A secretive expression vector of Pichia pastoris system which can be used for the direct cloning of PCR products was constructed,and was verified through the expression of recombinant cellobiohydrolase II in Pichia pastoris.A randomly selected fragment was amplified with properly designed primers by PCR.The XhoI and Eam1105Ⅰ restriction sites were included in the 5'end of the fragment,and the Eam1105Ⅰ and XbaI restriction sites were included in its 3'end.The PCR amplified product was inserted into the P.pastoris expression plasmid pPICZ?A through XhoI and XbaI restriction sites and the resultant plasmid was digested with Eam1105Ⅰ,and lastly the big fragment was recovered,generating the P.pastoris expressive Tvector pPICZ?T.Then the cellobiohydrolase II of T.reesei was successfully expressed in P.pastoris with this expressive Tvector.Such results indicated that the constructed expression Tvector was convenient for PCR product cloning,and was effective for heterologous protein expression in P.pastoris.On the other hand,the application of the expression Tvector avoided the introduction of additional amino acids at the Nterminus of the expressed protein,which generally occurred when normal expression vectors were used in secretive expression system.

OBJECTIVETo evaluate the efficacy and safety of three antithrombotic agents on venous thromboembolism (VTE) after unilateral total knee arthroplasty.

METHODSFrom November 2011 to March 2014, 149 patients undergoing unilateral total knee arthroplasty for knee osteoarthritis were reviewed. Among them, there were 66 males and 83 females, ranging in age from 48 to 76 years old. All the cases were randomly divided into three groups including Aspirin group, low-molecular-weight heparin (LMWH) group, and rivaroxaban group, according to antithrombotic agents. Deep vein thrombosis (DVT), pulmonary embolism (PE) and bleeding complication (including wound ecchymosis, hematoma and other local complications, gastrointestinal, cardiovascular, urinary hemorrhage and other major bleeding events) of antithrombotic agents were observed and analyzed statistically at the 6 week, 8 week, and 12 week after operation.

RESULTSAmong patients who received Aspirin (48 cases), 4 patients had DVT, in 1 patient had PE, and 2 patients had bleeding complication. Among 54 patients in low-molecular-weight heparin group, 3 patients had DVT, 1 patient had PE, and 3 patients had bleeding complication. While among those patients received the rivaroxaban (47 cases), 3 patients had DVT, 0 patient had PE, and 11 patients had bleeding complication. There were no statistically differences among three groups on DVT, and PE (P>0.05). The incidence of bleeding complication in rivaroxaban group was higher than the other two antithrombotic agents, and the difference among the three groups was statistically significant (P<0.05).

CONCLUSIONAspirin, low-molecular-weight heparin, and rivaroxaban could effectively reduce the incidence of VTE after total knee arthroplasty, and their efficacy was similar. Rivaroxaban has a higher incidence of bleeding complication and further clinical trials are required to be conducted to assess the safety of rivaroxaban in clinical.

Aged ; Arthroplasty, Replacement, Knee ; adverse effects ; Aspirin ; therapeutic use ; Case-Control Studies ; Female ; Fibrinolytic Agents ; therapeutic use ; Heparin, Low-Molecular-Weight ; therapeutic use ; Humans ; Male ; Middle Aged ; Postoperative Complications ; prevention & control ; Rivaroxaban ; therapeutic use ; Venous Thromboembolism ; prevention & control

Objective To compare the efficacy of endovenous laser treatment combined with transilluminated powered phlebectomy and traditional surgical treatment for great saphenous varicosity. Methods From January 2014 to January 2015,77 patients diagnosed as great saphenous varicosity were enrolled. They were randomly divided into two groups, 37 patients received endovenous laser treatment combined with transilluminated powered phlebectomy and the other 40 patients underwent traditional surgical treatment. The related indicators were compared.Results The mean operation time,average bleeding volume,operative incisionquantity and average hospitalization time of EVLT+ TIPP group were (66.1±14.7) min,(24.4±10.5) ml,5.7±1.7,(5.6±1.4) d respectively,of control group were (84.3±18.5) min,(59.0±15.6) ml,8.0±1.8,(10.1±3.1) d respectively,there were significant differences between the two groups(t =-4.749,-11.460,-5.714;P<0.001) .There were no significant complications in the two groups.In the EVLT+ TIPP group,6 patientsoccurred patsubcutaneous induration, 5 patients occurred subcutaneous hematoma and 7 patients had skinparesthesias.In the traditional operation group,2 cases occurred patsubcutaneous induration,3 cases occurredsubcutaneous hematoma and 2 cases had skin paresthesias.The EVLT+ TIPP group was slightly higher than thetraditional operation group in the complications, but there were no significant differences between them ( P>0.05).During a mean follow?up time of (18±7) months(12 months to 24 months),1 patient recurrence in theEVLT+ TIPP group,2 cases recurrence in the traditional operation group.The recurrence rates of the two groupswere similar,the difference was not significant between them(P=1.000) .Conclusion Endovenous lasertreatment combined with transilluminated powered phlebectomy for great saphenous varicosity is effective andsafe.It also has the advanta and better cosmetic results.

