培哚普利改善糖尿病大鼠急性心肌梗死后内皮祖细胞动员

目的 观察培哚普利对糖尿病大鼠急性心肌梗死后骨髓内皮祖细胞动员和血管新生障碍的影响,并探讨其可能的分子机制.方法 高脂饮食联合小剂量链脲霉素诱导雄性SD大鼠糖尿病模型,成模4周后结扎冠状动脉左前降支造成急性心肌梗死模型.术后将大鼠随机分至培哚普利治疗组和模型组(各组n=15).流式细胞术检测术前及术后不同时间点(1、3、5、7、14和28天)外周血CD45-/low+CD133+ KDR+内皮祖细胞数量,ELISA法检测不同时间点血浆血管内皮生长因子水平.CD31免疫荧光染色法评估心肌梗死1个月后心肌梗死周围区血管新生情况.超声心动图评估心功能改变.免疫印迹法测定骨髓细胞中内皮祖细胞动员相关通路蛋白的表达.结果 培哚普利治疗可显著改善糖尿病时缺血诱导的内皮祖细胞动员障碍,使循环内皮祖细胞峰值明显升高(103±37个/106单核细胞比58±19个/106单核细胞,P＜0.05),同时伴有血浆血管内皮生长因子水平升高,骨髓内皮祖细胞动员通路的信号分子蛋白激酶B与内皮型一氧化氮合酶磷酸化增加以及基质金属蛋白酶9的表达升高(P＜0.05).与模型组相比,培哚普利治疗后糖尿病大鼠心肌梗死周围区新生毛细血管密度显著增加,射血分数及左心室短轴缩短率明显改善(所有P＜0.05).结论 培哚普利能改善糖尿病大鼠缺血诱导的骨髓内皮祖细胞动员障碍,增加缺血区血管新生,最终改善心功能.这种作用可能通过活化骨髓内皮祖细胞动员相关通路介导.     

氨氯地平对糖尿病大鼠心肌梗死后骨髓内皮祖细胞动员及血管新生的改善作用

目的：观察氨氯地平对糖尿病大鼠心肌梗死（心梗）后骨髓内皮祖细胞(EPC)动员和血管新生障碍的改善作用以及对心功能的影响,并探讨其可能的分子机制。
　　方法：180~200g SPF级雄性Sprague-Dawley大鼠60只随机分为两部分：糖尿病大鼠（n=40）给予高脂饲料饲养四周后,腹腔注射30 mg/kg链脲佐菌素;非糖尿病大鼠(n=20)为正常饮食饲养。40只糖尿病大鼠结扎冠状动脉左前降支造成急性心梗模型。术后将大鼠随机分成对照组(n=20),每日予以0.5%羧甲基纤维素钠溶剂1ml灌胃,治疗组(n=20)每日予以氨氯地平2 mg/kg灌胃,继续高脂喂养四周。流式细胞术检测术前及术后不同时间点（第1、3、5、7、14和28 d）外周血CD45-/low+/CD133+/KDR+早期EPC数量,酶联免疫吸附法检测血浆血管内皮生长因子(VEGF)水平。CD31免疫荧光染色法评估心梗周围区血管新生情况。超声心动图评估心功能。免疫印迹法测定骨髓细胞中EPC动员相关信号通路蛋白的表达。
　　结果：外周血CD45-/low+/CD133+/KDR+ EPC水平术后峰值治疗组（第7 d,112±30/106单核细胞）较对照组（第5 d,55±10/106单核细胞）升高;血浆VEGF水平在心梗后峰值治疗组[第7 d ,（5.63±1.33）ng/L]较对照组[第5 d,(3.68±0.98) ng/L]升高;骨髓细胞蛋白激酶B与内皮型一氧化氮合酶的活化水平及基质金属蛋白酶-9的表达增加;心梗周围区新生毛细血管密度治疗组大鼠较对照组显著增加,左心室射血分数及左心室短轴缩短率明显提高。上述比较差异均有统计学意义（P<0.05~0.01）。
　　结论：氨氯地平治疗改善糖尿病大鼠缺血诱导的骨髓EPC动员障碍,缺血区血管新生以及心梗后心功能。这种作用可能通过改善VEGF/内皮型一氧化氮合酶信号通路活化而介导。     

