Objective:To explore the purification method of non- affinity chromatography for leptin expressed in Pichia Pastoris.Methods:Ion exchange chromatography (Sepharose Q fast flow) and hydrophobic interaction chromatography (Phenyl Sepharose 6 fast flow ) were used to purify human leptin in pH 7.5. Results: After purification by Q column,the purity of leptin increased from 42.3% to 89.6%.Subsequently, after hydrophobic interaction chromatography ,its purity reached 96.2%. In SDS-PAGE, leptin was shown as one specific band.Conclusion:The human recombinant leptin expressed by Pichia Pastoris yeast can be successfully purified by ion exchange chromatography plus hydrophobic interaction chromatography.

The key to deepen quality education in university is to integrate the education of humanities into speciality instruction.Research shows that the ways to combine them are varied: starting from knowledge,mental enlightenment, aesthetic pentration, interaction, classroom atmosphere creation, and anthropocentrism ideal promotion,etc.

Considering the character of biochemistry,this paper discussed teaching method and accounted for the importance of experiment teaching in biochemistry by some demonstrations including analyzing carefully for catching common ground;integrating theory with practice for helping understand and memorize;agitating the students' study interest etc.In addition,the importance of students was not to be ignored in teaching process.

Objective:To develop a high cell-density fermentation process in micro DCU-400 fermentor of Pichia pastoris GS115 containing a recombinant vector pPICZ ?/endostatin.Methods:Cells were grown in the basal salts medium(BSM) at 30℃,pO 2≥30%,pH 5.3(growing) or pH 5.0(inducing),and the feed operation using 50% glycerol was performed when dissolved oxygen(pO 2) increased(after approximately 16-18h).It lasted 3-5hrs.When cell density(OD 600 ) reached 450,the inducement was began.After the transition from glycerol-methanol mixture to methanol,methanol from 0.03ml/min/L to 0.08ml/min/L in 8h was used to induce the endostatin expression.The inducement lasted for approximately 60h.Then centrifugation,the supernatant was collected.The expression was examined using 12% SDS-PAGE and ELISA.Results:In inducing phase,OD 600 reached 580(wet wt 275g/L).The production of endostatin is 27.2mg/L,exceeding that in spinner flask(16.3mg/L).Conclusion:The human endostatin had been successfully expressed in 15L fermentor.

BACKGROUND: Leptin is a hormone produced predominantly by adipocytes and has a variety of physiological functions. It has been a hot spot in energy metabolism research. However, leptin presently used is usually produced by E coli, in which leptin cDNA is expressed and is in the form of insoluble inclusion body. Therefore, extremely complicated reactivating procedure is needed to obtain biologically activated human leptin.OBJECTIVE: To explore the condition for purification of leptin in non-affinity chromatography in order to obtain soluble human leptin.DESIGN: An observational experiment.SETTING: Molecular laboratory of biochemical department in a medical college.MATERIALS: The strong anion exchanger sepharose Q and hydrophobic phenyl sepharose 6 were used in different conditions for removal of as many contaminants as possible.METHODS: The supernatant of pichia pastoris yeast culture solution was first purified through Q column chromatography, the protein was collected and was further purified through hydrophobic support with 1 mol/ L (NH4) 2SO4.MAIN OUTCOME MEASURES: Gel scanning revealed that the purity of human leptin was 42. 3% before purification, 89.6% after Q column chromatography and 96.2% after hydrophobic interaction chromatography.RESULTS: The post-purification product presented a s. ingle band in SDS-PAGE. Gel scanning revealed that the purity of human leptin was 42.3% before purification, 89.6% after Q column chromatography and 96.2% after hydrophobic interaction chromatography.CONCLUSION: The combined use of strong anion exchange chromatography and hydrophobic interaction chromatography can effectively purify leptin expressed by pichia pastoris yeast and the purity is identical to that of nickel affinity column chromatography. It provides reliable evidence and method for possible manufacture of human leptin and lays experimental basis for leptin-related research.

Colon cancer is a common and deadly disease with high rising incidence rate.Proteomics has already become one of the hot topic and maken great progress in the research of colon cancer pathogenesis,early diagnosis,treatment and prognosis.In recent years,more and more colon cancer-associated biomarkers have been identified through proteomics-based approaches.This review will summarize the research progress of proteomics in colon cancer.

Objective: To study early diagnosis and treatment of multi-primary esophageal cancer and esophageal and cardiac double primary cancer. Methods: The data of 71 cases patients of multi-primary esophageal, double primary esophageal and cardiac cancer were collected. The diagnosis was made by dye staining through gastroscopy and X-ray. Pathological examination after operation was analyzed. Results: 14 patients were diagnosed by X-ray(14/71), 69 by endoscopy (69/71), 54 early foci and 6 early stage patients were found. All of them were operated. The resection rate is 100% with no operative death. 3-year survival rate was 40 5%. Conclusion: Routine X-ray examination of esophagus or stomach, and esophageal dye-staining and/or biopsy through endoscopy are important measures for early diagnosis of multi-primary cancer or esophageal and double primary cardiac cancer prompt surgery is advised.

