Objective To explore the value of human epididymis protein 4(HE4),cancer antigen 125(CA125) and the risk of ovarian malignancy algorithm(ROMA) in the diagnosis of ovarian cancer.Methods Electrochemical luminescence and Enzymelinked immunosorbent assay (ELISA) were used to determine the levels of serum HE4,CA125 in 56 patients with ovarian carcinoma,73 cases of ovarian benign tumor and 50 health women,and the ROMA was calculated by HE4 and CA125 levels depending on the menopause state,drawing the receiver operating characteristics(ROC) curve and calculating the area under the curve(AUC).Results The average levels of the HE4,CA125 and the value of the ROMA were (345.33±605.03)pmol/L,(701.46±1 500.30) U/mL,(58.72±31.00) ％ in the ovarian carcinoma group,(53.84± 14.68)pmol/L,(44.25±45.81)U/mL,(10.80± 6.75) ％ in the ovarian benign tumor group,and (46.03±10.26)pmol/L,(17.39±10.64)U/mL,(6.92±3.85)％ in the health control group respectively,compared with the benign tumor group and the health control group,the ovarian carcinoma group were higher in HE4,CA125 and the ROMA value,and the difference were significantly (P＜0.05),whereas compared in the ovarian benign group and the health group,except the CA125 was higher in the benign group and the difference had statistical significance(P＜0.05),the HE4 level and the value of the ROMA had no statistical significance(P＞0.05).The sensitivities of the HE4,CA125 and ROMA were 71.43％,76.79 ％,89.28％,the specificities were 93.15 ％,53.42％,94.52 ％ and the ROC-AUCs were 0.930,0.809,0.937 respectively.When the specificity for the diagnosis of the ovarian carcinoma was 95.00％,the sensitivities of the HE4,CA125 and ROMA were 80.40％,53.60％,83.90％ respectively.Conclusion HE4 and CA125 combined detection to calculate the ROMA can elevate the sensitivity and specificity for the ovarian carcinoma diagnosis.

Objective To establish a method for amplification of immature dendritic cells(DC) from murine bone marrow in vitro and investigate correlations between maturation degree of DC and varying dosages of granulocyte-macrophage-colony stimulating factor(GM-CSF).Methods Dendritic cells from murine bone marrow were cultured with different dosages of rm GM-CSF.The suspension cells were examined with scanning electronic microscope,and the non-sensitized T lymphocyte proliferation was observed by mixed lymphocyte reaction.Results DC cultured in lower dosage of rmGM-CSF(GMlow DC) exhibited typical characteristics of DC,and had immature characteristics in cell phenotype and cell functions with high expression of CD11c and low expression of CD80,CD86 and MHC II on the surface of the cells.The ability of GmlowDC to stimulate the proliferation of non-sensitized T lymphocyte in vitro was weaker than that of GmhighDC.Conclusions The methods of immature DCs culturing establised by allthors was feasible.The dosage of rm GM-CSF has a direct relationship with the maturation degree of DC.

