多药耐药相关蛋白1与人肝细胞耐砷性关系的研究

目的探讨多药耐药相关蛋白1（MRP1）与人肝细胞（L-02）耐砷性之间的关系。方法采用人肝细胞L-02细胞株,噻唑兰法（MTT）进行24h急性NaAsO2毒性试验,选择细胞生存率在90％～95％时的NaAsO2浓度为诱导浓度,同时设1组不加砷诱导的L-02细胞作为同步对照。置于细胞培养箱中37℃,5％CO2常规培养6周,定期换液传代,每周用MTT法计算半数致死浓度（LC50）及细胞的生存率;6周后,将加砷诱导的L-02肝细胞与同步对照细胞在培养基中培养至细胞80％融合,然后进行24h急性砷毒性试验（NaAsO2终浓度为0、2．5、5．0、10μmol／L）;采用real-time PCR检测细胞内MRP1mRNA的表达情况;MTT法检测细胞的存活率;石墨炉原子吸收光谱法检测各组细胞内总砷浓度。结果实验组细胞MRP1mRNA的表达明显高于对照组（P〈0．05）;24h急性砷毒性试验中,实验组不同砷浓度下细胞生存率和LC50明显高于对照组（均P〈0．001）;实验组细胞内总砷浓度明显较同步对照组低（P〈0．001）。结论L-02肝细胞具有可诱导的对砷的耐受性,其耐砷性与MRP1高表达有关。     

慢性砷暴露人骨髓间充质干细胞的生物学特性观察

目的 观察抗砷细胞模型慢性砷暴露人骨髓间充质干细胞(CAsE-hFBMSCs)的生物学特性,探讨长期低剂量砷暴露对人骨髓间充质干细胞(hFBMSCs)的影响.方法 常规条件培养hFBMSCs,48 h急性砷毒性实验MTS法检测不同剂量砷刺激条件下[0(对照)、0.25、0.50、1.00、2.00、4.00、8.00、20.00、40.00、80.00、120.00μmol/L]第2代(P2)hFBMSCs细胞生存率,根据检测结果用1.00μmol/L亚砷酸钠刺激hFBMSCs 14周,建立抗砷细胞模型CAsE-hFBMSCs,作为实验组,同时设立对照组,并采用荧光激活细胞分选术鉴定诱导前后细胞表型,流式细胞术检测第2、9、16代(P2、P9、P16)细胞周期,软琼脂克隆形成实验检测细胞是否发生恶性转化.结果 <10μmol/L时,急性砷刺激促进hFBMSCs增殖,而≥10μmol/L时,急性砷刺激则抑制细胞的生长.14周时实验组半数致死剂量(LC_(50))为(89.42±0.64)μmol/L,对照组为(52.48±0.71)μmol/L,组间比较差异有统计学意义(t=123.89,P<0.05);抗砷细胞模型CAsE-hFBMSCs的细胞表型CD34、CD45表达阴性,CD29、CD90、CD166表达阳性,与对照组比较细胞表型未见明显改变;CAsE-hFBMSCs的细胞周期变化较大,与对照组[(8.44±0.45)%、(9.14±0.14)%、(82.42±0.60)%]比较,P2实验组G2/M期[(17.72±5.47)%]和S期细胞[(25.34±3.36)%]增加,G0/G1期细胞减少[(56.96±8.83)%],P16细胞周期恢复至与对照组相近的水平:软琼脂克隆形成实验中,抗砷细胞CAsE-hFBMSCs未见克隆形成.结论 长期低剂量砷刺激对hFBMSCs生物学特性无明显影响.     

低剂量长期砷暴露对HaCat细胞增殖及凋亡水平的影响

目的探讨低剂量长期砷暴露对HaCat细胞增殖及凋亡水平的影响。方法 HaCat细胞暴露于浓度为0、0.05、0.10μmol/L的NaAsO215周后,用直接细胞计数法计数对照组及染砷组细胞数,检测细胞增殖水平;用流式细胞仪测定10 000个细胞的细胞凋亡发生水平。结果各染砷组细胞增殖速率与对照组相比均显著增高(P<0.05),且0.10μmol/L组细胞增殖率(245.00±8.66)%显著高于0.05μmol/L组(165.00±15.00)%(P<0.05),细胞增殖水平与染砷剂量间呈显著剂量-效应关系;0.05μmol/L组(0.28±0.08)%及0.10μmol/L组(0.34±0.09)%细胞凋亡率均明显低于对照组(0.74±0.18)%(P<0.05)。结论长期低剂量砷暴露可使HaCat细胞的增殖能力明显增强,并诱导细胞凋亡率显著降低。     

