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。     

人参多糖注射液体外诱导人白血病细胞株K562增殖抑制及分化

背景：既往研究表明,人参多糖既能促进正常血细胞生成,又能抑制白血病细胞的增殖,但这种双向调控的机制尚不清楚。目的：体外观察人参多糖注射液对人白血病细胞株K562增殖抑制及诱导分化的影响。方法：K562细胞由重庆医科大学临床检验系提供,人参多糖注射液为山西普德药业有限公司生产。取对数生长期的K562细胞,调整浓度为7×108 L-1。对照组予以常规培养;人参多糖组分别加入25,50,100,200,400,600,800 mg/L的人参多糖注射液。MTT比色法检测人参多糖注射液对K562细胞增殖情况的影响;血红蛋白测定及联苯胺、Wright’s染色检测K562细胞向红系细胞分化的特征;流式细胞仪测定细胞凋亡情况。结果与结论：人参多糖在体外对 K562细胞的增殖有明显抑制作用,并能诱导K562细胞向红系细胞分化,表现为K562细胞的增殖受到抑制。K562细胞形态学上可见细胞体积缩小,核直径减小,胞浆丰富,核浆比例降低;人参多糖在体外对K562细胞有诱导血红蛋白生成的作用,在100~800 mg/L范围内,呈浓度依赖性,且均在作用48 h时抑制率达高峰;细胞凋亡率增加。提示人参多糖注射液能抑制K562细胞增殖,诱导其凋亡,并使K562细胞向红系细胞方向分化。     

不同浓度人参皂甙Rd对次声性脑损害的保护

目的观察次声对大鼠记忆功能的影响及不同剂量人参皂甙Rd对其脑损害的治疗作用。方法通过Y型电迷宫训练将成绩相近的SD大鼠随机分为5个组，除正常对照组以外均接受16Hz 130dB的次声作用gh／d。3个药物组在次声作用前3d开始分别给予不同剂量（30mg／kg、10mg／kg、2mg／kg）的人参皂甙Rd。次声作用7d后再次评定每组大鼠的迷宫成绩，并用单链DNA（Single-stranded DNA，ssDNA）免疫标记法检测海马内ssDNA（凋亡细胞）数。结果与正常对照组相比单纯次声组大鼠学习记忆功能下降、标记的ssDNA阳细胞数明显增多（P〈0．05）；与单纯次声组相比人参皂甙（30mg/kg、10mg／kg组）有明显的保护作用，减轻了学习记忆功能下降，ssDNA阳性细胞数明显减少（P〈0．05）。结论16Hz 130dB次声可引发大鼠海马损伤、细胞凋亡、记忆功能减退，人参皂甙可明显减轻这些损害。     

人参皂甙Rg3-聚乳酸纳米粒诱导U87-MG凋亡和Caspase-3的表达

目的研究人参皂甙Rg3-聚乳酸纳米粒对U87胶质瘤细胞的作用。方法采用modified-SESD法制备人参皂甙Rg3-聚乳酸(PLA)纳米粒,采用四甲偶氮唑盐微量酶反应比色法(MTT),流式细胞技术(FCM)和Western blot研究人参皂甙Rg3-聚乳酸纳米粒对U87胶质瘤细胞周期和凋亡的影响。结果 Rg3-聚乳酸纳米粒抗肿瘤增殖作用呈剂量依赖性,人参皂甙Rg3-聚乳酸纳米粒通过与细胞核中DNA作用改变细胞生长的周期,造成在G0-G1期阻滞,引起细胞凋亡。结论表明人参皂甙Rg3-聚乳酸纳米粒在胶质瘤的综合治疗方面有广阔的应用前景。     

人参皂甙Rd对兔脊髓缺血性损伤保护作用的量效关系研究

目的探讨人参皂甙Rd对脊髓缺血性损伤保护作用的剂量效应关系。方法 40只雄性新西兰大白兔,随机分为5组（n=8）,采用肾下主动脉阻断法造成脊髓缺血（20min）。对照组：即单纯缺血再灌注组;保护组：即Rd-5组、Rd-10组、Rd-20组和Rd-40组,分别在缺血前1h从耳缘静脉注射人参皂甙Rd5mg/kg、10mg/kg、20mg/kg、40mg/kg;术后观察神经功能变化并记录再灌注4h、8h、12h、24h和48h神经功能学评分,再灌注48h处死动物后取脊髓（L5～7）标本行病理学观察。结果所有动物均存活,再灌注后48h,Rd-5组神经功能学评分和脊髓前角正常运动神经元计数与对照组相比均无显著性差异（P〉0.05）;Rd-10组神经功能学评分与对照组相比无显著性差异（P〉0.05）,但脊髓前角正常运动神经元计数明显高于对照组（P〈0.05）;Rd-20组、Rd-40组神经功能学评分和脊髓前角正常运动神经元计数均明显高于对照组（P〈0.01）,但这二组之间无显著性差异（P〉0.05）。且每组兔神经功能学评分与其对应脊髓前角正常神经元计数之间有显著相关性（r=0.769.P〈0.01）。结论人参皂甙Rd对脊髓缺血性损伤有保护作用,且呈一定的剂量效应关系。     

