CD133在食管鳞状细胞癌中的表达及临床意义

目的:研究食管鳞状细胞癌组织中CD133的表达与肿瘤细胞增殖及侵袭转移的关系。方法:取40例食管鳞状细胞癌患者的组织石蜡标本,用免疫组化方法检测CD133的表达,同时检测Ki67的表达,分析探讨CD133表达与食管鳞状细胞癌临床病理因素的相关性。结果:肿瘤组织中CD133及Ki67的表达率均较癌旁组织高,且CD133的表达与肿瘤的侵润深度、淋巴结转移情况及分化程度呈正相关,同时CD133的表达与患者术后远期生存率具有相关性。但CD133与Ki67的表达情况无显著相关性。结论:食管鳞状细胞癌组织中,CD133的表达与肿瘤细胞的增殖、侵袭及转移具有相关性,可以成为评价食管癌鳞状细胞癌肿瘤生物学特性及判断预后的的指标。

Generation of Antibodies Against DMRT1 and DMRT4 of Oreochromis aurea and Analysis of Their Expression Profile in Oreochromis aurea Tissues

Sex determination is composed of somatic and germ-line sex differentiation hierarchies whose interaction is poorly understood. A single gene known to control somatic sex determination, the DM-domain containing （Doublesex/Mab-3 DNA-binding motif） gene, is highly conserved across species. Vertebrate DMRT1 （DM-related transcription factor 1） expression occurs predominantly in the testis. Here, however, isolated two distinct DM-domain cDNA from Oreochromis aurea ovary and testis have been named DMRT4 （DM-related transcription factor 4） and DMRT1 by BLAST, respectively. Despite high homology in the DM-domain there is little similarity outside the DM-domain.To better understand the structure, function, and possible roles of DMRT4 and DMRT1 as potential candidates for sex differentiation and sex determination, the intact regions encoding DMRT4 and DMRT1 obtained by PCR were sub-cloned into the vector pMAL-c2x and introduced into the Escherichia coli TB1 cell for efficient fusion expression. After purification and cleavage, DMRT4 and DMRT1 proteins were used to immunize adult rabbits following standard protocols. Consequently, it was found by using Western blot analysis that polyclonal antibodies against DMRT4 and DMRT1 had high specificity. The relative expression levels of DMRT4 and DMRT1 mRNA were determined by fluorescent Real-time RT-PCR in female and male Oreochromis aurea with 13-actin as the internal standard. DMRT1 was expressed only in testis, whereas DMRT4 was over expressed in the ovary, but in both female and male, a slight expression in the brain was also detected. Statistical analysis showed that in the brain, mean DMRT4 mRNA levels in female were significantly higher than in male. Meanwhile, the expression of DMRT4 and DMRT1 protein was also analyzed using the purified antibodies through Western blot and immunohistochemistry. It was found that DMRT4 was exclusively expressed in the ovary and DMRT1 in the testis. Study on DMRT4 and DMRT1 expression facilitated the elucidation of their roles and the understanding of sex differentiation of fish.

DPD组织和Ki-67蛋白在青年结肠癌及癌前病变中表达及其临床意义

目的:分析二氢嘧啶脱氢酶(DPD)组织和细胞增殖核抗原(Ki-67)蛋白在青年结肠癌及癌前病变中的表达及其临床意义。方法:选取青年结肠癌患者67例(实验组)和癌旁正常组织(对照组)65例,采用免疫组化方法检测两组DPD组织和Ki-67蛋白的表达,并对比分析其临床意义。结果:实验组二氢嘧啶脱氢酶组织阳性率与细胞增殖核抗原Ki-67的阳性率显著高于对照组(P<0.05);二氢嘧啶脱氢酶组织与细胞增殖核抗原蛋白在青年结肠癌及癌前病变中的表达中呈正相关关系(P<0.05);二氢嘧啶脱氢酶、细胞增殖核抗原Ki-67二者之间与结肠癌及癌前病变中的病理特征关系:与PTNM分期、分化程度等比较有统计学意义(P<0.05);与性别、年龄等比较无统计学意义(P>0.05)。结论:DPD组织和Ki-67蛋白均参与了结肠癌以及癌变的发生发展。

Expression and Purification of E.coli NrfA Protein and Preparation of Polyclonal Antibody against NrfA

