Studies on the integration of hepatitis B virus DNA sequence in human sperm chromosomes

Aim: To study the integration of hepatitis B virus (HBV) DNA into sperm chromosomes in hepatitis B patients and the features of its integration. Methods: Sperm chromosomes of 14 subjects (5 healthy controls and 9 HB patients, including 1 acute hepatitis B, 2 chronic active hepatitis B, 4 chronic persistent hepatitis B, 2 HBsAg chronic carriers with no clinical symptoms) were prepared using interspecific in vitro fertilization between zona-free hamster oocytes and human spermatozoa. Fluorescence in situ hybridization (FISH) to sperm chromosome spreads was carried out with biotin-labeled full length HBV DNA probe to detect the specific HBV DNA sequences in the sperm chromosomes. Results: Specific fluorescent signal spots for HBV DNA were seen in sperm chromosomes of one patient with chronic persistent hepatitis B. In 9(9/42) sperm chromosome complements containing fluorescent signal spots, one presented 5 obvious FISH spots and the others 2 to 4 signals. The fluorescence intensity showed significant diffe     

肝门部胆管癌外科诊疗中存在争议的若干问题

认为肝门部胆管癌应与该区域其他良恶性疾病相鉴别,其分型问题仍需深入研究.肝门部胆管癌应开展多学科综合治疗,积极实施根治性切除术应为首选,对条件合适的首次仅行手术引流和初次手术已行病灶切除、但目前存在肿瘤局部复发的肝门部胆管癌,应积极争取再次手术实施根治性切除,有望获得长期存活.肝门部胆管癌行半肝切除、受累血管切除、实施再次手术切除等有望提高根治性切除率,但同时亦明显增加了出现严重手术并发症的风险,需根据实际病情审慎决定.

Abstract：

Hilar cholangiocarcinoma should be identified from other malignant tumors and/or benign disease involving the hilum which may present similar image features.Clinal stage need to be further studied. Radical resection should be recommended in multidisciplinary treatment of hilar cholangiocarcinoma. The patients who were received initial surgical drainage and/or local recurrence after initial surgical resection of hilar cholangiocarcinoma, should actively be given the opportunity of radical reoperation, with potential long-term survival. Extended surgical resection resulted in increased rate of R0 resections and significantly improved survival. However, it also significantly increased the risk of serious postoperative complications, which must be balanced between survival advantage and surgical risk.     

AA-861抑制5-LO途径对KC和HSC的影响

Objective To investigate the effects of AA-861, the specific inhibitor of 5-lipoxyge-nase (5-LO), on the activation, proliferation and gene expression of kupffer cell (KC) and hepatic stellate cell (HSC). Methods 1) After being exposed to AA-861, the gene expression, proliferative ability and apoptosis of activated KC induced by lipopolysaecharide (LPS) were detected; 2) After the activated KC being exposed to AA-861, the supernatant of KC was added into quiescent HSC, then the proliferative ability and gene expression of quiescent HSC were observed. Results 1) After being activated by LPS, the mRNA and protein expression of 5-LO in KC increased remarkably while the mRNA and protein expression of 5-LO and 5-LO production decreased significantly after KC being ex-posed to AA-861. 2) After the supernatant of activated KC was added into quiescent HSC, the prolif-erative ability and gene expression of Collagen-1, α-SMA and TIMP-1 were increased significantly. However, after the supernatant of activated KC being exposed to AA-861 was added to quiescent HSC, it inhibited the activation of quiescent HSC and the proliferative ability and gene expression of HSC were decreased. Conclusion Inhibition of the 5-LO pathway by AA-861 can induce the over-pro-liferated KC to undergo apoptosis, and then inhibit the activation and proliferation of HSC and de-crease the production of ECM.     

Reproductive characteristics of transgenic (TG) chickens caping an exogenous gene

An exogenous gene (lacZ/MiwZ) introduced into the germinal crescent region (GCR) of avian embryos was con-firmed to be successfully transferred to the gonads via the primordial germ cells (PGCs). Following hatching, the chickswere raised until the stage of sexual maturation. The incorporation of MiwZ DNA was detected in male and female trans-genic chickens, respectively. The normal male and female transgenic birds were subjected to artificial insemination ac-cording to routine methods. Fertilized eggs obtained from female transgenic chickens were incubated for 72 h and the em-bryos removed from the yolk were examined by X-gal staining to detect the introduction of MiwZ in the offspring. As aresult, the expression of MiwZ was detected in the offspring. Furthermore, the presence of MiwZ in the extracts fromembryos was also detected by polymerase chain reaction (PCR) analysis. In male transgenic chickens, the presence of in-jected MiwZ in the extracts from sperm was also confirmed. The exogenous gene intro     

