10.3969/j.issn.2095-4344.2013.26.014

Objective To investigate the dynamic variation of subsets of myeloid derived suppressor cells (MDSC) and their ratio in septic mice, and to discuss their role in the development of sepsis. Methods Male C57BL/6 mice were randomly divided into sepsis model group and sham group according to random number table. Polymicrobial sepsis was induced by using cecal ligation and puncture (CLP), while mice in sham group only underwent laparotomy and laparorrhaphy without CLP. Thirty mice in each group were used to observe living condition, and the 20-day survival rate was compared between the two groups. In addition, subsets of MDSC in peripheral blood, spleen and bone marrow were analyzed with flow cytometry for other 60 mice (12 mice at each time point, as 0, 3, 7, 12 and 20 days). Spleens were harvested at 7 days for weighing, and single cell suspension of spleen tissue was prepared for splenocyte counting. Histopathologic changes in spleen tissue and liver tissue were observed under light microscope after hematoxylin and eosin (HE) stain. Results ① No mice died in sham group within 20 days after the operation. On the other hand, 10 mice in model group died within 20 days, and the difference in survival rate between the two groups was statistically significant (100.0% vs. 66.7%, χ2 = 11.861, P = 0.001). ② The spleens in model group showed obvious enlargement and significantly outweighed as compared with those in sham group (mg: 413.33±41.63 vs. 111.67±17.56, t = 11.564, P = 0.000), and the total count of splenocytes was significantly higher than that in sham group (×109/L: 21.20±2.43 vs. 1.87±0.06, t = 13.578, P = 0.005). ③ Pathological sections with HE staining showed that the liver tissue and spleen tissue remained normal in sham group. In model group, the hepatic tissue showed acute inflammatory reaction, including tissue disruption, capillary congestion, infiltration of neutrophils, marked edema of hepatocytes and focal hepatocellular necrosis. Abnormalities were also found in the spleen tissue: the red pulp and white pulp were disordered, splenic sinus was congested with numerous red cells, the splenic capsule thickened, immature myeloid cells with circular nuclei proliferated in the subcapsular region and perivascular region, splenic cord and splenic sinus were infiltrated with a large number of hematopoietic cells. ④ No significant changes in the monocytic MDSC (M-MDSC) and granulocytic MDSC (G-MDSC), and their ratio were found in peripheral blood, spleen and bone marrow at every time point in sham group. On the other hand, in model group, the ratio of M-MDSC and G-MDSC was continuously increased in peripheral blood, spleen and bone marrow, and M-MDSC only slightly decreased at 20 days. On the other hand, the ratio of M-MDSC/G-MDSC rose at first followed by a decrease. The ratio of M-MDSC/G-MDSC in peripheral blood was higher than 1 from 3 days after the operation, reaching the peak at 12 days (compared with 0 day: 4.16±0.53 vs. 0.79±0.11, P < 0.05), while the ratio of M-MDSC/G-MDSC in spleen and bone marrow after CLP were lower than 1 at all time points, reaching the peak on 7 days after the operation (compared with 0 day: 0.70±0.06 vs. 0.25±0.02 in spleen, 0.39±0.06 vs. 0.11±0.01 in bone marrow, both P < 0.05), and then gradually decreased afterwards. Conclusion Subgroups of MDSCs were continuously aggregated in the peripheral blood, spleen and bone marrow, and their ratio rose first and decreased afterwards along with the development of sepsis, and the changes may reflect the change of immune status at different stages of sepsis.

Aim To study the mechanism of Escherichia coli DNA photolyase(EC 4.1.99.3) in repairing the ultraviolet induced pyrimidine dimmer lesions in DNA.Methods UV-Vis Specturum was used to measure the relationships between the enzyme activity and different state flavin ademine denucleotide(FAD).Results It was shown that the enzyme activity was the highest when FAD chromophore was in reduced state and lowest when it was in oxidated state,while the activity ranked between when the enzyme was in radical state.Partial denature was observed when the enzyme was in oxidated state and under over 30℃,yet the enzyme activity remained unchanged.The presence of MTHF chromophore enhanced the enzyme activity.Conclusion These findings provide clues for clinical application of DNA photolyase in the future.