叔丁基对苯二酚对亚砷酸钠致细胞毒性和氧化损伤的拮抗作用

目的 探讨叔丁基对苯二酚(tBHQ)对亚砷酸钠(NaAsO2)致细胞毒性和氧化损伤的拮抗作用。方法 Chang肝细胞用tBHQ[0(对照)、5、25μmol/L]预处理24h,再用5 μmol/L tBHQ和NaAsO2[0(对照)、30、40、50、60 μmol/L]共同作用24h,采用刃天青钠(Alamar blue)还原法检测细胞活力,结果用实验组Alamar blue还原率与对照组Alamar blue还原率的相对比值表示;Chang肝细胞用tBHQ[0(对照)、5、25 μmol/L]预处理24h,再用5μmol/L tBHQ和NaAso2[0(对照)、40、50 μmol/L]共同作用24h,采用荧光探针2′,7′-二乙酰二氯荧光素(DCFH-DA)检测细胞内活性氧(ROS)的生成,结果用实验组平均荧光强度与对照组平均荧光强度的相对比值表示。结果 30、40、50、60 μmol/L的NaAsO2暴露能够显著降低细胞活力,而tBHQ预处理(5、25 μmol/L)则可明显恢复细胞活力,NaAsO2和tBHQ两因素的主效应及其交互作用均有统计学意义（F值分别为566.57、55.09、14.50,P均＜0.05）;5、25 μmol/L tBHQ预处理的30、40、50、60 μmol/LNaAsO2组细胞活力(0.75±0.02、0.70±0.04、0.59±0.03、0.43±0.03和0.75±0.02、0.73±0.03、0.65±0.02、0.50±0.02)较相应NaAsO2单独作用组(0.70±0.03、0.64±0.03、0.43±0.03、0.33±0.01)显著升高（P均＜0.05）,25 μmol/L tBHQ 预处理的50、60μmol/L NaAsO2组细胞活力高于相应5μmol/L tBHQ预处理组（P均＜0.05）。40、50 μmol/L的NaAsO2能显著诱导Chang肝细胞内ROS的产生,而tBHQ预处理(5、25 μmol/L)则可使NaAsO2诱导产生的细胞内ROS水平显著下降,NaAsO2和tBHQ两因素的主效应及其交互作用均有统计学意义（F值分别为181.78、60.55、4.93,P均＜0.05）;5、25 μmol/L tBHQ预处理的40、50 μmol/L NaAsO2组细胞内ROS水平(1.87±0.09、1.80±0.07和1.36±0.11、1.44±0.12)较相应NaAsO2单独作用组(2.30±0.18、2.18±0.17)显著降低(P 均＜ 0.05),25 μmol/L tBHQ预处理的40、50 μmol/L NaAsO2组细胞内ROS水平低于相应5μmol/L tBHQ预处理组（P均＜ 0.05）。结论 tBHQ对NaAsO2诱导的细胞毒性和氧化损伤具有一定的拮抗作用。     

用单细胞凝胶电泳观察氟砷单独及联合对淋巴细胞DNA损伤

目的 观察氟、砷单独及联合作用对正常人淋巴细胞DNA的损伤作用及特点。方法 采用单细胞凝胶电泳（SCGE）实验，观察不同剂量氟、砷单独及联合应用对正常人淋巴细胞DNA的损伤。结果 人淋巴细胞在浓度为0．1、0．5μmol／L的NaAsO2及浓度为10、50μmol／L的NaF染毒24h后，均引起明显的DNA泳动（彗星尾）并呈现明显的剂量-效应关系；氟砷联合作用淋巴细胞，在0．5μmol／L NaAsO2＋50μmol／L NaF的剂量下，细胞DNA的损伤呈现出协同作用（P〈0．01）。结论 NaAsO2与NaF均可引起DNA损伤；低剂量NaAsO2与NaF联合作用时，则呈现协同作用。     

tBHQ对无机砷致HaCaT细胞细胞周期调节失控的拮抗作用

目的探讨叔丁基对苯二酚(tert-butylhydroquinone,tBHQ)对亚砷酸钠(sodium arsenite,NaAsO2)致人皮肤角质细胞系HaCaT细胞周期阻滞及其调控蛋白异常改变的拮抗作用。方法流式细胞仪法测定细胞周期;Western Blot检测细胞内Cyclin D1和CDK4的蛋白表达情况。结果 NaAsO2单独作用HaCaT细胞,G0/G1期细胞比例明显下降(P<0.01),S期和G2/M期细胞比例较对照组明显增加(P<0.01);tBHQ预处理后,tBHQ(50μmol/L)预处理的NaAsO2(25、50μmol/L)组细胞G0/G1期细胞比例有所上升(P<0.01);tBHQ(50μmol/L)预处理的NaAsO2(50μmol/L)组S期细胞比例明显下降(P<0.01);G2/M期细胞比例整体呈下降趋势。NaAsO2单独作用于HaCaT细胞,细胞周期相关蛋白Cyclin D1和CDK4的蛋白表达随NaAsO2染毒浓度的增加而明显下降(P<0.01);tBHQ预处理后,Cyclin D1和CDK4蛋白表达均明显高于相同浓度NaAsO2单独作用组(P<0.01)。结论 tBHQ对无机砷致人皮肤角质细胞细胞周期异常变化具有一定的拮抗作用。     

