拉米夫定耐药株感染者HBV基因型和逆转录酶区变异分析

目的了解拉米夫定耐药株感染者HBV基因型特征并分析耐药株HBV逆转录酶（RT）区变异位点和变异类型。方法应用PCR扩增和直接测序HBV逆转录酶区并与Genebank中90株不同基因型野毒株序列进行比较,确定54例耐药株感染者HBV基因型和HBVRT核苷酸的变异特点。结果在54例拉米夫定耐药株感染者中,HBVB基因型占27．78％,C型占70．37％,B／C混合型占1．85％;51例患者出现RT保守区氨基酸变异（包括550和526位氨基酸变异）;18例患者出现除主型区外HBVRT非主型区伴随变异;3例患者未检测到与拉米夫定相关性变异。结论拉米夫定耐药株感染者HBV基因型主要为B型和C型;拉米夫定耐药株的氨基酸变异不仅见于RT区的526和550两个位点,其他位点以及RT非保守区也可发生变异。     

不同基因型乙型肝炎病毒的HBx蛋白氨基酸序列的差异及意义

目的研究不同基因型HBV的HBx蛋白氨基酸序列差异及差异的意义。方法从美国生物信息中心（NCBI）搜索不同基因型HBV的全HBx蛋白氨基酸序列（154aa）,使用生物信息软件Clustalw2.0对搜索到的氨基酸序列进行比对,找出不同基因型HBV的HBx蛋白氨基酸序列差异及可能的差异规律,并分析差异的意义。结果 1.不同基因型HBV的HBx蛋白的氨基酸序列之间的同源性为73%～100%;2.HBV的各基因型（B、C、D、F、A、H）同型间的HBx蛋白氨基酸序列之间的同源性分别为87%～99%、88%～96%、87%～99%、86%～96%、96%～98%、99%～100%;3.在HBx蛋白A区（1～20aa）第5位和第12位的氨基酸在各基因型变异较大,其余位置的氨基酸很保守,很少发生变异。在HBx蛋白的B区（21～57aa）的氨基酸变异较大,即使在同一基因型别内部也出现了较大的变异,变异没有规律,呈现出了随机突变的特点。在C区（58～84aa）的氨基酸比较保守,除3个H型HBx的第60位氨基酸为A（丙氨酸）外,其余HBx蛋白的第60位氨基酸均为V（颉氨酸）,第60位以外的氨基酸很少突变。HBx蛋白D区的氨基酸（85～119aa）变异较大,并且变异呈现随机突变的特点。在HBx蛋白的E区（120～140aa）的氨基酸变异比较大的位置是130/131位置的氨基酸,其余位置的氨基酸较少发生变异,不同基因型130/131位置的氨基酸变异率是不一致的。在HBx蛋白F区（141～154aa）中,150～154aa区域的氨基酸比较保守,各基因型之间无变异,其余位点变异较大,没有一定的规律性。结论不同基因型的HBV的HBx蛋白氨基酸序列之间的同源性低于同种基因型HBV的HBx蛋白氨基酸序列之间的同源性;即使在比较保守的HBx蛋白区域也存在变异较大的氨基酸位点。     

云南地区慢性乙型肝炎病毒感染者病毒基因型、变异与肝细胞癌的关系

大量研究表明,HBV感染所致的肝细胞癌(hepatocellutar carcinoma,HCC)占全球HCC的75％～85％,但HBV导致HCC的具体发生机制尚未完全阐明[1].目前普遍认为HBVC基因型引起的炎性坏死及纤维化程度比B型更严重,且C型与肝硬化和HCC有关;B基因型的累积生存率要明显高于C型.近年来国内外学者对HBV基因型、变异位点与HCC关系进行了大量研究,云南地区近年也有关于HBV基因型分布的研究报道,但是关于HBV基因型、变异与HCC关系的研究尚鲜见报道.本研究旨在了解云南地区慢性HBV感染者病毒基因型及变异情况,探讨慢性HBV感染者病毒基因型、常见位点变异与HCC的关系.     

