Objective To investigate the related risk factors of gastroesophageal reflux disease (GERD).Methods 1 200 patients were chosen,including 99 GERD patients in the observation group,another 99 patients with non-GERD in control group.Questionnaires and esophageal and gastric pressure measurement method were used to analyze the related risk factors of GERD.LESP LESL,GP,the LESP-GP and LESP/GP's were compared between the observation group and the control group.Results In 1 200 patients participating in the survey,99 patients were confirmed GERD,with a ratio of 8.25％.Multivariate logistic regression analysis showed that eating too full,to eat before going to bed,like greasy food,spicy foods,heavy drinking and smoking were important factors affecting the incidence of GERD.2 LESL,LESP/GP and LESP-GP of the observation group were significantly less or lower than those of the control group,GP was significantly higher than that of the control group,the differences were statistically significant (t =12.216,12.515,8.973,3.437,all P ＜0.05).Conclusion Poor eating habits and lifestyle are important risk factors for GERD,the lower esophageal sphincter dysfunction and intragastric pressure increases in GERD incidence occupies an important position,should take positive interventions.

Objective: To investigate the expression of recombinant human intedeukin-18 (hIL-18) in 3.7 L fermenter with the constructed engineer train Pichia pastoris X-33/hIL-18, and the procedures of expression and purification thereof.Methods: The train X-33/hIL-18 was inoculated in BMGY medium and then inoculated into the fermenter until the A600 of the culture reached about 6. The supernatant of fermentation was isolated and purified with centrifugal fiher devices, hydrophobic chromatography column and anion exchange chromatography column. Results: The recombinant hIL-18 was expressed in 3.7 L fermenter with batch feed methanol and the concentration reached 202 mg/L. After the purification, the purity could be more than 97%. The recombinant hIL-18 was shown to induce interferon-gamma (IFN-γ) production by human peripheral blood mononuclear cells (PBMCs), and enhanced NK cell cytotoxicity synergistically with IL-2. Conclusion: A great deal of the recombinant hIL-18 with higher purity could be harvested by Pichiapastoris expression system. This study showed a new potential for further study of its function and activities.

Objective: To evaluate the anti-tumor and side effects of activated-HLA haploidentical peripheral blood tem cells (haplo PBSCs) in the treatment of advanced refractory solid tumor patients. Methods: Forty-two patients with advanced refractory tumor, who were diagnosed in our hospital from Oct. 2004 to Oct. 2007, were enrolled in this study (all patients signed informed consent), including 12 with ovarian cancer, 9 with renal cancer, 8 with lung cancer, 8 with breast cancer, 2 with colon cancer, 2 with gastric cancer, and 1 with melanoma. The donors were healthy direct relatives of the patients; the donors' haplo-PBSCs were mobilized, collected, and activated by rhIL-2 in vitro. The clinical efficacy and side effects of haplo-PBSCs therapy were assessed by CT/PET-CT scanning, RESIST standard, KPS score, and clinical response rates, etc. Results: All 42 patients received one episode of haplo-PBSCs treatment. The progression-free survivals (PFS) were 6 months and the clinical beneficial rate (CR+PR+SD) was 73.8%. The beneficial rate of life quality was 76.2% and the KPS increased by 20 (0-30) points on average after haplo-PBSCs treatment. The patients with KIR unmatched in GVH direction had better outcomes than those with KIR matched or KIR unmatched in HVG direction (P<0.05), and the clinical beneficial rate, PFS and total beneficial rate were 94.1% vs 60.0%, (13.4±1.3) vs (8.0±0.9) months, and 89.5% vs 65.2%, respectively (all P<0.05). The donor/recipient relation as the mother/child had a better outcome than that as the father/child (P<0.05). Patients with renal cancer or ovarian cancer had better outcomes than those with other cancers, with clinical beneficial rates being 90.0% and 81.8%, respectively. Conclusion: Activated haplo-PBSCs therapy can induce non-specific anti-tumor effect, and improve the clinical symptom and life quality of advanced tumor patients.

