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.     

巢式聚合酶链反应检测慢性粒细胞白血病bcr／abl融合基因

目的 探讨Ph染色体和bcr/abl融合基因在慢性粒细胞白血病（CGL）的发病机制、诊断、治疗、预后判断的价值。方法 对46例CGL患者作巢式逆转录聚合酶链反应（RT-PCR）检测bcr/abl融合基因,同时对其中28例作细胞遗传学检查。结果 46例CGL患者中,44例bcr/abl融合基因阳性,阳性率为95.7%;28例CGL患者作细胞遗传学检查。26例Ph染色体阳性,阳性率为92.9%;2例Ph染色体阴性的CGL患者,用RT-PCR检测出ber/abl融合基因。结论 巢式RT-PCR是一种快速、敏感而准确的检测方法,可以为部分Ph染色体阴性的CGL患者提供分子生物学的诊断依据。     

Three layer functional model and energy exchange concept of aging process

Relying on a certain degree of abstraction, we can propose that no particular distinction exists between animate or living matter and inanimate matter. While focusing attention on some specifics, the dividing line between the two can be drawn. The most apparent distinction is in the level of structural and functional organization with the dissimilar streams of ‘energy flow’ between the observed entity and the surrounding environment. In essence, living matter is created from inanimate matter which is organized to contain internal intense energy processes and maintain lower intensity energy exchange processes with the environment. Taking internal and external energy processes into account, we contend in this paper that living matter can be referred to as matter of dissipative structure, with this structure assumed to be a common quality of all living creatures and living matter in general. Interruption of internal energy conversion processes and terminating the controlled energy exchange with the environment leads to degeneration of dissipative structure and reduction of the same to inanimate matter, (gas, liquid and/or solid inanimate substances), and ultimately what can be called ‘death.’ This concept of what we call dissipative nature can be extended from living organisms to social groups of animals, to mankind. An analogy based on the organization of matter provides a basis for a functional model of living entities. The models relies on the parallels among the three central structures of any cell (nucleus, cytoplasm and outer membrane) and the human body (central organs, body fluids along with the connective tissues, and external skin integument). This three-part structural organization may be observed almost universally in nature. It can be observed from the atomic structure to the planetary and intergalactic organizations. This similarity is corroborated by the membrane theory applied to living organisms. According to the energy nature of living matter and the proposed functional model, the decreased integrity of a human body's external envelope membrane is a first cause of the structural degradation and aging of the entire organism. The aging process than progresses externally to internally, as in single cell organisms, suggesting that much of the efforts towards the restoration and maintenance of the mechanisms responsible for structural development should be focused accordingly, on the membrane, i.e., the skin. Numerous reports indicate that all parts of the human body, like: bones, blood with blood vessels, muscles, skin, and so on, have some ability for restoration. Therefore, actual revival of not only aging tissue of the human body's membrane, but the entire human body enclosed within, with all internal organs, might be expected. We assess several aging theories within the context of our model and provide suggestions on how to activate the body's own anti-aging mechanisms and increase longevity. This paper presents some analogies and some distinctions that exist between the living dissipative structure matter and inanimate matter, discusses the aging process and proposes certain aging reversal solutions.     

Unusual responses of nocturnal pineal melatonin synthesis and secretion to swimming: Attempts to define mechanisms

