5-Aza-CdR对人食管癌细胞株中T-cadherin基因表达和细胞增殖的影响

背景：T-cadherin在肿瘤的发生中可能扮演肿瘤抑制基因的角色．在食管癌等多种肿瘤中的表达明显下降。目的：探讨去甲基化制剂5-氮杂-2’-脱氧胞苷（5-Aza-CdR）对人食管癌细胞株EC109中T—cadherin基因表达和细胞增殖的影响。方法：常规培养人食管癌细胞株EC109．并分为5μmol／L5-Aza-CdR组和对照组。以甲基化特异性PCR（MSP）法检测T-cadherin基因启动子区甲基化状态．RT-PCR和蛋白质印迹法分别检测T-cadherinmRNA和蛋白表达,MTr法检测EC109细胞增殖。结果：对照组EC109细胞中T-cadhefin基因启动子区呈异常甲基化状态．5-Aza-CdR干预可逆转甲基化状态。与对照组相比,5-Aza-CdR组T-cadherin基因mRNA和蛋白表达均显著增高（P〈0．01）．细胞增殖明显受到抑制。结论：去甲基化制剂5-Aza-CdR通过逆转食管癌细胞中T-cadherin基因启动子区甲基化状态来增强其表达．并抑制肿瘤细胞增殖．     

p15INK4B和p21WAF1基因联合转染对人食管鳞癌细胞系EC109的协同抑制作用

目的:探讨p15INK4B和p21WAF1基因联合转染对人食管鳞癌细胞系EC109细胞增殖和凋亡的影响.方法:脂质体介导PcDNA3.1(+)-p15和pcDNA3.1(+)-p21转染EC109细胞,稳定筛选后用RT-PCR检测转染细胞p15与p21基因mRNA表达,Western blot检测转染细胞P15和P21蛋白的表达.用MTT法和透射电镜检测p15和p21基因分别及联合转染对EC109细胞增殖与凋亡的影响,流式细胞仪检测EC109细胞周期分布与凋亡率.结果:p15和p21转染组EC109细胞生长速度低于空载体组与未转染组,联合转染组与二者单独转染组相比.亦明显抑制EC109细胞体外生长速度.p15和p21转染组EC109细胞发生G1/S阻滞,G1期细胞比例显著高于空载体组和未转染组,S期则显著降低(G1期:60.52%±3.75%,63.12%±2.89% vs 42.17%±5.30%.41.38%±6.54%;S期:22.67%±1.25%,17.96%±2.03% vs 30.96%±3.33%,36.05%±1.78%,均P<0.01),并出现凋亡峰,透射电镜亦发现p15和p21转染组发生细胞凋亡,联合转染组发生更为明显的G1/S阻滞,G1期比例显著升高、S期比例明显降低(G1期:72.83%±2.31% vs60.52%±3.75%,63.12%±2.89%:S期:13.59%±2.59% vs 22.67%±1.25%,17.96%±2.03%.均P<0.05),凋亡率明显升高(21.21%±1.78%vs 4.32±1.74%,10.83%±2.40%,均P<0.01).结论:p15和p21基因联合转染在体外可以进一步增强对人食管鳞癌EC109细胞的抑制与诱导凋亡作用.     

p15INK4B基因转染联合亚砷酸对人食管鳞癌的协同抑制作用

目的探讨p15^INK4B（p15）基因转染与亚砷酸（As2O3）联合应用对人食管鳞癌细胞系EC109细胞增殖和凋亡的影响。方法脂质体介导将pcDNA3．1（＋）-p15转染EC109细胞，稳定筛选后加入2μmol／LAs2O3。PCR检测外源p15基因cDNA，Western印迹法检测转染细胞P15蛋白的表达。用MTT、集落形成实验和透射电镜检测p15基因转染联合As2O3对EC109细胞增殖和凋亡的影响，流式细胞仪检测EC109细胞周期分布和凋亡率。结果与二者单独应用相比，联合应用可明显抑制EC109细胞体外生长速度，集落形成率明显降低（P〈0．01），联合作用3d后发生更为明显的G1／S阻滞，G1期比例显著升高（P〈0．05），S期比例明显降低（P〈0．05），凋亡率明显升高（P〈0．01）。透射电镜发现二者联合应用诱导EC109发生更明显的细胞凋亡。结论p15基因转染与As2O3联合应用可以进一步增强对人食管鳞癌EC109细胞的抑制作用和诱导凋亡作用。     

