基于FAK-F-actin通路探讨岩藻糖基转移酶8对泡沫细胞运动能力的影响

目的探讨岩藻糖基转移酶8(Fut8)表达下调对泡沫细胞运动能力的影响及机制。方法建立泡沫细胞模型,检测Fut8表达的改变,并明确其与泡沫细胞运动能力之间的直接联系。检测Fut8过表达对细胞内纤维型肌动蛋白(F-actin)形成的影响及其上游分子局部黏着斑激酶(FAK)的活化情况。结果泡沫细胞形成过程中,Fut8的表达明显下调,而过表达Fut8能修复泡沫细胞受损的运动能力。F-actin的表达随着泡沫细胞运动能力下降而明显下调,且受到Fut8的调节。FAK的磷酸化在Fut8表达下调时受到明显抑制,过表达Fut8能导致FAK的磷酸化明显增强。结论 Fut8-FAK-F-actin通路受到抑制可能是泡沫细胞运动能力减弱的原因之一。     

化瘀解毒方含药血清体外抑制A549肿瘤细胞作用及其机制

目的研究化瘀解毒方提取物抗人肺癌A549细胞增殖、迁移、侵袭的作用及机制。方法培养人肺癌A549细胞株,利用MTT法分析肿瘤细胞增殖抑制率,利用Transwell细胞培养系统检测细胞迁移、侵袭作用,采用实时荧光定量PCR法检测化瘀解毒方提取物对磷脂酰肌醇3-激酶(PI3K),3-磷酸肌醇依赖性蛋白激酶(PDK)1和蛋白激酶B(Akt)mRNA的表达,进一步采用免疫印迹检测PI3K/Akt信号通路中蛋白及其磷酸化水平。结果化瘀解毒方提取物呈浓度依赖性抑制体外人肺癌A549肿瘤细胞增殖、迁移和侵袭,与生理盐水组相比,差异均有统计学意义(P0.05)。化瘀解毒方含药血清和阳性对照药环磷酰胺对PI3K,PDK1和Akt总蛋白水平无影响。然而,化瘀解毒方含药血清显著抑制A549细胞中Akt蛋白的磷酸化水平。结论化瘀解毒方含药血清抑制A549细胞增殖、黏附、迁移和侵袭与抑制Akt蛋白的磷酸化有关,其作用机制可能与抑制PI3K/Akt通路有关。     

百里醌对HUVECs细胞凋亡的影响及其机制

目的探究百里醌降低过氧化氢引起的人脐静脉内皮细胞(HUVECs)细胞凋亡及其潜在机制。方法使用百里醌预处理HUVECs细胞后加入过氧化氢共培养1 h,使用四甲基偶氮唑蓝(MTT)方法测定细胞增殖能力改变;应用流式细胞仪检测百里醌预处理后加入过氧化氢共培养的HUVECs细胞凋亡情况变化;使用GFP-LC3荧光体系检测百里醌对HUVECs细胞自噬的影响;应用Western印迹法测定不同处理后HUVECs细胞Caspase-3/Cleaved Caspase-3、LC3-Ⅰ/LC3-Ⅱ、Akt/p-Akt、mTOR/p-mTOR、Beclin1、Bcl-2、β-Actin蛋白表达情况改变。结果百里醌预处理HUVECs细胞,降低由过氧化氢引起的细胞凋亡并呈现剂量依赖性(P0.05);百里醌诱导HUVECs细胞发生自噬;3-MA阻断自噬途径后百里醌对过氧化氢处理的HUVECs细胞保护作用减弱(P0.05);百里醌抑制p-Akt、p-mTOR蛋白表达,促进Beclin1和Bcl-2蛋白表达(P0.05)。结论百里醌通过抑制Akt/mTOR信号通路和上调Beclin1蛋白激活自噬,促进Bcl-2蛋白表达抑制凋亡,降低过氧化氢引起的HUVECs细胞凋亡,为动脉粥样硬化的防治提供新思路。     