慢性乙型肝炎拉米夫定耐药患者HBV基因型及ALT变化的观察

观察慢性乙型肝炎患者用拉米夫定治疗后HBVP基因变异与不同HBV基因型感染及HBV DNA复升水平和转氨酶变化.收集51例慢性乙型肝炎患者用拉米夫定治疗52-78周后发生YMDD变异的血清标本,对照组128例未用拉米夫定治疗的慢性乙型肝炎患者血清标本,应用聚合酶链反应方法,测定HBV DNA基因型;用限制性片段长度多态性分析方法(PCR RELP)测定HBV DNA YMDD变异;同时进行HBV DNA定量分析.结果显示51例拉米夫定治疗后HBV DNA基因变异患者以B型和C型为主,分别为10例(19.6%)和39例(76.47%),B C混和型2例(3.92%),未见其它基因型.拉米夫定治疗引起HBVDNAYMDD变异可以发生在不同HBV基因型感染的慢性乙型肝炎患者中,与对照组比较二者没有显著性差异.     

HBV基因型及A1762T/G1764A双位点变异在肝癌患者中的临床意义

目的:研究肝细胞癌(HCC)患者乙型肝炎病毒(HBV)基因型与基本核心启动子(BCP)基因区A1762T/G1764A双位点变异(BCP区双突变)之间的关系,探讨HCC的发病机制。方法:选择40例HCC患者作为研究组,40例慢性乙型肝炎(CHB)患者作为对照组,采用多对型特异性引物PCR扩增法进行基因分型及HBV基因多态性芯片检测BCP区A1762T/G1764A双位点变异。结果:40例HCC患者中有30例HBV DNA定量阳性,平均对数值为(6·53±1·31)copy/ml,将30例HBV DNA定量阳性的HCC及40例CHB患者的血清进行HBV基因分型及基因变异检测,结果显示30例HCC中HBV B基因型5例(16·7%),C基因型25例(83·3%);BCP区双突变共有20例(67·7%),其中B基因型1例,C基因型19例,BCP双突变率在B基因型和C基因型中分别为20%(1/5)和76%(19/25);40例CHB患者中B基因型32例(80%),C基因型8例(20%);BCP区双突变共有13例,其中B基因型8例,C基因型5例,BCP双突率在B基因型和C基因型中分别是25%(8/32)和62·5%(5/8)。结论:肝细胞癌的发生与BCP区A1762T/G1764A双位点变异有关,多发生在HBV C基因型的患者。     

HBV基因型与干扰素抗病毒疗效的关系

目的:探讨HBV不同基因型对α-干扰素抗病毒疗效的影响.方法:选取应用α-干扰素进行抗病毒治疗的慢性乙型肝炎患者作为研究对象,观察其抗病毒疗效.患者的HBV基因型采用PCR微板核酸杂交-ELISA方法检测;血清HBV DNA复制水平采用荧光定量PCR检测;HBV前C区和BCP(基础核心启动子)区基因位点变异采用HBV基因多态性芯片进行检测.结果:94例慢性乙型肝炎患者的HBV基因型以C型、B型为主,未发现A、E、F基因型.HBV DNA高复制水平明显与C基因型及混合基因型有关.B基因型对α-干扰素抗病毒治疗的应答明显优于C、D型,而混合基因型对α-干扰素的应答最不敏感.仅B基因型对α-干扰素治疗产生完全应答.部分应答及无应答时HBeAg的转阴与HBV前C区nt 1 896位点变异、以及BCP区nt 1 762、nt 1 764双位点变异有关.C基因型HBV前C区及BCP区基因变异发生率明显高于B型.结论:HBV基因型与HBV DNA复制水平、HBV基因变异以及α-干扰素抗病毒疗效均有一定的相关性,提示HBV基因分型有重要的临床意义.     

