2004年 · 第117卷第10期

Background Dendritic cells (DCs) are the most powerful antigen-presenting cells to induce specific T-cell immunity, which plays an important role in the body's anti-tumor responses. In this study, we assessed the feasibility and efficacy of inducing T-cell immunity against Epsten-Barr virus (EBV)-associated tumors in vivo using dendritic cells transfected with EBV latent membrane 2A (LMP2A) recombinant adenovirus. Methods Cytokine-activated bone marrow-derived DCs transfected with EBV LMP2A recombinant adenovirus were infused into BALB/c mice. Splenic cytotoxic T-cell responses were evaluated by cytotoxicity and interferon-γ production assays. in vivo immune protection was then assessed in the mice tumor models implanted with tumor cells expressing EBVLMP2A. Results DCs transfected with EBV LMP2A recombinant adenovirus could strongly induce EBV LMP2A-specific cytotoxic T-cell responses and upregulate interferon-γ production in vivo. Vaccination using these DCs led to prolongation of overall survival rates in the mice tumor models and retarded tumor growth. Conclusions The results suggest that DCs transfected with EBV LMP2A recombinant adenovirus can serve as a feasible and effective tool for eliciting LMP2A-specific cytotoxic T-cell responses against EBV LMP2A in vivo in the treatment of EBV-associated tumors. Chin Med J 2004; 117(10):1558-1563

Background Much research has been focused on ischemia/ reperfusion injury (IRI) to the transplanted organs. As a free radical, nitric oxide (NO) plays an important role in IRI. In this study, the production of NO and its functions during IRI were monitored in rat models after allotransplantation of kidney grafts. Methods Of 75 male LEW rats, 30 served as donors, and the remaining 45 rats were divided into three groups (15 rats in each group): controls (group 1), kidney allotransplantation followed by bilateral nephrectomy during reperfusion (group 2), 2 hours before operation, donors and recipients were treated with NG-nitro L-arginine methyl ester (L-NAME), a NO synthase inhibitor, at a dose of 30 mg/kg (group 3). Bilateral nephrectomies were performed while kidney grafts were reperfused. The kidney grafts were hypothermically stored for 24 hours. The production of NO before and after reperfusion was measured by electron paramagnetic resonance (EPR). The creatinine level, the glomerular filtration rate (GFR) and the protein carbonyl content in tissue samples were recorded on the first and the fifth day after operation. The data were evaluated by one-way analysis of variance. Differences were considered to be statistically significant when a P value was less than 0. 05. Results After reperfusion for 15 minutes, the production of NO increased remarkably and kept increasing till 120 minutes, after which the level returned to normal. In group 3, which was pretreated with L-NAME, creatinine levels were higher than those in group 2 at the 24th hour (4. 10 ±0. 50 mg/ dl vs. 3. 77 ±0. 42 mg/ dl, P < 0. 05) and the 120th hour (3. 19 ±0. 79 mg/ dl vs. 2. 22 ±0. 53 mg/ dl, P < 0. 05). GFR levels in group 3 were lower than those in group 2 at the 24th hour (0. 50 ±0. 12 ml/ min vs. 0. 71 ±0. 19 ml/ min, P <0. 05) and the 120th hour (0. 59 ±0. 38 ml/ min vs. 1.27 ±0. 23 ml/ min, P < 0. 01). The content of protein carbonyl in tissue samples of group 3 was lower than that in group 2 at the 24th hour (29. 01 ±7. 02 nmol/ mg protein vs. 49. 39 ±13. 13 nmol/ mg protein, P < 0. 05), but was higher than that at the 120th hour (75. 71 ±16. 74 nmol/ mg protein vs. 57. 93 ±15. 32 nmol/ mg protein, P < 0. 05). Conclusions After transplantation of hypothemically stored kidney grafts, the increased NO production in the early stage has protective effects on the transplanted kidney. Application of L-NAME to inhibit NO production is harmful to the recovery of the renal functions of kidney grafts. Chin Med J 2004; 117(10):1552-1557