Objective To investigate vascular endothelial growth factor-c (VEGF-C) and vascular endothelial growth factor receptor-3(VEGFR-3) mRNA expression, microvessels density (MVD) and lymphatic microvessels density (LVD) in human hepatocellular carcinoma and normal liver tissue. Try to illuminate the relationship among VEGF-C,VEGFR-3,MVD,LVD and the clinical pathological features of hepatocellular carcinoma. Methods Liver tissue of 60 cases definitely diagnosed as hepatocellular carcinoma and 20 normal cases were collected. VEGF-C and VEGFR-3 mRNA expression were examined by RT-PCR, MVD and LVD were examined by immunohistochemistry staining. Relationship between these indexes and clinical pathological features of hepatocellular carcinoma was also analysed. Results VEGF-C and VEGFR-3 mRNA expression, MVD and LVD in hepatocellular carcinoma were higher than those in normal liver tissue (P<0.01); In hepatocellular carcinoma tissue, expression of VEGF-C mRNA positively related with VEGFR-3 mRNA, MVD and LVD(P<0.01). VEGF-C and VEGFR-3 expression positively related with portal vein tumor thrombus, intrahepatal metastasis and lymph node metastasis (P<0.01). MVD positively related with portal vein tumor thrombus and intrahepatal metastasis (P<0.01). LVD positively related with lymph node metastasis (P<0.01). Conclusion VEGF-C and VEGFR-3 expression increase in hepatocellular carcinoma tissue. They might play roles in tumor invasion and metastasis by inducing angiogenesis and lymphangiogenesis.

AIM:To investigate morphologic and functional changes of small intestinal mucosa and proliferating cell nuclear antigen in postoperative portal hypertension patients with single or combined administration of Gln and rhGH.METHODS:Twenty-nine portal hypertension patients with surgical treatment were prospectively randomized to four groups as follows:① Gln group(n=6);② rhGH group(n=8);③ Gln+rhGH group(n=7)and ④ control group(n=8).A standard solution for TPN was given three days after operation for a week.The concentration ratio of urinary lactulose and mannitol(L/M),the villus height and crypt depth and PCNA index of small intestinal mucosa were compared.RESULTS:A week after TPN postoperation,the increased ratios of L/M in Gln+rhGH group were less than those in control group(P0.05).CONCLUSION:This study suggest that Gln together with rhGH reduce the intestinal permeability and protect the mucosa integrality in postoperative portal hypertension patients,but not in single treatment.

Objective To identify the genotype of Toxoplasma gondii isolated strains from a congenital teras（KS strain）and an HIV⁃Toxoplasma co⁃infected patient in Jiangsu Province. Methods T. gondii DNA of tachyzoites of a isolate from a congenital teras（KS strain）and blood DNA of an HIV⁃Toxoplasma co⁃infected patient in Jiangsu Province were extracted，and 11 loci were identified for the genotype by polymerase chain reaction restriction fragment length polymorphism（PCR⁃RFLP）. Results The complete bands were obtained from the congenital teras（KS strain）and HIV⁃Toxoplasma co⁃infected patient in Jiangsu Province，and identified as T. gondii gene type I. Conclusion T. gondii gene type I may be the dominant genotype strain of T. gondii among the women who have the abnormal pregnant outcomes and HIV⁃Toxoplasma co⁃infected patients in Jiangsu Province.

Objective To evaluate the effect and safety of combined treatment by laparoscopic cholecystectomy and subsequent ablation in patients with HCC adjacent to the gallbladder. Methods From June 2005 to June 2009,13 patients with HCC nodules( less than 3 cm) adjacent to the gallbladder were treated by ablation after laparoscopic cholecystectomy. The rate of complete necrosis as well as postoperative complications were also analyzed. Results All the patients showed complete necrosis of their tumor lesions after treatment by ablation subsequence of laparoscopic cholecystectomy. During the follow-up period( nearly 2 years), recurrent nodules appeared in other subsegments but not at the original site treated by ablation. Of note, no fatal complications were observed in all the ablation treated patients. Conclusion Combined treatment by laparoscopic cholecystectomy and subsequent PMCT was an effective and safe method for patients with small HCC which was adjacent to gallhladder.

Objective To measure serum levels of 25-hydroxyvitamin D (25HVD) in patients with chronic urticaria (CU),and to explore its role in the occurrence of CU.Methods Peripheral blood samples were obtained from 50 patients with CU and 40 healthy controls.The urticaria activity score (UAS) was used to assess the severity of CU and to group the patients with CU.Enzyme-linked immunosorbent assay (ELISA) was performed to measure the serum levels of 25HVD,interferon-γ (IFN-γ),interleukin-4 (IL-4) and immunoglobulin E (IgE).Statistical analysis was carried out by t test,rank sum test and linear correlation analysis.Results The serum level of 25HVD was significantly lower in the patients with CU than in the healthy controls (15.20 ± 7.72 vs.21.54 ± 8.31 μg/L,t =3.75,P ＜ 0.05).Moreover,there was a significant difference in the distribution of serum 25HVD levels between the patients and healthy controls (H =17.9,P ＜ 0.05).However,no significant difference was observed in the serum level of 25HVD between severe and mild CU groups (15.57 ± 7.38 vs.14.86 ± 6.28 μg/L,t =0.37,P ＞ 0.05).Compared with the control group,the patient group showed significantly decreased serum levels of IFN-γ (t =15.34,P ＜ 0.05),but increased serum levels of IL-4 and IgE (t =6.54,4.88,respectively,both P ＜ 0.05).Among the patients with CU,the serum level of 25HVD was positively correlated with that of IFN-γ(r =0.738,P ＜ 0.05),but negatively correlated with that of IL-4 (r =-0.689,P ＜ 0.05),and uncorrelated with that of IgE (r =-0.271,P ＞ 0.05).Conclusion The serum level of 25HVD evidently decreased in patients with CU,and it may participate in the occurrence of CU by mediating the Th 1/Th2 imbalance.

