Objective To interpret genetic variation and population structure of Anopheles dirus A and D from China by molecular marker. Methods Samples included An. dirus A of Hainan laboratory colony (n=13), and field specimen from Mengla (n=17) and Jiangcheng (n=17) in Yunnan Province. The specimens were identified by PCR assay before study. mtDNA-COⅠ region was amplified and sequenced. Genetic variation and population structure was estimated according to sequence data. Results The mtDNA-COⅠ gene with a length of 959 bp was analyzed. There were three haplotypes in An. dirus A and six haplotypes in An. dirus D. The above haplotypes distributed in three populations unif-ormly. The average number of pairwise differences within Mengla population (7.441 2) was greater than that of Jiangcheng (1.279 4) and Hainan (1.051 3) populations, which suggested that the level of genetic divergence was the highest within Mengla population. The result of hierarchical AMOVA estimation showed a limited geneflow (Fst=0.799 9), therefore the variation level in a population (20.01%) was smaller than among the populations (79.99%). Conclusion The inter-specific genetic variation between An. dirus A and D in China was small and the level of divergence among individuals was high.

ObjectiveTo observe the effect of portal blood flow on rat's liver with prehepatic portal hypertension.MethodsSixty-six male Wistar rats were randomly divided into five groups:partial portal vein transfixation group( A,n =20),hepatic artery transfixation after partial portal veins transfixation group( B,n =20),hepatic artery transfixation group (C,n =10),SO group( D,n =10 ),hepatic artery and partial portal vein transfixation group ( E,n =6 ).The mortality as well as the biochemical examination wereobserved at the 12th week after operation.Then the density of the microvascular in portal area and proliferating cell nuclear antigen expression of intro-hepatic bile duct epithelium were determined by immunohistochemical staining method.The ultra microstructure changes of hepatocytes were observed under transmission electron microscope.Results Biochemical examination,including glutamic-oxa-lacetic transaminase,glutamic-pyruvic transaminase and total protein,showed no significant differences anong all groups (P ＞0.05) at the 12th week after operation.Glutamie-oxalacetic transaminase in group A,in group B,in group C and in group D are (132.69±21.03) U/L,(154.40±28.73) U/L,(125.84±26.60) U/L,(134.02±18.42) U/L; glutamic- pyruvic transaminase in group A,in group B,in group C and in group Dare (39.33±8.62) U/L,(44.84 t9.47)U/L,(40.41±8.04) U/L,(38.47±7.29) U/L.Compared with groups C and D,the mortality rate (group A 20％ ; group B 25％),and the microvascular number in portal area increased obviously in group B and A(P ＜0.05).The proliferating cell nuclear antigen expressions of intra-hepatic bile duct epithelium increased significantly in group B (mean optical density 0.345±0.027 compared with the rest groups mean optical density in group A,in group C and in group D are 0.264 ± 0.015,0.258±0.022,0.249±0.021 ) ( P ＜ 0.01 ) ; fat accumulation in the hepatocytes and mitochondria vacuolization were observed under transmission electron microscopic in group A and group B.ConclusionsThe portohepatic collateral circulation still has its significance in liver blood supply of prehepatio portal hypertensive rat; the epithelium of intra-hepatic bile duct was mildly damaged and the microvascular number in hepatic portal area increased as well as the fat accumulated in hepatocytes of prehepatic portal hypertensive rat; if the hepatic artery blood supply is lost,these changes would become more obviously.

Objective To investigate the effect of adrenomedullin on epithelial barrier function of Caco-2 cells under hypoxia/reoxygen-ation injury and the molecular mechanism.Methods Build hypoxia/reoxygenation injury model,Caco-2 cells were randomly divided into three groups:normal control group( N) ,hypoxia/reoxygenation group( H/R) and hypoxia/reoxygenation plus 1 μmol/L adrenomedullin group(H/R+AM).Transepithelial electrical resistance(TEER) was determined,and the laser confocal microscope was used to detect the construction of tight junction.The protein expressions of tight junction proteins (Occludin and ZO-1) and nuclear factor kappa B(NF-κBp65) were examined by using western blotting.Results Pretreatment of AM significantly attenuated the disruption of morphological structure of tight junction caused by H/R.The TEER of N group,H/R group and H/R+AM group were(157.68 ±5.54),(96.06 ±3.42),(134.56 ± 4.72) Ohm/cm2 respectively.After AM pretreatment, the TEER were significantly raised compared with that in H/R group by 40.00%(P<0.05).The relative protein expressions of Occludin and ZO-1 in N group,H/R group and H/R+AM group were [(0.43 ±0.03), (0.27 ±0.04)],[(0.20 ±0.03),(0.15 ±0.07)],[(0.32 ±0.15),(0.21 ±0.03)]respectively.After AM pretreatment,the protein ex-pression of Occludin and ZO-1 were significantly raised compared with that in H/R group(by 60.00% and 40.00%).Simultaneously,the relative protein expressions of NF-κBp65 in N group,H/R group and H/R+AM group were (0.53 ±0.30),(2.89 ±0.16),(1.75 ±0.25) respectively.And H/R induced NF-κBp65 activation was significantly inhibited by AM treatment (decreased as compared with H/R group by 40.00%).Conclusion AM can attenuate intestinal epithelial barrier dysfunction by regulating the expression of intestinal tight junction pro-teins.The inhibition of NF-κB activation may be involved in this mechanism.

