水飞蓟素在去血清条件下诱导G0／G1期人宫颈癌HeLa细胞凋亡

背景：宫颈癌是危害女性身体健康，降低生活质量的疾病之一。利用中药治疗宫颈癌，降低放射治疗、应用抗肿瘤药物治疗副作用对机体伤害已成为医药界的研究热点在欧洲，水飞蓟素曾广泛用于临床的肝炎治疗。它毒性小，在植物中含量高。因此，一些学者正在研究其抗肿瘤的活性。目的：探讨水飞蓟素对人子宫颈癌细胞的药理作用机制。设计：以体外10％血清正常培养的细胞作为阴性对照的实验对照研究。单位：沈阳药科大学中日医药研究所，昭和药科大学病态科学教研室。材料：人的宫预癌HeLa细胞为本实验室保存的美国ATCC细胞系实验于2003—01／07在沈阳药科大学中日医药研究所细胞室完成。方法：噻唑蓝还原法(MTT)分析HeLa细胞的死亡率。采用相差和荧光显微镜观察细胞形态学改变。用流式细胞术分析了细胞周期的变化。主要观察指标：细胞死亡率和细胞周期分化的变化。结果：水飞蓟素诱导的HeLa细胞死亡与培养液中血清含量有关。给予80μmol／L的水飞蓟素作用．去血清组的细胞死亡率达到85.27％．含5％．10％血清组的死亡率为35．53％，7.71％细胞的形态学变化表明水飞蓟素诱导的HeLa细胞发生凋亡流式细胞分析结果显示：水飞蓟素诱导70.04％的细胞发生凋亡，这些凋亡细胞主要来自G0／G1期。结论：水飞蓟素在去血清条件下．诱导处G0／G1期的HeLa细胞发生凋亡。     

5，7-二甲氧基白杨素对体外培养人宫颈癌HeLa细胞凋亡的影响

[目的】观察5,7-二甲氧基白杨素（dMchR）对体外培养人宫颈癌HeLa细胞凋亡的影响。【方法】运用A0／EB荧光双染色法观察HeLa细胞凋亡形态;采用PI单染流式细胞术检测HeLa细胞凋亡率;运用FITC荧光标记流式细胞术分析HeLa细胞的Bcl-2,Bax蛋白的表达。【结果]dMChR能有效地诱导HeLa细胞凋亡;dMChR能下调HeLa细胞的Bcl-2蛋白表达,上调Bax蛋白表达,且呈浓度依赖性变化。【结论]dM—ChR具有诱导人宫颈癌HeLa细胞凋亡作用,这种作用与上调细胞Bax／Bcl-2比值有关。     

20(S)-人参皂甙Rg3诱导宫颈癌Hela细胞凋亡的研究

目的 探讨20（S）-人参皂苷Rg3（SPG-Rg3）对宫颈癌Hela细胞的诱导凋亡作用。方法 采用MTT法观察SPG-Rg3对Hela细胞生长的抑制作用;流式细胞术观察SPG-Rg3作用后,Hela细胞之细胞周期的变化;AO/EB双染从形态学上观察SPG-Rg3对Hela细胞凋亡的作用。结果 经SPG-Rg3作用后,Hela细胞的生长受到明显的抑制（P〈0.05）,S期细胞数增加,G2/M期细胞数减少（P〈0.01）,凋亡细胞数增加。结论 SPG-Rg3可以诱导宫颈癌Hela细胞发生凋亡,可作为治疗宫颈癌的一种新途径。     

