重组腺病毒介导的人一氧化氮合酶基因转染对大鼠颈总动脉球囊损伤后新生内膜的影响

目的 :研究重组腺病毒介导的人内皮型一氧化氮合酶基因 (eNOS)表达生成的一氧化氮合酶 (NOS)对球囊损伤后大鼠颈总动脉新生内膜的抑制作用。方法 :在 2 93细胞内扩增、纯化Ad LacZ和Ad eNOS ,鉴定其是否携带有LacZ和eNOS基因。建立大鼠颈总动脉球囊损伤模型后 ,将磷酸缓冲液 (PBS)、Ad LacZ和Ad eNOS在体内分别转染到损伤血管段 ,以X gal染色、苏木精 伊红染色 ,免疫组化及计算机图像分析处理等方法观察转染动脉节段外源性eNOS蛋白表达及其对新生内膜的影响。结果 :重组腺病毒携带有eNOS基因 ,并且在损伤血管段得到有效表达。转染后PBS组、Ad LacZ组和Ad eNOS组的新生内膜面积分别为 (0 .187± 0 .0 18)、(0 .134± 0 .0 6 1)和 (0 .0 6 3± 0 .0 2 6 )mm 2 ,新生内膜与中膜面积比值 (I/M)分别为 1.5 76± 0 .2 73、1.342± 0 .35 7和 0 .5 6 0± 0 .16 1。与PBS组、Ad LacZ组相比Ad eNOS组无论新内膜面积 ,还是管腔狭窄程度都明显减小。结论 :腺病毒介导的eNOS基因转染能有效抑制球囊损伤后血管内膜的增生 ,可防治血管成形术后再狭窄     

野生型Rb基因导入促进血管平滑肌细胞衰老的观察

目的观察腺病毒载体介导的人野生型Ｒｂ基因导入后促进血管平滑肌细胞衰老的作用，并探讨Ｒｂ基因抑制平滑肌细胞增生的机理。方法用外源性Ｒｂ基因重组腺病毒载体感染体外培养的兔主动脉血管平滑肌细胞。以细胞超微结构、３Ｈ－胸腺嘧啶核苷掺入、流式细胞术分析细胞周期和β－半乳糖苷酶染色等方法观察平滑肌细胞的衰老变化。结果Ｒｂ基因导入可使平滑肌细胞发生衰老，表现为细胞线粒体肿胀，脂褐素堆积，ＤＮＡ合成减少，细胞停滞在Ｇ０／Ｇ１期，与衰老相关的β－半乳糖苷酶表达增加。结论野生型Ｒｂ基因通过促进细胞衰老而对平滑肌细胞增生起抑制作用     

腺病毒介导BTEB2基因转染对动脉损伤后新生内膜增生的影响

目的 :构建反义转录元件结合蛋白 2 (BTEB2 )重组腺病毒载体并研究BTEB2反义RNA对动脉损伤后新生内膜增生的影响。方法 :通过聚合酶链反应 (RT PCR)法从培养的大鼠血管平滑肌细胞中制备BTEB2cDNA ,将其反向克隆至腺病毒载体 ,构建反义BTEB2重组腺病毒 ;用重组腺病毒局部转染球囊损伤的大鼠颈动脉 ,观察反义BTEB2基因转染对BTEB2蛋白表达及损伤动脉新生内膜形成的影响。结果 :构建的BTEB2反义重组腺病毒经鉴定正确 ,其滴度为 5× 10 9/ml;反义BTEB2重组腺病毒转染可明显抑制BTEB2蛋白表达及新生内膜增生。结论 :成功构建了反义BTEB2重组腺病毒载体 ;BTEB2反义RNA可明显抑制大鼠颈动脉球囊损伤后的新生内膜增生。     

大鼠血管紧张素Ⅱ2型受体基因转染对颈动脉球囊损伤后新生内膜形成的影响

目的　探讨腺病毒载体介导的大鼠血管紧张素Ⅱ2型受体(AT2R)基因转染对大鼠颈动脉球囊损伤后新生内膜形成的影响。方法　应用带有绿色荧光蛋白报告基因的重组腺病毒载体将大鼠AT2R基因转移至颈动脉球囊损伤模型中,2 1d后取材,应用免疫组织化学方法检测AT2R在动脉壁中的表达,图像分析观察其对新生内膜形成的影响。结果　在体大鼠颈动脉腺病毒载体的转染率达4 0 % ,AT2R基因在血管壁的新生内膜、中膜、外膜稳定表达,转染AT2R基因的球囊损伤后颈动脉与绿色荧光蛋白基因对照组相比,血管新生内膜 中膜面积比降低了4 8%。结论　AT2R基因转染可抑制颈动脉球囊损伤后新生内膜的形成,对血管成形术后再狭窄可能有一定的预防作用。     

