BACKGROUND: Embryonic stem cells can be induced to differentiate into neural precursors under certain conditions, and they can effectively integrate with host cells after transplanted into healthy or injured central nervous system, and then repair and rebuild the injured nerve tissue.OBJECTIVE: To observe the effects of transplantation of neural precursors induced by embryonic stem cells on the recovery of neurological function in mice with spinal cord injury.DESIGN: A randomized controlled animal trial.SETTING: Research Center of Developmental Biology, Shanghai Second Medical University.MATERIALS: Twenty-eight C57/BL6J mice of 6-8 weeks old, both sexes, were used. Mice embryonic stem cell strain S8 and carrier LacZ labeling genes were provided by Shanghai Research Center of Developmental Biology. High-glucose Dulbecco's modified Eagle media (DMEM), β-mercaptoethanol (BME), mice leukemia inhibitory factor (LIF) and mitocin-C were all from GIBCO attachment induction medium, which were provided by Shanghai Research Center of Developmental BiologyMETHODS: The experiment was carried out in the central laboratory of developmental biology of Shanghai Second Medical University from April 2003 to April 2004. The embryonic stem cells were cultured and induced to differentiate into neural precursors by means of attachment induction. The mice were anesthetized and made into models of spinal cord hemisection on the T9-T10 plan. The mice were randomly divided into three groups: sham operated group (n =9): Only T9-T10 spinous process and corresponding lamina of vertebra were removed, then the skin was sutured layer by layer;ransplantation group (n =10): After spinal cord hemisection, embryonic stem cells were injected into the vertebral canal about 1 cm away from the injured site; model group (n =9): DMEM was injected into the region around the injured site.The mice were evaluated with Basso, Beattie, Bresnahan (BBB) locomotor rating scale to observe the recovery of neurological function at 1, 2, 4, 6 and 8 weeks (the score ranged from 1 to 21 points, the higher the scores, the better the recovery of neurological function). At 8 weeks, the survival and differentiation of embryonic stem cells at the injured site of spinal cord were observed using X-Gal staining in each group. The positively stained sections with X-Gal at the injured site of spinal cord were detected with fluorescent immunohistochemical staining.MAIN OUTCOME MEASURES: ① Recovery of neurological function; ② Survival and differentiation of embryonic stem cells at the injured site of spinal cord; ③ Results of fluorescent immunohistochemical staining.RESULTS: ① The BBB scores in the transplantation group at 1, 2 and 4 weeks were higher than those in the model group (P ＜ 0.01). ② Survival and differentiation of embryonic stem cells at the injured site of spinal cord: In the transplantation group, there were X-Gal positively stained cells in the tissue sections of the injured spinal cord of mice,the cytoplasm was blue with nucleoli in it, i.e. the cells derived from embryonic stem cells, which were not observed in the sham-operated group and model group. In the transplantation group, the cells derived from embryonic stem cells, which were implanted to the injured spinal cord, distributed around the injured sites, and integrated with the surrounding tissue and had similar form with the surrounding cells. ③ At the injured site, X-Gal positively stained cells in the transplantation group aiso expressed neurofilaments (the specific marker of neurons), but did not express GFAP.CONCLUSION:The embryonic stem cells were cultured and induced to differentiate into neural progenitors, and they could survive, migrate and differentiate into neurons after transplantation, but there was no obvious improvement of neurological function.

Objective:To establish a method for the determination of phenyl salicylate in compound titanium dioxide cream. Meth-ods:The HPLC assay was carried out on an Agilent Zorbax SB C18 (250 mm × 4. 6 mm, 5 μm) column with methanol -water (78∶22) as the mobile phase. The sample was detected at 308 nm with a UV detector. The column temperature was set at 25 ℃ and the flow rate was 1. 0 ml·min-1 . Results:Phenyl salicylate could isolate from the other materials by HPLC. The linear range of phenyl salicylate was 59.460-198.200 μg·ml-1(r=0.999 5). The recovery was 101.84% with RSD of 0.64%(n=9). The content of phenyl salicylate in 3 batches of compound titanium dioxide cream was 107. 7%, 107. 5% and 109. 8%, respectively. Conclusion:The method is simple, rapid, exclusive, accurate and sensitive, which is suitable for the determination of phenyl salicylate in com-pound titanium dioxide cream.