冬凌草乙素对白血病HL-60细胞的增殖抑制作用

背景:已有研究证明,冬凌草乙素可以通过诱导许多实体肿瘤的细胞凋亡而发挥抗肿瘤作用,目前,冬凌草乙素对白血病HL-60细胞的作用尚未见资料报道.目的:观察冬凌草乙素对白血病HL-60细胞的体外增殖抑制作用及其机制.方法:以不同浓度的冬凌草乙素(10-50 μmol/L)作用于体外培养的HL-60细胞24,48,72 h,应用MTT法检测细胞生长抑制率,流式细胞术检测细胞周期,免疫印迹法检测Caspase-3及其裂解底物多聚(ADP-核糖)聚合酶(PARP)表达水平的变化,并对细胞周期调节蛋白P21、P16的表达水平进行检测.结果与结论:冬凌草乙素可显著抑制细胞的生长及诱导细胞发生凋亡,呈现出明显的量-效与时-效关系.流式细胞术检测结果表明细胞主要被阻滞在G0/G1期,50 μmol/L的冬凌草乙素作用48 h后可以出现典型的亚G1期峰(细胞凋亡峰).免疫印迹法检测结果显示Mr32 000的Caspse-3酶被活化出现Mr20 000亚单位片段,同时Caspse-3的底物PARP被裂解出现Mr89 000的亚单位片段,免疫印迹法检测结果还显示50 μmol/L的冬凌草乙素作用不同时间后,细胞周期调节蛋白P21及P16的表达水平逐渐升高.结果提示冬凌草乙素在体外对HL-60细胞具有显著的细胞周期阻滞作用,并诱导细胞发生凋亡,通过上调细胞周期调节蛋白P21及P16的表达水平及激活Caspse-3可能是冬凌草乙素引起HL-60细胞G0/G1阻滞及诱导细胞发生凋亡的重要作用机制之一.     

TRPV4对HeLa细胞增殖的作用

目的 观察瞬时感受器电位离子通道香草素受体4（TRPV4）与宫颈癌HeLa细胞增殖的关系.方法 采用宫颈癌HeLa细胞系,用Western blot技术检测HeLa细胞中TRPV4的表达量;并分别用TRPV4阻滞剂RN1734干预体外培养HeLa细胞,通过细胞计数法、LDH检测及流式细胞技术观察TRPV4通道调控对HeLa增殖和细胞周期进展的影响.结果 与对照组W12细胞系相比,宫颈癌HeLa细胞系中TRPV4呈高表达.TRPV4阻滞剂干预48和72 h后,HeLa细胞增殖较未干预组降低（P＜0.05）;而TRPV4阻滞剂干预24h和48 h时,处于S期细胞的百分率明显低于未干预组;处于G0/G1期细胞的百分率则明显高于未干预组（P＜0.05）.结论 TRPV4抑制可以阻滞体外培养宫颈癌HeLa细胞增殖及细胞周期进展.     

刺参酸性黏多糖对宫颈癌HeLa细胞凋亡及Bax、Bcl-2基因表达的影响

目的:研究刺参酸性黏多糖(SJAMP)对人宫颈癌HeLa细胞的诱导凋亡作用并探讨其对凋亡相关基因Bax和Bcl-2表达的影响.方法:体外培养HeLa细胞:采用Hoechst 33258荧光染色检测SJAMP作用前后的细胞凋亡情况;流式细胞仪检测sJAMP作用HeLa细胞后线粒体膜电位(△ψm)的变化,Western Blot检测SJAMP对HeLa细胞作用后凋亡相关蛋白Bax,Bcl-2的表达.结果:荧光显微镜下可观察到细胞凋亡的形态学改变;与对照组比,各SJAMP浓度组均能降低HeLa细胞的线粒体膜电位;随着SJAMP作用浓度的增加,Bcl-2的表达减少,而Bax基因的表达增加.结论:SJAMP在体外可诱导HeLa细胞凋亡,其凋亡分子机制可能是因为对Bax、Bcl-2表达调控,改变线粒体跨膜电位诱导其凋亡.     

水飞蓟素抵抗血清撤除的脐带间充质干细胞凋亡

目的:探讨水飞蓟素抵抗血清引起的人间充质干细胞(MSC)凋亡及其相关机制。方法:培养人脐带MSC体系去血清,加入不同浓度(1-10μg/ml)的水飞蓟素,应用MTT实验检测细胞增殖状态。细胞培养72 h后,Annexin V-PI双标记流式细胞术观察处理组和对照组细胞凋亡情况。收集细胞,以未去血清组为阳性对照,Western blot分析细胞内凋亡蛋白BCL-2和BAX含量。结果:水飞蓟素可以促进脐带MSC的增殖,效应呈浓度依赖性,浓度为5μg/ml时其促增殖作用最为明显。水飞蓟素可抑制由血清撤除引起的MSC凋亡,浓度为5μg/ml时抑制率可达30%左右。血清撤除后,细胞内BAX蛋白含量明显上升,而BCL-2含量无显著变化。结论:水飞蓟素可促进脐带间充质干细胞的增殖,并可能通过线粒体途径抑制MSC凋亡。     

