RNA干扰治疗慢性心力衰竭大鼠的实验研究

目的利用RNA干扰(RNAi)技术抑制心力衰竭(心衰)大鼠受磷蛋白(PLN)mRNA的表达,改善心脏功能。方法选用SD大鼠40只,对照组7只;其余33只造心衰模型,存活21只,随机分为心衰组、rAAV2-phRi1组,rAAV2-phRi2组,每组7只;rAAV2-phRi导入后30天测定血流动力学,逆转录聚合酶链反应检测心肌PLN mRNA,定磷法测定肌浆网Ca2+-ATP酶(SERCA2a)活性。结果rAAV2-phRi导入30天时,与心衰组比较,rAAV2-phRi1及rAAV2-phRi2对PLN mRNA的抑制效率为85.32%和67.65%,SERCA2a活性增加了104%和74%,血流动力学显著改善(P<0.05),达到对照组水平(P>0.05);rAAV2-phRi1的抑制效率、对心功能的改善显著优于rAAV2-phRi2。结论体内水平上,RNAi能够抑制心衰大鼠PLN mRNA表达,改善心脏功能。     

压力超负荷下大鼠心肌细胞核钙调节系统的变化

目的：通过腹主动脉缩窄（abdominal aortic coaretation，AAC）心肌肥厚大鼠模型制备、差速离心提纯心肌细胞核、酶学方法测定Ca^2＋-ATPase活性、^45Ca^2＋同位素法测定核钙摄取和^3H放射配基受体分析心肌细胞核膜IP3R和RyR的动力学特性，初步揭示压力超负荷心肌肥厚大鼠心肌细胞核钙转导异常的环节。结果发现：AAC术后4周大鼠心肌显著肥厚，伴有明显的血流动力学异常，与对照组比较，AAC大鼠心肌细胞核Ca^2＋-ATPase活性减少51．93％（P〈0．001），但核^45Ca^2＋摄入量（核外[Ca^2＋]浓度为800-1600nmol／L时）明显增加（P〈0．05）；AAC大鼠心肌细胞核IP3R的Bmax和Kd与对照组比较分别增加1．217和2．149倍（P〈0．01），其细胞核RyR的最大结合（Bmax）较对照组减少57．8％（P〈0．001），解离常数（Kd）较对照组降低54．4％（P〈0．05）。结论为心肌细胞核Ca^2＋转运系统发生改变（Ca^2＋．ATPase活性减少、核^45Ca^2＋摄取增加、IP3R密度上调和亲和力降低，而细胞核RyR密度下调而亲和力增加），可能参与压力超负荷心肌肥厚的发生过程。     

益母草生物碱对心肌梗死大鼠心功能及肌浆网钙泵的活性及其蛋白水平的影响

目的 探讨益母草生物碱对大鼠心肌梗死后心功能保护作用的机制。方法 采用左冠状动脉前降支结扎制作大鼠心肌梗死模型，通过大鼠血流动力学指标以及心肌肌浆网钙泵的活性（SERCA2α）及其蛋白水平来反映益母草生物碱对大鼠心肌梗死后心功能的影响不明显（P〈0．05）。结果 益母草生物碱可以显著提高模型大鼠左室收缩压以及左室内压最大上升速率（P〈0．05），降低模型大鼠左室舒张末压以及明显提高左室内压最大下降速率（P〈0．05）；益母草生物碱对模型大鼠心肌组织SERCA2α活性及SERCA2α含量均有显著提高（P〈0．05）。结论 益母草生物碱能够提高冠状动脉结扎大鼠心肌收缩舒张功能，其可能机制是提高大鼠心肌组织SERCA2α活性及含量。     

腺相关病毒介导心肌肌浆网Ca2+-ATPase 2a基因转导治疗大鼠慢性心力衰竭

目的 研究腺相关病毒为载体的心肌肌浆网Ca^2＋ -ATPase 2a（sarcoplasmic reticulum Ca^2＋ -ATPase,SERCA2a）基因转导对慢性心力衰竭（HF）大鼠的治疗作用,并探讨其多种可能的机理.方法 采用腹主动脉缩窄术建立HF大鼠模型,应用经腹心包腔内注射术分别将生理盐水、携带eGFP基因和携带SERCA2a基因的重组腺相关病毒导入HF、HF＋EGFP和HF＋SERCA2a组大鼠心脏.于导入30天,检测各组大鼠的心脏功能、SERCA2a蛋白表达和活性;比较HF组和HF＋SERCA2a组大鼠心肌蛋白质组表达的差异;检测各组大鼠心肌肌球蛋白重链（MHC）亚型的表达.结果 HF大鼠心脏内转导入SERCA2a基因30天,心脏收缩和舒张功能达到对照组大鼠水平,并且HF＋SERCA2a组左室重/体重比值显著降低;SERCA2a蛋白表达和活性明显升高至对照组大鼠水平;多种能量代谢酶表达明显增加;α-MHC、β-MHC的表达以及α-MHC/β-MHC恢复至对照组大鼠水平.结论 以重组腺相关病毒2作为载体,SERCA2a基因转导可以增强衰竭心脏的SERCA2a功能,增加心脏能量代谢,纠正MHC亚型的异常表达;在临床方面表现为显著改善心脏收缩和舒张功能,可能能够减轻心脏肥厚等病理性结构改变.     

