OBJECTIVE： To develop a method for simultaneous determination of 7 components of Niuhuang qingwei pills as chlorogenic acid， geniposide， forsythoside A， narirutin， baicalin， ammonium glycyrrhetate， chrysophanol. METHODS： HPLC-wavelength switching method was adopted. The determination was performed on Agilent ZORBAX SB-C18 column with mobile phase consisted of acetonitrile （A）-0.1％ phosphoric acid （B） gradient elution at the flow rate of 1.0 mL/min. The detection wavelengths were set at 348 nm （chlorogenic acid）， 238 nm （geniposide）， 330 nm （forsythoside A）， 280 nm （narirutin and baicalin）， 237 nm （ammonium glycyrrhetate）， 254 nm （chrysophanol）. The column temperature was 30 ℃， and sample size was 10 μL. RESULTS： The linear ranges of chlorogenic acid， geniposide， forsythoside A， narirutin， baicalin， ammonium glycyrrhetate， chrysophanol were 0.011 67-0.233 4 μg （r＝0.999 4）， 0.042 91-0.858 1 μg （r＝0.999 4）， 0.125 0-2.500 μg （r＝0.999 9）， 0.118 0- 2.360 μg （r＝0.999 9）， 0.119 6-2.392 μg （r＝0.999 7）， 0.030 57-0.611 4 μg （r＝0.999 6）， 0.006 201-0.124 0 μg（r＝0.999 4）， respectively； the limits of quantitation were 1.167， 0.858， 1.250， 1.180， 1.196， 0.611， 0.620 μg/mL， respectively； RSDs of precision tests were 0.98％， 1.04％， 0.59％， 1.50％， 0.83％， 1.24％ and 1.32％ （n＝6）， respectively. RSDs of stability tests were 1.21％， 0.97％， 1.42％， 0.71％， 0.98％， 1.87％ and 1.63％ （n＝6， 12 h）， respectively. Average recoveries were 98.32％， 98.11％， 98.81％， 98.50％， 98.30％， 98.16％ and 97.83％， and the RSDs were 1.37％， 1.41％， 0.64％， 1.01％， 1.18％， 1.16％ and 1.16％ （n＝6）， respectively. CONCLUSIONS： Established method is easy and reproducible. It can be used for the quality control of Niuhuang qingwei pills.

Objective To determine the inhibitory effects of Ellipticine (ELL) on inflammation in lipopolysaccharide (LPS)-induced RAW264.7 cells of mouse and explore its molecular mechanism.Methods The RAW264.7 cells in log phase were challenged by LPS (10 mg/L) to induce inflammation and then treated with ELL (0.05, 0.5, 5μmol/L). At the same time the cells treated with ELL (5μmol/L) were considered as ELL control group while without any stimulation as control group. After 12 hours intervention, the content of inflammatory factors in cell supernatant was detected by enzyme linked immunosorbent assay (ELISA), and then confirmed the most suitable concentration for the next experiment. After LPS of 10 mg/L was used to challenged RAW264.7 cells to cause inflammation, 5μmol/L ELL was used for intervention, and the mRNA expressions of inflammatory cytokines were detected by reverse transcription-polymerase chain reaction (RT-PCR) after 2, 4, 6 and 12 hours; the nuclear translocation of nuclear factor-κB p65 (NF-κB p65) as well as the phosphorylation levels of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinases (p38MAPK), c-Jun N-terminal kinase (JNK), c-jun, c-fos were detected by Western Blot after 15 minutes, 30 minutes, 1 hour and 2 hours.Results ① The different proliferative potential of RAW264.7 treated with LPS (10 mg/L) and ELL (0.05, 0.5, 5μmol/L) had no significant difference comparing with control group, which indicated that ELL had no cytotoxicity with experimental concentration and had no effect on the cell proliferative potential as the result of drug interaction. The levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in supernatant were significantly increased after induced by LPS comparing with control group. However, the different concentrations of ELL could dose-dependently reverse the production of inflammatory factors, and 5μmol/L was the optimum concentration of anti-inflammatory. ② Compared with control group, the mRNA expressions of TNF-α, IL-6 were significantly increased, the nuclear translocation level of NF-κB p65 increased as well as the phosphorylation levels of ERK, p38MAPK, JNK, c-fos, c-jun after stimulated by LPS. While, the different concentration of ELL could reverse the mRNA of TNF-α, IL-6 and phosphorylation levels of JNK, c-fos, c-jun [TNF-αmRNA (2-ΔCt): 2.45± 0.19 vs. 3.41±0.32 after 2 hours, 1.20±0.11 vs. 2.11±0.21 after 4 hours, 1.68±0.09 vs. 2.51±0.31 after 6 hours;IL-6 mRNA (2-ΔCt): 3.41±0.52 vs. 4.10±0.38 after 6 hours, 1.61±0.08 vs. 3.91±0.25 after 12 hours; p-JNK/GAPDH:0.557±0.034 vs. 1.049±0.056 after 1 hour, 0.439±0.040 vs. 0.855±0.038 after 2 hours; p-c-fos/GAPDH: 0.158± 0.030 vs. 0.741±0.035 after 1 hour, 0.156±0.015 vs. 0.932±0.030 after 2 hours; p-c-jun/GAPDH: 0.425±0.036 vs. 0.802±0.059 after 1 hour, 0.345±0.075 vs. 0.952±0.068 after 2 hours; allP < 0.05]. However, it had no significant effect on the nuclear translocation level of NF-κB p65 and the phosphorylation level of ERK and p38MAPK. Conclusion ELL inhibited the production of IL-6, TNF-α inflammatory factors in LPS-induced RAW264.7 cells through suppression the phosphorylation of JNK and activator protein-1 (AP-1).