Survivin siRNA诱导人腺样囊性癌SACC-83细胞凋亡的实验研究

目的 应用RNA干扰技术靶向阻断Survivin基因表达,探讨其对人腺样囊性癌SACC-83细胞的诱导凋亡作用.方法 实验设空白对照组,RNA干扰阴性对照组和RNA干扰组.将RNA干扰载体通过Lipofection介导转染腺样囊性癌细胞.采用倒置显微镜观察细胞凋亡形态;流式细胞仪检测细胞凋亡峰;透射电镜观察细胞凋亡超微结构.结果 转染细胞48 h后RNA干扰组细胞形态学发生改变,呈典型的细胞凋亡形态.流式细胞仪检测在细胞周期G1/G0期前出现亚二倍体凋亡峰,电镜超微结构可见凋亡小体.空白对照组,RNA干扰阴性对照组无此变化.结论 Survivin siRNA干扰能诱导体外培养的人腺样囊性癌SACC-83细胞发生凋亡,以Survivin 为靶点有望成为肿瘤基因治疗的可行性.     

丹皮酚对人大肠癌细胞凋亡和细胞周期以及患者预后的影响

目的:探讨中药丹皮酚(paeonol,Pae)对人大肠癌细胞株HT-29凋亡和细胞周期的影响,及其与化疗药物的协同作用,为寻求新的大肠癌治疗策略提供理论依据。方法:实验于2004-05/09在武汉大学人民医院消化病研究所完成;应用MTT法、流式细胞仪技术检测不同浓度的丹皮酚对HT-29细胞的增殖抑制、凋亡诱导作用及对细胞周期分布的影响,同时观察低浓度的丹皮酚与化疗药物的协同作用。结果:丹皮酚(浓度7.81～250mg/L)可抑制HT-29细胞增殖,并且与药物浓度及作用时间呈正相依赖关系,最高抑制率为91.849%(P<0.01)。丹皮酚在15.63,62.5,250mg/L3种浓度下作用48h均可诱导HT-29细胞凋亡,流式细胞仪技术检测其凋亡率分别为7.6%,16.2%,和34.5%,有明显的剂量效应关系。同时丹皮酚使细胞周期分布发生明显变化,表现为S期细胞比例上升,G0/G1期和G2/M期细胞比例下降。低浓度的丹皮酚与化疗药物协同可产生较强的抑制细胞增殖作用,其中与5氟尿嘧啶的协同作用最为显著。结论:丹皮酚能抑制HT-29细胞增殖并诱导其发生凋亡,其机制可能与影响该细胞株的细胞周期有关。丹皮酚与细胞周期特异性化疗药联合使用具有协同抗肿瘤作用。     