tBHQ对NaAsO2诱导HaCaT细胞凋亡的拮抗作用

目的探讨叔丁基对苯二酚(tertiary butylhydroquinone,tBHQ)对亚砷酸钠(NaAsO2,sodium arsenite)诱导人角质上皮HaCaT细胞凋亡的拮抗作用。方法 tBHQ(10、25、50μmol/L)预处理12 h,再与NaAsO2(5、10、30μmol/L)共同处理HaCaT细胞24 h。用Annxin V/PI染色流式细胞术法检测细胞凋亡率;DAPI染色观察细胞形态学改变;JC-1法测定线粒体膜电位。结果将实验数据进行ANOVA分析处理后表明,5,10、30μmol/L NaAsO2单独作用时,细胞凋亡率与对照组相比显著升高,而线粒体膜电位则显著下降(P<0.05或P<0.01),形态学观察可见高强度DAPI染色细胞数目增加和凋亡小体出现;当给予tBHQ(10、25、50μmol/L)预处理后,NaAsO2致HaCaT细胞凋亡率升高和线粒体膜电位下降均明显受到抑制(P<0.05),形态学观察可见高强度DAPI染色细胞数目和细胞核碎片明显减少。结论实验结果表明tBHQ可能通过线粒体凋亡途径抑制NaAsO2引起的HaCaT细胞凋亡。     

氟砷单独及联合作用对正常人淋巴细胞增殖的影响观察

目的观察氟、砷单独及联合作用对正常人淋巴细胞增殖的影响.方法采用MTT实验测定亚砷酸钠及氟化钠对正常人淋巴细胞的LC50,然后以此为依据制定试验剂量(LC50的1/100～1/10),观察低剂量氟、砷单独及联合作用对正常人淋巴细胞增殖的影响.结果人淋巴细胞在经浓度为0.1,1μmol/L的NaAsO2及浓度为50mol/L的NaF染毒后,其MTT吸光度值与对照组相比,均呈下降趋势,且有统计学意义(P均＜0.01).结论氟砷联合作用对人淋巴细胞生长表现出明显的抑制作用,但经交互作用方差分析,尚不能认为氟砷联合作用对人淋巴细胞生长的抑制有交互作用(P＞0.05),低剂量亚砷酸钠及氟化钠对人淋巴细胞均存在明显的毒作用;氟与砷之间对人淋巴细胞的生长抑制作用并没有交互作用,仅表现为简单的相加作用.     

亚砷酸钠诱导人膀胱上皮永生化细胞氧化损伤作用观察

目的 观察亚砷酸钠(NaAsO2)诱导人膀胱上皮永生化细胞(SV-HUC-1)氧化损伤作用.方法 以不同浓度NaAsO2[0(对照)、1、2、4、8、10μmol/L]对SV-HUC-1细胞染砷24 h,利用流式细胞仪检测细胞内活性氧(ROS)水平,采用酶联免疫吸附实验(EHSA法)检测细胞内硝基酪氨酸(NT)含量和细胞培养液中8-羟基脱氧鸟嘌呤核苷(8-OHdG)水平.结果 1、2、4、8、10 μmol/L染砷组SV-HUC-1细胞ROS水平(81.76±4.91、95.23±2.17、126.61±17.95、126.74±27.77、114.18±9.65)明显高于对照组(69.84±1.28,P＜ 0.05或＜ 0.01),ROS水平与染砷剂量呈显著正相关(r=0.818,P＜ 0.01).10 μmol/L染砷组SV-HUC-1细胞内NT含量[ (919.66±206.33)μg/L]显著高于对照组[(238.19±38.28) μg/L,P＜ 0.01],NT含量与染砷剂量呈显著正相关(r=0.617,P＜0.01).各组细胞培养液中8-OHdG含量比较,差异无统计学意义(F=2.127,P＞0.05).结论 NaAsO2能够引起SV-HUC-1细胞氧化损伤.     

亚砷酸钠诱导外周血淋巴细胞凋亡的实验研究

目的探讨亚砷酸钠(NaAsO2)诱导淋巴细胞凋亡的作用机制。方法用倒置显微镜、透射电子显微镜、噻唑蓝(MTT)还原实验、细胞免疫组化法,分别对不同浓度的实验组及对照组淋巴细胞形态学改变,细胞存活率及Bcl-2蛋白的表达进行观察和测定。结果MTT实验显示,0.1、1.0、5.0μmol/L的NaAsO2均能抑制淋巴细胞的生长增殖(P<0.05);透射电镜观察到淋巴细胞表面的突起减少或脱失,部分细胞核染色质聚集、边移,线粒体结构模糊不清;Bcl-2在实验组中表达的强度均低于对照组。结论NaAsO2不仅抑制淋巴细胞增殖,而且诱导细胞凋亡,诱导细胞凋亡与Bcl-2表达有关。     

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.     

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.     

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.     

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.