Objective To develop and verify a high performance liquid chromatography(HPLC) method for the determination of ethylenediaminetetraacetic acid disodium salt(EDTA-2Na) residues in the bulk of NMM tumor DNA vaccine for the quality control of DNA vaccine.Methods After NMM tumor DNA vaccine bulk was complexed with copper sulfate,a HPLC method for the determination of EDTA-2Na residues was developed with Agilent ZORBA XSB-C18(150 mm × 4.6 mm,5 μm) as the chromatographic column,water,tetrabutylammonium hydroxide 10% and acetonitrile solution(74.5:0.5:25)as the mobile phase.The detection method was as follows:the detection wavelength was 254 nm,the flow rate was 0.8 mL/min,the column temperature was 20 ℃ and the injection volume was 20 μL.The method was verified for the specificity,linear range,limit of detection(LOD),limit of quantification(LOQ),solution stability,durability,accuracy and precision,and used to detect the EDTA-2Na residues in several batches of DNA vaccine bulk.Results When EDTA-2Na control and DNA vaccine bulk with EDTA-2Na reacted with copper sulfate,the absorption peak appeared at around 5.3 min,while no absorption peak was observed when DNA vaccine bulk reacted with copper sulfate;In the range of 4～400 μg/mL,the control solution concentration showed a good linear relationship with the peak area,R~2=0.999 9;The LOD of the method was 10 ng/mL,and the LOQ was 40 ng/mL;The solution of control and sample was stable after placed for 12 h;When the detection conditions changed slightly(different mobile phase ratio,flow rate and column temperature),the influence on the detection results was within acceptable range;The average recovery rate of EDTA-2Na in low,medium and high concentration standard added samples was 101.38% with the RSD of 0.39%;0.1 mg/mL control solution was injected continuously for 6 times,and the peak area RSD was 0.04%.EDTA-2Na was not detected in 6 sample solution,and the peak area RSD of DNA vaccine bulk with EDTA-2Na solution was 0.02%,indicating a good intermediate precision.EDTA-2Na residue was not detected in these batches of DNA vaccine bulk.Conclusion The developed method is simple,accurate,reliable with good specificity,which can be used for the determi-nation of EDTA-2Na residues in DNA vaccine bulk.

It is imperative to apply information technology in the area of management of clinical research so as to ensure the quality of clinical trials and to improve management efficiency.In this study,the based analysis method was the quality function deployment (QFD).This methodology is used to analysis the clinical trials management information system on a hospital directly under the Ministry of Health.It ensured user participation,lay a solid foundation for software engineers on system design.

Objective To investigate the expression characteristics of Golgi phosphoprotein 3(GOLPH3)in human hepatocellular carcinoma (HCC)and explore its clinicopathological significance. Methods The expressions of GOLPH3 protein was detected in 132 cases of paired paraf-fin embedded HCC specimens and pericarcinoma tissues using immunohistochemical staining ,and the relation of the expression of GOLPH3 to clinicopathologic features was analyzed. Meanwhile,the expression and distribution of GOLPH3 in HCC cells was observed by laser confocal mi-croscopy. Results The positive expression rates of GOLPH3 in HCC and pericarcinoma tissues were 70.0%(92/132)and 42.4%(56/132) (P<0.001),respectively. The incidence of portal vein tumor thrombus in high and low GOLPH3 expression groups of HCC were 21.2%(14/66) and 6.1%(4/66)(P<0.05),respectively. The expression rate of GOLPH3 in HCC was significantly higher than that in pericarcinoma tissues, and the expression of GOLPH3 in HCC was positively related to portal vein tumor thrombus. In addition ,GOLPH3 was mainly expressed in cyto-plasm of HCC cells,and there was also scattered distribution in the nucleus. Conclusion GOLPH3 acts as an oncogene and may play vital roles in the carcinogenesis and development of HCC.

Objective To investigate the effects of Xiao Yao San on intracellular Ca2 + concentration in cultured rat hippocampal neurons in the state of chronic stress and study the mechanism of chronic stress injuring and XiaoYao San protecting. Methods MK-801 acts as tool,cultured rat hippocampal neurons were divided into seven groups, those were group 1 (control), group 2 (normal serum), group 3 (normal serum + Glu), group 4 (model serum + Glu), group 5 (model serum + Glu + MK-801), group 6 (Xiaoyaosan + Glu), group 7 (Xiaoyaosan + Glu + MK-801). to detect intracellular Ca2+ concentration in cultured hippocampal neurons in the simulated micro - environment of chronic stress and after intervention with the serum treated with Xiao Yao San by confocal laser microscope at the same period of time. Results Compared with group 1 (779.97 ± 36.81), concentration of Ca2+ intracellular of group 2 (1092.38 ± 36.41), group 3 (1472.49 ± 76. 19), group 4 (1509.52 ±104.69) and group 5 (1186.97 ±41.92) all increased significantly (P＜0.01) ,group 6 (908.74 ±40.24) increased too (P ＜ 0.05), compared with group 2, concentration of Ca2 + intracellular of group 3,4 and 5 all increased significantly (P ＜ 0.01), but group 7 (721.99 ± 60.33) decreased significantly (P ＜ 0.01). Compared with group 4, concentration of Ca2+ intracellular of group 6 and 7 decreased significantly (P＜ 0.01), group 5 decreased too (P ＜ 0.05), compared with group 6, concentration of Ca2 + intracellular of group 5 increased significantly (P ＜ 0.01),when group 7 decreased significantly (P＜0.01). Conclusion Serum of chronic stress treated with Xiao Yao San has the effect of inhibiting Ca2+ overload in hippocampal neurons,it may work through a variety of signaling pathways including Glu-NR-Ca2+ to maintain the steady-state of Ca2+ concentration in hippocampal neurons, and then to protect neurons from the neurotoxic effects of excitatory.

