环磷酰胺致神经管畸形与细胞凋亡的关系

有研究证明环磷酰胺 (ＣＰ)有强烈地致神经管畸形(ＮＴＤ)和骨骼发育异常等作用 ;亦有学者对其ＮＴＤ的发生率和致畸原因作了初步探讨[1～ 3] 。但尚未见有关ＮＴＤ与细胞凋亡相关的研究报道。本研究利用光镜和透射电镜观察了ＣＰ致ＮＴＤ与神经上皮细胞凋亡的关系 ,从形态学方面阐明ＣＰ致ＮＴＤ的原因。1　材料与方法1 1　动物分组及处理　选用未孕ＳＤ大鼠 ,雌性 30只 ,雄性15只 (山东医科大学实验动物中心提供 ) ,体重 180～ 2 5 0 ｇ ,颗粒饲料喂养 ,普通饮水。雌、雄按 2∶1比例合笼过夜 ,次日晨作阴道涂片 ,查出精子者为受精第 0天。…     

全胚胎培养在环磷酰胺致畸与细胞凋亡研究中的应用

哺乳动物植入后全胚胎培养排除了母体等因素对胚胎发育的影响 ,并可人为控制实验条件 ,与组织、细胞培养及动物整体实验相比 ,具有许多明显的优点 ,可动态观察和研究药物致畸作用及机制。细胞凋亡是多细胞生物体内环境稳定和发育精确控制的基本条件 ,也是胚胎发生和发育过程中一定结构和功能器官形成的必要条件 ,过多的细胞凋亡或不适当的细胞凋亡最终导致先天性畸形的发生[1 ] 。本研究利用小鼠全胚胎培养方法观察了抗肿瘤药物环磷酰胺致畸作用 ,在获得畸形胚胎的基础上 ,对环磷酰胺致畸作用与细胞凋亡的关系进行了探讨。1　材料与方法1 1…     

利用小鼠全胚胎培养研究环磷酰胺致畸作用

本文应用小鼠全胚胎培养技术 ,通过妊娠 d8分别 ip5,1 0 ,1 5和 2 0 mg·kg-1环磷酰胺 ( CP) ,研究了该药对器官原基形成期胚胎的致畸作用 ,并对其致畸机理作了初步探索 .给药 4h后取胚胎进行培养 ,于 d 1 0 .5收获胚胎 ,测量其卵黄囊直径 ,头长及颅臀长并记录其大体形态的变化 .结果表明 ,1 5mg· kg-1组尾畸形率最高 ;2 0 mg· kg-1组生长迟缓率最高 .电镜观察所显示的细胞凋亡的形态学变化及 DNA琼脂糖凝胶电泳的结果均提示 CP致畸作用可能与其诱导的细胞凋亡有关 .     

三羟异黄酮对环磷酰胺致神经细胞早期凋亡的干预作用研究

目的观察三羟异黄酮对神经细胞早期凋亡的影响并对其可能机制进行探讨。方法在培养48h的神经细胞中加入高中低3个剂量的三羟异黄酮,30min后加入环磷酰胺8mg/L诱导其发生凋亡,在凋亡的早期阶段以流式细胞仪和扫描电镜进行检测观察,并与环磷酰胺单独给药组作比较。结果譹流式细胞仪检测结果提示三羟异黄酮在一定浓度下可使神经细胞凋亡发生率发生显著的下降,呈现明显的抑制作用;譺扫描电镜观察发现三羟异黄酮可抑制早期凋亡的神经细胞膜改变的程度,与流式细胞仪检测结果一致。结论一定剂量的三羟异黄酮可抑制环磷酰胺致神经细胞凋亡,对神经细胞具有较好的保护性作用。     

金纳多对大鼠急性脊髓损伤后神经细胞的影响

目的探讨银杏叶提取物(金纳多)对脊髓损伤大鼠损伤区凋亡细胞的作用。方法60只大鼠分正常组、损伤组和药物组,用Allen's改良法建立脊髓损伤模型,腹腔注射金纳多,损伤后1,3,7,14和21d取样,同时对大鼠损伤后双下肢功能作评估,对损伤区脊髓HE染色进行形态学观察,并用TUNEL法检测细胞凋亡。结果损伤组开始有片状出血,以后逐渐减少,7d全部消失。损伤组和药物组中均有凋亡细胞,损伤组凋亡率大于药物组。结论金纳多能抑制或保护大鼠脊髓损伤后神经细胞的凋亡,从而减轻脊髓损伤后的继发性损害。     