[Objective] This study aimed to clone the E. coil NrfA gene and construct the pET-28a （＋）-NrfA prokaryotic expression vector for preparation of polyclonal anti- body against E. coil NrfA. [Method] E. coil NrfA gene was cloned from the E. coli genome DNA by PCR and inserted into the vector pET-28a（＋） to construct prokary- otic expression vector pET-28a （＋）-NrfA. E. coil NrfA protein was expressed by IPTG induction and purified. Polyclonal antibody against NrfA protein was prepared by im- munizing rabbit with routine method. The specificity and titer of polyclonal antibody was confirmed by ELISA and Western Blotting. [Result] The constructed prokaryotic expression vector pET-28a（＋）-NrfA was induced by IPTG, the recombinant NrfA pro- tein could be expressed effectively. The titer of rabbit anti-NrfA polyclonal antibody obtained by immunization and purification was about 1：204 900. Western Blotting anal- ysis indicated that the obtained polyclonal antibody against E. coil NrfA protein had high titer and high specificity. [Conclusion] E. coil NrfA gene was cloned and the prokaryotic expression vector pET-28a （＋）-NrfA was constructed successfully, poly- clonal antibody with high titer and high specificity was prepared, which laid the foun- dation for the study of NrfA in different strains of bacteria.

Establishment of an Indirect ELISA with the Major Epitope Domain of ApxⅡ of Actinobacillus pleuropneumoniae Expressed in Prokaryotic Cells

[Objective] This study aimed to develop an indirect ELISA to detect the antibodies against Actinobacil us pleuropneumoniae （APP） using the recombinant ApxⅡA1 protein expressed in prokaryotic cells as the antigen. [Method] The major epi-tope domain of ApxⅡ was cloned into the prokaryotic expression vector pET-28a （＋） to obtain the recombinant plasmid pET-ApxⅡA1, which was then transformed into E. coli BL21 （DE3） for expression. The immunogenicity of the recombinant pro-tein was analyzed by western-blotting. After that, the purified recombinant protein was used as the coating antigen in the indirect ELISA for detecting the antibodies against APP. Final y, the concentration of coated antigen and the dilution of serum were optimized. [Result] Proved by enzyme digestion and sequencing, the recombi-nant plasmid pET-ApxⅡA1 was constructed successful y. The recombinant protein was highly expressed in prokaryotic cells, and Western-blotting analysis showed that it was recognized specifical y by positive serum of APP. The indirect ELISA could detect the antibody against APP with the purified recombinant protein as the coating antigen. The optimal concentration of coated antigen was 1.23 μg/ml and the opti-mal dilution of serum was 1：100. Compared with a commercial ELISA kit detecting antibody against ApxⅣ, the coincidence rate of the indirect ELISA was 90.4%. [Conclusion] Our results indicated that the indirect ELISA is sensitive and specific, and suitable for evaluating the effect of APP vaccine and epidemiological surveys.

Expression and clinical significance of TAp73α, p53, PCNA and apoptosis in hepatocellular carcinoma

AIM: To study the prognostic role of TAp73α, p53, proliferating cell nuclear antigen (PCNA) and apoptosis in patients with hepatocellular carcinoma (HCC) after surgical tumor ablation. METHODS: Forty-seven human resected HCC tissues and 42 adjacent non-cancerous tissues were studied with 10 normal liver tissues as control group. TAp73α, p53, and PCNA were detected with Elivision immunohistochemistry. Terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick-end labeling (TUNEL) method was used to detect the apoptosis cells. All clinical and pathological materials were analyzed by SPSS10.0 statistical package. RESULTS: TAp73α overexpressed in HCC tissues (36.2%) when compared with adjacent non-cancerous tissues (2.38%, P<0.005) and normal liver tissues (0, P<0.01). Mutant type p53 (mt-p53) overexpressed in HCC tissues (38.3%) when contracted with adjacent non-cancerous tissues (16.7%, P<0.05) and normal liver tissues (0, P<0.01). Proliferation index (PI) level in HCC tissues was significantly higher than that in adjacent non-cancerous tissues (30.34%±4.46% vs 27.88%±5.89%, t, P=0.028). Apoptosis index (AI) level in HCC tissues was higher than that in adjacent non-cancerous tissues (8.62%±2.28% vs 7.38%±2.61%, t, P=0.019). Expression of TAp73a was associated with lymph node metastasis and mt-p53, with r=0.407 and 0.265, respectively. Expression of mt-p53 was associated with Edmondson's stage and AFP, with r=0.295 and-0.357, respectively. In Kaplan-Meier univariant analysis, TAp73α, AFP, TNM stage, portal vein invasion, liver membrane invasion and HBsAg correlated with prognosis (log rank, P=0.039, 0.012, 0.002, 0.000, 0.014, 0.007, respectively). Multivariant Cox regression analysis showed that TAp73α, AFP, TNM stage, portal vein invasion, liver membrane invasion and age were independent factors of prognosis. CONCLUSION: These results suggest that TAp73α can be used as a prognostic indicator of patients with HCC undergoing surgical tumor ablation. AFP, TNM, portal vein invasion, liver membrane invasion and age also have a potency of predicting the prognosis of HCC.

