Objective To explore the effects and the molecular mechanism of synergistic hepatocarcinogenesis of HBV and AFB1 in the development of HCC by studying the difference in protein expression profiles in hepatocellular carcinoma exposed to hepatitis B virus and Aflatoxin B1.Method 32 HCC specimens were labeled under four categories based on their biomarkers of HBV and AFB1 exposure:group A:HBV(+)/AFB1 (+),10 cases,group B:HBV(+)/AFB1 (-),10 cases,group C:HBV(-)/AFB1(+),6 cases,groupD:HBV(-)/AFB1(-),6 cases.Normal hepatic tissues from 10 cases of hepatic hemangioma,liver resection and liver transplant donor were chosen as the normal control group.Isobaric Tagging Reagent Quantitative (iTRAQ) Proteomics together with 2DLC MS/MS were applied to analyze the differentially expressed proteins among the 4 groups.Result (1) A total of 117 unique differentially expressed proteins including 53 up-regulated proteins and 64 down-regulated proteins were identified in the four groups.The number of unique differentially expressed proteins,including up-regulated and down-regulated proteins,in group A,group B,group C and group D were 106,97,104 and 74 respectively.(2) Among the 117 differentially expressed proteins,9 proteins were heat shock proteins or chaperones,and they were up regulated in group A,B and C.Besides,15 proteins were detoxification and drug metabolism pathway related proteins,12 of them were down-regulated in group A,and more than half of them were also down-regulated in group B and C.(3) The Reverse Transcriptase PCR result showed the mean expression level of AKR1B10 mRNA in group A was significantly higher than group B,C and D (all P＜0.05,respectively).Group C also showed significantly a higher expression level of AKR1B10 mRNA than group D (P＜0.05).The Western-blot results showed the mean expression level of AKR1B10 protein in group A was significantly higher than group B and D (all P＜0.05,respectively).Conclusions The up-regulated expression of heat shock protein and the down-regulated expression of most protein enzymes related to detoxification and drug metabolism were the common molecular biological events of HCC associated with exposure to HBV and AFB1.This suggested the synergistic hepatocarcinogenesis effects of HBV and AFB1 may be related to dysregulation of protein enzymes related to detoxification and drug metabolism.The overexpression of AKR1B10 may be involved in the AFB1-related hepatocarcinogenesis process.

目的 观察p38信号通路(P38MAPK)在内皮细胞微粒(EMPs)诱导人脐静脉内皮细胞(HUVECs)表达细胞间黏附分子-1(ICAM-1)中的作用.方法 将体外培养的HUVECs随机分组:①EMPs不同时点观察组:用EMPs(终浓度105/ml)分别刺激细胞0、3、6、12、24 h；②EMPs不同剂量作用组:分别用终浓度为0、102、103、104、105/ml的EMPs刺激细胞24 h；③EMPs+p38MAPK特异性抑制剂SB203580组:在EMPs(终浓度105/ml)刺激前,与终浓度为5μmol/L的SB203580共同孵育30 min.用蛋白免疫印迹法(Western blot)测定p38MAPK磷酸化表达,实时荧光定量PCR测定ICAM-1mRNA的表达.结果 EMPs可激活p38MAPK,使磷酸化p38MAPK蛋白表达量及ICAM-1 mRNA表达量增加,且呈剂量和时间依赖性.p38MAPK特异性拮抗剂SB203580可显著抑制EMPs的此作用.结论 p38信号通路可能部分参与了对EMPs诱导HUVECs表达ICAM-1的调控.

Objective To explore genetic changes on chromosome 13 and particularly expression of the RB1 anti-oncogene,located on 13q14.2,in hepatocellular carcinoma(HCC) in patients exposed to both hepatitis B virus(HBV) and aflatoxin B1(AFB1).Meth-ods Thirty-two HCC tissue samples were divided into four subgroups according to HBV and AFB1 exposure:group A(n=10),HBV(+)/AFB1(+);group B(n=10),HBV(+)/AFB1(-);group C(n=6),HBV(-)/AFB1(+);group D(n=6),HBV(-)/AFB1(-).Genome-wide analysis was carried out using array-based comparative genomic hybridization(aCGH),and RT-PCR was used to detect RB1 mRNA expression.Results Deletion of 13q14.2 was found in 15 of 32(46.9%) samples overall,and deletion rates in each group were as follows:group A,80.0%(8/10);group B,50.0%(5/10);group C,33.3%(2/6);and group D,0%(0/6).Deletion rates differed signifi-cantly between groups A and C,A and D,and B and D(all P<0.05).RB1 mRNA expression was lower in the 15 cases with the 13q14.2 deletion than in the 17 cases possessing 13q14.2.Conclusions Genetic changes on chromosome 13,including deletion of 13q14.2,are frequent molecular characteristics of HCC.Deletion of 13q14.2 may result from the synergistic effects of HBV and AFB1,and it may contribute to RB1 gene inactivation in HCC.

