内皮祖细胞培养鉴定及其体外修复球囊损伤血管的实验研究

目的观察体外培养人脐带静脉血(脐血)内皮祖细胞(EPCs)对球囊损伤血管的修复作用。方法采用贴壁选择法培养人脐血内皮祖细胞,分别采用流式细胞术、DiIacLDL吞噬试验及Ⅷ因子相关抗原免疫组化对培养细胞进行鉴定。球囊损伤法制备去内膜兔腹主动脉段,与EPCs共孵育使再内皮化。结果体外成功培养出人脐血内皮祖细胞,流式细胞术分析显示培养6d后,白细胞分化抗原34(CD34)、血管内皮钙黏素(VECadherin)、血管内皮生长因子受体2(VEGFR2)及白细胞分化抗原133(AC133)阳性细胞比例分别为(65±12)%、(45±10)%、(31±10)%与(20±1)%,CD34VECadherin、VEGFR2VECadherin、CD34AC133及VEGFR2AC133双阳性细胞比例分别为(42±10)%、(28±15)%、(19±1)%与(15±7)%,DiIacLDL吞噬试验及Ⅷ因子相关抗原免疫组化阳性。人脐血EPCs与去内膜兔腹主动脉共培育使之再内皮化,形成新内膜。结论利用脐血可成功培养出内皮祖细胞,体外培养内皮祖细胞可修复内皮损伤血管。     

自体内皮祖细胞移植修复兔动脉内皮损伤

目的观察不同数量的内皮祖细胞移植对内膜球囊损伤血管修复的影响。方法密度梯度离心法分离兔外周血单个核细胞,以EGM-2培养基培养7天,获得兔外周血内皮祖细胞。分高细胞数量移植组(5×105个细胞)和低细胞数量移植组(2×105个细胞),将相应数量的内皮祖细胞重悬于100μL生理盐水中移植至兔内膜球囊损伤血管局部,对照组仅注入100μL生理盐水。同时用CM-DiI标记内皮祖细胞移植,进行细胞示踪。4周后,处死动物,荧光显微镜下观察CM-DiI标记细胞的分布,并测量各组内膜损伤血管再生内皮覆盖率和新生内膜增生程度。结果荧光显微镜下发现荧光标记的内皮祖细胞分布在血管的中膜层、新生内膜层和内膜表面。内皮祖细胞移植可显著促进内膜损伤血管的再内皮化,以高细胞数量移植组为著,同时内皮祖细胞移植也大大减少了新生内膜的形成。结论内皮祖细胞移植可修复内膜球囊损伤血管,高细胞数量移植有更显著的效果。     

吲哚美辛对受损颈动脉再内皮化的作用及机制

目的研究吲哚美辛对受损颈动脉再内皮化的作用及机制。方法在SD大鼠体内通过球囊法制备颈动脉损伤模型,腹腔注射吲哚美辛,观察其对血管内皮修复的影响;密度梯度离心法获得大鼠单核细胞,应用EGM-2培养基体外诱导培养获得内皮祖细胞(EPCs)。采用不同浓度的吲哚美辛处理晚期EPCs24 h后,MTT法检测细胞的增殖活性,细胞差异贴壁法检测EPCs的粘附能力,改良的Boyden小室检测EPCs的迁移能力,Matrigel基质胶检测EPCs的成血管能力。结果吲哚美辛可明显促进损伤血管内皮的修复,其机制可能与吲哚美辛增强EPCs的增殖、粘附、迁移及成血管能力有关。结论吲哚美辛促进了损伤血管内皮的再内皮化进程。     

肾毒性物质对甲酚抑制内皮祖细胞增殖和eNOS磷酸化

目的:观察对甲酚对人外周血晚期内皮祖细胞(EPCs)的体外增殖和内皮一氧化氮合酶(eNOS)活性的影响.方法:用密度梯度离心法分离健康成人外周血中的单个核细胞,在含有血管内皮生长因子等的培养基中培养.通过形态学、免疫荧光、流式细胞分析鉴定细胞,在贴壁细胞中加入不同浓度对甲酚,用细胞计数和集落生成实验法评价对甲酚对EPC...     