Single Nucieotide Polymorphisms in a male infertility-related cene CatSper

Aim: To identify the single nucleotide polymorphisms (SNPs) of human CatSper gene, the mouse homologous gene product. Methods: A systematic screening of SNPs in coding regions and flanking intronic regions of human CatSper gene in a sample subset from 210 Chinese Han males by DNA sequencing was demonstrated. PCR single-strand conformation polymorphism (SSCP) analysis was used to determine the allele frequencies of the possible SNPs. Results: Three SNPs, including two novels, were identified and their allele frequencies determined in 210 males. These SNPs were assembled into large SNP database that permits the analysis of the genetic basis of different diseases.     

雌激素和孕激素及其受体在肝门部胆管癌中的水平及意义

肝门部胆管癌(hilar cholangiocarcinoma,HCCA)发病率近年来有增加趋势,目前其致癌因子尚不明确。我们对42例HCCA组织进行雌激素(estrogen,E)、孕激素(progesterone,P)、雌激素受体(estrogen receptor,ER)及孕激素受体(progesterone receptor,PR)联合检测,旨在探讨它们在HCCA发     

免疫增强型胰腺癌MUC1-VNTR-C144 DNA疫苗的构建

Objective To construct new MUC1-VNTR-C144 DNA vaccine for pancreatic cancer and study both CTL responses and antibody responses specific to VNTR enhanced by the C144 gene. Methods DNA encoding the 144 amino acids of the N-terminus of HBV core gene was cloned and then inserted into the recombinant plasmid pcDNA3.1-VNTR/Myc-his( + ) A. C57BL/6 mice were randomly immunized intramusscularly into anterior tibialis muscle with 100 μg plasmid DNA encoding VNTR ( V group,n = 15) or VNTR/CI44 (VC group,n = 15). Mice injected with the plasmid pcDNA3.1-C144/ Myc-his( + ) A vector (C group, n = 15 ) were used as controls. Four weeks later, all mice were injected a-gain with the same solution as before. Two weeks after the last immunization, spleen cells were obtained and analyzed for cytotoxic activity 6 days after in vitro stimulation by the synthesized VNTR polypeptide (Calcein AM assay). Blood was collected for the ELISA assay of anti-VNTR antibodies. Results The specific CTL killing rate against VNTR polypeptide induced in the VC group (46.7±8.2)% was signifi-cantly higher than in the V group (34.8±3.1 ) % and the C group ( 12.9±1.2) % ( P < 0.01 ) ,indicating the C144 gene enhances the CTL response induced by the vaccine. However,CTL lysed much more VNTR positive EIA than VNTR negative EIA ( P < 0.01 ), while the anti-VNTR antibody VU 3C6 significantly restrained such activity ( P <0.01 ) ,which indicated the CTL response was specific to VNTR. The equiva-lent concentration of specific antibody against VNTR in the VC group (2810.2±308.3 ) mg/L was signif-icantly higher than the V group (2323.5±238.3) mg/L and the C group (2130.9±138.5) mg/L (P < 0.01 ),which implied that the C144 gene could enhance the antibody response induced by the vaccine. Conclusion The VNTR specific CTL response and antibody response induced in mice immunized with new MUCl-VNTR-C144 DNA vaccine for pancreatic cancer can be enhanced significantly by the C144 gene.     