Objective To explore gene mutations and mRNA expression of PTEN in cases of hepatocellular carcinoma(HCC) exvposed to both hepatitis B virus and aflatoxin B1(HBV/AFB1).Methods A total of 108 cases of HCC from different districts of Guangxi were divided into 4 groups according to their exposure to HBV and AFB1:group A,HBV(+)/AFB1-DNA(+),48 cases;group B,HBV(+)/AFB1-DNA(-),27 cases;group C,HBV(-)/AFB1-DNA(+),19 cases;and group D,HBV(-)/AFB1-DNA(-),14 cases.PTEN mutations in exons 4,5,and 8 were detected by PCR and direct sequencing,and mRNA expression was analyzed by RT-PCR.Results PTEN mutations in exons 4,5,or 8 were not found in any of the cases.However,a large deletion at the junction of exon 4 and intron 4 was found in 61 cases,corresponding to a deletion rate of 56.4%.The rate of PTEN deletion was 60.4% in group A,62.9% in group B,47.3% in group C and 46.6% in group D.The deletion rate was not statistically significant among the four sub-groups.Semi-quantitative gray values of the PTEN mRNA expression assay were as follows:group A,0.54 ± 0.13;group B,0.59± 0.16;group C,0.97 ± 0.16;group D,0.92 ± 0.13.The pair of groups A/B and the pair C/D differed significantly from each another in PTEN mRNA expression(PAC=0.002,PAD=0.032,PBC=0.000,PBD=0.011).Conclusions Down-regulation of PTEN mRNA expression is a frequent molecular event in HCC involving double exposure to HBV/AFB1 in Guangxi,and it may be related to HBV infection.AFB1 and HBV may act synergistically to down-regulate PTEN mRNA levels.

Objective To explore β-catenin gene mutations and protein expression in patients with hepatocellular carcinoma(HCC) in Guangxi.Methods β-catenin gene mutations and protein expression were analyzed in 108 HCC tissues by direct sequencing and immunohistochemistry,respectively.Results Mutations in the β-catenin gene were found in 12 of 108 HCC tissues,giving a mutation rate of 11.1%.The overall rate of positive β-catenin protein expression was 70.0%,including samples that were nuclear-positive(14.6%),cytoplasm-positive(57.3%)and membrane-positive(28.0%).The protein was expressed in 100% of the tissues carrying β-catenin gene mutations.Conclusions HCC patients in Guangxi show a relatively low rate of β-catenin gene mutation.These muta-tions may play an import role in hepatocarcinogenesis by causing over-expression of β-catenin protein.

以X精子和Y精子的DNA含量差别为基础的流式细胞仪分离精子技术是目前最有效的哺乳动物性别控制方法之一。本文从精子分离的生产工艺和生产实践出发,阐明精子分离的一些具体特征和分离工艺的效率,进而探讨提出了该技术的应用瓶颈及发展方向。

XY精子流式细胞分选技术是目前最有效的性别控制繁殖手段之一。本文使用冷冻后X精子人工授精,平均情期受胎率达63%,青年母牛的情期受胎率为77.45%;用性控冻精通过超排技术生产体内性控胚胎平均每头次达5.2枚。用PCR技术对生产的体内胚胎进行性别鉴定后,雌性胚胎的比率为92.98%,实际出生牛犊的母犊率在93%左右。由此表明,使用XY种畜(天津)有限公司生产的性控冻精在生产中应用效果良好,在受胎率不受影响的情况下其性控率可达90%以上。

The objective of this study was to develop an effective method to screen transgenic donor cells and improve the production efficiency of transgenic embryos. In the study, goat (Capra ibex) skin fibroblasts were co-transfected with two expression constructs, one containing the human α1 (I) procollagen cDNA fragment (4.3kb) and the neomycin resistance (Neor) gene, and the second containing the green fluorescent protein (GFP) gene. The Neor and GFP genes were used as markers for the screening of donor cells. Results revealed a significant difference (P<0.01) in the production rate of positive transgenic blastocysts (44.6% versus 82.8%) between donor fibroblasts selected using antibiotic G418 alone (Treatment A) and those from donor fibroblasts selected using antibiotic G418 and GFP (Treatment B), respectively. No significant differences were found between Treatments A and B in the total somatic cell nuclear transfer (SCNT) embryo cleavage rates (71.1% versus 65.9%) and total SCNT blastocyst formation rates (18.2% versus 15.7%). The research results indicate the Neor and GFP genes located at different constructs can be effectively combined by co-transfection to screen transgenic donor fibroblasts, and increase the yield of transgenic SCNT embryos carrying human α1 (I) procollagen cDNA in goats.