人外周血内皮祖细胞的分离培养及鉴定

目的:从人外周血中分离培养内皮祖细胞(EPCs),并探讨其体外诱导培养的条件。方法:密度梯度离心法分离正常人外周血单个核细胞,置于鼠尾胶原包被的培养瓶中进行体外培养,流式细胞术和免疫荧光法检测分化细胞表面特异性抗原标记物的表达。结果:人外周血单个核细胞在鼠尾胶原包被的培养瓶中培养后呈梭形,并表达内皮细胞的特异性抗原CD34和KDR,和干/祖细胞抗原CD133。提示这些培养细胞既具有内皮细胞的表面标志和功能,又具有祖细胞特性。结论:成功从外周血中培养出EPCs。鼠尾胶原可取代EPCs培养中常用的纤维连接蛋白,是体外分离培养外周血EPCs的更节省的一种方法。     

人外周血内皮祖细胞的分离培养及鉴定

目的从人外周血中分离培养内皮祖细胞(EPCs),并探讨其体外诱导培养的条件.方法密度梯度离心法分离正常人外周血单个核细胞,置于鼠尾胶原包被的培养瓶中进行体外培养,流式细胞术和免疫荧光法检测分化细胞表面特异性抗原标记物的表达.结果人外周血单个核细胞在鼠尾胶原包被的培养瓶中培养后呈梭形,并表达内皮细胞的特异性抗原CD34和KDR,和干/祖细胞抗原CD133.提示这些培养细胞既具有内皮细胞的表面标志和功能,又具有祖细胞特性.结论成功从外周血中培养出EPCs.鼠尾胶原可取代EPCs培养中常用的纤维连接蛋白,是体外分离培养外周血EPCs的更节省的一种方法.     

内皮祖细胞在血管新生和抑制血管损伤后再狭窄中的作用

内皮祖细胞（EPCs）也叫成血管细胞，是一种成体干细胞。日益增多的证据显示，通过各种途径向体内移植或动员机体自身的EPCs可以提高缺血组织的血管新生能力，促进损伤血管的再内皮化、抑制新生内膜的增生，从而为治疗缺血性心脏病和血管介入治疗后再狭窄提供了新思路。     

兔血管内皮祖细胞的分离及诱导分化

目的:探讨自兔骨髓中分离及诱导血管内皮祖细胞(EPCs)的方法。方法:抽取兔骨髓,Ficoll离心液梯度离心,分离出骨髓单个核细胞,用含有血管内皮生长因子(VEGF)、碱性纤维细胞生长因子(bFGF)、胰岛素样生长因子-1(IGF-1)的M199培养液诱导培养EPCs,透射电镜及免疫荧光法对所培养细胞进行鉴定。结果:细胞诱导培养48~72h后即可见有细胞贴壁,逐渐变成梭形。培养至8d左右,可观察到数条由梭形贴壁细胞直线生长连接而成的条索,7~10d出现多个细胞团,贴壁的梭形细胞开始从细胞团的边缘出芽长出,培养至14d左右,细胞逐渐融合,连接成大片条索状结构。透射电镜及免疫荧光检查证实为血管内皮祖细胞。结论:梯度离心法自兔骨髓获得单个核细胞,在体外经诱导分化可获得血管内皮祖细胞。     

姜黄素对内皮祖细胞功能及内皮型一氧化氮合酶的影响

目的观察姜黄素对体外培养的人外周血内皮祖细胞（EPCs）数量及功能的影响。方法采用贴壁选择法培养人外周EPCs,培养6 d后,收集贴壁细胞并加入姜黄素（0、5、10、15μmol/L）培养一定时间（6、12、24和48 h）。荧光显微镜鉴定EPCs,分别观察EPCs的体外增殖、黏附、迁移、体外血管生成能力。Griess法检测NO分泌量,并RT-PCR半定量测定eNOS基因表达。结果姜黄素增加外周血EPCs的数量,改善EPCs的体外增殖、黏附、迁移、体外血管生成能力（P<0.01）。并上调eNOS基因表达,促进EPCs来源的NO的释放（P<0.01）。结论姜黄素增加体外EPCs数量及改善其功能,上调eNOS基因表达并增加EPCs来源的NO分泌。     

移植到裸鼠体内的糖尿病患者外周血内皮祖细胞再内皮化能力的观察

目的探讨糖尿病患者外周血内皮祖细胞(EPCs)体内再内皮化能力的变化。方法培养、鉴定EPCs;构建裸鼠颈动脉内膜损伤模型。分组:健康组移植健康人EPCs,糖尿病组移植糖尿病患者EPCs,PBS对照组移植PBS。显微镜下观察伊文斯蓝染色损伤内膜再内皮化的情况,HE染色观察损伤内膜与中膜的面积的厚度,借助Image-Pro Plus 5.0图像分析系统计算再内皮化率及内膜/中膜比值。结果糖尿病组内膜损伤修复面积(23.78±1.95)%小于健康组(35.93±4.37)%(P0.05),内膜/中膜比值糖尿病组(74.50±6.80)%大于健康组(59.40±5.00)%(P0.05)。结论糖尿病患者EPCs体内再内皮化及抑制内膜增生能力降低,可能是其血管内皮损伤及内皮修复能力下降的重要机制。     

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.