Objective To isolate and purify the membrane protein of SeAx cells, which was derived from cutaneous T-cell lymphoma cell line (CTCL), and to prepare polyclonal antibodies against membrane proteins. CTCL antigens were screened with SEREX method from the cDNA expression library of SeAx cells. Methods SeAx cells were cultured to 2?1010 and membrane proteins were purified by means of differential centrifugation after homogenate was made, and organelle protein specific antibodies were used to perform Western blot. Rabbit was immunized 4 times by the membrane proteins, and the serum antibody titer was measured by ELISA after each immunization. The specific interactions between antibodies in serum and SeAx proteins were detected by Western blotting. E. coli were initially transfected by recombinant phages, and the recombinant proteins expressed were transferred onto the nitrocellulose membranes, which were then again reacted with diluted serum from the immunized rabbit. Positive clones were subcloned and the nucleotide sequence of the cDNA inserts was determined. Finally, cDNA sequence analysis via database searching was performed to define the gene. Results Membrane proteins of SeAx cells were purified by differential centrifugation and a 1?10-5 titer of antibody was obtained after 4 times of immunization. 1?106 phage clones were screened, 25 of which were positive. Nucleotide sequencing and database searching showed that 4 cDNA inserts were cell membrane proteins, respectively as integrin alpha 4,ligatin, matrix metalloproteinase 24 and MHC-I molecule HLA-A. Conclusion Membrane proteins of CTCL and SeAx cells, are purified and polyclonal antibodies against SeAx cell membrane proteins are successfully prepared. The 4 genes of membrane proteins are identified by means of SEREX from SeAx cDNA library, expressed by phages. These genic candidates are able to induce systemic humoral immune reactions and might therefore be promising targets for immunotherapeutic interventions of CTCL.

Objective To investigate the changes in Bcl-2-associated athanogene 1(BAG-1)expression,and the mechanism of nuclear translocation in rat alveolar macrophages(AMs)induced by lipopolysaccharide(LPS)and dexamethasone(Dex).Methods Primary culture AMs treated by LPS and Dex were divided randomly into three groups:6h group,2h group and 24h group.The BAG-1 expression in AMs was detected with Western blot.The interactions between BAG-1 and glucocorticoid receptor(GR)were detected with immune co-precipitation.The changes in GR expression in nuclear protein were evaluated with Western blotting after transfection of RNA interference recombinant plasmids(named psilencer 3.1-GR)targeting to GR gene.Results The expression of BAG-1L in total protein increased,and that of BAG-1S showed no changes.Only BAG-1L,with no BAG-1S,was detected in nuclear protein,and its expression increased gradually in 24h.Interaction between BAG-1L and GR was found in nucleolus after treatment.After transfection of plasmids psilencer 3.1-GR,the BAG-1L expression in nuclear protein decreased significantly compared with that of non-transfection group(P

Objective To investigate the change of the first-phase insulin secretion in impaired glucose regulation and its influencing factors.Methods The investigation was performed in 1024 subjects who were selected by cluster sampling.The body waist circumference (WC) was examined in these subjects.Oral glucose tolerance test was performed,and fasting plasma glucose,fasting insulin as well as 30-,60-,and 120-minute plasma glucose and insulin after glucose intake were also tested.All the 1024 subjects were divided into 5 groups based on the results of oral glucose tolerance test:normal glucose tolerance group,impaired fasting glucose group,impaired glucose tolerance group,impaired fasting glucos/impaired glucose tolerance group,and diabetes mellitus group.First-phase insulin secretion index (the ratio of change in insulin to change in glucose during the first 30 minutes after glucose ingestion was calculated.Results Compared with normal glucose tolerance group (1.96 ± 1.03),the first-phase insulin secretion index significantly decreased in the impaired fasting glucose group (1.79 ±0.91) (P =0.007),the impaired glucose tolerance group (1.81 ± 0.97) (P =0.007),the impaired fasting glucose / impaired glucose tolerance group (1.59 ± 0.85) (P =0.005),and the diabetes mellitus group (1.30 ± 0.60) (P =0.004).Logistic regression analysis showed that WC was the strongest influencing factor of first-phase insulin secretion (β =0.716,P =0.000).Conclusions The firstphase insulin secretion index has already dropped in the stage of impaired glucose regulation.WC can be a useful indicator for evaluating first-phase insulin secretion.

