体外转化生长因子—β1与表皮生长因子对人前列腺上皮细胞增作用调节

目的：体外观转化生长因子－β1(TGF-β1）、表皮生长因子（EGF）对前列腺上皮细胞增殖的调节作用。方法：胶原酶消化组织，建立原代前列腺上皮细胞系，细胞培养于无血清培养基中。前列腺特异抗原（PSA）、Pan-Keratin等免疫组织化学染色鉴定培养细胞。给制细胞生长曲线和运用细胞增殖ELISA染色（BrdU)方法观察传代后细胞的生长状况及不同剂量的生长因子TGF-β1、EGF对细胞增殖的作用。结果：培养细胞具有典型的上皮细胞形态特征，PSA、Pan-Keratin染色阳性，细胞传代5－7次开始衰退。不同培养基条件可明显地影响细胞生长活性。EGF促进细胞增殖活性，TGF-β1则抑制增殖，其作用与剂量成正比。此外，EGF与TGF-β1相经减弱对方的作用。结论：体外培养的人前列腺上皮细胞增殖受生长因子TGF-β1和EGF调节，结果提示TGF-β1、EGF可能在前列腺增生（BPH）发病中起重要作用。     

体外转化生长因子-β1与表皮生长因子对人前列腺上皮细胞增殖的作用调节

目的　体外观察转化生长因子 β1(TGF β1)、表皮生长因子 (EGF)对前列腺上皮细胞增殖的调节作用。　方法　胶原酶消化组织 ,建立原代前列腺上皮细胞系 ,细胞培养于无血清培养基中。前列腺特异抗原 (PSA)、Pan Keratin等免疫组织化学染色鉴定培养细胞。绘制细胞生长曲线和运用细胞增殖ELISA染色 (BrdU)方法观察传代后细胞的生长状况及不同剂量的生长因子TGF β1、EGF对细胞增殖的作用。　结果　培养细胞具有典型的上皮细胞形态特征 ,PSA、Pan Keratin染色阳性 ,细胞传代 5～ 7次后开始衰退。不同培养基条件可明显地影响细胞生长活性。EGF促进细胞增殖活性 ,TGF β1则抑制增殖 ,其作用与剂量成正比。此外 ,EGF与TGF β1相互减弱对方的作用。　结论　体外培养的人前列腺上皮细胞增殖受生长因子TGF β1和EGF调节 ,结果提示TGF β1、EGF可能在前列腺增生 (BPH)发病中起重要作用     

成人肾脏足细胞的原代培养和鉴定

目的建立一种重复性好、操作简便的成人肾脏足细胞原代培养方法。方法取肾脏肿瘤手术切除后远离患病部位的正常肾脏组织，分离出肾皮质，剪碎研磨并通过差异过筛法分别通过80目（孔径220μm）、40目（450μm）、120目（125μm）细胞筛网，收集120目筛网上肾小球利用植块法进行接种，将接种培养面向上放置，4h后添加培养液并将培养瓶翻转过来正常放置培养箱内孵育。采用形态学观察和细胞间接免疫荧光染色法，对去氧肾上腺素、第八因子（F8）蛋白、波形蛋白、角蛋白和肾母细胞瘤蛋白（WT-1）进行鉴定；并用流式细胞仪分析足细胞纯度。结果接种3d后可见绝大部分肾小球贴壁；5d后几乎全部肾小球贴壁，少许肾小球周围有细胞爬出，体积中等，呈多边形，无突起伸出；7～10d可见所有肾小球周围大量多边形细胞爬出，细胞迅速生长至融合状态，呈铺路石样外观，符合去分化状态足细胞特征；此时胰蛋白酶差异消化去除成纤维细胞传代培养。消化传代后的足细胞胞体、胞核逐渐增大，自细胞体伸出明显树枝状突起，常见双核，符合分化状态足细胞特征。免疫荧光染色发现传代7d后细胞表达足细胞特异性蛋白WT-1、去氧肾上腺素，不表达F8蛋白、波形蛋白、角蛋白，排除内皮细胞、系膜细胞和壁层上皮细胞污染。流式细胞仪分析足细胞纯度为98．3％。结论应用差异过筛法分离人肾小球，并结合植块法进行接种可促进肾小球贴壁，简便、高效地培养出原代足细胞。     