Mast cell density and the context of clinicopathological parameters and expression of p185,estrogen receptor,and proliferating cell nuclear antigen in gastric carcinoma

AIM:To investigate the relationship between the mast cell density(MCD)and the context of clinicopathological parameters and expression of p185,estrogen receptor(ER), and proliferating cell nuclear antigen(PCNA)in gastric carcinoma. METHODS:Mast cell,p185,ER,and PCNA were detected using immunohistochemical S-P labeling method.Mast cell was counted in tissue of gastric carcinoma and regional lymph nodes respectively,and involved lymph nodes(ILN)were examined as usual. RESULTS:MCD was significantly related to both age and depth of penetration(x^2=4.688,P<0.05 for age and x^2=9.350, P<0.01 for depth of penetration)between MCD>21/0.03 mm^2 and MCD≤21/0.03 mm^2 in 100 patients;MCD in 1-6 ILN group patients was significantly higher than that in 7-15 ILN or>15 ILN group patients(u=6.881,8.055,P<0.01); There were significant differences intergroup in positive expression rate of p185,ER and PCNA between MCD>21/ 0.03 mm^2 and MCD≤21/0.03 mm^2 in 100 patients. CONCLUSION:Mast cell may have effect on inhibiting invasive growth of tumor,especially in the aged patients; The number of mast cells,in certain degree,may predicate the number of involved lymph nodes,which is valuable for assessment of prognosis;MCD was related to the expression of p185,ER,and PCNA in gastric carcinoma.It suggests that mast cell accumulation may inhibit the proliferation and the dissemination of the gastric carcinoma. INTRODUCTION Recently,many studies have reported on the association of mast cell with various tumorst.In several malignancies,mast cell has been found to correlate with growth,penetration and prognosis of tumor.Therefore,our study was undertaken to investigate the relationship between the mast cell density (MCD)and the context of clinicopathological parameters and expression of p 185,estrogen receptor(ER),and proliferating cell nuclear antigen(PCNA)in gastric carcinoma.

Expression of cell cycle regulator p57^(kip2), cyclinE protein and proliferating cell nuclear antigen in human pancreatic cancer: An immunohistochemical study

AIM： To investigate the effects of p57^kip2, cyclinE protein and proliferating cell nuclear antigen （PCNA） on occurrence and progression of human pancreatic cancer. METHODS： The expression of p57^kip2, cyclinE protein and PCNA in tumor tissues and adjacent tissues from 32 patients with pancreatic cancer was detected by SP immunohistochemical technique. RESULTS： The positive expression rate of p57^kip2 protein in tumor tissues was 46.9%, which was lower than that in adjacent pancreatic tissues （χ^2 = 5.317, P〈0.05）. p57^kip2 protein positive expression remarkably correlated with tumor cell differentiation （P〈0.05）, but not with lymph node metastasis （P〉0.05）. The positive expression rate of cyclinE protein in tumor tissues was 68.8%, which was higher than that in adjacent pancreatic tissues （χ^2 = 4.063, P〈0.05）. CyclinE protein positive expression significantly correlated with tumor cell differentiation and lymph node metastasis （P〈0.05）. The positive expression rate of PCNA in the tumor tissues was 71.9%, which was higher than that in adjacent pancreatic tissues （χ^2 = 5.189, P〈0.05）. PCNA positive expression remarkably correlated with tumor cell differentiation and lymph node metastasis （P〈0.05）. CONCLUSION： The decreased expression of p57^kip2 and/or overexpression of cyclinE protein and PCNA may contribute to the occurrence and progression of pancreatic cancer. p57^kip2, cyclinE protein, and PCNA play an important role in occurrence and progression of pancreatic cancer.

