乙型肝炎病毒阳性血清体外感染Hep G2细胞的实验研究

目的　建立HBV阳性血清体外感染HepG2 细胞的实验方法。方法　培养HepG2 细胞传至6孔板中,2 4h后进行HBV阳性血清体外感染HepG2 细胞的实验。感染组用HBV阳性血清,阴性对照组用HBV阴性血清,空白对照组用DMEM培养基。实验开始后HepG2 细胞继续孵育2 4h ,而后用0 0 1mol LPBS清洗8次后加入2 %DMEM培养液。收集PBS第8次洗液,收集PBS洗后每隔12h各孔细胞培养上清。ELISA检测细胞培养上清中的HBsAg。PCR检测细胞培养上清和HepG2 细胞中的HBVDNA。结果　感染组在PBS洗后12h的细胞培养上清中ELISA检测HBsAg呈阳性。PCR检测显示感染组细胞培养上清和HepG2 细胞中HBVDNA呈阳性,阴性对照组和空白对照组HBVDNA呈阴性。结论　HBV阳性血清进行HBV感染体外培养HepG2 细胞是可行的。     

HBV体外感染卵巢颗粒细胞

目的建立HBV体外感染颗粒细胞模型,研究HBV在颗粒细胞中的复制情况,为深入研究HBV经卵细胞母婴垂直传播提供研究平台。方法原代颗粒细胞体外培养后用HBV阳性血清感染。收集培养上清,在不同时点检测HBsAg、HBeAg定量,实时定量PCR检测HBVDNA。免疫组化检测培养细胞中的HBsAg和HBcAg。巢式PCR检测细胞中的HBVDNA及HBV-mRNA。原位杂交检测细胞内的HBVDNA。结果成功建立了HBV体外感染颗粒细胞模型,在培养上清中可以持续96h检测到HBsAg和HBV DNA,在细胞内检测到HBsAg和HBcAg的阳性信号,PCR扩增显示细胞内有HBVD-NA及HBV-mRNA的存在,原位杂交证实细胞内HBVDNA阳性。结论 HBV能够在体外感染颗粒细胞,并在其内复制,该结果为深入研究HBV经卵细胞传播机制提供了很好的研究平台。     

胎盘组织TLR9分布与表达及与HBV宫内感染的关系

目的了解TLR9在外周血HBsAg阳性母亲胎盘组织中的表达及分布,探讨其与HBV宫内感染的关系。方法Abbott化学发光法检测新生儿外周血HBsAg;ELISA法检测母亲外周血HBV标志物;SABC免疫组织化学方法检测胎盘组织HBsAg和TLR9的表达和分布。结果胎盘HBV感染率77.27%(17/22),HBsAg分布于蜕膜细胞、滋养层细胞、间质细胞、绒毛毛细血管内皮细胞。TLR9在外周血HBsAg阴性母亲胎盘滋养层细胞表达,在外周血HBsAg阳性母亲胎盘滋养层细胞、间质细胞、绒毛毛细血管内皮细胞均有表达。外周血HBsAg阳性组比阴性组胎盘组织TLR9表达强,差异有统计学意义(P<0.05);而外周血HBsAg阳性母亲的胎盘,未感染HBV者比感染HBV者TLR9表达强,差异有统计学意义(P<0.05)。结论初步认为,胎盘组织TLR9表达与HBV宫内感染有一定关系,TLR9可能是HBV宫内感染的保护因素之一。     

HBV感染的CD68细胞在隐匿性HBV感染产妇胎盘中的分布及与宫内感染的关系

目的探讨HBV感染的外周血单个核细胞(peripheral blood mononuclear cells,PBMC)在乙肝宫内传播中的作用及机理。方法12例血清HBV DNA(-)、PBMC HBV DNA(+)产妇分娩的新生儿血清HBV DNA(+)和/或PBMC HBV DNA(+)的胎盘作为实验组,10例乙肝标志物均为阴性产妇的胎盘作为对照。采用SP法在连续切片上检测HBsAg和HBcAg在胎盘CD68细胞及各类细胞中的表达。结果8例新生儿血清HBV DNA(-)、PBMCsHBV DNA(+)胎盘绒毛间质5例CD68细胞HBsAg阳性,6例CD68细胞HBcAg阳性;毛细血管内5例CD68细胞HBsAg阳性,8例CD68细胞HBcAg阳性;滋养层细胞和血管内皮细胞均未见HBsAg、HBcAg阳性信号;2例新生儿血清HBV DNA(+)、PBMCs HBV DNA(-)的胎盘滋养层细胞、绒毛间质、毛细血管内皮细胞均有HBsAg、HBcAg的表达,而绒毛毛细血管内CD68细胞未见表达。2例新生儿血清和PBMC HBV DNA均阳性的的胎盘滋养层细胞、绒毛间质、CD68细胞和毛细血管内CD68细胞均有HBsAg、HBcAg的表达,毛细血管内皮细胞无表达。10例乙肝标志物全阴性产妇胎盘中均无阳性信号。结论HBV感染的外周血单个核细胞可作为宫内传播的载体。     

