心肌细胞感染病毒后钙、钾通道电流及其表达改变和牛磺酸的作用

目的 观察心肌细胞感染病毒后心肌细胞钙，钾通道电流及通道表达改变和牛磺酸的作用。方法 （1）体外心肌细胞感染病毒；用胰蛋白酶和胶原酶消化法分别获得大鼠，乳鼠培养和分离的心肌细胞并感染柯萨奇B3病毒，用于检测Ca^2 跨膜内流，电压依赖性钙通道电流（Lca)和外向钾通道电流（Iout)。（2）在体小鼠感染病毒；BALB/c小鼠腹腔内注射柯萨奇B3病毒7d后取出心脏用于检测电压堆呀性钙通道α1亚单位抗原和电压调控性钾通道（Kv)mRNA的表达。相应各组加入不同剂量的牛磺酸并观察其作用。结果 （1）感染柯萨奇B3病毒后Ca^2 跨膜内流量及Ica增加，牛磺酸对其有抑制作用。（3）柯萨奇B3病毒感染out明显增大。牛磺酸可使病毒感染后的Iout趋于恢复正常。（3）柯萨奇B3病毒感染后小鼠心肌出现强电压依赖性钙通道α1亚基抗原阳性信号。Kv1.2,Kv2.1,Kv4.2mRNA与3种探针的杂交信号亦明显增强，牛磺酸可使增强的阳性信号减弱。结论 柯萨奇B3病毒感染后心肌细胞钙，钾离子通道电流及表达均增加，牛磺酸可通过抑制其改变而发挥对感染病毒心肌细胞的保护作用。     

黄芪总黄酮与丹参酮ⅡA磺酸钠对病毒性心肌炎小鼠心肌细胞内质网伴侣蛋白及L-型钙通道表达的作用

目的:观察黄芪总黄酮(TFA)与丹参酮ⅡA磺酸钠对病毒性心肌炎(VMC)小鼠心肌细胞内质网伴侣蛋白GRP78及L-型钙通道(LCCs)mRNA表达的影响。方法:48只雄性Balb/c小鼠分为对照组、VMC组、TFA组和丹参酮ⅡA磺酸钠治疗组(丹参组),每组12只。注射柯萨奇(CV)B3病毒10d处死小鼠取心脏行心肌病理观察,采用半定量逆转录-聚合酶链式反应(RT-PCR)检测对照组、VMC组、TFA组、丹参组心肌细胞LCCsɑ1亚单位mRNA表达量,采用Western blotting技术检测4组心肌细胞内质网伴侣蛋白GRP78表达量。结果:1与对照组比较,VMC组心室肌LCCsɑ1亚单位的mRNA表达量明显增多(P0.01);与VMC组比较,TFA可使VMC小鼠心室肌细胞LCCsɑ1亚单位的mRNA表达量减少(P0.01),而丹参酮ⅡA磺酸钠可使VMC小鼠心室肌细胞LCCsɑ1亚单位的mRNA表达量减少(P0.01)。2与对照组比较,VMC组心室肌细胞内质网伴侣蛋白GRP78表达量明显增多(P0.01);与VMC组比较,TFA可使VMC小鼠心室肌细胞内质网伴侣蛋白GRP78表达量减少(P0.01),而丹参酮ⅡA磺酸钠可使VMC小鼠心室肌细胞内质网伴侣蛋白GRP78表达量减少(P0.05)。结论:TFA与丹参酮ⅡA磺酸钠可以降低VMC小鼠心室肌细胞LCCsɑ1亚单位的mRNA表达量,缓解VMC小鼠心肌细胞内质网应激,抑制心肌细胞钙超载介导的内质网应激,从而改善心肌损伤。     