Components of the mitogen-activated protein kinase cascade are activated in hepatic cells by Echinococcus multilocularis metacestode

AIM： To explore the effect of Echinococcusmultilocularis on the activation of mitogen-activated protein kinase （MAPK） signaling pathways and on livercell proliferation.METHODS： Changes in the phosphorylation of MAPKs and proliferating cell nuclear antigen （PCNA）expression were measured in the liver of patients withalveolar echinococcosis （AE）. MAPKs, MEK1/2 [MAPK/extracellular signal-regulated protein kinase （ERK）kinase] and ribosomal S6 kinase （RSK） phosphorylationwere detected in primary cultures of rat hepatocytesin contact in vitro with （1） E. multilocu/aris vesicle fluid（EmF）, （2）E. multilocularis-conditioned medium （EmCM）.RESULTS： In the liver of AE patients, ERK 1/2 andp38 MAPK were activated and PCNA expression wasincreased, especially in the vicinity of the metacestode.Upon exposure to EmF, p38, c-Jun N-terminal kinase（JNK） and ERK1/2 were also activated in hepatocytesin vitro, as well as MEK1/2 and RSK, in the absenceof any toxic effect. Upon exposure to EmCM, only JNKwas up-regulated.CONCLUSION： Previous studies have demonstratedan influence of the host on the MAPK cascade inE. multilocularis. Our data suggest that the reverse,i.e. parasite-derived signals efficiently acting onMAPK signaling pathways in host liver ceils, is actuallyoperating.

Neurogenesis by Activation of Inherent Neural Stem Cells in the Rat Hippocampus after Cerebral Infarction

Objective To investigate the changes of neural stem cells (NSCs) in the rat hippocampus after cerebral infarction (CI) and to evaluate the neurogenesis caused by the activation of NSCs. Methods CI models of rats were made and rats were assigned to 6 groups: sham-operated, 1 day, 3 days, 7 days, 14 days, and 28 days after CI. The dynamic expression of bromodeoxyuridine (BrdU), polysialylated neural cell adhesion molecule (PSA-NCAM), glial fibrillary acidic protein (GFAP), and neuronal nuclear antigen (NeuN) were determined by immunohistochemistry and immunofluorescence staining. BrdU was used to mark the proliferated NSCs. PSA-NCAM was used to mark the plasticity of activated NSCs. GFAP and NeuN were used to mark the differentiated NSCs. Results Compared with the controls, the number of BrdU+ cells in the hippocampus increased significantly at 1 day after CI (P<0.05), reached peak at 7 days after CI (P<0.05), decreased but still elevated compared with the controls at 14 days after CI (P<0.05), and nearly unchanged at 28 days after CI. The number of BrdU+/PSA-NCAM+ cells increased significantly at 7 days after CI (P<0.05), reached peak at 14 days after CI (P<0.05), and decreased but still elevated compared with the controls at 28 days after CI (P<0.05). The number of BrdU+/PSA-NCAM+ cells was equal to 60% of the number of BrdU+ cells in all the same period. The number of BrdU+/NeuN+ cells in the hippocampus increased significantly at 14 days after CI (P<0.05) and reached peak at 28 day after CI (P<0.05). The number of BrdU+/GFAP+cells in the hippocampus nearly unchanged after CI. Conclusion CI can stimulate the proliferation of inherent NSCs, and most proliferated NSCs may differentiate into neurons and represent neural plasticity.

Treatment of chronic proliferative cholangitis with c-myc shRNA

AIM： To investigate the feasibility and effectiveness of c-myc shRNA in inhibiting the hyperplastic behavior and lithogenic potentiality of chronic proliferative cholangitis （CPC）, in order to prevent stone recurrence and biliary restenosis. METHODS： An animal model of CPC was established by giving intralumenally 0.5 mL of c-myc shRNA. Then, the effects of c-myc shRNA on hyperplastic behavior and lithogenic potentiality of CPC were evaluated by histological observation, immunohistochemistry, real- time PCR and Western blotting for c-myc, proliferating cell nuclear antigen （PCNA）, procollagen m, mucin 5AC, enzymatic histochemistry for 13-glucuronidase, and biochemistry for hydroxyproline in the diseased bile duct. RESULTS： Treatment with c-myc shRNA efficiently suppressed the hyperplasia of biliary epithelium, submucosal gland, and collagen fiber by inhibiting mRNA and protein expression of c-myc. More importantly, it decreased the lithogenic potentiality of CPC by inhibiting the expression of mucin 5AC and the secretion of endogenous 13-glucuronidase. Further investigation indicated that c-myc shRNA-3 had a better inhibitory effect on CPC. CONCLUSION： Treatment with c-myc shRNA-3 can control CPC and reduce the lithogenic potentiality of CPC.