大鼠海马神经干细胞的原代培养方法

目的探索总结一种简单、高效的大鼠海马神经干细胞原代培养方法。方法选取新生24 h的SD大鼠,剥离出双侧海马,用机械吹打法制成细胞悬液,加入培养液,接种于培养皿中,3 d后半量换液,5~7 d后可传代。每天在荧光倒置显微镜下观察细胞的生长状况,采用免疫荧光法对第2代神经干细胞进行鉴定。结果本方法能准确分离出海马组织,培养出的神经干细胞中无其他混杂的细胞生长,经鉴定呈兔抗巢蛋白(Nestin)阳性和5-嗅-2-脱氧尿苷(BrdU)阳性。结论运用本方法获得的海马神经干细胞纯度高、细胞活力高、增殖能力强。     

正常胃黏膜上皮细胞原代培养方法

胃黏膜上皮细胞是进行胃生理功能、病变机制、药物治疗等研究的重要工具，已被广泛应用于科学实验。原代培养细胞能反映原始组织的特点，与体内姊妹细胞的生理状态相似，是进行相关研究的理想材料。但胃黏膜上皮细胞对内环境要求较高，分离纯化后并不象肝细胞等易于存活和生长，不少实验因这一环节而不能进行下去，不得不采用从肿瘤组织衍生的各种细胞株，此类细胞株与正常胃黏膜上皮细胞在生长特性、形态结构等方面存在较大差异，不能代表正常胃黏膜上皮。在我国，几乎所有的相关研究都是采用各种细胞株，因此本文较详细全面地介绍了文献报道中较成功的正常胃黏膜上皮细胞原代培养方法，以期对相关研究有所帮助。     

大鼠结膜杯状细胞的体外培养及鉴定

目的体外分离、培养大鼠结膜杯状细胞,观察其形态、结构等生物学特征。方法取大鼠穹隆部结膜,加入含10%胎牛血清的RPMI 1640培养液中行组织块培养,5~7 d后酶消化法传代。观察原代及传代细胞的形态及生长情况,并应用特殊免疫组化染色方法分别对原代、传代培养的杯状细胞进行鉴定,用Image J软件计算细胞纯度。结果倒置相差显微镜下可见杯状细胞呈鹅卵石样,5~7 d在组织块周围形成环状结节。免疫组化结果显示,培养的原代细胞角蛋白(CK)4染色阴性,CK7染色阳性,且CK7阳性细胞数占(97.3±2.0)%。传代杯状细胞中,绝大多数凝集素UEA-I染色为阳性,阳性细胞占(91.2±2.7)%。结论大鼠穹窿部结膜可以成功培养出杯状细胞,传代细胞仍保持原代细胞特性,且纯度较高。     

肺腺癌细胞系H114的建立

目的建立国人男性肺腺癌细胞系,为肺癌的防治研究提供理想的体外实验模型。方法以经组织学和／或细胞学确诊的标本进行原代培养,通过光镜观察、细胞苏木精．伊红（HE）染色、细胞生长曲线、异体移植实验及组织HE染色等对其进行分析鉴定,建立细胞系。结果细胞经液氮罐或-80℃低温冰箱反复冻存复苏后仍保持良好的增殖活性,复苏细胞经0．4％胎盘蓝染色,活细胞比率均保持在85％以上。在整个168h的观察时间里,肺腺癌H114细胞始终处于持续增殖状态,可见潜伏期和指数生长期,由于本细胞系传代次数较少,生长活跃,7d还没出现平顶期和退化衰亡期;体外培养细胞生长稳定,细胞形态学及浸润性生长结果表明该细胞系符合恶性细胞特性,裸鼠接种成功致瘤,瘤细胞形态与原患者的病理切片相似,命名为H114细胞系。结论该细胞系符合建系标准,是一株新建的人肺腺癌细胞系。     