Objective: To construct a hsa-microRNA-224(miR-224) lentiviral expression vector and to establish pancreatic mucinous cystadenocarcinoma MCC1 cell line with stable miR-224 over-expression. Methods: Pri-miR-224 gene fragment was designed and amplified by quantitative real-time polymerase chain reaction (qRT-PCR), and then loaded into GV369 lentiviral vectors (GV369-miR224) by gene recombination technology. GV369-miR-224 lentivrial expression vectors were then identified by PCR and DNA sequencing. The GV369-miR-224 vector fluid was then used to infect pancreatic mucinous cystadenocarcinoma MCC1 cell line to establish the MCC1 cell line stably over-expressing miR-224. The transfection efficiency of GV369-NC and GV369-miR-224 was observed under fluorescence microscopy; and the expression levels of miR-224 in MCC1, GV369-miR-224-MCC1 and GV369-NC-MCC1 cell lines were detected by RT-PCR. Results: The GV369-miR-224 lentiviral vectors were successfully constructed. GV369-miR-224-MCC1 and GV369-NC-MCC1 cells all emit green fluorescence under the fluorescence microscope. The expression level of miR-224 in GV369miR-224-MCC1 cell group was significantly higher than that in negative control GV369-miR-224-MCC1 group and blank control MCC1 cell group (23.45±1.94， 1.46±0.1 and 2.11±0.38, P<0.01), however, there was no significant difference between the two control groups (P>0.05). Conclusion: A pancreatic mucinous cystadenocarcinoma MCC1 cell line with stable miR-224 over-expression was successfully established, and this will provide a new cell model for exploring the function and pathogenesis of miR-224 in pancreatic mucinous cystadenocarcinoma.

Objective To summarize our experience with surgical management of renal neoplasm involving inferoir veno cava.Methods We review the data of 115 patients,including 74 male patients and 41 female patients,with renal neoplasm involving venous system between March 1993 and December 2015.The mean age was 52 years old,ranging 22 to 77 years old.The tumor was found in right side in 77 cases and left side in 38 cases.There were 15 patients (13.0％) with renal vein thrombus,38 (33.1％) with infrahepatic thrombus,29 (25.2％) with low retrohepatic thrombus,20 (17.4％) with high retrohepatic thrombus,and 13 (11.3％) with supradiaphragmatic thrombus.The mean age was 52 years old,ranging 22 to 77 years old.The tumor was found in right side in 77 cases and left side in 38 cases.All patients accepted the radical nephrectomy.Primary outcomes were overall survival (OS),and cancer special survival (CSS) in patients with renal cell carcinoma (RCC) estimated by Kaplan-Meier method.Secondary outcome included operative and oncological features,past-operative complications and hospital mortality.Cox proportional hazard model was used to univariate and multivariate analysis for risk factor impacting on OS of RCC patients.Results Complete resections of renal neoplasm with tumor thrombus were achieved in 113 patients (98.3％),2 patients died intraoperatively due to the dropping of thrombus.Postoperative complication rate was 16.8％.Hospital mortality was 2.6％.Mean follow-up interval was 48 months.OS rates at 5-,and 10-year in RCC patients were 66.5％,and 61.8％,respectively.Metastasis was an independent risk factor affecting on OS (P =0.000).However,the level of thrombus was not an risk factor affecting the prognosis.Conclusions Radical resection of renal tumor and caval thrombus is an effective treatment for prolonging survival in patients with RCC extending into venous system.Retrohepatic caval tumor thrombus below or above the main hepatic vein should be dichotomized and managed respectively with diverse techniques.Metastatic rather than the level of thrombus was a risk factor impacting on RCC patient survival.

