肝癌细胞系放射诱导的细胞凋亡和细胞周期的变化

目的：探讨肝癌细胞系放射诱导的细胞周期和细胞凋亡的变化特点．方法：研究细胞系为肝癌细胞系HepG2和SMMC-7721．对照细胞系为正常肝细胞系HL-7702、肺小细胞癌HCI-H460和肺腺癌A549．常规培养48h后接受4Gy射线照射,收获受照前(0h)和受照后6,12,24,36和48h的细胞,采用流式细胞术(FCM)检测各细胞系细胞周期和细胞凋亡．结果：4GyX线照射后,HepG2在照射后12h出现细胞凋亡高峰,射线诱导的细胞凋亡比率为45．16%(t=8．864,P＜0．0025),而SMMC-7721在24h达高峰,诱导的细胞凋亡比率为24．94％,HepG2较SMMC-7721射线诱导的细胞凋亡高峰出现早、比率高：HepG2和SMMC-7721与HCI-H460和A549变化较一致,凋亡变化的走势和峰值均与S期的相反,两株肝癌细胞可能均发生了射线诱导的有丝分裂前S期细胞凋亡．HepG2在照射后12h有明显的G_2/M期阻滞,可能有射线诱导的G_2/M期细胞损伤,发生了延迟的间期死亡．结论：两株肝癌细胞可能均发生了射线诱导的有丝分裂前S期细胞凋亡,HepG2可能伴有射线诱导的G_2/M期细胞损伤,发生了延迟的间期死亡．     

肝癌细胞放射敏感性与survivin蛋白表达的关系

目的 探讨肝癌细胞放射敏感性与survivin表达的关系.方法 肝癌细胞HepG2和SMMC-7721在接受不同剂量γ射线照射后,分别采用克隆形成法、免疫细胞化学法、流式细胞术、比色法等检测细胞存活率、survivin蛋白表达、细胞周期变化和Caspase-3活性.结果 在2Gy照射下HepG2和SMMC-7721细胞的存活分数分别为0.43±0.01与0.70±0.02,SMMC-7721较HepG2放射抗拒.γ射线对SMMC-7721细胞的G2/M期阻滞时间较HepG2细胞长(48 h对24 h),在阻滞峰各剂量点SMMC-7721细胞的G2/M期比例也更高.γ射线可上调两株肝癌细胞survivin蛋白的表达,照射后48～72 h,SMMC-7721细胞的survivin蛋白表达水平显著高于HepG2细胞(t值为2.81～5.20,P值均＜0.05).而Caspase-3的活化水平在放射敏感的HepG2细胞中更高(t值为6.05～6.72,P值均＜0.01).结论 射线诱导的survivin表达上调及survivin对Caspase-3的负调控可能是SMMC-7721细胞较HepG2细胞放射抗拒的原因之一.     

miR-26b通过信号通路Wnt调控肝癌细胞分化、增殖、凋亡

目的探讨miR-26b对肝癌细胞分化增殖凋亡的影响及机制。方法以qRT-PCR方法检测肝癌细胞SMMC-7721、HuH-7、HepG2和正常肝细胞HL-7702中miR-26b的表达水平。在肝癌细胞中转染miR-26b mimic、mimic control,qRT-PCR测定转染后细胞中miR-26b水平。CCK-8测定细胞增殖情况,流式细胞术测定细胞凋亡情况,Western印迹测定细胞中β-连环蛋白(catenin)、细胞周期蛋白(Cyclin)D1、剪切型含半胱氨酸的天冬氨酸蛋白水解酶(Cleaved Caspase)-3、Bcl-2相关X蛋白(Bax)表达水平。结果 HL-7702细胞中miR-26b表达水平显著高于SMMC-7721、HuH-7、HepG2细胞(P0.05)。SMMC-7721细胞中miR-26b表达水平显著低于HuH-7、HepG2细胞(P0.05)。选用SMMC-7721细胞作为研究对象。转染miR-26b mimic后的细胞中miR-26b水平显著升高(P0.05)。高表达miR-26b后的肝癌细胞存活率显著降低,细胞凋亡率显著提高,细胞中促凋亡蛋白Cleaved Caspase-3、Bax水平也显著升高,细胞中Wnt关键蛋白β-catenin、CyclinD1水平显著降低(均P0.05)。结论 miR-26b诱导肝癌肝癌细胞凋亡,抑制肝癌细胞增殖,作用机制与抑制Wnt信号通路有关。     

刺五加皂甙对肝癌SMMC-7721细胞凋亡的影响

目的研究刺五加皂甙(ASS)对人肝癌SMMC-7721细胞凋亡的诱导作用。方法体外培养人肝癌SMMC-7721细胞，用浓度0.25、0．5、1．0mg／ml的刺五加皂甙作用细胞，于12、24、48h后，用苏木素-伊红(HE)染色法及电镜技术观察凋亡细胞的形态，用琼脂糖电泳显示DNA凋亡梯带。结果ASS不促进肝癌细胞的调亡，且随着剂量和时间的增加诱发凋亡程度增高。结论ASS抑制肝癌SMMC-7721细胞增殖，诱导细胞凋亡，对指导临床药物治疗肝癌具有重要的意义。     