Objective To evaluate the impact of the recombined plasmid vector with enhanced green fluorescent protein (EGFP) encoding soluble tumor necrosis factor related apoptesis inducing ligand (pIRES-EGFP-sTRAIL) on proliferation and apoptosis of human hepatocellular carcinoma cell line HepG2, and investi-gate the feasibility and efficiency of the transfection of pIRES- EGFP- sTRAIL into HepG2 by ultrasound micro-bubble contrast agent. Methods pIRES-EGFP-sTRAIL was constructed and transfected into HepG2 cells by using different types of mediated methods: microbubble echocontrast agent combining appropriate dose of ultra-sound irradiation, liposome method, microbubble echocontrast agent only or blank medium treatment. Transfec-tion efficiency was evaluated by EGFP-expressed cell count; proliferation-lnhibiting rate and the apoptosis rate of HepG2 cells were determined by MTT method and flow cytometry analysis; changes of cell morphology were examined by microscopy with Hoechst33258 dyeing; expression of caspase-8 and caspase-3 was detected by Western blot. Results Ultrasound microbubbh enhanced pIKES-EGFP-sTRAIL uptake by HepG2 cells, and the transfection efficiency was significantly higher in ultrasound microlmbble group than that in other groups( P<0.05 ) ; pIRES- EGFP- sTBAIL effectively inhibited HepG2 cell proliferation and induced cell apoptosis by triggering caspase cascade. Both the inhibiting rate and apoptosis rate were significantly higher in ultrasound microbubble group than those in other groups(P<0.05). Conclusion pIRES-EGFP-sTRAIL expresses ef-fectively in HepG2 cells, sTRAIL has a potential role on the inhibiting proliferation and inducing apoptosis of HepG2 cells by triggering caspase cascade, and this role can be enhanced by the administration of low-intensity ultrasound and microbubble echecontrast agent.

BACKGROUND: There is no accepted treatment for liver fibrosis recently. Bone marrow meaenchymal stern cells (BMSCs) used in the treatment of liver fibrosis has been reported as an effectively treatment, but the mechanism is unclear.OBJECTIVE: To study the regulation of hepatic stellate cells mediated by human BMSCs in vitro.DESIGN, TIME AND SETTING: The cytological in vitro study was performed at the Center for Stem Cells and Tissue Engineering of Sun Yat-sen University and the Central Laboratory of Third Affiliated Hospital of Sun Yat-sen University from June to December 2008.MATERIALS: Human bone marrow masenchymal stem cells were collected from normal youth volunteers; Human hepatic stellate cells and normal liver call line L-O2 were supplied by the Animal Experimental Center of Sun Yat-sen University.METHODS: The purified human BMSCs and hepatic stellate calls were set up in Transwell co-culture system. The incubation density was 2×104cells/well. L-O2 was set up instead of human BMSCs as negative control. Hepatic stellate cells cultured alone served as blank control group. The culture was performed for 72 hours.MAIN OUTCOME MEASURES: Morphology of hepatic stellate cells and results of immunocytochemical staining. Apoptosis of hepatic stellte calls was determined by flow cytometry. Western blot were used to assay the expression of α-actin.RESULTS: Activated hepatic stellate cells presented fiat and thin shape under an inverted microscope. Fat drop was lack in cytoplasm, a -actin located in hepatic stellate calls, with the presence of high tension fibers. Compared with the L-O2 + hepatic stellate cell and hepatic stellate call groups, the apoptotic rate of hepatic stellate cells was significantly increased in the BMSC + hepatic stellate cell group (P < 0.05). α -actin expression was significantly down-regulated.CONCLUSION: Human BMSCs can inhibit activation of hepatic stellate ceils and promote them apoptosis, which may be the anti-hepatic fibrosis mechanism of BMSCs.