Acupuncture in Chinese medicine has been a treasure in Chinese traditional medicine for thousands of years. It is opposite to many basic theories in modern medicine in dynasties. Different from the cross compatibility and interactions among medical medicines, acupuncture in Chinese medicine has its own consolidation and inherent philosophical ideas. In view of this, how to discuss the philosophical ideas and its development of acupuncture in Chinese medicine becomes of great importance. It is crucial to clearly answer the three theoretical propositions in the development of acupuncture in Chinese medicine. Firstly, the differences in acupuncture should be identified between the ancient time and the modern time. The issues focus on the origin of() and its philosophical divergence. Secondly, the origin of acupuncture should be identified, whether it is from China or India. Thirdly, the differences in acupuncture should be identified between China and the west, focusing on the explanation and rectification of the interrelationship between the acupuncture in Chinese medicine and the western acupuncture. Hence, the basic features are discussed on the reality of acupuncture in Chinese medicine as well as its diversity. Finally, the proposition is extended on how to holistically grasp the philosophical foundation of acupuncture in Chinese medicine and its future trend.

Objective To study the effect of feto-matemal microchimerism in the treatment of activated human leukocyte antigen (HLA) haploidentical mobilized peripheral blood cells against solid tumors. Methods Genomic DNA samples of 25 pairs of HLA haploidentical donors and recipients were extracted. The donor-derived HLA-DRB loci were detected with nested PCR-sequence specific primer(SSP) typing. The mixed lymphocyte proliferation action between the patients and respective donors, the engraftment of donor's cells and the serum levels of Th1/Th2 type of cytokines were measured with MTT,FISH and EIJSA method respectively. The survival time of patients with or without feto-matemal microchimerism were compared as well. Results Using nested PCR-SSP typing, the positive rates of feto-maternal microchimerism in the 25 pairs of HLA haploidentical donors and recipients were 40% in the maternal/children pairs and 0 in the paternal/children pairs. The chimerism positive patients showed less proliferation activity when cocultured with respective donors as compared with unrelated ones (P=0.03).Only one chimerism positive patient experienced the engraft of donor's cell 3 months after treatment as the donor derived XX chromosome was identified with FISH. When the data of chimerism positive patients were deleted, the serum levels of IFNγ 1 month after treatment dropped dramatically from 171.4 (26. 3～258.4) ng/L to 29. 4(1.2～39.9)ng/L. The survival time in chimerism positive patients of the maternal/children pairs was significantly longer than that in chimerism negative patients, which was (31.2±4. 3) months and (11.1±3.3) months, respectively (P=0.036). Conclusion Feto-maternal microchimerism might induce anergy in the HLA haploidentical donors, favor the engraftment of donor's progenitors and maintenance of positive microenvironment and prolong the survival time.

AIM:To study the high-level HBV replication transgenic mice for evaluation of drugs treating hepatitis B virus.METHODS:The HBV transgenic mice were treated respectively with lamivudine,large dose recombinant hepatitis B protein vaccine,?-1b interferon,siRNA to evaluate their pharmacodynamics and mechanism of action.RESULTS:HBV DNA titre was reduced significantly in transgenic mice which were treated with lamivudine(100 mg?kg-1?d-1),recombinant hepatitis B protein vaccine(HBsAg 6 ?g/mouse),?-1b interferon(50 ?g /mouse),respectively.Recombinant hepatitis B protein vaccine and ?-1b interferon promoted the level of IL-2 and IFN-? and increased the Elispot number of spleen cells secreting IFN-? in the treated transgenic mice.HBV transgenic mice were treated with RNAi expression vector pU6-siHBV against HBV through vena caudalis by hydrodynamics technique.Five days later,the level of serum HBsAg was reduced by 56.7% and the inhibition lasted at least 14 days.The HbcAg(+)cells were decreased obviously by immunohistochemistry detection in liver tissue,but the RNAi did not reduce the serum HBV DNA titre.CONCLUSION:These inbreeding high-level HBV replication transgenic mice are reliable and feasible for evaluating the anti-HBV drugs and have its economical and convenient superiority.

