RNA 干扰沉默 IGF1R 表达对 A549细胞放射增敏作用的研究

目的：构建靶向胰岛素样生长因子1受体(IGF1R)-siRNA 重组慢病毒表达载体,观察其对人肺腺癌 A549细胞放疗增敏作用并探讨其机制。方法构建 IGF1R-siRNA 重组慢病毒,感染 A549细胞,蛋白免疫印迹法检测 IGF1R 沉默效率;荧光实时定量 PCR 法及 ELISA 法分别检测缺氧诱导因子1α(HIF-1α)、血管内皮生长因子(VEGF)和 Bad 基因及蛋白表达情况。克隆形成实验检测放射增敏作用。结果 IGF1R-siRNA 重组慢病毒使 A549细 胞 IGFlR 蛋白沉默效率达70.53%;HIF-1α、VEGF和 Bad 基因及蛋白表达量均显著降低;A549细胞对放射治疗的敏感性增加,平均致死剂量由1.20 Gy 下降至0.94 Gy,放射增敏比达1.28。结论沉默 IGF1R 对人肺腺癌 A549细胞具有放疗增敏效应,其机制可能与 HIF-1α、VEGF 和 Bad 表达下调相关。     

沉默血管内皮生长因子C对非小细胞肺癌A549细胞增殖和侵袭能力的影响

目的探讨沉默血管内皮生长因子(VEGF-C)基因对人非小细胞肺癌(NSCLC)A549细胞增殖和侵袭能力的影响。方法构建靶向VEGF-C基因的重组慢病毒Lv-siRNA-VEGF-C,将其感染A549细胞,建立VEGF-C基因低表达的A549细胞。Western印迹检测Lv-siRNA-VEGF-C细胞组、空载体Lv-Ctrl对照组及空白A549细胞中VEGF-C蛋白的表达噻唑蓝(MTT)法、Transwell侵袭实验和裸鼠移植瘤模型分别检测各组细胞的增殖和迁移能力。结果与对照相比,VEGF-C基因低表达组细胞中VEGF-C蛋白的表达显著降低,细胞体内、体外增殖活性也明显下降,细胞侵袭能力亦显著降低。结论成功构建的慢病毒介导的针对VEGF-C的干扰载体可有效抑制A549细胞表达VEGF-C蛋白表达,沉默VEGF-C能抑制人NSCLC A549细胞的增殖和侵袭能力。     

RNAi沉默VEGF-C对A549细胞VEGF家族因子及其受体和下游信号通路的影响

目的研究沉默血管内皮生长因子(VEGF)-C对人非小细胞肺癌(NSCLC)A549细胞VEGF家族因子及其受体和下游信号通路的影响。方法构建靶向VEGF-C基因的重组慢病毒Lv-siRNA-VEGF-C,将其感染A549细胞,建立VEGF-C基因低表达的A549细胞。ELISA法检测肿瘤细胞VEGF-C蛋白的分泌,Western印迹检测Lv-siRNA-VEGF-C细胞组、空载体Lv-Con对照组及空白A549细胞中VEGF家族因子VEGF-C、VEGF-A、VEGFR-2和VEGFR-3蛋白的表达,及下游ERK、AKT和p38信号通路的磷酸化活性。结果成功构建靶VEGF-C基因的干扰表达载体PSIH1-VEGF-C-siRNA,包装形成重组慢病毒Lv-siRNA-VEGF-C,以慢病毒转染A549建立VEGF-C基因低表达细胞。与对照组相比,VEGF-C低表达组细胞中VEGF-C蛋白的表达和分泌显著降低,VEGF-A、VEGFR-2和VEGFR-3的表达也显著降低。同时,VEGFR-2和VEGFR-3的磷酸化水平显著降低,并且VEGFR-2和VEGFR-3介导的下游信号分子ERK、AKT和p38的磷酸化水平亦明显降低(P<0.01)。结论慢病毒介导的VEGF-C基因沉默能抑制人非小细胞肺癌A549细胞VEGF家族因子VEGF-C、VEGF-A、VEGFR-2和VEGFR-3蛋白的表达及下游ERK、AKT和p38信号通路的磷酸化活性。     