39例成人急性乙型肝炎患者血清HBV基因变异检测及其临床意义探讨

目的了解成人急性乙型肝炎患者流行的病毒基因型及基因变异情况。方法在2010年12月~2014年1月北京佑安医院收治39例急性乙型肝炎患者,采用直接基因测序法和平行等位基因特异性检测技术检测HBV基因型、P区、S区、前C区(Pre C)和基本核心启动子(BCP)区序列。结果在39例患者中,成功测序35例。35例急性乙型肝炎患者感染HBV B基因型12例(36.4%),C基因型21例(57.6%,D基因型2例(6.0%);通过直接测序法和PASS法均检测到同1例患者存在A181S耐药变异,变异病毒占准种池比例达100%;直接测序法检测到1例患者存在S区S132F和W172C变异;1例患者存在Pre C/BCP区G1896A和T1758C变异。结论急性乙型肝炎患者感染病毒基因型与文献报道的慢性乙型肝炎患者感染病毒基因型分布一致,以C型和B型为主,存在P区、S区和Pre C/BCP区变异可能,未见不同病毒株感染引起转归的不同。     

39例成人急性乙型肝炎患者血清HBV基因变异检测及其临床意义探讨*

目的了解成人急性乙型肝炎患者流行的病毒基因型及基因变异情况。方法在2010年12月~2014年1月北京佑安医院收治39例急性乙型肝炎患者,采用直接基因测序法和平行等位基因特异性检测技术检测HBV基因型、P区、S区、前C区（PreC）和基本核心启动子（BCP） 区序列。结果在39例患者中,成功测序35例。35例急性乙型肝炎患者感染HBV B基因型12例（36.4%）,C基因型21例（57.6%,D基因型2例（6.0%）;通过直接测序法和PASS法均检测到同1例患者存在A181S耐药变异,变异病毒占准种池比例达100%;直接测序法检测到1例患者存在S区S132F和W172C变异;1例患者存在PreC/BCP 区G1896A和T1758C变异。结论急性乙型肝炎患者感染病毒基因型与文献报道的慢性乙型肝炎患者感染病毒基因型分布一致,以C型和B型为主,存在P区、S区和PreC/BCP区变异可能,未见不同病毒株感染引起转归的不同。     

河南省不同地区、不同肝病患者的HBV基因型分布

采用PCR扩增技术,对河南省部分地区315例乙肝感染患者的乙型肝炎病毒(HBV)基因进行分型,并分析其基因型与HBV相关肝病的相关性.结果 显示,本组HBV DNA阳性血清248份,其中HBV基因B型占28.6%,C型占70.2%,D型占1.2%.无症状携带者(ASC)、慢性肝炎(CHB)、重型肝炎(CSL)均以B型为主要基因型;C型在肝硬化中所占比例显著高于ASC和CSL;相关分析表明,肝细胞癌(HCC)与基因D型关系密切.提示河南省部分地区的HBV基因型多数为C型,少数为B型,极少数为D型;基因C型与较严重肝病有关,D型与HCC有一定相关性.     

乙型肝炎与肝癌关系

目的乙型肝炎病毒(HBV)持续感染和宿主免疫反应可导致肝细胞癌(HCC)发生.研究HBV感染致癌机制和乙肝免疫预防HCC具有重要的理论和实际意义.①从分子生物学观点出发探索HBV感染与HCC关系与HCC相关的最危险因素HBV的XORF常整合于宿主DNA并高度表达.现有越来越多的证据表明反式激活剂HBx可能通过封闭抑癌基因p53的功能而致癌.结构与功能分析表明HBx的远C末端为其抑制p53诱导凋亡的必需区.最近发现HBx基因反式激活功能区发生天然"突然”,即可消除其诱导细胞生长停顿和凋亡的效应.②HBx是一多功能调节因子近来研究相对集中在HBx对肝细胞凋亡的作用上,促凋亡或抑凋亡.发现HBx可抑制cospase3酶活性阻断细胞凋亡.但另有报告HBx可致敏细胞引起凋亡.联系上述HBx可封闭由于p53过表达而诱导的凋亡,可见病毒根据不同细胞环境或外来刺激采取不同的策略.③乙肝免疫预防HCCHBVDNA,多在HBx基因整合于宿主DNA,可激活或抑制与生长增殖有关的细胞基因.如整合得到阻遏,细胞转化不至发生.台湾儿童广行乙肝免疫使HBV携带率10%降至0.9%,HCC发生率0.52%降至0.13%,证明控制乙肝是有效防癌措施.     

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.