Objective To clone human pancreatic cancer gene PTCH,construct the recombinant(expression) plasmid PET22b/PTCH and express the fusion protein.Methods The PTCH gene was(amplified) by RT-PCR from the total RNA extracted from human pancreatic cancer strain SW1990. The amplified product was inserted into the vector PET22b to construct the recombinant expression plasmid PET22b/PTCH,which was transformed into E.coliBL21-CodonPlus~(TM)-RP and then identified by(sequence) analysis.The expression of fusion protein was induced with IPTG and verified by Western blot method.Results A human pancreatic cancer gene with a reading frame of 789 bp was successfully cloned from human pancreatic cancer strain SW1990,which had the same sequence as that of PTCH gene in Genbank.The expression of PET22b/PTCH was proved by Western blot.Conclusion Human(pancreatic) cancer gene PTCH was successfully cloned and constructed with PET22b plasmid.The(prepared) fusion protein lays the basis for further study.

BACKGROUND: Recently, Chinese herb and comprehensive therapy are widely adopted for the treatment of chronic atrophic gastritis (CAG), while infrared ray is widely used in the fields of physical therapy and scientific research. Therefore, some scholars suggest whether the physical characteristics of infrared ray have effects on the treatment of chronic atrophic gastritis.OBJECTIVE: To observe the effect of infrared ray on the changes of gastric mucosa tissue in rat models with chronic atrophic gastritis.DESIGN: Randomized controlled observation.SETTING: Hebei North University.MATERIALS: Thirty-five adult Wistar male rats weighing from 180 to 230 g were purchased from Hebei Experimental Animal Center [SCXK (ji) 2003-1-003]. The experiment was disposed with the ethical standard. Sodium salicylate powder produced by Beijing Fangcao Chemical Company (batch number: 890720). The drug was prepared with distilled water. Infrared lamp (220 V, 200 W) was bought by Equipment Division of our college.METHODS: The experiment was carried out in the Experimental Center of Hebei Beifang College from June 2005 to January 2006. ① Experimental intervention: Rats were fed with conventional standard granules for one week. Among them, 8 rats were selected as the normal control group, and other rats underwent model establishment. Rats were perfused with sodium salicylate and alcohol to stimulate gastric mucosa, and then chronic CAG models were established for 8 weeks based on exertion, irregular diet and other factors. Five rats were randomly selected for the check of histopathology before the end of model confirmedly making, and then the model rats were randomly divided into model group and infrared group with 11 in each group. Infrared lamp (220 V, 200 W, 0.76–1.5 μm in wavelength) was used to vertically radiate at the gastric projective area of rats in the infrared group, once a day, ten minutes once for twenty days. The rats in normal group and model group were regularly breed. ② Experimental evaluation: The body mass was weighed every week in 1, 4, 9 and 12 weeks after modeling. The infiltration of inflammatory cells and thickness of gastric mucosa were observed under optic microscope.MAIN OUTCOME MEASURES: ① Changes of body mass; ② pathohistological changes of gastric mucosa.RESULTS: All 30 rats were involved in the final analysis. ① Changes of body mass: From the end of the 4th week, increasing percentage of body mass in the model group and infrared group was decreased gradually as compared with that in the normal group, and there was significant difference (P < 0.05). ② Pathohistological changes of gastric mucosa: Gastric mucosa of rats in the model group was thinner, and atrophic glands, notable inflammatory infiltration and partial intestinal metaplasia were observed under optic microscope. The thickness of gastric mucosa in the infrared group was significantly thicker than that in model control group, and there was significant difference (P < 0.01); the inflammatory cells in the infrared group were less than those in the model group, and there was significant difference (P < 0.05). Morphologic structure and volume of the parietal cells were all recuperated or closed to normal.CONCLUSION: Infrared ray can decrease thickness of gastric mucosa and reduce inflammatory cells of rats with chronic atrophic gastritis, and it has greatly therapeutic achievements.