新型CpGODN对乙型肝炎病毒复制的抑制作用

目的:探讨我室自行设计的CpG ODN(CpG BW001)在体外对乙型肝炎病毒(HBV)复制的抑制作用.方法:CpG BW001刺激人外周血单个核细胞(PBMC)48小时,用VSV病毒保护实验及ELISA法检测培养上清的病毒保护能力及其中的IFN-α含量.将上述CpG BW001刺激人PBMC的上清与HepG2.2.15细胞共孵育12天后收集培养上清,用放射免疫法检测该培养上清液中HBsAg及HBeAg的含量,用点杂交法检测HBV DNA的含量.结果:CpG BW001刺激人PBMC产生以IFN-α为主的抗病毒物质;CpG BW001刺激人PBMC的培养上清作用于HepG2.2.15细胞后,HepG2.2.15细胞培养上清中HBsAg和HBeAg的含量明显减少,且细胞内HBV DNA的含量也显著降低.结论:CpG BW001能通过刺激细胞产生抗病毒物质如IFN-α等,从而在体外抑制HBsAg和HBeAg的表达及HBV的复制.     

人类乙肝病毒体外感染树鼩肝原代细胞的研究

目的 为进一步研究人类乙肝病毒(HBV)提供接近自然感染状态的理想细胞模型.方法 体外二步灌流法分离树鼩原代肝细胞,纯化后的乙肝患者血清感染上述肝细胞,Southern blot和Noahem blot检测感染后细胞内的DNA和RNA,ELISA方法 检测细胞上清的HBsAg,免疫组化检测细胞内HBsAg的表达.结果 可检测出肝细胞内cccDNA(共价闭合环状DNA)、pgRNA(前基因组RNA)和sgRNA(亚基因组mRNA),感染后第7天,信号开始增强,持续到实验结束的第14天.细胞上清中HBsAg自第1天到第5天S/CO值逐渐下降,随后S/CO值逐渐升高.结论 HBV可在原代树鼩肝细胞中复制和表达.     

HBsAg携带产妇初乳FQ-PCR检测HBV DNA结果与分析

目的了解HBsAg携带产妇初乳HBV DNA的含量.方法对94例HBsAg携带产妇初乳同时进行ELISA方法测定HBV-M和FQ-DNA方法检测HBV DNA.结果28例初乳中HBsAg阳性组中19例HBVDNA＞1.0×10拷贝/ml,阳性率为67.86%(19/28);其中"大三阳"组为82.35%(14/17);"小三阳"组为37.5%(3/8).在定量方面,初乳中"大三阳"的DNA含量与血清相比,相对较低.在103-105拷贝/ml之间,平均2.92×104拷贝/ml.同期随机留取的20份HBsAg阴性产妇初乳作为对照,结果均HBVDNA＜1.0×103拷贝/ml.结论在HBsAg携带产妇的初乳存在有HBV,含有HBV的初乳是一传染因子,有可能会将HBV传播给乳儿.     

HBV adr亚型体外感染肝细胞模型的建立

目的:构建1.1倍乙型肝炎病毒(HBV)全基因的真核表达载体,稳定转染L-02细胞,建立HBVadr亚型体外感染的细胞模型。方法:以pVUⅡ酶切质粒p3.6Ⅱ获得1.1倍HBV DNA片段,用牛小肠碱性磷酸酶将经EcoRV线性化的pcDNA3去磷酸化,以T4DNA连接酶连接1.1倍HBV DNA片段和线性化的pcDNA3,将构建的pcDNA3-1.1HBV以脂质体转染L-02肝癌细胞,经G418稳定筛选。ELISA检测转染细胞培养上清中HB-sAg、HBeAg的表达。RT-PCR检测转染细胞中HBpreS2、HBX的表达。结果:成功构建了1.1倍HBV全基因真核表达载体,稳定转染L-02后,建立了新的肝细胞系L-02.1Z,其培养上清中可检测到HBsAg、HBeAg的稳定表达,并可检测到HBpreS2、HBX RNA在转染细胞中表达。结论:该表达载体可介导病毒复制,其稳定转染的细胞可作为一种新型的HBV体外感染模型。     

