外源性基因在血管球囊损伤后新生内膜中的表达

目的：应用重组腺病毒载体介导的β-半乳糖苷酶基因（LacZ）标记基因,探讨外源性基因在股动脉血管球囊损伤后,新生内膜中的表达。方法：用犬的股动脉内皮损伤模型,4周后经犬颈外动脉插管至股动脉内皮损伤处,并导入重组腺病毒载体介导的LacZ标记基因。转基因7天后,处死动物。β-半乳糖苷酶表达由x-Gal染色评价。经显微镜观察：在血管球囊损伤后,新生内膜中所见的蓝染细胞即为β-半乳糖苷酶表达阳性。结果：β-半乳糖苷酶表达出现在新生内膜、部分中膜和部分修复的内皮细胞。血管损伤后,新生内膜中平滑肌细胞转染率为100％。对照血管和远处脏器未显示β-半乳糖苷酶的表达。结论：在重组腺病毒载体的介导下,外源性基因可以在新生内膜中高效率地表达,新生内膜中的平滑肌细胞为基因治疗的靶细胞。     

改进的明胶蛋白涂层支架用于小型猪冠状动脉转染一氧化氮合酶基因的可行性研究

目的为了判定明胶蛋白涂层支架携带质粒介导的一氧化氮合酶(iNOS)基因转染小型猪冠状动脉的可行性.方法将自行构建的pcDNA3.1/iNOS(L24553)质粒以浸泡(15～60min)及涂抹(60min)的方法携带在明胶蛋白涂层支架(由上海微创公司提供)上,采用标准球囊导管技术置入小型猪(n=5)冠状动脉前降支及右冠近中段,支架与置入处血管直径之比1.1～1.31.结果支架置入术后7天,在支架置入处血管段使用对人特异性引物完成的逆转录多聚酶链反应(RT-PCR)反应表明有iNOS基因转染,免疫组织化学检测表明有iNOS蛋白生成.结论明胶蛋白涂层支架携带质粒介导的iNOS基因可有效转染小型猪冠状动脉成形部位.     

蛋白涂层支架携带一氧化氮合酶基因转染

目的探索蛋白涂层支架携带质粒介导人肝脏诱导型一氧化氮合酶(iNOS)基因转染小型猪冠状动脉可行性。 方法使用蛋白支架吸附去内毒素纯化质粒,以常规支架置入技术置入小型猪冠状动脉前降支中段。置入后第7天取出前降支置入段,分别提取总核糖核酸(RAN)并进行逆向多聚酶链反应(RT-PCR),免疫组化染色检测导入人肝脏iNOS蛋白的表达。 结果小型猪前降支置入支架处显示人iNOS基因信使核糖核酸(mRNA)转录,免疫组化染色显示中膜、内膜人iNOS基因表达人iNOS蛋白的颗粒,以平滑肌细胞最明显。 结论蛋白涂层支架吸附去内毒素携带人iNOS基因质粒植入小型猪前降支冠状动脉,RT-PCR显示人iNOS基因的mRNA转录,免疫组化显示人iNOS蛋白的表达。     

质粒介导下转染血管内皮生长因子基因不能防治小型猪冠状动脉再狭窄

目的 :评估质粒介导下蛋白涂层金属支架局部转染血管内皮生长因子 (VEGF)基因对小型猪冠状动脉再狭窄的作用。　　方法 :选用 1 4只正常小型猪作为实验动物 ,载体为携带有VEGF基因的PcDNA3质粒。用常规球囊导管技术 ,将携带有VEGF基因的蛋白涂层支架置入冠状动脉前降支中段作为VEGF转基因组 (n =7) ,以相同方法置入单纯蛋白涂层支架作为对照组 (n =7) ,球囊与血管直径之比为 1 1∶1。在基因转染后 7天 ,行反转录聚合酶链反应和免疫组化染色。在支架置入后 3个月重复冠状动脉造影 ,测定管腔狭窄直径 ;同时行组织病理学检查。　　结果 :支架被成功置入 1 4只动物冠状动脉前降支中段。支架置入后 7天 ,反转录聚合酶链反应结果证实 ,在转染部位的动脉壁内有VEGF信使核糖核酸的表达。免疫组化染色显示在中层平滑肌细胞浆内及外膜有VEGF蛋白质生成。在支架置入后 3个月重复冠状动脉造影显示 :转基因组和对照组均发生再狭窄 ,平均管腔狭窄直径分别是 ( 6 9±1 0 ) %和 ( 80± 1 4 ) % ,无显著性差异 (P =0 2 4 7)。组织病理学形态分析结果显示 :转基因组和对照组 ,在平均损伤积分、新生内膜厚度、平均新生内膜面积、百分狭窄面积和内膜 /中膜值等各项参数上均无显著性差别。　　结论 :在本实验的再狭窄动物模     

