人参皂苷单体Rb1及Rg1对白血病细胞K562增殖的影响

背景：国内外研究发现人参皂苷及其单体在抑制白血病细胞增殖、增加化疗药物的敏感性和逆转耐药等方面均有疗效，目前主要集中于对实体瘤和急性白血病的研究，涉及慢性白血病的报道非常少。 目的：探讨人参皂苷单体Rb1，Rg1对人慢性粒细胞白血病细胞K562增殖的影响。 设计、时间及地点：细胞学体外观察，于2008-03/2009-03在重庆医科大学干细胞与组织工程研究室和眼科实验室完成。 材料：K562细胞由重庆医科大学临床检验系提供。纯度为98.6%的人参皂苷单体Rb1，Rg1由南京艾斯替么中药研究所提供。 方法：取对数生长期的K562细胞，调整密度为7×108 L-1，空白对照组予以常规培养；人参皂苷单体Rb1组分别加入20，40，80，160 μmol/L的Rb1；人参皂苷单体Rg1组分别加入5，10，20，40，80 μmol/L的Rg1。 主要观察指标：MTT比色法、锥虫蓝拒染法检测K562细胞增殖情况，流式细胞仪测定细胞周期分布的变化。 结果：人参皂苷单体Rb1，Rg1在体外对K562细胞增殖均有明显的抑制作用，在20~160 μmol/L Rb1和5~20 μmol/L Rg1浓度范围内，其抑制作用呈浓度依赖性，且均在作用48 h时抑制率达高峰。与空白对照组比较，体外培养48 h后160 μmol/L Rb1组细胞周期分布无变化；20 μmol/L Rg1组S期细胞比例明显增加(P < 0.05)，G1期细胞比例明显下降(P < 0.05)，且160 μmol/L Rb1组、20 μmol/L Rg1组均未出现“亚二倍体”峰。 结论：人参皂苷单体Rb1，Rg1均可抑制K562细胞增殖，Rg1增殖抑制作用可能是通过将K562细胞阻滞于S期而实现的，而Rb1的增殖抑制作用与细胞周期的关系有待进一步分析。     

人参皂甙Rb_1对大鼠海马脑片缺血损伤的保护作用

目的　研究人参皂甙 Rb1 对脑缺血损的保护作用及其机制。方法　实验利用离体海马脑片模型 ,结合电生理学技术进行研究 :(1)观察大鼠海马脑片在缺血条件下顺向群峰电位 (orthodrom ic population spike,OPS)的变化及人参皂甙 Rb1 对它的影响。 (2 )观察谷氨酸对海马脑片 OPS的影响及人参皂甙 Rb1 抗谷氨酸毒性作用。结果　 (1)使用人参皂甙 Rb1 (6 0 0 μm ol/ L)后 ,可使缺氧损伤电位 (hypoxic injury potential,HIP)出现率明显减少 ,OPS恢复率和 OPS恢复程度与对照组比较有显著性差异。 (2 )使用人参皂甙 Rb1 (6 0 0 μm ol/ L)后 ,OPS恢复率和OPS恢复程度与对照组比较有显著性差异。结论　人参皂甙 Rb1 有明显的抗脑缺血损伤作用 ,其作用机制与抗谷氨酸的神经毒性作用有关。     

人参皂甙Rg1对大鼠脑源性神经干细胞分化作用的实验研究

目的 探讨人参皂甙Rg1对体外培养大鼠大脑皮质神经干细胞(NSC)分化作用的影响.方法 利用无血清DMEM/F12(含20ng/mL bFGF和EGF及2% B27)体外培养技术从新生Wistar大鼠大脑皮质中培养NSC,在NSC分化过程中添加不同剂量(20mg/L、40mg/L和80mg/L)人参皂甙Rg1进行干预,以免疫荧光及细胞化学方法对NSC及其分化后的细胞进行鉴定.结果 从大脑皮质分离培养的NSC能够表达巢蛋白(Nestin),并具有分化为神经元、星形胶质细胞及少突胶质细胞的能力.在同一接种密度条件下,不同剂量人参皂甙Rg1干预组的Nestin阳性细胞表达数分别为20mg/L组(21.5±2.9);40mg/L组(18.6±2.4);80mg/L组(16.7±2.2),与对照组(24.8±3.1)比较明显减少(P＜0.05).而人参皂甙Rg1干预组表达神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)、半乳糖苷酶(GalC)的阳性细胞数较对照组明显增加(P＜0.05).结论 人参皂甙Rg1可能有促进新生大鼠大脑皮质NSC的分化的作用.     

