乙型肝炎病毒共价闭合环状DNA套式-实时荧光定量聚合酶链反应的建立

目的 建立检测HBV共价闭合环状DNA(cccDNA)的套式一实时荧光定量PCR法.方法 根据HBV cccDNA与松环DNA(rcDNA)结构上的差异,设计2对跨缺口的特异引物及1条位于负链缺口下游的特异TaqMan荧光探针.根据Plasmid-SafeTM ATP-Dependent Dnasc(PSAD)对rcDNA与cccDNA作用的不同,对模板DNA进行酶切纯化,降解reDNA,再进行套式PCR扩增,先用外引物和模板进行第一轮常规PCR,再用内引物、荧光探针和第一轮PCR产物进行实时荧光定量PCR,根据阳性参照标准品,得出待检标本定量值.结果 检测阳性参照标准品.得出该方法灵敏度可达2 lg拷贝/mL.用上述方法检测34份乙型肝炎患者血清HBV DNA阳性标本,25份血清HBVcccDNA阳性,28份外周血单个核细胞HBV cccDNA阳性.27份健康对照者血清HBV DNA阴性标本,6份HBV cccDNA阳性.对5份HBV cccDNA阳性标本扩增产物进行克隆测序,无碱基缺失、突变.与HBV不同基因型序列(A～G)比较,同源性为90.6%～99.1%,其中,与B、C基因型同源性为95.3%～99.1%,验证了方法的特异度.结论 套式-实时定量PCR法可检测乙型肝炎患者血清、PBMC中的HBV CCCDNA,且具有敏感、特异性.     

A～D基因型乙型肝炎病毒全长逆转录酶区PCR方法的建立及应用

目的 建立适用于扩增我国常见的乙型肝炎病毒(HBV)A～D基因型全长逆转录酶(RT)区(包含全长HBsAg编码区)的聚合酶链反应(PCR)法,并确定其在分析临床标本中的应用价值.方法 建立我国HBV基因序列库,设计适用于扩增A～D基因型HBV全长RT区引物,以A～D基因型HBV重组质粒为模板建立半巢式PCR(snPCR)法,并确定该法灵敏度.用所建立的snPCR对44份HBV DNA定量阳性(>5.0×102 拷贝/ml)慢性乙型肝炎患者血清标本进行扩增及PCR产物直接测序.结果 琼脂糖凝胶电泳及测序证实,snPCR可扩增A～D基因型HBV全长RT区,对A、B、C和D基因型HBV质粒扩增的灵敏度分别为1.2×103、7.0×102、6.0×102和6.0×102 拷贝/ml.snPCR检测HBV DNA >5×102 拷贝/ml血清标本的阳性率为88.64% (39/44),扩增阴性血清标本HBV DNA滴度均处于103 拷贝/ml水平.扩增目的 产物经直接测序确证无误.生物信息学分析显示,样品中B和C基因型分别占35.90%(14/39)和64.10%(25/39);其中15.38%(6/39)发生RT区变异,包括4例为已知耐药变异;7例(17.95%)存在HBsAg编码区变异,其中2例为已知免疫逃逸变异;6例(15.38%)发生RT区和HBsAg编码区"镜像改变".结论 建立了一种新的扩增全长RT区(涵盖全长HBsAg编码区)的snPCR.该法结合直接测序法,可同时分析我国HBV A～D基因型已知和潜在的耐药变异位点.     

