粘着斑激酶相关非激酶质粒转染抑制肝星状细胞周期进程及机制研究

粘着斑激酶（focal adhesion kinase，FAK）磷酸化后，对多种类型细胞的生物学行为具有重要影响，如促进增殖，正性调控细胞周期，增强粘附、迁移，控制凋亡等。粘着斑激酶相关非激酶（FAK—related non-kinase，FRNK）是FAK的内源性抑制剂。本研究应用体外细胞培养技术，以纤维连接蛋白（fibronectin，FN）刺激肝星状细胞（hepatic stellate cell，HSC）增殖，在脂质体介导下瞬时转染FRNK质粒，探讨选择性阻断FAK磷酸化对HSC增殖周期及细胞周期相关蛋白的影响。     

黏着斑激酶相关非激酶对肝星状细胞凋亡及细胞外信号调节激酶的影响

目的 应用黏着斑激酶相关非激酶(FRNK)表达质粒瞬时转染纤维连接蛋白(FN)预刺激的肝星状细胞(HSC),探讨FRNK对HSC凋亡及细胞外信号调节激酶(ERK)的影响.方法 在体外,以FN诱导HSC增殖,采用脂质体介导的方法用FRNK表达质粒瞬时转染HSC,应用膜联蛋白/碘化丙啶双标记流式细胞术、DNA凝胶电泳技术和透射电镜技术检测细胞的凋亡,Western blot及RT-PCR方法检测FRNK、黏着斑激酶(FAK),p FAK(Tyr397)、半胱氨酸天冬氨酸特异性蛋白酶-3(caspase-3)、ERK1、p-ERK蛋白及其mRNA表达. 结果FRNK表达质粒成功转染HSC,在翻译后水平抑制FAK磷酸化.与空质粒组比较,FRNK表达质粒转染HSC48 h后,HSC凋亡率由9.28%±1.05%增至25.37%±1.92%(P<0.01),caspase-3蛋白由185.82±9.69增至264.17±12.60(P<0.01),caspase-3 mRNA由1.07±0.27增至4.19±0.48(P<0.01).FRNK抑制FAK磷酸化和在翻译和转录水平抑制ERK1、p-ERK的表达,而FN则促进FAK和ERK1,p-ERK在翻译和转录水平的表达. 结论在脂质体介导下瞬时转染FRNK表达质粒,可使外源性的FRNK在HSC内大量表达,在翻译后水平抑制FAK磷酸化;并可能通过FAK-ERK信号转导通路诱导FN刺激的HSC发生凋亡.     

缺氧促进人肺动脉平滑肌细胞黏着斑激酶表达

黏着斑激酶（FAK）与人肺动脉平滑肌细胞（HPASMC）增殖有关。FAK是否参与了缺氧状态下HPASMC的增殖尚少报道。我们的研究对此进行了缺氧情况下FAK在HPASMC增殖中的作用。     

FAK-ERK信号转导通路在FRNK抑制肝星状细胞胶原合成中的作用

目的:探讨FAK-ERK信号转导通路在黏着斑激酶相关非激酶(FAK-related non-kinase,FRNK)质粒转染抑制肝星状细胞(hepatic stellate cells,HSCs)胶原合成中的作用.方法:在体外,以FN诱导HSCs增殖,采用脂质体介导的方法用FRNK表达质粒瞬时转染HSCs,利用3H-Pro掺入技术测定HSCsⅠ型胶原的合成,Western blot及RT-PCR方法检测FRNK、FAK、p-FAK(Tyr397)、ERK蛋白和mRNA的表达.结果:FRNK表达质粒成功转染HSCs,在翻译后水平抑制FAK磷酸化.在FRNK转染HSC48 h后胶原合成能力较空质粒组显著下降(498.17±73.20 vs 748.33±61.30,P<0.01).FRNK抑制FAK磷酸化和在翻译和转录水平抑制ERK1、p-ERK的表达,而FN则促进FAK和ERK1、p-ERK在翻译和转录水平的表达.结论:FRNK可以使HSCs胶原合成能力降低,FAK-ERK信号转导通路可能发挥了负调控作用.     

粘附斑激酶家族新成员Pyk2的研究进展

富含脯氨酸的酪氨酸激酶2（proline—rich tyrosine kinase 2，Pyk2），又称细胞粘附激酶β（CAKβ）、相关粘附聚焦酪氨酸激酶（RAFTK），是粘附斑激酶（focal adhesion kinase．FAK）家族的新成员，与FAK具有高度同源性。Pyk2活化可催化多种含SH2结构域的底物蛋白磷酸化，涉及到多条信号传导通路，包括离子通道的调节、细胞生长增殖分化和细胞洲亡等，但其功能机制至今尚不清楚。     

黏着斑激酶在结直肠癌中的研究进展

黏着斑激酶(focal adhe sion kinase,FAK)是一种非受体酪氨酸激酶,他作为整合素介导的信号传导过程中的中心分子与多个信号通路相交通,参与细胞的生长、增殖、损伤修复及凋亡等多种生物学行为.FAK在结直肠癌中高表达,在正常大肠组织中呈弱阳性或阴性.FAK可能成为结直肠癌治疗的一个重要靶点,抑制FAK的功能可以阻断多条与肿瘤相关的信号通路.     