Objective:To have an experimental investigation on teaching reform of Orthopedics of TCM based on network resources.Method:Two comparison groups were set and observed when we were teaching the orthopedics students of 2003 grade.One was taught by traditional problem-based learning(PBL) method,the other was taught by PBL method based on network resources.Results:The creative thought and research ability of the students who had been taught by PBL method based on network resources were greatly improved.Conclusion:The teaching method based on network resources has so many advantages such as students being able to self-learn and research without time and space limit that it can be used more in medical students teaching.

Objective:To observe the effects of AFP gene silencing by siRNA on the Survivin mRNA of hepatocellular carcino-ma cell line HepG2. Methods:AFP gene expression was downregulated in HepG2 cell by RNAi, and the AFP content in the superna-tant was detected by ELISA. Survivin mRNA level was tested by RT-PCR. MTT was applied to evaluate cell proliferation. Flow cytom-etry was employed to observe cell apoptosis. Results:At 48 h after transfection, AFP expression was almost completely inhibited, cell proliferation activity was decreased by 43.1%, cell apoptosis rate was increased by 24.3%, and the Survivin mRNA expression was re-duced to 22.0%in the experimental group. No evident changes were observed in negative control and blank groups. Conclusion:AFP gene silenced by RNAi induces growth inhibition and apoptosis promotion of hepatocellular carcinoma cell line HepG2. This gene may be associated with the suppression of Survivin mRNA.

Objective:To approach the distinctive histopathological chages of nontuberculous mycobacteria lymphadenitis. Methods: The experimental animal model of nontuberculous mycobacteria lymphadenitis was developed and the histopathological changes were observed under the light microscope.Results:The distinctive histopathological changes of nontuberculous mycobacteria lymphadenitis were identified as following: ①Granulomas were found in lymph nodes which were infected with nontuberculous mycobacteria.The coagulation necrosis was located at the center of the granuloma and it was not caseation.Neutrophils were distributed over the necrosis area.Surrounding the center necrosis area,many epithelioid cells,lymphocytes and mononuclear cells could be found.The periphery of the granuloma was surrounded by the fibrous tissues.Outside the granuloma,Langhans giant cells could be found.②Serpiginous necrosis was found in the lymph nodes which were infected with nontuberculous mycobacteria.The shape of serpiginous necrosis was a long strip.In the center area,the strip of coagulation necrosis and the strip of the space which was remnant after the desquamation of necrosis tissues could be found.Many neutrophils were distributed over the necrosis area.Around the center necrosis area,many epithelioid cells,lymphocytes and mononuclear cells could be found.The fibrous tissues were in the borderline.Conclusion:Serpiginous necrosis was one of the distinctive histopathological change of nontuberculous mycobacteria lymphadenitis.

Objective To investigate the effect of clotrimazole on apoptosis of hepatic cells after ischemia-reperfusion injury and its mechanism. Methods Hepatic ischemia-reperfusion rat model was established. Thirty-two male Sprague-Dawley rats were randomly allocated into sham-operated group, model control group, low dose clotrimazole group and high dose clotrimazole group. Apoptosis in hepatic tissue was assessed by TUNEL method. Protein expression levels of CYP3A1,Bcl-2,Bax and PARP were measured by Western blotting. Results As compared with model control group, the apoptosis rate, tissue injury,activity of plasma enzymes and the Bax/Bcl-2 expression ratio were reduced in low and high dose clotrimazole groups. The apoptotic index in both clotrimazole-treated groups was lower than that of model control group with statistically significant difference. CYP3A1 expression was significantly induced by clotrimazole compared to the sham-operated group. Conclusion Clotrimazole may inhibit apoptosis of hepatic cells by up-regulating Bcl-2 and down-regulating Bax, thus produce a protective effect on hepatic ischemia-reperfusion injury and it is also related to the inhibition of PARP shear.