siRNA阻断NF-κB信号通路联合5-FU对食管鳞癌细胞凋亡的促进作用

目的: 利用RNAi技术特异性的抑制NF-κB亚单位p65的表达,观察其对p65表达的抑制作用及联合5-FU对食管鳞癌细胞Eca109和EC9706的影响.方法:将终浓度为50 nmol/L的p65 siRNA转染到食管鳞癌细胞EC9706和Eca109中,通过RTPCR检测0、24、48和72 h时段p65 mRNA的表达水平.Western blotting法检测p65和Bcl-2蛋白表达,Annexin V/PI复染结合流式细胞仪检测细胞凋亡,显微镜下观察p65 siRNA与5-FU单独或联合应用对食管鳞癌细胞形态学特性的影响.结果:EC9706和Eca109细胞转染p65 siRNA 24、48和72 h后,p65 mRNA的表达水平随时间的延长逐渐下调,在72 h的阻断效率最为明显,与0 h相比,差异有显著性(0.12±0.01 vs 0.28±0.05,0.1±0.01 vs 0.38±0.04,均P<0.05),转染72 h后,p65和Bcl-2蛋白表达水平下调.EC9706和Eca109转染p65siRNA后,细胞凋亡指数明显升高(6.65%±0.27% vs 2.03%±0.08%,8.03%±0.06% vs 2.66%±0.25%,均P<0.05);p65 siRNA转染72 h后,EC9706和Eca109细胞增殖较慢;当p65 siRNA与5-FU联合作用,细胞增殖明显受到抑制.结论:p65 siRNA可阻断NF-κB信号通路,下调NF-κB下游基因中抗凋亡蛋白Bcl-2的表达,表明活化的NF-κB信号通路可成为食管鳞癌基因治疗中一个重要的分子靶点.     

三氧化二砷对肾癌GRC-1细胞凋亡及p53、Survivin基因表达的影响

目的观察三氧化二砷(As_2O_3)对人肾癌GRC-1细胞凋亡及p53、Survivin基因表达的影响,探讨其作用机制。方法将体外培养的人肾癌GRC-1细胞分为对照组(N)和实验组,实验组根据加入As_2O_3浓度的不同分为A、B、C、D、E(2、4、6、8、10μmol/L)组。48h后,四甲基偶氮唑蓝(MTT)法测定细胞增殖,流式细胞仪检测细胞凋亡和细胞周期变化．免疫细胞化学和RT-PCR方法测定p53和Survivin基因表达。结果As_2O_3可抑制GRC-1细胞的增殖．当As_2O_3由2μmol/L增加到10μmol/L时,细胞增殖率由88．3%降低到18．7%(F= 7546．587,P＜0．01)。各实验组p53、Survivin基因表达均较对照组减少,且随着As_2O_3的增加表达呈递减改变(F=1120．741、F=6 296．535,P＜0．01)。结论As_2O_3能够抑制GRC-1细胞增殖,并且具有浓度依赖性。能将细胞阻滞在G_2/M期,诱导细胞凋亡,其作用机制与p53和Survivin基因表达水平下降有关。     

三氧化二砷在类风湿关节炎治疗中作用机制的探讨

目的观察佐剂关节炎(AA)动物模型滑膜组织在诱导凋亡前后的苏木素-伊红(HE)染色及核因子(NF)-κB表达活性的差异,探讨三氧化二砷(As_2O_3)在治疗类风湿关节炎(RA)的可能作用机制。方法将Wistar大鼠造模成功后随机分为两组：AA模型组和As_2O_3治疗组。治疗组每日于发病鼠腹腔注射As_2O_3连续1周,观察3d全部动物处死取材。再经固定、脱钙、包埋,制成切片。然后进行HE染色及免疫组织化学检测。结果HE染色光镜下观察：与正常对照组相比,AA模型组大鼠的滑膜细胞层次增多,6～8层,排列紊乱,有大量炎性细胞浸润；而As_2O_3治疗组可达3～4层,但仍有炎性细胞浸润。免疫组织化学检测结果：AA模型组大鼠关节滑膜的NF-κB(p65)的阳性染色强度明显高于正常对照组,以胞核染色为深,有的成团块状。而As_2O_3治疗组滑膜的NF-κB表达及活性明显下调,但未恢复到正常对照组水平,平均灰度值计算结果显示三组之间差异有统计学意义(P＜0．05)。结论As_2O_3可以抑制分化,诱导滑膜细胞凋亡．而抑制NF-κB的活性和表达可能是As_2O_3发挥治疗作用的重要机制。     