补中益气汤对肺腺癌顺铂耐药细胞株与顺铂敏感细胞株荷瘤BALB/c小鼠移植瘤P-gp蛋白表达的影响

目的探讨补中益气汤对肺腺癌顺铂(DDP)耐药与敏感细胞株荷瘤BALB/c小鼠移植瘤P-gp蛋白表达的影响。方法体外培养肺腺癌顺铂耐药株A549/DDP及敏感细胞株A549,接种于BALB/c小鼠腋窝皮下,荷瘤成功后给予顺铂或顺铂联合低、中、高剂量补中益气汤治疗。实验共分为10组:A549/DDP或A549荷瘤对照组、A549/DDP或A549荷瘤+顺铂组、A549/DDP或A549荷瘤+顺铂+低剂量补中益气汤组、A549/DDP或A549荷瘤+顺铂+中剂量补中益气汤组、A549/DDP或A549荷瘤+顺铂+高剂量补中益气汤组。给药结束后,测量移植瘤体积并计算药物抑瘤率及抑瘤作用提高率,蛋白质印迹法检测移植瘤组织P-gp蛋白的表达。结果顺铂对A549/DDP移植瘤体积无显著影响(P0.05),而使A549移植瘤体积显著缩小(P0.05);联合低、中、高剂量补中益气汤的A549/DDP及A549移植瘤体积均缩小(P0.05),抑瘤率均增加(P0.05),随着中药剂量的增加,移植瘤体积随之缩小,抑瘤率随之增加,抑瘤作用提高率也随之增加;单独顺铂可上调A549/DDP移植瘤组织中P-gp蛋白表达(P0.05),下调A549移植瘤组织中P-gp蛋白表达(P0.05);联合低剂量补中益气汤并未显著下调A549/DDP及A549移植瘤组织中P-gp蛋白表达(P0.05);联合中、高剂量补中益气汤的A549/DDP及A549移植瘤组织中P-gp蛋白表达均显著下调(P0.05),两剂量间无差异(P0.05)。结论在顺铂耐药与顺铂敏感的肺腺癌移植瘤组织中,补中益气汤均有缩小移植瘤体积、提高顺铂抑瘤率、下调P-gp蛋白表达的作用,尤以中、高剂量作用最显著。     

RNAi技术对A549细胞survivin基因表达及顺铂敏感性的影响

目的 利用RNA干扰(RNAi)技术沉默抗凋亡基因survivin,观察其对人肺腺癌细胞A549增殖以及顺铂药物敏感性的影响.方法 将靶向survivin的基因片段插入到载体后构建重组质粒,将其导入A549细胞,RT-PCR及免疫荧光法分析转染前后A549细胞survivin mRNA及蛋白的表达情况,MTT法检测转染后A549细胞对顺铂的敏感性变化.结果 成功构建pGenesil1.1-survivin重组质粒.转染重组质粒后,A549细胞survivin mRNA和蛋白的表达明显降低;转染前顺铂对A549细胞的IC50明显高于转染后(P＜0.05).结论 靶向survivin的RNAi表达载体能下调survivin基因表达,增强顺铂对A549细胞的药物敏感性.     

