实时定量RT-PCR检测急性早幼粒细胞白血病PML/RARα融合基因的临床意义

目的:探讨实时定量RT-PCR方法检测急性早幼粒细胞白血病(APL) PML/RARα融合基因的临床意义.方法:采用实时定量RT-PCR方法,用Taqman探针检测32例初治APL患者的PML/RARα融合基因3种异构体表达水平,并对治疗过程中的20例患者融合基因表达水平进行动态观察. 结果:①实时定量RT-PCR的敏感度为101拷贝数/μl,标准品日间差异及日内差异平均变异系数均<5%;②32例PML/RARα融合基因阳性的初治APL患者中,21例为PML/RARα融合基因长型异构体,11例为PML/RARα融合基因短型异构体;③32例初治患者PML/RARα融合基因表达量中位数和±s分别为1.44%,(1.29±1.46)%.比较异构体长型及短型标准拷贝数(NCN)中位数和±s分别为1.40%,(1.46±1.18)%和1.28%,(1.39±1.51)%,差异无统计学意义P<0.05;④20例治疗后APL患者PML/RARα融合基因表达水平随着临床治疗而改变.结论:①建立了检测APL微小残留病的高敏感性、高特异性的实时定量RT-PCR方法;②32例初治APL患者PML/RARα融合基因长型及短型异构体mRNA NCN差异无统计学意义;③PML-RARα融合基因表达水平的变化与临床疾病发展相一致,有助于疗效评价、微小残留病的检测和预后判断.     

荧光原位杂交检测维A酸受体α基因重排对急性早幼粒细胞白血病的诊断价值

目的:研究荧光原位杂交(fluorescence in situhybridization,FISH)检测维A酸受体α(RARα)基因重排对于急性早幼粒细胞白血病(acute promyelocytic leukemia,APL)的诊断价值。方法:对骨髓形态学检查呈典型的APL改变而常规染色体R显带核型分析t(15;17)及RT-PCR检测RARα基因重排两者皆为阴性的4例急性白血病患者采用双色荧光原位杂交检测RARα基因的重排。结果:2例FISH检测为阴性,1例80%的细胞中显示早幼粒白血病基因(PML)/RARα融合信号,1例PML/RARα融合基因信号阴性,但99.8%的细胞显示RARα基因17q21断裂点以下基因的复制及重排。结论:应用FISH技术可以在部分APL患者中检出核型及RT-PCR技术无法检出的RARα基因重排,有助于APL的确诊。     

白血病MLL基因重排及其融合基因检测的临床意义

目的:研究白血病MLL(mixed lineage leukemia)基因重排及其融合基因的检测及其临床意义。方法:采用荧光原位杂交(FISH)检测70例白血病患者MLL基因重排,对于MLL基因重排的患者,用巢式RT-PCR方法检测常见6种MLL融合基因类型。结果:9例白血病有MLL基因重排,发生率为12.86,5例B细胞系急性淋巴细胞白血病(B-ALL),其中融合基因2例为MLL/ENL,1例MLL/AF4,2例未扩出融合基因产物;3例为急性髓细胞白血病M5(AML-M5),其中2例融合基因均为MLL/AF9,1例未扩出融合基因产物;1例为幼年型粒单核细胞白血病(JMML),其融合基因为MLL/ENL。结论:巢式RT-PCR是检测MLL基因重排及其融合基因类型简便有效的方法,MLL基因重排见于B-ALL、AML-M5、JMML,预后差。     

实时定量PCR监测急性早幼粒细胞白血病患者PML/RARα融合基因的临床意义

目的 探讨实时定量PCR检测急性早幼粒细胞白血病（APL） PML/RARα融合基因表达水平的意义.方法 用扩增培养的NB4细胞的PML/RARα融合基因进行梯度稀释作为标准品,ABL作为内参,建立标准曲线.采用TaqMan法进行实时定量PCR,并对方法的可靠性、可重复性及灵敏性进行测定.对14例APL患者在初治、诱导缓解及巩固治疗3个时期的PML/RARα mRNA转录本水平进行动态定量监测,结果以NQ表示,NQ=PML/RARα mRNA的拷贝数/ABL mRNA的拷贝数×105.结果 实时定量PCR方法可以检测出1×10-5μ g NB4细胞cDNA中PML/RARα的融合基因,其重复性和稳定性Ct值变异系数分别为1.77％和2.11％.14例患者初治时骨髓PML/RARα融合基因平均水平为3 731,经全反式维甲酸、三氧化二砷双诱导缓解时PML/RARα融合基因平均水平为231,化疗与维甲酸序贯巩固治疗2个疗程后PML/RARα融合基因平均水平为116.治疗前后比较,PML/RARα基因转录本水平明显下降（P＜0.05）.结论 实时定量PCR检测PML/RARα融合基因表达敏感性好、特异性高、重复性好,可用于急性早幼粒细胞的诊断和微小残留病的检测.     

