.06.In stathmin-positive patients,the survival rate or time showed no difference between groups with paclitaxel and vinblastine.The differentiated grade,metastasis to lymph node and expression of stathmin were independent risk factors influencing survival rate.Conclusion Our study suggested that the detection of stathmin in resected NSCLC tumor tissues may be helpful for prediction of prognosis,but helpless for making a choice between paclitaxel and vinblastine.NSCLC patients with stathmin-negative,no metastasis to lymph node or good-differentiated grade may have a better prognosis.

Objective To investigate the expression of sphingosine kinase 1(SphK1)and NF-κB in colon carcinoma tissues and their correlation with clinicopathologic features.Methods Sixty-six paraffinembedded colon carcinoma samples and 66 fresh colon carcinoma samples were tested using immunohistochemistry,RT-PCR and Western blot,respectively.Results In 66 fresh colon carcinoma samples,the positive rate of SphK1 and NF-κB mRNA expression were 84.85％(56/66)and 74.24％(49/66),while the positive rate of SphK1 and NF-κB protein detected by Western blot were 78.79％(52/66)and 69.70％(46/66).The positive rates were higher than those in the adjacent tissues[mRNA:63.64％(42/66),48.49％(32/66);protein:57.58％(38/66),45.45％(30/66)]and the normal mucosa [mRNA:42.42％(28/66),25.76％(17/66); protein:36.36％(24/66),24.24％(16/66)],with statistical significances(all P values ＜ 0.05).The mean expressive levels of SphK1 and NF-kB mRNA and protein in colon carcinoma were both significantly higher than those in the adjacent tissues and the normal mucosa(mRNA:0.55±0.06 vs0.35 ±0.05 vs0.25±0.05,0.75 ±0.06 vs0.43±0.05 vs0.30±0.04 ; protein:0.77 ± 0.05 vs 0.38 ± 0.06 vs 0.12 ± 0.03,0.45 ± 0.08 vs 0.23 ± 0.05 vs 0.13 ± 0.03 ;all P values ＜ 0.05).There was a close correlation between SphK1 and NF-kB expression levels (r =0.459,P =0.036).The results of immunohistochemistry were similar to those of RT-PCR and Western blot.Overexpression of SphK1 and NF-κB in colon carcinoma was related with depth of invasion,distant and lymph node metastasis and Dukes'stages(all P values ＜0.05).The expression of SphK1 was also related with differentiation(P ＜ 0.05).Conclusions Overexpression of SphK1 and NF-κB may be involved in the pathogenesis and progression of colon carcinoma.Moreover,SphK1 and NF-κB may be correlated with the invasion and metastasis of colon carcinoma.

Objective A large number of injured earthquake patients were accepted by the hospital whilethe professional surgeons were relatively lack. This article introduced the hospital emergency management in 2008Sichuan Wenchuan Earthquake, China. Method Within 3 days, Central Hospital of Mianyang accepted andtreated over 1000 patients after Wenchuan Earthquake jolted on 12 May 2008,and within 2 weeks, the number ofpatients reached 1500. The hospital carded out emergency management plan: (1) emergency comprehensive treat-ment district was established, which was divided into traumatic surgery district, general surgery district, and gen-eral medical district. Traumatic surgery district is responsible for treating traumatic patients, and most doctors andnurses were in this district. The district also had preview, contamination, operation, isolation, monitoring sec-tions, and the tents were numbered and labeled. General surgery district and general medical district were responsi-ble for patients not from earthquake, and only few doctors and nurses were in the two districts. According to the in-jury degree, all the wounded were classified into acute and severe, moderate and minor injuries, and wore red,yellow and blue label on the wrists, respectively. The name, gender, age and diagnosis of each patient and thename of doctor were written on the label. The infectious patients and non-infectious patients were separated.Results Near 200 operations and near 300 operations were performed at one night and at one day, respectively.Within one week, only 1 patient had the lung infection, and one patient with gangrenous emphysema was effective-ly treated. In-hospital cross infection and epidemic of infection disease didn't happen. Conclusions Emergencymanagement model and mechanism, which referred to the model of the battlefield ambulance, played an importantrole in treating a large number of injured patients.

Objective To evaluate the therapeutic effect of endoscopic management of plastic stents of post-liver transplantation anastomotic biliary stricture. Methods From January 2010 to October 2015, clinical data of patients with post-liver transplantation anastomotic biliary stricture and received endoscopic retrograde cholangiopancreatog﹣raphy and plastic stents management was collected. The technical success rate, ERCP-related complications, clinical remission rate and long-term complications were main outcome measurements to compare the efficacy and safety of different number of stents in managing post-liver transplantation anastomotic biliary stricture. Results Among the 18 patients (0.5 ~ 60.0 months) with post-liver transplantation ABSs, seven patients received less plastic stents treat﹣ment (< 3 stents), nine patients with persistent anastomotic or recurrent stricture received multiple plastic stents treatment (≥ 3 stents), two patients received multiple plastic stents treatment once suffered with post-liver trans﹣plantation ABSs. The endoscopic technical success rate was seventy-six over eighty (95.0 %). Among the seven pa﹣tients received less plastic stents treatment, one loss to follow-up, two were still under treatment, one died of acute hepatic failure, one died of septic stock, one combined with biliary fistula resulted in treatment failure, one achieved clinical remission, the clinical remission rate was one third (33.3 %). Among the eleven patients received multiple plastic stents treatment, two loss to follow-up, one was still under treatment, two received surgery because of failed treatment, six achieved clinical remission, the clinical remission rate was 75.0 % (6/8). The average diameters and stent durations of management of 1 stent, 2 stents, 3 stents, 4 stents, 5 stents, 7 stents were 8.5 F, 17.0 F, 24.0 F, 28.0 F, 36.0 F, 50.0 F. Among the six early postoperative complications, five cases occurred in less stent manage﹣ment and one occurred in MPSs management, the early postoperative complication rate was 7.5 %(6/80). No severe ERCP-related complications and procedure-related deaths. Conclusions Endoscopic management of plastic stents is safe and effective for post-liver transplantation ABSs. Providing larger biliary support, the multiple plastic stents treatment was superior to less plastic stents treatment in view of clinical remission rate, especially for refractory one. Multiple plastic stents did not increase the incidence of complications, it could be used as the first-line treatment of post-liver transplantation duct-to-duct biliary anastomosis for its safety and effectivity.