酒精对丙烯腈致神经细胞畸形及凋亡的影响

饮酒是许多人的一种生活嗜好。有报道认为酒精有辅助致癌作用 ,可使一些致死原因 (包括毒物 )的危险性增加[1] 。丙烯腈是重要的化工原料 ,也是高毒性化学物 ,是致突变剂和潜在的人类致癌剂。本实验用电镜观察了亚急性口服酒精和丙烯腈大鼠的脑组织 ,研究酒精对丙烯腈致脑微循环及神经细胞凋亡和畸形的影响 ,旨在探讨酒精对毒物效应尤其是对致突变剂和可疑致癌物毒效应的影响。1　材料与方法1 1　动物模型　Ｓｐｒａｑｕｅ Ｄａｗｌｅｙ大鼠 12只 ,体重 (2 85±71 8) ｇ(上海市实验动物中心提供 ) ,雌雄各半 ,随机分丙烯腈组和丙烯腈加酒精…     

甲氨蝶呤联合环磷酰胺对胶原诱导性关节炎大鼠滑膜细胞凋亡作用的研究

目的通过对类风湿关节炎(RA)动物模型胶原诱导性关节炎(CIA)大鼠的实验研究,从滑膜细胞凋亡的角度,探讨甲氨蝶呤(MTX)联合环磷酰胺(CTX)的协同作用和机制。方法建立Ⅱ型胶原诱导性雌性Wistar大鼠CIA模型,将造模成功的60只大鼠随机分成4组:CIA模型对照组、小剂量MTX治疗组(MTX0.9mg/kg,每周1次)、小剂量CTX治疗组(24mg/kg,每3周1次)及小剂量MTX联合小剂量CTX治疗组(0.9mg/kg,每周1次,CTX24mg/kg,每3周1次);再选8只为正常对照组。治疗24周后全部动物处死取材,再经固定、脱钙、包埋,通过TUNEL法检测滑膜细胞凋亡。结果各治疗组滑膜细胞凋亡均较CIA模型组增加,联合治疗组凋亡程度最高,各组间平均灰度值差异有统计学意义(P<0.05)。结论提示MTX和CTX联合治疗RA为协同作用。     

雌激素影响急性大鼠脊髓损伤后细胞凋亡的相关性实验研究

目的 在建立大鼠脊髓损伤模型基础上,观察不同时间点雌激素对脊髓胶质细胞,神经元凋亡的影响,以期为临床急性脊髓损伤的治疗提供理论依据.方法 健康成年大鼠72只,随机分为两组,其中单纯损伤组(单纯打击损伤)36只,雌激素组(手术加雌激素)36只.脊髓损伤动物模型的制备:用自制Allen打击器25gcm致伤力撞击脊髓,制成脊髓损伤动物模型.雌激素组每日肌注雌激素(100μg/kg)直至处死,单纯损伤组仍每日肌注生理盐水0.5ml直至处死.组织切片的制备:脊髓损伤后1d、3d、5d、8d、14d、21d共6个时间段分批处死动物.4%多聚甲醛心肌灌注固定24h,切取每只大鼠T5～T13节段脊髓组织,常规制成切片,给于Bcl-2检测,TUNEL原位末端标记.检测大鼠脊髓损伤后细胞凋亡的情况.脊髓损伤后2周给予Gale评分及斜板维持试验.结果 Bcl-2蛋白检测:在脊髓损伤后的第1天,脊髓组织即开始较高表达Bcl-2蛋白.单纯损伤组Bcl-2高峰发生在损伤后3天,而雌激素组伤后8天达到高峰,此时Bcl-2蛋白不仅表达在神经细胞中,更多的在胶质细胞中大量表达,且此状态一直维持到伤后14天才开始下降,伤后21天仅少量表达(P＜0.05).TUNEL原位标记检测:单纯损伤组24小时已出现不少阳性细胞,以胶质细胞为主,3～8天达到高峰,此后逐渐回落,但21天时仍有阳性细胞,应用雌激素治疗后,凋亡细胞明显减少(P＜0.05).脊髓损伤后2周Gale评分及斜板维持率雌激素组优于单纯损伤组(P＜0.01).结论 雌激素在脊髓损伤中通过改善微循环,促进Bcl-2蛋白的表达,清除氧自由基,抗氧化作用能够抑制脊髓损伤早期神经细胞和胶质细胞的凋亡,从而减轻脊髓继发性损伤,促进脊髓神经功能的恢复.所以脊髓损伤后早期应用雌激素配合其它药物阻止神经细胞和胶质细胞的凋亡可能有助于减轻脊髓损伤的损害程度,促进脊髓神经功能的恢复,为SCI的治疗提供一个新的途径.     