人参皂甙Rb1对大鼠海马脑片缺血损伤的保护作用

目的 研究人参皂甙Rb1对脑缺血损的保护作用及其机制。方法 实验利用离体海马脑片模型，结合电生理学技术进行研究：(1)观察大鼠海马脑片在缺血条件下顺向群峰电位(orthodromic population spike，OPS)的变化及人参皂甙Rb1对它的影响。(2)观察谷氨酸对海马脑片OPS的影响及人参皂甙Rb1抗谷氨酸毒性作用。结果 (1)使用人参皂甙Rbl(600μmol／L)后，可使缺氧损伤电位(hypoxic injury potential，HIP)出现率明显减少，OPS恢复率和OPS恢复程度与对照组比较有显著性差异。(2)使用人参皂甙Rb1(600μmol／L)后，OPS恢复率和OPS恢复程度与对照组比较有显著性差异。结论 人参皂甙Rb1有明显的抗脑缺血损伤作用，其作用机制与抗谷氨酸的神经毒性作用有关。     

人参皂甙Rb1对体外培养雪旺细胞作用的实验研究

目的 研究人参皂甙Rb1对体外培养雪旺细胞增殖分化的影响,探讨其促进神经再生的作用和机制.方法 取8个月月龄的新西兰兔的坐骨神经雪旺细胞体外培养第2代,加入不同浓度的人参皂甙Rb1,继续培养10d,相差显微镜观察计数,绘制各自的增殖曲线.在36h和72h对各组细胞进行流式细胞分析.结果活细胞计数显示：含20μg/ml人参皂甙Rb1组的倍增时间为5.3d,明显优于对照组（P＜0.01）.用含20μg/ml人参皂甙Rb1培养36h、72h后雪旺细胞增殖的流式细胞分析,处于S期的雪旺细胞较对照组明显增高（P＜0.01）.结论 人参皂甙Rb1具有促进体外培养雪旺细胞快速增殖分化的作用,有助于损伤神经的再生.     

甲泼尼龙琥珀酸钠对许旺细胞分泌功能的影响*☆

背景：许旺细胞分泌多种神经营养因子在神经再生中发挥关键作用，但其分泌能力受诸多因素影响，寻找可行方法促进许旺细胞分泌神经生长因子是神经缺损后再生的重要环节。 目的：观察不同浓度甲泼尼龙琥珀酸钠对许旺细胞分泌功能的影响。 方法：酶消化法分离培养SD乳鼠许旺细胞，倒置相差显微镜下观察细胞生长情况；传代后，一部分进行S-100蛋白免疫鉴定许旺细胞的纯度；另一部分用细胞计数板调整细胞浓度为1×109 L-1，移入到6孔平底培养板(接种15个孔)继续培养。培养板中培养4 d后4个孔分别加入不同浓度的甲泼尼龙琥珀酸钠(10-3，10-4，10-6，10-8 mol/L)，并设立1个孔为不加药物的空白对照组，分别作用于细胞24，48，72 h后行RT-PCR检测，观察许旺细胞24，48，72 h神经生长因子mRNA水平的变化。 结果与结论：原代细胞培养至第7天数量明显增加，细胞铺满培养瓶底部80%以上；传代细胞形态为梭形，有2个细长的突起，荧光染色阳性，成纤维细胞为圆形或扁圆形，荧光染色阴性。RT-PCR检测结果显示，10-8 mol/L的甲基强地松龙作用72 h分泌的神经生长因子与空白对照组及其他浓度、时间组相比均表达增加(P < 0.05)；10-3 mol/L甲基强地松龙在各时间段分泌的神经生长因子与空白对照组及其他浓度、时间组相比表达均减少(P < 0.05)。提示高浓度的甲泼尼龙琥珀酸钠抑制许旺细胞分泌神经生长因子，长时间、低浓度的甲泼尼龙琥珀酸钠则促进许旺细胞分泌神经生长因子。     