Multiple biomarkers of colorectal tumor in a differential diagnosis model:A quantitative study

AIM:To evaluate the multiple biomarkers of colorectal tumor and their potential usage in early diagnosis of colorectal cancers. METHODS:Multiple biomarkers (DNA contents,AgNOR, PCNA,p53,c-erbB-2) in 10 normal colorectal mucosae,37 colorectal adenomas and 55 colorectal cancers were analyzed quantitatively in the computed processing imaging system. Discrimination patterns were employed to evaluate the significance of single and multiple indices in diagnosis of colorectal cancers. RESULTS:The mean values of the analyzed parameters increased in order of the normal mucosa,adenoma and adenocarcinoma,and this tendency reflected the progression of colorectal malignancy.The parameters including DNA index,positive rates,densities of AgNOR,c-erbB-2,and p53, shape and density of nucleus were relatively valuable for diagnoses.Then a diagnostic discrimination model was established.The samples were confirmed with the model, the sensitivity rates in cancer group and adenoma group were 96.36% and 89.19%,respectively.The value of proliferating cell nuclear antigen (PCNA) in early diagnosis of colorectal cancers was uncertain. CONCLUSION:The quantitative evaluation of some parameters for colorectal tumor can provide reproducible data for differential diagnosis.The established diagnostic discrimination model may be of clinicopathological value, and can make the early diagnosis of colorectal cancer possible.

Effects of recombinant human growth hormone on enterocutaneous fistula patients

AIM： To explore the effects of recombinant human growth hormone （rhGH） on intestinal mucosal epithelial cell proliferation and nutritional status in patients with enterocutaneous fistula. METHODS： Eight patients with enterocutaneous fistulas received recombinant human growth hormone （10 ug/d） for 7 d. Image analysis and immunohistochemical techniques were used to analyse the expression of proliferating cell nuclear antigen （PCNA） in intestinal mucosal epithelial cells in biopsy samples from the patients who had undergone an endoscopic biopsy through the fistula at day 0, 4 and 7. Body weights, nitrogen excretion, serum levels of total proteins, albumin, prealbumin, transferrin and fibronectin were measured at day 0, 4 and 7. RESULTS： Significant improvements occurred in the expression of PCNA in the intestinal mucosal epithelial cells at day 4 and 7 compared to day 0 （24.93 ± 3.41%, 30.46 ± 5.24% vs 12.92 ± 4.20%, p 〈 0.01）. These changes were accompanied by the significant improvement of villus height （500.54 ± 53.79 um, 459.03 ± 88.98um vs 210.94 ± 49.16 um, P 〈 0.01）, serum levels of total proteins （70.52 ± 5.13 g/L, 74.89 ± 5.16 g/L vs 63.51 ± 2.47 g/L, P 〈 0.01）, albumin （39.44 ± 1.18 g/L, 42.39 ± 1.68 g/L vs 35.74 ± 1.75 g/L, P 〈 0.01） and fibronectin （236.3 4- 16.5 mg/L, 275.8± 16.9 mg/L vs 172.5 ± 21.4 mg/L, P 〈 0.01） at day 4 and 7, and prealbumin （286.38 ± 65.61 mg/L vs 180.88 ± 48.28 mg/L, P 〈 0.05）, transferrin （2.61 ± 0.12 g/L vs 2.41 ±0.14 g/L, P 〈 0.05） at day 7. Nitrogen excretion was significantly decreased at day 7 （3.40 ± 1.65 g/d vs 7.25 ± 3.92 g/d, P 〈 0.05）. No change was observed in the body weight. CONCLUSION： Recombinant human growth hormone could promote intestinal mucosal epithelial cell proliferation and protein synthesis in patients with enterocutaneous fistula.