大鼠局灶性脑缺血时人参皂甙Rbl抑制细胞凋亡和调控凋亡基因的表达

目的 研究人参皂甙Rbl(Ginsenoside Rb1,GRb1)对大鼠脑缺血再灌注时神经细胞凋亡及神经细胞凋亡抑制蛋白(NAIP)、Bcl-2和Bax蛋白表达的影响,探讨人参皂甙Rbl的神经保护作用机制.方法 阻塞Wistar大鼠大脑中动脉制备短暂性脑缺血模型,出现神经功能缺失症状的大鼠随机分为缺血组和GRb1组,GRb1组大鼠在再灌注后立即腹腔注射人参皂甙Rb1(40mg/kg).每组按不同的再灌注时间(3h、12h、1d、2d、3d、5d和10d,每时间点4只)分为7个亚组.分别用原位未端标记法和免疫组织化学方法观察凋亡细胞、神经元凋亡抑制蛋白(NAIP)、Bcl-2和Bax的表达.结果 与缺血组相比,GRb1组的各亚组凋亡细胞数下降,但只在再灌注12h～3d时有显著差异;GRb1组的NAIP阳性细胞数在再灌注12h～10d时明显高于缺血组;GRb1组的Bcl-2阳性细胞数在再灌注12h～10d时显著上升,Bax阳性细胞数则在相同时间点下降.结论 人参皂甙Rbl通过促进NAIP、Bcl-2表达和抑制Bax表达发挥神经保护作用.     

人参皂甙、CD3与IL-2协同诱导PBMC治疗恶性脑胶质瘤的实验研究

目的　提高人脑胶质瘤过继免疫治疗的效果 ,探索治疗脑胶质瘤的新途径。方法　用人参皂甙 (GS)、抗CD3单抗 (CD3 )和 IL-2共同诱导人外周血单个核细胞 (PBMC) ,诱导、扩增新型抗胶质瘤效应细胞 GS-CD3 AK细胞 ,并与 CD3 AK细胞在某些生物学方面进行了比较。结果　两组效应细胞增殖曲线均于第 6天达高峰 ,峰值可见 GS-CD3 AK细胞 >CD3 AK细胞 (P <0 .0 5 ) ;GS-CD3 AK细胞扩增倍数明显高于 CD3 AK细胞 (P <0 .0 5 ) ;两组效应细胞于培养的第 6天所测得的杀伤恶性脑胶质瘤细胞 BT3 2 5活性可见 GS-CD3 AK细胞 >CD3 AK细胞 (P <0 .0 5 )。结论　GS、CD3和 IL-2具有协同增强作用 ,使 GS-CD3 AK细胞成为较 CD3 AK细胞增殖能力、杀伤活性更强的免疫效应细胞 ,且 IL-2用量减少 ,为胶质瘤的过继免疫治疗打下了理论基础。     

microRNA-183通过下调Bcl-2诱导人神经母细胞瘤细胞株SK-N-SH细胞凋亡的研究

目的研究microRNA-183对神经母细胞瘤细胞的调控作用,为神经母细胞瘤的治疗提供新策略。方法购买microRNA-183和对照microRNA,转染至人神经母细胞瘤细胞株SK-N-SH细胞,再使用MTT实验,Caspase-3活性测定试剂盒和流式检测细胞生长和凋亡的影响。合成Bcl-2siRNA,检测Bcl-2对人神经母细胞瘤细胞株SK-N-SH细胞凋亡的影响。转染Bcl-2质粒后,再转染microRNA-183至人神经母细胞瘤细胞株SK-N-SH细胞,分析Bcl-2的表达水平和人神经母细胞瘤细胞株SK-N-SH细胞的凋亡。结果转染microRNA-183降低SK-N-SH细胞的生长(P=0.005 9),发生磷脂酰丝氨酸膜表面表达(P=0.008)和Caspase-3的激活(P=0.014),Bcl-2的表达降低(P=0.015)。干扰SK-N-SH细胞中Bcl-2增强了microRNA-183诱导的细胞凋亡(P=0.005 8),而过表达Bcl-2抑制了microRNA-183诱导的细胞凋亡(P=0.007 3)。结论转染microRNA-183抑制SK-N-SH细胞的生长和增殖。microRNA-183通过下调Bcl-2而诱导SK-N-SH细胞的凋亡,提示Bcl-2可能是神经母细胞瘤潜在的候选治疗靶点。     

