胰岛素改善糖尿病小鼠骨髓内皮祖细胞动员障碍研究

目的:探讨胰岛素对糖尿病小鼠骨髓内皮祖细胞(EPC)动员的影响及其机制.方法:链脲霉素诱导C57BL/6雄鼠糖尿病,随机分为非糖尿病组、糖尿病组和糖尿病胰岛素治疗组(胰岛素组)3组(各组n=32).入组小鼠饲养2个月后行左侧股动脉高位结扎离断术造成急性单侧后肢缺血模型.胰岛素组皮下注射胰岛素控制血糖.流式细胞术检测术后(0,1,3,7天,n=6-10)外周血单个核细胞中干细胞因子受体/干细胞抗原-1/胎肝激酶-1阳性早期EPC比例.酶联免疫吸附法测定血浆及骨髓血管内皮生长因子(VEGF)及间充质衍生因子1α(SDF-1α)水平.免疫印迹法测定骨髓VEGF、蛋白激酶B、内皮一氧化氮合酶及基质金属蛋白酶-9的蛋白表达及磷酸化水平,酶谱法测定基质金属蛋白酶-9酶活性.结果:糖尿病组呈现缺血诱导的骨髓EPC动员障碍,动员高峰期其外周血早期EPC数量较非糖尿病组显著减少(P<0.01);并伴随血浆及骨髓VEGF及SDF-1α释放减少(P<0.01);骨髓VEGF蛋白表达受抑制、蛋白激酶B与内皮一氧化氮合酶磷酸化减弱及基质金属蛋白酶-9酶活性受损(P<0.05),差异均有统计学意义.胰岛素组缺血术后骨髓EPC动员能力、VEGF及SDF-1α释放水平及骨髓蛋白激酶B/内皮一氧化氮合酶/基质金属蛋白酶-9信号通路活化水平较糖尿病组均明显提高(P<0.05),差异均有统计学意义.结论:胰岛素改善糖尿病动物缺血诱导的骨髓EPC动员障碍,这种作用可能通过恢复受损的SDF-1α/VEGF信号通路活化而介导.     

静脉注射携带缺氧诱导因子的内皮祖细胞对裸鼠缺血下肢血管新生的作用

目的探讨静脉注射携带缺氧诱导因子1α的内皮祖细胞对裸鼠缺血下肢血管新生的作用及机制。方法在体外将腺病毒介导人缺氧诱导因子1α基因入人外周血内皮祖细胞,观察转染后的内皮祖细胞在裸鼠缺血下肢局部的促血管新生作用,并探讨其可能的作用机制。结果转染腺病毒—缺氧诱导因子1α后的内皮祖细胞在细胞内有效并持续表达;移植异种腺病毒—缺氧诱导因子1α内皮祖细胞至Balb/c鼠缺血下肢后可见外源性内皮祖细胞定向作用于缺血部位;移植腺病毒—缺氧诱导因子1α内皮祖细胞组较对照组明显促进体内毛细血管数目增加(P<0.05);mRNA检测提示过表达缺氧诱导因子1α基因可上调其下游的基质细胞衍生因子、CXCR4因子(P<0.05),增加内皮祖细胞招募,同时促血管内皮生长因子水平上调(P<0.05)。结论在体内,转染腺病毒—缺氧诱导因子1α的内皮祖细胞可促进缺血下肢的局部血管新生,这种内皮祖细胞数量的增加可能与基质细胞衍生因子、CXCR4的招募及血管内皮生长因子的分泌增加有关。     