Inhalation of hydrogen gas reduces liver injury during major hepatotectomy in swine

AIM： To study the effect of H2 gas on liver injury in massive hepatectomy using the Intermittent Pringle maneuver in swine. METHODS： Male Bama pigs （n = 14） treated with ketamine hydrochloride and Sumianxin Ⅱ as induc- tion drugs followed by inhalation anesthesia with 2% isoflurane, underwent 70% hepatotectomy with loss of bleeding less than 50 mL, and with hepatic pedicle occlusion for 20 min, were divided into two groups： Hydrogen-group （n = 7）, the pigs with inhalation of 2% hydrogen by the tracheal intubation during major hepa- totectomy; Contrast-group （n= 7）, underwent 70% hepatotectomy without inhalation of hydrogen. Hemo- dynamic changes and plasma concentrations of alanine aminotransferase （ALT）, aspartate aminotransferase （AST）, hyaluronic acid （HA）, tumor necrosis factor-α （TNF-α）, interleukin-6 （IL-6）, and malondialdehyde （MDA） in liver tissue were measured at pre-operation, post-hepatotectomy （PH） 1 h and 3 h. The apoptosis and proliferating cell nuclear antigen （PCNA） expres- sion in liver remnant were evaluated at PH 3 h. Then we compared the two groups by these marks to evalu- ate the effect of the hydrogen in the liver injury during major hepatotectomy with the Pringle Maneuver in the swine. RESULTS： There were no significant differences in body weight, blood loss and removal liver weight be- tween the two groups. There was no significant differ- ence in changes of portal vein pressure between two groups at pre-operation, PH 30 min, but in hydrogen gas treated-group it slightly decrease and lower than its in Contrast-group at PH 3 h, although there were no significant difference （P = 0.655）. ALT and AST in Hydrogen-group was significantly lower comparing to Contrast-group （P = 0.036, P = 0.011, vs P = 0.032, P = 0.013） at PH 1 h and 3 h, although the two groups all increased. The MDA level increased between the two group at PH i h and 3 h. In the hydrogen gas treated- group, the MDA level was not significantly significant at pre-operation and significantly low at PH 1 h and 3 h comparing to Contrast-group （P = 0.0005, P = 0.0004）. In Hydrogen-group, the HA level was also significantly low to Contrast-group （P = 0.0005, P = 0.0005） al- though the two groups all increased at PH 1 h and 3 h. The expression of cluster of differentiation molecule 31 molecules Hydrogen-group was low to Contrast-group. However, PCNA index （%） was not statistically signifi- cant between the two groups （P = 0.802）. Micropho- tometric evaluation of apoptotic index （AI） in terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-stained tissue after hepatotectomy for 3h, the AI% level in the hydrogen was significantly low to Contrast-group （P = 0.012）. There were no significant difference between Hydrogen-group and Contrast- group at pre-operation （P = 0.653, P = 0.423）, but after massive hepatotectomy, the TNF-α and IL-6 levels increase, and its in Hydrogen-group was significantly low compared with Contrast-group （P = 0.022, P = 0.013, vs P = 0.016, P= 0.012）, respectively. Hydro- gen-gas inhalation reduce levels of these markers and relieved morphological liver injury and apoptosis.CONCLUSION： H2 gas attenuates markedly ischemia and portal hyperperfusion injury in pigs with massive hepatotectomy, possibly by the reduction of inflamma- tion and oxidative stress, maybe a potential agent for treatment in clinic.

Effect of Herpes Simplex Virus-2 Infection in Vitro on the Expression of HLA Class II Antigen of Monocytes

Objectives: In order to investigate the role of mono-cytes and human leukocyte antigen (HLA) class II an-tigen in herpes simplex virus-2 (HSV-2) infection, westudied the effect of HSV-2 infection in vitro on theexpression of HLA class II antigen on monocytes.Methods: Monocytes were infected with HSV-2(Strain 333). Culture cells were collected 1, 3, 5 and 7days after infection. The levels of expression of HLAclass II antigen were measured by using alkaline phos-phatase antialkaline phosphatase method (APAAP).Results: The levels of the expression of HLA class IIantigen on monocytes significantly decreased on thefirst day of post-infection, and then gradually returnedto levels found in the controls by the 7th day post-infection.Conclusion: HLA class II antigen expression onmonocytes was inhibited with HSV-2 infection, whichmight be one means of virus escape at an early phase.The expression of HLA class II antigen may play animportant role in herpes simplex viurs-2 pathogenic-ity and immunity.

