pEGFP-C1-反义生存素重组质粒的构建和转染

目的 构建pEGFP-C1-反义生存素（survivin)重组质粒，并转染入人MKN-45低分化胃腺癌细胞株，观察转染前后生存素基因的表达及对凋亡的影响。从基因水平探讨反义生存素核酸治疗胃癌的分子生物学机制。为今后利用反义基因治疗肿瘤提供实验和理论依据。方法 应用基因重组技术构建 pEGFP-C1-反义生存素重组质粒，通过脂质体转染法转染人MKN-45胃腺癌细胞株，以流式细胞仪方法检测细胞凋亡，以RT-PCR技术检测质粒转染前后生存素基因mRNA的表达。结果 pEGFP-C1-反义生存素重组质粒转染MKN-45低分化胃腺癌细胞株后细胞凋亡明显增加，G2/M期细胞减少，同时生存素基因在mRNA水平被抑制。结论 反义生存素核酸能抑制细胞增殖。减少生存素基因的mRNA表达而促进胃癌细胞凋亡。     

重组DNA甲基化酶1表达质粒对结肠癌细胞相关基因表达的影响

目的 分析DNA甲基化酶1(DNMT1)基因的真核表达质粒对人结肠癌细胞肿瘤相关基因的表达影响。方法 分别构建并转染含有人正义和反义DNMT1的真核表达质粒入结肠癌SW1116细胞，PCR和限制性内切酶证实转染结果，以Western印迹法分析各组细胞DNMT1蛋白的表达情况。定量PCR检测hMLH1、hMSH2及c—myc、p16^INK4A基因的表达。结果 经G418筛选得到稳定转染DNMT1基因的结肠癌细胞系，且分别在该转染有正义和反义质粒的细胞系中，DNMT1蛋白表达上调和下调。同时发现转染正义DNMT1的细胞中hMLH1、hMSH2及c—myc的表达降低，而转染反义DNMT1的细胞中hMSH2的表达明显增强。各组细胞p16^INK4A基因的表达差异不明显。结论 DNMT1基因调控人结肠癌细胞中肿瘤相关基因的表达。     

生存素反义核酸诱导肝癌细胞株SMMC-7721凋亡和增加其对抗肿瘤药物敏感性的研究

背景：生存素是凋亡抑制蛋白家族的重要成员，生存素基因有可能成为肿瘤反义基因治疗的理想靶基因。目的：研究生存素反义核酸诱导肝癌细胞株SMMC-7721凋亡和增加其对常用抗肿瘤药物敏感性的作用，探讨生存素反义核酸用于肿瘤基因治疗的可能性。方法：应用基因重组技术构建pEGFP-C1-生存素反义核酸重组质粒，以脂质体转染法转染SMMC．7721细胞，用逆转录聚合酶链反应(RT-PCR)检测生存素mRNA的表达，用流式细胞仪检测细胞凋亡。将生存素反义核酸分别与7种常用抗肿瘤药物共同作用于SMMC-7721细胞，用四唑蓝(MTT)比色法测定细胞杀伤率。结果：生存素反义核酸可抑制SMMC-7721细胞中生存素mRNA的表达，从而导致细胞凋亡增加，其作用呈剂量依赖性。生存素反义核酸可增加SMMC-7721细胞对7种常用抗肿瘤药物的敏感性，明显增强药物的杀伤作用。结论：生存素反义核酸能靶向抑制野生型生存素基因的表达，提高肝癌细胞对常用抗肿瘤药物的敏感性，有可能成为肿瘤基因治疗的新方法。     

结直肠癌缺失基因功能区诱导结肠癌细胞凋亡

目的检测结直肠癌缺失(DCC)基因功能区转染表达后对结肠癌细胞SW1116凋亡的诱导作用。方法通过脂质体法将含有DCC基因胞内区功能域(3 727～3 792 bp)的重组表达载体pIRES2-EGFP-DCC转染到结肠癌细胞SW1116,MTT比色法检测细胞增殖情况,TUNEL、AO/EB染色法检测细胞凋亡情况。结果转染SW1116细胞36 h后,MTT比色法检测结果表明实验组细胞数目低于对照组,并且具有显著性差异;AO/EB染色及TUNEL法检测结果表明实验组有大量凋亡细胞,对照组未见凋亡细胞。结论 DCC基因的胞内功能域(3 727～3 792 bp)具有诱导结肠癌细胞凋亡的作用。     