免疫增强型胰腺癌MUC1-VNTR-C144 DNA疫苗的构建

Objective To construct new MUC1-VNTR-C144 DNA vaccine for pancreatic cancer and study both CTL responses and antibody responses specific to VNTR enhanced by the C144 gene. Methods DNA encoding the 144 amino acids of the N-terminus of HBV core gene was cloned and then inserted into the recombinant plasmid pcDNA3.1-VNTR/Myc-his( + ) A. C57BL/6 mice were randomly immunized intramusscularly into anterior tibialis muscle with 100 μg plasmid DNA encoding VNTR ( V group,n = 15) or VNTR/CI44 (VC group,n = 15). Mice injected with the plasmid pcDNA3.1-C144/ Myc-his( + ) A vector (C group, n = 15 ) were used as controls. Four weeks later, all mice were injected a-gain with the same solution as before. Two weeks after the last immunization, spleen cells were obtained and analyzed for cytotoxic activity 6 days after in vitro stimulation by the synthesized VNTR polypeptide (Calcein AM assay). Blood was collected for the ELISA assay of anti-VNTR antibodies. Results The specific CTL killing rate against VNTR polypeptide induced in the VC group (46.7±8.2)% was signifi-cantly higher than in the V group (34.8±3.1 ) % and the C group ( 12.9±1.2) % ( P < 0.01 ) ,indicating the C144 gene enhances the CTL response induced by the vaccine. However,CTL lysed much more VNTR positive EIA than VNTR negative EIA ( P < 0.01 ), while the anti-VNTR antibody VU 3C6 significantly restrained such activity ( P <0.01 ) ,which indicated the CTL response was specific to VNTR. The equiva-lent concentration of specific antibody against VNTR in the VC group (2810.2±308.3 ) mg/L was signif-icantly higher than the V group (2323.5±238.3) mg/L and the C group (2130.9±138.5) mg/L (P < 0.01 ),which implied that the C144 gene could enhance the antibody response induced by the vaccine. Conclusion The VNTR specific CTL response and antibody response induced in mice immunized with new MUCl-VNTR-C144 DNA vaccine for pancreatic cancer can be enhanced significantly by the C144 gene.     

免疫增强型胰腺癌MUC1-VNTR-C144 DNA疫苗的构建

Objective To construct new MUC1-VNTR-C144 DNA vaccine for pancreatic cancer and study both CTL responses and antibody responses specific to VNTR enhanced by the C144 gene. Methods DNA encoding the 144 amino acids of the N-terminus of HBV core gene was cloned and then inserted into the recombinant plasmid pcDNA3.1-VNTR/Myc-his( + ) A. C57BL/6 mice were randomly immunized intramusscularly into anterior tibialis muscle with 100 μg plasmid DNA encoding VNTR ( V group,n = 15) or VNTR/CI44 (VC group,n = 15). Mice injected with the plasmid pcDNA3.1-C144/ Myc-his( + ) A vector (C group, n = 15 ) were used as controls. Four weeks later, all mice were injected a-gain with the same solution as before. Two weeks after the last immunization, spleen cells were obtained and analyzed for cytotoxic activity 6 days after in vitro stimulation by the synthesized VNTR polypeptide (Calcein AM assay). Blood was collected for the ELISA assay of anti-VNTR antibodies. Results The specific CTL killing rate against VNTR polypeptide induced in the VC group (46.7±8.2)% was signifi-cantly higher than in the V group (34.8±3.1 ) % and the C group ( 12.9±1.2) % ( P < 0.01 ) ,indicating the C144 gene enhances the CTL response induced by the vaccine. However,CTL lysed much more VNTR positive EIA than VNTR negative EIA ( P < 0.01 ), while the anti-VNTR antibody VU 3C6 significantly restrained such activity ( P <0.01 ) ,which indicated the CTL response was specific to VNTR. The equiva-lent concentration of specific antibody against VNTR in the VC group (2810.2±308.3 ) mg/L was signif-icantly higher than the V group (2323.5±238.3) mg/L and the C group (2130.9±138.5) mg/L (P < 0.01 ),which implied that the C144 gene could enhance the antibody response induced by the vaccine. Conclusion The VNTR specific CTL response and antibody response induced in mice immunized with new MUCl-VNTR-C144 DNA vaccine for pancreatic cancer can be enhanced significantly by the C144 gene.     

原发性肝细胞肝癌患者癌组织和血清中HBV前C区基因的检测

目的 乙型肝炎炎病毒（ＨＢＶ）是原发性肝细胞肝癌（ＨＣＣ）的主要诱导因素之一。为探讨ＨＢＶ在ＨＣＣ中的重要性，我们检测了ＨＣＣ患者癌组织及血清中的ＨＢＶ前Ｃ区基因。方法 根据ＨＢＶ前Ｃ区ＤＮＡ序列，采用聚合酶链式反应（ＰＣＲ）对２３例手术切除后病理确诊有ＨＣＣ患者癌组织以及血清进行了检测。结果 ＰＣＲ显示２３例ＨＣＣ患者癌组织和血清中ＨＢＶ前Ｃ区基因的阳性率分别为５２．２％（１２／２３）和３０．４     

Expression of hepatitis B virus genes in early embryonic cells originated from hamster ova and human spermatozoa transfected with the complete viral genome