TSPAN1 protein expression:A significant prognostic indicator for patients with colorectal adenocarcinoma

AIM:To determine if TSPAN1 overexpression is associated with clinicopathological and prognostic factors in human colorectal adenocarcinoma.METHODS:Total RNA was extracted in 20 human adenocarcinoma tissues for TSPAN1 mRNA assay by RT-PCR.Eighty-eight specimens of human colorectal adenocarcinoma were surgically removed.TSPAN1 protein levels in cancer tissues were determined by immunohistochemistry using a polyclonal antibody against self-prepared TSPAN1.The correlation between TSPAN1 expression and the clinicopathological factors and the overall survival rate was analyzed by univariate and multivariate assay.RESULTS:TSPAN1 mRNA was detected in 90.0%(18/20) of cancerous tissues.The light density of TSPAN1 mRNA expression levels was 0.89 ± 0.30 in adenocarcinoma by gel-image system.TSPAN1 protein expression was detected in 78.41%(69/88) and weakly expressed in 40% normal colorectal tissues.There were significant differences between colorectal adenocarcinoma and normal control epithelium(P < 0.05).TSPAN1 protein expression in colorectal cancerous tissue was significantly correlated with the histological grade,cell expression PCNA,lymph nodal metastasis and TNM staging of the disease.Patients with TSPAN1 protein overexpression had a significantly shorter survival period than that in patients with TSPAN1 protein negative or weak expression,respectively(P < 0.05).Furthermore,by multivariate analysis,TSPAN1 protein expression demonstrated an independent prognostic factor for human colorectal cancers(P < 0.05,relative risk 0.755;95% confidence interval 0.302-1.208).CONCLUSION:The expression of TSPAN1 gene is increased in colorectal carcinoma,suggesting that TSPAN1 might serve as an independent prognostic factor for the colorectal adenocarcinoma patients.

Enhancement of CTLs induced by DCs loaded with ubiquitinated hepatitis B virus core antigen

AIM： To investigate whether hepatitis B virus （HBV） could induce a hepatitis B virus core antigen （HBcAg）specific cytotoxic T lymphocyte （CTL） response in vitro by dendritic cells （DCs） transduced with lentiviral vector-encoding ubiquitinated hepatitis B virus core antigen （LV-Ub-HBcAg）.METHODS： Recombinant LV-Ub-HBcAg were transfected into highly susceptible 293 T cells to obtain high virus titres, Bone marrow-derived DCs isolated from BALB/c mice were cultured with recombinant granulocyte-macrophage colony-stimulating factor and recombinant interleukin （IL）-4. LV-Ub-HBcAg, lentiviral vector-encoding hepatitis B virus core antigen （LV-HBcAg）, lentiviral vector （LV） or lipopolysaccharide were added to induce DC maturation, and the DC phenotypes were analyzed by flow cytometry. The level of IL-12 in the supernatant was detected by enzyme-linked immunosorbent assay （ELISA）. T lymphocytes were proliferated using Cell Counting Kit-8. DCs were cultured and induced to mature using different LVs, and co-cultured with allogeneic T cells to detect the secretion levels of IL-2, IL-4, IL-10and interferon-γ in the supernatants of T cells by ELISA. Intracellular cytokines of proliferative T cells were analyzed by flow cytometry, and specific CTL activity was measured by a lactate dehydrogenase release assay.RESULTS： LV-Ub-HBcAg-induced DCs secreted more IL-12 and upregulated the expression of CD80, CD86 and major histocompatibility class ]I, DCs sensitised by different LVs effectively promoted cytokine secretion; the levels of IL-2 and interferon-y induced by LV-Ub- HBcAg were higher than those induced by LV-HBcAg, Compared with LV-HBcAg-transduced DCs, LV-Ub- HBcAg-transduced DCs more efficiently stimulated the proliferation of T lymphocytes and generated HBcAgspecific cytotoxic T lymphocytes.CONCLUSION： LV-Ub-HBcAg effectively induced DC maturation. The mature DCs efficiently induced T cell polarisation to Thl and generated HBcAg-specific CTLs.

Detection of hepatitis C virus core antigen for early diagnosis of hepatitis C virus infection in plasma donor in China

AIM： To evaluate the efficacy of a new hepatitis C virus （HCV） core antigen assay developed in China. METHODS： After the determination of HCV infection, 49 serial samples were selected from II regular plasma donors in 5 different plasma stations. To compare the performance of HCV core antigen detection and HCV PCR, these samples were genotyped, and each specimen was analyzed by ELISA for the detection of HCV core antigen and by qualitative HCV PCR. RESULTS： Among all of the sequential samples, the original 23 specimens were HCV RNA-negative, and 36 samples were HCV RNA-positive. Twenty-seven samples （75%） were HCV core antigen-positive from these HCV RNA-positive specimens. Conversely, 27 samples （93.2%） were found HCV RNA-positive in HCV core antigen- positive samples. Intervals between HCV RNA and HCV core antigen-positive, as well as between HCV core antigen-positive and HCV antibody-positive were 36.0 and 32.8 d, respectively. CONCLUSION： This HCV core antigen assay, developed in China, is able to detect much of anti-HCV-negative, HCV RNA-positive preseroconversion window period （PWP） plasma donations.

