AIM To observe the change of Ca 2+ /calmodulin dependent protein kinaseⅡ (CaMKⅡ) signal pathway in opioids dependent NG108-15 cells and the relationships between Ca 2+ /calmodulin dependent protein kinaseⅡ (CaMKⅡ) signal pathway and cAMP accumulation. METHODS NG108-15 cells were used as an in vitro model system. Competitive protein binding assay and radioimmunoassay were used to examine the intracellular cAMP accumulation. Calmodulin activity was assayed by PDE method. CaMKⅡ activity was assayed by ?- 32 P incorporation of syntide-2. mRNA expression of mDOR was measured by RT-PCR. RESULTS DPDPE long-term treatment also increased cAMP accumulation, and resulted in opioids dependence in NG108-15 cells; DPDPE long-term treatment increased calmodulin activity and CaMKⅡ activity in NG108-15 cells. Specific calmodulin antagonist W-7 was found to inhibit significantly the elevation of calmodulin and CaMKⅡ activity which resulted from DPDPE long-term treatment, and CaMKⅡ inhibitor KN-62 also inhibited elevation of CaMKⅡ activity by DPDPE long-term treatment. DPDPE long-term treatment increased cAMP accumulation, when W-7 or KN-62 was added at the same time, cAMP accumulation decreased. When naloxone was added to NG108-15 cells which were long-term treated by DPDPE, calmodulin and CaMKⅡactivity increased more. mRNA level of mDOR did not change significantly in opioids dependent NG108-15 cells. CONCLUSION Ca 2+ /CaMKⅡ signal pathway was involved in the mechanisms of opioids dependence through regulating cAMP level when DPDPE is long-term administered to NG108-15 cells.

As for the ? opioid receptor, there are some disputes about the domains involved in the selective recognition of ligands, the relative spatial orientation of the amino acids within the TM affect the affinities of selective ligands. Regulation of opioid receptor activities does not appear to involve in their ability to promote the association of GTP onto the G proteins and the subsequent dissociation of heterotrimers. It is not very clear if phosphorylation correlates with agonist induced receptor desensitization. The celluar processing of ? opioid receptors requires the formation of multiple protein complexes, it is clear that interactions of ubiquitous transcriptional factors determine gene transcription.

Objective: To clone porcine IL-4 cDNA and observe its adjuvanticity of vaccine against T. solium cysticerco-sis. Methods:The cDNA of porcine IL-4 was amplified by RT-PCR, which had 5' AUG initiatory codon with optimized trans-lational initiation. After sequencing,the cDNA was intergrated into expression vector pcDNA and transient expression was performed. Then newborn pigs were immunized with IL-4 expression vector and protective antigen DNA vaccine against T. solium cysticercosis. Results:The obtained sequence of porcine IL-4 cDNA was the same as reported. IL-4 and protective antigen could effectively promote the humoral response. Conclusion:An efficient expression plasmid containing porcine IL-4 cDNA is constructed. Its adjuvanticity of DNA vaccine against T. solium cysticercosis is preliminarily conformed.

BACKGROUND:Studies have found that erythropoietin has a protective effect on embryonic development of the retina, but there are rare studies concerning the embryonic development of the lens. OBJECTIVE: To observe the difference in erythropoietin expression in the lens from Wistar rats at different embryonic periods and to investigate the effect and significance of erythropoietin in the embryonic development of the lens. METHODS:Clean Wistar rats with pregnancy for 10 days (n=5), 12 days (n=5), 14 days (n=5), 16 days (n=5), 18 days (n=5), 20 days (n=5) were randomly colected and divided into six groups. Every two embryonic ratsof the different 30 pregnant rats were obtained randomly by the caesarean operation under ketamine-induced anesthesia. The eye tissues of al the embryonic rats were isolated and cut into sections. The expression of erythropoietin protein and mRNA in rat lens was detected by immunohistochemistry and reverse t ranscription-PCR, respectively. RESULTS AND CONCLUSION:Erythropoietin was distinctly expressed at the six different embryo stages, and the expression of erythropoietin protein and mRNA gradualy increased from embryonic day 10 to embryonic day 16, and decreased from embryonic day 10 to embryonic day 20. There were significant differences between the six groups. These findings indicate that the expression of embryonic appears in a low to high to low fashion during the embryonic development of Wistar rats, which may be closely associated with the developing procedure of lens.