BACKGROUND: During pancreas transplantation, ischemia/reperfusion (I/R) injury can lead to many complications, which directly threaten the survival of the donor pancreas and the receptor itself, and the serious ones may result in the failure of transplantation. Ischemic preconditioning can protect the target organs in the following ischemia, which has become one of the hot spots in investigating organ transplantation.OBJECTIVE: To observe the early protective effect of ischemic preconditioning on the I/R injury of the grafted pancreas in the rat, and analyze its correlation with apoptosis.DESIGN: A randomized control animal experiment.SETTINGS: Department of Gastrointestinal Surgery, Department of Anesthesiology, Xijing Hospital of the Fourth Military Medical University of Chinese PLA.MATERIALS: Seventy male SD rats of 3-6 months, weighing 250-320 g, were used.METHODS: The experiments were conducted in the laboratory of Department of Gastrointestinal Surgery between September 2001 and April 2004.Six normal rats were taken as the control group, and 24 successful diabetic models were divided into I/R group and 1, 2 and 3-time ischemic preconditioning groups (n=18) according to the method of random number table,with 6 rats in each. The rats in the latter three groups were treated with 5-minute ischemia and 5-minute reperfusion for once, twice and three times respectively, all the rats underwent the pancreas transplantation. Twentyfour SD rats served as donors.MAIN OUTCOME MEASURES: ① Blood glucose before and after reperfusion in each group; Serum contents of tumor necrosis factor alpha (TNF-α) and nitric oxide; Activities of superoxide dismutase (SOD) and myeloperoxidase (MPD) and content of malondialdehyde (MDA) in the grafted pancreatic tissue; ② Apoptosis in the grafted pancreatic tissue observed by means of in situ end-labeling; Expressions of Bax and Bcl-2 proteins in the grafted pancreatic tissue with the method of Western blotting.RESULTS: ① Changes of blood glucose before and after reperfusion: The levels of blood glucose were decreased as compared with those before reperfusion in the I/R group and ischemic preconditioning groups. It was significantly lower in the 2-time ischemic preconditioning group than in the I/R group, 1 and 3-time ischemic preconditioning groups (P ＜ 0.05). ②Serum content of TNF-α at 2 hours after reperfusion: It was lower in the ischemic preconditioning groups than in the I/R group; It was lower in the 2-time ischemic preconditioning group than in the 1 and 3-time ischemic preconditioning groups (P ＜ 0.05). ③ Serum content of nitric oxide after reperfusion: It was higher in the ischemic preconditioning groups than in the I/R group; It was higher in the 2-time ischemic preconditioning group than in the 1 and 3-time ischemic preconditioning groups (P ＜ 0.05). ④SOD activity in the grafted pancreatic tissue after perfusion: It was higher in the ischemic preconditioning groups than in the I/R group; It was higher in the 2-time ischemic preconditioning group than in the 1 and 3-time ischemic preconditioning groups (P ＜ 0.05). ⑤ MAD content and MPD activity in the grafted pancreatic tissue after perfusion: Those were lower in the ischemic preconditioning groups than in the I/R group, also lower in the 2-time ischemic preconditioning group than in the 1 and 3-time ischemic preconditioning groups. ⑥ Apoptosis in the grafted pancreatic tissue: The apoptosis index after perfusion was lower in the ischemic preconditioning groups than in the I/R group; It was significantly lower in the 2-time ischemic preconditioning group than in the 1 and 3-time ischemic preconditioning groups (P ＜ 0.05). ⑦ Expressions of Bax and Bcl-2proteins in the grafted pancreatic tissue: There was high expression of Bax protein and low expression of Bcl-2 protein in the grafted pancreatic tissue after perfusion in the I/R group; Low expression of Bax protein and high expression of Bcl-2 protein in the grafted pancreatic tissue after perfusion were observed in the ischemic preconditioning groups; In the 2-time ischemic preconditioning group, the expression of Bcl-2 protein was the highest but that of Bax protein was the lowest.CONCLUSION: Ischemic preconditioning can protect the grafted pancreas from I/R injury at early pancreas transplantation, which maybe correlated with the elevation of SOD activity, increase of the synthesis of endogenous nitric oxide, down-regulation of TNF-α and the alleviations of the adhesion and aggregation of polymorphonuclear leukocytes. Ischemic preconditioning can reduce the apoptosis of the grafted pancreas, and the the possible mechanism may be correlated with the alleviations of the adhesion and aggregation of polymorphonuclear leukocytes, reduce of oxygen-derived free radicals, up-regulation of Bcl-2 protein and the down-regulation of Bax protein. 5-mintue ischemia and 5-minute reperfusion for twice is the best way to induce ischemic preconditioning in rat pancreas transplantation.