过氧化氢体外诱导人血管内皮细胞损伤与人参皂苷Ｒｂ１的保护效应★

目的: 内皮细胞损伤可以导致心血管疾病及经皮冠状动脉介入术后再狭窄的发生。实验以过氧化氢（H2O2）体外诱导的人脐静脉损伤内皮细胞为对象，观察人参皂苷Rb1的保护作用，并探讨其可能的作用途径。 方法：实验于2006-09/11在扬州大学医学院中西医结合肿瘤研究所完成。①实验材料：人脐静脉血管内皮细胞（武汉大学培养物保存中心）；人参皂苷Rb1(含量>95%，HPLC；由吉林大学化学学院提供)。②实验过程及分组：在体外培养的人脐静脉内皮细胞上建立H2O2损伤模型，实验分为正常对照组； H2O2损伤对照组：在培养基中加入2 mmol/L的H2O2诱导损伤4 h；人参皂苷Rb1 50，100，200 μmol/L组：分别在培养基中先加入终浓度为50，100，200 μmol/L的人参皂苷Rb1孵育24 h，再加入相同的H2O2诱导损伤。试验重复6次。③实验评估：采用四甲基偶氮唑盐比色法测定各组细胞活力；硫代巴比妥酸法测定丙二醛含量；黄嘌呤氧化酶法测定超氧化物歧化酶活性；酶联免疫吸附法检测血管内皮生长因子蛋白表达。 结果：①与损伤对照组比较，不同剂量组人参皂苷Rb1可以提高H2O2诱导损伤的人脐静脉内皮细胞活性（P < 0.01），并随着浓度的增高，细胞活力（A值）也增高，呈一定的剂量相关性。②与损伤对照组比较，不同剂量组人参皂苷Rb1可以降低H2O2诱导损伤的人脐静脉内皮细胞丙二醛含量、增加超氧化物歧化酶活性，随着浓度的增高，丙二醛含量降低，超氧化物歧化酶活性升高呈一定的剂量相关性。③与损伤对照组比较，不同剂量组人参皂苷Rb1可以促进H2O2诱导损伤的人脐静脉内皮细胞血管内皮生长因子蛋白的分泌，且随着人参皂苷Rb1浓度的增高，血管内皮生长因子蛋白表达也增高，呈一定的剂量相关性。 结论：人参皂苷Rb1对氧化损伤的内皮细胞具有保护作用，其作用途径可能与保护了细胞的线粒体，提高了该细胞的抗氧化酶活性，上调了血管内皮生长因子的表达有关。     

ShRNA-AKT2对人脑胶质瘤U87细胞株增殖及凋亡的影响

目的研究通过慢病毒载体靶向抑制丝/苏氨酸蛋白激酶2(AKT2)基因表达对胶质瘤细胞株U87增殖、凋亡的影响。方法利用慢病毒介导的短发夹RNA(shRNA)干扰载体感染U87细胞株,逆转录酶-聚合酶链式反应(RT-PCR)和蛋白印迹技术(Western blot)分别检测转染后细胞株AKT2 mRNA和蛋白表达水平变化,四唑盐(MTT)法检测细胞增殖的影响,流式细胞术检测细胞凋亡率、细胞周期改变。结果转染AKT2-shRNA可有效降低U87细胞株内的AKT2表达。干扰组细胞从第3 d开始增殖明显降低(P0.05);AKT2-shRNA干扰组细胞的凋亡率为11.80%±1.83%,明显高于阴性对照组的2.01%±0.20%和未转染组的2.13%±0.32%(P0.05);AKT2-shRNA干扰组细胞周期S期细胞百分比显著低于对照组,而G0/G1期细胞比分比显著高于对照组(P0.05)。结论 AKT2-shRNA可抑制胶质瘤细胞株的增殖,并导致肿瘤细胞凋亡增加。     

Dose- and time-dependent scopolamine-induced recovery of an inhibitory avoidance response after its extinction in rats

