Objective To prepare centromere-associated protein E(CENP-E)polyclonal antibody with specificity by using New Zealand white rabbits.Methods Prokaryotic expression plasmid of pHis-CENPEC410 was constructed by molecular cloning technique and then transformed into competent cells of E.coli BL-21 (DE3).HisCENPEC410 fusion protein was induced by isopropyl β-D-thiogalactoside (IPTG) and purified by affinity chromatography using Ni-NTA beads.The purified protein was used as antigen to immune New Zealand white rabbits to produce spccific polyclonal antibody of CENP-E.The antibodies serum was detected by immunoblotting and co-immunoprecipitation,and the purified antibodies were detected by immunofluorescene staining.Results The results of immunoblotting and co-immunoprecipitation demonstrated that the antibody serum was effective and the purified antibody could be applied to immunofluorescene test.Conclusions CENP-E polyclonal antibody with high specificity and sensitivity was obtained,which lay the foundation for the follow-up study of CENP-E.

Objective To establish KIF4A 3'UTR esiRNA library and analyze the advantage of method in studying the function of KIF4A in SGC-7901 cells.Methods The GST-fusion protein of Escherichia coli endoribonuclease Ⅲ (GST-RNase Ⅲ) was used to digest the double strand RNA (dsRNA),which was transcribed in vitro from a 502bp template of KIF4A genome (T7-KIF4A 3'UTR).The KIF4A esiRNA library generated from the above method and chemically synthesized KIF4A siRNA were then used to transfect SGC-7901 cells at 10 nmol/L and 20 nmol/L.Real-time quantitative PCR and Western Blot were used to detect the mRNA and protein level of KIF4A,respectively.Results The KIF4A esiRNA library was effectively established from dsRNA digestion using GST-RNase Ⅲ.KIF4A expression was significantly reduced in SGC-7901 cells transfected with KIF4A esiRNA or siRNA.In addition,the inhibitory effect of KIF4A esiRNA was more effective than that of chemically synthesized siRNA.Conclusion KIF4A esiRNA library which was obtained using biological method can more effectively inhibited the expression of KIF4A more effectively in SGC-7901 cells than chemically synthesized siRNA.Therefore,esiRNA library can be used as a new and more effective method in studying the function of KIF4A in SGC-7901 cells.

Objective To explore the role of chromokinesin KIF4A in gastric cancer cell invasion using gastric cancer cells SGC-7901 and chromokinesin KIF4A deficient gastric cancer cells (SGC-shKIF4A).Methods Expression levels of KIF4A in controlling gastric cancer cells (SGC-shNC) and SGC-shKIF4A cells were determined by Western Blot.Invasion of gastric cancer cells were assessed using Transwell invasion assay and the number of cells passing through the matrigel was counted.Changing numbers of cortactin in SGC-7901,SGC-shNC,and SGC-shKIF4A cells were analyzed by immunofluorescence staining.Results Compared to the SGC-shNC cells,invasion ability of SGC-shKIF4A cells was increased.Compared to other cells,the numbers of cortactin in SGC-shKlF4A cells was also increased suggesting invadopodia in these cells was increased(P＜0.01).Conclusions Chromokinesin KIF4A acts as a tumor suppressor by inhibiting gastric cancer cells invasion and the results provides strong evidences for KIF4A serving as one of potent targets for gastric cancer prognostics and treatment in clinic.

Objective KIF18A is a protein that has close relation with mitotic regulation and tumor development.This study aimed to establish KIF18A protein expression system in baculovirus,which may help to realize high efficient synthesis of KIF1 8A protein in vitro.Methods Transfer vector was constructed with molecular cloning method,and recombinant baculovirus were obtained through gene transposition.KIF18A protein was expressed by transforming recombinant baculovirus into infected insect Sf-9 cells to realize the synthesis efficiency.Results It was confirmed by DNA sequencing,microscopic observation and Western Blot that the KIF18A protein expression system in baculovirus was successfully established.Conclusions Conditions for transfer vector and recombinant virus transfection are defined,and high efficient KIF18A protein baculovirus expression system are successfully constructed.