Objective: To investigate the expression changes of Bcl-2-associated athanogene 1(BAG-1) and its regulatory effect on the glucocorticoid receptor(GR) activity in rat alveolar macrophages in conditions of cell inflammation and glucocorticoid therapy.Methods: The expression changes of BAG-1 were detected by Western blot after lipopolysaccharide(LPS) and Dexamethasone(Dex) treatment of rat alveolar macrophages(AMs),the interaction between BAG-1 and GR determined by immune coprecipitation experiment,and the transcriptional activation of GR measured by relative luciferase activity assay.Results:After LPS and Dex treatment,the expression of BAG-1L in total protein increased but that of BAG-1S remained changed,BAG-1L rather than BAG-1S was detected in nuclear protein and its expression increased gradually within 24 hours,the interaction between BAG-1L and GR was observed in nucleoli,and the transcriptional activation of GR decreased,with a negative correlation between BAG-1L expression and GR activity.Conclusion:LPS and Dex acting on rat alveolar macrophages,the expression of BAG-1L increases,which,coupled with GR,translocates into nucleoli and inhibits GR activity.This might be the important mechanism that underlies glucocorticoid resistance in inflammation.

AIM:To investigate the activation of nuclear factor ?B(NF-?B) induced by lipopolysaccharides(LPS) in rat alveolar macrophages(AMs) and its regulatory role in tumor necrosis factor(TNF-?) secretion.METHODS:The dynamic activity changes of NF-?B induced by LPS were determined with electrophoretic mobility shift assay(EMSA).Antisense oligonucleotides of NF-?B subunit(p65) were transfected into AMs prior to LPS stimulation.The effect of antisense oligonucleotide transfection on expressions of p65 and TNF-? in supernatant were measured with Western blotting and enzyme linked immunosorbent assay(ELISA),respectively.RESULTS:NF-?B activity increased markedly and reached its peak level at 4 h after LPS stimulation.After transfected with antisense oligonucleotides of NF-?B subunit(p65),expression of p65 and TNF-? in supernatant decreased markedly.CONCLUSION:NF-?B activity has a positive effect on regulating secretion of TNF-? in AMs induced by LPS.

Objective: Vascular endothelial growth factor A (VEGFA) was investigated as the key protein which might promote the specific metastasis progress of nasopharyngeal carcinoma. Methods: Sixteen specimens of pulmonary metastasis carcinoma and counterparts in primary nasopharyngeal carcinoma tissue were collected from patients. The expression of VEGFA through immunohistochemistry was investigated.VEGFA was knocked down by siRNA in two cell lines of nasopharyngeal carcinoma (CNE-1 and 5-8F), MTT and Transwell test were used to explore the role of VEGFA in praxiology. Then shRNA was used to cultivate the stable CNE-1 cell line with down-regulated-expression of VEGFA. The nude mice models were built through tail vein injection of specific nasopharyngeal carcinoma cells, and lungs were collected to perform further metastasis analysis. Results: Previous genetic studies showed that VEGFA had higher expression in metastasis tissue, and the result was validated in the present study using immunohistochemistry. The percentage of positive cells was 84.8% in pulmonary metastasis group, 51.5% in primary tissue group (t=8.639, P<0.05), average optical density was 0.154 in pulmonary metastasis group, 0.061 in primary tissue group (t=18.791, P<0.05). Low expression of VEGFA inhibited cell viability of optical density value of CNE-1 in siRNA gourp was 0.715, 0.902 in control group (t=7.274, P<0.05); 5-8F in siRNA group was 0.715, 0.935 in control group (t=7.751, P<0.05). Number counting suppressed migration of CNE-1 in siRNA group was 52 per high-power lens, 124 per high-power lens in control group (t=29.380, P<0.05), 5-8F in siRNA group was 65 per high-power lens, 155 per high-power lens in control group (t=18.181, P<0.05). Number counting invasion of CNE-1 in siRNA gourp was 38 per high-power lens, 86 per high-power lens in control group (t=27.665, P<0.05), 5-8F in siRNA group was 52 per high-power lens, 116 per high-power lens in control group (t=40.972, P<0.05) in vitro. Furthermore, knock-down of VEGFA in nasopharyngeal carcinoma reduced the pulmonary metastasis in vivo. Number counting of tumor volumes in shRNA group was 2.4, and 11.0 in control group (t=6.143, P<0.05); average optical density of immunohistochemistry in shRNA group was 0.033, and 0.176 in control group (t=15.734, P<0.05). Conclusions Results above reveal the overexpression of VEGFA in nasopharyngeal carcinoma can facilitate the pulmonary metastasis. Targeting VEGFA may provide a new biomarker of clinical study.