p21WAF1基因对人食管鳞癌细胞增殖的抑制作用

目的:探讨p21WAF1( p21)基因转染对食管鳞癌细胞系EC109细胞增殖的影响.方法:根据转染质粒的不同和是否进行质粒转染分为3组.p21转染组:用脂质体Lipofectamine2000介导将pCDNA3.1(+)- p21质粒转染入EC109细胞; 空载体转染组:同样方法将pCDNA3.1(+)-neo质粒转染入EC109细胞; 未转染组:未转染的EC109细胞.应用RTPCR、Western blot分别检测p21基因mRNA、P21蛋白变化; 流式细胞仪分析细胞周期变化,应用MTT、流式细胞仪和透射电镜检测转染外源p21基因对EC109细胞增殖和凋亡的影响.结果:p21转染细胞中p21 mRNA和P21蛋白高表达; p21转染组EC109细胞生长速度低于空载体组和未转染组; 流式细胞仪观察到P21蛋白高表达使EC109细胞发生G1/S阻滞,G1期细胞比例显著高于空载体组和未转染组(63.120%±2.893% vs 41.380%±6.536%,42.173%±5.301%,均P<0.01),S期比例显著低于空载体组和未转染组(18.923%±3.084%vs 22.573%±5.463%,26.867%±2.922%,均P<0.01),并出现亚G1峰(凋亡峰).透射电镜亦发现p21转染组发生细胞凋亡.结论:p21基因转染可以抑制人食管鳞癌细胞系EC109细胞增殖并能诱导其发生细胞凋亡.     

曲古菌素A对食管癌EC1细胞增殖和细胞周期的影响及其分子机制

目的:探讨不同浓度曲古菌素A对食管癌细胞系EC1 细胞增殖、细胞周期的影响及其对细胞周期调控基因p21 WAF1/CIP1 表达的影响. 方法:用0.3,0.5,1.0 μmol/L 的TSA 处理EC1 细胞,MTT 检测TSA 作用24 、48 h 对EC1 细胞的抑制作用,流式细胞仪检测0.3,0.5,1.0 μmol/L 的TSA 作用24 h 后EC1 细胞周期的改变,Western blot 法检测p21 WAF1/CIP1 变化. 结果:TSA 在0.5 μmol/L 以上时对EC1 细胞有抑制作用;0.3 μmol/L TSA 处理细胞后细胞周期与对照组相比,无明显变化;0.5 μmol/L TSA 处理EC1 细胞后,G0/G1期细胞较对照组明显增加,S 期细胞较对照组明显减少(74.56% ±1.34% vs 62.12%±0.52%;14.52%±1.81% vs 27.50%±0.66%,均P <0.05);0.5,1.0 μmol/L TSA 处理细胞后p21 WAF1/CIP1 表达明显增加(均P<0.05). 结论:一定浓度的TSA 对人食管癌细胞EC 具有的增殖抑制作用,引起EC1 细胞发生G0/G1 期阻滞,其部分机制与p21 WAF1/CIP1 上调有关.     

多发性骨髓瘤患者p16基因甲基化及砷剂诱导去甲基化的研究

目的探讨p16基因启动子区CpG岛甲基化在多发性骨髓瘤(MM)患者发病中的作用以及三氧化二砷(As2O3)诱导p16基因的去甲基化作用。方法采用巢式甲基特异性PCR法检测MM患者以及利用As2O3作用前后人MM细胞株U266的p16基因的甲基化状态,RTPCR检测U266细胞用药前后p16基因mRNA的表达变化,生长曲线、MTT法检测As2O3对细胞的生长和增殖抑制。利用流式细胞仪检测DNA含量分析法探讨As2O3对骨髓瘤细胞系U266周期的影响。结果MM患者p16基因的甲基化比例为54.8%,U266细胞存在p16基因甲基化,p16基因不表达,As2O3作用后p16基因甲基化程度明显减弱至消失,与未处理组相比,不同剂量作用72h后p16基因表达阳性条带灰度值与βactin比值分别为0.22±0.10、0.59±0.11、0.68±0.09,阳性对照灰度比值为0.77±0.13,其差异有统计学意义(P<0.01)。结论p16基因甲基化在MM患者中较为常见,这可能为MM的诊断和治疗提供借鉴;As2O3可诱导p16基因去甲基化,使p16基因表达上调,恢复其活性,为去甲基化治疗提供新思路。     

叶酸治疗萎缩性胃炎并改变P16蛋白表达

目的:研究叶酸干预萎缩性胃炎后的黏膜改变、P16蛋白表达变化及p16基因甲基化状态．方法:根据胃镜及病理结果确诊为萎缩性胃炎的患者56例,随机分为叶酸治疗组(n=28) 和非叶酸治疗组(n=28),治疗前后观察临床症状、血浆叶酸水平、黏膜病理、P16蛋白表达变化,并采用甲基化敏感的内切酶酶切后 PCR法检测p16基因甲基化状态．结果:叶酸治疗组临床症状缓解快而持久,患者血浆叶酸水平升高(47．98±2．68 μg／L vs 14.37±3．56 μg／L,P<0．01),病理改变逆转(有效率:80．8％vs39．3％,P<0．01),P16蛋白表达增强,有效率达61．6％(P<0．01),而p16基因甲基化状态两组在治疗前后均正常．结论:萎缩性胃炎的发生与叶酸缺乏有关,叶酸治疗可以提高血浆叶酸水平,有效改善临床症状,一定程度上逆转胃黏膜的萎缩、肠化和不典型增生,能提高P16蛋白表达．萎缩性胃炎中不存在甲基化异常．     

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.