OBJECTIVETo evaluate the feasibility of the pedicle screw pilot holes placement in thoracic spine using the spiral wires as the guide pin.

METHODSThe pedicle screw pilot holes were drilled within the center of the pedicle and the lateral and medial pedicle walls were violated in 9 human dried thoracic vertebrae. Kirschner wires or spiral wires were separately placed in the holes, and then the posteroanterior and lateral radiographs were taken. The radiographs were evaluated by 3 experienced spine surgeons and 3 young orthopedists. After radiographs were shown to these observers, they combined the posteroanterior and lateral radiographs in each place and determined whether the pedicle screw pilot hole violated the pedicle cortex or not. The results were analyzed by a statistical software.

RESULTSSensitivity, specificity and accuracy of the method using spiral wires to detect pedicle pilot hole placement were significantly higher than those of using Kirschner wires. With a true posteroanterior radiograph, the sensitivity, specificity and accuracy of the method using spiral wires approximated or attained 100%.

CONCLUSIONSThe method of intrapedicular pilot hole placement verification using spiral wires is effective for guiding the accurate placement of pedicle screws.

Bone Screws ; Bone Wires ; Cadaver ; Feasibility Studies ; Humans ; Internal Fixators ; Predictive Value of Tests ; Radiography ; Reproducibility of Results ; Sensitivity and Specificity ; Spinal Fusion ; instrumentation ; Thoracic Vertebrae ; diagnostic imaging ; surgery ; Treatment Outcome

This study was carried out to examine the effect of different donor cell type and micro-manipulation on the development of reconstituted embryos. Cultured mural cumulus cells or fibroblast cells from an adult transgenic goat expressing human erythropoietin(rhEPO) were used as the donor cells in nuclear transfer experiments. The reconstituted eggs were generated by transferring fibroblast cells or cumulus cells into the perivitelline space of enucleated M II oocytes and then followed by electrofusion and activation. After 6 days' incubation in vivo, the reconstructed embryos developed into morulae or blastocysts were transferred into 6 foster recipients. Two of the foster-mothers were pregnant and gave birth to two offspring, which were derived from the fibroblast cell and cumulus cell, respectively. Fingerprint analysis showed that the PCR-RFLP patterns of the two offspring were identical to that of donor goats. PCR results indicated that these cloned goats carried hEPO gene as same as their donor cells.
Animals ; Animals, Genetically Modified ; genetics ; Cell Fusion ; methods ; Cloning, Organism ; Embryo Transfer ; trends ; Erythropoietin ; biosynthesis ; genetics ; Fibroblasts ; cytology ; Goats ; embryology ; genetics ; Humans ; Microinjections ; methods ; Nuclear Transfer Techniques ; Oocytes ; cytology

Objective:To study the expression of FOXP3 in gastric cancer and its prognostic significance.Methods:One hundred and six specimens of gastric adenocarcinoma confirmed by pathology from the Department of Gastrointestinal Surgery of the Second People′s Hospital of Lianyungang in Jan. 2005 to Dec. 2015 were selected as the observation group and 85 normal para-cancer tissues as the control group. The expression of FOXP3 in 106 cases of gastric cancer was detected by tissue microarray and immunohistochemical technology. Cox regression model was used to analyze the correlation between survival data and prognosis, and Kaplan-Meiers was used to analyze the relationship between FOXP3 expression and the prognosis of gastric cancer patients.Results:The positive rate of FOXP3 expression was 53.8% (57/106) in gastric cancer tissues and 28.2% (24/85) in paraneoplastic tissues, and the difference was statistically significant ( χ2=12.597, P<0.001). The level of FOXP3 expression in gastric cancer tissues was significantly correlated with the TNM stage of patients′ tumors ( χ2=4.402, P<0.05). Single-factor Cox survival analysis showed that age, tumor pathological grade, tumor size, whether it invaded lymph nodes, TNM stage, FOXP3 expression level and prognosis were correlated.Kaplan-Meier survival analysis showed that the survival rate of tumor cell FOXP3-positive group was lower than that of FOXP3-negative group; in the subgroup type, among the subgroups, any age group, any sex group, stage Ⅲ and Ⅳ tumor cell subgroups, low- and undifferentiated groups, tumor size <16 cm 3 group, and tumor cell FOXP3 expression-positive group invading lymph nodes had lower survival than tumor cell FOXP3 expression-negative group. Conclusions:The expression of FOXP3 is up-regulated in gastric cancer. The expression of FOXP3 in gastric cancer may be a prognostic factor, and the positive expression of FOXP3 may indicate a worse prognosis.