灯盏花素对肺癌A549细胞增殖、凋亡及细胞周期的影响

目的灯盏花素对肺癌A549细胞增殖、凋亡及细胞周期的影响。方法利用MTT法研究不同浓度(0 mM、1 mM、3. 125 mM、6. 25 mM、12. 5 mM、25 mM、50 mM、100 mM)灯盏花素对肺癌A549细胞的生长抑制作用并计算半数抑制浓度(IC50)值;用半数抑制浓度的灯盏花素处理肺癌A549细胞12 h、24 h、48 h后,通过Annexin V-FITC形态学染色法和流式细胞术分析细胞死亡类型和细胞周期变化; Western blot方法检测细胞内p-53/p53、p21、cyclin B1、p-cdc2/cdc2、Bcl-2/Bad和cleaved-caspase-3蛋白表达变化。结果 MTT检测发现灯盏花素处理后的A549细胞生长受到显著抑制(IC50为25. 03 mM),且表现出明显的剂量依赖性(P 0. 05);形态学染色和流式细胞术表明灯盏花素能够诱导肺癌A549细胞时间依赖性凋亡(P 0. 05),并发生G2/M细胞周期阻滞; Western blot结果显示与细胞周期相关蛋白p-p53、p21、p-pcdc2随着灯盏花素作用时间的增加,表达量显著提高(P 0. 05),而cyclin B1的表达明显受到抑制(P0. 05);凋亡相关蛋白分析发现:促凋亡蛋白Bad和cleaved-caspase-3的表达显著增加,抑凋亡蛋白Bcl-2的表达明显降低(P 0. 05)。结论灯盏花素能够使肺癌A549细胞发生G2/M期阻滞,上调细胞周期相关蛋白表达,改变细胞周期,抑制细胞增殖;同时通过调控Bcl-2和caspase家族蛋白诱导并执行肺癌A549细胞凋亡。     

Tumor growth inhibition in vivo and G2/M cell cycle arrest induced by antisense oligodeoxynucleotide targeting thymidylate synthase.

Chemotherapeutic agents targeting thymidylate synthase (TS) are effective against human tumors. Efficacy is limited by drug resistance, often mediated by TS overexpression. Treatment of HeLa cells in vitro with an antisense oligodeoxynucleotide (ODN 83) targeting human TS mRNA reduces TS mRNA and protein levels, inhibits cell proliferation, and sensitizes cells to TS-targeting drugs (Ferguson et al., 1999). The present study investigates the mechanism by which ODN 83 inhibits cell proliferation and examines its antitumor efficacy in vivo. ODN 83 treatment did not induce apoptosis in HeLa cells in vitro but caused accumulation of cells at G2/M. In contrast, TS-targeting chemotherapeutics arrest at G1 or S. Antisense down-regulation reduced TS mRNA levels in human colon cancer (HT29) cells by 40% in vitro, resulted in G2/M arrest, and reduced proliferation without enhanced cell death. Growth of HT29 tumors in immunocompromised mice was significantly inhibited when antisense ODN 83 treatment began promptly after tumor implantation and was accompanied by a 40% reduction in TS protein levels. Growth of tumors allowed to reach 400 mm3 prior to ODN administration was unaffected by antisense ODN 83. Radiolabeled ODNs were localized to the tumor periphery but evenly distributed in normal tissue. Thus, down-regulation of TS mRNA and protein by antisense ODN treatment exerts a novel G2/M cell cycle block without increasing cell death and inhibits HT29 tumor cell growth in vivo. Antisense ODN 83 may be an effective therapy for colon carcinoma, alone or in combination with TS-targeting cytotoxic drugs.     