目的:使去透明带金黄地鼠的卵母细胞与携带乙肝病毒 DNA(HBV DNA)的人类精子体外受精,检测乙肝病毒基因(S 基因与 C 基因)在早期胚胎细胞中的复制与表达。方法:通过异种体外受精,由人类精子将乙肝病毒基因导入去透明带地鼠卵母细胞中;用聚合酶链反应(PCR)检测单细胞和2-细胞胚胎细胞的基因组中是否有 S 基因和前 C/C 基因;用逆转录聚合酶链反应(RT-PCR)研究上述基因是否在胚胎细胞中表达。以全长 HBV DNA 制备探针,与胚胎细胞制片进行荧光原位杂交(FISH),观察胚胎细胞基因组中是否有 HBV DNA整合。结果:PCR、RT-PCR 和 FISH 分析均在待测样本中获得阳性结果。结论:以精子为载体、携带到卵内的 HBV 基因能够在早期胚胎细胞中复制和表达,该结果为 HBV 有可能通过男性生殖细胞垂直传递给子代提供了直接证据。     

反义MBD1基因片段真核表达载体的构建及鉴定

目的构建反义MBD1基因片段真核表达载体,为研究MBD1基因功能提供工具.方法根据MBD1基因cDNA序列中编码序列,设计PCR引物,在引物5＇端分别添加Xba Ⅰ和Kpn Ⅰ酶切位点,将PCR片段反向插入真核表达载体pcDNA3.1（＋）的多克隆位点上,构建反义MBD1基因片段真核表达载体,并用PCR、酶切法和DNA测序鉴定.结果PCR鉴定得到322 bp特异性条带,双酶切得到327 bp目的基因片段和5.4kb载体片段,DNA测序说明插入片段序列是正确的.结论采用基因克隆技术,成功构建了反义MBD1基因片段真核表达载体,为研究MBD1基因在DNA甲基化和肿瘤发生中的功能提供了实验工具.     

In situ hybridization of hepatitis B DNA in hepatitis B-associated glomerulonephritis

Renal tissues from 43 of 49 children with hepatitis B virus-associated glomerulonephritis (HBV-GN) were examined for HBV DNA by in situ hybridization (ISH) assay within the last 10 years. HBV DNA was identified in 41 of the 43 cases (95.3%). HBV DNA was distributed generally in the nucleus and cytoplasm of epithelial cells and mesangial cells of glomeruli, and epithelial cells of renal tubules. HBV DNA also existed simultaneously in renal interstitial tissues in some of these cases. The positive results from HBV DNA ISH correlated well with HBV antigen assays. The analyses implied that the more extensive the existence of HBV DNA in the nephron unit and interstitial tissue, the more severe the clinical manifestation. The duration of proteinuria in cases with HBV DNA in renal tubules was much longer than in those with no HBV DNA in renal tubules. The persistence of the HBV genome or genes in the kidney could lead to the expression of viral antigens in renal tissues and might cause cellular pathological alteration. This would support utilization of antiviral therapy, such as cytokines, in the treatment of HBV-GN. Received December 10, 1996; received in revised form March 7, 1997; accepted July 30, 1997     

转乙型肝炎病毒x基因肝癌细胞株的建立

目的 建立表达转乙型肝炎病毒Ｘ基因（ＨＢＸ）的人肝癌细胞株,为研究ＨＢＸ基因与肝癌生物学行为间的关系提供模型。方法 以聚合酶链反应（ＰＣＲ）法从３．２ｋｂ 型肝炎病毒（ＨＢＶ）全基因组中主增ＨＢＸ基因,亚克隆至逆转 功体,磷酸钙共沉淀法将其导入包装细胞系,以含病毒的培养上清感染人肝癌细胞系ＱＧＹ７７０１,新霉素（Ｇ４１８）筛选,ＰＣＲ与反转录ＰＣＲ（ＲＴ－ＰＣＲ）鉴定。结果 ＰＣＲ法从ＨＢＶ基因     

