转铁蛋白SA阳离子脂质体介导DNA转染肝癌细胞 SMMC-7721的实验研究

目的检测HTf(Fe)2SA阳离子脂质体是否具有靶向转染肝癌细胞的能力.方法在有血清和无血清两种条件下检测HTf(Fe)2SA阳离子脂质体和SA阳离子脂质体介导DNA进入肝癌细胞SMMC-7721和小鼠B16黑色素瘤细胞内的量.结果在有血清和无血清两种转染条件下HTf(Fe)2SA阳离子脂质体介导入SMMC-7721细胞内DNA的量明显高于SA阳离子脂质体,而对小鼠B16黑色素瘤细胞两者无明显差异.结论HTf(Fe)2SA阳离子脂质体在体外具有良好的靶向介导DNA转染肝癌细胞的能力.     

阳离子脂质体联合转铁蛋白对人肿瘤细胞的靶向基因转染

目的研究阳离子脂质体联合转铁蛋白(transferrin ,Tf)介导报告基因虫荧光素酶基因对人肿瘤细胞的靶向转染。方法比较阳离子脂质体Lipofectin、Lipofectin联合含双铁的人转铁蛋白[HTf(Fe) 2 ]介导质粒pDR2 luc转染人肝癌细胞株HepG2 、SMMC772 1、人肾癌细胞株GRC及非洲绿猴肾细胞COS7的转染效率,转染效率通过以液体闪烁计数仪单光子计数法测定荧光素酶活性估计。结果除COS7外,其余3株细胞以Lipofectin联合HTf(Fe) 2 介导pDR2 luc的转染效率均比单用Lipofectin的转染效率高(P <0 0 1) ,其中HepG2 与SMMC772 1中的转染效率增高8倍多,GRC中的转染效率增高4倍多。结论Lipofectin联合HTf(Fe) 2 介导质粒pDR2 luc转染细胞时对人肿瘤细胸株有一定的靶向作用。     

转铁蛋白受体介导的自杀基因转移系统的建立及其体外抗瘤效应

目的 构建转铁蛋白受体介导的自杀基因系统并观察其对肝癌细胞的体外杀伤效应。方法 SPDP法制备抗转铁蛋白受体与多聚赖氯酸(PLL)的复合物并用分子筛层析纯化。根据DNA阻断试验结果，pEBAF／tk重组质粒与Ab-PLL按1：6混合使二者结合形成PEBAF／tk—Ab PLL复合物。将此复合物转入人肝癌细胞株SMMC7721、HepG2和肺癌细胞株A549，并以脂质体转移为对照。加入不同浓度的更昔洛韦(GCV)以观察细胞的自杀效应。结果 加入GCV后HepG2／tk的增殖受抑制最明显。100mg／L和1mg／L时抑制率分别为60．5％和24．3％。SMMC7721／tk受抑制较低，而A549的增殖不受抑制。结论 本基因治疗体系具仃很好的靶向性和杀肿瘤效果。     

乳糖脂修饰的阿霉素脂质体体外杀伤肝癌细胞的实验研究

在ＮａＢＨ４还原作用下制备乳糖酰磷脂酸乙醇胺（Ｌａｃ－ＰＥ）,反应４００ｈ测定仍联率为４８．６％,硅酸柱纯化得纯品。用该脂质修饰阿霉素脂质体表面,观察体外对人肝癌细胞系ＳＭＭＣ－７７２１的杀伤作用。方法乳糖和磷脂酰乙醇胺经化学反应制备Ｌａｃ－ＰＥ,用１０％摩尔的糖脂整合到超声法制备的小单层阿霉素脂质体脂膜上进行体外杀瘤研究,ＮＴＴ法检测杀瘤活性。结果４８小时细胞毒试验糖脂阿霉素脂质体对人肝癌细胞系ＳＭＭＣ－７７２１的杀伤作用优于无糖脂阿霉素脂质体（Ｐ＜０．０１）,而对非靶细胞（Ｂ１６黑色素瘤细胞）的杀伤作用优于无糖脂阿霉素脂质体两者相近。０．５ｈ预处理实验表明：缩短药物与细胞接触时间后,糖脂阿霉素脂质体仍保持较强的杀伤肝癌细胞作用。结论乳糖脂阿霉素脂质体的杀伤作用来自乳糖中的半乳糖基配体的特异性介导和人肝癌细胞上肝半乳糖受体的识别内吞作用。     