金属蛋白涂层支架用于小型猪冠状动脉质粒介导下转基因研究

目的 :评价在质粒介导下 ,金属蛋白涂层支架向血管内局部转基因的可行性、效率和选择性。　　方法 :金属支架由 316 L不锈钢丝编织而成 ,其涂层是通过把支架放入有交联剂的明胶溶液中浸泡而成。基因载体为Pc DNA2质粒 ,并携带有β-半乳糖苷酶标记基因 ,该基因编码核特异性β-半乳糖苷酶。首先将蛋白涂层支架分别固定在3.0 mm或 3.5 mm经皮冠状动脉腔内成形术球囊上并在浓度为 8μg/μl的基因原液中浸泡 3分钟 ,然后通过 8F大腔引导导管将支架送入小型猪冠状动脉前降支中段 (转基因组 ,n=3) ,另外把没有浸泡过基因的支架也送入小型猪冠状动脉前降支中段 (对照组 ,n=3)。在支架植入后 7天处死动物。β-半乳糖苷酶表达由 X- Gal染色评估。　　结果 :所有转基因动物均有基因表达。转基因表达出现在内膜、中层和外膜。中层平滑肌细胞转染率为 3.0 %。远处器官和对照组冠状动脉均未显示核特异性β-半乳糖苷酶阳性表达。　　结论 :蛋白涂层支架在质粒介导下向血管内转基因有效、可行 ,因此 ,它有可能成为冠状动脉腔内成形术后再狭窄基因治疗的有效转基因系统。     

外源性Rb基因对神经细胞衰老的影响

目的　观察外源性 Rb基因对神经细胞衰老的影响 ,并探讨 Rb基因对神经细胞 P2 1基因表达的调控。方法　用外源性 Rb基因重组腺病毒载体感染体外培养的胚胎大鼠神经细胞 ,以β-半乳糖苷酶染色观察神经细胞的衰老变化 ,用免疫组化法测定 P2 1蛋白表达水平。结果　外源性Rb基因导入神经细胞后 ,P2 1蛋白水平显著增高 ,与衰老相关的 β-半乳糖苷酶表达也增加。结论　 Rb基因可能通过上调 P2 1基因表达促进神经细胞衰老。     

Fas配体基因导入对大鼠颈动脉损伤后新生内膜的影响

目的 :探讨腺病毒载体介导 Fas配体 （Fas L）基因导入对大鼠颈动脉损伤后新生内膜的影响。方法 :利用重组腺病毒载体将 Fas L 导入大鼠颈动脉球囊损伤的内膜 ,14d后观察其对新生内膜形成的影响。结果 :通过腺病毒载体介导导入 Fas L 基因的被球囊损伤的大鼠颈动脉 ,与 Ad- β gal基因的对照组相比较 ,14d后损伤血管新生内膜 /中膜面积比降低了 73%（P<0 .0 1）。结论 :Fas L 可抑制新生内膜的增生。     

过表达p27基因抑制血管损伤后新生内膜的形成

目的　探讨p2 7基因转染对血管损伤后新生内膜形成的影响。方法　以腺病毒为载体转染p2 7基因 ,用MTT法观察其对血管平滑肌细胞增殖的影响 ,用流式细胞仪检测细胞周期 ;在兔颈总动脉球囊损伤模型上转染p2 7基因 ,采用免疫组织化学法检测p2 7基因的表达 ,检测其对新生内膜形成的影响。结果　p2 7可明显抑制培养的血管平滑肌细胞增殖 ,细胞停滞于G1期。兔颈总动脉球囊损伤后转染p2 7基因可以减少新生内膜的形成达 2 7 4 3%。结论　以腺病毒为载体转染p2 7基因可以有效地抑制血管平滑肌细胞增殖及血管损伤后新生内膜的形成。     

组织因子途径抑制物基因转移对血管平滑肌细胞迁移的影响

目的研究组织因子途径抑制物基因转移对兔血管平滑肌细胞迁移的影响,探讨其抑制再狭窄的机制。方法将含有人组织因子途径抑制物基因的重组腺病毒和含β-半乳糖苷酶基因的重组腺病毒在体外分别转染兔血管平滑肌细胞,用X-gal染色法检测腺病毒的转染率,用逆转录聚合酶链反应方法检测转染后平滑肌细胞中组织因子途径抑制物mRNA的表达,于第13、、5及7天用玻片法检测血管平滑肌细胞迁移距离。结果感染指数为100,腺病毒的转染率可达到95%;基因转移后3天在血管平滑肌细胞中检测到组织因子途径抑制物mRNA的表达;同一浓度下转染组织因子途径抑制物基因重组腺病毒组和对照组相比迁移距离显著减少(P<0.001),并具有浓度依赖性。结论腺病毒具有较高的转染培养的血管平滑肌细胞的能力,组织因子途径抑制物基因转移能够显著抑制体外培养的兔血管平滑肌细胞迁移,并且这种抑制作用具有浓度依赖性。     

双球囊导管法基因导入血管的实验研究

目的　探讨双球囊导管法腺病毒介导性基因导入犬股动脉的效率及时间曲线。方法　选成年杂交犬 2 0只 ,经股动脉分支逆行插入 7F双球囊导管至髂动脉 ,采用气囊法剥脱血管内皮后 ,充盈双球囊 ,经球囊间侧孔注入 0 3mL约含 3 0× 10 9pfu的含有细菌lacZ基因的重组腺病毒入间腔 ,培养 30分钟。术后 1、3、7、14、2 1、2 8及 35天处死动物 ,组织化学分析法检测lacZ基因在股动脉的表达。结果　导入基因后 2 4小时即见lacZ基因在血管内膜表达 ,7天达高峰 ,持续 4周左右。高峰时 ,培养间腔血管内侧近 10 0 %表达了外源基因 ,深度达血管中膜 2 /3。结论　双球囊导管法腺病毒介导性基因导入犬股动脉呈高效及一过性特点。     

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.