新生期树鼩接种人乙型肝炎病毒的长期实验观察

目的 观察新生期树鼩接种HBV后体内HBV感染标志物的长期动态.方法 6只树鼩于新生期接种人HBV DNA阳性血清,每4-6周抽血1次和每6～12周做肝活体组织检查1次,应用巢式聚合酶链反应(nPCR)、荧光定量聚合酶链反应(FQ-PCR)、Southern blot、酶联免疫吸附试验和免疫组织化学染色等方法动态观察血清和肝组织中HBV感染标志物的消长,用电镜寻找肝组织内的HBV颗粒和用光镜观察肝组织病理变化.结果 新生期树鼩接种后48周,3只动物(1、2和6号)血清和肝组织标本经多对引物进行的nPCR,均稳定显示HBV DNA阳性,肝组织HBVcccDNA阳性;FQ-PCR显示血清和肝组织HBV DNA的拷贝数分别为103-104/ml和每微克肝组织总DNA 107～108拷贝;Southern blot检测显示肝组织存在HBV复制中间体HBV cccDNA和HBV单链DNA;酶联免疫吸附试验检测显示血清HBsAg持续阳性;免疫组织化学染色可见数量逐步增多的HBsAg阳性肝细胞.其中的1号动物至接种后2年每微克肝组织总DNA仍可测得107～108拷贝的HBV DNA,电镜下可见疑似HBV颗粒.另3只动物除nPCR显示肝组织HBV DNA阳性条带和FQ-PCR显示低拷贝数(每微克肝组织总DNA103拷贝)HBV DNA外,其余的HBV感染标志物均为阴性或一过性阳性.结论 新生树鼩能够长期感染HBV,并且HBV能够在树鼩体内稳定复制和长期存在.     

乙型肝炎病毒表面抗原基因的克隆及序列分析

目的克隆并鉴定乙型肝炎病毒表面抗原基因,为下一步构建该重组腺病毒载体以及进一步研究腺病毒载体的包装及在乙型肝炎病毒基因治疗中的作用。方法参照人 HBV adr 亚型序列,设计和合成 S 基因特异引物。应用 PCR 技术,从含有 HBsAg 的 HBV DNA 中扩增目的 DNA,通过 TA 连接将其克隆人 pGEM-T easy 载体,经限制性内切酶 BglⅡ/SalⅠ鉴定后,进一步测序鉴定。结果从乙肝表面抗原阳性(HBsAg )血清中成功提取 HBV DNA,并以此 DNA 为模板,成功扩增出 S 基因,测序结果与 GenBank 中注册的相应序列比对,核苷酸序列的同源性高达92％～99％,预测氨基酸序列同源性亦达82％。结论从 HBsAg 阳性血清中成功克隆出 S 基因序列,为进一步构建重组腺病毒及后续实验奠定了基础。     

乙型肝炎病毒cccDNA巢式-实时荧光定量PCR法的建立及应用

目的建立检测HBV共价闭合环状（cccDNA）的巢式-荧光定量PCR法,检测外周血单核细胞（PBMC）及骨髓单核细胞（MMNC）中cccDNA。方法根据HBV cccDNA与松弛结构DNA（rcDNA）结构上的差异,设计2对跨缺口的特异性引物及下游的特异性TaqMan探针。根据Mung Bean Nuclease对rcDNA与cccDNA作用的不同,使cccDNA扩增而使rcDNA降解,分别用外引物及内引物进行PCR反应,再用荧光探针进行实时荧光定量PCR,根据阳性参照物,计算出检测标本定量值。结果成功建立了HBV cccDNA巢式-荧光定量PCR的检测方法,线性范围为5.0×102～3.9×107拷贝/ml。用上述方法检测25例慢性乙型肝炎（CHB）及肝硬化血清HBV DNA阳性患者PBMC中cccDNA,7例MMNC中cccDNA,21例健康献血者PBMC cccDNA,骨髓标本中有3例cccDNA阳性,25例外周血标本中有9例cccDNA阳性。结论巢式-荧光定量PCR法可检测乙型肝炎患者PBMC及MMNC中的HBV cccDNA含量。PBMC及MMNC中可检测到HBV cccDNA。     