柯萨奇B病毒性心肌炎小鼠急性期心肌组织微小RNA1初始体和连接蛋白43的表达

目的观察柯萨奇B病毒性心肌炎小鼠急性期心肌组织微小RNA1初始体(pri-miRNA-1)和连接蛋白43(Cx43)表达变化,探讨病毒性心肌炎(VMC)室性心律失常发生机制。方法40只4周龄雄性Balb/c小鼠随机分为VMC组(n=20)和对照组(n=20)。VMC组小鼠腹腔注射柯萨奇病毒B3(CVB3)Nancy株悬液0.1ml,对照组小鼠腹腔注射不含病毒的RPMI1640培养基0.1ml,分别于接种病毒后第14天无痛苦处死全部小鼠并留取心脏。逆转录-聚合酶链反应(RT-PCR)检测心室肌组织pri-miRNA-1的表达,免疫组织化学法检测Cx43蛋白水平表达,并进行半定量分析。结果①VMC组小鼠心室肌组织pri-miRNA-1表达量明显高于对照组(0.82±0.04vs0.63±0.07,P(0.01);②VMC组小鼠心室肌组织炎症病灶中变性、坏死周围心肌细胞Cx43表达明显减弱,甚至阴性,分布不规则,Cx43蛋白表达明显低于对照组(0.27±0.01vs0.42±0.02,P(0.01);③VMC组小鼠心室肌组织的pri-miRNA-1表达量与Cx43蛋白水平呈显著负相关(r=-0.868,P(0.01)。结论CVB心肌炎小鼠急性期心肌组织pri-miRNA-1表达上调,Cx43蛋白表达下降,miRNA-1可能通过抑制Cx43表达促进室性心律失常的发生。     

黄芪总黄酮对柯萨奇B3病毒感染心肌细胞钠电流的作用

目的探讨黄芪总黄酮（TFA）对嗜心性柯萨奇B3病毒（CVB3m）感染心肌细胞钠电流的作用。方法采用体外培养小鼠新生心肌细胞方法,建立柯萨奇B3病毒感染心肌细胞模型,采用全细胞膜片钳记录技术记录心室肌细胞钠电流（INa）。结果应用黄芪总黄酮（TFA）组与柯萨奇B3病毒感染模型组相比较,INa峰值从（-7．79±1．53）nA下降到（-5．64±1．67）nA,差异有统计学意义（P〈0．01,n=6）。结论黄芪总黄酮可显著降低柯萨奇B3病毒感染心肌细胞钠电流的幅值。     

黄芪总黄酮对病毒性心肌炎小鼠心脏血流动力学及心肌细胞钙电流的作用

目的研究黄芪总黄酮(TFA)对BALB/c小鼠病毒性心肌炎急性期心脏血流动力学及心肌细胞钙电流的作用。方法应用柯萨奇B3病毒制作BALB/c小鼠病毒性心肌炎模型7只,腹腔注射TFA溶液(20mg/kg)1次/d,并设正常对照组及病毒感染对照组各7只,7d后行血流动力学检查;应用胶原酶酶解法急性分离病毒性心肌炎BALB/c小鼠心室肌细胞,利用全细胞膜片钳的方法记录TFA对BALB/c小鼠病毒性心肌炎模型心室肌细胞Ca2+电流的作用。结果(1)TFA组与正常对照组和病毒感染组相比血流动力学指标明显改善。(2)TFA组与对照组比较,L-型钙电流(L-ICa)由给药前的(0.28±1.07)nA增加到给药后的(0.39±0.84)nA(P<0.05)。结论TFA可以明显改善BALB/c小鼠病毒性心肌炎急性期心脏血流动力学,TFA可增加病毒性心肌炎BALB/c小鼠心室肌细胞L-ICa的幅值。     

克山病尸检心肌标本柯萨奇B组病毒RNA片段的序列分析

目的通过原位核酸杂交技术和RT-PCR技术探讨柯萨奇B组病毒感染与吉林亚急型克山病的关系.方法以柯萨奇B组病毒基因组的一段共同序列做寡核苷酸探针,于5′端行生物素标记,检测亚急型克山病患者尸检心肌组织标本中柯萨奇病毒的感染率;选取阳性标本做RT-PCR,并对PCR产物进行核苷酸序列分析,比较其与柯萨奇B组病毒各血清型之间的差异.结果 9例亚急型克山病患者尸检心肌组织标本中有7例原位核酸杂交结果阳性,阳性率为77.78%;所选3例原位核酸杂交结果阳性标本RT-PCR均有长541 bp的DNA条带出现,测序结果表明该片段与柯萨奇病毒B3同区域序列一致,与B1、B4、B5的同源性较大.结论亚急型克山病患者体内有柯萨奇B组病毒RNA存在,柯萨奇病毒可能在克山病的发生发展中扮演重要的角色.     

快速起搏大鼠心房肌细胞L-型钙通道及钾通道Kv4.3的表达变化

目的 利用原代培养的心房肌细胞建立快速起搏模型,研究L-型钙通道及钾通道Kv4.3在快速起搏早期的表达变化.方法 原代培养大鼠心房肌细胞,并建立快速起搏细胞模型,利用RT-PCR以及Western-blot方法检测L-型钙通道α1c及钾通道Kv4.3在快速起搏3、6、12、24 h后mRNA和蛋白的表达变化.结果 快速起搏6 h后L-型钙通道α1c的mRNA和蛋白表达较起搏前持续降低,并于24 h时达到最低值;而钾通道Kv4.3 mRNA和蛋白的表达在快速起搏12 h后降低,并且在其后保持相对稳定的水平.结论 快速起搏早期,原代培养心房肌细胞L-型钙通道α1c及钾通道Kv4.3的mRNA和蛋白表达均出现不同程度的降低,提示其发生了离子通道重构,并且可能是电重构的分子基础.     