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。     

骨髓间充质干细胞对球囊损伤的大鼠颈总动脉内皮修复的影响

目的 研究骨髓间充质干细胞对球囊损伤的大鼠颈总动脉内皮修复的影响.方法 球囊损伤24只SD大鼠颈总动脉,建立动脉内皮损伤模型,随机分为:(1)治疗组12只SD大鼠,球囊损伤颈总动脉即刻注射1 mL骨髓间充质干细胞溶液;(2)对照组12只SD大鼠,球囊损伤颈总动脉即刻注射1 mL磷酸盐缓冲液.14 d及28 d后,取颈总动脉标本作组织病理切片,行苏木精伊红及血管内皮生长因子受体2、α-平滑肌肌动蛋白免疫组化染色.结果 用苏木精伊红染色观察新生内膜的厚度,14 d及28 d治疗组血管内膜增生均较对照组轻(14 d时0.57±0.06 cm比1.09±0.06 cm,P<0.05;28 d时0.43±0.09 cm比4.72±0.15 cm,P<0.05);用血管内皮生长因子受体-2免疫组化染色观察内皮化程度,治疗组血管内皮细胞覆盖程度高于对照组(14 d时70.8%±1.3%比20.4%±1.1%,P<0.05;28 d时90.2%±1.3%比10.7%±0.4%,P<0.05),治疗组可见血管内皮生长因子受体-2/5-溴脱氧尿核苷双阳性细胞约占血管内皮生长因子受体-2单阳性细胞的50%.α-平滑肌肌动蛋白免疫组化染色观察平滑肌细胞浸润积分,14 d时治疗组平滑肌细胞浸润1.5分,对照组2分;28 d时两组分别为1分和3分.结论 骨髓间充质干细胞可减轻新生内膜增生,加快损伤血管的内皮化进程,减少平滑肌细胞浸润,从而促进球囊损伤的大鼠颈总动脉内皮完整性修复.     

肢体缺血后代偿性血管新生及相关基因表达的动态变化和意义

目的 探讨肢体缺血后代偿性血管新生及相关基因表达的动态变化和意义.方法 股动脉结扎法建立裸鼠肢体缺血模型,分别于术后3天及术后1、2、3、4周观察裸鼠肢体缺血的改变,应用苏木素-伊红染色和CD34免疫组织化学染色观察缺血肌肉组织形态学的改变,应用Western Blotting和逆转录聚合酶链反应检测低氧诱导因子1α、肝细胞生长因子和血管内皮生长因子蛋白和基因在缺血肌肉中表达的动态变化.结果 裸鼠肢体坏疽于术后1～2周最为严重,至术后3～4周时有所改善.缺血后的肌肉纤维萎缩、变形,3～4周时逐渐好转.微血管数于缺血后2周时最多.低氧诱导因子1α和肝细胞生长因子的基因表达于缺血后3天时最为强烈,血管内皮生长因子的基因表达于缺血后1周时达高峰,至缺血后3～4周时,各基因的表述均接近正常对照.结论 肢体缺血发生后,低氧诱导因子1α、肝细胞生长因子和血管内皮生长因子基因表达的变化介导了短暂的血管新生过程,但微血管的生成数量有限,尚不足以代偿肢体缺血的状态.     

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.     

AMD3100对胰腺癌细胞AsPC-1及其移植瘤血管生成的影响

目的 探讨非肽类特异性CXCR4拮抗剂AMD3100对胰腺癌细胞株AsPC-1细胞增殖及其移植瘤生长的影响.方法 AsPC-1细胞分为对照组、基质细胞衍生因子-1(SDF-1α)处理组、AMD3100处理组和SDF-1α+AMD3100处理组,MTT法检测细胞增殖,Western blotting检测血管内皮生长因子(VEGF)表达.建立AsPC-1细胞裸鼠移植瘤模型,应用AMD3100瘤内及瘤周注射,观察移植瘤的生长,免疫组化法检测裸鼠移植瘤的微血管密度(microvessel density,MVD).结果 SDF-1α可明显促进AsPC-1细胞增殖(1.430±0.122对1.002±0.001,P<0.05),而同时应用AMD3100处理能抑制这种增殖反应(0.983±0.068对1.430±0.122,P<0.05);SDF-1α亦可促进AsPC-1细胞VEGF的表达(0.565±0.047对0.439±0.034,P<0.05),而同时用AMD3100处理可抑制这种效应(0.450±0.071对0.565±0.047,P<0.05).AMD3100可抑制AsPC-1细胞皮下移植瘤的生长,第24天的抑瘤率达59.5%,移植瘤的MVD显著减少(28.56±6.94对98.75±20.60,P<0.01).结论 AMD3100在体外和体内均可抑制AsPC-1细胞增殖及其移植瘤的血管生成.     