Exendin-4 对大鼠脊髓损伤后氧化应激和神经细胞凋亡的影响

目的研究Exendin-4 对大鼠脊髓损伤后氧化应激和神经细胞凋亡的作用。方法成年雄性SD 大鼠36 只（体质量200~250 g）随机分为3 组（n=12）：假手术组（Sham 组）、单纯脊髓损伤组（SCI 组）、Exendin-4 组（Ex-4 组）。Sham 组只暴露脊髓,不打击;SCI 组和Ex-4 组均采用Allen′s 重物打击法制作脊髓损伤模型;Ex-4 组在脊髓损伤后立即腹腔内注射Exendin-4 溶液,剂量10 μg/只;SCI 组和Sham 组均注射等体积生理盐水。在24 h 后检测各组脊髓组织中丙二醛（MDA）含量和过氧化氢酶（CAT）活性,TUNEL 法检测神经细胞凋亡情况,Western blot 检测 caspase-9 和凋亡诱导因子（AIF）的表达情况。结果与Sham 组相比,SCI 组脊髓组织中的MDA 含量明显增加, caspase-9 和AIF 表达水平明显增加,神经细胞凋亡比例明显升高,CAT 活性明显降低（P < 0.01）。与SCI 组相比, Ex-4 组脊髓组织中MDA 含量明显降低,caspase-9 和AIF 表达水平明显降低,神经细胞凋亡比例明显降低,CAT 活性明显升高（P < 0.01）。结论大鼠脊髓损伤后,Exendin-4 可通过减轻氧化损伤抑制神经细胞凋亡。     

Hematoprotective effect of seleno-L-methionine on cyclophosphamide toxicity in rats

Cyclophosphamide (CP) is a widely used antineoplastic drug that causes toxicity in the normal cell due to its metabolites. The major drawback of this drug is an undesirable myelosuppression. Selenium (Se) is a potent nutritional antioxidant that carries out biological effects by its incorporation into selenoproteins, such as glutathione peroxidase (GPx). The possible protective effects of seleno-l-methionine (SLM) against CP-related toxicity of blood cells and bone marrow of rats were investigated in this study. Intraperitoneal (i.p) administration of 50, 100, or 150?mg/kg of CP caused, in a dose-dependent manner, reductions in the number of leukocytes (78, 89, and 92%, respectively), thrombocytes (22, 33, and 52%, respectively), and bone marrow–nucleated cells (72, 90, and 94%, respectively). The groups that had CP treatment alone were killed 3 days after the CP injection. For the groups having CP+SLM, SLM (0.4 or 0.8?mg/kg i.p) administration was started 3 days earlier than the CP administration and continued to the end of the experiment (6 days). On day 4, the animals were weighed again, relative doses of CP were estimated, and CP+SLM was administered together. On day 7, blood samples were collected and bone marrow of animals were resected under anesthesia. The results indicated that treatment of rats within a select dose range of SLM could reduce CP-induced toxicity on blood cells and bone marrow.     

Protective effect of lipoic acid on micronuclei induction by cyclophosphamide

The present study investigated the protective efficacy of DL-α-lipoic acid on the cyclophosphamide (CP)-induced clastogenicity using the in vivo micronucleus assay. Male Wistar rats of 140 ± 20 g were categorized into eight groups. Five groups were administered CP (40 mg/kg body weight, intraperitonealy) to induce genotoxicity; four of these groups received a single intraperitoneal injection of lipoic acid at a dose of either 100 or 200 mg/kg body weight, and either 30 or 60 min prior to CP administration. A vehicle-treated control group and lipoic acid control groups were also included. The number of micronucleated polychromatic erythrocytes (MNPCEs) was determined at 24 h after CP administration. In rats injected with CP, the frequency of MNPCEs in bone marrow and peripheral blood was increased significantly in comparison with the controls, and in rats treated with lipoic acid and CP, the number of MNPCEs was decreased significantly in comparison to those given CP alone. The chemoprotective effect was found to be stronger after the administration of lipoic acid at a dose of 200 mg/kg body weight than 100 mg/kg body weight dosage, indicating the dose-dependent protective effect of lipoic acid. However, the protection by lipoic acid was not dependent on the time intervals between lipoic acid and CP administration. Our results illustrate the protective effect of lipoic acid on the in vivo clastogenicity induced by CP.     