BACKGROUND: Ginkgo biloba extract (GBE) has the pharmacological actions of antioxidation, eliminating free radicals and anti-platelet activating factors, it also can relieve the ischemia/reperfusion injury of various organs.OBJECTIVE: Toobserve whether GBE can relieve the ischemia/reperfusion injury of transplanted pancreas in diabetic rats or not.DESIGN: A complete randomized grouping design, controlled study.SETTINGS: Department of Gastrointestinal Surgery and Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University of Chinese PLA Hospital.MATERIALS: Totally 128 male SD rats of clean grade, aged 3-6 months,weighing 250-320 g, were used. GBE was produced by Dr. Willmar Schwabe Pharmaceuti - cals (Ginaton parenteral solution, 5 mL/piece, containing 17.5 mg GBE, including 4.2 mg ginkgo flavone glycosides, batch number: 1511102).METHODS: The experiments were carried out in the laboratory of Department of Gastrointestinal Surgery from September 2001 to April 2004.① Eighty rats were injected with STZ (65 mg/kg) via penile vein, and 60 of them with fasting blood glucose exceeding 17.4 mmol/L for 2weeks were taken as the diabetic rats, and the other 48 normal rats were taken as donors. ② The 60 diabetic rats were randomly divided into two groups: ischemia/reperfusion group (n=30) and GBE group (n=30), and pancreas transplantation was performed in both groups. In the ischemia/reperfusion group, the rats were douched with 4 ℃ iced balanced salt solution containing heparin (1.5×105 U/L) for 20 minutes. In the GBE group, the recipients were given intravenous injection of GBE (1.5 mL/kg) at 1 day and 30 minutes before transplantation, and those in the ischemia/reperfusion group were intravenously injected with saline of the same volume. The donor pancreases were all reserved in 4 ℃ iced balanced salt solution containing heparin (1.5×105 U/L), the cold and hot ischemia times were kept for 180 and 15 minutes in each group to induce ischemia/reperfusion injury of transplanted pancreas. ③ Six randomly selected rats were killed at 2 days before transplantation and at 3 and 7days after transplantation respectively to detect fasting blood glucose; The activity of amylase was determined with corresponding kit provided by Nanjing Jiancheng Bioengineering Institute; Pancreas tissues were removed for hematoxylin and eosin (HE) staining; Six rats were used to observe the metabolic indexes; The other 6 rats were used to observe the survival rate within 1 month. ④ The differences of the measurement data were compared with the paired t test.MAIN OUTCOME MEASURES: ① Changes of fasting blood glucose level, metabolic indexes and activity of amylase before and after pancreas transplantation in the rat recipients of both groups; ② Pathological changes at 3 and 7 days after transplantation in the rat recipients of both groups.RESULTS: All the 60 rat as recipients finished the detections of blood glucose, food intake, water intake, urinary output and blood amylase. ①The survival rate within 1 month after transplantation was obviously higher in the GBE group than in the ischemia/reperfusion group (83%, 33%, P＜ 0.01). ② The blood glucose, water intake, food intake and the urinary output at 3 and 7 days after transplantation were obviously decreased as compared with those at 2 days before transplantation in both theischemia/reperfusion group and GBE group (P ＜ 0.05-0.01), and those at 3 and 7days after transplantation were obviously lower in the GBE group than in the ischemia/reperfusion group (P ＜ 0.05-0.01). ③ The activity of blood amylase at 3 days after transplantation was obviously increased as compared with that before transplantation in both the ischemia/reperfusion group and the GBE group (P ＜ 0.01, 0.05), it was still obviously higher at 7 days after transplantation than at 2 days before transplantation in the ischemia/reperfusion group (P ＜ 0.01), and it had almost recovered to normal in the GBE group. The activities of blood amylase at 3 and 7 days after transplantation were obviously lower in the GBE group than in the ischemia/reperfusion group (P ＜ 0.01). ④ The results of the pathological observation showed that the damaged severity of the transplanted pancreas was greater in the ischemia/reperfusion group than in the GEB group.CONCLUSION: GBE pretreatment can improve the survival rate of pancreas transplantation in rats, reduce the activity of blood amylase, ameliorate the metabolism, relieve the severity of reperfusion injury of pancreas,and plays a protective role in the pancreas transplantation.

OBJECTIVETo determine the influence of Helicobacter pylori (H. pylori) on gastric cancer-related antigen MG7 expression.

METHODSThe H. pylori infection and the expression level of antigen MG7 in gastric mucosa were determined by HE stain, PCR, ELISA and immunohistochemistry in 291 patients with H. pylori-related conditions, among whom 34 were followed-up.