雌激素对人肝癌细胞增殖和凋亡的影响

目的探究雌激素对人肝癌细胞株增殖和凋亡的影响。方法采用普通聚合酶链反应(PCR)技术、Western印迹实验检测肝癌细胞株中雌激素受体(ER)α、ERβ的mRNA和蛋白质的表达情况;细胞增殖实验检测雌激素对肝癌细胞增殖的影响;流式细胞术检测雌激素对肝癌细胞凋亡率的影响;Tunel染色法检测雌激素对肝癌细胞晚期凋亡率的影响;雌激素处理肝癌细胞株后,基因芯片技术检测表达增加的凋亡基因。结果 ERα和ERβ在肝癌细胞株SMMC7721、HepG2、Bel-7404、huh7、MHCC-97L、MHCC-97H、PLC、LM3、SK-HEP-1及Bel-7402中均有表达;不同浓度的雌激素处理肝癌细胞不同时间后,可显著抑制肝癌细胞增殖,且抑制效果存在时间和剂量依赖性;与0.00μmol/L组相比,不同浓度的雌激素(1.25、2.50、5.00、10.00、20.00、40.00、80.00和160.00μmol/L)处理HepG2、SMMC-7721细胞24 h、48 h后,HepG2和SMMC-7721细胞的细胞凋亡率增加,存在剂量和时间的依赖性;Tunel结果显示,与0.00μmol/L组相比,10.00μmol/L雌激素处理HepG2、SMMC-7721细胞48 h后,细胞晚期凋亡率均显著增加;20.00μmol/L雌激素处理SMMC-7721细胞48 h后,凋亡基因BAK1、GADD45A、HRK、LTA、TNFRSF-10A均表达增加。结论雌激素对人肝癌细胞具有抑制生长、诱导凋亡的作用。     

人端粒酶逆转录酶干扰对肿瘤坏死因子相关凋亡诱导配体诱导肝癌细胞凋亡的影响

目的研究人端粒酶逆转录酶(hTERT)干扰对肝癌．HepG2、SMMC-7221细胞生物学形为的影响和对肿瘤坏死因子相关的凋亡诱导配体(TRAIL)诱导凋亡的影响。方法将HepG2细胞和SMMC-7721细胞分为转染组 (转染重组质粒真核表达载体)、对照组(转染空载体质粒)和未转染组。采用聚合酶链反应方法检测hTERT干扰序列, 逆转录聚合酶链反应方法检测hTERT表达,HE染色、生长曲线和流式细胞术方法分别检测细胞形态、增殖情况和细胞周期,β-半乳糖苷酶染色方法检测细胞状态,Armexin V／PI染色流式细胞术检测细胞凋亡。结果转染组细胞内均存在hTERT干扰序列,HepG2和SMMC 7221细胞hTERT干扰率分别为100％和43．3％;与未转染组细胞相比, 转染细胞核质比明显缩小,增殖率下降差异有统计学意义(P<0．05),老化细胞和G2-M期细胞明显增加(P<0．05)。细胞老化率分别由未转染组的0增加到转染组的20．4％,由3．60%,增加到10．O％;G2-M期分别由未转染组的7．1％、6．9％增加到转染组的10．6％、7．9％。hTERT干扰显著增加肝癌细胞凋亡和TRAIL诱导凋亡敏感性(P<0．05)。两株肝癌细胞凋亡率分别由未转染组的3．5％、4．8％增至转染组的5．2%、7．9％;100 ng／ml TRAIL作用24 h后两株肝癌细胞凋亡率分别由未转染组的5．3％、13．9％增加到转染组的10．4％、77．2％,而对照组细胞各指标均无显著变化。结论 hTERT干扰明显影响肝癌细胞的生物学行为,显著增加细胞凋亡和TRAIL诱导凋亡的敏感性。     

survivin反义寡核苷酸对SMMC-7721细胞增殖和凋亡的影响

目的 观察survivin反义寡核苷酸(ASODN)对人肝癌细胞株SMMC-7721增殖和凋亡的影响.方法 人工合成survivin基因ASODN和正义ODN(SODN),并行硫代磷酸化修饰,通过脂质体途径转染SMMC-7721;分别用RT-PCR和Western blot检测survivin mRNA和蛋白表达;用MTT法检测ASODN对SMMC-7721增殖的影响;流式细胞仪检测细胞周期变化及细胞凋亡率;倒置显微镜观察细胞形态变化.结果 SMMC-7721可强表达survivin mRNA和蛋白;ASODN呈浓度依赖性抑制survivin mRNA和蛋白表达及SMMC-7721增殖,诱导细胞凋亡,使细胞阻滞于G2/M期.SODN对survivin mRNA和蛋白及SMMC-7721的增殖、细胞周期无明显抑制作用.结论 脂质体介导转染survivin ASODN可抑制细胞增殖、使细胞阻滞于G2/M期,从而促进细胞凋亡.     