CpG-ODN对慢性乙型肝炎患者PBMC免疫刺激作用的观察

目的 观察CpG-ODN体外对慢性乙型肝炎患者PBMC的免疫刺激效应.方法 CpG-ODN体外刺激慢性乙肝患者外周血单个核细胞(PBMC),用ELISA检测培养液中IFN-α的水平;将不同比例的PBMC培养上清与HepG2.2.15细胞共孵育4和8 d后,用ELISA和荧光定量PCR法,分别检测培养上清液中HBsAg、HBeAg和HBV DNA的水平;MTT法观察活化的PBMC培养上清液对HepG2.2.15的杀伤作用.结果 CpG-ODN可有效诱导慢性乙肝患者PBMC分泌IFN-α,增强其活化的PBMC培养上清液对HepG2.2.15的杀伤作用;CpG-ODN本身虽不能直接抑制HBV的复制,但可通过其活化的PBMC的培养上清抑制HepG2.2.15细胞产生HBsAg、HBeAg和HBV DNA.结论 CpG-ODN可有效激活慢性乙肝患者的免疫细胞,发挥抗病毒效应.     

DC-SIGNR在人胎盘滋养层细胞中的表达及意义

目的:探讨DC-SIGNR在人胎盘绒毛组织中的定位及在体外培养滋养层细胞上的表达.方法:建立稳定的滋养层细胞原代培养体系,采用免疫组化单染及荧光双染的方法检测不同孕期正常胎盘绒毛组织及体外培养滋养层细胞上DC-SIGNR的表达.结果:DC-SIGNR主要表达于胎盘滋养层细胞、Hofbauer细胞及胎盘微血管内皮细胞的胞质及包膜,在原代培养的人绒毛膜滋养层细胞中的DC-SIGNR的表达与在体组织表达一致.结论:DC-SIGNR表达于不同孕期的胎盘组织及体外分离培养滋养层细胞,为研究滋养层细胞上该受体在宫内感染中的作用提供体外实验的细胞学基础.     

Hepatitis B virus infection and replication in primary cultured human granulosa cells

To investigate the infection and replication of hepatitis B virus (HBV) in primary cultured human granulosa cells. Human granulosa cells were cultured with HBV-positive serum. Media were collected and assayed for HBsAg and HBeAg by ELISA, and HBV DNA by quantitative PCR. HBsAg and HBcAg were detected by immunocytochemistry in cultured cells. HBV DNA and RNA were extracted and amplified by nested PCR. Intracellular HBV DNA was localized by in situ hybridization. By co-cultivation of human GCs with HBV-positive serum, a system was established to study HBV infection and replication in GCs. HBsAg in medium could be detected from 4 to 96 h, and HBV DNA could be detected from 12 to 96 h after exposure. HBsAg and HBcAg showed positive signals by immunocytochemistry. A 206-bp fragment was amplified by nested PCR to detect HBV DNA and RNA in granulosa cells. HBV DNA was detected in GC nuclei by in situ hybridization. HBV can infect and replicate in human primary granulosa cells. This culture system could enable us to study infection of ova by HBV.     

奉贤地区HBsAg阴性的HBV自然感染母亲对新生儿影响的研究

目的　为揭示HBsAg阴性的乙型肝炎病毒 (HBV)自然感染孕妇的宫内感染及其危险因素。方法　采用多聚酶链反应 (PCR)技术结合酶联免疫吸附法 (ELISA) ,对奉贤地区 131例HBsAg阴性的HBV自然感染孕妇外周血 ,及其分娩后的脐带血进行HBV血清学标志物 (HBVM)和HBVDNA检测。结果　HBsAg阴性的HBV自然感染孕妇宫内的感染率 (除外单一抗 -HBs阳性 )为 5 2 6 7% ;脐血中不同HBVM组合的HBVDNA检出率依次为 :抗 -HBe( )、抗 -HBc( ) >抗 -HBs( )、抗 -HBe( )、抗 -HBc( ) >抗 -HBs( )、抗 -HBe( ) >抗 -HBs( )、抗 -HBc( ) >抗 -HBs( ) ;脐血HBVDNA总检出率为 16 79%。结论　HBsAg阴性的HBV自然感染孕妇也可能发生宫内感染。提议HBsAg阴性的HBV自然感染孕妇和新生儿有进行自动和被动免疫接种的必要性     