人参皂甙Rb1对体外培养雪旺细胞作用的实验研究

目的研究人参皂甙Rb1对体外培养雪旺细胞增殖分化的影响,探讨其促进神经再生的作用和机制。方法取8个月月龄的新西兰兔的坐骨神经雪旺细胞体外培养第2代,加入不同浓度的人参皂甙Rb1,继续培养10d,显微镜观察计数,绘制各自的增殖曲线。在36h和72h对各组细胞进行流式细胞分析。结果活细胞计数显示:含20μg/ml人参皂甙Rb1组的倍增时间为5.3d,明显优于对照组(P<0.01)。用含20μg/ml人参皂甙Rb1培养36h、72h后雪旺细胞增殖的流式细胞分析,处于S期的雪旺细胞较对照组明显增高(P<0.01)。结论人参皂甙Rb1具有促进体外培养雪旺细胞快速增殖分化的作用,有助于损伤神经的再生。     

人参皂甙Rg1对局灶性脑缺血大鼠脑组织巢蛋白表达的影响及意义

目的探讨局灶性脑缺血后脑组织巢蛋白（Nestin）的表达以及人参皂甙Rg1对其表达的影响。方法采用改良的线栓法制备大鼠大脑中动脉阻塞脑缺血模型，应用免疫组织化学技术检测脑缺血以及预先给予人参皂甙Rg1后不同时间段（1、3、7、14d）脑组织Nestin的表达水平。结果给予人参皂甙Rg1后大鼠脑组织Nestin标记细胞的数目及阳性部位明显增多。结论人参皂甙Rg1可使大鼠脑缺血后脑组织Nestin表达水平上调，提示人参皂甙Rg1可刺激脑组织神经干细胞的增殖及分化，这也可能是其发挥脑保护及抗衰老作用的机制之一。     

人参皂甙Rh2对人胶质瘤细胞U87MG凋亡的影响

目的 观察人参皂甙Rh2对胶质瘤细胞凋亡的影响并初步探讨其可能机制。方法 将培养的人胶质瘤细胞U87MG随机分为人参皂甙Rh2组、人参皂甙Rh2+尼莫地平组和对照组。人参皂甙Rh2组在常规培养细胞时加入20 μg/ml的人参皂甙Rh2，人参皂甙Rh2+尼莫地平组在人参皂甙Rh2组培养细胞时加入浓度为10 μmol/L的尼莫地平。利用流式细胞仪检测U87MG细胞凋亡，利用激光共聚焦显微镜和流式细胞仪检测U87MG细胞内钙离子浓度。结果 与对照组相比，人参皂甙Rh2促进U87MG细胞凋亡（P<0.05），且增加细胞内游离钙离子浓度（P<0.05）；尼莫地平显著减少人参皂甙Rh2引起的U87MG细胞凋亡（P<0.05）。结论 人参皂甙Rh2可以通过增加细胞内游离钙离子浓度促进U87MG细胞凋亡。     

人参皂甙Rg1对局灶性脑缺血大鼠脑组织神经元特异性烯醇化酶表达的影响及其意义

目的 探讨人参皂甙Rg1对局灶性脑缺血大鼠脑组织神经元特异性烯醇化酶(NSE)表达的影响及其意义.方法 给大鼠腹腔注射人参皂甙Rg1 20mg/kg,每日2次,连续5d;然后采用改良的线栓法制备大鼠局灶性脑缺血模型,观察大鼠脑缺血后不同时间段(3h、24h、48h、72h)神经功能缺损评分;应用免疫组化法检测脑组织NSE的表达;并与对照组(生理盐水)和假手术组比较.结果 人参皂甙Rg1组大鼠脑缺血后各时间点神经功能缺损评分明显低于对照组(均P＜0.05).与假手术组相比,对照组脑缺血后3h NSE表达即明显降低;人参皂甙Rg1组各时间点NSE阳性细胞数明显多于对照组(均P＜0.01).结论 人参皂甙Rg1可使缺血脑组织的NSE表达上调,这可能是其发挥脑保护作用的机制之一.     