胰腺干细胞体外诱导分化为胰岛素分泌细胞及对1型糖尿病模型鼠的治疗作用

背景：有实验证实人和动物起源的胚胎干细胞或成体干细胞可分化为胰岛素分泌细胞，但其分泌胰岛素的功能及对实验性糖尿病的治疗价值尚需要验证。 目的：观察从大鼠的胰腺组织分离纯化干细胞在体外将诱导分化胰岛素分泌细胞后胰岛素mRNA的表达、分泌胰岛素的能力及对1型糖尿病模型鼠的治疗作用。 设计：随机对照观察。 单位：中日友好医院临床医学研究所。 材料：实验于2004-08／2007—12在北京中日友好医院临床医学研究所病理生理研究室完成。选用8—10周龄雄性SD大鼠及雌性裸鼠各10只，均购自北京维通利华实验动物技术有限公司。实验过程中对动物的处置符合动物伦理学标准。尼克酰胺、链脲菌素均为Sigma公司产品，nestin抗体为BD Biosciences产品。大鼠胰岛素放射免疫检测试剂盒购自Linco research。胰岛素抗体及胰高糖素抗体为Santa Cruz产品。 方法：应用nestin结合的免疫磁珠从SD大鼠胰腺导管细胞中分离和纯化干细胞，经体外扩增及诱导分化形成胰岛素分泌细胞。①采用RT-PCR法检测细胞分化过程中胰岛素mRNA的表达。②将干细胞经诱导分化形成的胰岛进行冰冻切片，采用免疫荧光染色观察胰岛素及胰高糖素阳性细胞表达。③对干细胞分化胰岛的胰岛素释放功能进行评价，放射免疫法测定上清中胰岛素检测干细胞分化形成的胰岛分泌胰岛素的能力。④鼠按220mg／kg链脲菌素腹腔注射法制备为1型糖尿病模型，造模后随机摸球分为天然胰岛组及干细胞胰岛组，每组5只。将大鼠天然胰岛（SD大鼠分离）及干细胞分化形成的胰岛分别移植于糖尿病裸鼠左肾包膜下，观察移植后鼠尾静脉血糖变化。 主要观察指标：①细胞分化过程胰岛素mRNA表达。②胰岛样结构中胰岛素及胰高糖素阳性细胞表达。③干细胞分化形成的胰岛分泌胰岛素情况。④移植后鼠尾静脉血糖变化。 结果：①RT-PCR检测结果表明胰岛素mRNA的表达随诱导时间延长而明显升高。②免疫荧光染色结果显示胰岛结构的中间存在大量胰岛素阳性细胞，胰高糖素阳性细胞主要分布于胰岛样结构的周边。③干细胞分化的胰岛在高浓度葡萄糖刺激后明显缺乏快速相的胰岛素释放，而是表现细胞外上清中胰岛素浓度的缓慢升高。④天然胰岛组移植后3d使血糖降低到10mmol／L以下，至观察到60d仍维持在正常水平；干细胞胰岛在移植后8d使血糖降低至10mmol／L以下，且在移植35d后血糖逐渐升高并恢复到胰岛移植前的水平。 结论：大鼠胰腺干细胞经体外扩增和诱导分化后可分化胰岛素分泌细胞，但对葡萄糖的刺激没有快速相的胰岛素分泌。将胰岛素分泌细胞移植到1型糖尿病模型裸鼠后可一定程度地改善糖代谢的紊乱。     

In vitro and in vivo evaluation of novel cationic liposomes utilized for cancer gene therapy.

Advanced peritoneal carcinomatoses is very difficult to treat. We have explored the potential therapeutic application of gene therapy using cationic liposomes in this disease. The lacZ gene was introduced in vitro into ovarian and endometrial cancer cells using cationic liposomes. The transfection efficiency was similar to that of commercially available liposomes in serum-free medium (11.0-20.9% vs. 5.4-26.0%). In serum-containing medium, the efficiency was 1.9-18.1%, which is comparable with the efficiency in serum-free medium. However, the efficiency of commercial liposomes decreased drastically to between 0.1% and 4.7% in the serum-containing medium. When cultured cells were transfected with the herpes simplex virus thymidine kinase (HSV-tk) gene and ganciclovir (GCV) was added, the anti-tumor effect of GCV was 47-640 times greater than when the same experiment was performed with lacZ gene. Evaluation of anti-tumor effect was performed with the MTT assay. In vivo, the HRA and mEIIL ascitic mice were treated with HSV-tk gene and GCV using the peritoneal route, a significant prolongation of the mean survival time was observed by Kaplan-Meier analysis (16-18 days and 15-30 days, respectively, p < 0.05). These results indicate a potential role for gene therapy in the treatment of advanced intraperitoneal carcinomatoses using the novel cationic liposomes.     