高表达整合素连接激酶对大鼠骨髓间充质干细胞旁分泌功能的影响

目的 研究转染腺病毒介导的整合素连接激酶(ILK)对大鼠骨髓间充质干细胞旁分泌作用的影响.方法 构建携带人野生型整合素连接激酶质粒cDNA和人重组绿色荧光蛋白的腺病毒载体(adeno-ILK)和空载腺病毒(MSC),通过全骨髓贴壁法分离培养骨髓间充质干细胞.采用无血清培养转染adeno-ILK(MSC-ILK组)或空载腺病毒(MSC组),通过流式细胞术检测人重组绿色荧光蛋白从而测定转染效率,确定最佳的病毒感染复数.提取骨髓间充质干细胞mRNA,用Real-time PCR测定血管内皮生长因子、成纤维细胞生长因子2和胰岛素样生长因子1的基因表达量.结果 MSC-ILK组旁分泌因子血管内皮生长因子、成纤维细胞生长因子2和胰岛素样生长因子1的基因表达量分别是MSC组的 8.2±0.4、2.6±0.2及2.4±0.2倍(P＜0.05或P＜0.01).结论 整合素连接激酶基因转染可明显促进骨髓间充质干细胞表达旁分泌因子血管内皮生长因子、成纤维细胞生长因子2和胰岛素样生长因子1.     

内皮型一氧化氮合酶在骨髓内皮祖细胞治疗缺血性脑血管病中的作用

内皮型一氧化氮合酶( eNOS)/NO在血管新生和血管发生中起着重要作用.脑缺血时,缺血组织内的eNOS表达增加,促进NO的释放,有利于骨髓内皮祖细胞(EPCs)的动员、迁移、归巢.而EPCs能在缺血组织分化为成熟的血管内皮细胞,修复损伤的内皮,参与新生血管的形成,改善组织细胞的供血.本文就eNOS在EPCs治疗缺血性脑血管病中的可能作用做一综述.     

缺血预适应对大鼠急性心肌梗死缺氧诱导因子1α表达的影响及蛋白激酶C信号通路的作用

目的研究缺血预适应后急性心肌梗死缺氧诱导因子1α表达的变化及蛋白激酶C的信号传导作用。方法选择雄性Wistar大鼠32只,随机分为4组:缺血预适应组、单纯急性心肌梗死组、缺血预适应加蛋白激酶C抑制剂组和假手术组。缺血预适应加蛋白激酶C抑制剂组预适应前10min静脉注射蛋白激酶C抑制剂白屈菜季铵碱5mg/kg,然后结扎左前降支;假手术组不结扎左前降支。1天后,10%水合氯醛腹腔注射麻醉,活体取出心脏,测量心肌梗死面积,测定缺氧诱导因子1α和血管内皮生长因子mRNA表达,蛋白印记分析检测缺氧诱导因子1α和血管内皮生长因子蛋白的表达,术前术后测定血管内皮生长因子含量。结果缺氧诱导因子1α mRNA表达,缺血预适应组明显增加(P<0.01),缺血预适应加蛋白激酶C抑制剂组与单纯急性心肌梗死组差异无显著性(P>0.05),其蛋白表达有相同的变化。缺血预适应加蛋白激酶C抑制剂后,缺氧诱导因子1α mRNA表达明显减少(P<0.01),其靶基因血管内皮生长因子mRNA表达也明显减少,其蛋白表达有相同的变化。缺血预适应组心肌梗死面积(24.18%±2.25%)明显小于缺血预适应加蛋白激酶C抑制剂组(38.11%±2.94%)和单纯急性心肌梗死组(37.73%±3.32%;P<0.01)。结论缺氧诱导因子1α的表达是缺血预适应重要的内源性保护机制,其表达是依赖于蛋白激酶C的激活,蛋白激酶C是缺氧诱导因子1α表达的重要信号传导通路。     

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.     

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.     

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.     

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.