Background The purpose of the study was to evaluate the role of neoadjuvant chemotherapy and embolization via the anterior branches of the bilateral internal iliac arteries in treating patients with advanced ovarian epithelial carcinoma. Methods Forty-two patients with advanced ovarian epithelial carcinoma (study group) were treated via the anterior branches of the bilateral internal iliac arteries after cytoreductive surgery and 7 courses of adjuvant platinum-based combination chemotherapy. Primary cytoreductive surgery was performed in 43 patients with advanced ovarian epithelial carcinoma (control group), and then followed by 8 courses of adjuvant platinum-based combination chemotherapy. The rate of optimal cytoreductive surgery, survival rate, blood loss during operation and operative time were investigated in the two groups. Statistical significance was asessed using Student's t test, the Chi-squre test and the log-rank test. Results In the study group, the rate of optimum debulking after platinum-based chemotherapy and embolization via the anterior branches of the bilateral internal iliac arteries was 71. 43 %(30/42) (χ2 = 10. 06, P <0. 005), and 9 (21. 43 %) of the 42 patients showed no gross residual disease after surgery. Blood loss and operative time were significantly decreased in the study group as compared with those in the control group (665.24 ±37.61 ml: 849.31 ±41. 20 ml, t1 = 33. 21, P1 < 0. 001; 4. 23 ±0. 21 hours: 6. 15 ±0. 38 hours, t2 = 28. 92, P2 <0. 01). In the study group,the mean survival time and the median overall survival were 33. 66 months (95 % CI, 24. 73 to 42. 58) and 26. 00 months (95 % CI, 19. 22 to 32. 78), respectively. The median disease-free interval was 18. 20 months. In the control group, the mean survival time and the median overall survival were 32. 38 months (95 % CI, 24. 92 to 39. 84) and 25. 00 months (95 % CI, 22. 80 to 27. 20), respectively. The median disease-free interval was 14. 20 months. The overall survival rates were not significantly different between the two groups (χ2 = 6. 48, P >0. 05). Conclusions Neoadjuvant platinum-based combination chemotherapy and embolization via the anterior branches of the bilateral internal iliac arteries is an alternative treatment for patients with advanced ovarian epithelial carcinoma, in whom the chance of optimal cytoreductive surgery is low. The treatment can reduce blood loss, decrease operative time, and increase the rate of optimal cytoreductive surgery; but the median survival can't be improved significantly. Chin Med J 2004; 117(10):1547-1551

Background Hypoxia-inducible factor 1 (HIF-1), a transcription factor, is overexpressed in common human cancers and their metastases. This study aimed at determining the expression levels of HIF-1α and vascular endothelial growth factor (VEGF) in SW480 cells and in colorectal adenocarcinoma tissue and ascertaining whether HIF-1α and VEGF play important roles in tumor angiogenesis. Methods HIF-1α mRNA expression was analyzed using in situ hybridization and RT-PCR. HIF-1α and VEGF protein were detected in SW480 cells and colorectal adenomas and adenocarcinomas by immunohistochemistry using streptavidin/peroxidase (SP). Western blot was used to detect HIF-1α protein extracted from SW480 cells. Microvessel density (MVD) in colorectal carcinomas was determined by anti-CD34 immuno staining in colorectal carcinomas. Results Optical density values representing HIP-1α mRNA expression levels were found to be significantly higher in SW480 cells in hypoxic conditions than in cells under normoxic conditions (P < 0. 05) or in hypoxic conditions but treated with genistein (P < 0. 05). The levels of HIF-1α and VEGF protein expression in SW480 cells were significantly higher in the hypoxia group than in the normoxia group (P < 0. 01, P < 0. 05, respectively) and hypoxia/genistein group (P < 0. 01, P < 0. 05, respectively). The positive expression rates of HIF-1α mRNA changed dramatically when comparing colorectal adenomas with adenocarcinomas of different Dukes' stages (P < 0. 05). HIF-1α mRNA was also expressed at higher levels in adenocarcinomas than that in adenomas (P < 0. 01). HIF-1α protein expression correlated significantly with VEGF protein expression and MVD. Conclusions Hypoxia induces the expression of HIF-1α and VEGF in colorectal adenocarcinomas. HIF-1α may play an important role in angiogenesis and tumor progression by regulating the expression of VEGF in human colorectal carcinomas. Chin Med J 2004; 117(10):1541-1546