Aim To elucidate the polymorphism of hepatic lipasegene gene and the relation to coronary heart disease. Methods CHD group had one hundred and fifty-six patients, and each subgroup was: myocardium infarction CHD subgroup included eighty-four patients; non-myocardium infarction subgroup included seventy-two patients; pure CHD subgroup comprised sixty-five patients and hypertension and CHD subgroup comprised ninety-one patients. Phenol-Chloroform method was used to extract DNA from human peripheral blood, and a combination of polymerasechain reaction and restriction fragment length polymorphism were used to analyze the distribution of genotypes and alleles of the polymorphism site of hepatic lipase. Results The genotype and allele distribution of HL-514C/T polymorphism were significantly different between the whole CHD group and control group(P

Objective To study the effects of He-Ne laser irradiation dosage on the expression of heat shock protein ( HSP70) and CyclinD_1 in rats with chronic atrophic gastritis ( CAG). Method Fifty-two adult male Wistar rats were randomly divided into five groups: a normal control group, model group and three groups receiving different doses of He-Ne laser irradiation. CAC was induced using an enema of 2% sodium salicylate and 30% alcohol combined with irregular fasting and forced exercise as pathogenic factors. Laser irradiation was applied for 20 days (large dose 6.24 J/cm~2 , medium dose 4. 80 J/cm~2, small dose 3. 36 J/cm~2). The changes in HSP70 and CyclinD_1 expression were observed. Results The expression of HSP70 and CylinD_1 were highest in the normal control group and the small dose laser group. Compared with the model group, the average expression of HSP70 and CyclinD_1 increased significantly in the small dose group. Conclusions Irradiation with a He-Ne laser at 3. 36 J/cm~2 provides good adjuvant therapeutic effect for CAG in rats. After irradiation, the expression of HSP 70 and CyclinD_1 increased. HSP is important in improving mucosal defenses and promoting cell proliferation in CAG, and it can be promoted through small doses of He-Ne laser irradiation.

Objective To construct small hairpin RNA(shRNA) lentiviral vectors targeting human a proliferation-inducing ligand(APRIL) gene and detect the titer of virus and suitable multiplicity of infection (MOI) after 293T cells were infected by the lentival vectors. Methods Three RNA interference targeting sequences of APRIL gene were screened including shAPRIL1210, shAPRIL1754 and shAPRIL1604. Both sense and antisense Oligo DNA of the targeting sequences were synthesized and cloned into the pGCL-GFP vector, respectively. The resulting lentiviral vectors containing shAPRIL were named LV-shAPRIL1210, LV-shAPRIL1754, LV-shAPRILI604. Then they were confirmed by PCR and DNA sequencing. 293T cells were co-transfected with LV-shAPRIL, pHelper 1.0 and pHelper 2. 0 to product lentivirus, respectively. The titer of virus and suitable MOI were tested according to the expression level of GFP in the 293T cells. Results PCR analysis and DNA sequencing confirmed that three shAPRIL DNA were successfully inserted into the lentiviral vectors. The titers of concentrated virus were 5 × 107, 6 × 107 and 4 × 107(transduction units )TU/ml, respectively, and the suitable MOI was 5. Conclusions Three shRNA lentiviral vectors targeting human APRIL gene have been successfully constructed, which lays a foundation for future studying APRIL's gene silencing in related target cells.

Objective To investigate the inhibitive effect of shRNA (short hairpin RNA) targeting APRIL gene on the pancreatic cancer cells in vitro and in vivo, in order to explore the feasibility of gene therapy for pancreatic cancer. Methods The LV-shAPRIL targeting APRIL gene had been constructed before, and was used to infect the CFPAC-1 cells. Cell proliferation and apoptosis were examined by MTT and flow cytometry. Then CFPAC-1 cells were used to construct the model of transplantation tumor into the nude mice, the tumor growth was assessed after LV-shAPRIL treatment. Results 96 hours after the LV-shAPRIL infection into CFPAC-1 cells, the cell proliferation was significantly inhibited when compared with control group and lentivirus infection group (P＜0.05 ). Flow cytometry showed the apoptosis ratio of the CFPAC-1 cells was (17.35±0.96)% in LV-shAPRIL group, which was higher than that in control group and lentivirus infection group (P＜0.05 ). After LV-shAPRIL injection into the model of nude mice, the tumor growth was slower than that in the two control groups. The tumor's volume of the LV-shAPRIL group was(821.8±123.3) mm3 and the mass was (2.16±0.18)g at 27 day, and were obviously depressed, when compared with two control groups (P±0.05). Conclusions LV-shAPRIL targeting APRIL gene can inhibit the growth of the CFPAC-1 cells in vitro and vivo. This may provide a new gene therapy approach for pancreatic cancer.