蛋白涂层支架携带一氧化氮合酶基因转染小型猪冠状动脉的可行性研究

目的 :探索蛋白涂层支架携带质粒介导人肝脏诱导型一氧化氮合酶 (i NOS)基因转染小型猪冠状动脉可行性。　　方法 :使用蛋白支架吸附去内毒素纯化质粒 ,以常规支架置入技术置入小型猪冠状动脉前降支中段。置入后第 7天取出前降支置入段 ,分别提取总核糖核酸 (RAN)并进行逆向多聚酶链反应 (RT- PCR) ,免疫组化染色检测导入人肝脏i NOS蛋白的表达。　　结果 :小型猪前降支置入支架处显示人 i NOS基因信使核糖核酸 (m RNA)转录 ,免疫组化染色显示中膜、内膜人i NOS基因表达人 i NOS蛋白的颗粒 ,以平滑肌细胞最明显。　　结论 :蛋白涂层支架吸附去内毒素携带人 i NOS基因质粒植入小型猪前降支冠状动脉 ,RT- PCR显示人 i NOS基因的 m RNA转录 ,免疫组化显示人 i NOS蛋白的表达。     

蛋白涂层支架携带质粒介导的一氧化氮 合酶基因预防冠状动脉球囊扩张后 再狭窄的实验研究

目的　为评价蛋白涂层金属支架携带质粒介导的诱导性一氧化氮合酶 (iNOS)基因局部转染血管壁 ,预防冠状动脉内血管成形术后再狭窄的效果。方法　金属支架涂层为胶联明胶制成。载体为去内毒素纯化pcDNA3。采用标准球囊导管技术 ,将吸附有质粒介导的人肝脏的iNOS基因(pcDNA3hepiNOS)涂层支架置入小型猪 (n =9)冠状动脉前降支中段 ,以相同方法置入单纯蛋白涂层支架做为对照组 (n =9) ,支架与血管直径之比为 1.1～ 1.3:1。结果　在支架置入后 7d ,RT PCR检测和免疫组织化学染色 ,证实在pcDNA3hepiNOS转染的血管段有iNOSmRNA的表达和iNOS蛋白生成 ,而远离器官则无基因的表达。 3个月时冠状动脉造影显示 :转染pcDNA3hepiNOS组 (n =5 )无再狭窄发生 ,而对照组均发生显著的再狭窄。组织病理学形态分析结果显示 :pcDNA3hepiNOS组新生内膜面积 (1.7± 0 .8)mm2 、平均百分狭窄面积 (2 6 .5± 7.5 ) %、平均管腔狭窄百分数 (4 1.2± 16 .5 ) % ,均较对照组小 ,对照组分别为 (2 .8± 0 .8)mm2 ,P <0 .0 5 ;(94.2± 14.3) % ,P <0 .0 0 1;(88.0± 16 .6 ) % ,P <0 .0 0 1;比较内膜面积 /中膜面积比值 (I/M)治疗组较对照组减少了 5 9.8%。结论　在小型猪模型使用蛋白涂层支架携带纯化质粒介导的iNOS基因可直接导入血管     

钩藤总生物碱对D-半乳糖诱导的内皮细胞衰老的干预

目的探讨钩藤总生物碱保护血管内皮细胞、抑制血管内皮细胞衰老的作用。方法采用D-半乳糖诱导的大鼠主动脉内皮细胞建立衰老模型,扫描电镜技术观察衰老血管内皮细胞的形态,β-半乳糖苷酶染色法测定衰老血管内皮细胞发生率以间接反映β-半乳糖苷酶表达,PCR-ELISA法测定衰老血管内皮细胞端粒酶活性。结果钩藤总生物碱可以改善血管内皮细胞形态、降低内皮细胞中β-半乳糖苷酶表达和端粒酶活性相对表达量(P<0.05)。结论钩藤总生物碱具有抑制内皮细胞衰老、保护血管内皮的效用。     

在小型猪模型由蛋白涂层金属支架局部转染尿激酶前体基因 …

目的为了评价蛋白涂层金属支架局部转染尿激酶前体基因对冠状动脉内血小板沉积、早期血栓形成和平滑肌细胞增生的影响。方法 金属支架涂层为交联明胶制成。载体为复制缺陷的、携载Ｐｒｏ＝ＵＫ基因的重组腺病毒。采用标准球囊导管技术,将携带有Ｐｒｏ－ＵＫ基因的涂层支架置入小型猪冠状动脉前降支中段,以相同方法置入单纯蛋白涂层支架或裸露支架做为对照。结果 支架置入后３天,Ｐｒｏ－ＵＫ基因转染血管段（ｎ＝６）＾１１１Ｉ     

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。     

药物涂层支架的研究进展

经皮冠状动脉腔内成形术和支架置入术是解决冠状动脉腔内狭窄的一种新型手段,但其有30%左右的再狭窄率.药物涂层支架通过其药物基质混合物对支架涂层,实行抗血栓涂层、抗炎症涂层、抗血管平滑肌细胞增殖和迁移涂层,从而达到抑制新生内膜增殖,进一步减少PTCA术后冠状动脉血管内再狭窄的发生.     

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.