The present investigation was aimed at elucidating the dose and time dependency of scopolamine-induced recovery of inhibitory avoidance after its extinction. Two experiments were conducted: in the first, we analyzed the effects of four doses (1, 2, 4, and 8 mg/kg) of the musacrinic receptor antagonist scopolamine, on the expression of this conditioned response once it had been extinguished. Independent groups of rats were trained in a one-trial, step-through inhibitory avoidance task and submitted to daily retention (extinction) tests. After extinction had occurred, animals were injected intraperitoneally 10 min before retention testing, either with saline or scopolamine. Results show that scopolamine produced a dose-dependent recovery of the avoidance response. The second experiment was carried out in the same animals, which were now tested for retention of inhibitory avoidance at 1, 2, 3, 6, and 9 months after completion of the first experiment. All rats received counterbalanced injections of saline or scopolamine 10 min before testing at each time interval. Reliable recovery of the avoidance response was observed at the 1-month interval with a clear dose dependency while, after the second month, only the groups treated with the two higher doses continued responding. The results indicate that recovery of the extinguished response produced by muscarinic blockade follows dose- and time-dependent curves, and can be achieved long after a single training session. These data suggest that the inhibitory avoidance memory trace is retained in the brain after behavioural extinction of this response, thus supporting the view of extinction as new learning that affects the retrieval of the original memory, but does not modify its storage.     

Dose- and time-dependent, context-induced elevation of dopamine and its metabolites in the nucleus accumbens of morphine-induced CPP rats

The dopamine (DA) projections from the ventral tegmental area to the nucleus accumbens (NAc) are the key component of the brain reward circuitry. The encoded information by DA in reward-related memory within this circuit during opiate reinforcement requires further clarification. The present study was designed to explore the correlations between morphine dose, retention of morphine-induced conditioned place preference (CPP), morphine-induced changes in levels of DA and its metabolites in the NAc in expression and retention of CPP in Sprague–Dawley male rats. A dose-effect curve for morphine-induced CPP (0.01–10 mg/kg, i.p.) was obtained using 4-day conditioning sessions followed by a CPP test; the retention of morphine CPP was measured with CPP tests after the development of CPP. We found a dose-dependent effect of morphine (from 0.01 to 10.0 mg/kg, i.p.) on both the magnitude and the retention of CPP. During the retention of morphine-induced CPP, a morphine-dose- and time-dependent elevation of DA and its metabolites was observed in the NAc. These changes were absent if the same dose of morphine was injected outside of the conditioning environment (i.e., in the home cage). These results suggest that that the long-lasting elevation of DA and its metabolites in the NAc is attributable mainly to drug-associated context, rather than the residual effect of morphine.     

Dose- and time-dependent selective and non-selective effects of ricin (RCA 120) on rat primary sensory neurons

The distribution of degenerating fibers in the spinal cord was studied in Fink-Heimer-stained sections following treatment of the tibial nerve with ricinus communis agglutinin (RCA 120). The ricin was either injected into the nerve or applied in a capsule on the transected nerve. Short survival times and low doses of ricin resulted in degeneration in somatotopically appropriate parts of the medial dorsal horn. Longer survival times and higher doses resulted in degeneration which progressively expanded into inappropriate areas in the central and lateral parts of the dorsal horn and in deeper laminae regardless of the mode of application. Furthermore, the effect of a ricin injection into the tibial nerve on transganglionic transport of choleragenoid horseradish peroxidase (B-HRP) in the peroneal nerve was studied following a simultaneous or delayed B-HRP injection. A simultaneous ricin and a B-HRP injection resulted in primary afferent HRP labeling in the gray matter, regardless of the dose of ricin. Following a delayed B-HRP injection almost no primary afferent labeling was seen in the gray matter, unless a very low dose of ricin was injected. This study shows that treatment of a peripheral nerve with a high dose of ricin and a long survival time may result in a considerable non-selective degeneration of fibers in the spinal cord. A selective degeneration may, however, be obtained by using lower doses or shorter survival times.     

