不同冻存条件对细胞因子诱导的杀伤细胞的细胞表型及杀伤活性的影响

目的 探讨不同冻存条件下细胞因子诱导的杀伤细胞(CIK)细胞表型的变化及对K562细胞杀伤活性的影响.方法 收集培养12 d的CIK细胞,分别冻存于-80℃冰箱及液氮中,于冻存后4、12、24周复苏,通过流式细胞仪检测CIK细胞表型变化,CCK-8法测定其对K562细胞的杀伤活性,并与未冻存CIK细胞进行比较.结果 液氮冻存4、12、24周及-80℃冻存4周后复苏培养的CIK细胞增殖、细胞存活率、免疫细胞表型及对K562的杀伤活性与未冻存组比较差异无统计学意义(P＞0.05).-80℃冻存12周及24后复苏培养的CIK细胞与未冻存组比较,细胞增殖明显抑制,细胞存活率低,CD3+、CD3 +/CD4+、CD3 +/CD8+、CD3 +/CD56+细胞比例显著降低,对K562细胞杀伤活性显著抑制(P＜0.05).结论 临床治疗中CIK细胞应尽量冻存于液氮中,如冻存于-80℃时冻存时间应≤4周.     

蛇床子素对L02细胞的毒性作用及其机制研究

目的 探究蛇床子素（osthole,Ost）对L02细胞的毒性损伤和作用机制,为中药蛇床子及其制剂的安全合理应用提供实验依据。方法 以不同浓度Ost作用于L02细胞,MTT法检测细胞活性;乳酸脱氢酶（LDH）试剂盒测定细胞LDH释放率;Hoechst 33342染色法检测细胞核形态;Annexin V/PI双染法检测细胞凋亡;Western blot检测Bcl-2、Bax、pro-caspase-3、cleaved-caspase-3（p17）、p-Histon H3（Ser10）的表达。结果 L02细胞在Ost作用下活性下降,LDH释放率提高,且呈浓度依赖;Hoechst 33342染色荧光下可见细胞核皱缩碎裂;AnnexinV/PI双染法结果表明凋亡率随浓度提高而上升。与对照组比较,50,100,200 μmol·L^-1 Ost作用24 h后,Bcl-2、pro-caspase-3、p-Histon H3（Ser10）表达水平降低,Bax、cleaved-caspase-3表达水平升高。结论 Ost对L02细胞有毒性损伤作用,呈一定的时间和浓度依赖性,可促进细胞凋亡,抑制细胞增殖。     

人脑胶质瘤细胞的分离、培养、冻存与复苏

目的 研究分离、培养人脑胶质瘤细胞方法.方法 手术获得切除胶质瘤新鲜标本30例,酶消化法分离培养获得人脑胶质瘤细胞.利用含10%胎牛血清的DMEM培养液进行体外原代培养及传代,观察肿瘤细胞生长特点.运用冻存液冻存培养的胶质瘤细胞,分别于冻存后1、4、8、12、16、20、24周复苏,计数复苏后活细胞比例,进行再培养并观察肿瘤细胞生存情况.结果 从脑胶质瘤标本分离出的胶质瘤细胞可进行原代培养及传代.冻存1、4、8、12、16、20、24周后脑胶质瘤细胞复苏的成活比例分别为54%、52%、52%、50%、48%、46%、40%.复苏后再培养的肿瘤细胞生长良好.结论 通过分离培养手术切除胶质瘤标本可获得脑胶质瘤细胞株,肿瘤细胞可通过冻存的方式进行保存,冻存后复苏的细胞可再培养.     

泽漆抑制三阴乳腺癌MDA-MB-231细胞及其凋亡机制研究

目的 研究泽漆对三阴乳腺癌MDA-MB-231细胞的作用及其作用机制。方法 用MTT法检测细胞活力;用荧光显微镜法测定MDA-MB-231细胞的活性氧（ROS）生成量;用流式细胞仪检测细胞的凋亡率;用TUNEL检测法检测细胞凋亡DNA碎片;用Western blot检测caspase-9、caspase-3、PARP等凋亡相关因子的水平变化。结果 MTT试验显示泽漆提取物对MDA-MB-231细胞具有显著的抑制作用,但作用可被ROS抑制剂NAC及caspase抑制剂Z-VAD-FMK所消除;荧光显微镜检测显示泽漆提取物能显著提高ROS的生成;流式细胞仪检测显示泽漆提取物处理后,PI染色阳性细胞明显增加,但被NAC减弱。Caspase-9、caspase-3在提取物处理后均转为激活形式,PARP被剪切。TUNEL法显示,提取物处理后细胞凋亡碎片明显增多,而提前加入ROS抑制剂NAC和caspase抑制剂Z-VAD-FMK能使泽漆提取物诱导凋亡的DNA碎片明显减少。结论 泽漆乙酸乙酯提取物可以有效抑制MDA-MB-231细胞的生长,诱导其凋亡,其作用机制可能与ROS过量生成所致的线粒体损伤途径有关。     