丝裂霉素诱导的肝癌细胞凋亡致敏树突状细胞

目的:建立丝裂霉素诱导癌细胞凋亡致敏从人外周血诱导扩增的树突状细胞的方法。设计:以癌细胞凋亡致敏树突状细胞为观察对象的随机对照实验。单位:解放军第三军医大学大坪医院野战外科研究所,解放军军事医学科学院放射医学研究所。材料:实验于1998-04/1999-05在解放军军事医学科学院放射医学研究所完成。细胞株为QBC939胆管癌细胞株,药物为抗肿瘤药物丝裂霉素。方法:从正常人外周血分离获得单核细胞,加入50μg/L重组人粒细胞集落刺激因子,1000U/mL白细胞介素4,隔天一次,共4次,培养第3天,加入丝裂霉素诱导凋亡的胆管癌细胞,再继续体外培养4d后,用树突细胞富集柱收集树突状细胞。主要观察指标:①培养的树突状细胞的鉴定。②树突状细胞和坏死胆管癌细胞以及正常培养的胆管癌细胞共培养,观察其吞噬凋亡小体负载抗原。③检测不同密度的树突状细胞(1×103/孔,5×103/孔,1×104/孔)的免疫刺激活性及负载抗原后的免疫刺激活性,以单核细胞作对照组。结果:①培养、扩增得到的树突状细胞高表达共刺激分子B7和CDla,表面具有典型不规则突起。②丝裂霉素诱导癌细胞凋亡形成的凋亡小体被树突状细胞捕捉、吞噬。③负载抗原的树突状细胞其激发同种异体T淋巴细胞增殖的能力进一步增强。结论:丝裂霉素诱导癌细胞凋亡可以致敏重组人粒细胞集落刺激因子加重组人白细胞介素4从人外周血单核细胞诱导、扩增出的树突状细胞,树突状细胞可以有效提呈凋亡胆管癌细胞的抗原,可望成为有效的肿瘤抗原致敏树突状细胞的新途径。     

In vitro culture of human acute myelogenous leukemia (AML) cells in serum-free media: studies of native AML blasts and AML cell lines

The functional characteristics were compared for acute myelogenous leukemia (AML) cells (native blasts and AML cell lines) cultured in three serum-free media (X-vivo 10, X-vivo 15, [Bio-Whitacker, Walkersville, MD] and StemSpan [Stem Cell Technologies, Vancouver, BC, Canada]) and in medium containing 10% inactivated fetal calf serum (FCS). For native AML blasts the following functions were compared: (1) autonomous and cytokine-dependent proliferation; (2) frequency of clonogenic cell; and (3) constitutive cytokine secretion. AML blast proliferation differed between patients independent of the culture medium used, and clonogenic cells were maintained after in vitro culture in all media. In contrast, constitutive cytokine secretion was higher for cells cultured in StemSpan and FCS-containing medium than for cells cultured in the X-vivo media. Native AML blasts incubated in StemSpan also showed a low frequency of apoptotic cells. The three serum-free media could also be used for long-term expansion of well-characterized AML cell lines, but the optimal medium for cell expansion and cytokine secretion differed between cell lines. We conclude that standardized serum-free culture conditions can be used for in vitro studies of native AML blasts and AML cell lines.     

Effect of naringenin and its combination with cisplatin in cell death,proliferation and invasion of cervical cancer spheroids

The main treatment alternative for cervical cancer is cisplatin chemotherapy. However, the resistance of tumor cells to cisplatin, in addition to side effects, limits its use. The flavonoid naringenin has shown cytotoxic effects on tumor cells and may be considered as a coadjuvant in the treatment of cervical cancer. In the present study, the effect of naringenin on cell viability, cytotoxicity, proliferation, apoptosis and invasion was evaluated in HeLa spheroid cultures. Naringenin impaired the cell viability as indicated by low ATP levels and caused concentration- and time-dependent cytotoxicity via the loss of cell membrane integrity. Furthermore, it did not activate caspases 3, 7, 8, and 9, suggesting that the cytotoxic effect was by necrotic cell death instead of apoptosis. Additionally, proliferation in the G0/G1 phase of the cell cycle was inhibited. Cell invasion also decreased as time progressed. Later, we determined if naringenin could improve the anti-tumor effect of cisplatin. The combination of naringenin with low concentrations of cisplatin improved the effect of the drug by significantly decreasing cell viability, potentiating the induction of cytotoxicity and decreasing the invasive capacity of the spheroids. Since these effects are regulated by some key proteins, molecular docking results indicated the interaction of naringenin with RIP3 and MLKL, cyclin B and with matrix metalloproteases 2 and 9. The results showed the anti-tumor effect of naringenin on the HeLa spheroids and improved effect of the cisplatin at low concentrations in combination with naringenin, placing flavonoids as a potential adjuvant in the therapy against cervical cancer.

