逆转录病毒介导HSV-TK基因治疗胰腺癌——实验研究

目的:探讨应用逆转录病毒载体介导HSV-TK基因治疗实验性人胰腺癌细胞系8988的价值。方法:HSV-TK被定向克隆入逆转录病毒载体pMNSM的SV_(40) 下游。重组逆转录病毒载体pMNS-TK-M转染至逆转录病毒包装细胞PA317细胞,产生的重组病毒将HSV-TK转入人胰腺癌细胞系8988细胞内。结果:Southern-blot试验及药敏试验均证实HSV-TK基因已整合至细胞DNA中并完全表达。体外试验证实,HSV-TK阳性8988细胞对ACV的敏感性较母细胞明显为高;裸鼠移植瘤试验证实,腹腔注射ACV有明显阻止移植瘤的形成以及对移植瘤的治疗作用。结论:HSV-TK/ACV有体内治疗胰腺癌的作用,可作为胰腺癌基因治疗的潜在方法之一。     

病毒载体介导的帕金森病基因治疗研究进展

帕金森病 (PD)是一种常见的神经系统变性疾病 ,其病理改变主要为中脑黑质致密区多巴胺能神经元变性和消失 ,以及体内多巴胺水平的下降。 80年代末 ,随着基因治疗概念的不断深入 ,基因治疗技术日趋成熟 ,人们开始了对PD基因治疗的尝试。近年来PD动物模型基因治疗已经取得了不少进展。在基因治疗的体外及体内两种途径中 ,研究最多的是病毒载体相关的基因导入系统。目前研究较多病毒载体有 :腺病毒载体、腺相关病毒载体、单纯疱疹病毒载体、慢病毒载体、其他逆转录病毒载体。　　一、病毒载体的制备　　用于基因治疗的病毒载体应具备以下条…     

HSV-TK逆转录病毒包装细胞系建立及病毒滴度测定

目的 建立HSV-TK逆转录病毒包装细胞系并获得高产病毒的细胞株.方法 将TK基因与逆转录表达载体PLEGFP-N1连接后转染到包装细胞PA317中,经G418及荧光蛋白双重筛选得到高产病毒的细胞株.结果 经酶切鉴定成功构建PIEGFP-N1-TK重组载体,含HSV-TK基因重组逆转录病毒包装细胞PA317成功建立,经病毒滴度测定获得高产病毒的PA317/TK细胞株.结论 制备的重组病毒具有感染靶细胞的活性,为进一步应用HSV-TK基因进行肺癌的自杀基因治疗研究奠定基础.     

心血管疾病的基因治疗

随着人类基因组序列研究的不断深入,不同心血管疾病的易感基因的揭示,为基因治疗提供了明确方向。基因治疗将为心血管疾病更加有效的防治带来更大的希望[1～4\〗。本文仅就基因治疗心血管疾病的现状作简要介绍。1　技术方面1.1　载体　携带基因到所期望的作用部位的行为者谓之载体。因此心脏最适宜的载体对于实现有效的基因转移是十分重要的。载体通常分成两类:病毒的和非病毒的。1.1.1　病毒载体　采用的主要病毒是逆转录病毒和腺病毒[5] 。(1)逆转录病毒:所携带的基因片段短,只能转染增殖生长的细胞,可稳定高效地把携带的基因整合到靶细胞…     

胞嘧啶脱氨酶基因的体外前药转换胃癌细胞的杀伤作用研究

目的建立胃癌组织细胞自杀基因体外前药转换基因治疗方法。方法和结果首先将含ＣＤ基因的重组逆转录病毒载体ｐＣＤ２在ＰＡ３１７细胞中包装、筛选、收获病毒上清及进行病毒滴度测定后，体外转导Ｍ８５胃癌细胞，以ＰＣＲ方法证明ＣＤ基因稳定整合到宿主细胞基因组中，Ｎｏｒｔｈｅｒｎｂｌｏｔ分析了ＣＤ基因的表达；转基因胃癌细胞在前药５－ＦＣ作用下呈现明显的选择性生长抑制效应。且观察到强效的“旁观者效应”。结论为胃癌组织基因治疗提供了极有意义的资料。     