大黄素诱导人肝癌HepG2细胞线粒体凋亡作用研究

目的 研究大黄素对人肝癌HepG2细胞线粒体凋亡的影响。方法 培养人肝癌HepG2细胞,与5、10、20、40、60、80、100 μmol/L的大黄素作用24、48 h,MTS法检测细胞增殖;40、80、160 μmol/L大黄素作用HepG2细胞24 h,AO/EB双荧光染色法观察细胞凋亡的形态学改变;Annexin V/PI染色经流式细胞仪检测细胞凋亡;分光光度法检测caspase 3活性;ATP试剂盒检测细胞ATP含量,不同荧光探针加载后流式细胞仪测定大黄素对HepG2细胞内活性氧（ROS）含量、Ca²⁺浓度、线粒体膜电位（MMP）变化的影响。结果 大黄素抑制HepG2细胞生长,且呈时间、浓度相关性,半数抑制浓度（IC₅₀）为（77.42±1.25）μmol/L;随着大黄素浓度升高,AO/EB双染观察到细胞核浓缩、碎裂、凋亡小体等凋亡形态;与对照组比较,大黄素40、80、160 μmol/L作用于HepG2细胞24 h后细胞凋亡率显著增加,caspase 3活性显著增强,ROS水平、Ca²⁺浓度明显增加（P<0.05、0.01、0.001）,80、160 μmol/L组线粒体膜电位明显降低,ATP含量显著下降（P<0.05、0.01、0.001）。结论 大黄素造成HepG2细胞内ROS堆积,ATP合成功能障碍,线粒体膜电位明显下降,进而诱导线粒体通透转运孔开放,导致钙离子和细胞色素C外流,活化caspase蛋白家族,导致细胞凋亡。     

败酱草单萜环烯醚酯类对HepG2、MCF7细胞增殖及凋亡的影响

目的 探讨败酱草单萜环烯醚酯类（patrinia monoterpene iridoid ether esters,PMIEE）对HepG2和MCF7细胞增殖抑制和凋亡的影响。方法 HepG2和MCF7细胞经PMIEE作用后,采用CCK8法检测细胞增殖抑制情况;Annexin V-FITC/PI双标记流式细胞术检测细胞凋亡及周期情况;细胞划痕实验检测细胞迁移状况;Western blot法检测Bcl-2、Bax、caspase3、cdc2和CyclinB1的表达情况。结果 CCK8、划痕实验和Annexin V-FITC/PI流式细胞术检测显示,PMIEE对HepG2和MCF7细胞均有显著的增殖抑制、促凋亡率和降低迁移率作用（P<0.05）,呈一定量效关系,且PMIEE对HepG2细胞的周期阻滞以G2/M期为主,MCF7细胞以G0/G1期为主;Western blot结果显示,PMIEE可显著下调2种细胞Bcl-2、cdc2、CyclinB1的表达,上调Bax和caspase3的表达水平。结论 PMIEE可诱导HepG2和MCF7细胞增殖抑制和凋亡,下调Bcl-2、cdc2和CyclinB1表达及上调Bax和caspase3表达,其抗癌的潜在机制可能与此有关。     

西黄丸组分中药调节肿瘤微环境中Treg细胞PI3K/AKT通路的抗肿瘤作用机制研究

目的 研究西黄丸组分中药是否与西黄丸具有相似的抗癌作用及机制。方法 4T1乳腺癌细胞荷瘤建立动物模型,西黄丸低、中、高剂量（0.39、0.78、1.95 g/kg）和西黄丸组分中药0.3 mL （74.5 mg/kg榄香烯、2.73 mg/kg牛磺酸、0.52mg/kg麝香酮和4.75 mg/kg 11-羰基-β乙酯乳香酸混合物,对应于西黄丸高剂量组） ig给药14 d,剥离肿瘤组织,称质量,匀浆,制备单细胞悬液,免疫磁硃分离Treg细胞。流式细胞术和免疫组化检测肿瘤微环境中Treg细胞数量变化情况,Western Blotting检测肿瘤微环境中Treg细胞磷脂酰肌醇3-激酶/蛋白激酶B （PI3K/AKT）蛋白表达情况,实时荧光定量PCR （qRT-PCR）检测肿瘤微环境中Treg细胞PI3K、AKT mRNA表达情况。结果 与模型组及西黄丸低、中剂量组比较,西黄丸组分中药组肿瘤质量显著减少（P<0.05）;流式细胞术和免疫组化结果显示,肿瘤微环境中Treg细胞数量显著减少（P<0.05）;Western Blotting和qRT-PCR结果显示,肿瘤微环境中Treg细胞PI3K、AKT蛋白及mRNA表达显著下降（P<0.05）。与西黄丸高剂量组比较,西黄丸组分中药组肿瘤质量、Treg细胞数量和PI3K、AKT蛋白及mRNA表达均明显升高（P<0.05）。结论 西黄丸组分中药通过抑制肿瘤微环境中Treg细胞PI3K/AKT信号通路的表达减少Treg细胞数量,进而表现与西黄丸相似的抗癌作用和机制。     