Objective To construct of shRNA lentiviral expression vector targeting APRIL (aproliferation-inducing ligand) gene in CFPAC-1 cell of human pancreatic cancer. Methods We used gene engineering to screen RNA interference targeting sequence of APRIL gene. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the pGCL-GFP vector. The resulting lentiviral vector containing shAPRIL were named LV-shAPRIL. Then it was conformed by PCR and DNA sequencing identification. 293T cells were eotransfected with LV-shAPRIL,pHelper 1.0 and pHelper 2.0 to product ientivirus. The titer of virus was tested according to the expression level of GFP in the 293T cells. After recombinant lentivirus infection into CFPAC-1 cells, we used real-time RT-PCR and Western blotting to examine APRIL mRNA and protein expression at different cell culture period.Results PCR analysis and DNA sequencing conformed that shAPRIL DNA was successfully inserted into the lentiviral vector. The titer of concentrated virus were 5 × 107 TU/ml. APRIL expression in CFPAC-1 cells were inhibited significantly at both mRNA and protein level. APRIL mRNA expression were decreased 73%, 70%and 71% , respectively, after the infection of 4 days, 4 weeks and 8 weeks by LV-shAPRIL. APRIL protein expression were decreased 66%, 63% and 62%, respectively , after the infection of 4 days , 4 weeks and 8weeks by LV-shAPRIL. Conclusions ShRNA lentiviral expression vector targeting APRIL gene has been successully constructed, and it can effectively inhibit the expression of APRIL gene in CFPAC-1 ceils. This study lays a foundatin for in vivo research APRIL gene scilence in pancreatic cancer cell using the model of nude mice.

Objective To study the effects of He-Ne laser irradiation on histological changes and the expression of proliferating cell nuclear antigen ( PCNA ) and B-cell lymphoma/leukemia-2 ( Bcl-2 ) in the gastric mucosa of rats with chronic atrophic gastritis (CAG) so as to elucidate the relationship of He-Ne laser irradiation with precancerous lesions and apoptosis in the gastric mucosa. Methods The rats were divided into a normal group,a model group and a laser group.A model of CAG was established by gastric perfusion with a mixture of sodium salicylate and alcohol combined with irregular fasting and forced exercise.A He-Ne laser was used to irradiate the rats at 3.36 J/cm2 for 7 min daily for 20 d.Histopathological changes including the severity of inflammation in the gastric mucosa and the morphology and structure of the parietal cells were observed with a light microscope,and the expression of PCNA and Bcl-2 was detected with immunohistochemical methods. Results The pathologic morphological changes in the gastric mucosa of the model group were atrophy of the glands of the gastric mucosa and notable inflammatory infiltration.But in the laser group inflamed cells decreased,and the morphology,structure and volume of the cells all recovered close to normal.The immunohistochemistry results showed that during the atrophy of the gastric mucosa the expression of PCNA and Bcl-2 was elevated,and it was significantly higher in the model group than in the normal group.After irradiation the expression of PCNA and Bcl-2 was significantly lower. Conclusions There was hyper-proliferation in the gastric mucosa of the CAG model rats,with high expression of apoptosis suppressor PCNA and Bcl-2 proteins.Laser irradiation can reduce the expression of PCNA and Bcl-2,enhance cell proliferation and induce apoptosis,preventing the development of cancer.Laser irradiation has a good adjuvant therapeutic effect for all the pathological changes observed.