复方黄黛片及其与化疗序贯应用清除APL—PML／RARα融合基因的临床研究

目的:探讨复方黄黛片及其与化疗交替序贯应用对急性早幼粒细胞白血病(APL)患者PML/RARα融合基因的影响。方法:采用复方黄黛片及其与化疗序贯治疗42例APL患者,并采用RT-PCR技术监测PML/RARα融合基因。结果:①CR后1个月内融合基因转阴率达92.3%(12/13),12个月内达100%(34/34);②长期监测表明86%(31/36)的患者处于持续阴性状态。③治疗过程中融合基因转为阳性的患者加强治疗可再度转为阴性,处于持续的血液学和分子学缓解状态。结论:复方黄黛片治疗APL有很高的分子学缓解率,联合化疗可使86%以上患者处于持续血液学和分子学缓解状态。     

急性髓系白血病患者AML1-ETO融合基因动态监测及分析

目的 观察急性髓系白血病(AML)患者AML1-ETO融合基因表达水平及实时定量RT-PCR (RQ-PCR) 动态监测的临床意义.方法 对15例AML患者采用化疗药物进行诱导缓解、巩固和维持治疗,分别于初诊、诱导化疗结束及复发时采集骨髓标本,采用RQ-PCR法检测AML1-ETO融合基因表达水平,并采用Pearson相关分析法分析其与患者临床参数的关系.结果 15例患者初诊时AML1-ETO融合基因表达水平为168％～694％,其与患者骨髓幼稚细胞比例、年龄、性别等临床参数均无明显相关性(P＞ 0.05);AML1-ETO融合基因表达水平在完全缓解时降低、复发时明显升高(并早于骨髓形态学复发).结论 AML患者AML1-ETO融合基因表达水平与病情有关,采用RQ-PCR对其动态监测有利于发现分子水平复发及进行诊断、疗效判定、微小残留病(MRD)监测.     

急性早幼粒细胞白血病融合基因荧光定量动态检测的临床意义

目的利用逆转录荧光实时定量聚合酶链反应(RQ-PCR)检测急性早幼粒细胞白血病(APL)PML-RARα融合基因的方法,并观察其敏感度。方法建立荧光染料SYBR-Green1RQ-PCR技术,同时检测56例APL融合基因PML-RARα的表达水平,并且与传统巢式PCR方法进行敏感度比较。结果 RQ-PCR的灵敏度均可达到50拷贝,稍低于巢式PCR,但RQ-PCR定量准确,不易污染。分别取103和107拷贝的PML-RARα质粒以及患者cDNA同时作8次平行扩增,批内变异系数分别为7.31%、8.26%和5.02%,表明方法稳定。不同APL患者PML-RARα表达水平有较大差异,初诊患者PML-RARα/GAPDH比值介于0.018～0.799,中位值为0.070。结论RQ-PCR可以准确检测微小残留病(MRD)融合基因表达水平;监测融合基因表达可以有效观察治疗效果。     

PML/RARα融合基因检测早幼粒细胞白血病微小残留病

目的:PML/RARα融合基因在监测急性早幼粒细胞白血病(APL)微小残留病(MRD)中的意义.方法:诱导缓解及巩固维持治疗期间,采用筑巢式逆转录-聚合酶链反应(RT-PCR)技术检测患者骨髓细胞中PML-RARα融合基因的变化.结果:长期随访的18例完全缓解(CR)患者,2例分子学复发.其中1例发生于CRI后4个月,诱导缓解治疗后获CR2,CR2后2个月再次分子学与血液学的复发,诱导治疗1个疗程获得CR3;1例发生于CR1后74个月,诱导缓解治疗后获得CR2,随访结束时生存期已达106个月.结论:在CR期定期监测PML-RARα融合基因,可尽早发现分子学复发,及时治疗可避免血液学复发.     

复方黄黛片及其与化疗序贯应用清除APL-PML/RARα融合基因的临床研究

目的探讨复方黄黛片及其与化疗交替序贯应用对急性早幼粒细胞白血病(APL)患者PML/RARα融合基因的影响.方法采用复方黄黛片及其与化疗序贯治疗42例APL患者,并采用RT-PCR技术监测PML/RARα融合基因.结果①CR后1个月内融合基因转阴率达92.3%(12/13),12个月内达100%(34/34);②长期监测表明86%(31/36)的患者处于持续阴性状态.③治疗过程中融合基因转为阳性的患者加强治疗可再度转为阴性,处于持续的血液学和分子学缓解状态.结论复方黄黛片治疗APL有很高的分子学缓解率,联合化疗可使86%以上患者处于持续血液学和分子学缓解状态.     

急性髓性白血病患者外周血Notch信号相关基因的表达变化及意义

目的 观察急性髓性白血病(AML)患者外周血Notch信号相关基因的表达变化,并探讨其临床意义.方法 初诊AML患者30例(初诊组)、完全缓解AML患者30例(缓解组)、健康体检者24例(正常组),采用实时荧光定量逆转录聚合酶链反应法检测其外周血Notch1 mRNA、Delta4 mRNA、Jagged2 mRNA、HES1 mRNA.结果 与正常组比较,初诊组Notch1 mRNA、De1ta4 mRNA、Jagged2 mRNA、HES1 mRNA表达水平升高(P均＜0.05),缓解组HES1 mRNA表达水平亦升高(P＜0.05).结论 AML患者外周血Notch信号相关基因表达水平高于正常体检者,外周血Notch信号相关基因检测有助于AML的诊断.     

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.