慢病毒载体在RNA干扰中的应用

慢病毒载体是以人类免疫缺陷1型病毒为基础发展起来的基因治疗载体.区别一般的逆转录病毒载体,它对分裂细胞和非分裂细胞均具有感染能力.该载体可以将外源基因有效地整合到宿主染色体上,从而达到持久件表达.在感染能力方面可有效地感染神经元细胞、肝细胞、心肌细胞、肿瘤细胞、内皮细胞、干细胞等多种类型的细胞,从而达到良好的基因治疗效果.RNA干扰技术是近年来发展起来的新技术,通过产生针对特定基因的小分子干扰RNA,能高效特异地抑制特定基因的表达,在探查基因功能和治疗人类疾病方面有广阔的应用前景.     

消化疾病的基因治疗

基因治疗旨在从分子水平纠正疾病,将靶基因传递进宿主细胞以提供新的基因功能,或阻止有害基因表达的合成。靶基因可以是ＤＮＡ或ｍＲＮＡ水平。基因治疗可通过体外或体内途径达到。载体一、病毒载体１．逆转录病毒载体：逆转录病毒载体已被广泛运用,操作简便,病毒产量高,转染各型分裂相细胞的效率高。已成功地应用这一系统于体外,将ＩＬ－７和新霉素磷酸转移酶转移入大鼠肝细胞,以及在体内,将ＩＸ因子传递入血友病Ｂ犬模型的肝细胞。转基因的表达分别维持在期望水平２１天和５个月。除肝细胞外,肠道上皮细胞也是基因转移的可行目标…     

逆转录病毒介导单纯疱疹病毒-胸苷激酶基因治疗胰腺癌的实验研究

目的 探讨应用逆转录病毒载体介导单纯疮疹病毒－胸苷激酶（herpes simplex virus type Ⅰthymidine kinase gene,ＨＳＶ－ＴＫ）基因治疗实验性人胰腺癌细胞系８９８８的价值。方法 ＨＳＶ－ＴＫ被定向克隆入逆转录病毒载体pＭＮＤＭ的ＤＶ４０下游。重组逆转录病毒包装细胞ＰＡ３１７细胞,产生的重组病毒将ＨＳＶ－ＴＫ转入人胰腺癌细胞系８９８８细胞内。结果 Ｓorthern blot试验及药敏试验均证实ＨＳＶ－ＴＫ基因已整合至细     

pSLX-CMV／TpRII-IgG1 Fc载体的构建

目的 构建表达人TGF-β111型受体细胞外结合区和人IgG1 Fc的融合蛋白逆转录病毒载体，为进一步肝纤维化的基因治疗奠定实验基础。方法以RT-PCR方法扩增目的基因TβRII-IgG1 Fc，扩增产物纯化后克隆至测序载体pGEM-T-Easy，挑取阳性克隆酶切鉴定后测序；利用重组DNA技术，将TβRII-IgG1 Fc基因亚克隆至逆转录病毒载体pSLX-GMV中，重组质粒pSLX-CMV／TβRII-IgG1 Fc在脂质体介导F转染PA317包装细胞，G418筛选，直至出现抗性克隆，扩大培养，测定病毒滴度。结果经测序、限制性酶切分析及PCR方法鉴定，载体插入基因序列、大小、位置均正确无误，并用PA317细胞进行包装、病毒滴度测定、筛选，建立具有较高滴度的感染性重组病毒产生细胞系。结论成功构建了重组质粒pSLX-CMV／TβRII-IgG1 Fc，可望为肝纤维化的基因治疗提供有效途径。     

研究表明基因治疗有希望用于HIV感染患者

据医业网11月7日报道（原载Proc Natl Acad Sci USA 2006），使用基于HIV慢病毒载体的基因治疗，在对至少2种抗逆转录病毒治疗方案无反应的慢性感染患，可降低病毒负荷和增加CD4 T细胞计数。     

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.