Objective To cultivate,identify and sort bronchoalveolar stem cells(BASC)derived from normal adult mouse lung.Methods After enzymatic digestion of lung tissue with dispase and collagenase in combination,the Sca-1+ cells were isolated from the obtained pulmonary cells by magnetic cell sorting.These Sca-1+ cells were cultured in dishes coated with collagen and mouse fibroblast cell line Swiss-3T3 under a serum-free culture system for BASC,which were identified by the dual-color immunofluorescent staining clara cell specific antigen(CCA)and surfactant protein C(SP-C).Finally,these pure BASC were isolated by the flow cytometry.Results One lung of normal adult mouse could yield(1.6-1.8)?107 nucleated cells in this enzyme digestion procedure.The percentage of Sca-1+ cells we sorted from lung tissue was much higher than the unsorted [(87.3?5.9)% and(9.6?1.8)%,P

Objective: To explore the effects of recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) gel on treatment of thefull-thickness frostbite wounds on foot and hand. Methods: From November 2013 to April 2017, a total of 45 patients of 71 full-thickness frostbite wounds on foot and hand meeting the inclusion criteria were admitted to the First Hospital of Jilin University and the prospective randomized controlled study was done. The patients were divided into rhGM-CSF group of 24 patients with 35 wounds and control group of 21 patients with 36 wounds according to the random number table. There were 20 males and 4 females, aged (38±13) years among patients in rhGM-CSF group, and there were 19 males and 2 females, aged (36±14) years among patients in control group. Patients in 2 groups were performed with the same systemic treatment of rewarming, anti-inflammation, pain relief, anti-infection, anti-coagulation, and thrombolysis. Wounds of patients in rhGM-CSF group and control group were respectively treated with rhGM-CSF gel and aloe vera gel for external usage with 10 mg for every square centimeter and dressing change once every 24 hours, until wounds healed completely. The wound inflammatory response was scored on treatment day (TD) 1, 3, 7, 14, wound secretion was collected for bacteria culture and positive bacteria detection rate was calculated before treatment and on TD 6 and 12, adverse drug reaction after drug use was observed, and the complete wound healing time was recorded. Data were processed with Fisher′s exact probability test, analysis of variance for repeated measurement, t test, and Bonferroni correction. Results: The scores of wound inflammatory response of patients in 2 groups on TD 1 and 3 were close (t=0.37, 2.93, P>0.05). The scores of wound inflammatory response of patients on TD 7 and 14 in rhGM-CSF group were significantly higher than those in control group (t=5.77, 5.83, P<0.01). The results of bacteria culture of wound secretion of patients in 2 groups before treatment were negative. The positive bacteria detection rates of wound secretion of patients in rhGM-CSF group on TD 6 and 12 were 5.71% (2/35) and 22.86% (8/35), which were slightly lower than 13.89% (5/36) and 30.56%(11/36) in control group respectively, but there was no significantly statistical difference (P>0.05). No adverse drug response occurred in patients in rhGM-CSF group, while 1 patient in control group had adverse drug response, with symptoms of redness and swelling of wounds and patchy erythema on skin around wounds, which were alleviated by irrigating with normal saline. The complete wound healing time of patients in rhGM-CSF was (12.3±0.5) d, which was significantly shorter than (16.5±0.8) d in control group (t=24.89, P<0.05). Conclusions The topical rhGM-CSF gel has effects of shortening time of wound healing and reducing inflammatory response of wound on treatment of full-thickness frostbite wounds on foot and hand, which is safe in clinical application.