包裹阿霉素的VEGF-脂质体体外靶向杀伤人胃癌血管内皮细胞

目的:利用包裹阿霉素的血管内皮生长因子(VEGF)-脂质体于体外观察其对肿瘤血管内皮细胞的杀伤作用。方法:利用体外细胞毒实验(MTT)方法,检测VEGF-阿霉素脂质体对肿瘤血管内皮细胞的杀伤作用。结果:48h细胞毒试验连有VEGF的阿霉素脂质体对肿瘤血管内皮细胞的杀伤作用优于无VEGF的阿霉素脂质体(P<0.05),而对非靶细胞(人血管平滑肌细胞)的杀伤作用两者相近。0.5 h预处理试验表明:缩短药物与细胞接触时间后,VEGF-阿霉素脂质体仍保持较强的杀伤肿瘤血管内皮细胞的作用。结论:VEGF修饰的阿霉素脂质体对肿瘤血管内皮细胞的杀伤作用较无VEGF组和非靶细胞组强,可望成为一种有效的抗肿瘤物质。     

转铁蛋白与细胞穿膜肽共修饰脂质体的制备及其抗肿瘤效果

目的制备转铁蛋白(TF)与细胞穿膜肽(TAT)共修饰载紫杉醇(PTX)脂质体(TF/TATLP-PTX),对其体外性质进行评价。方法采用薄膜分散法制备TF与TAT共修饰TF/TATLP-PTX,考察其粒径、电位、包封率等理化特征。通过MTT实验考察脂质体对肝癌HepG2细胞的毒性,流式细胞仪检测肿瘤细胞对脂质体的摄取能力。构建HepG2肝癌异位瘤模型,考察不同脂质体对肿瘤的生长抑制能力。结果制备的TF/TATLP-PTX粒径为(137.8±13.5)nm,Zeta电位(17.55±3.85)mV,紫杉醇的包封率为85.3%。MTT实验结果显示,给药72 h后,以生理盐水组为对照,未修饰紫杉醇脂质体(LP-PTX)、转铁蛋白修饰紫杉醇脂质体(TFLP-PTX)、TAT修饰紫杉醇脂质体(TATLP-PTX)和TF/TATLP-PTX肝癌HepG2细胞存活率分别为62.4%、45.5%、39.2%和17.7%,组间比较差异显著(P<0.01);体外细胞摄取实验表明,肝癌HepG2细胞对TF/TATLP的摄取效率分别是TFLP、TATLP和LP的2.7倍、2.2倍和3.9倍,组间比较差异显著(P<0.01);荷瘤裸鼠治疗实验以生理盐水组为对照,给药20 d后,生理盐水组体积变为给药前的2.55倍,LP-PTX、TFLP-PTX、TATLP-PTX和TF/TATLP-PTX组肿瘤体积分别增长到原体积的2.22、1.73、1.89、1.29倍,与其他组间差异显著(P<0.01)。结论 TF/TATLP-PTX制备工艺简单,与肝癌HepG2细胞具有良好的亲和性,是一种潜在高效的肿瘤靶向给药系统。     

B7转染人肝癌细胞株的建立及生物学特性的实验研究

研究共刺激信号在肿瘤免疫中的作用。脂质体介导DNA转移法将入B7基因转入人肝癌细胞株Smmc7721,建立新的细胞株B7^ Smmc7721,PCR及逆转录PCR（RT-PCR）法鉴定。四唑盐（MTT）比色试验体外检测白细胞介素-2（IL-2）激活的LAK细胞（LAK-C）对两种肝癌细胞的杀伤活性。PCR,RT-PCR法证实B7^ Smmc7721细胞稳定表达B7基因,LAK细胞对B7^ Smmc7721细胞的杀伤活性明显高于Smmc7721,结果有统计学意义。B7基因可以体外转染人肝癌细胞,并表达有活性的B7分子,并可明显增强IL-2激活的LAK细胞的肿瘤杀伤作用,为进一步开展临床应用奠定基础。     