内皮祖细胞与冠心病患者CD14~(++)CD16~+单核细胞共培养后移植心肌梗死大鼠对血管密度及心肌梗死面积的影响

目的研究内皮祖细胞(endothelial progenitor cell,EPC)与冠心病患者CD14~(++)CD16~+单核细胞共培养后移植心肌梗死大鼠对血管密度及心肌梗死面积的影响。方法比较冠心病患者及健康志愿者外周血中CD14~(++)CD16~+单核细胞水平,将冠心病患者外周血中分选出的CD14~(++)CD16~+单核细胞与健康志愿者的EPC体外共培养观察其对EPC的增殖、迁移、黏附的影响后,将CD14~(++)CD16~+单核细胞与EPC共培养的细胞移植进入心肌梗死大鼠模型,观察其对大鼠缺血心肌新生血管密度、坏死心肌面积及心功能指标的影响。结果冠心病患者外周血中CD14~(++)CD16~+单核细胞水平显著高于健康志愿者(P 0. 05);冠心病患者外周血中CD14~(++)CD16~+单核细胞与健康志愿者的EPC体外共培养后增殖能力、黏附细胞数、迁移细胞数均显著降低(P 0. 05);与假手术组大鼠相比,移植4周后模型组、EPC组、CD14~(++)CD16~+共培养组左心室梗死面积均显著升高(P 0. 05),且EPC组CD14~(++)CD16~+共培养组模型组(P 0. 05),新生毛细血管密度、左心室射血分数、左心室短轴缩短率均显著降低(P 0. 05),且EPC组 CD14~(++)CD16~+共培养组模型组(P 0. 05)。结论冠心病患者外周血中CD14~(++)CD16~+单核细胞水平升高,并抑制EPC修复及逆转左室重塑,改善心肌功能的生理作用。     

急性心肌梗死伴2型糖尿病患者内皮祖细胞动员障碍

目的观察急性心肌梗死伴2型糖尿病患者血浆缺血相关因子血管内皮生长因子和基质细胞衍生因子水平,骨髓内皮祖细胞动员是否存在障碍,以及基质细胞衍生因子、血管内皮生长因子-内皮祖细胞动员通路是否存在异常。方法采用密度梯度离心法分离外周血单个核细胞,用流式细胞仪检测急性心肌梗死后不同时间点(1、3、5、7、14和28天)外周血CD45-/low /CD34 /CD133 /KDR 早期内皮祖细胞数量。酶联免疫吸附法检测血浆中血管内皮生长因子、基质细胞衍生因子以及高敏C反应蛋白的浓度。结果急性心肌梗死伴2型糖尿病患者外周血内皮祖细胞动员高峰(第7天)较急性心肌梗死非糖尿病患者(第5天)延迟且显著减少[(140±48)/106比(246±100)/106,P<0.05]。糖尿病组血浆血管内皮生长因子(第5天:277±95ng/L比168±35ng/L,P<0.05)、基质细胞衍生因子(第5天:3835±402ng/L比3287±384ng/L,P<0.05)以及高敏C反应蛋白(第3天:55.55±14.88mg/L比36.92±14.83mg/L,P<0.05)在高峰点的水平显著高于非糖尿病组。结论糖尿病患者心肌梗死后组织缺血程度较重,但组织缺血后基质细胞衍生因子、血管内皮生长因子-内皮祖细胞动员通路存在障碍,这可能是糖尿病患者缺血后血管新生功能障碍,发生急性心肌梗死后预后较差的原因之一。     