STAT3靶向短发夹RNA干扰重组载体对结肠癌SW480细胞的沉默作用

目的 探讨STAT3基因短发夹RNA(shRNA)表达质粒对结肠癌SW480细胞STAT3基因的干扰作用.方法 根据siRNA设计原则,构建靶向STAT3基因的短发夹RNA干扰质粒(pGPU6/GFP/STAT3),使用脂质体法转染人结肠癌细胞系(SW480),通过RT-PCR和 Western 印迹检测结肠癌SW480细胞STAT3基因mRNA和蛋白表达水平.结果 pGPU6/GFP/STAT3重组质粒经限制性内切酶酶切及测序证明基因插入正确.质粒成功转染SW480细胞后,该细胞的STAT3 mRNA和蛋白表达明显下降(P<0.05).结论 成功构建靶向STAT3基因的shRNA表达载体,转染SW480细胞,能有效抑制细胞的STAT3 mRNA和蛋白表达,为STAT3基因靶向治疗提供前期的实验依据.     

siRNA沉默PPARα减弱羟喜树碱诱导的人结肠癌细胞凋亡

背景:多药耐药一直是遏制提高结肠癌化疗有效性的瓶颈问题。目的:探讨沉默PPARα能否减弱羟喜树碱诱导的人结肠癌细胞凋亡,从而阐明PPARα在羟喜树碱耐药中的潜在作用。方法:构建针对PPARα基因的siRNA干扰载体,并转染人结肠癌SW1116细胞,经G418筛选出稳定转染的细胞,以RT-PCR和蛋白质印迹法验证干扰效果。经羟喜树碱干预后,以MTT法检测细胞增殖,Annexin V-FITC/PI检测细胞凋亡。以定量RT-PCR检测不同浓度miR-506前体转染SW1116细胞后对PPARα下游调控靶基因mRNA表达的影响。结果:siRNA干扰载体Ⅲ的沉默效果最佳,PPARα mRNA和蛋白表达分别下调了约94.1%±3.4%和70.8%±8.6%。稳定转染siRNA的SW1116细胞的半数抑制浓度(IC_(50))为(332±19)μg/L,亲本SW1116细胞为(152±12)μg/L。在相同浓度羟喜树碱的作用下,亲本SW1116细胞的凋亡率显著高于稳定转染siRNA的SW1116细胞(150μg/L:7.0%±2.5%对2.8%±1.6%;300μg/L:32.4%±7.1%对18.5%±2.7%)。miR-506前体下调HMGCS-2表达,上调FABP-2、CCND1和TGF-β1表达,其中上调CCND1的作用最为明显。结论:成功构建了针对PPARα的siRNA干扰载体;沉默PPARα能增强SW1116细胞对羟喜树碱的抵抗性,可能与下调HMGCS-2表达并上调FABP-2、CCND1和TGF-β1表达有关。     

生存素基因突变体诱导胃癌细胞凋亡的实验研究

目的 生存素(survivin)基因在胃癌组织中高水平表达，而在正常胃黏膜中无表达。生存素表达的水平与胃癌的预后密切相关。通过基因重组构建生存素基因突变体(pcDNA3—survivin—mutant，Cys84Ala)质粒，并以脂质体转染的方法将质粒DNA转入胃癌细胞株，观察其对胃癌细胞的生物学特性及多药耐药性的影响。方法 应用RT—PCR、Western blot、免疫组化等方法检测胃癌细胞中生存素基因mRNA和蛋白的表达，流式细胞仪和吖啶橙染色检测胃癌细胞凋亡。结果 通过RT—PCR检测，在所有的胃癌细胞株中均有不同程度生存素基因表达，生存素基因突变体Cys84Ala通过抑制野生型生存素基因的功能诱导胃癌细胞凋亡，增加caspase—3活性和促进细胞色素C的释放，增加胃癌细胞对化疗药物的敏感性。结论 突变体Cys84A1a能诱导胃癌细胞凋亡和增加胃癌细胞对化疗药物的敏感性。因此，生存素基因突变体Cys84Ala有可能成为胃癌治疗的一个候选基因。     

