三肽化合物酪丝缬肽对人肝癌BEL-7402细胞增殖抑制作用的实验研究

目的观察三肽化合物酪丝缬肽(tyroservaltide,YSV)对体外培养人肝癌BEL-7402细胞增殖的抑制作用.方法建立人肝癌BEL-7402及Chang氏肝细胞体外培养体系,用BrdU法、MTT法及LDH法,观察YSV对体外培养的BEL-7402细胞、Chang氏肝细胞DNA分裂指数、MTT代谢率、LDH释放量的影响,用琼脂糖凝胶电泳观察药物对体外培养BEL-7402细胞DNA片段化的影响.结果 YSV对BEL-7402细胞作用48 h、72 h时药物浓度为1 mg·L-1和0.1 mg·L-1组与阴性对照组比较:①能显著抑制肿瘤细胞DNA的合成(P<0.01),抑制率高为药物浓度1 mg·L-1作用72 h时可达32.53%,②能表现出明显抑制细胞代谢的作用(P<0.05)其中YSV 0.1 mg·L-1作用72 h时抑制率高为19.12%,③能增加胞浆内LDH的释放(P<0.05),YSV(1 mg·L-1)作用72 h时抑制率高,为36.13%,④能诱导BEL-7402肝癌细胞DNA片断化成低分子量DNA,经1%琼脂糖电泳后显示为DNA Ladder.对正常肝细胞系Chang氏肝仅0.1 mg·L-1在48 h表现出抑制DNA合成能力的作用.结论 YSV抑制人肝癌BEL-7402细胞的增殖,对正常肝细胞系Chang氏肝没有影响.

人参皂苷对体外培养的缺氧海马神经元的预防保护作用

取新生Wistar大鼠海马神经元体外培养14d,随机分为对照组、尼莫地平5μmol/L组、人参皂苷Rg20.025mmol/L组、Rg20.05mmol/L组.将药物加入到培养液中4h后,连续冲以95%N2+95%CO2混合气体,建立急性缺氧细胞模型.台盼蓝染色细胞计数,吉姆萨染色观察细胞形态并计算其凋亡率,DNA Ladder测定细胞凋亡程度.结果提示,人参皂苷Rg2可显著降低体外培养大鼠缺氧海马神经元的细胞凋亡率而发挥对其保护作用.

Microvascular endothelial cell (MVEC) is one of the target cells of TNFα (TNF effect). The dysfunction of MVEC induced by TNF plays an important role in some cardio-cerebral vascular diseases. ① Cell proliferation kinetic: Using flow cytometry, we found cell count ［(4.30±0.34)×107／L)］ in TNF group (4×105 U/L) was obviously less than that in control ［(5.23±0.50)×107／L, P＜0.01］. The cells of G1 phase were more than those of the control, while the cells of G2, S and M phase became less (P＜0.05). ② Coagulant and anticoagulant: 72 h after MVEC cultued in the media, the content of 6-keto-PGF1α (RIA) and activity of PAI decreased significantly in TNF (4×105 U/L) group (P＜0.01, vs control). The difference between TXB2 content and t-PA activity in groups was not significant (P＞0.05). ③ Adhesive molecule: The effect of low concentration TNF (＜4×105 U/L) on adhesion between cultured MVEC and leukocytes was not signficant, but when the concentration of TNF reached 8×105 U/L or more, 12 h after culture the adhesion rate between MVEC and neutrophil increased 30.8%±4.5%. If adding monoclonal antibody of ICAM-1/CD11 into media, the adhesion rate of leukocytes decreased significantly (from 31.2% to 63.4%). ④ NO: The level of nitrite in culture media (Griess reaction) was higher than that of control (P＜0.05) after pretreatment of TNF (2×106 U/L) for 6 h. Adding L-NMMA, Dexamethasone or Cycloheximide in media could block the increase of nitrite induced by TNF, while L-Arg could enhance it. The expression of iNOS mRNA of PMVEC increased significantly after treated with TNF (2×106 U/L) for 24 h (quantitative RT/PCR). Pretreatment with Dexamethasone or Cycloheximide could block the increase (P＜0.05). Meanwhile, the expression of eNOS mRNA decreased significantly compared with control, the decrease can be blocked by Cycloheximide but not by Dexamethasone. So that TNF can induce the expression of iNOS mRNA in PMVEC, but inhibited the expression of eNOS mRNA. NO production in PMVEC can be time-dependently induced by TNF. ⑤ Apoptosis: Adding high concentration TNF(＞1.2×106 U/L) in culture media for 12 h can induce apoptosis of PMVEC with electron microscopy, flow cytometry (PI/AnnexinV stain), TUNEL and DNA ladder eletrophoresis. Meanwhile, expression of apoptosis-associated gene Bcl-2 mRNA decreased and that of Fas mRNA increased (Northern blot). The expression of FADD, caspase 3 and caspase 8 enhanced too. So that signal of apoptosis induced by TNF may be transmitted following the cascade of Fas→FADD→caspase8→caspase3. In conclusion, it should be paid attention to metabolism inability and dysfunction induced by TNF which can not found easyily using light microscope. High concentration TNF can induce apoptosis of endothelial cells regulated by apoptosis-associated genes. The changes mentioned above are common pathway in pathogenesis of some diseases related to TNF.