Abstract: The effect of swimming at night on rat pineal melatonin synthesis was compared with that of light exposure at night. Rats were forced to swim at 0030 hr (lights out at 2000 hr) and sacrificed by decapitation 15 and 30 min later, immediately after swimming. Other groups of animals were exposed to white light (650μW/cm²) for 15 and 30 min at same time. Swimming caused a rapid and highly significant drop in the melatonin content in the pineal gland; however, the activity of N-acetyltransferase (NAT), the supposed rate limiting enzyme in the melatonin production, was not changed. Despite the drop in pineal melatonin levels, serum concentrations of the indole remained elevated in the rats that swam. In contrast, melatonin levels in the pineal and serum of light exposed rats fell precipitously, accompanied by a significant suppression of NAT activity. Since we anticipated that the strenuous exercise associated with swimming may induce release of artrial natriuretic peptide (ANP) from the heart, which in turn could cause the release of pineal melatonin, in a second study we injected physiological saline intravenously to stretch the cardiac muscle and release ANP. Three milliliters of normal saline was injected during the day into the jugular vein of anesthetized rats that were pretreated with isoproterenol to stimulate pineal melatonin production. Animals were killed 15 min after the saline injection, and pineal NAT activity and pineal melatonin levels were measured. The saline injections caused no alteration in the elevated levels of either NAT or melatonin. These data suggest that the disparity in pineal NAT activity (which was high) and pineal melatonin (which was low), in animals swum at night, may not be caused by ANP which is released during strenuous exercise such as swimming.     

Melatonin and photoperiodic time measurement: Seasonal breeding in the sheep

Abstract: Well-established circadian physiology supports the view that photoperiodic time measurement utilizes the coincidence between the presence of light and a photosensitive phase of a 'biological clock' to alter reproductive status—the so-called external coincidence model of seasonal breeding. In this review, we examine the mechanism whereby photoperiod interacts with presumed suprachiasmatic nuclei activity to allow endogenous melatonin to normally synchronize reproductive activity to the optimal time of year. The Romney Marsh sheep is particularly explored as an experimental model. It is suggested that the on/off activity of seasonal reproduction may be a robust mechanism able to be predictably manipulated by the judicious use of the light/dark cycle and exogenous melatonin, but firmly based on circadian principles.     

Serological survey of HIV and syphilis in pregnant women in Madagascar

Objectives Peripartal transmission of human immunodeficiency virus (HIV) and Treponema pallidum, the causative agent of syphilis, leads to severe consequences for newborns. Preventive measures require awareness of the maternal infection. Although HIV and syphilis testing in Madagascar could be theoretically carried out within the framework of the national pregnancy follow‐up scheme, the required test kits are rarely available at peripheral health centres. In this study, we screened blood samples of pregnant Madagascan women for HIV and syphilis seroprevalence to estimate the demand for systemic screening in pregnancy. Methods Retrospective anonymous serological analysis for HIV and syphilis was performed in plasma samples from 1232 pregnant women that were taken between May and July 2010 in Ambositra, Ifanadiana, Manakara, Mananjary, Moramanga and Tsiroanomandidy (Madagascar) during pregnancy follow‐up. Screening was based on Treponema pallidum haemagglutination tests for syphilis and rapid tests for HIV, with confirmation of positive screening results on line assays. Results Out of 1232 pregnant women, none were seropositive for HIV and 37 (3%) were seropositive for Treponema pallidum. Conclusions Our findings are in line with previous studies that describe considerable syphilis prevalence in the rural Madagascan population. The results suggest a need for screening to prevent peripartal Treponema pallidum transmission, while HIV is still rare. If they are known, Treponema pallidum infections can be easily, safely and inexpensively treated even in pregnancy to reduce the risk of transmission.     

Variabilité des concentrations plasmatiques de l’angiotensinogène et risque d’hypertension artérielle chez le rat transgénique

Aim

Genetic polymorphisms of the human angiotensinogen gene are frequent and may induce up to 30% increase of plasma angiotensinogen concentrations with a blood pressure increase of up to 5 mmHg. Their role for the pathogenesis of human arterial hypertension remains unclear. High plasma angiotensinogen levels could increase the sensitivity to other blood pressure stressors.

Methods

Male transgenic rats with a 9-fold increase of plasma angiotensinogen concentrations and male non-transgenic rats aged 10 weeks were treated or not with NG-Nitro-L-arginine-methyl ester for 3 weeks in their drinking water (n = 3/group). Systolic blood pressure and body weight were measured at baseline and at the end of the study when left ventricular weight and ventricular expression of angiotensin I-converting enzyme and procollagen Iα1 were determined (polymerase chain reaction).