纤维连接蛋白对人结肠癌细胞侵袭力的影响

目的研究纤维连接蛋白（fibronectin，FN）对人结肠癌细胞侵袭力的影响，并探讨其中的信号传导机制。方法以递增浓度的FN刺激结肠癌细胞株Colo320，以免疫沉淀和蛋白质印迹法检测结肠癌细胞内黏着斑激酶（focal adhesion kinase，FAK）第397位酪氨酸（tyr-397）磷酸化的状况．以改良Boyden小室法检测相应的细胞侵袭力变化。设计反义寡核苷酸阻断FAK蛋白质表达，再次观察接受FN刺激后．结肠癌细胞内FAK tyr-397磷酸化状况及细胞侵袭力的改变。结果FN能够促进Colo320 FAK tyr-397磷酸化，在一定范围内具有剂量依赖性。FN浓度达10nmol／L时．此作用最显著，继续增加FN浓度．FAK tyr-397磷酸化不再呈现继续增强趋势．当浓度达到100nmol／L时，磷酸化程度反而有所下降；FN可以增强细胞侵袭力，同样在一定范围内具有剂量依赖性；反义寡核苷酸干预后．结肠癌细胞内FAK tyr-397磷酸化及细胞侵袭力均有显著降低（P〈0.01）。结论FN可以有效增强结肠癌细胞的侵袭力，其作用是通过FN—FAK信号通路实现的，FAK的活性形式是其酪氨酸位点的磷酸化．阻断FAK的表达町以减弱FN促进细胞侵袭的作用。     

反义局部粘着斑激酶脱氧寡核苷酸对肝癌细胞侵袭性生长的抑制作用

目的 观察反义局部粘着斑激酶脱氧寡核苷酸(FAK ODN)转染对肝癌细胞侵袭性生长的影响,并探讨其作用机制。 方法 以LipofecTAMINE介导的反义FAK ODN转染Bel 7402肝癌细胞株,测定Bel 7402肝癌细胞株体外生长曲线、细胞活力,测定不同时间点该细胞体外黏附能力变化,以Transwell小室测定细胞的体外侵袭能力,同时行FAK表达与细胞DNA含量的双参数流式细胞仪检测及细胞凋亡的流式细胞仪检测。 结果 p125FAK表达在反义转染组(6.49％±0.10％)显著低于正义转染组(14.33％±1.88％)与对照组(16.68％±1.62％),F=7.66,P<0.01;反义FAK ODN转染显著抑制Bel 7402肝癌细胞株的生长,其细胞活力显著下降,肿瘤细胞抑制率在30％-60％之间;细胞体外黏附能力受到显著抑制,黏附抑制率在25％-55％间;细胞的体外侵袭能力显著下降,侵袭抑制率在15％-25％之间;细胞凋亡显著增加;细胞周期分析显示S期细胞比率显著降低,细胞生长主要阻滞在G2/M期。 结论 FAK在Bel 7402肝癌细胞的黏附与迁移运动中发挥重要作用,其表达阻断显著抑制肝癌细胞的体外黏附与侵袭活性。FAK表达阻断显著抑制肝癌细胞的体外增殖,促进细胞凋亡。     

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.     

肝细胞生长因子对肝细胞癌细胞系SMMC-7721黏着斑激酶的影响

目的:探讨肝细胞生长因子(HGF)对黏着斑激酶(FAK)、Src表达及活性的影响以及PI3K在该过程中的作用.方法:LY294002预处理阻断PI3K的活性、HGF处理SMMC-7721后检测FAK和Src的表达、磷酸化及在细胞内的分布. 应用免疫印迹技术检测FAK、Src的表达及磷酸化, 免疫荧光技术检测FAK在SMMC-7721细胞中的分布.结果:HGF(50 μg/L)可以促进FAK Y397位点的磷酸化, 对FAK的表达没有影响. 应用PI3K抑制物LY294002处理后, FAK Y397的磷酸化水平显著降低. HGF处理后, FAK主要位于细胞边缘, 呈簇状分布, 应用PI3K抑制物, FAK在细胞内弥散分布. HGF处理后, Src激酶和SrcY416的磷酸化水平明显增加, 而经LY294002处理后, Src激酶与Src Y416的磷酸化水平明显降低.结论:肝细胞癌中, HGF以PI3K依赖性的方式促进FAK和Src的活化.     