肝门部胆管癌手术治疗：附44例报告

背景与目的：对于肝门部胆管癌（HCCA）而言,R0切除率仍然很低。目前对术前评估、术前胆道引流、门静脉栓塞、手术切除范围、手术方式、血管切除、淋巴结清扫、化疗等问题仍有很多争议。R0切除被认为是HCCA患者获取长期生存的最重要的治疗手段。笔者总结HCCA的治疗体会,并分析不同术式的有效性及近远期疗效。 方法：回顾性分析2015年1月—2020年1月行手术治疗的44例HCCA患者的临床资料。 结果：44例患者中,Bismuth-Corlette分型I型5例,II型7例,IIIa型8例,IIIb型13例,IV型11例;29例行半肝/扩大半肝+全尾叶切除（联合半肝切除）,13例行肝门部/围肝门区+全尾叶切除（围肝门切除）,术中包括门静脉部分切除修补2例,门静脉切除重建2例,肝动脉切除重建2例,另2例因肿瘤转移无法切除行T管引流。全组均完成手术,无手术死亡。术后病理结果显示,镜下切缘阴性（R0）切除37例（联合半肝切除组26例,围肝门切除组11例）,镜下切缘阳性（R1）切除5例（半肝切除2例、围肝门切除3例）。临床指标分析结果显示,联合半肝切除组的手术时间（240.4 min vs. 358.1 min）、术中出血量（705.5 mL vs. 809.9 mL）明显少于围肝门切除组,肿瘤标本切缘阳性率（6.9% vs. 23.1%）明显低于围肝门切除组（均P<0.05）;生存分析结果显示,联合半肝切除组术后与无复发生存期及1年累积生存率明显优于围肝门切除组（均P<0.05）。 结论：根治性R0切除是HCCA患者可能获得治愈的唯一方法,与围肝门切除术比较,联合半肝、尾状叶切除的大范围肝切除术,能提高R0切除率,改善无复发生存期及1年生存率。术前精确评估、合理的围手术期治疗、选择个体化的手术方案可提高HCCA的疗效。     

Differential diagnosis of proximal biliary obstruction

BACKGROUND: Obstruction at the hepatic duct confluence is generally due to hilar cholangiocarcinoma (HCCA). However, in up to 15% of patients, hilar obstruction could be due to alternative diagnoses other than HCCA. The aim of this study was to determine preoperative criteria that could differentiate HCCA from the alternative diagnoses. METHODS: All patients with hilar obstruction presumed to represent HCCA were included (1997-2001). The extent of disease was assessed preoperatively with computed tomography, magnetic resonance cholangiopancreatography, and Duplex ultrasonography, and these findings were correlated to the final histopathology. RESULTS: A total of 171 patients were included in the study, with HCCA being the most common diagnosis (141 patients [82.4%], group I). Alternative diagnoses other than HCCA were encountered in 30 patients (17.5%, group II) and included benign stricture (9 patients [5.2%]) and other malignancy (21 patients [12%]). There was a higher incidence of involvement of the second-order bile ducts in group I (26% vs 3% in group II, P<.01). Vascular involvement and lobar atrophy were more common in group I (58% and 41%) when compared with group II (16% and 6%, P<.005 and P<.002). The combination of these 2 findings (vascular invasion+lobar atrophy) was reliable for discriminating patients with HCCA from the alternative diagnoses. (38% in group I and 3.3% in group II, P<.001). CONCLUSIONS: Involvement of second-order bile ducts, vascular invasion, and lobar atrophy are more likely in patients with HCCA. The combination of vascular invasion and lobar atrophy significantly increases the diagnostic likelihood of HCCA. The absence of these findings should raise awareness of the possibility of an alternative diagnosis.     

靶向过氧化物酶体增殖子活化受体-γ基因siRNA腺病毒载体的构建与鉴定

目的 构建、鉴定靶向过氧化物酶体增殖子活化受体-γ(PPAR-γ)基因的siRNA腺病毒载体.方法 遵循siRNA片段的设计原则,依据GenBank中PPARγ基因(AF013266)的序列,设计3个特异性靶序列(36～54位、73～91位和404～422位);体外合成对应发卡样DNA片段,经退火后,将其克入pSIREN-Shuttle载体,通过PCR和I-Ceu I和PI-Sce I酶切鉴定后,再亚克隆入pAdeno-X腺病毒载体.对插入序列进行DNA序列分析.结果 发卡样siRNA单链DNA寡核苷酸退火后,电泳可见明亮靶条带;PCR扩增和酶切鉴定得到阳性克隆pSIREN-Shuttle-siPPARγ-1、pSIREN-Shuttle-siPPARγ-2和pSIREN-Shuttle-siPPARγ-3.亚克隆重组腺病毒载体pAdeno-X siPPARγ1、pAdeno-X-siPPART-2和pAdeno-X-siPPARγ-3,经测序鉴定证实插入DNA序列与设计完全一致.结论 成功构建3个靶向PPARγ基因的siRNA重组腺病毒载体,为进一步的基因治疗研究奠定了实验基础.     

根治性切除治疗肝门部胆管癌

目的 探讨肝门部胆管癌根治性切除的疗效及围手术期处理要点.方法 回顾性分析2000年1月至2010年12月安徽医科大学第一附属医院肝胆外科收治的50例肝门部胆管行根治性切除(RO)患者的影像学资料、临床分型、病理特征、手术方式及随访情况.结果 50例患者中,依据Bismuth-Corlette分型,I型者4例,Ⅱ型者...