Results

At baseline, transgenic rats had +18 mmHg higher bood pressure and –8% lower body weight compared to non-transgenic rats (P < 0.05) without significant changes for the vehicle groups throughout the study (P > 0.05). NG-Nitro-L-arginine-methyl ester increased blood pressure, left ventricular weight and left ventricular weight indexed for body weight by +41%, +17.6% and +18.6% (P < 0.05) in transgenic and +25%, +5.3% and +6.7% (P > 0.05) in non-transgenic rats compared to untreated animals, respectively. Cardiac gene expression showed no differences between groups (P > 0.05).

Conclusion

Increased plasma angiotensinogen levels may sensitize to additional blood pressure stressors. Our preliminary results point towards an independent role of angiotensinogen in the pathogenesis of human hypertension and associated end-organ damage.     

Morphological and immunocytochemical heterogeneity of cultured pinealocytes from one-week-and two-month-old rats: Planimetric and densitometric investigations

Abstract: In vitro preparations of rat pinealocytes are widely used for biochemical analyses of signal transduction processes. This paper deals with morphological and immunocytochemical features of such preparations. Special attention was paid to the problems of whether pinealocytes represent a heterogeneous cell population and how such heterogeneity may develop during ontogeny. The investigations were performed with cells which were obtained from the pineal organ of one-week-and two-month-old rats, attached to synthetic peptide-coated coverslips or tissue culture chamber slides, and maintained under in vitro conditions overnight. The attached cells were then fixed with paraformaldehyde. These preparations yielded monolayers of spherical cells of different sizes; most cells were isolated, but some of them were aggregated and formed small clusters. On the average, the cells from the one-week-old animals were smaller than the cells from the two-month-old animals. Immunocytochemical demonstration of S-antigen, a pinealocyte-specific marker, showed that the majority of the cells from two-month-old animals were intensely or moderately labelled. Pinealocytes from one-week-old animals were less S-antigen immunoreactive. Only very few cells (less than 1% displayed glial fibrillary acidic protein (GFAP)-immunoreactivity. Planimetric investigations of the cell size and semiquantitative densitometric investigations of the intensity of the S-antigen immunoreaction revealed that (i) pinealocytes kept in vitro form a heterogeneous cell population, and that (ii) this heterogeneity increases during postnatal development from one-week-old to two-month-old animals. Two groups of pinealocytes can be distinguished based on their developmental fate: pinealocytes of one group grow dramatically, but show only a moderate increase in S-antigen immunoreactivity, and pinealocytes of the other group retain their size, but display a distinct increment in S-antigen immunoreacti vitv.     

Effects of pinealectomy and melatonin administration on certain indices of ovarian hyperplasia and/or hypertrophy in rats with both ovaries intact or after unilateral ovariectomy

Abstract: In earlier studies from other laboratories it was shown that melatonin decreased ovarian weight in rats and inhibited compensatory hypertrophy of the remaining ovary after unilateral ovariectomy. This study was designed to examine the influence of melatonin on certain indices of ovarian hyperplasia and/or hypertrophy in adult female rats with both ovaries preserved and with either an intact pineal gland or with the pineal gland removed (pinealectomy, PX) or, finally, in sham-PX animals. Similar studies were conducted on rats after unilateral ovariectomy, referring the examined parameters to the remaining intact ovary. The studies included mitotic activity of granulosa layer cells and corpus luteum cells, ovarian weight, ovarian cross-sectional area, cross-sectional area of the granulosa layer of all the Graafian follicles and the cross-sectional areas of the corpora lutea, visible on the ovarian cross-section. On the basis of results, we conclude that: 1) the effect of PX on the processes of ovarian hyperplasia and hypertrophy may vary; analogously, exogenous melatonin administration may influence ovarian hyperplasia and hypertrophy in different ways; 2) PX and exogenous melatonin may, under certain conditions, exert similar biological effects, even synergistic effects; 3) melatonin inhibits ovarian growth processes, while the effects of PX are variable; 4) the results indicate that in experiments performed on rats, with the use of two control groups, i.e., intact and sham-PX, melatonin effects on these two groups may differ.