人结肠癌细胞胃泌素-黏着斑激酶通路的研究

目的研究胃泌素对人结肠癌细胞信号分子黏着斑激酶(FAK)酪氨酸磷酸化和蛋白质表达的影响。方法使用胆囊收缩素2受体(CCK2R)的真核表达载体pCR3.1/CCK2R,转染人结肠癌细胞株Colo320,上调胃泌素作用;使用胃泌素拮抗剂下调胃泌素作用。使用胃泌素按浓度和时间梯度刺激细胞,以免疫沉淀和蛋白质印迹法检测FAK酪氨酸磷酸化和蛋白质表达情况。结果胃泌素能够引起FAK酪氨酸磷酸化,具有时间和剂量依赖性;CCK2R表达上调可以增强此作用;胃泌素拮抗剂具有相反作用。结论FAK是胃泌素发挥效应的下游信号分子,以酪氨酸磷酸化的形式发挥作用。胃泌素CCKRFAK信号通路在胃泌素引起的肿瘤细胞增殖过程中发挥重要作用。     

VEGF基因沉默对胰腺癌细胞化疗敏感性和Akt信号通路的影响

目的 观察靶向血管内皮生长因子(vascular endothelial growth factor,VEGF)基因小干扰RNA(small interfering RNA,siRNA)对胰腺癌BxPC3细胞化疗药物敏感性的影响,并探讨其可能机制.方法 将培养后的BxPC3细胞分为单纯BxPC3细胞组、脂质体处理组、错配siRNA转染组和VEGFsiRNA转染组.以5、10、20、100、200 nmol/L的VEGF siRNA转染BxPC3细胞.采用实时定量RT-PCR和ELASA法检测细胞VEGF mRNA和蛋白的表达;MTT法检测吉西他滨对各组细胞生长的影响;蛋白质印迹法检测BxPC3细胞Akt蛋白的磷酸化.结果 VEGF siRNA转染后,BxPC3细胞VEGF mRNA和蛋白呈浓度和时间依赖性明显下调,但对BxPC3细胞的增殖无明显影响.用0.2 μmol/L吉西他滨处理细胞48 h后,BxPC3细胞组、错配siRNA组及5、10、20 nmol/L siRNA组细胞的生长抑制率分别为(16.9±0.3)%、(17.3±0.3)%、(28.8±0.4)%、(52.2±0.3)%、(75.4±0.4)%,抑制率与siRNA浓度相关(r=0.928).同时,siRNA转染后细胞Akt蛋白的磷酸化被明显抑制.结论 VEGF基因在胰腺癌BxPC3细胞耐药中起着重要的作用,其机制可能与Akt蛋白磷酸化有关.     

半乳凝素3在胰腺癌细胞中的表达及对胰腺癌SW1990细胞增殖和侵袭的影响

目的 研究半乳凝素3( galectin-3)在胰腺癌细胞株中的表达及对人胰腺癌细胞株SW1990增殖和侵袭能力的影响.方法 采用免疫细胞化学法和RT-PCR方法检测胰腺癌细胞株SW1990、PANC1、AsPC-1的galectin-3蛋白和mRNA表达.分别应用1、2、3、5μg/ml galectin-3单抗处理SW1990细胞24、48、72 h,采用CCK-8试剂盒检测细胞的增殖,Transwell小室检测细胞的侵袭能力.结果 胰腺癌SW1990.PANC1、AsPC-1细胞均有galectin-3 mRNA和蛋白表达.galectin-3单抗呈浓度和时间依赖性抑制SW1990细胞的增殖及穿膜细胞数.培养72 h时2、3、5μg/ml galectin-3单抗的细胞生长抑制率分别为19.8％、29.9％和42.7％,与对照组比较差异均有统计学意义(P值均＜0.05).3 μg/ml galectin-3单抗组的穿膜细胞抑制率为37.1％,与对照组的10.4％差异有统计学意义(P＜0.05).结论 galectin-3在胰腺癌细胞中高表达,用抗体中和galectin-3能抑制SW1990细胞的增殖和侵袭能力     

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.     