绞股蓝总皂苷对阿霉素致心力衰竭大鼠心功能的影响

目的观察绞股蓝总皂苷(GP)对阿霉素(ADR)致心衰大鼠心肌ATPase活性和肌浆网Ca2+-ATPase(SERCA2a)的影响。方法 50只SD大鼠随机分为5组,正常对照组(NC),阿霉素组(ADR),绞股蓝总皂苷低、中、高剂量组(L-GP,MPGP and H-GP)。大鼠腹腔注射ADR2.5 mg·kg-1·d-1,隔日1次,共6次,复制心衰模型。绞股蓝总皂苷低、中、高剂量组分别给予绞股蓝总皂苷50、100和200 mg·kg-1·d-1灌胃。第7周末,采集大鼠心室内压,分析心功能;电镜观察心肌组织超微结构变化,同时测定心肌组织ATPase活性、琥珀酸脱氢酶(SDH)和SERCA2a活性;RT-PCR检测心肌SERCA2a mRNA表达。结果 (1)与NC组相比,ADR组大鼠LVSP和±dP/dtmax均明显下降,LVEDP上抬(P<0.05或P<0.01),心肌ATPase活性和SDH活性显著下降(P<0.01);SERCA2a活性和SERCA2a mRNA表达水平显著下降(P<0.01);电镜显示心肌细胞变性坏死;(2)与ADR组比较,给予中、高剂量GP后大鼠心功能均有改善,电镜下心肌细胞变性坏死程度及心肌细胞超微结构形态改变显著减轻,心肌ATPase活性、SDH活性和SERCA2a活性明显升高,SERCA2a mRNA表达水平增加(P<0.05或P<0.01)。结论绞股蓝总皂苷改善阿霉素致心衰大鼠心肌功能,其机制可能与升高心肌组织ATPase活性、SERCA2a活性有关。     

钠-钙交换与慢性心力衰竭

钠－钙交换（NCX）是心肌细胞膜上钠、钙双向转运蛋白，以正向和反向模式调节细胞内的钙离子平衡。在慢性心力衰竭（CHF）的过程中，NCX在信使核糖核酸（mRNA)和蛋白水平上均升高，肌质网（SR）Ca^2 -ATPase(SERCA)活性则降低，SERCA／NCX转运活性比增大，从而影响心衰心肌的收缩和舒张功能。NCX抑制剂的研制将有助于保护心肌，减少心肌损伤，在未来用于慢性心衰和心律失常的治疗。     

骨髓间充质干细胞移植对大鼠梗死心脏核因子KB活性的影响

目的观察骨髓间充质干细胞对心肌梗死的治疗效果，以及心肌梗死后炎症因子表达的影响，探讨其治疗心肌梗死的可能机制。方法提取SD大鼠骨髓间充质干细胞，体外培养、纯化扩增。采用结扎冠状动脉前降支方法复制大鼠心肌梗死模型，将建立的模型随机分为：1）假手术组（仅穿线不结扎血管，n=8）；2）PBS溶液注射组（n=8）；3）干细胞移植组（n=8）。模型制作成功即刻，将骨髓间充质干细胞通过心外膜分4点注射到梗死周边部位。4周后测定血流动力学指标。随后通过Western blot方法测定核因子KB活性，RT-PCR和western blot法测定肿瘤坏死因子α、白介素6 mRNA和蛋白表达。结果1）4周后，假手术组大鼠死亡率20％（2／10），治疗阴性对照组大鼠死亡率33．3％（4／12），干细胞移植组大鼠死亡率20％（2／10），3组死亡率没有统计学差异（P=0．646）；2）与注射PBS溶液相比，骨髓间充质干细胞移植可以改善心肌梗死大鼠血流动力学指标；3）骨髓间充质干细胞可以抑制大鼠梗死心肌核因子kB活性，下调肿瘤坏死因子α和白介素6 mRNA和蛋白表达水平。结论骨髓间充质干细胞可以改善心肌梗死大鼠的心功能，同时可调节大鼠梗死心脏炎症反应。     