联合检测HBV前S1抗原和核心抗原的临床意义

探讨联合检测血清HBV前s1抗原（preSl）和核心抗原（HBcAg）（均为HBV核酸相关抗原，nucleic acids related antigen，HBV NRAg）的意义及临床价值。方法：采用ELISA法对393份HBsAg、HBV DNA双阳性的血清和612份HBsAg阴性血清进行HBV NRAg检测，所有标本均采用多区段巢式PCR确认阳性、阴性，采用荧光定量PCR法进行HBV DNA定量分析。结果：393份HBsAg、HBV DNA双阳性血清中，HBV NRAg阳性为382份，其阳性率为97．2％；612份HBsAg阴性的血清标本中，609份确认为HBV DNA阴性，其中检出2份HBV NRAg阳性，607份为阴性，其HBV NRAg的阴性率为99．7％（607／609），另3份HBsAg阴性血清HBV DNA阳性者，其HBV NRAg均为阳性。结论：联合检测preSl和HBcAg的HBV NRAg可作为临床HBV感染的筛选及判断HBV复制的有意义的补充项目。     

HBsAb阳性隐匿性HBV感染者血清、肝组织HBV全基因序列分析

目的 了解HBsAb阳性隐匿性HBV感染者血清和肝组织中的HBV基因序列,并比较其差异性.方法 以1例长期随访HBsAb阳性隐匿性HBV感染者作为研究对象,用多种试剂盒检测其血清HBsAg、HBsAb,提取外周血血清和肝组织HBV DNA进行全基因组分段扩增,行序列测定及同源性比较.结果 多种试剂盒检测均提示该例患者HBsAg阴性、HBsAb阳性;血清HBV DNA为103～ 105拷贝/mL;血清和肝组织来源的HBV DNA全基因测序完全相同,均为3 215个碱基、B基因型,与参照序列核苷酸同源性为98.82％,各编码区均没有缺失或移码突变,不同编码区的核苷酸序列同源性为98.37％～ 100％,氨基酸序列同源性为98.18％～ 100％,在S区存在几种变异如PreS1的Q80H、S的C64Y、E164G、L175S,但前S区、“a”决定簇、1 762/1 764、1 896位点均未见变异.结论 HBsAb阳性隐匿性HBV感染者血清和肝组织来源的HBV基因序列无明显差异.     

定量PCR检测血清中乙型肝炎病毒DNA及其意义

目的检测血清中乙型肝炎病毒DNA拷贝数,并了解HBV感染的不同血清学指标组合相应的HBV DNA含量分布,以指导临床。方法 采用定量PCR和定性PCR方法,检测216份不同临床类型血清标本的HBV DNA,再用ELISA方法测定HBV-M,统计不同免疫指标组合的HBV DNA平均含量。结果病毒量分为高、中、低三度,大于107拷贝mL-1为高滴度:107-105拷贝mL-1为中等滴度;105拷贝mL-1以下为低滴度。60例HBsAg(+)HBeAg(+)HBcAb(+)血清,HBV DNA全部阳性,平均含量为1.9×108拷贝mL-1。51例HBsAg(+)HBeAb(+)HBcAb(+)血清,HBV DNA平均含量为5.4×106拷贝mL-1;33例HBsAg(+)HBeAb(+)血清,HBV DNA平均含量为7.5×105拷贝mL-1;114例HBsAb(+)HBeAb(+)HBeAb(+)血清,HBV DNA平均含量为1.8×105拷贝mL-1。定性和定量PCR阳生率分别为59.3％和61.6％。两种方法相对符合率为94.5％。结论定量PCR可真实反应HBV感染、复制及病程变化,对乙型肝炎临床诊断及治疗均有较大的指导意义。     

乙肝病毒标志物与微观辨证

结合本人经验和近年的文献报道,拟出慢乙肝患者各证型的 HBVM 微观辨证标准,以供临床参考。1.肝郁脾虚型:血清 HBsAg 阳性,滴度较低;HBeAg 或 HBV DNA 阳性,亦可阴性;肝组织内 HBVDNA 阳性率较低。2.肝肾阴虚型:血清 HBsAg 阳性,滴度较高;HBeAg 或 HBV DNA 的阳性率高,滴度较高;肝组织内 HBsAg 及 HBcAg 阳性率较高。3.湿热留恋型:血清 HBsAg 阳性,滴度最高;HBeAg 及 HBV DNA 多数阳性,且滴度高;本型患者     