人膀胱移行细胞癌中生存素mRNA的表达及与细胞凋亡的关系

目的检测膀胱癌中生存素(Survivin)基因表达,探讨其与细胞凋亡的关系。方法采用原位杂交、免疫组织化学技术和DNA原位末端标记技术,检测46例膀胱癌组织、15例正常膀胱组织生存素mRNA及膀胱癌中caspase3蛋白和细胞凋亡。结果与正常膀胱组织相比,生存素mRNA显著表达于膀胱癌中,并与病理分级有关(P<0.05);膀胱癌中caspase3蛋白与凋亡细胞阳性率分别为76.1%和67.4%,凋亡指数均值为8.1%;生存素阴性组AI显著高于阳性组(P<0.01)。结论生存素选择性表达于膀胱癌中,通过抑制细胞凋亡,参与膀胱癌发生发展。     

miR-506在人结肠癌羟基喜树碱多药耐药中的功能研究

目的探讨miR-506在人结肠癌羟基喜树碱多药耐药中的潜在作用。方法首先通过PCR从基因组DNA中克隆出包含miR-506前体的片段,将其连接至miRNA表达载体pMIF-cGFP-Zeo中,测序进行验证。将构建成功的pMIF-miR-506转染至低表达miR-506的亲本细胞SW1116中,加入含G418的选择性培养基进行选择性培养。运用RT-PCR验证稳定转染pMIF-miR-506的SW1116细胞株中的miR-506的表达情况,MTT法检测稳定转染pMIF-miR-506的SW1116细胞株和亲本细胞SW1116对羟基喜树碱的敏感性差异,Annexin V/PI检测各自对羟基喜树碱的凋亡情况。结果测序结果表明hsa-miR-506的前体序列被成功地构建至miRNA表达载体pMIF-cGFP-Zeo中。TaqMan荧光定量PCR技术检测发现,相比亲本细胞SW1116,稳定表达绿色荧光的SW1116细胞克隆中miR-506的含量显著升高,约为33.7±8.3倍。细胞增殖试验表明稳定转染pMIF-miR-506的SW1116细胞株的IC50为(280±13)μg/L,而对照组SW1116的IC50为(152±12)μg/L。同时还发现在相同浓度的羟基喜树碱作用下,亲本细胞SW1116较稳定转染pMIF-miR-506的SW1116细胞株的凋亡细胞比率增高(150μg/L:7.8%±1.7%vs5.4%±1.2%;300μg/L:33.6%±3.9%vs18.2%±4.7%)。结论成功地构建了过表达miR-506的载体pMIF-miR-506;过表达miR-506能够增加SW1116对羟基喜树碱的抵抗性。     

热休克蛋白70通过Bcl-2抑制氧化应激所致C2C12细胞凋亡

目的 探讨C2C12肌原细胞内热休克蛋白70对Bcl-2表达的影响及Bcl-2对热休克蛋白70抗细胞凋亡作用的影响.方法 应用Western Blotting观察Bcl-2在转染热休克蛋白70真核表达质粒(pcDNA3.1-HSP70)或其反义寡核苷酸C2C12肌原细胞中的表达;采用基因瞬间转染技术使热休克蛋白70过表达的C2C12肌原细胞内Bcl-2表达抑制,应用流式细胞术检测H2O2处理所致细胞凋亡的发生情况.结果 转染热休克蛋白70真核表达质粒的C2C12肌原细胞中,热休克蛋白70和Bcl-2的表达明显高于空载体转染组(P<0.01);而转染热休克蛋白70反义寡核苷酸后,热休克蛋白70和Bcl-2的表达明显低于随机寡核苷酸转染组 (P<0.01);热休克蛋白70过表达的C2C12肌原细胞分别转染Bcl-2反义寡核苷酸及随机寡核苷酸,0.5 mmol/L H2O2处理24 h,Bcl-2反义寡核苷酸转染组的细胞凋亡率明显高于随机寡核苷酸转染细胞组.结论 热休克蛋白70能上调C2C12肌原细胞内Bcl-2的表达;热休克蛋白70的抗细胞凋亡功能可能与其上调Bcl-2表达相关.     

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.     

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.