The combination of naringenin–cis-platin prevents the invasion of cancer, at a lower concentration of cis-platin.     

黄芪对N-甲基-N-亚硝脲诱导大鼠光感受器细胞凋亡的预防途径

背景:视网膜色素变性是非炎症性、双侧进行性的视网膜变性,其特征是光感受器细胞因发生凋亡而丢失,最终导致失明。中药黄芪在阻止该病的进程上显示出了良好的前景。目的:观察黄芪对N-甲基-N-亚硝脲致SD大鼠视网膜损伤的保护作用。设计:随机对照动物实验。单位:新乡医学院药学院。材料:实验于2004-03/12在中山大学中山眼科中心药理实验室完成。雌性SD大鼠114只,由中山大学中山医学院动物中心提供,N-甲基-N-亚硝脲为美国Sigma公司产品,黄芪注射液为成都地奥九泓制药厂生产(生产批号:国药准字Z99060535,2mL/支,主要成分为黄芪)。方法:选用雌性SD大鼠114只,30只用于视网膜层形态学分析;30只用于细胞凋亡检测;54只用于胞核内核因子κBp65活性的检测。采用抽签法随机分组,每组6只。于大鼠生后47d,分别经腹腔注射不同剂量的黄芪(2.5,5和10g/kg),1次/d,除对照组外,其余组的大鼠同时于生后50d腹腔注射N-甲基-N-亚硝脲60mg/kg。在N-甲基-N-亚硝脲处理不同时间后处死动物,取眼球,视网膜形态学分析测量周边视网膜的总厚度,TUNEL试剂盒检测光感受器细胞凋亡,转录因子试剂盒分析核因子κBp65的活性。主要观察指标:各组视网膜厚度、凋亡指数和胞核内核因子κBp65的活性比较。结果:黄芪可剂量依赖性地显著增加周边视网膜总厚度和降低N-甲基-N-亚硝脲引起的光感受器细胞凋亡指数。黄芪注射液10g/kg还可时间依赖性地上调视网膜细胞胞核内核因子κBp65的活性。但其对N-甲基-N-亚硝脲引起的中心视网膜损伤无明显的保护作用。结论:黄芪通过上调视网膜细胞胞核内核因子κBp65的活性,抑制光感受器细胞凋亡,从而部分地保护N-甲基-N-亚硝脲引起的视网膜损伤。     

具G418抗性NIH3T3细胞饲养层的制备

背景:建立具有抗药(新霉素或潮霉素)性的饲养层细胞是转染外源目的基因胚胎干细胞阳性克隆的筛选必备条件。建成的具有抗药性的NIH3T3细胞系可用于胚胎干细胞其他目的基因的筛选。目的:建立具有G418抗性的NIH3T3细胞系,用于转染pTet-on基因的胚胎干细胞阳性细胞克隆筛选的饲养层。设计:细胞培养观察及DNA检测。单位:中国医科大学实验动物部。材料:NIH3T3细胞由中国医科大学细胞生物教研室惠赠,pWL/neo质粒为美国哈佛大学医学院金壮教授惠赠。G418、白血病抑制因子(胚胎干细胞GRO106U/mL)、DMEM为GIBCOBRL产品,丝裂霉素-C、DIG标记及检测试剂盒为Roche产品,脂质体为Invitrogen产品,均购自沈阳联星生物技术有限公司。方法:①通过脂质体转染的方法,将含有neo基因的质粒pWL/neo导入NIH3T3细胞中。②将细胞传至25mL培养瓶中,加入梯度浓度的G418继续培养,通过观察细胞生长及死亡情况测定G418对NIH3T3细胞最小致死量。③将转染的细胞进行传代,挑取单个细胞的克隆至24孔培养板继续进行筛选扩增,选用正常的NIH3T3细胞加入相同剂量的G418的选择性培养基作为筛选的阴性对照。④采用较低的细胞密度铺板,加入含最小致死量G418的DMEM培养基进行再次筛选;同时选用正常的NIH3T3细胞为对照,使用含有最小致死量G418及未加G418的DMEM培养基进行培养,采用光学显微镜对G418抗性NIH3T3细胞进行观察。⑤选用具有G418抗性的NIH3T3细胞和小鼠胚胎成纤维细胞分别作为饲养层细胞进行传代,采用光学显微镜对培养后胚胎干细胞-D3细胞进行观察,并制备细胞DNA,对其进行PCR和southernblot鉴定。主要观察指标:①G418对NIH3T3细胞最小致死量的测定结果。②G418抗性NIH3T3细胞的筛选结果。③G418抗性NIH3T3细胞的生长观察结果。④胚胎干细胞-D3细胞生长观察结果及G418抗性NIH3T3细胞的鉴定。结果:①G418对NIH3T3细胞最小致死量为500mg/L。②经过500mg/LG418压力筛选后,获得了抗G418细胞克隆。③抗性NIH3T3细胞的形态和生长速度与正常NIH3T3细胞没有差异。④饲养层培养的胚胎干细胞呈集落形状生长,边缘光滑,保持未分化的状态。用特异性核苷酸引物检测抗性细胞基因组DNA,可以扩增出对应的核苷酸片段,Southernblot鉴定结果表明neo基因片段已整合入G418抗性NIH3T3细胞中。结论:成功地培育了G418抗性NIH3T3细胞,生长在G418抗性NIH3T3细胞饲养层上胚胎干细胞细胞基本保持正常胚胎干细胞细胞特征。     