抗-HBc阳性患者中的隐匿性HBV感染的调查研究

<正>隐匿性乙型肝炎是指血清HBsA g为阴性,而血清和肝脏HBV DNA仍有低水平(200 IU/ml)复制。来自新南威尔士大学的Martinez MC等描述了澳大利亚高危人群中隐匿性HBV感染的发生率,同时比较了研究对象和对照组HBV S基因序列。在SEALS血清实验室应用雅培试剂检测HBV DNA阳性、HBsA g阴性、抗-HBc阳性患者的血清标本。共回顾分析228 108个样本,1451例患者标本测试了隐匿性HBV感染的3个血清学指标,其中仅有10例患者(0.69%)符合隐匿性HBV感染的标准。对5个可疑隐匿性HBV感染者HBV S基因测序,与对照序列相比,发现主要亲水区域"a"表位变异。另外,共8个连续核苷酸转换(替换)导致7个氨基酸序列     

The immunoneuroendocrine role of melatonin

Abstract: A tight, physiological link between the pineal gland and the immune system is emerging from a series of experimental studies. This link might reflect the evolutionary connection between self-recognition and reproduction. Pinealectomy or other experimental methods which inhibit melatonin synthesis and secretion induce a state of immunodepression which is counteracted by melatonin. In general, melatonin seems to have an immunoenhancing effect that is particularly apparent in immunodepressive states. The negative effect of acute stress or immunosuppressive pharmacological treatments on various immune parameters are counteracted by melatonin. It seems important to note that one of the main targets of melatonin is the thymus, i.e., the central organ of the immune system. The clinical use of melatonin as an immunotherapeutic agent seems promising in primary and secondary immunodeficiencies as well as in cancer immunotherapy. The immunoenhancing action of melatonin seems to be mediated by T-helper cell-derived opioid peptides as well as by lymphokines and, perhaps, by pituitary hormones. Melatonin-induced-immuno-opioids (MHO) and lymphokines imply the presence of specific binding sites or melatonin receptors on cells of the immune system. On the other hand, lymphokines such as -γ-interferon and interleukin-2 as well as thymic hormones can modulate the synthesis of melatonin in the pineal gland. The pineal gland might thus be viewed as the crux of a sophisticated immunoneuroendocrine network which functions as an unconscious, diffuse sensory organ.     

Response of rat pineal melatonin to calcium, magnesium, and lithium is circadian stage dependent

Abstract: Herein we documented the response of pineal melatonin production to electrolytes known to be effective on pineal function in view of a possible circadian stage dependence. We studied the release of melatonin by perifused rat pineal glands at 2 different circadian stages corresponding to the middle of the light and dark periods, i.e., respectively, 7 and 19 HALO (Hours After Light Onset, L:D = 12:12). The initial efflux rates were, as expected, much higher in the perifusates of glands removed from rats sacrificed during the dark phase than of those removed during the light phase. After 3 hr of perifusion, melatonin release reached similar levels which were found constant up to the 8th hr of perifusion, whatever the circadian stage. Perifusion of the glands with physiological concentrations for the rat of calcium (5.2 mmol/1) and magnesium (1.34 mmol/1) resulted in a stimulatory effect on the pineal glands removed from rats sacrificed in the middle of the dark period (19 HALO), whereas no effects were observed on the pineal glands removed from rats sacrificed during the light (7 HALO). Lithium (0.28 and 0.55 mmol/1) was ineffective on melatonin release in pineal glands removed 7 and 19 HALO. Our results show differences in the initial efflux rates of melatonin and in the response of perifused pineal glands to calcium and magnesium according to the circadian stage.     

