乳鼠窦房结细胞取材与纯化培养方法的探讨

本研究旨在建立一套可靠的窦房结细胞取材分离、纯化培养技术。取Wistar乳鼠的窦房结组织进行原代细胞培养 ,随机分两组 :常规方法组与差速贴壁结合 5 溴脱氧尿核苷 (BrdU)处理组。结果 :①在培养的窦房结细胞中观察到三种不同形态的细胞 ,即梭形细胞、三角形细胞与不规则形细胞。其中梭形细胞最多 ,且形态结构与电生理特征符合窦房结起搏细胞的特点。三角形细胞与心房肌三角形细胞特征相似。②常规方法培养中窦房结梭形细胞所占比例为 45 % ,而差速贴壁技术结合BrdU处理的纯化培养中梭形细胞达 6 8%左右 (P <0 .0 1)。结论 :用差速贴壁结合BrdU处理的纯化培养可明显提高窦房结梭形细胞的比例 ,是一种可靠的窦房结细胞培养技术     

乳鼠窦房结细胞的原代培养与鉴定

目的 :建立一套可靠的窦房结细胞培养与鉴定技术。方法 :取Wistar乳鼠的窦房结组织 ,用差速贴壁技术及BrdU处理法进行原代细胞培养 ;对培养细胞进行形态学观察 ,用电生理技术记录细胞的动作电位。结果 :在培养的窦房结细胞中观察到有 3种不同形态的细胞 ,即梭形细胞、三角形细胞与不规则形细胞 ,其中梭形细胞最多 ,且形态结构与电生理特征符合窦房结起搏细胞的特点。三角形细胞与心房肌三角形细胞特征相似。结论 :①用差速贴壁技术及BrdU处理法进行乳鼠的窦房结细胞培养 ,是一种可靠的窦房结细胞培养技术。②培养乳鼠窦房结细胞中的梭形细胞即为窦房结的起搏细胞     

乳鼠窦房结细胞的原代培养和形态学特征

目的比较差速贴壁技术和常规培养方法在体外获取窦房结自律细胞的密度,并观察其形态学特征。旨在建立可靠的窦房结自律细胞体外取材分离、纯化培养及形态学鉴定。方法取24 h内新生Wistar乳鼠1 20只,随机分为实验组(差速贴壁技术培养方法)和对照组(常规培养方法),每组60只。每次随机各取5只乳鼠行窦房结取材,分别用2种方法进行原代细胞纯化培养。通过光、电镜观察比较2种方法体外窦房结自律细胞的密度比及形态学特征。结果体外培养窦房结细胞有3种不同形态,梭形细胞、三角形细胞和不规则形细胞,其中梭形细胞最多,搏动频率最快;三角形细胞与心房肌三角形细胞特征相似;实验组窦房结梭形细胞比例明显高于对照组[(65±4)%vs(45±3)%,P<0.01]。结论采用差速贴壁技术纯化培养方法较常规培养可更明显提高窦房结梭形细胞的比例,是一种可靠的窦房结自律细胞培养、纯化技术。在培养的乳鼠窦房结细胞中,细胞体积小、搏动频率快的梭形细胞即是窦房结的起搏细胞。     

乳兔窦房结细胞的分离及鉴定

目的 乳兔窦房结细胞（sinus node cell,SNC）的分离、纯化、培养与鉴定.方法 选用新生新西兰乳兔5只,采用双酶解法对细胞进行消化分离,差速贴壁结合5-BrdU对分离的细胞进行纯化培养,观察SNC形态变化并采用全细胞膜片钳技术对SNC进行动作电位的记录.结果 培养得到的SNC主要有3种形态：梭形、三角形与不规则形,而梭形细胞最多,搏动频率最快,符合窦房结细胞的特征.采用膜片钳技术记录10个梭形细胞的动作电位中,平均最大舒张电位为（-50.9±5.3）mV,动作电位幅度为（61.9±4.8）mV.结论 采用双酶解、差速贴壁及BrdU对乳兔窦房结细胞进行消化、分离可得到纯化的SNC,此种方法得到的SNC状态活性良好,且具有典型特征的动作电位.     

乳鼠窦房结细胞原代培养方法的优化

目的：对乳鼠窦房结细胞原代培养方法进行改进,并观察超极化激活环核苷酸门控阳离子通道（HCN4）在心脏细胞上的表达特点. 方法：取新生SD乳鼠的窦房结组织,在传统乳鼠窦房结细胞原代培养方法的基础上进行优化改进,选用0.1％胰蛋白酶与0.1％Ⅱ型胶原酶按照1∶1制成的混合消化酶,以减少窦房结自律细胞的损伤,混合酶消化后采用差速贴壁法及5-溴脱氧尿嘧啶核苷（5-Brdu）纯化处理.通过免疫荧光实验观察HCN4在乳鼠心脏细胞上的表达情况. 结果：（1）本方法培养的心脏细胞存活率为95.8％.（2）经差逮贴壁、5-Brdu处理,窦房结细胞所占比例明显提高.（3）培养3～5 d,视野中绝大多数为搏动的心脏细胞,成纤维细胞无明显增殖,可观察到梭形、三角形和不规则形3种细胞.其中梭形细胞所占比例最高,其细胞器不发达,糖原颗粒不丰富,肌原纤维较少,搏动频率最快,表达HCN4;三角形细胞微量表达HCN4. 结论：（1）混合消化酶结合差速贴壁及5-Brdu处理,可以显著提高细胞成活率及窦房结梭形细胞的比例,是可靠的窦房结细胞的分离纯化方法.（2）搏动频率快、细胞器不发达、糖原颗粒少、HCN4表达阳性的梭形细胞可能就是窦房结的起搏细胞.     

