Objective; To express and purify HCA518 protein and prepare its monoclonal antibody ( McAb). Methods: The HCA518 protein was expressed with gene recombinant technique in prokaryotic system and purified with nickel chelate nitrilotriacetic acid(Ni-NTA) affinity chromatography column. Hybridoma cell lines that secreted anti-HCA518 McAb were established by cells fusion and screened by enzyme linked immunosorbent assay( ELISA). The specificity of anti-HCA518 McAb wa3 identified by Western blot assay. The HCA518 protein in tumor cells was stained by immunoflourescence assay. Results: Rcombinant HCA518 protein was expressed with a purity of 98%. Two hybridoma cell lines was selected and anti-HCA518 McAb was purified from mice ascites. The titers of anti HCA518 McAb in ascites were 1?10-4 and 5?10-4 respectively. The antibody belonged to IgG2b subtype and IgM. Anti-HCA518 McAb specifically reacted with recombinant HCA518 protein and tumor cells'nuclear protein (P100). The HCA518 protein was mainly located in cell nucleus. Conclusion: Stable hybridoma cell lines that secreted anti-HCA518 McAb have been established and anti HCA518 McAb was prepared with high specificity. It has important significance for detecting HCA518 protein in tumor tissues and determining malignant proliferation status of tumor cells and predicting its prognosis.

Objective To evaluate the curative methods of periampullary diverticula(PAD) complicated with biliopancreatic disease.Methods The clinical data of 32 cases of PAD complicated with biliopancreatic disease treated by surgical or endoscopic measures were retrospectively analyzed.Results Of the 32 cases,16 cases were treated with gastroectomy and Roux-en-Y gastrojejunostomy,and 8 with gastroectomy and Billroth Ⅱ gastrojejunostomy,8 with endoscopic sphincterotomy (EST).Postoperative complication occured in 4 cases,but no operative death.Twenty-seven cases were followed up for 5 months-8 years, and the results were excellent in 20 cases, good in 7 cases.Conclusions Gastroectomy and Roux-en-Y gastrojejunostomy is a better surgical method for PAD complicated with biliopancreatic disease.EST has become a new effective measure to deal with the diseases;It will be safe to use the first method for patients with intradiverticular papilla if it treated by EST.

ObjectiveTo explore the relationship of body fat distribution with serum lipid and its potentially predictive value for dyslipidemia.MethodsA total of 784 Beijing rural residents were enrolled in this study using a cluster sampling method.The body height,weight,waist circumference (WC),hip circumference,body composition,high-density lipoprotein cholesterol ( HDL-C ),low-density lipoprotein cholesterol ( LDL-C),total cholesterol ( TC),and triglycerides (TG) were measured.The body mass index (BMI) and waist to hip ratio (WHR) were calculated.ResultsThe age-adjusted partial correlation analysis showed that WC had the best correlation with HDL-C ( r =- 0.310) and LDL-C ( r =0.204 ),while WHR with TC ( r =0.151 ) and TG ( r =0.271 ).Subgroup analysis with different BMI,WC,WHR,and trunk fat mass (TFM) showed that WC,WHR,and TFM sensitively reflected the changes of body lipids,whereas BMI,WC,WHR,and TFM sensitively reflected the low HDL,high TG,and risk of dyslipidemia.Receiver operating characteristic curve analysis showed that the predictive curves of WC,WHR,BMI,and TFM were above the reference line,and the areas under the receiver operating characteristic curves of WHR (0.684,0.630),WC (0.667,0.616),and TFM (0.661,0.604) showed high tendencies than BMI (0.629,0.597) for both male and female subjects,although no statistically significant differences were found ( all P ＞ 0.05 ).ConclusionsCompared with BMI,the body fat distribution indicators including WHR,WC,and TFM have higher predictive values in evaluating the risk of dyslipidemia.When the maximum Youden index for predicting the risk of dyslipidemia is applied,the ideal cutoff points was 24 kg/m2 for BMI,0.91 for WHR,85cm for WC,7.5kg for TFM in males,and 25 kg/m2,0.91,87cm,and 9.5 kg,respectively,in females.

Objective To evaluate the efficacy and safety of induction concurrent chemoradiation therapy with weekly docetaxel and cisplatin(DP) for stage Ⅲ A-N2 lung cancer.Methods Eighteen patients diagnosed of stage Ⅲ A-N2 NSCLC in our center were enrolled from March,2011 to November,2013.The induction regimen consisted of 5 cycles of docetaxel(20 mg/m2) and cisplatin(20 mg/m2) administered intravenously on days 1,8,15,22 and 29 with concurrent thoracic radiotherapy in fractions of 1.8Gy,to a total dose of 45Gy.Patients proceeded to surgery,if no progressive disease occurred,followed by adjuvant chemotherapy with DP strategy.Results Eighteen patients were enrolled and 12 underwent surgery.The tumor response for the induction therapy was 1 CR,10 PRs,6 SDs and 1 PD.Five of 18 patients presented with level 3 or above adverse effects,among which were 2 neutropenia,1 liver toxicity,1 anemia and 1 lymph node infection.The median operation time was 290 min,intraoperative blood loss was 350 ml,length for postoperative drainage was 5 d,and time to discharge was 7 d.The mediastinal lymphnodedownstaging rate was 50％ (3 pN0 cases and 3 pN1 ones),92％ of the operated patients reached complete resection.One-year survival was 75.9％ and 1-year progression free survival was 49.2％.Conclusion Weekly docetaxel and cisplatin strategy in induction concurrent chemoradiotherapy for stage Ⅲ A-N2 NSCLC patients has been validated to be safe and effective.