Objective To evaluate the significance of weight-bearing lateral radiographs in evaluation of malunited frac-tures of the ankle. Methods 17 patients with malunited fractures of the ankle were treated by different reconstructive operations be-tween March 2010 and October 2012, including 9 females and 8 males, aged from 17 to 64 years. According to the Takakura classifi-cation of ankle arthritis, there were 7 patients in grade 1, 4 in grade 2 and 6 in grade 3. Simple open reduction and internal fixation were performed in 5 patients, supramalleolar tibial osteotomy in 5, lengthening osteotomy of the fibula in 2, supramalleolar tibial and fibular osteotomy in 5. Tibiofibular clear space on the anteroposterior radiograph. Medial clear space, tibiofibular clear space and tib-iofibular overlap on ankle mortise radiographs were compared preoperatively and at last follow-up. Tibial lateral surface angle (TLS), the offset of the center of talar rotation from the tibial axis, and the displacement of tibiotalar articular surface center on weight-bear-ing lateral X-ray were also compared preoperatively and at last follow-up. AOFAS score was used to evaluate the function of the ankle. Results The duration of follow-up was 9 to 32 months. Bone healing was observed in all patients, and the average healing time was 11 to 14 weeks. Ankle joint degeneration grade had no exacerbation. The medial clear space, tibiofibular clear space and tibiofibular overlap had no significant difference between the pre and postoperative. Statistically significant change was seen postoperatively in the values of TLS (76.9°±4.1° vs 80.9°±5.2°). The offset of the center of rotation from the tibial axis and the displacement of tibiotalar articular surface center were changed from 10.8 ± 2.1 mm to 2.0 ± 0.5 mm and 4.5 ± 1.5 mm to 2.2 ± 1.0 mm, respectively. The average AOFAS score was improved from 45.7 ± 15.9 points preoperatively to 82.0 ± 9.9 points postoperatively. Conclusion Weight-bearing lateral radiographs can be used to judge the ankle restoration. Even if the mortise radiograph had relative good realignment, it may ap-pear obvious deformity on lateral radiographs. Good reduction of lateral radiographs requires that the mid axis of the tibia should pass through the center of talar rotation and tibiotalar articular surface be parallel on the lateral radiograph.

Objective To investigate the clinical curative result of the new method of postermedial deepen of the fibular groove in the treatment of chronic peroneal tendon subluxation.Methods From March 2006 to October 2012,21 patients (15 male,6 female) with chronic peroneal tendon subluxation via a novel method of postermedial deepen of the fibular groove.In this group:Ⅰ grade 5 cases;Ⅱ grade 9 cases;Ⅲ grade 5 cases;Ⅳ grade 2 cases.All patients were followed up for at least 24 months.The American Orthopaedic Foot and Ankle Society (AOFAS) Ankle-Hindfoot scale score and visual analogue score (VAS) were used to evaluate the clinical outcomes.Operation time,time to heal and complications were also recorded.Results The average operation time was 50 minutes (30-70 minutes).The blood loss was 30-60 ml,average 40 ml.All incisions healed after operation,no case of incision infection,skin necrosis and sural nerve,vascular injuryed.Mean AOFAS score was significantly increased from (55.2±7.1) preoperative to (93.6±5.6) postoperative.15 patients with excellent,good for 1 cases.The excellent and good rate was 100％ (16/16).VAS score by an average of (5.3±2.1) points down to (1.2±1.1) points postperative.All the patients can wear normal shoes postoperative,waking with normal gait.No patients had peroneal tendon tenosynovitis,tendon adhesion,fracture of lateral malleolus and other serious complications,no dislocation recurrent,strength of the peroneal muscle returns to normal.Conclusion The novel method of postermedial deepen of the fibular groove in the treatment of chronic peroneal tendon subluxation was safe and effective for ankle instability with a relatively short time.