羽扇豆醇影响肝癌细胞株SMMC-7721增殖与凋亡的机制

目的:讨论羽扇豆醇影响肝癌细胞株SMMC-7721增殖及促进其凋亡作用机制.方法:使用不同浓度(0、2、5、10、20μg/m L)的羽扇豆醇在不同处理时间(24、36、48 h)下,处理S M M C-7721细胞株后,通过MTT法检测SMMC-7721细胞增殖情况;对使用不同浓度的羽扇豆醇处理48 h后的SMMC-7721细胞,利用流式细胞仪检测S M M C-7721细胞的细胞周期以及细胞凋亡情况,还使用Western blot检测细胞细胞增殖细胞核抗原(proliferating cell nuclear a n t i g e n,P C N A)、B淋巴细胞瘤-2(B-c e l l lymphoma-2,Bcl-2)基因及Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)蛋白的表达情况,最后我们通过免疫组织化学的方式检测在裸鼠体内接种的SMMC-7721细胞瘤体的微血管密度(microvessel density,M V D)值,检测羽扇豆醇对抑制肿瘤血管生成的作用.结果:相较于对照组,通过不同浓度的羽扇豆醇处理后的SMMC-7721细胞增殖水平均受到不同程度的抑制,而且既显示出量效关系又显示出时效关系;受到羽扇豆醇体外诱导处理后的SMMC-7721细胞能够使G0/G1期受到阻滞,甚至出现凋亡现象,SMMC-7721细胞的PCNA和Bcl-2蛋白表达情况下调,Bax蛋白的表达情况上调(P0.05).肿瘤瘤体MVD值下降(P0.01).结论:SMMC-7721细胞的增殖会因为羽扇豆醇的处理而受到抑制,并且还会因为羽扇豆醇的处理而出现凋亡.羽扇豆醇还可以抑制肿瘤瘤体血管生成.     

DEK基因在肝癌中的表达及靶向抑制其表达对肝癌细胞增殖及凋亡的影响

目的探讨DEK基因在肝癌中的表达及RNA干扰抑制其表达对肝癌细胞增殖及凋亡的影响及机制。方法以人正常肝细胞HL-7702作为对照,RT-PCR检测人肝癌HepG2、Huh-7、SMMC-7721、SNU-475细胞中DEK mRNA表达;DEK-siRNA转染HepG2细胞,同时转染阴性对照和空白对照,转染48 h后Western blotting检测各组细胞中DEK、Cleaved caspase 3、β-catenin、Cyclin D1蛋白表达;CCK8实验和流式细胞仪分别检测细胞的增殖及凋亡情况。结果各个肝癌细胞中DEK mRNA表达均显著高于正常细胞,差异有统计学意义(P0.01);DEK基因在HepG2细胞中表达最高,选择作为后续研究对象;转染DEK-siRNA后DEK蛋白表达显著降低;DEK-siRNA组细胞存活率及β-catenin、Cyclin D1蛋白表达显著低于空白对照组和阴性对照组,细胞凋亡率和Cleaved caspase 3蛋白表达显著高于空白对照组和阴性对照组,差异有统计学意义(P0.01)。结论抑制肝癌HepG2细胞中DEK基因可降低癌细胞的增殖及诱导细胞凋亡,其机制与Wnt/β-catenin信号通路的调控有关。     

RNA干扰GRP78基因对肝癌细胞株SMMC-7721增殖和凋亡的影响

目的 研究shRNA干扰GRP78对肝癌细胞株SMMC-7721增殖和凋亡的影响。方法 将GRP78特异性shRNA质粒载体Pla-anti-GRP78转染SMMC-7721,采用流式细胞术(FCM)检测转染效率、分析细胞周期分布和凋亡,从蛋白和mRNA水平检测干扰GRP78效果,MTT 法检测干扰GRP78对SMMC-7721增殖的影响。结果Pla-anti-GRP78转染SMMC-7721细胞48h后,GRP78表达下降,空白对照组GRP78 mRNA的表达量为1,无关shRNA组为0.95,而Pla-anti-GRP78组为0.25(P<0.05);转染Pla-anti-GRP78的SMMC-7721细胞G2期阻滞(55.2%,P<0.05);空白对照组凋亡率为6.6%,无关shRNA组为8.1%,而Pla-anti-GRP78组高达58.2%(P<0.05)。 结论　shRNA干扰GRP78表达抑制SMMC-7721的增殖,并诱导其凋亡。     

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.     

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.     

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.     

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.