大鼠胸主动脉平滑肌细胞的培养与鉴定

目的探讨平滑肌细胞培养方法,了解生长特性。方法用组织贴块法培养大鼠胸主动脉平滑肌细胞,用相差显微镜观察其生长情况,用免疫荧光染色分析其抗原的表达。结果培养3日可见组织块周边有细胞长出,2周达亚融合,传代后细胞“谷峰”生长明显。SM-α-肌动蛋白和Calponin抗体免疫荧光染色呈阳性。结论组织贴块法是简单、经济、高效的平滑肌细胞培养方法,为心血管疾病发病机制的研究提供了理想的细胞模型。     

大鼠冠状动脉平滑肌细胞的原代培养及鉴定

目的探讨大鼠冠状动脉平滑肌细胞的原代培养方法,为冠状动脉疾病的研究提供理想的细胞模型。方法无菌条件下分离大鼠冠状动脉,采用酶消化法分离培养平滑肌细胞,进行形态学观察及台盼蓝染色测定细胞活力,采用免疫荧光染色技术对平滑肌α肌动蛋白进行鉴定。结果形态学、免疫荧光染色鉴定表明培养的细胞为血管平滑肌细胞,细胞存活率达97%。原代培养14d左右即可进行传代,可以传6代以上,且细胞形态及生长状态良好。结论酶消化法分离大鼠冠状动脉平滑肌细胞,方法简单、高效,细胞纯度高、且活性好、生长迅速。     

组织块贴壁法培养SD大鼠腹主动脉平滑肌细胞及鉴定

目的培养、鉴定SD大鼠腹主动脉平滑肌细胞。方法采用组织块贴壁法分离培养SD大鼠腹主动脉平滑肌细胞,胰蛋白酶消化传代,并应用相差显微镜和平滑肌肌动蛋白对细胞进行形态学和免疫组化鉴定。结果培养出的平滑肌细胞呈长梭形生长,镜下可见培养细胞呈典型的"峰-谷"状生长,高倍镜下可见胞质内大量棕色、与细胞长轴平行的纤维细丝,免疫组化染色显示胞浆内平滑肌肌动蛋白阳性表达。结论组织块贴壁法体外培养SD大鼠腹主动脉平滑肌细胞操作简单、纯度较高、可行性高。     

Ⅱ型胶原酶/弹性蛋白酶消化法培养大鼠血管平滑肌细胞

为快速、成功培养大鼠胸主动脉血管平滑肌细胞,在无菌条件下分离大鼠胸主动脉,并剥离外膜及内皮,然后用Ⅱ型胶原酶／弹性蛋白酶消化血管中层,2h左右完全消化成单个细胞时,离心收集细胞,然后重悬,接种细胞,24h后便可见细胞贴,4－5天便可传代,传至第3代时,用台盼蓝检查细胞存活率为96％,光镜和免疫组织化学鉴定培养细胞,培养细胞纯度为97％,与国内报道的贴块法及消化法相比,用酶解法培养血管平滑肌细胞的培养周期缩短4－5天,能更好的保护细胞,维持细胞形状。     

大鼠胸主动脉血管平滑肌细胞的原代培养和鉴定

目的探讨大鼠胸主动脉血管平滑肌细胞的原代培养方法,了解生长特性,为血管增生性疾病的治疗研究提供试验材料。方法采用组织贴块法培养大鼠胸主动脉血管平滑肌细胞,用倒置相差显微镜观察其生长情况,用免疫组化染色做鉴定,台盼蓝染色进行活力检测。结果85%的组织块接种存活,原代培养后2周左右即可传代,至少可以传8代以上,并且细胞形态、生长特点不发生明显的改变,6代的血管平滑肌细胞纯度达97%以上。台盼蓝染色约有94%以上的细胞为活细胞。镜下培养的血管平滑肌细胞呈典型的"谷峰状"生长,免疫组化染色显示胞浆内α平滑肌肌动蛋白阳性表达。结论正确地利用组织贴块法可以简单、经济、高效地培养出血管平滑肌细胞,为血管增生性疾病的治疗研究提供了理想的细胞模型。     

一套系统培养鼠主动脉血管壁细胞简单可靠的方法

目的 探讨一套系统培养鼠主动脉血管壁成分细胞的简单、可重复的方法.方法 分别采用植块贴壁法进行主动脉血管平滑肌细胞和血管外膜成纤维细胞、结扎贴壁法进行血管内皮细胞的原代培养,胰酶消化传代;差速贴壁法及自然纯化法进行细胞纯化;转化生长因子β1诱导血管外膜成纤维细胞转化为肌成纤维细胞;相差显微镜及α-平滑肌肌动蛋白(α-SMA)、血小板/内皮细胞黏附分子(CD31)、波形蛋白(Vimentin)抗体两两联合的方式分别进行形态学和免疫细胞化学鉴定.结果 组织及细胞活性良好,血管平滑肌细胞及血管外膜成纤维细胞原代培养周期为10～12天,血管内皮细胞为12～14天.传代培养周期为7～10天.经纯化传代后的细胞纯度达95%～100%.血管平滑肌细胞呈典型的"峰-谷"状生长,α-SMA(+)/Vimentin(-);血管内皮细胞呈"铺路石"样外观,CD31(+)/α-SMA(-);血管外膜成纤维细胞形态和血管平滑肌细胞不易区分,Vimentin(+)/α-SMA(-),诱导的肌成纤维细胞Vimentin(+)/α-SMA(+).原代细胞传至10代以上仍未见生长活力减退.结论 我们建立了一套系统培养纯度高、生长状态良好的大鼠主动脉血管内皮细胞、血管平滑肌细胞、血管外膜成纤维细胞及肌成纤维细胞的简单可靠、重复性好的方法.该法对培养小鼠主动脉壁成分细胞仍然适用.     