内皮祖细胞移植治疗大鼠急性心肌梗死的实验研究

目的探讨大鼠内皮祖细胞(EPCs)移植于梗死心肌的增殖分化情况及对心功能的影响。方法分离培养大鼠EPCs,免疫荧光法检测其CD34+、CD133+和Flk-1+的表达。将SD大鼠冠状动脉左前降支结扎制造急性心肌梗死模型后,在梗死心肌处植入DAPI标记的EPCs(实验组)或M 199培养液(对照组)。移植后1周及4周,心脏超声检查心功能,并对梗死区心肌组织进行移植细胞形态学检查及毛细血管密度测定,逆转录聚合酶链反应及酶联免疫吸附测定梗死周边区血管内皮生长因子(VEGF)的表达。结果培养获得EPCs,其表型为CD34+、CD133+和Flk-1+。植入梗死区的EPCs可以分化为血管内皮细胞,实验组梗死心肌处血管密度较对照组明显增高(P<0.01),并且VEGF基因及蛋白表达在移植后1周均较对照组明显增高(P<0.01)。移植后4周,实验组大鼠左心室射血分数及左心室短轴缩短率较对照组明显提高(P<0.01);而移植后1周,两组大鼠超声检测心功能各指标变化不明显。结论同种异体EPCs移植到梗死心肌大鼠缺血心肌能分化为毛细血管内皮细胞,促进梗死后心肌血管新生,改善心功能。     

心肌梗死后Akt介导的间质细胞源因子1调控循环血内皮祖细胞归巢

目的 探讨急性心肌梗死引起的缺血心肌中Akt-SDF-1信号分子变化情况及其与内皮祖细胞归巢之间的关系.方法 利用左前降支冠状动脉结扎术制备大鼠心肌梗死模型后,局部心肌注射Akt抑制剂或等体积二甲基亚砜,分别于术后1、7、14、28天取材,用酶联免疫吸附测定法检测Akt及SDF-1表达变化情况,用流式细胞仪(FACS)及免疫组织化学法检测循环血及局部心肌内皮祖细胞数量变化情况,免疫组织化学法计数血管数量.结果 酶联免疫吸附测定结果显示,急性缺血诱导局部心肌Akt和SDF-1表达水平呈进行性升高,FACS及免疫组织化学检测也显示循环血及心肌局部内皮祖细胞数量呈进行性增加,14天达高峰,28天后以上各项指标明显回落,血管计数也显示相同的动态变化,Akt抑制剂显著抑制上述各项指标的动态演变,使其在心肌梗死后各时间点无显著性差异.结论 心肌梗死后缺血心肌可通过Akt-SDF-1信号通路调控循环血内皮祖细胞归巢、梗死心脏局部干细胞生存和血管新生.     

重组腺相关病毒介导CD151转染心肌梗死小型猪促进心肌功能性血管形成

目的 观察重组腺相关病毒(rAAV)介导的CD151基因转染促小型猪心肌梗死后心肌血管形成的有效性.方法 结扎小型猪冠状动脉左前降支建立心肌梗死模型,梗死区及梗死周围心肌分别注射rAAV-CD151、rAAV-anti-CD151和rAAV-绿色荧光蛋白(rAAV-GVP).8周后用Western blot分析心肌组织CD151蛋白表达,免疫组化检测心肌微血管密度和小动脉密度,用13N-NH3电子发射计算机断层心脏显影(PET)评价心肌血流灌注.结果 CD151基因转染促进心肌组织局部CD151高表达.rAAV-CD151组心肌微血管密度为(83.8±6.7)个/mm2,明显高于正常对照组的(33.2±4.5)个/mm2和rAAV-GFP组的(41.6±5.6)个/mm2,均P＜0.05;rAAV-CD151组小动脉密度为(16.4±2.5)个/mm2,也明显高于正常对照组的(6.6±2.3)个/mm2和rAAV-GFP组(8.4±1.6)个/mm2,均P＜0.05.rAAV-anti-CD151组心肌微血管密度和小动脉密度明显低于其他各组.13N-NH3 PET心肌血流灌注显像示rAAV-CD151组缺血区心肌血流灌注明显增加,rAAV-anti-CD151组血流灌注减少.结论 CD151基因转染能增加小型猪局部心肌组织的CD151表达,明显增加缺血心肌微血管和小动脉的生成,并显著增加心肌血流灌注.     