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.     

N-acetyl-L-cysteine combined with mesalamine in the treatment of ulcerative colitis： Randomized,placebo-controlled pilot study

AIM： To evaluate the effectiveness and safety of oral N-acetyl-L-cysteine （NAC） co-administration with mesalamine in ulcerative colitis （UC） patients.
METHODS： Thirty seven patients with mild to moderate UC were randomized to receive a four-wk course of oral mesalamine （2.4 g/d） plus N-acetyl-L-cysteine （0.8 g/d） （group A） or mesalamine plus placebo （group B）. Patients were monitored using the Modified Truelove-Witts Severity Index （MTWSI）. The primary endpoint was clinical remission （MTWSI ≤ 2） at 4 wk. Secondary endpoints were clinical response （defined as a reduction from baseline in the MTWSI of ≥ 2 points） and drug safety. The serum TNF-α, interleukin-6, interleukin-8 and MCP-1 were evaluated at baseline and at 4 wk of treatment. RESULTS： Analysis per-protocol criteria showed clinical remission rates of 63% and 50% after 4 wk treatment with mesalamine plus N-acetyl-L-cysteine （group A） and mesalamine plus placebo （group B） respectively （OR = 1.71; 95% CI： 0.46 to 6.36; P = 0.19; NNT = 7.7）. Analysis of variance （ANOVA） of data indicated a significant reduction of MTWSI in group A （P = 0.046） with respect to basal condition without significant changes in the group B （P = 0.735） during treatment. Clinical responses were 66% （group A） vs 44% （group B） after 4 wk of treatment （OR = 2.5; 95% CI： 0.64 to 9.65; P = 0.11; NNT = 4.5）. Clinical improvement in group A correlated with a decrease of IL-8 and MCP-1. Rates of adverse events did not differ significantly between both groups.
CONCLUSION： In group A （oral NAC combined with mesalamine） contrarily to group B （mesalamine alone）, the clinical improvement correlates with a decrease of chemokines such as MCP-1 and IL-8. NAC addition not produced any side effects.     

Delorme's transrectal excision for internal rectal prolapse

Surgical therapy of functional outlet obstruction in patients with internal rectal intussusception may include abdominal, perineal, or transrectal procedures. Because abdominal procedures often result in significant physiologic impact but unrelieved constipation, the authors have elected Delorme's transrectal excision for management of these patients. Since a short-term placebo effect attends many therapies, this report describes results of transrectal excision only after a threeyear postoperative period. Delorme's transrectal excision of internal intussusception accomplished sustained symptomatic relief in over 70 percent of otherwise refractory constipated patients. The association of internal intussusception with other abnormalities underscores the importance of defining both anatomic and functional components when selecting patients whose constipation may require surgical therapy. Critical technical elements, surgical pitfalls, and potential complications of the procedure are discussed.Poster presentation at the meeting of The American Society of Colon and Rectal Surgeons, Toronto, Canada, June 11 to 16, 1989.     

The oral glucose tolerance test: A comparison of the time points on the basis of limit values,normal dispersion,and reproducibility

Summary Time points in the glucose tolerance test (GTT) are compared on the basis of limit values, dispersion within a reference population, and reproducibility. We suggest using the distance between a limit value and the median reference value as a measure of the magnitude of abnormality. The distance between 140 mg/100 ml and the median fasting plasma glucose value is chosen as a standard distance and limits for other points in the GTT are calculated to equal this standard distance of abnormality. We suggest that the probability of correctly interpreting an inividual result is directly related to the reproducibility of the test and inversely related to the percentage of the total range of values which is dispersed among the normal population. The ratio of reproducibility to percentage normal dispersion is proposed as an index of the probability of correctly interpreting an individual result. According to this index, the probability of correct interpretation varies in order: fasting plasma glucose concentration>3-h>2-h>0.5-h>1-h plasma glucose concentration.