Objective To investigate the relation between a set of single nucleotide polymorphisms (SNP) in core gene of HBV in chronic hepatitis B patients and HBV DNA levels. Methods PCR restriction fragment length polymorphism(PCR-RFLP) assay and restriction enzyme Tsp509I were adopted to determine HBV SNP in HBV core gene. Nucleotide sequences of core gene were determined using the dideoxy chain termination method. HBV DNA levels were quantitated with real-time PCR. Results Five typical RFLP patterns, RFLP-C, RFLP-D, RFLP-E, RFLP-G and RFLP-C/G mixture were found and the distribution of HBV RFLP patterns was as follows: C, 61. 5% ; D, 2. 6% ; E, 9.6%; G, 16.7%; C/G mixture, 9.6%. Five SNPs, A165T, A336C, A336T, T337C and T385C, were found to be associated with RFLP patterns change and only SNP A336C or A336T caused the substitution of Glu-83 with Asp in HBcAg. The serum HBV DNA levels in RFLP-C group were higher than that in RFLP-G (P =0. 02) and RFLP-C/G group(P = 0. 006) , respectively, furthermore, the positive rate of serum ALT in RFLP-C/G group was lower than that in RFLP-C, RFLP-E and RFLP-G group, respectively. Under the condition of HBeAg-positive, the serum HBV DNA levels in RFLP-C group were higher than that in RFLP-G (P = 0. 015) and RFLP-C/G group(P =0.008) , respectively. Conclusion PCR-RFLP used in this study can be adopted to determine HBV SNPs, not genotypes in Chinese patients with chronic hepatitis B. The serum HBV DNA level in RFLP-C group higher than that in RFLP-G or RFLP-C/G group maybe associated with amino acid mutation, Glu83 Asp.

Objective To investigate the ability of proton magnetic resonance spectroscopy (1H-MRS) for differentiating benign from malignant solid adnexal tumors. Methods One-hundred and six patients (114 tumors) with surgically and histologically proven solid adnexal tumors (44 benign, 70 malignant) underwent conventional MR imaging and 1H-MRS. Single-voxel spectroscopy was performed using the point resolved spectroscopy localization technique with a voxel size of 2.0 cm × 2.0 cm × 2.0 cm. Resonance peak integrals of choline (Cho), N-acetyl aspartate (NAA), creatine (Cr), lactate (Lac), and lipid (Lip) were analyzed and the Cho/Cr, NAA/Cr, Lac/Cr and Lip/Cr ratios were recorded and compared between benign and malignant tumors using independent two-sample t test. Receiver operating characteristic (ROC) curve analysis was used to evaluate the diagnostic performance of Cho/Cr ratio for differentiating benign from malignant tumors. Results A Cho peak was detected in all 114 tumors, NAA peak in 112 tumors (43 benign and 69 malignant), Lip peak in 70 tumors (21 benign and 49 malignant), and Lac peak in 16 tumors (7 benign and 9 malignant). The Cho/Cr and Lip/Cr ratios were 4.8±2.5, 6.4±4.0 in benign versus 9.6 ± 3.3, 10.5 ± 4.6 in malignant solid adnexal tumors, respectively, with a statistically significant difference (t values were-8.826 and-2.915,P<0.05). The NAA/Cr ratio were 1.4 ± 0.7 in benign versus 1.6 ± 1.0 in
malignant solid adnexal tumors, with no statistically significant difference (t=-1.523,P>0.05). When the Cho/Cr threshold was 7.2 for differentiating between benign and malignant tumors, the sensitivity, specificity, accuracy were 80.0%(56/70),88.6%(39/44) and 83.3%(95/114) respectively. Conclusions The 1H-MRS patterns of benign and malignant solid adnexal tumors differ. The Cho/Cr ratio can help clinicians differentiate benign from malignant tumors.