稳心颗粒对心力衰竭兔心肌细胞肌浆网钙泵基因和蛋白表达的影响

目的探讨稳心颗粒对心力衰竭（简称心衰）兔肌浆网钙泵基因和蛋白表达的影响及其意义。方法采用主动脉瓣返流联合腹主动脉缩窄制作家兔慢性心衰模型。36只日本大耳白兔随机分为假手术组（Sham）、假手术稳心颗粒组（Sham—drug）、心衰组（HF）、心衰稳心颗粒组（HF—drug）。仅游离结扎右颈总动脉和分离腹主动脉,并不造成主动脉瓣返流和腹主动脉缩窄为假手术处理,稳心颗粒组于主动脉瓣返流术后第二天开始喂药稳心颗粒。连续给药8周后行心脏超声检查观察兔心脏结构和功能的变化,并检测肌浆网钙泵（SERCA2a）和受磷蛋白（PLB）的mRNA和蛋白的表达水平。结果与Sham组比较,HF组左室舒张末期内径（LVEDD）、左室收缩末期内径（LVESD）、左室舒张末期容积（LVEDV）和左室收缩末期容积（LVESV）增大（P〈0．05）,而左室射血分数（LVEF）和左室短轴缩短率（LVFS）降低（P〈0．05）,HF—drug组的LVEDD、LVESD、LVEDV、LVESV均低于HF组（P〈0．05）,LVEF、LVFS高于HF组（P〈0．05）。与Sham组相比,HF组心肌SERCA2amRNA和蛋白表达明显降低（P〈O．05）;然而,HF-drug组心肌SERCA2amRNA和蛋白表达显著高与HF组（P〈O．05）。而PLB的mRNA表达和蛋白表达水平4组间无差异（P〉0．05）。结论稳心颗粒能一定程度上提高心衰家兔SERCA2amRNA和蛋白的表达,增加SERCA2a的活性,可能是稳心颗粒改善心功能的原因。     

黄芪总黄酮对急性心肌梗死大鼠心脏血流动力学及心肌细胞钙电流的作用

目的探讨黄芪总黄酮（TFA）对急性心肌梗死（AMI）大鼠心脏血流动力学及心肌细胞钙电流的作用。方法大鼠开胸左前降支结扎造成AMI，开胸前5min实验组大鼠舌静脉注射剂量为（20mg／kg）TFA溶液50μl，并设正常对照组，AMI组及TFA实验组，后行血流动力学检查；酶解分离心室肌细胞，采用全细胞膜片钳记录技术记录左前降支供血区心外膜细胞的L型Ca^2＋电流（L—Ica）的作用。结果①应用TFA（20mg／kg）后与AMI组比较，血流动力学指标明显改善（P〈0．05，n=7）。与正常对照组相比差异无统计学意义（P〉0．05，n=7）。②应用TFA（20mg／kg）后与AMI对照组比较，L-型钙电流（L—ICa）由给药前的（0．31±1．09）nA增加到给药后的（0．49±0．89）nA（P〈0．01，n=7）。结论TFA可以明显改善AMI大鼠心脏血流动力学，增加心室肌细胞L-型钙电流的幅值。     

β1-AR持久兴奋通过CaMKIIδ-内质网应激凋亡通路导致心力衰竭的机制

目的研究β1-AR持久兴奋通过CaMKIIδ内质网应激（ERS）凋亡通路导致心力衰竭的机制。方法30只SD大鼠随机分成三组，正常对照组（Control）、异丙肾上腺组（Iso）和、异丙肾上腺＋美托洛尔组（Iso＋Meto），每组10只，所有动物均自由进食进水。（1）Iso组大鼠背部皮下注射Iso5mg／（kg·d），连续10d；Control组背部皮下注射相同体积的生理盐水；Iso＋Meto组大鼠背部皮下注射Iso5mg／（kg·d），连续10d，在背部皮下注射Iso前一天开始Meto 10mg／（kg·d）灌胃，连续4周；（2）所有大鼠饲养4周后，采用美国Millar公司P—V Loop导管经颈动脉插管至左心室，使用Pow—erlab生理记录系统测量血流动力学相关指标；统计各组大鼠心脏重量和心脏重量／体重比值；（3）TUNEL法和Caspase-3活性检测心肌细胞凋亡；（4）Western blot分析CaMKIIδERS相关基因（GRP78、CHOP和caspase-12）和凋亡相关基因Bcl-2／Bax的表达水平。结果30只SD大鼠实验过程精神状态好，进食进水正常。（1）与Control组比较，Iso组SD大鼠心脏重塑和血流动力学指标有显著性差异（P〈0．05）；而Iso＋Meto组心脏重塑和血流动力学指标与Iso组相比明显改善（P〈0．05）；（2）TUNEL法原位检测各组大鼠心肌细胞凋亡示与Control组比较，Iso组TUNEL阳性细胞数明显增高（P〈0．05）；而Iso＋Met组明显低于Iso组（P〈0．05）。心肌细胞Caspase-3活性和TUNEL凋亡阳性细胞核指数各组变化一致。Western blot检测心肌细胞凋亡相关基因bcl-2／Bax蛋白表达。与Control组相比，Iso组bcl-2蛋白表达明显降低和Bax蛋白表达明显增加（P〈0．05）；而Iso＋Met组与Iso组相比,bcl-2明显增高和Bax明显降低（P〈0．05）；（3）Western blot分析CaMKIIδp-CaMKIIδ白表达显示，与Control组比较，Iso组CAMKII活性和p-CaMKIIδ蛋白表达明显增加；而Iso＋Meto组与Iso组相比，CAMKII活性和p-     

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.