Dose- and time-dependent action of morphine, tramadol and flupirtine as studied by radioelectroencephalography in the freely behaving rat

The necessity of testing psychoactive drugs in awake freely moving animals has led to the development of a telemetry-based system which enables the pharmacologist to follow centrally active molecules in their time- and dose-dependent effects on electric brain activity in terms of changes in spectral power density of extracellularly recorded field potentials (tele-EEG). This report describes the effect of three analgesics with respect to bioelectric changes in frontal cortex, thalamus, striatum and reticular formation. Two opiate drugs, morphine and tramadol, behaved very similarly despite a tenfold difference in dosage, whereas flupirtine, a nonopiate analgesic, changed the frequency content of the EEG signals in an entirely different manner. The frequency pattern produced by the opiates closely resembles that of centrally acting serotonin uptake inhibitors and thus is consistent with the view of a serotonergic prevalence of neurochemical interactions within the recorded brain areas. In contrast, the action of flupirtine obviously can be attributed to a clonidine-like effect on noradrenergic alpha 2-receptors. The results are discussed with respect to already known influences of these drugs on indoleaminergic and catecholaminergic transmission.     

CDC2敲低治疗人脑胶质瘤的实验研究

目的 提供CDC2作为胶质瘤发生发展相关新分子的实验依据.方法 构建针对目的 基因的shRNA逆转录病毒表达载体;对体外培养的人脑胶质瘤细胞及其裸小鼠移植瘤进行转染,观察与目标基因相关的表型变化;荧光实时定量PCR检测mRNA表达,Western印迹检测蛋白表达;流式细胞仪检测细胞周期和凋亡的变化.结果 shRNA表达载体使人脑胶质瘤细胞株SHG44 CDC2的mRNA、蛋白表达显著下调,同时出现了明显的细胞G2/M期阻滞,细胞生长抑制,凋亡增加.裸小鼠皮下移植瘤明显缩小;肿瘤颅内移植裸小鼠生存期明显延长.结论 CDC2基因过表达是胶质瘤发生发展病因分子之一,敲低其表达可使肿瘤的恶性增殖得到控制.     

载脂蛋白E和Ⅱ型髓系细胞受体在脂多糖诱导的小胶质细胞炎症反应中的作用

目的 探讨载脂蛋白E(ApoE)、Ⅱ型髓系细胞受体(TREM2)在脂多糖(LPS)诱导的小胶质细胞炎症反应中的作用.方法 原代培养野生型小鼠及基因敲除(ApoE-/-、TREM2-/-、ApoE-/-TREM2-/-)小鼠的小胶质细胞.用0 ng/ml、100 ng/ml、500 ng/ml LPS刺激野生型小鼠小胶质...     

依达拉奉对大鼠脑外伤后细胞凋亡率及Bcl-2／Bax表达的影响

目的研究依达拉奉对大鼠脑外伤（TBI）后细胞凋亡率及Bcl-2／Bax表达的影响,探讨其对脑外伤后脑组织损害的保护作用。方法成年SD大鼠36只随机分为假手术组、脑外伤组、依达拉奉组。Allen＇s改良法制作脑外伤的模型;流式细胞仪检测伤侧海马细胞凋亡率的变化,免疫组化法检测海马细胞Bcl-2和Bax蛋白的表达,图象分析仪进行灰度定量分析。结果假手术组没检测到明显的细胞凋亡;脑外伤组检测到较高的细胞凋亡率;依达拉奉组细胞凋亡率较外伤组明显下降。外伤组脑细胞Bcl-2、Bax蛋白表达水平均明显高于假手术组;与外伤组相比,依达拉奉组Bcl-2蛋白表达水平显著增高,Bax蛋白表达水平明显下降。结论依达拉奉可有效抑制大鼠外伤后神经细胞凋亡,此作用可能与其有效清除氧自由基、上调Bcl-2和下调Bax蛋白表达水平有关。     

脑源性神经营养因子对脑梗死大鼠神经细胞凋亡及Bcl-2、Bax蛋白表达的影响

目的 探讨侧脑室内立体定向注射脑源性神经营养因子(BDNF)对脑梗死大鼠神经细胞凋亡及Bcl-2、Bax蛋白表达的影响.方法 32只SD大鼠成功制作大脑中动脉闭塞模型,随机分为BDNF组(n=16)和对照组(n=16),在两组大鼠侧脑室内分别注射10μ1 BDNF溶液(0.5 μmol)和10μl磷酸盐缓冲液(PBS液).注射2周后,2组大鼠分别行神经功能严重性评分(mNSS)及梗死面积测定,应用免疫组化染色、Western blot分析脑组织Bcl-2、Bax蛋白的表达,Tunel检测细胞凋亡.结果 与对照组比较,BDNF组脑梗死面积缩小,神经细胞凋亡数少,Bax蛋白表达低,Bcl-2蛋白表达高,短期内神经功能恢复好,差异均具有统计学意义(P＜0.05).结论 BDNF可能通过调节Bcl-2、Bax蛋白表达,减少神经细胞凋亡,改善脑缺血大鼠的功能.