Objective To discuss rational diagnosis and treatment of acute infrarenal abdominal aortic occlusion. Methods Retrospective analysis was made on 6 cases with acute infrarenal abdominal aortic occlusion from January 2005 to December 2008. Emergency operations of retrograde catheter were done on 3 cases, 2 cases received transaortic embolectomy, 1 case received anticoagulation therapy successfully. Results Two cases were cured, 2 cases with 3 legs received amputation, 2 cases died. The time in hospital was 4 hours to 122 days, averaged (24±55) days. Conclusions A prompt thrombolytic, anticoagulation therapy and operation are suggested. It is emphasized to prevent reperfusion injury after arterial ischemia during the peri-and post-operation. Conservative treatment may be used in the patients incorporated with seriously multiple organ failure.

Objective Mitotic spindle and midbody are both microtubule-based temporary structures during cell growth and play essential roles in mitosis.The purpose of this study was to establish a mature and efficient method to extract mitotic spindle and midbody.Methods Through the cell cycle synchronization method,mitotic spindle or midbody was made appear inside cells.Low permeability swelling and glycerol gradient centrifugation principles were then used to extract spindle and midbody.Results By Western Blot and immunofluorescence staining,the extracts were identified as mitotic spindle and midbody.Conclusions The successful extraction of mitotic spindle and midbody from synchronized Hela cells will provide foundation for identifying the proteins located in cell during mitosis,and be of great significance to the study of molecular regulation mechanisms of mitosis and tumorigenesis.

Objective To construct stable chromokinesin KIF4A knockdown gastric cancer cell lines (SGC-7901) and study the mitotic spindle midzone formation in these cells.Methods Short hairpin RNA (shRNA) expression plasmids of pGPU-GFP-shKIF4A,pGPU-GFP-shNC specific targetting KIF4A and non-sense DNA sequences were constructed respectively and then transfected into SGC-7901 cells.The cells were cultured in selection medium containing G418 to form single clones.Stable KIF4A shRNA expressing SGC-7901 cells were picked up under an fluoroscent microscope and identified by Western Blot.The spindle midzone in these cells was analyzed after immunofluorescence staining.Results Three cell lines stably expressing KIF4A shRNA and one with shNC were successfully constructed.Compare to the control cell line,KIF4A knockdown cell lines represented remarkable elongation of spindle midzone and the less the KIF4A,the longer spindle midzone.Conclusions Stable KIF4A knockdown SGC-7901 cell lines were successfully constructed that can serve for further study of KIF4A ' s function and its role in proliferation and development of gastric cancer.

Objective To explore the expressions of cyclooxygenase-2 (COX-2), phosphatase and tensin homolog deleted on chromosome ten (PTEN) in hepatobiliary calculus associated with cholangiocarcinoma (HCWC) and their clinical significance. The relationship between the expressions of COX-2, PTEN and the onset and progression of HCWC was investigated to form an experimental base for the prevention and treatment of HCWC. Methods Thirty seven patients with tumor tissues of HCWC (group C), thirty patients with tissues of bile duct surrounding intrahepatic calculus (group B), and ten patients with normal tissues of bile duct from operations of hemangiomas of liver or liver trauma as the control (group A) were sampled and collected. A two-step immunohistochemistry (SP method) was employed to detect and statistically analyze the expressions of COX-2 and PTEN in each of the 3 groups. Results In groups A, B, C, the positive rate of the expression of COX-2 was 10%,33.3%, and 70.3%, respectively. The positive rates of expression of COX-2 in the carcinoma tissues of HCWC was significantly higher compared with the control group (P＜0. 01). In groups A, B, C the positive rates of the expression of PTEN was 90. 0%, 80. 0%, and 35.0%, respectively. The positive rate of expression of PTEN in the carcinoma tissues of HCWC was significantly lower than the control group (P＜0. 01). The expression of COX-2 was followed by a low expression of PTEN in the tissues of HCWC. Kendall's related analysis showed a strong negative correlation between the expression of COX-2 and PTEN in HCWC (r=-0. 323, P＜0. 05). Conclusions A high expression of COX-2 was related to HCWC. There was a negative correlation between the expressions of COX-2 and PTEN in HCWC. A high expression of COX-2 and a low expression of PTEN suggested a high chance of HCWC in extrahepatic or lymphatic metastasis.