Objective To analyze the investigation and treatment of signet ring cell carcinoma of pancreas.Method The clinical data of patients with histopathologically confirmed signet ring cell carcinoma of pancreas were analyzed retrospectively.Results There were 4 patients with signet ring cell carcinoma.There was no patient who presented with a typical carcinoid syndrome.Most patients presented with upper abdominal pain,backache or jaundice caused by bile duct obstruction.In one patient CA19-9 and CEA were raised.All patients received palliative biliary bypass and needle biopsy of the tumour.The median survival was 2.8 months.Conclusions Pancreatic signet ring cell carcinoma is a rare disease with poor prognosis.Surgery is the only effective treatment but the resectability rate is low.Whether this tumour responds to chemotherapy requires further studies.

Objective To establish a stable and feasible animal model of acute cerebral infarction,and to evaluate it with functional magnetic resonance imaging and pathology.Methods Twenty-five S-D rats were randomly divided into five groups,and there were 5 rats in each group.Rats in group A were sham-operated for control study.Unilateral middle cerebral artery occlusion(MCAO) was performed with improved thread in group B,C,D,E and were enrolled for MRI and MRS study at 3,6,12,24 hours after MCAO.All rats were examined by 1-hydrogen magnetic resonance spectroscopy(~1H MRS).Two or three rats in each group were sacrificed for 2,3,5-triphenyltetrazolium chloride(TTC) staining of the brain and two rats for pathological examination after MRS.Results Rats in group A showed no change in brain on ~1 H MRS or pathological study.~1H MRS of the rat brains after right MCAO showed a decrease of N-acetyl-aspartate(NAA),an increase of Lactate(Lac) in all groups.There was no significant change of Choline(Cho) and Creatine(Cr) peaks onrats in group B,C,D.The peaks of Cho and Cr were slightly dropped in group E.Conclusion The acute regional cerebral ischemic model in rats made by our approach is stable and reproducible,and it is suitable for evaluation and study with functional magnetic resonancespectroscopy accurately and sensitively.

Aim To investigate the effects of cholecystokinin-octopeptide on IL-12 secreted in murine bone marrow-derived dendritic cells induced by lipopolysaccharide.Methods The CCK receptor subtypes were investigated by immunofluorescence in murine bone marrow-derived dendritic cells.Enzyme linked immunosorbent assay and Western blot were used to estimate the contents of IL-12 and p38MAPK activity.Results CCK-1R and CCK-2R were detected in BM-DC;CCK-8(at concentrations of 10-10,10-8,10-6 mol?L-1)could significantly increase the secretion of IL-12 in the LPS-induced DC, and LPS-activated p38MAPK activity in a dose-dependent manner.The effect of CCK-8 was reduced partially by CR1409(a CCK-1R antagonist) and CR2945(a CCK-2R antagonist).Conclusion CCK-8 could dose-dependently increase the expressions of IL-12 in LPS-induced DC,probably by promoting p38MAPK activity and the effect was mediated by CCK1 and CCK2 receptors.

AIM: To study the effects of cholecystokinin octapeptide (CCK-8) on T-lymphocyte subsets in keyhole limpet haemocyanin (KLH)-immunized mice.METHODS: Female BALB/c mice were immunized with KLH and injected with different dosages of CCK-8 or saline simultaneously. Positive CD4+and CD8+ T cells in peripheral blood or splenocytes were measured by flow cytometry. The production and mRNA level of Th1 cytokine, IFN-? and Th2 cytokine, IL-4 in the splenocytes were evaluated by ELISA and RT-PCR, respectively. In addition, lung tissue sections were made with HE staining.RESULTS: CCK-8 downregulated the positive CD4+and CD8+ T lymphocytes both in peripheral blood and in splenocytes in KLH-immunized mice, resulting in the reduction of CD4+/CD8+ ratio.CCK-8 improved the production of IFN-?, elevated IFN-? gene transcription, whereas cut down the production of IL-4, decreased IL-4 gene transcription. CCK-8 lightened the pulmonary inflammation induced by KLH.CONCLUSION: CCK-8 inhibits CD4+T lymphocytes activation, Th2 cytokine mRNA expression and protein production in KLH-immunized mice, indicating that CCK-8 modulates adaptive immune response.