4种甲胎蛋白抗原表位肽段致敏树突状细胞诱导细胞毒性T淋巴细胞对肝癌细胞的杀伤效应比较

目的 通过体外杀伤试验,比较4种人肝癌特异性甲胎蛋白抗原表位肽段PLFQVEPV[hAFP(137～145)],FMNKFIYEI[hAFP(158～166)]、GLSPNLNRFL[hAFP(325-334)]和GVAL,QTMKQ[hAFP(542～550)]修饰树突状细胞(DC)后诱导的细胞毒性T淋巴细胞(CTL)对人肝癌细胞的特异性杀伤效应. 方法 选取健康人外周血单个核细胞,体外诱导成熟DC;合成4种hAFP抗原表位肽段分别修饰和诱导DC,体外刺激CTL,通过流式细胞仪法和细胞毒性检测法检测该修饰后的DC所诱导的CTL在体外对肝癌细胞株SMMC-7721细胞的杀伤作用.组间比较应用t检验进行统计学分析. 结果 4种人肝癌特异性甲胎蛋白片段均能在体外修饰和诱导DC细胞的增殖和成熟;这些成熟DC在体外均能诱导特异性CTL,CTL对SMMC-7721细胞均可产生杀伤效应.其中FMNKFIYEI[hAFP(158～166)]肽段诱导的CTL在效靶比分别为80:1、40:1及10:1时对肿瘤细胞的杀伤效应分别达到了78.1%±9.8%、43.9%±5.9%和28.2%±4.9%,比其他3种肽段的杀伤效应高(P<0.05). 结论 短肽段具有单独体外诱导特异CTL的作用,其诱导效果和杀伤效果均优于完整hAFP蛋白,其诱导的CTL有特异地杀伤SMM C-7721细胞的作用.     

4种甲胎蛋白抗原表位肽段致敏树突状细胞诱导细胞毒性T淋巴细胞对肝癌细胞的杀伤效应比较

目的 通过体外杀伤试验,比较4种人肝癌特异性甲胎蛋白抗原表位肽段PLFQVEPV[hAFP(137～145)],FMNKFIYEI[hAFP(158～166)]、GLSPNLNRFL[hAFP(325-334)]和GVAL,QTMKQ[hAFP(542～550)]修饰树突状细胞(DC)后诱导的细胞毒性T淋巴细胞(CTL)对人肝癌细胞的特异性杀伤效应. 方法 选取健康人外周血单个核细胞,体外诱导成熟DC;合成4种hAFP抗原表位肽段分别修饰和诱导DC,体外刺激CTL,通过流式细胞仪法和细胞毒性检测法检测该修饰后的DC所诱导的CTL在体外对肝癌细胞株SMMC-7721细胞的杀伤作用.组间比较应用t检验进行统计学分析. 结果 4种人肝癌特异性甲胎蛋白片段均能在体外修饰和诱导DC细胞的增殖和成熟;这些成熟DC在体外均能诱导特异性CTL,CTL对SMMC-7721细胞均可产生杀伤效应.其中FMNKFIYEI[hAFP(158～166)]肽段诱导的CTL在效靶比分别为80:1、40:1及10:1时对肿瘤细胞的杀伤效应分别达到了78.1%±9.8%、43.9%±5.9%和28.2%±4.9%,比其他3种肽段的杀伤效应高(P<0.05). 结论 短肽段具有单独体外诱导特异CTL的作用,其诱导效果和杀伤效果均优于完整hAFP蛋白,其诱导的CTL有特异地杀伤SMM C-7721细胞的作用.     

4种甲胎蛋白抗原表位肽段致敏树突状细胞诱导细胞毒性T淋巴细胞对肝癌细胞的杀伤效应比较

目的 通过体外杀伤试验,比较4种人肝癌特异性甲胎蛋白抗原表位肽段PLFQVEPV[hAFP(137～145)],FMNKFIYEI[hAFP(158～166)]、GLSPNLNRFL[hAFP(325-334)]和GVAL,QTMKQ[hAFP(542～550)]修饰树突状细胞(DC)后诱导的细胞毒性T淋巴细胞(CTL)对人肝癌细胞的特异性杀伤效应. 方法 选取健康人外周血单个核细胞,体外诱导成熟DC;合成4种hAFP抗原表位肽段分别修饰和诱导DC,体外刺激CTL,通过流式细胞仪法和细胞毒性检测法检测该修饰后的DC所诱导的CTL在体外对肝癌细胞株SMMC-7721细胞的杀伤作用.组间比较应用t检验进行统计学分析. 结果 4种人肝癌特异性甲胎蛋白片段均能在体外修饰和诱导DC细胞的增殖和成熟;这些成熟DC在体外均能诱导特异性CTL,CTL对SMMC-7721细胞均可产生杀伤效应.其中FMNKFIYEI[hAFP(158～166)]肽段诱导的CTL在效靶比分别为80:1、40:1及10:1时对肿瘤细胞的杀伤效应分别达到了78.1%±9.8%、43.9%±5.9%和28.2%±4.9%,比其他3种肽段的杀伤效应高(P<0.05). 结论 短肽段具有单独体外诱导特异CTL的作用,其诱导效果和杀伤效果均优于完整hAFP蛋白,其诱导的CTL有特异地杀伤SMM C-7721细胞的作用.     

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.     

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.     

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.     

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.