Objective To study the genetic basis for biovar conversion of Y. pestis. Methods In silico comparative genomic analysis was conducted and some critical genetic variations of Yersinia pestis were comparatively analyzed by means of PCR and DNA sequencing. Results A 93bp in-frame deletion in glpD gene results in the glycerol negative characteristic of Orientalis strains. A point mutation in the napA gene may cause the negative characteristic of nitrate reduction in Mediaevalis and Microus strains. A 122-bp frameshift deletion in the araC gene may lead to the arabinose negative phenotype of Microus strains. Conclusion In this study, Microtus strains with their unique pathogenic, biochemical and molecular features, were proposed as a novel biovar Microtus. In the light of its differential ability to ferment glycerol and arabinose and to reduce nitrate, Y. pestis can be classified into four biovars-Antiqua(glycerol positive, arabinose positive and nitrate positive), Mediaevalis(glycerol positive, arabinose positive and nitrate negative), Orientalis(glycerol negative, arabinose positive and nitrate positive), and Microtus(glycerol positive, arabinose negative and nitrate negative).

Objective To study the genome dynamics of Y. pestis and look for the relationship between its genome microevolution and niche adaption.Methods The DNA microarray combined with PCR was used to perform comparative genomic analysis of natural populations of Y.pestis.Results It was revealed that considerable genome dynamics of Y. pestis were the result of gene acquisition and loss in genome. We established a genomotyping system to group homologous isolates of Y. pestis, drew an outline of parallel microevolution of the Y. pestis genome, and established the link between the bacterial niche adaptation and genome microevolution.Conclusion The transmission, colonization and expansion of Y. pestis in natural foci are the results of its parallel, directional and gradual adaptation to the complex interactions among the environment, the hosts, and the pathogen itself.

Objective To identify the DNA tag sequences with the purpose of rapid and specific characterization of Y. pestis. Methods DNA microarray hybridization combined with PCR was used to perform genomic comparison between strains of Y. pestis and Y. pseudotuberculosis in order to screen and identify Y. pestis-specific genes. Results Twenty eight signature genes of Y. pestis were discovered. Three pairs of Y. pestis-specific primers were designed according to tag genes and proved to amplify the specific sequences of the target bacterium, showing no cross-reaction with the closely related Y. pseudotuberculosis and a large collection of genomic DNAs from other organisms. Conclusion DNA tag sequence is an ideal target for the rapid detection and identification of Y. pestis by PCR method.

Objective To study genetic variations of pgm locus and its flanking regions in Yersinia pestis isolated from Chinese natural focus so as to understand differences in virulence between different strains and to improve the prevention of plague. Methods We analysed the sequence variations of pgm locus and its flanking regions by using PCR-sequencing and allele-specific PCR among 260 strains of Y. pestis isolated from different foci and 7 strains of Yersinia pseudotuberculosis. Results For YP1666, only the strains of Xilin Gol grassland type and Microtus fuscus type had intact transmembrane helix, and the T-deletion at base 1406 was unique in strains of Orientalis. Same as Y. pseudotuberculosis, there was no IS100-insertion at the 3′ end of pgm locus of strains from Eastern section of North Tianshan Mountain type, Western section of North Tianshan Mountain type A and B, Xilin Gol grassland type and M. fuscus type. Only the strains of Xilin Gol grassland type and M. fuscus type had IS285-insertion in pigmentation segment and IS100-insertion at its downstream flanking region. pgm locus was deleted entirely from 36 strains, most of which came from Ordos plateau type, Song-liao plain type B , Kunlun Mountain type A and B. Conclusion Strains of Eastern section of North Tianshan Mountain type, Western section of North Tianshan Mountain type A and B, Xilin Gol grassland type and M. fuscus type are the oldest lineage of Chinese isolates. The pgm locus of the strains of these three types is very stable because there is no IS100-insertion at its 3′ terminal. We suggest that the strains of Xilin Gol grassland type and M. fuscus type should be grouped into a fourth biovar: Microtus. pgm locus is highly conserved among strains of different ecotypes, and its variations are well correlated with biovar, focus and ecotype, which indicates that pgm locus has played a role in strains′ adaptation to their environment.