骨髓间质干细胞移植治疗急性心肌梗死的实验研究

目的 研究同种异体骨髓间质干细胞（bone marrow mesenchymak stem cells MSCs）移植至梗死心肌后对兔的一般情况、心功能、梗死面积和血管新生的影响.方法 开胸结扎冠状动脉左前降支建立急性心肌梗死动物模型,将5-溴脱氧尿核苷（bromodeoxyuridine,BrdU）标记的MSCs注入心肌梗死区.测定各组手术前后体重和心功能并取出心脏作相关检查.结果 与急性心肌梗死组比较,MSCs组术后一般情况好,6周时体重明显增加（P＜0.05）,术后3周、6周左心室射血分数（left ventricular ejection fraction,LVEF）均明显增加（P＜0.05）,梗死面积明显缩小（P＜0.05）,毛细血管数目显著增多（P＜0.01）.MSCs组心肌梗死区可见BrdU阳性细胞,电镜下可见不成熟的心肌样细胞.结论 同种异体MSCs移植治疗急性心肌梗死除提高心功能外,尚可改善兔子的一般情况,增加体重.     

心复康口服液对心力衰竭大心功能及心肌中链酰基辅酶A脱氢酶基因表达的影响

目的 观察心复康口服液对心肌梗死后心力衰竭大鼠血流动力学、心功能及梗死周围心肌中链酰基辅酶A脱氢酶基因表达的影响.方法 采用结扎SD大鼠左冠状动脉前降支致心肌梗死后心力衰竭大鼠模型,用心复康口服液于术后24 h灌胃给药至6周,以卡托普利作为阳性对照药.给药4周后观察大鼠心脏功能、血流动力学及梗死周围心肌中链酰基辅酶A脱氢酶基因表达的变化.结果 模型组与假手术对照组比较,左心室舒张末压升高,左心室收缩压、心排血量、左心室内压最大上升速率、左心室内压最大下降速率均显著降低,梗死周围心肌中链酰基辅酶A脱氢酶基因表达下调(P＜0.01).与模型组比较,心复康口服液和卡托普利皆可显著降低心梗后心衰大鼠左心室舒张末压(P＜0.05),升高左心室收缩压、心排血量、左心室内压最大上升速率、左心室内压最大下降速率(P＜0.05或P＜0.01);梗死周围心肌中链酰基辅酶A脱氢酶基因表达上调(P＜0.01).结论 心复康口服液可改善心肌梗死后心力衰竭模型大鼠心脏功能,并可上调心肌中链酰基辅酶A脱氢酶基因表达.     

2型糖尿病大鼠急性心肌缺血对心肌新生血管的影响

目的探讨糖尿病急性心肌缺血对心肌新生血管生成的影响及其机制。方法清洁级健康雄性8周龄sD大鼠52只,任意选取其中20只以高脂喂养联合小剂量链脲佐菌素建立糖尿病大鼠模型;以糖尿病建模成功的大鼠与16只正常大鼠以结扎冠状动脉前降支制作急性心肌梗死模型;另外16只大鼠只开胸不造模、不给药作为假手术组。心肌梗死造模术后2周,处死各组动物,取左心室梗死区及相邻区域心肌,采用免疫组化方法检测各组大鼠缺血周边区微血管密度（MVD）,Western blotting法检测血管内皮生长因子（VEGF）及其信号转导蛋白[磷酸化蛋白激酶B（p-Akt）、内皮型一氧化氮合酶（eNOS）、磷酸化eNOS（p-eNOS）]、内皮抑素蛋白的表达。组间数据比较采用单因素方差分析,样本均数间两两比较采用q检验,两组计量资料比较采用t检验。结果3组成活大鼠分别为：糖尿病心肌梗死组12只,非糖尿病心肌梗死组（对照组）13只,假手术组14只。与对照组相比,糖尿病组缺血心肌新生血管明显减少（19．7±3．8比14．2±3．6,q=2．98,P〈0．05）。糖尿病组、假手术组VEGF表达显著低于对照组（分别为0．89±0．12、0．65±0．23和1．53±0．20,F=6．52,P〈0．01）。糖尿病组和对照组p-Akt和eNOS蛋白表达差异无统计学意义（t值分别为3．02、2．78,P〉0．05）,但糖尿病组p-eNOS蛋白表达显著减少（0．49±0．09比1．16±0．12,t=5．68,P〈0．05）。糖尿病组内皮抑素表达显著高于对照组（4．6±0．6比2．3±0．4,t=8．63,P〈0．05）。结论糖尿病大鼠急性缺血心肌的新生血管生成低下,VEGF转导通路在多个水平上参与其中,并与内皮抑素共同作用。     

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.