Objective: To separate and identify the exosomes derived from MCF-7 tumor cells,and observe their function on IL-2 induced lymphocyte proliferation.Methods:The exosomes were separated by serial centrifugation,concentration and density gradient centrifugation, and then identified by transmission electron microscope.The effects of exosomes on IL-2 induced proliferation were studied by MTT method.Results:Total 20 mL exosomes from 2×10~7 MCF-7 cells with concentration of protein were(1.84±0.01) g/L. The diameters of these purified bi-layer membrane exosomes were 55-70 nm, which were consistent with the internal vesicles in multi-vesicular endosomes.The result of MTT test showed that proliferation of donor peripheral blood lymphocytes was inhibited by tumor exosomes at a dose-dependent manner in response to IL-2.Conclusion:It is feasible to separate high-purity exosomes from tumor cell culture medium supernatant by serial centrifugation and concentrate method. These tumor derived exosomes have inhibitory effect on IL-2 induced proliferation.

Objective: To investigate the effect of RetroNectin on CIKs cells and the related mechanisms. Methods: Peripheral blood mononuclear cells (PBMC) were collected from patients and divided into two groups: group Ⅰ and group Ⅱ. Samples in group Ⅰ were seeded into culture flask precoated with RetreNec-tin and CD3mAb to induce CIKs. While samples in group Ⅱ were seeded into common culture flask. The pro-liferation of CIKs was detected by cytometric analysis. The cytotoxic activity of CIKs was determined by LDH assays. The phenotype changes and cell cycle of CIKs were identified by flow cytometry. The apoptosis of cells was detected by Annexin V/PI. Western blot was employed to detect the level of protein Vav1. The CD49d and CD49e were blocked by anti-CD49d and anti-CD49e and the proliferation of cells was tested by cytometric analysis after the blockage. The phenotype changes of cells were identified by flow cytometry after the blockage. Results: RetroNectin enhanced the proliferation of CIKs (P<0.05). Flow cytometric analysis showed that RetroNectin significantly increased the number of CD25+ T cells (P<0.05). RN-CIK was more ac-tive than CIK in killing HCT-8 cell lines in vitro (P<0.05). RetroNectin could block the CIKs at G_1 phase (P<0.05) and resist apoptosis. There was no significant difference in the proliferation between the two groups af-ter the blockage with CD49d and CD49e (P>0.05). The expression of protein Vavl was associated with CD25+T cells. Conclusion: RetroNectin enhances the proliferation of CIKs by influencing the cell cycle, resist-ing apoptosis possibly through the site of CD49d and CD49e, and inducing T cell activation as the second sig-naling through Vav1.

Objective To screen the splicing isoforms of estrogen receptor βin the Beagle hypothalamic -pituitary -gonadal axis.Methods For ERβmRNA CDS sequence of eight exons, primers were designed confined to the CDS sequences of two sequential exons.Beagle hypothalamus, pituitary, ovary and uterus tissue cDNA were used as template, and corresponding sequences were amplified by PCR.PCR products were sequenced and aligned in the NCBI web site.The correct gene was then analyzed with DNAMAN comparative analysis software and handwork checking up, thus got the ERβsplicing isoforms of Beagle. Results Four beagle ER beta splicing isomers were obtained:exon 4 complete skipping ER βisomer (300 bp missing), two kind of Beagle ERβisoforms with partial exon 4 and partial exon 5 complicated missing (isoformⅠ334 bp missing and isoformⅡ265 bp missing), and exon 7 complete missing ERβsplicing isoforms (181 bp missing).Exon 4 complete skipping and exon 7 complete missing isomers had been obtained full length coding sequence, and the other two splicing isomers were partial coding sequence.Conclusion This project gained four ERβsplicing isomers of Beagle, and that will lay an important foundation for further study of their roles in the Beagle reproductive regulation mechanism.

Case recruitment of large-scale clinical trials should be strictly checked in quality and quantity for it is the key to clinical trial. This study discusses the main difficulties and countermeasures in the case recruitment of large sample, multi-center clinical trials according to the national research project "Myocardial Infarction Secondary Prevention Study in Traditional Chinese Medicine".