The ibex (Capra ibex) is currently listed as a priority protected animal by the Chinese government. Due to the limited availability of ibex oocytes, the objective of this study was to explore the feasibility of using domestic goat (Capra hircus) cytoplasts to reprogram nuclei from ibex donor fibroblasts in interspecies nuclear transfer (iSCNT). An effort was thus made to produce ibex embryos via iSCNT. The trial was performed using three treatment groups. The embryo cleavage rates on day 3 of in vitro culture and the blastocyst development rates on day 7 were recorded. In Treatment A, the female ibex fibroblasts were used as donor cells and of the fused cell-cytoplast couplets, 68% cleaved and 11% reached the blastocyst stage. In Treatment B, domestic adult dairy doe fibroblasts were used as donor cells and as comparison of homologous nuclear transfer (NT). Of fused cell-cytoplast couplets, 76% cleaved and 31% reached the blastocyst stage. In Treatment C, the development of parthenogenetic goat oocytes was used as the control and 90% of the embryos cleaved and 43% reached the blastocyst stage. No significant difference was found between treatment groups A and B in embryo cleavage rate, but a significant difference (P<0.01) was recorded between treatment groups A and B in the blastocyst production rate. There was a significant difference (P<0.01) between Treatment C and Treatment A/B in embryo cleavage rate and blastocyst production rate. The results indicated that although both cleavage rate (68%) and blastocyst yield (11%) of iSCNT embryos derived from female ibex fibroblasts (Treatment A) were lower than those of nuclear transfer embryos (Treatment B) and parthenogenetic development embryos (Treatment C). The domestic goat (C. hircus) cytoplasts supported the mitotic cleavage of ibex karyoplasts and were capable of reprogramming the nucleus to achieve a blastocyst stage embryo in exotic Capra which has the potential of alleviating species on the endangered list.

Through isolation and pure culture of Holstein milk cow ear fibroblasts and analyses research on several cryopreservation methods in this studing, the results show the satisfactory primary culture of Holstein milk cow ear fibroblasts had been obtained by small tissue cubes culture with complete medium (DMEM/F12+20%FCS+100IU/mL(P/S). Most fibroblasts could be collected in the digestion of primary fibroblast mass cultures by TrypsinEDTA (0.25% Trypsin,1mM EDTA.4Na) , through 2 to 3 subcultures the pure culture of Holstein milk cow ear fibroblasts could be implemented .On the conditions of the same cryoprotectant and FCS concentration Holstein milk cow ear fibroblasts were cryopreservated in five cryopreservation methods, through the contrast and analyses of cell vigor after five months cryopreservation , method 3 (70% cell suspension +20%FBS+10% DMSO ; cryopreservation procedure: First fibroblasts were pre-cooled and balanced for half hour at 4 ℃, and second hanged in gaseous nitrogen in close to the mouth of nitrogen canister for 4 hours ,and finally sunk into liquid nitrogen.) was the best cryopreservation method , it could maintain 84.8% of living rate after cells were revived and stained by Trypan Blue and 85.4% of adherence rate after cells were revived and cultured for 48 hours.

实现奶牛出生性别控制,多产母牛犊是广大奶牛养殖人员的美好愿望,是奶牛业发展的必然要求,是实现奶牛业经济效益的重要手段,是实现我国畜牧业快速发展的必然需要.

In this study using the transgenic(GFP) fibroblasts of Saanen dairy goat as donor cells and using the abattoir-derived oocytes of normal goat as recipient cells the nuclear transfer experiments had been processed.During experiments the optimization of electricfusion experiment(parameters) of transgenic nuclear transfer embryos had been done.For the treatment of transgenic reconstruction embryos of Saanen dairy goat,the electricfusion parameter of group B in(experiment) plans(1.5kV/cm for 30 μs and pulse interval for 1s) is most suitable for the(transgenic) reconstruction embryos of Saanen dairy goat,the electricfusion rate of 89.4% for cell-cytoplast complexes and the cleavage rate of 68.0% for reconstructed embryos have been obtained.

以新疆野生盘羊（Ovis ammon）和萨能奶山羊转基因（GFP）成纤维细胞为核供体细胞,分别以绵羊卵母细胞和普通山羊卵母细胞为受体细胞进行了核移植,对不同种类核移植重构卵进行电融合处理,发现盘羊重构卵电融合参数为1.4 kV/cm、20μs/次、间隔1 s时的重构卵融合率最高;对于转基因（GFP）萨能奶山羊重构卵的处理,电融合前先放入融合液中于38.5℃下平衡5 min,融合时电融合参数为1.5 kV/cm、20μs/次、间隔1 s时的融合率最高。实验中发现将融合后的两种重构胚胎先置入IVM培养液中于38.5℃下平衡4 h,可降低重构胚胎的死亡率,提高发育质量和卵裂率。

Through isolation and pure culture of Shaneng milk goat fibroblasts with skin tissue, fibroblast line was established. On conditions of the same cryoprotectant and FCS concentration Pure Shaneng milk goat fibroblasts cultured were cryopreservated in five cryopreservation methods with different pre-cool and balance ways and pre-cool time, through the contrast and analyses of cell vigor after five months cryopreservation, method 3 (70% cell suspension + 20%FCS + 10% DMSO; cryopreservation procedure: first fibroblasts were pre-cooled and balanced for half hour at 4 C , and second hanged in gaseous nitrogen in close to the mouth of nitrogen canister for 4 hours, and finally sunk into liquid nitrogen. ) was the best cryopreservation method, it could maintain 84. 8% of living cell rate through cell vigor test after cells were revived and stained by Trypan Blue and 85. 4% of cell adherence rate after cells were revived and cultured for 48 hours, living cell rate and cell adherence rate of method 3 superior to other four methods.