Objective To explore the effects of chlamydia and mycoplasma infections on the healing of wounds after surgical operation for haemorrhoids,fistula and anoschisis.Methods Sixty-two patients with haemorrhoids,fistula and anoschisis undergoing the surgical operation during 2000.1 to 2008.10 were collected,and wounds did not heal 40 days after operation and the wound's surface infection occurred after postoperative anti-inflammatory therapy and dressing change.All patients were positive at least for laboratory Ct or Uu,and those with infections caused by other fungi,bacteria,viruses and the other systemic diseases were excluded.The correlation between the infections in 62 cases and sex,surgical types was analyzed.Results In 62 patients,there were 30 cases (48.4%) positive for both Ct and Uu,22 cases for single Ct (35.5%),and 10 cases for single Uu (16.1%),respectively.The infection rate in females was higher than in males (P<0.01).The surgical types included:surgery for 8 cases of haemorrhoids (12.9%),for 18 cases of fistula(29.0%),and for 36 cases of anoschisis(58.1%).respectively.Conclusion There is the possibility of chlamydia and mycoplasma infections in the patients with delayed healing of wounds following the surgical treatment on the anal region.Early diagnosis,early treatment,avoidance of ineffective medicines and repeatedly management of the wounds can shorten the healing period.

BACKGROUND: Troponin I (Tn I ) could inhibit the growth of vascular endothelial cells, inhibit neovascularization,through which to inhibit the development and metastasis of solid tumor. Similar to Tn I, TnC also exists in non-muscular tissue, but does it has the analogous activity of anticancer like Tn I ?OBJECTIVE: To explore the effect of recombinant human TnC (rhTnC) on the growth of human umbilical vein endothelial cells (HUV-EC) and mouse xenograft tumor.DESIGN: Controlled observation in vivo and in vitro.SETTING: Research Institute of Medicine, Chongqing K.E.W Pharmaceutical Co., Ltd. and Department of Biochemistry of Chongqing Medical University.MATERIALS: The experiment was conducted in the Biochemical Laboratory of Research Institute of Medicine,Chongqing K.E.W Pharmaceutical Co., Ltd. from March 2003 to December 2004. 100 Kunming mice either male or female of 15-22 g purchased from Chongqing Academy of Chinese Materia Medica. E.coli BL21 (DE3)pLysS/pET3b-TnC provided by Chongqing K.E.W Pharmaceutical Co., Ltd. HUV-EC (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences).METHODS: Human TnC cDNA was obtained from human thymus cDNA library using PCR. The colony was cloned in E.coli and a bacterial strain of gene engineering E.coli BL21 (DE3) pLysS/pET3b-TnC was obtained, which could express hTnC. The recombinant human TnC (rhTnC) was purified with affinity chromatography of Ni-NTA agarose. ①In vitro cell experiment: HUV-ECs were seeded in the 96-well plates at density of 2×103 cells per well and co-cultured with rhTnC of 1, 5, 10, and 50 mg/L for 3 days. The absorption (A value) was detected with microplate reader at 540 nm and the inhibition rate of cell growth was calculated. Meanwhile, the 50% inhibiting dose (IC50 value) was assayed by LOGIT method. ②In vivo animal experiment: Ascites tumor (S-180) that had been inoculated for 7-8 days was harvested. The tumor cells were diluted to 1 ×1010 L-1 and 0.2 rnL was subaxillarily and intraperitoneally injected into each mouse (50 mice in each group). The next day, the mice were randomly divided into 5 groups: rhTnC 20 mg/kg group, rhTnC 10 mg/kg group, rhTnC 5 mg/kg group, Cyclophosphamide (Cy) group and control group with 10 mice in each group. The rhTnC 20,10 and 5 mg/kg groups were given administration at the corresponding doses, once a day for 7 days; 50 mg/kg Cy was given the Cy group one after an interval of day, and the same volume normal saline was given to the control group. One day after the last time of administration, all mice were killed and the tumor was harvested and weighed. The inhibition rate of tumor growth was calculated: tumor inhibition rate=[(Average weight of tumor in control group-Average weight of tumor in drug group)/Average weight of control group]×100%.MAIN OUTCOME MEASURES: Inhibition rate of rhTnC to HUV-EC proliferation in cell experiment in vitro and mouse xenograft tumor in animal experiment in vivo.RESULTS: ①In vitro cell culture showed that rhTnC suppressed HUV-EC proliferation in a dose-dependent manner (IC50=7.5 mg/L). ②Similar to the result of in vitro cell experiment, after intraperitoneal administration, the inhibition rate of rhTnC 5, 10, and 20 mg/kg groups was higher than that of control group (P ＜ 0.01); after subaxillary administration, the inhibition rate of rhTnC 5, 10, and 20 mg/kg groups was also higher than that of control group (P ＜ 0.05-0.01). There was no significant difference in the inhibition rate between two administration approaches (P ＞ 0.05).CONCLUSION: rhTnC is capable of inhibiting the proliferation of HUV-EC dose-dependently, and displays the activities of inhibiting the proliferation of HUV-EC and anti-tumor.

Objective: To analyze the status and general trend of urban residents’ health equity and health performance. Methods:Adopting standardization of concentration index, the extended concentration index and health performance index. Results: Chronic disease and self-assessed health of urban residents in China gradually improved from 2007 to 2011, while disability of those people did not improve significantly. Health inequity exists among urban residents with different incomes. Chronic disease and disability are inclined to the poor while self-assessed health is inclined to the rich, but the unfair degree has decreased gradually by year; the self-assessed health and the health performance of chronic disease has been improved from the comprehensive health level and equity, the disability sustains serious condition. Conclusion: China’s new health care reform does not significantly improve the domestic urban residents’ health equity and health performance, the objective set of the health care reform in China should pay more attention to health equity.