Hepatitis B virus (HBV) infections in turtles

Thirty turtles (15 Clemys mutica and 15 Geoclemys reevesii) which were inoculated with human sera those were positive for hepatitis B surface antigen (HBsAg) and hepatitis B "e" antigen (HBeAg) were found to be infected with hepatitis B virus (HBV). The levels of HBV infection markers, such as HBsAg and antibody to HBsAg (anti-HBsAg), were retinely monitored in the turtles' serum for 46 weeks. Within two weeks of the inoculation, 42% of the turtles tested were positive for HBsAg, and their reciprocal titers as measured by reverse passive hemagglutination (RPHA) and enzyme linked immunoabsorbance assay (ELISA) ranged from 16 to 96. Within 20 weeks, the remaining turtles tested HBsAg positive, as confirmed by ELISA. At 20 weeks, all but one of the turtles exhibited changes in HBV blood marker from HBsAg to anti-HBs; the one exception was positive for both HBsAg and anti-HBs. At the 47th week, 7 animals were killed and their organs were examined for HBV infected cells utilizing an immunofluorescent technique. Numerous fluorescent cells which reacted with human anti-HBs nad anti-HBc were observed in the following organs: pancreas, liver, kidney, and brain. Histopathologically, edematous changes in hepatocytes and minor cellular infiltration attributed to an inflammatory response were noted. Liver and kidney cells from the infected animals were cultured, and HBV antigen positive cells for HBsAg and HBcAg were detected in the cultures. Throughout the experiment, HBsAg was detected in the supernatant by ELISA. Virus particles which were indistinguishable from Dane particles were seen in the cytoplasmic vacuoles of the cultured cells by electron microscopy. Finally, the presence of HBV DNA was established by molecular hybridization techniques in the culture supernatants of kidney cells from the infected turtles.     

Cellular chromosome DNA interferes with fluorescence quantitative real-time PCR detection of HBV DNA in culture medium

Fluorescence quantitative real-time PCR (FQ-PCR) is a recently developed technique increasingly used for clinical diagnosis by detection of hepatitis B virus (HBV) DNA in serum. FQ-PCR is also used in scientific research for detection of HBV DNA in cell culture. Understanding potential FQ-PCR interference factors can improve the accuracy of HBV DNA quantification in cell culture medium. HBV positive serum was diluted with culture medium to produce three test groups with HBV DNA levels of 5 x 10(7) copies/ml (high), 5 x 10(5) copies/ml (medium), and 5 x 10(3) copies/ml (low). Chromosome DNA was extracted from HepG2 cells and then added to high, medium, and low group samples at final concentrations of 0, 12.5, 25, 50, and 100 microg/ml. The samples were quantified by FQ-PCR and data were evaluated using statistical software. No marked changes were seen in the quantitative curves for high level HBV DNA samples when the samples were supplemented with 0-100 microg/ml of chromosome DNA. Interference was observed in medium level samples when 50 and 100 microg/ml of chromosome DNA was added. Interference was also observed in low level HBV DNA samples when the concentration of added chromosome DNA was greater than 25 microg/ml. The interference was eliminated when samples were digested by DNase I prior to PCR detection. In Conclusions, the presence of cellular chromosome DNA can interfere with the detection of HBV DNA by FQ-PCR. Removal of cellular chromosome DNA from culture media prior to FQ-PCR is necessary for reliable HBV DNA quantitative detection.     