Changes in connexin43, the gap junction protein of astrocytes, during development of the rat pineal gland

Abstract: The abundance of gap junctions between rat pineal astrocytes formed by connexin43 (Cx43) was studied during development. Levels and distribution of Cx43 were measured by immunoblotting and indirect immunofluorescence, respectively. The amount of Cx43 in cells located within the gland was low until about the 7th postnatal day and increased to adult values between the 14th and 21st days postpartum. Although astrocytes, recognized by their vimentin immunoreactivity, were scarce before birth, they were abundant by the 7th postnatal day suggesting that the low levels of Cx43 found at this age corresponded to a low expression of this protein. Localization of the immunoreactivity to Cx43 and vimentin showed a close correlation, indicating that mature or immature pineal astrocytes form gap junctions made of Cx43. Since Cx43 levels attained their adult values at about the time the innervation and the functional state of the gland reached maturity (2–3 weeks after birth), it is proposed that astrocyte gap junctions are involved in the function of the adult rat pineal gland.     

Conservative management of perforated duodenal diverticulum： A case report and review of the literature

Duodenal diverticula are a relatively common condition. They are asymptomatic, unless they become complicated, with perforation being the rarest but most severe complication. Surgical treatment is the most frequently performed approach. We report the case of a patient with a perforated duodenal diverticulum, which was diagnosed early and treated conservatively with antibiotics and percutaneous drainage of secondary retroperitoneal abscesses. We suggest this method could be an acceptable option for the management of similar cases, provided that the patient is in good general condition and without septic signs.     

Presence of immunoreactive growth hormone and prolactin in the ovine pineal gland

Abstract: The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M_r range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [¹²⁵I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (K_a of 4.7 ± 0.2 × 10⁹ M^-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M_r 24,000. The functional status of PRL-and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.     

Microbiology of human immunodeficiency virus anorectal disease

PURPOSE: Individuals who are seropositive for the human immunodeficiency virus are at high risk for opportunistic infection and anorectal disorders. Little prospective information is available regarding anorectal pathogens in these patients. METHODS: One hundred sixty-three HIV-seropositive patients presented to the colorectal clinic between 1989 and 1992. Forty-seven (29 percent) patients were thought to have an infectious process and were prospectively studied using a standardized multiculture protocol. RESULTS: Mean age was 33 (range, 19–59) years. All were male; high-risk behavior accounted for 87 percent of HIV transmissions. Presenting complaints included anorectal pain (79 percent), pus per anum (28 percent), and blood per anum (26 percent). Examination revealed perianal tenderness (60 percent), condyloma (38 percent), perianal ulcers (38 percent), and anal fissures (34 percent). Sixty-six sets of cultures were performed; 28 patients had one set, 15 had two sets, and 4 had three sets. Thirty-two of these 47 patients (68 percent) had positive cultures including herpes (50 percent), cytomegalovirus (25 percent),Neisseria gonorrhoeae (16 percent), chlamydia (16 percent), acidfast bacilli (2 percent), and others (9 percent). Six of 32 patients with positive cultures had more than one organism cultured. Sixteen (50 percent) patients with positive cultures were treated medically, 8 (25 percent) were treated surgically and 8 (25 percent) were treated with both modalities. Sixty-one procedures were performed on 17 patients for condylomata. Eighteen patients had 20 procedures for abscesses, 50 percent of whom had positive cultures for other than common bowel flora; all improved. Fourteen patients underwent 33 procedures for perianal fistulas.Mycobacterium fortuitum was cultured from one patient who required 13 procedures for abscesses and fistulas. Forty-five (96 percent) patients were followed for an average of 12.5 months ±2.9 SEM (range, 1–94 months). Symptoms were improved or resolved in 22 of 32 (69 percent) patients with positive cultures and in 11 of 13 (84 percent) with negative cultures. CONCLUSIONS: Specific pathogens may often be identified in human immunodeficiency virus-seropositive patients with anorectal disorders if aggressively sought. Although patients without specific pathogens identified may be expected to improve with planned empiric treatment, positive identification allows more directed therapy.