Objective To explore the clinical effectiveness and safety of selective usage of embolic protection device to prevent distal embolization during SilverHawk atherectomy for atherosclerotic femoropo-pliteal artery disease. Methods From Jan 2014 to December 2015, 45 femoropopliteal artery atherosclerot-ic patients were treated with SilverHawk atherectomy and selective embolic protection device (EPD). The indication for EPD was instent restenosis, highly calcified lesion, suspicious of thrombosis, ulcerated le-sion, and single below-the-knee runoff. All cases who met the indication were treated with atherectomy and EPD, and those who did not meet the indication were treated with or without EPD according to the patient's choice. The embolic related complications were analyzed. Results Twenty three out of 45 patients who met the EPD indication were all treated with SilverHawk atherectomy under EPD protection, filter captured deb-ris in 17 patients (73. 9％) of the patients. The other 22 patients who did not meet the indication were di-vided into 2 groups according to the patient's choice of EPD usage, 11 were treated by atherectomy with EPD and 11 without EPD. One case out of 11 unindicated patients without EPD suffered a tibioperoneal trunk embolization and restored with catheter aspiration. For 1/11 (9. 1％) unindicated cases with EPD protec-tion, the filter captured embolization. There was a significant difference of distal embolization rate between the indicated and unindicated patients (χ2 =19. 368,P =0. 000). All filters were retrieved successfully without any distal embolization and any complications except arterial spasm occurred in 2 patients and re-stored well with nitroglycerin. Conclusions It is safe and effective for selective usage of embolic protection device to prevent distal embolization during SilverHawk atherectomy for atherosclerotic femoropopliteal artery disease.

Objective: To investigate the relationship between serum levels of high sensitivity cardiac troponin T (hs-cTnT) and the severity of coronary lesions in patients with stable coronary artery disease (SCAD). Methods: A total of 450 SCAD patients with coronary angiography (CAG) conifrmed diagnosis in our hospital were studied, and serum levels of hs-cTnT were examined at 3 days prior CAG in all patients. Based on tertiles of Gensini score, the patients were divided into 3 groups: Low score group,n=153 patients with Gensini score<14, Intermediate score group, n=145 patients with Gensini score at 14-28 and High score group,n=152 patients with Gensini score>28. The relationships between Gensini score and hs-cTnT levels were analyzed among 3 groups. The optimal cut-off value of hs-cTnT for predicting high Gensini score and the need of revascularization were studied by ROC curve, the relationships between hs-cTnT and high Gensini score, the need of revascularization were further detected by Logistic regression analysis. Results: The median values (25%-75%) of hs-cTnT in Low score group, Intermediate score group and High score group were 6.72 (4.20, 8.93) pg/ml, 7.90 (5.74, 12.68) pg/ml and 14.99 (10.26, 24.30) pg/ml respectively, allP<0.01. ROC curve analysis indicated that the area under curve (AUC) of hs-cTnT for predicting high Gensini score was 0.837 (95% CI 0.803-0.874), for the need of revascularization was 0.772 (95% CI 0.728-0.817); the best cut-off value of hs-TnT for predicting high Gensini score was 10.04 pg/ml and for the need of revascularization was 8.56 pg/ml. Logistic regression analysis suggested that with adjusted age, gender, the history of hypertension, diabetes, smoking, blood levels of creatinine, LDL-C and hs-CRP, hs-cTnT was still an independent predictor for high Gensini score (OR=1.13, 95% CI 1.06-1.20,P<0.001) and for the need of revascularization (OR=1.19, 95% CI 1.14-1.24,P<0.001). Conclusion: Serum level of hs-cTnT has been related to severity of coronary lesions in SCAD patients, hs-cTnT might be used as one of the pre-operative predictor for severe coronary disease and for the need of revascularization.