RESULTSNo significant difference was found between H. pylori-negative and H. pylori-positive intestinal metaplasia, atrophic gastritis and dysplasia of gastric epithelium in positive rate of antigen MG7 expression. There was significant difference between H. pylori-negative and H. pylori-positive superficial gastritis in the positive rate of MG7 expression (P < 0.05). During follow-up, one of 3 H. pylori-negative cases turned to be H. pylori-positive, and its MG7 expression turned to be higher at the same time. Three of 31 H. pylori-positive patients were discovered as having early gastric cancer, among whom one with antigen MG7 expression (+ + +) was found to have a reduced Mg7 expression accompanied with H. pylori eliminutied after operation.

CONCLUSIONThere is correlationship between H. pylori infection and MG7 expression in superficial gastritis. Although the MG7-positive lesions with H. pylori infection shows a benign nature in morphology, they also have the potential risk of developing into gastric cancer. Therefore, they should be followed up, during which special attention should be paid to patients with increased MG7 expression.

Antibodies, Bacterial ; blood ; Antigens, Neoplasm ; biosynthesis ; DNA, Bacterial ; genetics ; Enzyme-Linked Immunosorbent Assay ; Gastric Mucosa ; metabolism ; microbiology ; pathology ; Gastritis ; metabolism ; microbiology ; Helicobacter Infections ; metabolism ; microbiology ; Helicobacter pylori ; genetics ; growth & development ; immunology ; Humans ; Immunohistochemistry ; Polymerase Chain Reaction ; Stomach Diseases ; metabolism ; microbiology ; Stomach Ulcer ; metabolism ; microbiology

ObjectiveTo investigate the immune tolerance function and significance of allogene bone marrow injection to the small intestines transplantation of rats.MethodsInbreeding line rat F344/N and Wistar/A were selected to perform heterotopic graft of the whole small intestine. 7 days before allogene transplantation, donator bone marrow cells (BMC) were injected into thymus of acceptor (the testing group). According to the isogene and allogene rat transplant model, it was comprehended whether injecting allogene donator marrow into acceptor thymus could decrease the acute rejection after transplantation.Results3, 5 or 7 days after allogeneic rat dystopia whole small intestine transplantation, typical reject reaction appeared, but there was no reject reaction in isogenome and testing group. 3 days after allotransplantation, serum soluble interleukin-2 receptor (sIL-2R) and tumor necrotic factor-α (TNF-α) levels were significantly higher than the other groups (P<0.01). The level of serum sIL-2R and TNF-α in the allogene marrow injecting group were only slight higher on the 3rd or 5th day, and getting downtrend, and there was no significant difference compared with isogenic transplantation group.ConclusionAllogenic donator bone marrow intrathymic injecting into acceptor 7 days before small intestina transplantation, can reduce the reject reaction after the grafting. The levels of serum sIL-2R and TNF-α can be selected as a sensitive early diagnosis index of acute rejection after small intestine transplantation.