差速贴壁联合应用5-BrdU对心肌细胞纯度及活力的影响

目的研究多次差速贴壁并联合应用5-溴脱氧尿苷（5-B rdU）对分离的心肌细胞纯度及活性的影响。方法以酶消化法分离乳鼠心肌细胞,分别进行13次差速贴壁并联用或不联用5-B rdU以纯化心肌细胞。正常培养3 d后,观察细胞的形态特征。用流式细胞技术分析各实验组心肌细胞的纯度。以台盼蓝染色、MTT比色法及细胞线粒体内膜功能检测（流式细胞术）来反映各实验组心肌细胞的活性。结果随着差速贴壁次数的增加,心肌细胞得以纯化,但细胞的活性有所下降。联用5-B rdU后细胞纯度进一步提高,且对细胞的活性无明显影响;其中两次差速贴壁并联合应用5-B rdU后,可获得纯度高、活性好的心肌细胞。结论两次差速贴壁并联合应用5-B rdU为纯化心肌细胞的有效方法。     

大鼠乳鼠原代心房肌细胞培养的方法及鉴定

目的:介绍大鼠乳鼠心房肌细胞的分离、纯化,以及培养和鉴定方法. 方法:取1d龄SD大鼠心房组织,用0.1％胰蛋白酶和0.025％Ⅱ型胶原酶消化,将心房肌细胞收集在含10％胎牛血清的DMEM培养基中,用差速贴壁和5-溴脱氧尿嘧啶核苷纯化心房肌细胞.观察细胞形态,结合α-横纹肌肌动蛋白抗体免疫荧光鉴定心房肌细胞.结果:细胞培养24 h已完全贴壁,成梭形或三角形,无细胞搏动,细胞体积可逐渐增大.培养细胞经免疫荧光鉴定,95％为心房肌细胞 结论:该研究是一种较好的乳鼠心房肌细胞原代培养及鉴定方法.     

人胚胎窦房结细胞原代培养及纯化方法探讨

目的:探讨人胚胎窦房结细胞的原代培养及纯化方法,寻找一种较为可行的适宜于人胚胎窦房结细胞生长的方法。方法:取人工水囊引产的12～16周人胚胎心脏,随机分为实验组及对照组,取其窦房结组织分别进行细胞培养,观察和比较两个培养组中各种细胞的数量及生长状况,以细胞的搏动频率评价细胞的活性,以梭形细胞的数量评价窦房结细胞的纯度。结果:实验组简化了操作环节,优化了细胞的培养环境,可提高细胞的活性;经过差速贴壁阿糖胞苷纯化处理,可明显减少不规则细胞的数量,窦房结细胞的数量明显上升(P<0.05)。结论:纯化培养法是一种较为可行的人胚胎窦房结细胞原代培养方法,可为进一步研究提供生长较稳定的窦房结细胞。     

免疫磁珠法分离培养小鼠原代骨骼肌微血管内皮细胞及鉴定

目的 旨在建立一种有效分离、培养、扩增小鼠原代骨骼肌微血管内皮细胞（MMECs）的方法。方法 采用胶原酶消化法、差速贴壁法以及免疫磁珠法分离纯化骨骼肌微血管内皮细胞,贴壁培养进行体外扩增。利用相差显微镜观察培养细胞的形态、MTT法测定细胞的生长情况、CD31免疫荧光染色对其表型进行鉴定、流式细胞术鉴定细胞纯度。结果 实验成功获取小鼠骨骼肌微血管内皮细胞,小鼠原代骨骼肌微血管内皮细胞在培养24 h后迅速贴壁,(3～6) d内迅速生长,呈梭形,单层排列,培养(6～8) d可长满培养皿底,呈铺路石样排布。MTT法测得培养第2代小鼠骨骼肌微血管内皮细胞的生长曲线呈倒“S”形。流式细胞术检测经差速贴壁联合磁珠纯化的CD31+的MMECs细胞可达97%以上。结论 成功建立了一种简单、高效的分离小鼠骨骼肌微血管内皮细胞的方法,为开展与骨骼肌微血管内皮细胞相关研究提供了良好的实验方法和技术手段。     

体外原代培养脐带基质间充质细胞和人皮肤成纤维细胞

目的探讨体外原代培养脐带基质间充质细胞(UMC)和人皮肤成纤维细胞(HSF)的方法。方法用Ⅳ型胶原蛋白酶、Ⅰ型脱氧核糖核酸酶和0.25%胰蛋白酶消化法原代分离培养UMC细胞,用组织块贴壁法原代分离培养HSF细胞,镜下观察细胞的培养过程和生长状态。结果脐带组织分离培养第3天,有少量UMC贴壁生长,细胞膜周围有折光性,形态类似于成纤维细胞;皮肤组织块贴壁培养第7天,有HSF爬出,呈长梭形、不规则三角形。随着细胞培养时间延长,UMC和HSF数量逐渐增多,细胞生长状态良好。结论应用酶消化法和组织块贴壁法可成功分离培养出UMC和HSF。     

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.