Objective To evaluate the clinical effect of acupuncture combined with estazolam for the treatment of primary insomnia with heart and spleen deficiency type. Methods Seventy qualified patients were randomized into treatment group and control group, 35 in each group. Both groups received oral use of estazolam before bed time for 6 continuous weeks, and acupuncture group was additionally given acupuncture for regulating governor vessel and tranquilizing spirit on acupoints of Baihui(GV20), Yintang(GV29), Shenting(GV25), Anmian (EX-HN22), Shenmen(H7), Sanyinjiao(SP6), 3 times a week and lasting for 6 continuous weeks. The sleep state of the patients was estimated according to Pittsburgh Sleep Quality Index (PSQI), Epworth Sleepiness Scale (ESS) and Athens Insomnia Scale(AIS) before treatment, on the third and the sixth week of treatment, and one month after the completion of treatment. Results (1) Five cases in the treatment group and 3 cases in the control group were dropped out for not completing the treatment timely. In the end , 30 cases in the treatment group and 32 cases in the control group finished the trial. (2) The total effective rate in the treatment group was up to 90.0%, and was 71.9% in control group, the difference being significant(P<0.01).(3) Compared to the control group, the scores of PSQI, ESS and AIS were much decreased in treatment group on the third and the sixth week of treatment , and one month after treatment(P<0 . 05 or P<0 . 01). Conclusion Acupuncture for regulating governor vessel and tranquilizing mind combined with oral use of estazolam exerts certain therapeutic effect for the treatment of heart and spleen deficiency type insomnia, and the effect was superior to that of estazolam alone.

Objective To investigate the safety and feasibility of carotid endarterectomy (CEA) combined with carotid artery stent angioplasty (CASA) in treating tandem stenosis of carotid artery. Methods The clinical data of 9 patients with tandem stenosis of carotid artery, who were treated at authors' hospital during the period from January 2013 to October 2014, were retrospectively analyzed. The patients included 7 males and 2 females, with a mean age of (66.0 ±4.2) years. The disease course ranged from 2 months to 36 months, with a mean of 7 months. Clinically, all patients had cerebral ischemia symptoms. Transient ischemia attack was seen in 5 patients and history of cerebral infarction was present in 2 patients. Coronary artery disease was found in 2 patients, hypertension in 6 patients and lower limb ischemia in one patient. After receiving adequate antiplatelet therapy, CEA and CASA were carried out in all patients. Results The technical success rate was 100%, postoperative residual stenosis was less than 30%, no death occurred in perioperative period. After the treatment, the clinical symptoms were improved in all 9 patients;no new stroke or cerebral hemorrhage occurred. After the treatment, 2 patients developed cerebral hyperperfusion-related symptoms such as headache and dizziness, which were much relieved at the time of discharge. The patients were followed up for 4-19 months, with a mean of (10.5±6.2) months. No recurrence of symptoms was observed . In one patient , transcranial Doppler ultrasound performed at 6 months after treatment showed that the carotid artery became moderate restenosis (50%-70%). No death occurred. Conclusion For the treatment of tandem stenosis of carotid artery, CEA combined with CASA is safe and effective, although larger sample and long-term follow-up studies are still needed to further confirm the effect.

BACKGROUNDTotally laparoscopic aortoiliac surgery has been newly developed in China. It is known as the most complex laparoscopic technique to learn because of its high-risk procedures. Analysis of the operation-related complications of this surgery is supposed to be helpful for the early success of this technique.

METHODSTwelve male patients (56-70 years old) with aortoiliac occlusive disease underwent totally laparoscopic aortoiliac bypass surgery (TLABS) in our institute. Clinical data and operation-related complications were retrospectively analyzed.

RESULTSOf the 12 patients, TLABS succeeded in nine and conversion to open surgery occurred in three. One of the converted patients finally died of pulmonary infection. Operation-related complications included bleeding from arterial injury, perforation from colonic injury, graft embolism, residual aortic stenosis, and hydronephrosis. Bleeding in two patients and colonic perforation in one patient resulted in three conversions to open surgery. Intraoperative graft embolectomy and postoperative aortic stenting were performed to resolve the thrombus/embolus-referring complications. Left hydronephrosis, which was thought to result from intraoperative injury and treated with ureteric intubation drainage, recovered 6 months after TLABS.

CONCLUSIONSGood understanding and avoidance of operation-related complications are important to guarantee the technical success of TLABS. Immediate conversion to open surgery is necessary for saving the patient's life in case of life-threatening complications.

Aged ; Arterial Occlusive Diseases ; surgery ; Humans ; Iliac Artery ; surgery ; Laparoscopy ; methods ; Male ; Middle Aged ; Retrospective Studies ; Treatment Outcome

Stem cells have self-renewal and differentiation potential.Cyclin-dependent kinase (CDK) plays an important role in promoting pluripotency and self-renewal.The crucial activity of S-phase,DNA replication,presents a unique opportunity during the cell cycle for the genetic and epigenetic regulation that may be involved in stabilizing the pluripotent state.It is also clear that Myc acts to coordinate both the cell cycle and the pluripotency transcription network in stem cells.Here we review the regulating mechanisms of stem cell cycles and pluripotency by CDK and Myc to help researchers obtain a better understanding of mutual regulation of the cell cycle and the pluripotent state by CDK and Myc which may be exploited in regenerative medicine.