Effects of cyclophosphamide on the phenotypes and functions of THP-1 cells

Early and accurate evaluation of immunotoxicity is crucial. However, there are few in vitro models for immunosuppressive evaluation. THP-1 cells has long been used for in vitro sensitivity evaluation. Whether it can be used for immunosuppressive evaluation remains unclear. In this study, effects of immunosuppressant cyclophosphamide (CY) on THP-1 cells were observed while 2, 4-Dinitrochlorobenzene (DNCB) was used as a control. The phenotypes of THP-1 cells, the ability to activate naïve T cells, intracellular reactive oxygen species (ROS) level, gene markers, phagocytic ability and cell apoptosis were detected after THP-1 cells being exposed to different concentrations of CY and DNCB. Both CY and DNCB were able to activate THP-1 cells, but there were a lot of differences in their effects on THP-1 cells, such as the changes in phenotypes, in the ability to activate naïve T cells, in ROS production and in marker gene expression. Firstly, CY down-regulated the expression of CD86 on THP-1 cells while DNCB up-regulated its expression. Secondly, the ability of THP-1 cells to activate naïve T cells was enhanced by CY and suppressed by DNCB. Thirdly, CY raised rapid and transient elevation of ROS level in THP-1 cells, while the effects of DNCB were slower and longer-lasting. Finally, only CY could lead to an increase in heme oxygenase 1 (HMOX1) expression. Taken all these results into account, we suggested that THP-1 cell line possesses the potency to be an in vitro model of immunosuppressive evaluation. And the surface molecule CD86, the ability to activate naïve T cells, the ROS production and the gene marker HMOX1 of THP-1 cells are promising markers.     

Nuclear aberrations in hair follicle cells of patients receiving cyclophosphamide

The toxic effect of cyclophosphamide on the proliferative cell population of hair follicles plucked from the human scalp was examined by the in vivo nuclear aberration assay. Patients participating in an independent clinical trial received oral low dose cyclophosphamide, intravenous high dose cyclophosphamide or oral placebo treatment. The percent of cells with nuclear aberrations (indicating apoptosis, a special form of cell death) and the percent of mitotic cells, in the hair matrix, were calculated for each patient before treatment and at several time points following cyclophosphamide or placebo treatment. The mean percentages of nuclear aberrations in both the treated Low dose and High dose cyclophosphamide patients were significantly higher than those for the pre-treatment and Placebo patients. The nuclear aberrations in hair follicle cells increased from pre-treatment (and Placebo) to treated Low dose and finally to treated High dose patients. The average percentage for pre-treatment samples from all patients was 0.06 ±0.03 SE. For 1 week and 1 month samples from Low dose patients it was 0.35 ±0.08 SE, and for combined 2,3 and 4 day samples from High dose patients it was 1.08 ±0.12 SE. Cyclophosphamide also had a significant effect on mitosis. A decrease in mitotic activity was observed at 1 month following the initial low dose cyclophosphamide treatment and at 24±2 h following each of the first two high dose cyclophosphamide treatments. The observed increase in nuclear aberrations following low dose as well as high dose cyclophosphamide suggests that it is feasible to use the nuclear aberration assay for in vivo human genotoxicity testing, using proliferating hair follicle cells.     

环磷酰胺和紫杉醇联合化疗诱导小鼠Lewis肺癌细胞的凋亡

目的观察小剂量环磷酰胺(Ctx)和紫杉醇(Px)联合化疗对小鼠Lewis肺癌细胞凋亡的影响并探讨其可能的机制。方法建立小鼠Lewis肺癌模型,将38只C57BL/6♂小鼠随机分为4组:生理盐水对照组(A)、Ctx单药组(B)、Px单药组(C)、Ctx Px组(D)。采用免疫组化技术检测各组肿瘤组织中Bcl-2、Bax及Caspase-9的蛋白表达水平。结果D组Bcl-2蛋白表达水平明显低于A、B、C组,D组Bax、Caspase-9蛋白表达水平及Bax/Bcl-2则明显高于A、B、C组。结论小剂量Ctx和Px联合化疗比单用具有更强的促进小鼠Lewis肺癌细胞凋亡的作用,其机制可能与增加Bax/Bcl-2比值和激活Caspase-9等有关。     