重组腺相关病毒介导的RNA干扰抑制血管生长素表达对肺腺癌细胞成瘤能力的影响

目的 通过腺相关病毒(AAV)介导的RNA干扰(RNAi)抑制肺腺癌细胞A549血管生长素(ANG)表达,观察其对癌细胞生长和成瘤能力的影响.方法 构建H1启动子驱动的表达针对ANG的小干扰RNA(siRNA)重组腺相关病毒(AAV-shANG),转染A549细胞,同时以正常A549细胞以及转染AAV-Null的A549细胞作为对照,观察重组腺相关病毒介导的RNAi抑制ANG表达对A549细胞生长、致瘤、肿瘤细胞增殖、凋亡及肿瘤微血管密度的影响.用t检验分析各组间差别,所有数据均用x±s表示.结果 体外实验结果表明重组腺相关病毒AAV-shANG成功构建,重组腺相关病毒转染A549细胞72 h后ANG蛋白表达水平明显低于A549细胞组及AAV-Null组;转基因A549细胞细胞周期分析结果提示,正常A549细胞、AAV-Null转染细胞和AAV-shANG转染细胞的增殖指数(PI)分别为0.32±0.29、0.35±0.38和0.31±0.43,差异不明显.体内实验结果表明,AAV-shANG转染细胞组胸腺缺陷小鼠的成瘤体积、瘤质最均明显低于两对照组.各组微血管密度分别为9.4±1.5、9.8±2.1和5.7±1.9,提示AAV-shANG转染细胞组与正常A549细胞和AAV-Null转染细胞组有明显差异.各组凋亡细胞的百分率分别为(7.7±3.1)%、(8.5±5.4)%和(17.1±8.6)%.AAV-shANG转染细胞组凋亡细胞的百分率明显高于正常A549细胞组和AAV-Null转染细胞组,各组细胞增殖核抗原(PCNA)阳性表达率分别为(84.8±9.7)%、(85.8±9.8)%、(70.4±10.1)%,AAV-shANG转染细胞组PCNA表达率低于两对照组.结论 AVV-siRNA表达能显著抑制肿瘤细胞ANG表达及肿瘤细胞增殖,促进肿瘤细胞凋亡,抑制肿瘤生长.     

RNAi沉默HMGN5基因对肺癌A549细胞增殖的影响

目的构建HMGN5基因的shRNA慢病毒载体,并在肺癌A549细胞上鉴定其沉默效率,观察其对细胞增殖能力的影响。方法筛选HMGN5基因特异性siRNA靶点,合成短发卡结构shRNA,与LentiLox3.7慢病毒载体重组形成shRNA表达载体,包装shRNA慢病毒颗粒,感染肺癌A549细胞,设阴性组及干涉组,应用Real-time PCR和Western印迹的方法检测HMGN5在mRNA和蛋白水平的沉默效率,MTT和克隆形成实验观察HMGN5基因沉默对A549细胞增殖的影响。结果构建的shRNA慢病毒颗粒感染A549细胞后,干涉组HMGN5基因的mRNA表达量较阴性对照载体慢病毒感染组下降了71.7%,显著抑制其蛋白表达;MTT结果表明细胞增殖能力明显降低,干涉组克隆形成能力较阴性组明显减弱。结论成功构建了HMGN5基因的shRNA慢病毒表达载体,其能够在肺癌A549细胞上有效沉默靶基因。HMGN5基因具有影响肺癌细胞恶性增殖的能力,为肺癌的基因治疗奠定了实验基础。     

黑色素瘤相关抗原-A3基因重组慢病毒载体的构建与鉴定及其在肺癌细胞株中的表达

目的 构建重组慢病毒载体PLV-MAGE--A3-GFP,并检测其在人肺癌细胞系A549的表达情况.方法 利用DNA重组技术将MAGE-A3基因克隆到带GFP的慢病毒表达载体质粒PLV中,经限制性酶切和测序鉴定重组载体.将重组慢病毒载体PLV- MAGE- A3 -GFP与包装质粒pCMV-dR 8.91及pCMV-VSV-G共转染人胚肾上皮细胞株293TAB,包装重组慢病毒PL V-MAGE-A3-GFP,并感染人肺癌细胞株A549,用Western blot法检测MAGE-A3蛋白的表达情况.结果 限制性内切酶酶切和DNA测序分析证实,构建了重组慢病毒载体PLV-MAGE-A3-GFP,免疫荧光显微镜下观察显示慢病毒感染A549细胞后,MAGE-A3基因能够在细胞内正确地转录、翻译并稳定表达MAGE-A3基因.结论 成功构建了慢病毒载体PLV-MAGE-A3-GFP,包装的病毒能够成功感染人肺癌细胞系A549,并使MAGE-A3基因得以稳定表达,为进一步对非小细胞肺癌进行免疫治疗提供了适合的稳定转染的载体.     

