HBV preS2反义锁核酸在HepG22.2.15细胞内的抗病毒效果

目的:探讨针对HBV preS2基因mRNA翻译起始区的反义锁核酸(LNA)片段在2.2.15细胞内抗HBV复制和表达的作用.方法:分别合成三段互补于HBV preS2基因mRNA翻译起始区同一靶位的反义锁核酸、全硫代反义寡核苷酸、未修饰寡核苷酸及无关对照序列,以阳离子脂质体作为载药体系,作用于HepG22.2.15细胞,采用时间分辨免疫荧光技术(TRFIA)和荧光定量聚合酶链技术(FQ-PCR)动态检测细胞上清液中HBsAg和HBV DNA的含量,并比较其抑制HBV DNA复制与表达的作用;以四甲基偶氮唑蓝(MTT)法检测LNA对细胞的毒性.结果:加入LNA后第1天,即出现对HBsAg表达和HBV DNA复制的抑制作用,第7天,未修饰反义寡核苷酸组、全硫代修饰反义寡核苷酸组、反义锁核酸组对HBsAg表达的抑制率分别达45.79%、52.92%和67.21%;对HBVDNA复制的抑制率分别达35.15%、40.69%和52.16%.其中LNA抑制病毒活性最强且对细胞代谢无影响.各组与对照组比较均有显著性差异(均P<0.01),且反义LNA组与其他ASODN组比较也有显著性差异(均P<0.05).结论:针对preS2基因的反义锁核酸体外能有效抑制HBV的复制与表达,故preS2基因可作为乙型肝炎基因治疗的有效靶位.     

针对HBV S基因mRNA反义锁核酸设计及其在2.2.15细胞内的抗病毒作用

目的:探讨针对乙型肝炎病毒(HBV)S 基因翻译起始区反义锁核酸(LNA) 片段在HepG22.2.15 细胞内抗病毒效果及有效LNA 序列筛选. 方法:设计合成互补于HBV S 基因mRNA 翻译起始区同一位点的4条不同序列长度LNA 片段及无关对照序列,以阳离子脂质体介导,作用于HepG22.2.15 细胞,采用ELISA 法和FQPCR 法分别监测24 、48 和72 h 细胞培养上清液中HBsAg 和HBV DNA 的含量;MTT 法检测LNA 对细胞代谢的影响. 结果:4条不同序列长度(10 、15 、20 及25 个碱基)的反义LNA 对HBsAg 的表达和HBV DNA 的复制均有显著性抑制作用,72h 后的抑制率分别为46.58% 、54.38% 、72.43% 、69.92% 及27.09% 、28.77% 、34.71% 、32.68%,且抑制率随时间呈增高趋势. LNA 对细胞代谢无明显影响. 结论:针对HBV S 基因mRNA 翻译起始区的反义LNA 短序列体外能显著抑制HBV 基因的表达,且抑制作用最强的序列长度应在15-25 个碱基之间.     

锁核酸核酶抑制丙型肝炎病毒5'非编码区内部核糖体进入位点的基因表达

锁核酸核酶(LNAzyme)是在脱氧核酶(DNAzyme)的两个结合臂上引入1个或多个锁核酸单体构建而成的新型核酸酶[1].锁核酸(LNA)是一种新型的带双环状结构的寡核苷酸衍生物,具有稳定性好、分子杂交能力强、抗核酸酶降解能力强和无毒性等优势[2-3].LNA的引入极大增加了LNAzyme与底物RNA的杂交亲和力,增强了切割反应的酶促活力,使LNAzyme较相应的DNAzyme切割效率明显提高.针对HBV S基因的反义LNA能有效抑制病毒的表达[4].本研究针对HCV 5'非编码区(NCR)内部核糖体进入位点(IPES)设计合成LNAzyme,以HepG2.9706为对象,观察LNAzyme对病毒复制与表达的抑制作用,旨在为丙型肝炎治疗寻找专一性强、高效的新型分子药物.     

HBV S基因的反义锁核酸对乙型肝炎转基因小鼠HBV复制和表达的影响

目的: 探讨乙型肝炎病毒(hepatitis B virus,HBV)S基因的反义锁核酸(LNA)对乙型肝炎转基因小鼠HBV复制和表达的影响.方法: 将30只HBV转基因小鼠随机分为5组,每组6只. 第1组为5% GLU液对照组, 第2组为空脂质体对照组, 第3组为单LNA组, 第4组为S-ASODN脂质体组, 第5组LNA脂质体组. 反义LNA经尾静脉注入小鼠体内, 采用ELISA法检测血清HBsAg;PCR定量检测血清HBV DNA含量;免疫组织化学法检测肝细胞HBsAg的表达;自动生化分析仪检测ALB、ALT、BUN、CR、ApoA1、ApoB等指标;小鼠肝脏、肾脏做常规病理切片HE染色, 观察反义LNA对小鼠脏器的影响.结果: 注射反义LNA 1 d、3 d、7 d、14 d后,LNA-脂质体组对血清HBsAg的表达抑制率分别为41.7%、52.8%、57.8%及30.5%. 对HBV DNA的抑制率分别为18.5%、36.1%、52.9%和32.7%, 与对照组比较均有显著性差异(P <0.05);全自动生化分析仪检测血清中ALB、ALT、BUN、CR、ApoA1、ApoB等指标, 各组结果与对照组比较均无显著性差异(P >0.05);小鼠肝细胞HBsAg的表达显著低于对照组. HE染色显示小鼠肝肾功能及组织学未见异常.结论: HBV S基因反义LNA对乙型肝炎病毒转基因鼠HBV复制和表达有显著抑制作用.     