Cervical angina has been defined as chest pain that resembles true cardiac angina but originates from the disorders of the cervical spine. Thus, physicians and spine surgeons alike should raise awareness of this unusual condition for diagnosis and treatment. Particularly when neurologic signs and symptoms are present, there should be a strong suspicion for cervical angina in any patient with inadequately explained noncardiac chest pain. Cervical angina can be diagnosed according to negative cardiac workups, positive neurologic examination, and cervical radiographic findings (herniated disk, spinal cord compression, or foraminal encroachment). However, the mechanisms of pain production in cervical angina remain unclear. Previous studies attributed the pain to cervical nerve root compression, cervical sympathetic afferent fibers, referred pain, or lesions of the posterior horn of the spinal cord. Conservative treatments, which include neck collar fixation, head traction, and nonsteroidal anti-inflammatory drugs, have been determined to be successful in most patients with cervical angina. But when conservative treatment fails, anterior cervical surgery with complete decompression of the spinal cord and/or nerve root has been identified to effectively relieve cervical angina symptoms.

Cervical angina has been defined as chest pain that resembles true cardiac angina but originates from the disorders of the cervical spine. Thus, physicians and spine surgeons alike should raise awareness of this unusual condition for diagnosis and treatment. Particularly when neurologic signs and symptoms are present, there should be a strong suspicion for cervical angina in any patient with inadequately explained noncardiac chest pain. Cervical angina can be diagnosed according to negative cardiac workups, positive neurologic examination, and cervical radiographic findings (herniated disk, spinal cord compression, or foraminal encroachment). However, the mechanisms of pain production in cervical angina remain unclear. Previous studies attributed the pain to cervical nerve root compression, cervical sympathetic afferent fibers, referred pain, or lesions of the posterior horn of the spinal cord. Conservative treatments, which include neck collar fixation, head traction, and nonsteroidal anti-inflammatory drugs, have been determined to be successful in most patients with cervical angina. But when conservative treatment fails, anterior cervical surgery with complete decompression of the spinal cord and/or nerve root has been identified to effectively relieve cervical angina symptoms.

Aim To investigate the effeet of Eehinaeo- side ( ECH ) regulating the expression of prohibitin (PHB) on MPP+ -induced apoptosis of SH-SY5Y eells and the underlying mechanism.Methods SH-SY5Y eells were seleeted and divided into control group, MPP+ group, MPP+ + ECH group, NC + MPP + group, NC + MPP+ + ECH group, PHB-RNAi + MPP + + ECH group.Cell survival rate was determined by CCK-8 assay.Cell morphology was observed using an inverted phase contrast mieroscope; the apoptotie eells were observed by Hoechst33342 fluorescence staining, whereas apoptotie rate, reactive oxygen speeies eon- tent, and mitochondrial membrane potential were ana¬lyzed by flow eytometry.The relative protein expres¬sions of PHB, Akt, p-Akt, Bel-2, Bax, and cleaved- easpase3 were determined by Western blot.Results Compared with eontrol group, the eell survival rate of MPP+ group signifieantly deereased.The growth state of the eells beeame significantly worse.Intracellular ROS content inereased, mitoehondrial membrane po tential decreased, apoptosis-related protein expression increased and the apoptotic rate increased.Compared with MPP+ group, MPP+ + ECH group significantly increased cell viability.The growth status of cells was significantly improved.Intracellular ROS content de¬creased, mitochondrial membrane potential increased, apoptosis-related protein expression decreased, and the apoptotic rate decreased significantly.The expression levels of PHB and p-Akt significantly increased.Com¬pared with NC + MPP+ + ECH group, p-Akt level de¬creased and the cell apoptotic rate increased in PHB- RNAi +MPP+ + ECH group.Conclusions Echino- side can reduce MPP + - induced apoptosis of SH-SY5Y cells, which may be realized by upregulating PHB ex¬pression and phosphorylation of Akt to protect mito¬chondrial function.