Objective o observe the expression of Notch1 and Delta-like ligand 4 (Dll4) on the fibrovascular membranes in proliferative diabetic retinopathy (PDR),and investigate its relationship with vascular endothelial growth factor receptor 2 (VEGFR2).Methods Fifty-seven PDR patients (60 eyes) who underwent vitrectomy were enrolled in this study.The PDR patients were divided into non-injection group (30 patients,32 eyes) and injection group (27 patients,28 eyes).The eyes in injection group received intravitreal injection with ranibizumab at 2 to 7 days before surgery.The preretinal fibrovascular membranes were obtained from the PDR patients during vitrectomy.Eighteen epiretinal membranes were obtained from the non-diabetic patients was served as controls.The real-time polymerase chain reaction (RT-PCR) and immunohistochemical methods were used to detecting the expression ofNotch1,Dll4 and VEGFR2.In the meantime,the numbers of the nucleus of vascular endothelial cells in the membranes stained with hematoxylin were counted.Results The immunohistochemical staining revealed that there were positive expression ofNotch1,Dll4 and VEGFR2 in all PDR membranes,regardless of the injection of the ranibizumab.The levels ofNotch1,Dll4 and VEGFR2 protein in non-injection group were higher than those of injection group (t=3.45,6.01,4.08;P=0.030,0.008,0.023).In injection group,the number of endothelial cells in the membranes reduced (17.17 ± 2.48) compared with that of the non-injection group (41.50± 5.57).There was significant difference in the number of endothelial cells in the membranes between the two groups (t=9.58,P＜0.05).RT-PCR showed that the differences of the mRNA expression ofNotch1,Dll4 and VEGFR2 were all statistically significant among the PDR group and control group (H=12.50,12.50,12.02;P＜0.05).The expression ofNotch1,Dll4 and VEGFR2 in the PDR membranes was higher than that of epiretinal membranes from non-diabetic patients.In the PDR group,the expression of Notch1,Dll4 and VEGFR2 of non-injection group was higher than that of injection group.Spearman correlation analysis showed that the expression of mRNA between VEGFR2 and Dll4 (r=0.83),VEGFR2 and Notch1 (r=0.81),Notch1 and Dll4 (r=0.87) were all significantly correlated (P＜0.05).Conclusions The expression of Notch1 and Dll4 in the PDR membranes are higher than that of the control group,and it is positively correlated with the expression of the VEGFR2.Notchl and Dll4 play a regulatory rule in the neovascularization in PDR,the acting way may be correlated with VEGFR2.

Objective To analyse the immunostimulatory activity of CpG sequences in cysticercus cellulosae paramyosin (also named Antigen B,AgB)cDNA. Methods C57BL/6 mice were immunized with pcDNA3 AgB plasmid,pcDNA3 AgB′(CpG sequences were mutated),pcDNA3 or AgB protein and two weeks later,immune response was assayed by ELISA. Results IgG and IgG 2a were detectable at week 2 after immunization and continually increased until week 4.The antibody levels elicited by pcDNA3 AgB were significantly higher( P

[Abstract ] Objective Difficulties with the preparation of gastric cancer stem cells (CSCs) have been a main obstacle to the studies of gastric cancer .This article addresses the technology of the serum-free medium suspension cultivation of the MKN-45 gastric cancer cell line and screening of stem cells from the cell line based on the biomarkers of gastric CSCs . Methods MKN-45 cells were cultured in serum-free medium for 8 weeks and those in the logarithmic phase cultivated with hoechst 33342 followed by detection of the side cells by flow cytometry .When the side population cells reached 25%, all the cell microspheres were collected , hatched with CD133 and CD44, and subjected to fluorescence-activated cell sorting.The CDl33 +and CD44 +cells were selected as gastric CSCs . Results About 40%of the MKN-45 gastric CSCs were alive , prolif-erated, and formed floating cell balls .Side population cells constitu-ted 3.4% of the MKN-45 cells and 26.9% of the cell balls.The CDl33 +and CD44 +cells made up 11.2% of the MKN-45 cells and 90.3%of the cell balls. Conclusion Cell balls rich in CSCs can be successfully obtained by serum-free medium suspension culti-vation and CSCs can be screened out with hoechst 33342 and surface markers , which may serve as an experimental ground for the stud-ies of gastric CSCs .

AIM: To study the expression and the role of neurokinin-1 receptor(NK-1R) and neurokinin-2 receptor (NK-2R) in distal ileum during acute necrotizing pancreatitis(ANP).METHODS: 130 adult Sprague-Dawley rats were divided into 2 groups: the rats in ANP group( n=80 ) were induced by the retrograde intraductal infusion of 5% sodium taurocholate. The rats in sham operation control group ( n=50 ) received laparotomy only. Reverse transcription polymerase chain reaction (RT-PCR) was used to determine the mRNA expression of NK-1R and NK-2R, Western blot was used to investigate expression level of NK-1R.RESULTS: NK-1R and NK-2R mRNA levels were enhanced in the distal ileum of ANP rats compared with controls. Western blot revealed stronger NK-1R immunoreactivity exited in intestinal mucosa in ANP rats. The overexpression of NK-1R was associated with mucosal pathological score( r=0.77,P

Objective: To study the correlation between Erα gene polymorphism and the distribution pattern of TCM meridian syndrome in women with knee osteoarthritis. Methods: A total of 120 Knee osteoarthritis (KOA) female patients admitted to our hospital between January 2018 and December 2018 were selected as the observation group, and 120 healthy female subjects accepted physical examination at our hospital over the same period were selected as the control group. The Erα gene distribution of estrogen receptor was compared between the two groups to identify the differences between KOA women and healthy women. The TCM syndromes and meridians types of the patients in the observation group were defined to evaluate the degree of KOA. The correlation among KOA status, Erαgenotypes, TCM syndromes and meridians was analyzed. Results: The distribution of Erα gene XbaI was significantly different between the observation group and the control group (χ²=35.339, P<0.001). There was a statistical difference in the distribution of XbaI genotypes between mild to moderate and severe patients in the observation group (χ²=29.088, P<0.001). There was a moderate correlation between the Lequesne score and the fascia type in KOA women, and a high correlation between the Lequesne score and the TCM syndrome differentiation type, and a moderate correlation between the Lequesne score and the Erα gene and the XbaI genotype (P<0.05). The Erα gene polymorphisms, meridians and syndrome differentiation were moderately correlated (P<0.05). Conclusions In female patients with knee osteoarthritis, the conditions are more serious in patients with complex type of tendons, deficiency of liver and kidney, phlegm and blood stasis, Erα gene xx genotype, and there is a clear correlation between the TCM type of tendons and ER gene polymorphism.