Objective To investigate the effects of p300/CBP on histone acetylation of Foxp3 gene and its roles in the immunological pathogenesis of Kawasaki disease (KD).Methods Forty-six children with KD and twenty-eight age-matched health children were consented to participate in this study.Co-immunoprecipitation and real-time PCR were performed to detect Foxp3-associated acetylation levels of histone H4 and binding abilities of p300, CBP, pSmad3 (phosphorylated mothers against decapentaplegic homolog 3) and NF-AT (nuclear factor of activated T cells) with Foxp3 gene in CD4+ T cells.The percentages of CD4+CD25high Foxp3+ cells (Treg) and the expression of Foxp3, CTLA4 (cytotoxic T-lymphocyte-associated protein 4), p300, CBP, TGF-βRⅡ (transforming growth factor β receptor Ⅱ) and pLAT1 at protein level were analyzed by flow cytometry.Quantitative real-time PCR was used to measure the expression of Foxp3, IL-10, TGF-β, TGF-βRⅠ, Egr-1 (early growth response protein 1), RARα (retinoic acid receptor α) and PLCγ1 (phospholipase C-γ1) in Treg cells at mRNA level.Plasma concentrations of TGF-β and retinol acid (RA) were measured by enzyme-linked immunosorbent assay.Results (1) The percentages of Treg cells, levels of Foxp3 and molecules associated with suppressive function of Treg cells (TGF-β, IL-10 and CTLA4), acetylation levels of histone H4 associated with promoter, conserved non-coding DNA sequence 1 (CNS1) and CNS2 of Foxp3 gene decreased remarkably during acute KD (P<0.05), but were restored after IVIG therapy (P<0.05).Meanwhile, all of the aforementioned items in KD patients with coronary artery lesions (KD-CAL+) were lower than those without coronary artery lesions (KD-CAL-) (P<0.05).No significant differences in histone H4 acetylation associated with CNS3 were found among different groups (P>0.05).(2) The levels of p300 and CBP in Treg cells and their binding abilities with Foxp3 gene were down-regulated significantly during acute KD (P<0.05), but were restored to some extent after IVIG treatment (P<0.05).The Foxp3-associated histone acetylation was positively correlated with the expression of p300 and CBP at mRNA level during acute KD (r=0.65, 0.42, P<0.05).Furthermore, the expression of p300 and CBP and their binding abilities with Foxp3 gene in KD-CAL+ group were lower than those in KD-CAL-group (P<0.05).(3) Compared with healthy subjects, plasma concentrations of TGF-β and RA and the expression of TGF-βRⅠ/Ⅱ/Egr-1, RARα and pLAT1/PLCγ1 were down-regulated during acute KD (P<0.05);the binding abilities of pSmad3 and NFAT with Foxp3 gene were reduced remarkably in patients with acute KD (P<0.05).All the items mentioned above were restored after IVIG treatment (P<0.05).Moreover, the ten items aforementioned in KD-CAL+ group were lower than those in KD-CAL-group (P<0.05).(4) Higher acetylation levels of histone H4 associated with promoter, CNS1 and CNS2, and enhanced binding abilities of p300 and CBP with Foxp3 gene were found in CD4+ T cells isolated from patients with acute KD after co-stimulation with TGF-β, RA and anti-CD3/CD28 antibodies as compared with those in CD4+ T cells without stimulation (P<0.05).However, no statistical difference in the acetylation level of histone H4 associated with CNS3 was found between the two groups (P>0.05).Conclusion Hypoacetylation of histone H4 associated with Foxp3 gene caused by insufficient expression of p300/CBP and their impaired binding abilities might be involved with immune dysfunction in KD.IVIG therapy regulates the expression of p300/CBP and their binding abilities with Foxp3 gene through up-regulating TGF-β signal.