Background The aim of this study was to investigate DNA content and expression of c-erbB-2, PS2, and prostate-specific antigen (PSA) proteins in breast carcinomas with neuroendocrine (NE) cell differentiation. Methods Chromogramin, c-erbB-2, PS2, and PSA in 131 samples of breast cancer were detected immunohistochemically. Classic Feulgen staining image analysis techniques were used to quantify DNA content in 81 of the breast cancer samples. Results The c-erbB-2 positive rate in breast carcinoma samples containing neuroendocrine cells was 37. 5 % and the rate of high expression of c-erbB-2 (+ + or + + +) was 33. 3 %, both significantly lower than that in breast carcinomas without neuroendocrine cells (62. 6 % and 68. 7 %, respectively, P < 0. 05). The rates of po sitive PS2 and PSA expression in breast carcinoma samples containing neuroendocrine cells were 72. 2 % and 55.0 %, respectively, both significantly higher than that in breast carcinoma samples without neuroendocrine cells (45. 0 % and 16.4 %, respectively, P <0. 05). In NE (+) samples, the integral optical density, DNA index, DNA stemline peak, > 5c aneuploidy cells, and rate of aneuploidy among cells were all lower than that in NE (-) breast carcinomas (P < 0. 01). In NE (+) grade I or Ⅱbreast carcinomas, these indices were also all lower than that in the NE (-) breast carcinoma samples (P <0. 01). Conclusion Breast carcinomas with neuroendocrine differentiation have a lower rate of malignancy. Neuroendocrine differentiation could serve as a prognostic marker in clinical practice. Chin Med J 2004; 117(10):1536-1540

Background This study was to evaluate bivariate bromodeoxyuridine (BrdUrd)/DNA flow cytometric analysis in detection of gastric carcinoma and to study the relations of cellular BrdUrd labeling indices (LI), G2/M-phase fraction (G2/MPF) and DNA ploidy pattern to lymphatic involvement, venous invasion and prognosis. Methods Fresh tumor samples from 60 patients with gastric carcinoma were analyzed by bivariate BrdUrd/DNA flow cytometry. The results were correlated with lymphatic vessel invasion, lymphatic node metastasis, the number of matastatic lymphatic nodes, and venous invasion. Propidium iodide (PI) was used as a fluorescent probe for total cellular DNA, and a monoclonal antibody against BrdUrd was used as a probe for BrdUrd incorporated into DNA. Fluorescent-labeled goat anti-mouse antibody was used as a second antibody. S-phase fractions were measured by in vitro BrdUrd labeling, and DNA ploidy and G2/MPF were also measured. Comparison of survival was performed with the log-rank test, the Chi-square test for qualitative data, and Student's t test for quantu data. Results BrdUrd LI and G2/MPF values were significantly higher in tumors with lymphatic vessel invasion than in those without invasion respectively (P <0. 01); the patients who had tumors with lymphatic vessel invasion showed a significantly poor prognosis (P < 0. 01). Both BrdUrd LI and G2/MPF values were significantly higher in tumors with lymphatic node metastasis than in those without metastasis (P <0. 01). A statistical significant difference was noted in the 5-year survival rates between the patients with lymph node metastasis and those without metastasis. Compared with diploid carcinoma, the incidence of lymph node metastasis was significantly higher in aneuploid carcinoma (P < 0. 05), and the patients with aneuploid carcinoma showed a significantly poor prognosis (P <0. 05). BrdUrd LI was significantly higher in patients with more than 5 metastatic lymph nodes than those with 1-4 metastatic lymph nodes (P < 0. 05) and those without metastasis (P < 0. 01). G2/MPF values in those patients either with more than 5 metastatic lymph nodes or 1 - 4 metastatic lymph nodes were higher than those without metastasis (P <0. 01 and P < 0. 05). A statistical significance was seen in the 5-year survival rates among the patients with no metastatic lymph node, 1-4 metastatic nodes and more than 5 metastatic nodes (P < 0. 01). G2/MPF values were significantly higher in patients with venous invasion than in those without invasion (P <0. 01). Conclusions Positive correlations exist between cellular BrdUrd LI, G2/MPF with lymphatic involvement and prognosis, and DNA aneuploid with lymphatic involvement and prognosis. The same was true between G2/MPF value and venous invasion in gastric carcinoma. Chin Med J 2004; 117(10):1530-1535