Objective:To analyze 916 cases of endoscopically diagnozed reflux esophagitis(RE) and to explore the endoscopic and clinicopathologic characteristics of RE.Methods:Totally 916 RE patients from Lixiahe district in Jiangsu province(1997 2002) were graded with criterion made in Chinese Yantai in 1999.Some cases were subjected to pathology,age,sex,symptoms,endoscopic manifestations and pathology variety examination.Results:The incidence of RE was for 7.4% in endoscopic checked patients(916/12 376).The male to female ratio was 2.2∶1,mean age was (54.42?15.05),patients above 60 years old accounted for 46.5%.Only 32.6% of RE patients had typical reflux symptoms(426/916,299/916).Endoscopic diagnosis focused on Chinese Yantai standardⅠ Ⅱ (85.3%,781/916),pathology diagnosis focused on light to moderate degree,secondary RE was common in severe degree.Some patients were accompanied by bile reflux,esophagus gap hernia and peptic ulcer.Conclusion:The incidence of RE in Lixiahe district in Jiangsu province is rather high,it is common in middle aged and senior male patients(Ⅰ Ⅱ).Pathology variety is chiefly scalelike epithelial proliferation filled with chronic inflammatory cells.It was partly accompanied by atypical proliferation,erosion and ulcer,appearance of early cancer was occasionally found.The sensitivity of clinic symptoms diagnosis is lower than endoscopic diagnosis which is simple and of great value.

ObjectiveTo investigate the protective effect of transfected microRNA-146a (miR-146a) on mice with sepsis-induced acute lung injury (ALI) in vivo.Methods Twenty-four healthy male BALB/C mice were randomly divided into sham group, sepsis group, transfection group and transfection control group, eachn = 6. Mice in transfection group were given miR-146a agomir loaded by in vivo-jetPEITM via airway before reproduction of model, and mice in transfection control group were given negative control loaded by in vivo-jetPEITM only via airway. The septic model was reproduced by cecal ligation and puncture (CLP) 12 hours after transfection , and the mice in the sham group underwent laparotomy and closure only without ligation and puncture of the cecum. The mice of each group were sacrificed at 24 hours post-operation. The expression of miR-146a in lung tissue was determined by real time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and the quantity of tumor necrosis factor-α (TNF-α) in the bronchial alveolar lavage fluid (BALF) was determined with enzyme-linked immunosorbent assay (ELISA). The wet/dry ratio of lung (W/D) was determined. The pathohistological changes in the lung were observed and scored. Results The expression of miR-146a showed a significant increase in sepsis group, transfection group and transfection control group, which were (3.56±0.43), (27.64±3.46) and (3.72±0.54) folds of that in sham group, respectively (P< 0.05 orP< 0.01). The miR-146a expression in transfection group was significantly increased compared with sepsis group and transfection control group (bothP< 0.01), but no statistical difference in the expression was found between sepsis group and transfection control group (P> 0.05). Compared with the sham group, higher level of TNF-αin the BALF was found in the sepsis group, transfection group and transfection control group (ng/L: 511.65±43.47, 305.74±34.76, 492.27±42.21 vs. 50.72±7.23, allP< 0.01). The level of TNF-α in transfection group was significantly lower than that in sepsis group and transfection control group (bothP< 0.01). Compared with the sham group, the W/D ratio of lung in sepsis group, transfection group and transfection control group showed a significant increase (6.11±0.32, 5.02±0.29, 6.05±0.43 vs. 4.18±0.10, allP< 0.01). The W/D ratio of lung in transfection group was significantly lower than that of sepsis group and transfection control group (bothP< 0.01). The lung injury score of transfection group was significantly lower than that of sepsis group and transfection control group (6.12±0.75 vs. 10.53±1.52, 9.73±1.08, bothP< 0.01).Conclusions miR-146a agomir loaded by in vivo-jetPEITM instillation into airway was able to increase the expression of miR-146a in the lung tissue of septic mice. Up-regulation of miR-146a inhibit the release of the inflammatory cytokine TNF-α stimulated by sepsis, and alleviate inflammatory reaction and lung tissue injury in mice with sepsis-induced ALI.