肿瘤坏死因子α抑制豚鼠急性缺血心室肌细胞L型钙通道电流

为探讨肿瘤坏死因子α对正常及模拟缺血状态下豚鼠心室肌细胞L型钙通道电流的影响。全细胞膜片技术记录豚鼠心室肌细胞单个L型钙通道电流。肿瘤坏死因子α(100、300和500 ku/L)对豚鼠心室肌细胞L型钙通道电流峰值的影响与对照组均无明显差异(P>0.05),但模拟急性缺血时,同浓度的肿瘤坏死因子α却明显抑制豚鼠心室肌细胞L型钙通道电流峰值并呈浓度依赖性,抑制率分别为46.5％±3.5％、62.7％±3.0％和80,7％±3.7％,与对照组和单纯缺血组相比差异明显(均P<0.05)。正常灌流液中不同浓度的肿瘤坏死因子α对豚鼠心室肌细胞峰值L型钙通道电流无明显影响;但模拟急性缺血时其却呈浓度依赖性地增强缺血对豚鼠心室肌细胞的L型钙通道电流峰值抑制作用。     

原位核酸杂交法探讨柯萨奇B组病毒与云南亚急型克山病的关系

用光敏生物素标记的柯萨奇 B_3病毒 cDNA 探针,与云南病区20例经临床和病理检查确诊为亚急型克山病的心肌组织切片进行原位核酸杂交,以检测柯萨奇 B 组病毒 RNA。结果有18例出现阳性杂交信号,阳性率达90%。说明云南亚急型克山病心肌组织中有柯萨奇 B 组病毒 RNA 的存在。提示云南亚急型克山病的发生与柯萨奇 B 组病毒的持续感染密切相关。     

自发性高血压大鼠左心室肌细胞动作电位延长的离子机制

目的:研究自发性高血压大鼠(SHR)左心室肌细胞动作电位时程延长的膜离子流基础.方法:应用酶解方法分离获得正常血压Wistar大鼠和SHR的左心室肌细胞,采用玻璃微电极技术记录动作电位,膜片钳全细胞记录膜离子流,对比正常心室肌细胞和肥大心室肌细胞间动作电位及膜离子流差别.结果:(1)SHR和Wistar大鼠的心脏/体重比分别为5.66±0.46 mg/g和3.7±0.29 mg/g (P<0.001) ,细胞平均膜电容分别为280.68±67.98 pF 和189.94±56.59 pF(P<0.05).提示SHR 心脏肥厚、心肌细胞增大;(2)SHR动作电位APD50和APD90较Wistar大鼠明显延长(21.33±1.56 ms vs 14.91±2.95 ms,P<0.001; 164.6±74 ms vs 93.27±10.59 ms,P<0.00 1),说明SHR心室肌细胞存在复极延迟;(3)SHR的平均ICa-L幅值显著大于Wistar大鼠,分别为1944±466.8 pA和1136±33.3 pA(P<0.001),电流密度二者间无差异(6.932±1.7 1 pA/pF vs 6.19±2.85 pA/pF) ,但SHR的慢失活时间常数明显延长(56.01±13.36 ms vs 43.63±17.89 ms,P<0.001);(4)S HR的Ik1内向电流密度显著小于Wistar大鼠(11.3±2.26 pA/pF vs 14.33 pA/pF,P<0.05),外向电流密度二者间差异无显著性(2.36±0.86 pA/pF vs 2.96±1.27 pA/pF);(5)SHR的Ik密度与Wista r大鼠间无差别(12.38±5.46 pA/pF vs 11.86±3.59 pA/pF);(6)SHR的Ito密度显著地低于Wistar 大鼠(+70 mV时, 8.21±6.64 pA/pF vs 19.16±6.17 pA/pF, P<0.001).但通道的激活和失活时间常数二者无差异,提示Ito的降低可能仅是通道数减少所致.结论:SHR左心室肌细胞动作电位时程延长系外向复极钾流(Ito、Ik1)减小和慢钙通道失活时间常数延长所致.     

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.