Background Insulin treatment plays a key role in management of diabetes mellitus. Clinical researches showed that extra improvements in restoration of insulin secretion of pancreatic β cells were found in patients with newly diagnosed type 2 diabetes. The purpose of this study was to investigate the effects of early insulin treatment on insulin mRNA expression and morphological alterations of β cells in a Sprague Dawley (SD) rat model of type 2 diabetes. Methods A rat model of type 2 diabetes mellitus (T2DM) was induced by a high fat diet (high energy, HE) and low doses of streptozotoxin (STZ, 40 mg/kg). A group of diabetic rats was then injected with protamine zinc insulin (PZI, 1 - 2 U · kg-1 · d-1) for one week. Insulin mRNA expression, morphological features of pancreatic islets, and metabolic parameters were examined in rats using reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemistry, and other techniques. Results In insulin-treated diabetic rats, insulin mRNA levels prominently increased by 81. 3 % (P < 0. 05), as compared with untreated diabetic rats. Moreover, timely insulin treatment noticeably improved the insulin content of β cells, with an increase of 10. 2 % (P < 0. 05), despite a slight reduction in fasting blood glucose (FBG), triglyceride (TG), and free fatty acid (FFA) levels, as compared to an untreated diabetic group. Conclusion Insulin treatment at the onset of T2DM effectively improves insulin synthesis, as confirmed by morphological changes to β cells in a rat model of type 2 diabetes. Chin Med J 2004; 117(10):1523-1529

Background Reports are increasingly appearing on the side effects caused by excessive iodine intake. Our objective was to find out whether iodine excess would impair the thyroid function and intelligence of schoolchildren in rural areas of China. Methods A comparative epidemiological study was made on thyroid function and intelligence of the schoolchildren in the areas of low, moderate or excessive intake of iodine. In the area of low intake of iodine (Panshan, Liaoning province, median urinary iodine (MUI) was 99 μg/L), of moderate intake of iodine (Zhangwu, Liaoning Province, MUI was 338 μg/L) and of excessive intake of iodine (Huanghua, Hebei Province, MUI was 631 μg/L). The numbers of schoolchildren from each area selected to take part in a Chinese version of Raven's Test were 190, 236 and 313, respectively, and then 116, 110 and 112 of them were tested for thyroid function, thyroid autoantibody (TAA) and urinary iodine (UI). Results There were no significant differences in the incidences of overt hyperthyroidism, subclinical hyperthyroidism and overt hypothyroidism in Panshan, Zhangwu and Huanghua. But significant differences were found in the incidences of subclinical hypothyroidism (P = 0. 001) in these three areas. The incidences of subclinical hypothyroidism in Huanghua and Zhangwu were 4. 76 and 3. 37 times higher than that in Panshan. TAA were negative in all the schoolchildren with subclinical hypothyroidism except for one. No significant difference was found among the rates of thyroid peroxidase antibody (TPOAb) and thyroglobulin antibody (TGAb) in these three areas. Mean serum thyroglobulin (TG) value of Huanghua was markedly higher than those of the other two (P = 0. 02). Mean serum TG value of Zhangwu was higher than that of Panshan but the difference was not significant. Mean IQ value of the schoolchildren in Huanghua was markedly higher than that for Zhangwu (P = 0. 001). Mean IQ value of the schoolchildren in Panshan was lower than that of Huanghua and higher than that of Zhangwu but, again, the differences were not significant. Conclusions The increase of iodine intake may increase the risk for schoolchildren of subclinical hypothyroidism. In the area of iodine excess, most of the subclinical hypothyroidism cases are not of autoimmune origin. No obvious effect of excess iodine was found on mental development of schoolchildren. Chin Med J 2004; 117(10):1518-1522