还原型谷胱甘肽对抗硫芥诱导大鼠脾淋巴细胞毒性作用

目的观察还原型谷胱甘肽(glutathione,GSH)对硫芥(sulfur mustard,SM)诱导大鼠脾淋巴细胞线粒体功能的保护作用.方法用密度梯度离心法分离大鼠脾淋巴细胞,与硫芥共同进行体外培养.用MTT法检测线粒体功能状态;用荧光分光法检测细胞内GSH含量和用琼脂糖DNA凝胶电泳观察DNA的损伤.结果硫芥作用的线粒体功能变化具有剂量和时间依赖性,它与细胞内GSH含量下降呈直线正相关(P＜0.01).使用GSH后,对不同剂量组的线粒体功能早期均有保护作用.保护效果好时间的DNA断裂情况有所减轻,甚至可以逆转坏死向凋亡发展.结论GSH可部分保护线粒体功能.

硫芥对大鼠脾脏淋巴细胞线粒体的损伤作用

目的观察硫芥对大鼠脾脏淋巴细胞线粒体的损伤作用.方法用密度梯度离心法分离大鼠脾淋巴细胞,与硫芥共同进行体外培养.用琼脂糖DNA凝胶电泳观察细胞凋亡的发生;用Western blot观察Cyt-c释放.用MTT法检测线粒体功能状态,以罗丹明123标记的荧光探针检测线粒体跨膜电位.结果 100 μmol/L硫芥作用4 h线粒体功能就有降低,线粒体跨膜电位降低,二者间呈直线相关.硫芥中毒早期即可引起淋巴细胞线粒体细胞色素C释放和细胞凋亡(DNA ladder).结论硫芥可引起大鼠脾淋巴细胞线粒体明显损伤,线粒体参与了硫芥的细胞毒性作用过程.

ATM在人胃癌细胞系的表达及其在细胞DNA损伤中的作用

目的:研究DNA损伤剂对ATM基因缺陷和正常的胃癌细胞系的作用机制.方法:Western-blot方法检测6株胃癌细胞系ATM蛋白的表达情况,并用DNA损伤剂顺铂作用于ATM基因缺陷的MKN28细胞系和ATM基因正常的BGC823细胞系,采用Western-blot、DNAladder、Hoechest 33258 /PI双荧光染色等方法观察凋亡相关激酶的表达和细胞的应答反应.结果:Western-blot显示,ATM蛋白在胃癌细胞系MGC803、MKN28、MKN45、SGC7901的表达水平显著低于BGC823和AGS细胞系,BGC823细胞在CDDP作用后,ATM蛋白表达水平升高,磷酸化chk1和chk2活性24小时后增强,而Cdc25C表达减少;MKN28细胞中G2期检测点蛋白ATM、p-chk1、p-chk2表达较低,Cdc25C表达较高,但在CDDP作用前后无明显变化.MKN28细胞在DNA损伤剂作用后出现显著凋亡,而BGC823细胞在DNA损伤剂作用48小时后未见显著凋亡.结论:ATM在细胞周期的多个环节均有调控作用,特别是在细胞周期检测点和DNA损伤的修复中有重要的监视和启动作用.

钬元素对小鼠肝脏细胞DNA损伤的影响

背景与目的:通过研究钬离子溶液对小鼠肝脏细胞DNA的损伤,探讨钬元素对诱导动物细胞凋亡的影响.材料与方法:处理组1:小鼠定时灌胃氯化钬溶液50 mg/(kg·d),连续5 d;组2～3:小鼠分别定时腹腔注射氯化钬溶液60mg/(kg·d)和120 mg/(kg·d),连续2 d;组4:小鼠一次性腹腔注射氯化钬溶液320 mg/kg;每次染毒相间24h,组1～4:小鼠均在末次染毒24 h后处死,提取肝脏DNA.组5:小鼠一次性腹腔注射等体积生理盐水;组6～9:小鼠一次性腹腔注射氯化钬溶液80mg/kg,分别于注射后12、24、48和96 h处死小鼠提取肝脏DNA.通过琼脂糖凝胶电泳研究各组DNA带型变化.结果:处理组7DNA电泳中出现连续的弥散带型,其它各组均未观察到明显的拖尾现象.也未观察到"DNA ladder".结论:钬离子对小鼠肝脏细胞DNA的断裂作用可能与其剂量大小、处理时间及DNA修复作用有关,而且无特异性.本实验结果表明,钬元素未诱导小鼠肝脏细胞凋亡.