BAG-1基因沉默对非小细胞肺癌放射敏感性的影响及机制

目的探讨BAG-1基因与非小细胞肺癌放射敏感性的关系,为非小细胞肺癌个体化治疗提供理论依据。方法对A549肺腺癌细胞、H460大细胞癌及NCL-H520鳞癌细胞三株非小细胞肺癌细胞株及人胚肾细胞株HEK293行细胞放射敏感性试验,采用Western blot法检测各细胞株中BAG-1蛋白表达情况。选择其中对放射线相对较抗拒、BAG-1蛋白表达最高的A549细胞株,构建干扰载体pGCsi-BAG-1并筛选BAG-1基因沉默细胞株,用shRNA-2转染,酶标仪检测细胞生长情况,流式细胞仪检测细胞凋亡率,放射敏感性试验观察细胞对放射线的敏感性。结果 shRNA-2转染的细胞株生长受到抑制,且随时间延长抑制作用明显,而阴性对照质粒转染的A549细胞株生长无明显变化;6 Gy射线照射后24 h,shRNA-2转染细胞凋亡率为(49.6±4.23)%,未转染的A549细胞为(15.3±2.11)%,阴性对照质粒转染细胞为(14.8±3.55)%,shRNA-2转染细胞凋亡率较未转染细胞增加了3.2倍(P<0.01),而阴性对照转染细胞与未转染细胞间相比差异无统计学意义;亲本A549细胞和阴性对照转染细胞之间的放射敏感性无差异(P>0.05)。而shRNA-2转染细胞的放射敏感性明显增加,与前二者比较差异有显著性(P<0.05)。结论 BAG-1基因沉默可增强非小细胞肺癌放射敏感性,可能机制为BAG-1基因沉默后抑制细胞生长,抗凋亡作用减弱,细胞内BAG-1分布发生改变,细胞对射线更敏感。     

内皮抑素基因转移联合电离辐射对肺腺癌移植瘤生长的影响

目的探讨内皮抑素(endostatin)基因转移联合电离辐射对肺腺癌移植瘤生长的抑制作用. 方法以逆转录病毒为载体,转导内皮抑素基因至肺腺癌A549细胞,获得含内皮抑素基因的A549/Endo细胞.20只裸鼠平均分为4组(A549 、A549/Endo、A549+辐照、A549/Endo+辐照组),A549、A549+辐照组裸鼠皮下种植A549细胞,A549/Endo、A549/Endo+辐照组裸鼠皮下种植A549/Endo细胞,建立移植瘤模型.A549、A549/Endo组裸鼠用于观察成瘤时间,并计算抑瘤率;另2组裸鼠于种植瘤细胞后第28天,用直线加速器电离辐照移植瘤体,剂量为20 Gy,3 d后重复照射20 Gy;照射后继续饲养荷瘤鼠,定期观察移植瘤体积变化.于瘤细胞移植后第42天(照射后第14天)处死荷瘤鼠,用免疫组化法测定4组裸鼠瘤体的微血管密度(MVD).结果聚合酶链测定(PCR)法证实,A549/Endo细胞基因组中整合有内皮抑素基因.裸鼠成瘤时间A549组(7.8±1.6)d、A549/Endo组(12.2±1.7 )d ,两组比较差异有显著性(P<0.05);瘤细胞移植后42 d,移植瘤体积A549组(927.8±269.2)mm3 、A549/Endo组(217.5±81.5)mm3,两组比较差异有显著性(P<0.01),抑瘤率为76.5% .电离辐照后第14天,移植瘤体积A549+辐照组为(157.7±49.0)mm3,A549/Endo+辐照组为(4.6±2.9)mm3, 两组比较差异有显著性(P<0.01).裸鼠移植瘤组织MVDA549组35.78±5.67 、A549/Endo组21.62±3.55、 A549+辐照组31.52±4.43、 A549/Endo+辐照组11.32±2.78,与A549组比较,A549/Endo组MVD明显减少(P<0.01),联合电离辐照后MVD进一步降低(P<0.01).结论逆转录病毒能有效介导内皮抑素基因转移;内皮抑素基因转移能通过抑制血管生成而抑制肺腺癌移植瘤的生长;内皮抑素基因转移联合电离辐射对肺腺癌血管形成和瘤体生长具有协同抑制作用.     

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.     

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.