Aim To investigate the effect of neferine on proliferation and invasion of human hepatocellular car-cinoma. Methods HepG2 and Bel-7402 cells were cultured in vitro with different concentrations of nefer-ine, then cell proliferation was observed by CCK-8 as-say; cell invasion was observed by transwell invasion assay; the protein expression of RhoA, RhoC and ROCK was detected by Western blot. Results CCK-8 results showed that neferine could significantly inhibit cell proliferation in a dose-dependent manner compared with the control group. Transwell invasion assay showed that cell invasion was significantly decreased with neferine 3 μmol · L-1 . Western blot results showed that RhoA, RhoC and ROCK protein expres-sion was decreased when neferine was co-incubated with hepatocellular carcinoma cells. Conclusion Nef-erine can inhibit proliferation and invasion of HepG2 and Bel-7402 cells, which is mediated mainly by the inhibition of RhoA, RhoC and ROCK protein expres-sion.

Objective To explore the expression of annexin A10 (ANXA10) in human hepatocellular carcinoma (HCC),and analyze its correlation with matrix metalloproteinases-9 (MMP-9) and vascular endothelial growth factor (VEGF),thus to provide the basis for clinical diagnosis and assessment of HCC.Methods 88 HCC patients were selected,and they were all performed surgical treatment,HCC stage Ⅰ in 11 cases,stage Ⅱ 25 cases,stage Ⅲ 31 cases,stage ⅣV 21 cases.The expressions of ANXA10,MMP-9,VEGF of HCC cancer tissues,adjacent liver tissues and normal liver tissues were tested by immunohistochemical method.The correlation of ANXA10 with VEGF and MMP-9 was analyzed.Results The absorbance value of ANXA10 expression in the HCC cancer tissue was (0.074 ± 0.012),which was lower than that in the adjacent liver tissues [(0.091 ± 0.013)] and normal liver tissues[(0.131 ±0.025)],and ANXA10 expression of the adjacent liver tissue was lower than that of the normal liver tissues,the differences were statistically significant (t =8.96,9.44,8.71,all P ＜ 0.05).The absorbance values of MMP-9 and VEGF expression in the HCC cancer tissue were (0.147 ± 0.017) and (0.127 ± 0.028),respectively,which were higher than those in the adjacent liver tissues [(0.096 ± 0.012),(0.091 ± 0.015)] and normal liver tissues [(0.075 ± 0.014),(0.077 ± 0.019)].The absorbance values of MMP-9 and VEGF of the adjacent liver tissues were higher than those of the normal liver tissues,the differences were statistically significant (t =8.05,9.30,8.11;8.28,9.51,8.02,all P ＜ 0.05).The absorbance value of ANXA 10 expression in the HCC stage Ⅲ + Ⅳ cancer tissue was (0.056 ± 0.010),which was lower than that in the stage Ⅰ + Ⅱ cancer tissue [(0.082 ±0.016)],the difference was statistically significant (t =8.90,P ＜ 0.05).The absorbance values of MMP-9 and VEGF expression in the stage Ⅲ + Ⅳ cancer tissue were (0.157 ± 0.022) and (0.169 ± 0.033),respectively,which were higher than those in the stage Ⅰ + Ⅱ cancer tissue [(0.114 ±0.015),(0.091 ±0.021)],the differences were statistically significant (t =9.13,9.72,all P ＜ 0.05).ANXA10 was correlated with MMP-9 and VEGF (r =0.324,0.295,all P ＜ 0.05).Conclusion ANXA10 presents lower expression in HCC cell,and the expression decreased with the increase of staging.It is negatively related with the MMP-9 and VEGF.ANXA10 expression missing or inactivation of malignant change may be one of the most important features in HCC.

Objective To establish the molecular identification of five members in Anopheles maculatus complex from China. Methods Different rDNA-ITS2 regions of An. maculatus complex were sequenced and analyzed. The species specific primers were designed, and PCR assay was used for the identification. Results The length and GC contents of ITS2 were 328 bp, 58.54% in An. pseudowillmori, 330 bp, 57.85% in An. maculatus, 337 bp, 59.05% in An. willmori, 334 bp, 58.68% in An. dravidicus, and 338 bp, 57.69% in An. sawadwongporni, respectively. The intra-species ITS2 sequences were conservative. The ranges of divergence level among five members were from 9.7% to 18.9% . Five distinct specific fragments were amplified by PCR assay using five species specific primers and 5. 8S primer. The length was 119, 186, 231, 327 and 406 bp respectively. Conclusion The diagnostic PCR assay based on ITS2 divergence to distinguish five members of An. maculatus complex was simple and reliable.