Objective To investigate the effects of SMYD3 and MLL5 on histone methylation of Transcription factor forkhead box protein 3 (Foxp3) gene and its roles in the immunological pathogenesis of Kawasaki disease (KD). Methods Forty-two children with KD and 26 age-matched healthy children were consented to participate in this study. Co-Immunoprec-ipitation and real-time polymerase chain reaction (PCR) was performed to determine Foxp3-associated histone methylation levels of H3K4me3 and H3K27me3, and binding levels of SMYD3 and MLL5 with Foxp3 gene in CD4+T cells. The proportion of CD4+CD25high Foxp3+cells (Treg) and protein levels of Foxp3, cytotoxic T lymphocyte associated antigen-4 (CTLA4), TGF-βRⅡand pSmad3 were analyzed by flow cytometry. Quantitative real-time PCR was used to evaluate levels of Foxp3, interleukin (IL)-10, GITR, TGF-βRⅠand RARαmRNA in CD4+T cells. Plasma concentrations of TGF-βand retinol acid (RA) were measured by enzyme-linked Immunosorbent assay. Independent-samples t-test was used as the statistical method in this study. Results ① The proportion of Treg, expression levels of Foxp3 and molecules associated with suppressive function of Treg cells(IL-10, GITR and CTLA4), and histone methylation levels of H3K4me3 associating with promoter, conserved non-coding DNA sequence (CNS) 1 and CNS2 of Foxp3 gene decreased remarkably during acute KD [Promoter:(5.4±1.8)%vs (9.1±2.2)%;CNS1:(2.6±0.9)% vs (3.8±1.1)%; CNS2: (2.4±0.8)% vs (4.2±1.0)%; t=5.50, 6.02, 9.56, 7.92, 7.97, 4.76, 7.73, 5.01, 8.66; P<0.05], and restored after intravenous immunoglobulins (IVIG) therapy [Promoter: (7.2 ±2.1)% vs (5.4 ±1.8)%; CNS1:(3.6±1.4)% vs (2.6±0.9)%; CNS2: (3.6±1.4)% vs (2.4±0.8)%; t=5.56, 4.59, 7.01, 6.04, 5.89, 4.83, 4.45, 4.00, 5.12; P<0.05]. Meanwhile, the nine former items in KD patients with coronary artery lesions (KD-CAL+) were lower than those without coronary artery lesions (KD-CAL-) [Promoter: (4.11±1.45)% vs (6.16±1.93)%; CNS1:(1.99±0.87)%vs (2.96±1.10)%;CNS2: (1.75±0.63)%vs (2.72±1.16)%;t=6.28, 3.24, 4.56, 3.69, 3.38, 4.40, 3.65, 3.00, 3.51; P<0.05]. No significant difference of H3K4me3 associated with CNS3 and H3K27me3 were found among the groups (t=1.03, 0.91, 1.48 and 0.79, 0.82, 1.53; P>0.05). ② Binding levels of SMYD3 and MLL5 with Foxp3 gene in CD4+T cells were down-regulated significantly during acute KD (t=6.63, 6.15; P<0.05), and restored to some extent after IVIG treatment (t=5.36, 4.56; P<0.05). Positive correlations between binding levels of SMYD3 and MLL5 and expression level of Foxp3 mRNA were detected in patients with acute KD (r=0.62、0.45, P<0.05). Furthermore, Binding levels of SMYD3 and MLL5 with Foxp3 gene in KD-CAL+group were lower than those in KD-CAL- group (t=4.11, 4.31; P<0.05). ③ Compared with healthy controls, plasma concentration of TGF-β and RA, and expressions of TGF-βRⅡ, TGF-βRⅠ, pSmad3 and RARα were down-regulated during acute KD (t=11.54, 12.81, 7.43, 16.10, 8.25, 12.06; P<0.05), and elevated remarkably after IVIG treatment (t=8.40, 6.24, 5.94, 11.78, 6.27, 8.30; P<0.05). Simultaneously, all the items aforementioned in KD-CAL+ group were found to be lower than those in KD-CAL-group (t=3.58, 3.30, 3.82, 5.27, 4.71, 3.78; P<0.05). Conclusion Hypomethylation of H3K4me3 associated with Foxp3 gene caused by insufficient binding levels of SMYD3/MLL5 may be involved with immune dysfunction in Kawasaki disease.

Objective: To observe the therapeutic effect of acupuncture therapy for treatment of chronic atrophic gastritis. Methods: According to the theory of TCM, 36 cases of chronic atrophic gastritis were differentiated and treated by acupuncture. Gastroscopy and electrogastrograph (EGG) of the body surface, gastric acid and pepsin, blood gastrin, blood prostaglandin E (PGE), gastric mucosal cells and G cells are recorded. Results: Acupuncture therapy had a better effect in improving clinical symptoms, the markedly effective rate was 97.2% and the disappearing rate of clinical manifestations 97.02%;after acupuncture treatment,blood PGE concentration increased; gastric acid secretion increased; gastric dynamics was enhanced and the amplitude of EGG rose; acupuncture could significantly facilitate secretion of gastrin. Conclusion: Acupuncture is a convenient treatment method for atrophic gastritis with quick effect and without side effects.

Tumor-derived exosomes (TEXs) are small membrane vesicles secreted by tumor cells.They contain various proteins and RNA which make they serve as functional mediators in cell interaction.TEXs can alter the components of extracellular matrix and induces epithelial-mesenchymal transition of tumor cells,which enhance the invasiveness of tumor cells.TEXs regulate immunity through multiple pathways,allowing circulating tumor cells to escape immune surveillance.TEXs promote pre-metastatic microenvironment in target organ before metastasis and induce angiogenesis after circulating tumor cells colonization.Understanding the role and mechanism of TEXs in this process can effectively block relevant signaling pathways which may provide new targeted therapies for clinic.