萘哌地尔衍生物YMⅢ对血管紧张素Ⅱ诱导大鼠胸主动脉平滑肌细胞增殖的抑制作用

目的 研究萘哌地尔衍生物YMⅢ对血管紧张素Ⅱ诱导Wistar大鼠和自发性高血压大鼠胸主动脉平滑肌细胞增殖的抑制作用及机制.方法 将Wistar大鼠和自发性高血压大鼠胸主动脉平滑肌细胞进行体外培养,采用四甲基偶氮唑盐比色法检测YMⅢ对胸主动脉平滑肌细胞和血管紧张素Ⅱ诱导胸主动脉平滑肌细胞增殖的影响;用实时逆转录聚合酶链反应技术检测血管紧张素原、c-myc mRNA表达.结果 未经血管紧张素Ⅱ处理的胸主动脉平滑肌细胞.0.1 μmol/L YMⅢ能抑制Wistar大鼠和自发性高血压大鼠胸主动脉平滑肌细胞增殖;YMⅢ(0.01、0.05、0.1 μmol/L)能呈浓度依赖性抑制血管紧张素Ⅱ所致Wistar大鼠和自发性高血压大鼠胸主动脉平滑肌细胞的增殖;YMⅢ(0.05～0.1 μmol/L)作用能使血管紧张素Ⅱ所致Wistar大鼠血管紧张素原、c-myc mRNA表达水平下调,而各浓度YMⅢ均能下调血管紧张素Ⅱ所致自发性高血压大鼠胸主动脉平滑肌细胞的血管紧张素原、c-myc mRNA表达.结论 YMⅢ明显抑制血管紧张素Ⅱ诱导大鼠胸主动脉平滑肌细胞的增殖,且抑制增殖作用在自发性高血压大鼠比在Wistar大鼠明显,其作用机制可能与下调血管紧张素原、c-myc mRNA的表达有关.     

高糖刺激血管平滑肌细胞血管内皮生长因子的表达

平滑肌细胞可分泌血管内皮生长因子 (ＶＥＧＦ) 〔1〕,在糖尿病视网膜组织中ＶＥＧＦ表达明显增高〔2〕。ＶＥＧＦ的主要功能是增加血管通透性、促进内皮细胞增生和促进新血管增殖。推测ＶＥＧＦ与糖尿病视网膜血管病的病理变化直接相关。本研究探讨Ｄ 葡萄糖浓度、作用时间以及波动糖浓度刺激对ＶＥＧＦ表达的影响。一、材料和方法1.血管平滑肌细胞培养 :无菌条件下取大鼠胸主动脉段 ,按Ｈｏｆｍａｎ的贴片法原代培养血管平滑肌细胞。本实验选用 5～ 10代细胞进行实验。2 .细胞总ＲＮＡ的提取 ,ＲＮＡ变性电泳 ,转膜 :采用异硫氰酸胍一步法…     

小鼠主动脉平滑肌细胞培养及钙化模型的优化

目的 通过改良后的酶消化法进行小鼠主动脉血管平滑肌细胞（vascular smooth muscle cells,VSMCs）原代培养，提高VSMCs原代培养效率及质量，进而建立更快速有效的体外钙化模型。方法 剥离小鼠主动脉中膜并将其剪碎，分别采用改良组织贴块法及改良酶消化法进行小鼠VSMCs原代培养，免疫荧光染色鉴定细胞纯度后，分组（经典钙化组、改良钙化组）进行体外钙化诱导，采用vonKossa染色法评估各组钙化差异。结果 与改良组织贴块法相比，改良后的酶消化法培养原代VSMCs可在短时间内获得大量细胞，鉴定VSMCs纯度>98%，且细胞不易老化，传代存活率高。与传统体外钙化方法比，改良组的钙化更容易且稳定。结论 本研究通过改良的酶消化法提高了原代平滑肌细胞的存活率及培养效率，同时通过改良体外钙化诱导液配方，建立了良好的体外钙化模型。     

不同方法对血管平滑肌细胞表型特性影响的实验研究

目的 通过两种分离方法体外获取血管平滑肌细胞（VSMCs）,比较细胞表型差异及生物学特性,为血管性疾病研究提供精确实验基础.方法 分别采用组织块法及酶消化法获取SD大鼠胸主动脉VSMCs,分为组织块组与酶消化组.倒置显微镜、结晶紫染色观察形态学改变并定量分析;MTT比色法及TransweU细胞迁移实验分别检测分析细胞增殖与迁移活性;流式细胞周期测定细胞周期分布;细胞免疫荧光染色鉴定VSMCs并测定分析收缩型标记蛋白α-平滑肌肌动蛋白（SMA）以及合成型标记蛋白平滑肌胚胎型肌球蛋白重链（SMemb）、原肌球蛋白-4（TPM-4）表达量的变化.结果 两组细胞SMA细胞免疫荧光染色阳性率≥95％,细胞呈现谷峰状结构生长.组织块组与酶消化组两组细胞长径径值分别为（95.10±16.23）μm和（114.67±15.92）μm.与酶消化组相比,组织块组细胞增殖及迁移活性分别增加23.04％和1.54倍（P＜0.05）,S＋G2期所占比例增加49.68％（P＜0.05）,SMA蛋白表达量下降（P＜0.05）,SMemb及TPM-4蛋白表达均增加（P＜0.05）.结论 组织块法获取细胞多以合成表型为主,酶消化法则主要呈收缩表型.伴随细胞形态学、增殖及迁移活性、表型特异性蛋白表达等不同改变,两种细胞获取方法可为不同目的研究提供精确的体外模型.     

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.