Different forms of hepatitis B virus DNA and expression of HBV antigens in peripheral blood mononuclear cells in chronic hepatitis B

The presence of both hepatitis B virus (HBV) DNA and HBV antigens (HBsAg, HBeAg) was assessed in peripheral blood mononuclear cells (PBMC) from 32 patients chronically infected with HBV. Three different molecular forms of HBV DNA were observed: free monomers (5), high-molecular-weight free concatemers (11), and integrated HBV DNA (9). The HBV DNA patterns in the PBMC were different from those found in liver and did not correlate with any specific profile of serum HBV markers. When the same PBMC were assayed for HBsAg, 22 of the 25 HBV DNA positive samples, but only three of the seven HBV DNA negative samples, were positive. By contrast, none of the PBMC samples from five healthy HBV vaccine recipients gave any positive signal in the HBV DNA or HBsAg assays. In some patients, T and B cells, monocytes, and polymorphonuclear (PMN) cells were assayed separately, showing that the DNA pattern was similar for these different leucocytes subsets and ruling out the possibility that these patterns might reflect PMN cell contamination. Thus, in chronic HBV infection, 87.5% (28/32) of patients were found to contain at least one HBV marker in their PBMC, and a strong correlation was found between the presence of HBV DNA and viral antigens, suggesting a specific expression of HBV encoded proteins.     

In vitro infection of primary cultures of cryopreserved adult human hepatocytes with hepatitis B virus

Infection with HBeAg and HBV DNA positive serum in primary cultures of cryopreserved human hepatocytes in the presence of human whole blood in the medium was performed in 8 consecutive experiments. HBsAg and HBV DNA release into the medium was increased in the second week after infection. Via immunostaining, HBcAg was first observed in the nucleus of hepatocytes approximately 3 days after infection. A maximal percentage of HBcAg positive cells in 0.1% of cultured hepatocytes was detected on the 7th day. HBsAg was also first demonstrated on the 3rd day, and predominantly localized in the cytoplasm. About 5% of hepatocytes were HBsAg positive on the 12th day after infection. The percentage of positive cells did not appear to increase after this time. Using in situ cytohybridization and agarose gel electrophoresis and Southern blot analysis, HBV DNA was first detected on the 4th day. In addition, electron microscopic studies revealed the presence of 42 nm virus-like particles in the cytoplasm of infected cells in the second week after infection. This in vitro system provides a model for studying the mechanism of HBV infection, viral replication and maturation. However, further improvement of culture systems is needed, to increase the number of infected cells and for active HBV replication.     

乙肝免疫球蛋白不能阻断乙肝病毒感染人绒毛膜癌细胞

目的: 观察不同浓度乙肝免疫球蛋白(HBIG)在体外对乙肝病毒(HBV)感染人绒毛膜癌细胞系JAR细胞的影响。方法: 实验分为HBIG实验组、空白对照组(胎牛血清)、阴性对照组(健康人血清)。HBIG实验组又分为高浓度HBIG组(HBV+10³IU/L HBIG、HBV+10² IU/L HBIG)、最低保护浓度HBIG组(HBV+10 IU/L HBIG)、低浓度HBIG组(HBV+1IU/L、HBV+0.1 IU/L)和HBV组;采用MTT法检测HBIG对细胞生长的影响;采用实时荧光定量PCR检测细胞内HBV-DNA的表达;采用细胞免疫荧光染色法检测细胞内乙肝表面抗原(HBsAg)的表达变化。结果: (1) 不同浓度的HBIG对JAR细胞生长无影响;(2) 不同浓度HBIG组的细胞内HBsAg的荧光强度无显著差异(P>0.05);(3)不同浓度HBIG组的细胞内HBV-DNA无显著差异(P> 0.05)。结论: HBIG不能阻断HBV感染体外培养的JAR细胞。     

CpG-ODN体外抑制乙型肝炎病毒复制的研究

目的:探讨CpG-ODN体外对HBV复制和HBV抗原表达的抑制作用。方法:CpG-ODN体外刺激人外周血单个核细胞(PBMC),用ELISA检测培养液中IFN-α及IFN-γ的水平。将不同比例的PBMC培养上清与HepG2. 2. 15细胞共孵育 2、4、6、8d后,用ELISA和荧光定量PCR法,分别检测培养上清液中HB sAg、HBeAg和HBVDNA的水平。结果:CpG-ODN可诱导PB MC分泌IFN-α和IFN-γ。CpG-ODN本身虽不能直接抑制HBV的复制,但经刺激活的化的PBMC的培养上清,却能显著抑制 2. 2. 15细胞产生HBsAg、HBeAg和HBVDNA,在培养的第 8天,抑制率最高分别达 90. 8%、31. 3%和 32. 2%。结论:CpG-ODN作为一种新型的免疫调节剂,可诱发机体的免疫效应,抑制HBV复制和HBV抗原的表达。