Objective To investigate the value of remnant stomach-jejunal dual pathways reconstruction after laparoscope-assisted radical proximal gastrectomy in the treatment of upper gastric cancer. Methods Twenty-five patients with upper gastric cancer underwent laparoscope-assisted radical proximal gastrectomy and the remnant distal stomach was preserved for side-to-side remnant stomach-jejunal anastomosis and end-to-side jejuno-jejunal anastomosis to reconstruct dual pathways. Results The mean operation time was (240±35) minutes, the mean number of lymph nodes dissected were 22±5, and all the incised margins were negative. No anastomotic leakage, obstruction or stenosis occurred. All patients received postoperative barium meal examination. A large amount of barium directly entered the jejunum, leaving a small amount of barium entered the jejunnum via the route of remnant stomach-duodenum, and was detained in the remnant stomach for 30-60 minutes. No esophageal reflux of barium was observed. All the patients were followed up for 4-18 months, no reflux esophagitis was detected and the short-term life quality was satisfactory. Conclusions Remnant stomach-jejunal dual pathways reconstruction prevents the reflux esophagitis and dumping syndrome, preserves the pathway of duodenum and promotes the life quality of patients.

BACKGROUND:Peripheral nerve tissue engineering needs a large number of Schwann cells.In previous studies,lack of normal human nervous tissue,so animal(rat,rabbit,et al)nervous tissues are commonly used to isolate Schwann cells,but as xeno-cells it is limited in clinical application.OBJECTIVE:To investigate an effective technique for isolation,cultivation and purification of human Schwann cells of normal peripheral nerves cultured in vitro.METHODS:Normal peripheral nerves were obtained from the surgery of cerebral palsy patients.Schwann cells were cultured with enzymatic digestion culture method and differential attachment method.Tissues were cut into pieces and incubated in medium supplemented with fetal bovine serum,collagenase and Oispase enzyme,centrifuged.Tissue blocks were placed in the medium,triturated into monoplast suspension,and then moved into a DMEM Petri dish containing polylysine,supplemented with basic fibroblast growth factor.When adherent cells were confluent about 85% 90%,cells could subculture.Schwann cells were counted by Trypan Blue coloring method at 2,3,4,5,6,7,8,9,10.The purity of Schwann cells was identified through S-100 protein immunohistochemistry staining.RESULTS AND CONCLUSION:More than 0.5×10~8/L Schwann cells were detected after four days under a microscope.Following the third passage,the number of Schwann cells was over 9×10~8/L.The purity of Schwann cell population was up to 85%.Results suggested that plenty and purified human Schwann cells could be obtained by enzymatic digestion culture and differential attachment methods in a short time,which can be used for the source of peripheral nerve tissue engineering.

Objective To evaluate the changes in the expression of protein interacting with Cα kinase 1 (PICK1) in the spinal cord and dorsal root ganglion (DRG) neurons during remifentanil-induced hyperalgesia in rats with incisional pain.Methods Thirty-two male Sprague-Dawley rats, weighing 240-260 g, aged 42-49 days, were randomly divided into 4 groups (n =8 each) using a random number table: control group (group C) , incisional pain group (group Ⅰ) , remifentanil group (group R), and remifentanil + incisional pain group (group R + Ⅰ).In R and R+Ⅰ groups, remifentanil was infused intravenously for 60 min at the rate of 1.2 p,g · kg-1 · min-1.In C and Ⅰ groups, normal saline was infused intravenously for 60 min at the rate of 0.12 ml · kg-1 · min-1.In Ⅰ and R+Ⅰ groups, the model of incisional pain was established, and remifentanil and normal saline were infused intravenously, respectively, at the same time.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured before normal saline or remifentanil infusion, and at 2, 6, 24, and 48 h after the end of normal saline or remifentanil infusion (T1-4).The rats were sacrificed after the last measurement of pain threshold.The lumbar segment (L4-6) of the spinal cord and left DRGs were removed for determination of the expression of PICKl mRNA (by quantitative real-time reverse transcriptase-polymerase chain reaction) and PICK1 protein (by Western blot).Results Compared with group C, the MWT was significantly decreased, the TWL was shortened, and the expression of PICK1 protein and mRNA was up-regulated in R and R+Ⅰ groups, and the MWT was significantly decreased, the TWL was shortened (P＜0.05) , and no significant change was found in the expression of PICK1 protein and mRNA in group Ⅰ (P＞0.05).Compared with group Ⅰ, the MWT was significantly decreased, the TWL was shortened, and the expression of PICK1 protein and mRNA was up-regulated in group R+Ⅰ (P＜0.05).Compared with group R, the MWT was significantly decreased, the TWL was shortened (P＜0.05) , and no significant change was found in the expression of PICK1 protein and mRNA in group R+ Ⅰ (P＞0.05).Conclusion The mechanism by which remifentanil induces hyperalgesia may be related to up-regulation of PICK1 expression in the spinal cord and DRG neurons of rats with incisional pain.

The deep fermentation technique of Tricholoma matsutake is systemically studied in this paper firstly. The best culture determined by orthogonal test is 3g/L of cornflour, 1g/L of glucose, 1g/L of bean cake flour, 1mL/L of corn steep liquid, 1g/L of KH 2PO 4. The best fermenting condition is: 25℃, rotating speed 160 r/min, pH5.0,inoculating amount 10%, 120mL culture medium per 500mL flask. Under these conditions, the mycelia reach 12.94g/L after fermenting 12d.