Background Lipoprotein glomerulopathy (LPG), once recognized as a rare glomerular disease, has been reported around the world in recent years. In this study, we reported 8 patients of LPG and aimed to explore the clinical features and pathological characteristics of LPG under light microscope, immunofluorescence staining, and electron microscope. Methods Clinical manifestations were recorded on the day of renal biopsy. Biochemical patterns of lipids and lipoproteins were detected by routine examination. Plasma concentrations of apo B and apo E were determined by radial immunodiffusion assays. Biopsy specimens were then processed for light microscopy, immunohistochemical staining for immunoglobulins and complement components, and electron microscopy. Glomerular deposition of apo A, B, and E were detected using monoclonal antibodies on cryostatic sections. Results All of the eight patients presented with edema, microscopic hematuria, severe proteinuria, anemia, and enlarged kidney size. Biochemical profiles revealed high levels of triglycerides, apo B, and apo E. We noted increments of glomerular size and lipoprotein thrombi occupying capillary lumina in the glomeruli of all patients. Immunofluorescence staining showed that the thrombi were strongly positive for apo A, B, and E. Granules and various sizes of vacuoles were observed in the thrombi under electron microscope. Conclusion Compared with previous reports on LPG in other countries, unique clinical and pathological features were found in this group of Chinese LPG patients. Chin Med J 2004; 117(10):1513-1517

Background Cerebral ischemia is a significant clinical problem, and cerebral ischemia usually causes neuron irjury such as apoptosis in various brain areas, including hippocampus. Cysteinyl aspartate-specific protease (Caspases) are fundamental factors of apoptotic mechanism. Caspase-3 inhibitors show effect in attenuating brain injury after ischemia. But all the results were from animal models in research laboratories. This study aimed at investigating the correlation between the change of ischemic neuronal injury and Caspase-3 post-ischemia in human hippocampus. Methods We selected and systematized 48 post-mortem specimens from 48 patients, who died of cerebral infarction. Morphological change was firstly analyzed by observing hematoxyline/eosin-staining hippocampal sections. The expression of Caspase-3 was investigated using the methods of in situ hybridization and immunohistochemistry. Terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5’-triphosphate-biotin nick-end labeling (TUNEL) method was used to clarify the involvement of Caspase-3 in neuron death. The loss of MAP 2 (MAP-2) was applied to judging the damaged area and degree of neuronal injury caused by ischemia. Results In the CA1 sector of hippocampus, Caspase-3 immunostaining modestly increased at 8 hours [8. 05/ high-power field (hpf)], dramatically increased at 24 hours (24. 85/hpf), decreased somewhat after 72 hours. Caspase-3 mRNA was detectable at 4 hours (6. 75/ hpf), reached a maximum at 16 hours (17. 60/ hpf), faded at 72 hours. TUNEL-positive cells were detectable at 24 hours (10. 76/hpf), markedly increased at 48 - 72 hours. The loss of MAP-2 was obviously detected at 4 hours, progressed significantly between 24 and 72 hours; MAP-2 immunoreactivity was barely detectable at 72 hours. Before 72 hours, the Caspase-3 evolution was related with the upregulation of TUNEL and the loss of MAP-2. The positive correlation between Caspase-3 mRNA and TUNEL was significant at the 0. 05 level (correlation coefficient was 0. 721); the negative correlation between Caspase-3 mRNA and MAP-2 was significant at the 0. 05 level (correlation coefficient is 0. 857). In the early stage (before 72 hours), the staining of Caspase-3 mRNA and immunohistochemistry was predominantly present in cytoplasm; the staining of TUNEL was predominantly localized in nucleus. At 4 - 16 hours, most neurons in hippocampal CA1 areas had relatively normal morphology; at 24 - 48 hours, neurons showed apoptotic morphology; at 72 hours, most cells showed significantly pathological morphology. Conclusions There exist a time-dependent evolution of neuronal damage after hippocampal ischemia in human brain, which was characterized by its close correspondence to Caspase-3. Chin Med J 2004; 117(10):1507-1512