黏着斑激酶在结肠癌细胞中的表达及对其迁移的影响

[目的]观察局部黏着斑激酶（FAK）在结肠癌细胞中的表达及对结肠癌细胞侵袭动力和生长的影响。[方法]应用RT-PCR、Western blot方法测定结肠癌细胞株（HT-29）和正常肠上皮细胞中FAK的表达；在血小板源性生长因子（PDGF）刺激肿瘤细胞后，应用细胞黏附分析和体外侵袭试验方法，测定不同时间点细胞的体外黏附能力变化及侵袭能力，同时行FAK表达测定。[结果]结肠癌细胞较正常上皮细胞存在较高水平的FAK表达。PDGF干预组与非干预组比较，能促进HT-29的黏附功能（90．0％）和侵袭能力（54．5％），在该过程中，FAK的表达和活性升高（P〈0．05）。[结论]FAK对结肠癌细胞的体外黏附与迁移发挥重要的作用。     

黏着斑激酶相关非激酶对CFSC细胞基质金属蛋白酶-2及其抑制因子mRNA表达的影响

目的 探讨黏着斑激酶相关非激酶(FRNK)对纤维连接蛋白刺激的肝星状细胞CFSC胶原代谢的影响及其分子机制. 方法用体外细胞培养技术,脂质体介导法进行FRNK质粒瞬时转染;采用Western blot方法测定FRNK蛋白表达,鉴定转染效果;用3H-Pro掺入技术测定CFSCI型胶原的合成;用RT-PCR方法测定FRNK转染前后基质金属蛋白酶2(MMP-2)及其抑制因子(TIMP-2)基因在CFSC中表达的变化情况. 结果 FRNK质粒成功转染CFSC,Ⅰ型胶原合成下降;MMP-2基因表达上升,TIMP-2基因表达下降,MMP 2/TIMP-2比值明显上升.结论 外源性FRNK在CFSC内大量表达后,CFSC胶原表达减少;FRNK可能通过调节MMP-2/TIMP-2比值来促进CFSC的胶原降解.     

黏着斑激酶在人肺动脉平滑肌细胞增殖中的作用机制探讨

研究表明，黏着斑激酶(focal adhesion kinase，FAK)参与了多样细胞黏附、扩散、迁延、存活、生长等过程。然而，FAK是否参与了人肺动脉平滑肌细胞(HPASMCs)增生报道尚较少。本组研究采用人FAK反义寡核苷酸(ODNs)选择性抑制FAK蛋白表达的方法，旨在探讨FAK是否参与了HPASMCs的增生。     

黏着斑激酶在大鼠胸主动脉球囊损伤后内膜增殖中的作用

目的　在大鼠主动脉球囊损伤模型上 ,研究血管黏着斑激酶在内膜增殖过程中表达与活性的变化 ,以及氯沙坦对其的影响。方法　 54只Wistar大鼠被分成 3组。损伤组 :行单纯球囊拉伤 ,术后 1、3、7、1 4d取主动脉 ;氯沙坦组 :大鼠主动脉球囊损伤前 5d至术后 1 4d采用氯沙坦1 0mg·kg- 1 ·d- 1 灌胃 ,术后 1 4d取主动脉 ;对照组 :不进行球囊损伤 ,用饮用水代替氯沙坦灌胃。应用蛋白印迹杂交和免疫沉淀的方法 ,观察主动脉损伤后内膜增殖过程中黏着斑激酶表达与活性的变化 ;同时采用放射免疫法对血浆和主动脉组织中血管紧张素Ⅱ进行检测。结果　术后第 1天 ,黏着斑激酶表达与活性分别增加 1 4 %和 99% ,并持续至术后 1 4d。氯沙坦可抑制血管内膜增殖 ,并降低黏着斑激酶的表达 (35 % )与活化 (84% ) ,但对血浆与主动脉组织中血管紧张素Ⅱ水平无显著影响。结论大鼠主动脉球囊损伤后 ,黏着斑激酶表达与活性增加。氯沙坦抑制血管内膜增殖 ,并可显著降低黏着斑激酶的表达与活化 ,提示黏着斑激酶在血管内膜增殖中起重要作用     

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.