反义核酸对乙型肝炎患者外周血单个核细胞免疫刺激作用

反义寡核苷酸(asON)可刺激小鼠淋巴细胞增殖和分化。为探索其对人体免疫功能的影响,我们选择轻度慢性乙型肝炎(乙肝)患者,观察乙型肝炎病毒(HBV)DNA特异性asON对其外周血单个核细胞(PBMC)的体外细胞增殖、杀伤和CD40抗原表达的变化。材料和方法　　一、试剂　　(一)致丝裂原或细胞激活物　PHA-m(Sigma公司);葡萄球菌Cowan-I(SAC,卫生部上海生物制品所);重组人粒细胞-巨噬细胞集落刺激因子(rhuGM-CSF,本校秦都医院生物工程研究室)。　　(二)化学合成硫化磷酸寡核苷酸　反义序列I为GATGACTGTCTCTTA(针对HBV基因组3188～32…     

反义核酸对乙型肝炎患者外周血单个核细胞免疫刺激作用

采用3H-TdR掺入试验、~(51)Cr释放试验和流式细胞仪分析技术检测了针对HBV基因的硫代磷酸反义寡核苷酸对乙型肝炎患者外周血单个核细胞的增殖、杀伤和CD_(40)抗原表达。结果发现反义核酸能刺激正常人和乙型肝炎患者的淋巴细胞增殖,且不影响单核细胞的杀伤作用;在T、B细胞混合培养中,反义核酸使患者的CD_(40)~+细胞百分率提高的幅度不如对正常人或正义     

脂质体介导的VEGF-C反义寡核苷酸对肺腺癌细胞系 A549生长、凋亡的影响

目的 探讨脂质体介导的血管内皮生长因子C(VEGF-C) 反义寡核苷酸对肺腺癌细胞系 A549生长、凋亡的作用.方法 将 A549 细胞随机分为磷酸盐缓冲液对照组(PBS组)、正义寡核苷酸对照组(SODN组)和反义寡核苷酸转染组(AODN组),应用细胞计数方法计数每组细胞数,苏木精-伊红(HE)染色法观察细胞形态及分布,流式细胞仪分析A549细胞凋亡情况.应用Western印迹方法检测转染后各组A549细胞中VEGF-C蛋白表达.结果 VEGF-C反义寡核苷酸转染后,AODN组细胞生长受抑程度显著高于对照组,差异有统计学意义(P<0.01),生长缓慢且核分裂像较对照组明显减少.流式细胞仪检测结果显示AODN组细胞凋亡率明显高于对照组,差异有统计学意义(P<0.01).Western印迹结果显示VEGF-C反义寡核苷酸可明显抑制VEGF-C基因表达.结论 脂质体介导的VEGF-C反义寡核苷酸能有效地干扰VEGF-C基因表达,并可抑制肺腺癌A549细胞生长并促进其凋亡.     

反义磷酸硫化寡聚脱氧核苷抑制乙型肝炎病毒的基因表达

利用乙型肝炎病毒(HBV)DNA 转染的Hep G2细胞,研究了反义磷硫化寡核苷酸(S-oligos)对乙肝表面抗原(HBsAg)和 e 抗原(HBeAg)的产生所具有的抑制作用。人工合成了直接作用于由 S 基因的转录起始点区     

反义锁核酸与拉米夫定在HepG2.2.15细胞中抗HBV作用的比较

目的:应用反义锁核酸与拉米夫定作用HepG2.2.15细胞,对他们抗乙型肝炎病毒(hepatitis B virus,HBV)效果进行比较.方法:设计针对HBV翻译起始区S基因mRNA的反义寡核苷酸,并进行锁核酸修饰,以阳离子脂质体介导反义锁核酸转染HepG2.2.15细胞;拉米夫定组直接作用HepG2.2.15细胞;分别于用药后第2、4、6、8、10天收集细胞培养上清液.用ELISA法和FQ-PCR法检测收集上清液HBsAg、HBeAg和HBVDNA的含量.MTT法分别检测反义锁核酸与拉米夫定对细胞存活率的影响.结果:拉米夫定对HBVDNA具有明显抑制作用,最高可达46.52%,但对HBsAg、HBeAg影响较小;反义锁核酸对HBsAg、HBeAg及HBVDNA均有较强抑制作用,对HBsAg、HBeAg和HBVDNA的最高抑制率分别达67.69%、59.71%和62.96%(P<0.05),且抑制随时间呈增高趋势.反义锁核酸与拉米夫定对细胞代谢均无明显影响.结论:反义锁核酸抗HBV作用机制与拉米夫定不同,反义锁核酸抗HBV作用明显优于拉米夫定.     

反义核酸对乙型肝炎患者外周血单个核细胞免疫刺激作用的观察

采用3H—TdR掺入试验、^51Cr释放试验和流式细胞仪分析技术检测了针对HBV基因的硫代磷酸反义寡核苷酸对乙型肝炎患者外周血单个核细胞的增殖、杀伤和CD40抗原表选。结果发现，反义核酸能刺激正常人和乙型肝炎患者的淋巴细胞(主要是T细胞)增殖．但无提高单核细胞杀伤作用,在T、B细胞混合培养中．反义核酸使患者的CD40^ 细胞百分率提高的幅度不如对正常人或正义核酸对患者的高；在单纯B细胞培养时．反义核酸不能阻止CD40分子的消失。研究提示,抗HBV反义核酸能非特异性刺激人淋巴细胞增殖，并以T细胞依赖方式增强淋巴细胞CD40抗原的表达，这对应用反义药物抗病毒治疗机理研究及机体免疫功能变化将有重要意义。     

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.     

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.     

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.     

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.