Objective: To observe the effect of poloxamer 188 (P188) on megakaryocyte cultivation and induction from cord blood mononuclear cells in order to obtain more megakaryocyte progenitor cells (MPC). Methods: The cord blood mononuclear cells were isolated and inoculated in cell culture bag or cell culture flask respectively. The WIGGENS shaker and cell culture bags were used to mimick WAVE Bioreactor for three-dimensional (3D) cell culture, and the P188 was added to induction medium, The cells were detected for morphology, surface marker, viability, and number on day 14. Results: In the two-dimensional (2D) culture, CD41⁺, CD41⁺/CD61⁺, CD61⁺ megakaryocytic numbers increased significantly after adding P188 (all P<0.01). And in the 3D culture of adding P188, the cell volume became larger and the nuclear shape was irregular, the cytoplasm appeared magenta granules, and the megakaryocyte cells became more mature. By 3D culture, the expression of CD41/CD61 was (36.30±1.27)% vs (23.95±1.34)%, hence the differentiation for MPC was significantly higher than that in the 2D group (P<0.01). Furthermore, adding P188 in 3D culture resulted in highest differentiation efficiency for MPC [(59.45±1.20)%]. There were no significantly differences in terms of cell viability and cell number among 3D culture containing P188, 2D and 3D culture groups (all P>0.05). Conclusion 3D culture was beneficial for the differentiation of MPC, but the cell viability was lower than 2D group; However, the satisfied cell growth and better induction efficiency were obtained by adding of P188, which might provide a new method of megakaryocytes production for clinical application.

OBJECTIVE To provide reference for scientifi c and standardized development of clinical comprehensive evaluation of drugs in China. METHODS Guided by the theory of total quality management （TQM），drawing lessons from the successful experience of the British and German conducting evaluation ，combining with plan-do-check-act cycle and other quality management methods and tools ，drug clinical comprehensive evaluation of total quality management system was constructed in accordance with the requirements for our country related policy and local practice. RESULTS & CONCLUSIONS To construct total quality management system of clinical comprehensive evaluation of drugs in China from 5 aspects of organization system ，management process，assessment system ，evaluation and supervision platform ，support and guarantee mechanism. The organization system included national ，provincial and medical institutions ；management process should focus on the key links in the 3 stages of theme selection，evaluation and implementation ，and result transformation and application ；assessment system ，evaluation and supervision platform，support and guarantee mechanism should be established together so as to further improve the scientificity ，rationality， practicality and standardization of total quality management of clinical comprehensive evaluation of drugs. The development of total quality management is an effective starting point to promote the continual improvement of the drug clinical comprehensive evaluation；relevant government departments and the implementation of evaluation of medical institutions should further set up quality management consciousness ，establish report quality feedback mechanism and the results co-constructing and sharing mechanism and strengthen professional personnel training and innovation synergy regulation mode to ensure that the authenticity and reliability of evaluation results.

OBJECTIVETo build a protocol of separation and induction of megakaryocytes derived from cord blood mononuclear cells.

METHODSRed blood cells were precipitated by hydroxyethyl starch (HES). Mononuclear cells were obtained by density gradient centrifugation with Ficoll. The inducing efficiencies of megakaryocytes by using of different cytokine cocktails and culture media were analyzed.

RESULTSThe best choice for erythrocyte sedimentation and high efficiency of nucleated cells retrieving were obtained by using of 1.5% HES. The isolated cord blood mononuclear cells were cultured with domestic serum-free medium supplemented with 116t (IL-11, IL-6, TPO), st36(SCF, TPO, IL-3, IL-6), pt36 (PDGF,TPO,IL-3,IL-6) or pst36 for 7 days. St36 group (50 ng/ml SCF, 50 ng/ml TPO, 20 ng/ml IL-3 and 50 ng/ml IL-6) yielded the most CD41/CD61 positive [(6.79±1.97)×10⁴]. The cell viability [(82.85 ± 0.64)%] of st36 group by using of imported serum-free medium was better than [(60.90±6.93)%] that in domestic medium on day 7 after induction, and CD41/CD61 positive cells count [(18.60±1.97)×10⁴] were more than domestic serum-free medium group. Therefore, we chose imported serum-free medium containing st36 to induce cord blood mononuclear cells. After a prolonged culture, the total cell numbers increased accompanied with an elevated percentage of CD41/CD61 positive cells, which reached (54.27 ± 6.31)% on day 14. Wright-Giemsa staining showed that different phase cells, such as megakaryoblast, promegakaryocyte and granular megakaryocyte, occurred after 10 days'culture. Clone forming unit-megakarocytes (CFU-MK) assay showed that the colonies count increased with the prolonged incubation. CFU-MK colonies were [1 236.0±32.9] on day 14, which was higher than that in medium without induction (P<0.01). Platelets from megakaryocytes showed agglutination function after 10 days'culture.

CONCLUSION1.5% HES was the best solution to precipitate erythrocytes. The combination of an imported serum-free medium with IL-3, IL-6, SCF and TPO showed better induction efficiency than domestic medium or other cytokine cocktails. Meanwhile, induced megakaryocytes produced functional platelets.

Cell Culture Techniques ; Cell Differentiation ; Cell Division ; Cell Separation ; methods ; Cells, Cultured ; Culture Media, Serum-Free ; Fetal Blood ; cytology ; Humans ; Megakaryocyte Progenitor Cells ; cytology