Expression of intestinal trefoil factor, proliferating cell nuclear antigen and histological changes in intestine of rats after intrauterine asphyxia

AIM:To study the expressions of intestinal trefoil factor (ITF) and proliferating cell nuclear antigen (PCNA) and histologic changes in intestine, to investigate the relationship between ITF and intestinal damage and repair after intrauterine hypoxia so as to understand the mechanism of intestinal injury and to find a new way to prevent and treat gastrointestinal diseases. METHODS: Wistar rats, pregnant for 21 d, were used to establish animal models of intrauterine asphyxia by clamping one side of vessels supplying blood to uterus for 20 min, another side was regarded as sham operation group. Intestinal tissues were taken away at 0, 24, 48 and 72 h after birth and stored in different styles. ITF mRNA was detected by RT-PCR. PCNA expression was measured by immunohistochemistry. Intestinal tissues were studied histologically by HE staining in order to observe the areas and degree of injury and to value the intestinal mucosa injury index (IMDI). RESULTS: ITF mRNA appeared in full-term rats and increased with age. After ischemia, ITF mRNA was decreased to the minimum (0.59?.032) 24 h after birth, then began to increase higher after 72 h than it was in the control group (P<0.01). PCNA positive staining located in goblet cell nuclei. The PCNA level had a remarkable decline (53.29±1.97) 48 h after ischemia. Structure changes were obvious in 48-h group, IMDI (3.40±0.16) was significantly increased. Correlation analyses showed that IMDI had a negative correlation with ITF mRNA and PCNA (r= -0.543, P<0.05; r= -0.794, P<0.01, respectively). CONCLUSION: Intrauterine ischemia can result in an early decrease of ITF mRNA expression. ITF and PCNA may play an important role in the damage and repair of intestinal mucosa.

Role of TGF-β1/Smads pathway in carotid artery remodeling in renovascular hypertensive rats and prevention by Enalapril and Amlodipine

Objective To investigate the role of transforming growth factor-β1 （TGF-β1）, Smad2/3 and Smad7 expressions in carotid artery remodeling in renovascular hypertensive rats, and also the therapeutic effect of Enalapril and Amlodipine. Methods The renovascular hypertensive rat （RHR） models with ＂two-kidney and one-clip＂ were established, including model group （n = 6）, sham-operated group （n = 6）, Enalapril group （10 mg/kg per day, n = 6）, Amlodipine group （5 mg/kg per day, n = 6） and combination group （Amlodipine 2.5 mg/kg per day ＋ Enalapril 5mg/kg per day, n = 6）. The medication were continuous administrated for six weeks. Carotid artery morphological and structural changes in the media were observed by HE staining, Masson staining and immuno histochemical staining. Media thickness （MT）, MT and lumen diameter ratio （MT/LD）, and the expression levels of media a-smooth muscle actin （α-actin）, proliferating cell nuclear antigen （PCNA）, TGF-β1, phosphorylated Smad2/3 （p-Smad2/3） and Smad7 in carotid arteries were measured. Results The media of carotid arteries in RHR model group was significantly thickened, the volume of smooth muscle cell was increased, and the array was in disorder; MT, MT/LD, the proliferation index of smooth muscle cell and collagen fiber area percentage of carotid arteries in the model group were significantly higher than those in the sham-operated group （P 〈 0.01）. Compared to sham-operated group, the model group had significantly higher expressions of TGF-β1 and p-Smad2/3 （P 〈 0.05） and lower Smad7 expression. Both Enalapril and Amlodipine improved smooth muscle hypertrophy and collagen deposition, reduced RHR carotid MT, MT/LD, proliferation index of smooth muscle cell, collagen fiber area percentage and the expressions of TGF-β1 and p-Smad2/3 （P 〈 0.05）, increased Smad7 expression （P 〈 0.05）. Moreover, the combination treatment of Enalapril and Amlodipine had significantly better effects than single Amlodipine group （P 〈 0.05）, but not single Enalapril group. Conclusions TGF-β1/Smads pathway may participate in the mechanism of carotid artery remodeling in RHR; the role of Amlodipine and Enalapril in inversing carotid artery remodeling may be related to the change of TGF-β1/Smads pathway, the combination treatment of Amlodipine and Enalapril had better effects than single administration of Amlodipine.