Objective The aim of this study was to search for a better individualized clinical radiotherapy scheme for breast cancer patients after modified radical mastectomy by comparing the dosimetric data on the radiotherapeutic techniques of three-dimensional conformal radiation therapy (3D-CRT) and volumetric modulated arc therapy (VMAT). Methods This retrospective study included 77 cases of breast cancer treated by modified radical mastectomy followed by radiotherapy in Hebei General Hospital from November 2015 to February 2019. According to the radiotherapeutic techniques used, we divided the patients into a 3D-CRT (n = 54) and a VMAT group (n = 23), Using the dose-volume histogram, we evaluated the dose parameters of the clinical target volume (CTV) and the organs at risk (OAR). Results Compared with the patients in the 3D-CRT group, those in the VMAT group showed significantly decreased V110% ([12.14 ± 14.03]% vs [7.18 ± 6.36]%, P < 0.05), increased conformity index (0.62 ± 0.11 vs 0.66 ± 0.09, P < 0.05), reduced ipsilateral lung dose ([1308.42 ± 276.49] vs [1114.34 ± 233.71] cGy, P < 0.05), V5 (P < 0.05) and V10 (P < 0.05), and elevated contralateral breast dose (P < 0.05) and V5 in those with left breast cancer ([63.49 ± 17.49]% vs [76.97 ± 11.81]%, P = 0.028). Conclusion VMAT can effectively reduce the average dose and the dose in the low-dose area of the ipsilateral lung of the breast cancer patient after modified radical mastectomy. VMAT and 3D-CRT each have its own advantages for specific patients.

Objective: To study the effect of Rosae Chinensis Flos total flavones(RCTF) on the focal cerebral ischemia-reperfusion model in rats, in order to preliminarily explore the mechanism of action. Method: Rats were randomly divided into sham-operated group, model group, large, medium, and low-dose RCTF group(200,100,50 mg ·kg^-1) and positive group[Nimodipine group(20 mg ·kg^-1) and Naoluotong group (500 mg ·kg^-1)]. After 7 days of continuous administration, 1 hour later after the last administration, the middle cerebral artery middle cerebral artery occlusion (MCAO) model was duplicated. After 2 hours of modeling, perfusion was performed for 22 hours. Mortality and neurological deficits were scored. Serum S-100β was detected; brain tissue malondialdehyde(MDA), superoxide dismutase (SOD), nitric oxide (NO), nitric oxide synthase (NOS), tumour necrosis factor-α(TNF-α), interleukin-1β(IL-1β), intercellular adhesion molecule-1(ICAM-1), adenosine triphosphate (ATP)ase were measured. The brain tissue morphological changes were observed. Result: The rat model of focal cerebral ischemia and reperfusion was successfully replicated. Compared with the model group, RCTF in large, medium, and low-dose RCTF group significantly decreased the score of neurological deficit in rats (P<0.01), significantly decreased the content of S-100β in serum (P<0.01), significantly reduce the levels of MDA, NO and NOS in brain tissue (P<0.01), significantly increase the level of SOD in brain tissue (P<0.01), and significantly increase Na⁺K⁺-ATPase, Mg²⁺-ATPase, and Ca²⁺ in brain tissue (P<0.01), significantly reduced TNF-α content, IL-1β, ICAM-1 content in brain tissue (P<0.01), and significantly improved brain tissue damage (P<0.01). Conclusion: RCTF have a protective effect on cerebral ischemia-reperfusion injury in rats. The mechanism may be related to the resistance of anti-free radicals, the reduction of inflammation in brain tissue and the improvement of brain energy metabolism after cerebral ischemia reperfusion injury.

OBJECTIVETo investigate the optimal time window for intervention of BK virus (BKV) replication and its effect on the outcomes of kidney transplant recipients (KTRs).