AIM: To investigate in vitro effects of cholecystokinin octapeptide(CCK-8) on the expressions of B7.1 and B7.2 and the costimulatory activity of T lymphocytes in unstimulated macrophages.METHODS: Mouse peritoneal macrophages were isolated and incubated with CCK-8(10-12-10-6 mol/L) for indicated time.The B7.1 and B7.2 expressions of murine peritoneal macrophages were analyzed by flow cytometry.CD4+T cells were isolated from mouse spleen using immunomagnetic beads,and cultured with 1/4 numbers of macrophages which were pretreated with CCK-8 and/or anti-B7.1 antibody,anti-B7.2 antibody,CCK1R antagonist CR1409,CCK2R antagonist CR2945 for 24 h.ConA was added into the culture medium to stimulate CD4+T cell proliferation.The proliferation was determined by measuring -TdR incorporation in a ?-scintillation counter.RESULTS: B7.1 and B7.2 expressions and costimulatory activity of peritoneal macrophages were enhanced by CCK-8 in a dose-dependent manner,and the maximal effects occurred at the concentrations of 10-9 mol/L to 10-7 mol/L.Anti-B7.2 antibody,but not anti-B7.1 antibody,reduced the modulatory role of CCK-8 on costimulatory activity.Both CR1409 and CR2945 reversed the effect of CCK-8 on costimulation,and the role of CR1409 was more significant.CONCLUSION: CCK-8 enhances macrophage costimulatory activity by upregulating B7.2 expression,which is mediated by CCK1R and CCK2R.CCK1R might be the major receptor responsible for the modulation of CCK-8 on costimulation.

OBJECTIVETo investigate the effect of IL-12 on IFN-gamma and IL-10 production of peripheral blood mononuclear cells (PBMC) in chronic hepatitis B virus infection patients during IFN-alpha treatment.

METHODSBefore and after IFN-alpha treatment of 3 months and 6 months, PBMC of 20 patients with chronic hepatitis B virus infection were collected and cultured in vitro in the culture fluid containing PHA (100 microg/ml), HBcAg (1 microg/ml), or HBeAg (1 microg/ml) for 48 h under the condition of the presence or absence of IL-12 (10 ng/ml). Then the levels of IFN-gamma and IL-10 were determined by ELISA.

RESULTSThere were 12 responders and 8 nonresponders to IFN-alpha treatment. In the responders, the enhancing effect of IL-12 on IFN-gamma production was significantly greater after IFN-alpha treatment than before the treatment. The production of IL-10 was suppressed in the presence of IL-12 after 3 months and 6 months of IFN-alpha treatment. In the same treatment time, the level of IFN-gamma in the presence of IL-12 was significantly higher than that in the absence of IL-12. To the nonresponders, the enhancing effect of IL-12 on IFN-gamma production was also significantly increased after IFN-alpha treatment. Moreover, in the same treatment time, the level of IFN-gamma in the presence of IL-12 was significantly higher than that in the absence of IL-12.

CONCLUSIONSThe enhancing effect of IL-12 on IFN-gamma production of PBMC in patients with chronic hepatitis B virus infection is increased during IFN-alpha treatment. IFN-alpha and IL-12 may enhance the efficacy for the treatment of chronic hepatitis B virus infection.

Adolescent ; Adult ; Antiviral Agents ; therapeutic use ; Enzyme-Linked Immunosorbent Assay ; Female ; Hepatitis B, Chronic ; blood ; drug therapy ; Humans ; Interferon-alpha ; therapeutic use ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-12 ; pharmacology ; Leukocytes, Mononuclear ; cytology ; drug effects ; metabolism ; Male ; Middle Aged ; Time Factors