Adult GM1 gangliosidosis--biochemical studies--1]

To address the pathogenesis of GM1 gangliosidosis, especially adult form, intracellular signal transduction pathway of EGF in skin fibroblasts from patients with this disorder was examined. For this purpose, skin fibroblasts from 2 different patients with adult form of the disorder and from 4 different normal controls were used. The results showed that 1) EGF-receptor autophosphorylation was diminished in skin fibroblasts from patients with altered time course of phosphorylation-dephosphorylation reaction. 2) The amount of EGF-receptor protein was decreased in cells from patients compared with that of controls. 3) 125I-EGF binding + internalization studies revealed decreased rate of EGF binding and internalization in patient cells. 4) Ribosomal S6 protein phosphorylation was strongly enhanced in naive cells from patients, but the reactivity to EGF was diminished compared with control cells. These data strongly suggest that patient fibroblasts have abnormalities in the intracellular signal transduction pathway of EGF. This paper is considered to be the first report demonstrating abnormalities in EGF-signal transducing system in human disorders.     

CYP1A1 and GSTM1/T1 Genetic Variation in Predicting Risk for Cerebral Infarction

Cytochrome P450 1A1 (CYP1A1) is involved in the production of arachidonic acid-derived vasoactive substance. We hypothesized that CYP1A1 polymorphism might be related to pathological conditions associated with cerebral infarction (CI). We investigated the effect of genetic polymorphism in the 3′-flanking region (T6235C) of CYP1A1 gene in 353 patients with CI and 376 controls. The distributions of T6235C CYP1A1 genotypes in patients with (TT: 36.0%; TC/CT: 64.0%; n = 353) and without CI (TT: 44.7%; TC/CT: 55.3%; n = 376) indicate that the C allele is associated with CI (P = 0.017, odds ratio (O.R.) = 1.44; 95% confidence interval (C. I.) = 1.07–1.94). Furthermore, we examined whether the glutathione S-transferase (GST) gene, which is one of detoxification enzyme, influence the risk of CI. GST M1 null genotype increased the relative risk for the CI in the subjects with the CYP1A1 C allele (P = 0.015, O.R. = 1.47; C. I. = 1.08–2.00). We conclude that T6235C CYP1A1 polymorphism is a risk factor for the development of CI and suggest that GST polymorphism contribute to the odds of CI.     

DRB1*1502-DQB1*0601-DQA1*0103 and DRB1*04-DQB1*0302 in Jewish hypersomnolent patients

OBJECTIVES: Narcolepsy is a sleep disorder with a genetic association with the haplotype DRB1*1501, DQA1*0102, DQB1*0602. This haplotype has been described in different ethnic groups suffering from narcolepsy (Japanese, Caucasian, African Americans, Jews). In a recent study we have found the haplotype DRB1*1502, DQB1*0601, DQA1*0103 in three patients with hypersomnolence. The similarity of this haplotype to the narcoleptic haplotype DRB1*1501, DQB1*0602 and DQA1*0102 has raised the question of whether this haplotype is a marker for sleepiness, or rather indicates a variant of non-cataplectic narcolepsy. This study was conducted to further investigate this question. METHODS: HLA-DNA analysis was carried out in 20 healthy Jewish patients (age 23.9+/-6.3 years; 13 Ashkenazi, seven non-Ashkenazi) who had objective measures of hypersomnolence. All underwent whole-night polysomnography, multiple sleep latency test and tissue typing. RESULTS: HLA-DNA analysis revealed HLA-DR2 in eight patients of whom five (25%) carried the haplotype DRB1*1502, DQB1*0601, DQA1*0103 (vs. 1.4% in the Israeli population, P<0.0001). Six patients were diagnosed as non-cataplectic narcoleptics. Five of them carried the haplotype DRB1*1502, DQB1*0601, DQA1*0103. Forty percent of the patients carried the haplotype DRB1*04, DQB1*0302, which was not statistically different from its prevalence in the healthy Israeli population (25%). CONCLUSIONS: This is the first report describing the haplotype DRB1*1502, DQB1*0601, DQA1*0103 in narcoleptic patients (non-cataplectic). This haplotype is close but different from the already known narcoleptic haplotype DRB1*1501, DQA1*0102, DQB1*0602. We assume that this haplotype represents a variant of non-cataplectic narcolepsy rather than association with hypersomnolence. However, in order to conclude whether this haplotype is a marker for the lack of cataplexy, or represents a variant of non-cataplectic narcolepsy, a larger group of patients should be investigated.     