Objective:To express human epididymis protein 4 (HE4) in prokaryotic cells,purify the expressed product and de-termine its activity by immunoassay kit.Methods: The gene encoding HE 4 was cloned using RT-PCR technique from total RNA of ovarian carcinoma cells ES-2,the amplified HE4 gene was cloned into prokaryotic expression vector pGEX-4T-1 and PET26b respective-ly.The recombinant plasmid pGEX-4T-1-HE4 and PET26b-HE4 were constructed and transformed into E.coli BL21 cells respectively, and protein ( GST-HE4 and His-HE4 ) expressions were induced by IPTG and identified by SDS-PAGE and commercial ELISA kit.Results:Restriction analysis and sequencing proved that recombinant plasmid pGEX -4T-1-HE4 and PET26b-HE4 were constructed correctly.The expressed recombinant proteins ,with the relative molecular mass of about 38 000 and 12 000 ,showed specific binding to monoclonal antibody against HE 4.Conclusion:Two kinds of recombinant HE 4 protein are successfully expressed in prokaryotic cells , which laid a foundation of preparation of immunoassay reagents.

[ ABSTRACT] AIM:To investigate the effects of sphingosine kinase l ( SphK1) and focal adhesion kinase ( FAK) on the epithelial-mesenchymal transition ( EMT) of human colon cancer HCT 116 cells.METHODS:Human colon cancer HCT116 cells were divided into 3 groups.N, N-dimethylsphingosine (DMS) was used to suppress the activity of SphK1. PF573228 was used to suppress the activation of FAK .The cells treated with equal volume of culture medium severed as control group.The cell viability was measured by MTT assay .The protein expression of SphK1, FAK and the EMT relative protein E-cadherin, N-cadherin, vimentin and matrix metalloproteinase (MMP) 2 was analyzed by Western blot.The mR-NA expression of SphK1, sphingosine-1-phosphate (S1P), FAK, E-cadherin and vimentin was detected by real-time PCR. The ability of tumor cell migration was measured by wound-healing assay.RESULTS:The cell viability of HCT116 cells was suppressed by DMS and PF 573228 in dose and time dependent manners .DMS significantly suppressed the expression of SphK1, FAK, N-cadherin, vimentin and MMP2, meanwhile enhanced the expression of E-cadherin.PF573228 reduced the expression of FAK , SphK1, N-cadherin, vimentin and MMP2, meanwhile increased the expression of E-cadherin (P<0.01).In addition, the migration ability of HCT116 cells was significantly decreased by treating with DMS and PF573228 (P<0.01).Compared with control group , the mRNA expression of FAK, SphK1, S1P and vimentin was de-creased, while the expression of E-cadherin was increased significantly in PF573228 group and DMS group (P<0.05). CONCLUSION:SphK1 and FAK signaling pathways may play an important role in the occurrence of EMT in the colon cancer HCT116 cells.

Objective To construct an eukaryotic expression vector of human telomerase reverso transcriptase (hTERT) gene specific shRNA, and investigate the effect of pshRNA-hTERT combined with γ-irradiation on telomerase activity and DNA damage. Methods The recombinant expression plasmid pshRNA-hTERT was constructed and transfected into Hep-2 cells. The telomerase activity was examined by the PCR-hased telomeric repeat amplification protocol (TRAP). DNA single-stranded break (SSB) and the DNA double-stranded break (DSB) were detected by Comet assay. The xenograft model of human laryngeal carcinoma with the same genetic background and different radiosensitivity (Hep-2 and Hep-2R) was established in nude mice. The mixture of pshRNA-hTERT and liposome was injected to the transplanted tumor to observe the inhibition of the tumor growth. The cell apoptosis was detected by TUNEL. The hTERT protein expression was determined by streptavidin-peroxidase conjugated method (AP). Results Recombinant expression plasmid pshRNA-hTERT was successfully constructed and transfected into Hep-2 cells. The hTERT expression inhibition rate reached 60.78 %. pshRNA-hTERT not only inhibited telomerase activity of Hep-2 inehiding the increase of telomerase activity induced by γ-irradiation, but also inhibited the repair of the SSB and DSB induced by irradiation in the human laryngeal carcinoma xenograft in nude mice with the same genetic background and different radiosensitivity. The pshRNA-hTERT combined with γ-irradiation could inhibit the growth of transplanted tumor (Hep-2: EPO = 1.79; Hep-2R: EPO = 2.01) with reduced telomerase activity and hTERT protein expression. Conclusions The eukaryotic expression vector pshRNA-hTERT could enhance the radiosensitivity of Hep-2 cells in vitro and the human laryngeal carcinoma xenograft in nude mice which had the same genetic background with different radiosensitivity.