Silymarin induces apoptosis primarily through a p53-dependent pathway involving Bcl-2/Bax, cytochrome c release, and caspase activation

Silymarin, a plant flavonoid, has been shown to inhibit skin carcinogenesis in mice. However, the mechanism responsible for the anti-skin carcinogenic effects of silymarin is not clearly understood. Here, we report that treatment of JB6 C141 cells (preneoplastic epidermal keratinocytes) and p53+/+ fibroblasts with silymarin and silibinin (a major constituent of silymarin) resulted in a dose-dependent inhibition of cell viability and induction of apoptosis in an identical manner. Silymarin-induced apoptosis was determined by fluorescence staining (8-64% apoptosis) and flow cytometry (12-76% apoptosis). The silymarin-induced apoptosis was primarily p53 dependent because apoptosis occurred to a much greater extent in the cells expressing wild-type p53 (p53+/+, 9-61%) than in p53-deficient cells (p53-/-, 6-20%). The induction of apoptosis in JB6 C141 cells was associated with increased expression of the tumor suppressor protein, p53, and its phosphorylation at Ser15. The constitutive expression of antiapoptotic proteins Bcl-2 and Bcl-xl were decreased after silymarin treatment, whereas the expression of the proapoptotic protein Bax was increased. There was a shift in Bax/Bcl-2 ratio in favor of apoptotic signal in silymarin-treated cells, which resulted in increased levels of cytochrome c release, apoptotic protease-activating factor-1, and cleaved caspase-3 and poly(ADP-ribose) polymerase in JB6 C141 cells. The shift in Bax/Bcl-2 ratio was more prominent in p53+/+ fibroblasts than in p53-/- cells. Silymarin-induced apoptosis was blocked by the caspase inhibitor (Z-VAD-FMK) in JB6 C141 cells which suggested the role of caspase activation in the induction of apoptosis. These observations show that silymarin-induced apoptosis is primarily p53 dependent and mediated through the activation of caspase-3.     

Molecular growth requirements of single mammalian cells. III. Quantitative colonial growth of single S3 cells in a medium containing synthetic small molecular constituents and two purified protein fractions

Two purified serum protein fractions, fetuin and serum albumin, will replace whole or dialyzed serum in supporting the growth of single S3 HeLa cells in an otherwise chemically defined nutrient solution. In the serum-free medium, single S3 cells will form macroscopic colonies with essentially 100 per cent efficiency. The generation time of S3 cells in the serum-free medium is approximately 50 per cent greater than that observed in an optimal, serum-containing medium. All components of the serum-free medium are available commercially, except fetuin, which can easily be prepared in substantial quantities. The problem of the purity of the protein preparations and of their possible roles in promoting cell growth is discussed.