大鼠骨髓基质细胞培养方法的改良

目的　对体外分离培养大鼠骨髓基质细胞 (BMSC)方法进行改良 ,观察其生物学特点。方法　分离大鼠胫骨、股骨 ,以 IMDM培养基冲洗骨髓 ,与培养液混合后直接接种至培养瓶中 ,接种后 7～ 14 d形成单层贴壁的成纤维状细胞。检测传代细胞接种贴壁率、生长曲线、细胞周期 ,电镜观察细胞超微结构。结果　分离培养的原代 BMSC生长良好 ,倍增时间约为 4 2 h。 BMSC传代后 12 h贴壁率达 80 %以上 ,82 %的细胞处于 G0 、G1 期 ,超微结构呈现较早期细胞特点。结论　改良法培养的 BMSC生长稳定 ,传代细胞适应性强。与传统培养方法比 ,该法操作简单、实用性强 ,可以用于 BMSC的体外实验研究     

人肛瘘管壁成纤维细胞的体外分离培养和鉴定

通过组织块法分离并原代培养人肛瘘管壁组织，用胰蛋白酶和乙二胺四乙酸（EDTA）消化成纤维细胞、上皮细胞，获得更加纯化的成纤维细胞；以DMEM培养液为基础培养蒸，添加胎牛血清（体积分数10％）、青霉素（100U／L）和硫酸链霉素（100U／L），置37℃、5％CO2培养箱中培养。倒置相差显微镜和细胞爬片HE染色。观察成纤维细胞形态结构，并对培养成纤维细胞行角蛋白、波形蛋白免疫细胞化学鉴定．结果分离后的成纤维细胞在第4～12h于体外快速贴壁，第2～4天进入对数期生长、增殖期。细胞起源鉴定波形蛋白免疫细胞化学染色为阳性，角蛋白免疫细胞化学染色为阴性。提示该方法所获得的肛瘘管壁成纤维细胞可在体外稳定培养，不含有杂质细胞。可为在细胞分子水平上研究肛瘘瘘管愈合的机制提供可靠的细胞模型。     

BrdU作为脂肪干细胞标记示踪法的可行性

目的:研究BrdU标记猪脂肪干细胞(adipose-derived stem cells,ADSCs)的最佳剂量及时间,探讨其作为干细胞标记示踪方法的可行性.方法:自中华实验猪背部提取脂肪组织,采用贴壁法分离培养ADSCs,取第3代细胞以终浓度分别为10、15、20、25及30 μmol/L BrdU进行标记:另一孔不含BrdU作为对照组,分别培养12、24、48、72及96 h.免疫荧光法检测各组细胞BrdU标记率,找出BrdU的最佳标记方法,通过台盼蓝排斥试验、MTT及细胞凋亡检测,观察BrdU对ADSCs生长情况的影响.对第3代的ADSCs采用最佳标记方法后更换普通培养基继续培养,适时传代,连续检测第4、5、6、7、8代ADSCs的BrdU标记率.结果:原代培养的ADSCs的形态主要为长梭形,第3代ADSCs经BrdU标记后胞核呈红色荧光,随浓度的升高及时间的延长,BrdU阳性标记率逐渐升高,以终浓度20μmol/L BrdU标记48 h后阳性率达90%以上,且连续传5代标记率仍达40%.MTT、台盼蓝排斥试验及细胞凋亡检测发现BrdU对ADSCs生长增殖基本无影响.结论:BrdU标记ADSCs的最佳剂量和时间为20 μmol/L和48 h,该方法标记率高,对细胞影响小,可用于动态研究ADSCs在移植体内生长、分化.     

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.