METHODSA retrospective analysis of the clinical data and treatment regimens was conducted among KTRs whose urine BKV load was ≥1.0×10copies/mL following the operation between April, 2000 and April, 2015. KTRs with urine BKV load <1.0×10copies/mL matched for transplantation time served as the control group.

RESULTSA total of 54 recipients positive for urine BKV were included in the analysis. According to urine BKV load, the recipients were divided into 3 groups: group A with urine BKV load of 1.0×10-1.0×10copies/mL (n=22), group B with urine BKV load >1.0×10copies/mL (n=24), and group C with plasma BKV load ≥1.0×10copies/mL (n=8); 47 recipients were included in the control group. During the follow-up for 3.2-34.5 months, the urine and plasma BKV load was obviously lowered after intervention in all the 54 BKV-positive recipients (P<0.05). Eighteen (81.82%) of the recipients in group A and 19 (79.17%) in group B showed stable or improved estimated glomerular filtration rate (eGFR) after the intervention; in group C, 4 recipients (50%) showed stable eGFR after the intervention. In the last follow-up, the recipients in groups A and B showed similar eGFR with the control group (P>0.05), but in group C, eGFR was significantly lower than that of the control group (P=0.001). The recipients in group A and the control group had the best allograft outcome with stable or improved eGFR.

CONCLUSIONEarly intervention of BKV replication (urine BKV load ≥1.0×10copies/mL) in KTRs with appropriate immunosuppression reduction can be helpful for stabilizing the allograft function and improving the long-term outcomes.

BACKGROUNDCoronary artery in-stent restenosis (ISR) and late stent thrombosis remain as important complications of stenting. The inflammation reactions to sirolimus and paclitaxel-eluting stents were investigated in a swine stenosis model induced by interleukin (IL)-1β.

METHODSMini pigs (n = 12; 2-3 months old and weighing 25-30 kg) were subjected to thoracotomy. Segments (10 mm) of the mid left anterior descending coronary artery and left circumflex coronary artery were exposed and aseptically wrapped with a cotton mesh soaked with IL-1β (5 µg). After 2 weeks, the animals were anesthetized and quantitative coronary arteriography (QCA) was performed. The stenosis sites were randomized into three groups for stent insertion: a sirolimus-eluting stent (SES) group (Firebird(TM), n = 7), a paclitaxel-eluting stent (PES) group (TAXUS(TM), n = 9), and a bare-metal stent (BMS) group (YINYITM, Dalian Yinyi Biomaterials Development Co., Ltd, China, n = 8). The three different stents were randomly implanted into stenosis segments. Expression of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-α), P-selectin and vascular cell adhesion molecule-1 (VCAM-1) was determined by reverse transcription-coupled polymerase chain reaction (RT-PCR).

RESULTSQCA showed severe stenosis in IL-1β treated segments. The SES and PES groups showed lower 1-month angiographic late lumen loss (LLL) within the stent and the lesion compared with BMS (P < 0.05) by follow-up QCA. The SES showed lower LLL than that of PES in reducing 1-month inflammation lesions in pigs by follow-up QCA ((0.15 ± 0.06) mm vs. (0.33 ± 0.01) mm, P < 0.0001). The neointimal hyperplasia areas in SES and PES showed lower than those of BMS (SES (11.6 ± 1.7) mm(2), PES (27.2 ± 1.6) mm(2) vs. BMS (76.2 ± 1.3) mm(2), P < 0.0001). The mRNA expression of MCP-1 by RT-PCR in SES and PES showed lower than that of BMS at 30 days after stenting (SES 0.20 ± 0.03, PES 0.48 ± 0.49 vs. BMS 0.58 ± 0.07, P < 0.05). Levels of VCAM-1 in SES were significantly lower than those of PES and BMS (SES 0.35 ± 0.08 vs. PES 0.65 ± 0.13, BMS 0.70 ± 0.06, P < 0.05). Histochemical immunostaining of vessel walls showed lower inflammatory chemokine MCP-1 expression in the SES and PES groups compared with BMS.

CONCLUSIONSESs were superior in reducing 1-month angiographic LLL in inflammation lesions in pigs, strongly suggesting that SESs can suppress inflammatory reactions in ISR at multiple points.

Angioplasty, Balloon, Coronary ; adverse effects ; Animals ; Coronary Restenosis ; prevention & control ; Drug-Eluting Stents ; adverse effects ; Inflammation ; prevention & control ; Interleukin-1beta ; pharmacology ; Male ; Paclitaxel ; administration & dosage ; Sirolimus ; administration & dosage ; Swine