IL-1Rrp2 expression and IL-1F9 (IL-1H1) actions in brain cells

The recently discovered IL-1F9 (IL-1H1) is a putative member of the interleukin (IL)-1 family of cytokines that has been shown to activate nuclear factor-kappa B (NFkappaB) in Jurkat cells transfected with the orphan receptor IL-1 receptor-related protein (IL-1Rrp)2. The aim of the present study was therefore to investigate expression of IL-1Rrp2 and to determine if IL-1F9 induces known IL-1 signaling pathways in the different cell types of the mouse brain in culture. Messenger RNA for IL-1Rrp2 was not detected in primary neurones by RT-PCR, but significant constitutive expression was found in mixed glial cells, particularly in astrocytes and microglia, which was strongly decreased by exposure to bacterial lipopolysaccharide (LPS). LPS induced the release of IL-6, and activated NFkappaB and the mitogen-activated protein kinases (MAPKs) p38, extracellular signal-regulated protein kinase (ERK1/2) and c-Jun N-terminal kinase (JNK) in microglial cultures. IL-1beta induced release of IL-6 and activated NFkappaB, p38, JNK and ERK1/2 in mixed glial cultures, which was completely abolished in the presence of IL-1 receptor antagonist (IL-1ra). When injected intracerebroventrically in the rat, IL-1beta increased core body temperature, and reduced body weight and food intake. In contrast, IL-1F9 failed to induce any of these responses either in vivo or in vitro. These results demonstrate that glial cells may be a target for the new ligand IL-1F9, since high expression of IL-1Rrp2 mRNA was detected in these cells. However, IL-1F9 failed to induce any of the classical IL-1beta responses, suggesting that it may trigger alternative pathway(s).     

Polycystin-1 can interact with homer 1/Vesl-1 in postnatal hippocampal neurons

Polycystin-1 (PC-1) has been identified as critical to development of the nervous system, but the significance of PC-1 expression in neurons remains undefined, and little is known of its roles outside the kidney, where mutation results in autosomal dominant polycystic kidney disease (ADPKD). In kidney, PC-1 interacts with cadherins, catenins, and its cognate calcium channel polycystin-2 (PC-2), which in turn interacts with a number of actin-regulatory proteins. Because some of the proteins that interact with PC-1 in kidney also participate in synaptic remodeling and plasticity in the hippocampus, we decided to test PC-1's potential to interact with a recently discovered type of plasticity-associated protein (homer 1a/Vesl-1S) in postnatal mouse hippocampus. Homer 1a/Vesl-1S is an activity-induced protein believed to participate in synaptic remodeling/plasticity responses to temporal lobe seizure and learning. Here we report the following. 1) PC-1 contains a homer-binding motif (PPxxF), which lies within its purported cytoplasmic domain. 2) Immunoreactivity for PC-1 (PC-1-ir) is highly colocalized with homer 1a immunoreactivity (H1a-ir) in primary cultured hippocampal neurons. 3) PC-1-ir and H1a-ir are present and appear to be colocalized in mouse hippocampus but not cortex on postnatal day 2 (P2), when higher frequencies of spontaneous activity are normal for hippocampus compared with cortex. 4) An endogenous PC-1-ir band with the correct size for the reported C-terminal G-protein-sensitive domain cleavage product of PC-1 (approximately 150 kDa) coimmunoprecipitates with endogenous homer 1/Vesl-1 proteins from mouse brain, suggesting that PC-1 can interact with homer 1/Vesl-1 proteins in postnatal hippocampal neurons.