短期益赛普与甲氨蝶呤 环磷酰胺联合治疗类风湿关节炎的安全性研究

目的探讨短期使用重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白(rhTNFR:Fc,益塞普)与甲氨蝶呤﹙MTX﹚、环磷酰胺(CTX)联合治疗类风湿关节炎(RA)的安全性。方法将84例患者随机分为实验组和对照组,其中74例患者完成了本研究,实验组给予益赛普皮下注射治疗3个月,每周2次,每次25mg;同时给予MTX治疗,每周1次,每次7.5￣15mg;CTX每3周1次,每次200mg;3个月后给予空白模拟益赛普,继续接受MTX和CTX维持治疗。对照组给予MTX和CTX治疗,剂量同实验组,同时给予益赛普空白模拟对照,两组疗程均为1年。随时观察并记录治疗过程中的任何不良事件,在第0、2、4、8、12、24、36、52周监测其血常规、红细胞沉降率、肝肾功能。结果整个试验过程中未发生严重不良事件,治疗组和对照组不良事件发生率比较,差异无统计学意义(P>0.05)。结论短期益赛普与MTX、CTX联合治疗RA,可以减少益赛普的疗程,并不增加不良反应,具有较好的安全性。     

来氟米特与环磷酰胺治疗狼疮肾炎的对照观察

目的比较来氟米特（LEF）和环磷酰胺（CTX）治疗狼疮肾炎（LN）的疗效、不良反应及安全性。方法43例活动性LN患者在使用激素的基础上随机分为两组,LEF组22例和CTX组21例,LEF组给予LEF20mg／d,半年为一疗程;CTX组间断给予CTX12mg·k^-1·d^-2加入10％葡萄糖注射液250ml中静脉滴注,每2周为一疗程,环磷酰胺总累计量控制在150mg／kg以内,随访6个月,监测血常规、血清白蛋白（Alb）、血肌酐（SCr）、ANA、ds—DNA、血24h尿蛋白定量、尿沉渣红细胞计数和白细胞计数和可能伴随的不良反应。结果治疗总有效率LEF组81．8％,CTX组85．7％,组间差异无统计学意义。不良反应CTX组13例（61．9％）,LEF组3例（13．6％）,组间差异有统计学意义。结论LEF治疗LN疗效与CTX相当,但其不良反应相对较轻,耐受性较好。     

川芎嗪对小鼠环磷酰胺损伤的保护作用

目的：考察川芎嗪对小鼠环磷酰胺损伤的保护作用。方法：通过对小鼠灌胃给药川芎嗪，腹腔注射环磷酰胺，检测血液白细胞，总蛋白，球蛋白水平以及心脏，脑，肝脏组织脂质过氧化物产物丙二醛含量。结果：川芎嗪能够抑制环磷酰胺对血液中白细胞，总蛋白，球蛋白的降低，同时，与模型组比较，明显降低心脏，脑，肝脏组织丙二醛含量。结论：川芎嗪对小鼠环磷酰胺损伤有一定保护作用。     

周期联合甲氨蝶呤 环磷酰胺对类风湿关节炎患者外周血淋巴细胞周期及凋亡的影响

目的探讨甲氨蝶呤(MTX)和环磷酰胺(CTX)及两者联合治疗对类风湿关节炎(RA)患者外周血淋巴细胞(PBL)周期及凋亡的影响。方法入选活动期RA患者30例,随机分为3组(MTX组、CTX组及MTX+CTX联合组),应用流式细胞术检测MTX、CTX及联合治疗前后PBL周期及凋亡的变化,同时对10名健康志愿者PBL的周期及凋亡情况进行检测。结果活动期RA患者基线PBL各期细胞比例与正常人相比,G0/G1期细胞比例低于正常对照组,S期细胞比例高于正常对照组,差异均有统计学意义;基线早期凋亡率与正常人相比差异无统计学意义。MTX组患者用药后较用药前G0/G1期细胞比例增加,S期细胞比例减少,PBL早期凋亡率明显增加,差异均有统计学意义。CTX组患者用药前后PBL各期细胞比例差异无统计学意义;用药后较用药前PBL早期凋亡率明显增加,差异有统计学意义。MTX+CTX联合组用药后较用药前PBLG0/G1期细胞比例增加,S期细胞比例减少,早期凋亡率较MTX组、CTX组明显增加,差异均有统计学意义。MTX组及MTX+CTX联合组用药前后外周血淋巴细胞G0/G1期细胞比例变化与早期凋亡率变化之间呈显著正相关。结论MTX+CTX联合治疗能更加有效地诱导阻滞于G0/G1期的PBL发生凋亡。