大鼠外周血内皮祖细胞分离培养与鉴定

目的 获得大鼠外周血内皮祖细胞（EPCs）并进行鉴定。方法 首先采用密度梯度离心法获得大鼠外周血单个核细胞，然后用EGM-2完全培养基体外培养条件下诱导单个核细胞分化为靶标细胞，最后分别用EPCs特异性标记物CD133和Flk-1检测及内吞乙酰低密度脂蛋白（ac-LDL）和荆豆凝集素-1（UEA-1）的能力，识别和鉴定靶标细胞。结果 靶标细胞培养至第6天呈纺锤形，梭状排列，具有与典型EPCs形态一致的生物学特征，CD133及FLK-1双抗体荧光检测结果阳性，内吞ac-LDL及UEA-1的能力实验结果阳性，证明该纺锤形细胞为EPCs。结论 通过密度梯度离心法及多细胞因子诱导大鼠外周血单个核细胞获得EPCs的方法可行，该方法可为EPCs的基础研究及临床应用提供理论与实验方法学基础。     

自体CIK细胞对晚期不同阶段肿瘤患者免疫状态和生活质量的影响

目的 探讨自体细胞因子诱导的杀伤细胞（CIK）治疗晚期恶性肿瘤患者,对其免疫状态和生活质量的影响。方法 选取2013年2月至2016年3月合肥市第一人民医院血液肿瘤科收治的42例晚期肿瘤患者,按照预计生存期分为A组（预计生存期<3个月）20例、B组（预计生存期≥3个月）22例,检测治疗前后A、B组患者外周血T细胞亚群的变化。观察治疗前后A、B组患者免疫功能的变化,生活质量改善情况及治疗相关不良反应。结果 CIK细胞治疗后,A组患者CD₃⁺T细胞水平较治疗前降低（P<0.05）;B组治疗后CD₃⁺、CD₄⁺和CD₄⁺/CD₈⁺水平较治疗前明显升高（P<0.05）,CD₈⁺T淋巴细胞亚群水平较治疗前降低（P<0.05）。A、B两组患者治疗后KPS评分分别较治疗前均有提高,其中A组差异无统计学意义（P>0.05）,而B组差异有统计学意义（P<0.05）。42例患者在输注CIK过程中未出现明显不良反应。结论 CIK细胞回输安全、副作用小,自体CIK细胞治疗可提高晚期恶性肿瘤患者的免疫功能,改善其生活质量,但应尽早进行。     

五味子提取工艺优化及乙醇提取物的皮肤细胞抗氧化作用研究

目的 探究五味子提取物对人皮肤细胞氧化损伤的保护作用,开发其在皮肤抗氧化方面的应用。方法 分别采用热水蒸煮和水蒸气蒸馏的方法进行脱色和除味;另外,采用叔丁基过氧化氢（tBHP）诱导的HaCaT细胞氧化应激模型评价五味子提取物对人皮肤细胞氧化损伤的保护作用,采用CCK-8试剂盒检测细胞存活率,AnnexinV-FITC/PI双标记流式细胞仪检测细胞凋亡率,2,7-二氯荧光黄双乙酸盐法检测细胞内活性氧的水平。结果 采用优化后的提取工艺得到了气味变淡且颜色明显变浅的五味子提取物;用北五味子提取物预处理HaCaT细胞6 h后,可有效降低tBHP引起的细胞凋亡,提高细胞存活率,降低细胞内ROS水平。结论 优化后的乙醇提取工艺显著改善了五味子提取物的颜色和气味,并保持了提取物的抗氧化活性,因而有望成为一种应对皮肤氧化损伤的外用保护剂。     

青藤碱通过调控NF-kB信号通路抑制胰腺癌Capan-1细胞增殖并诱导细胞凋亡

目的 探讨青藤碱(Sinomenine, SIN)对胰腺癌Capan-1细胞增殖及凋亡的影响。方法 CCK8法检测细胞活力,光学显微镜下观察细胞凋亡形态,DAPI/TUNEL双染检测细胞凋亡,流式细胞术Annexin V-FITC/PI法检测细胞凋亡率,Western blot检测裂解型Caspase-3蛋白(cleaved caspase-3)、细胞核内NF-κB(Nuclear factor-kappa-binding)蛋白表达。结果 CCK8结果表明青藤碱抑制胰腺癌Capan-1细胞增殖,并具有时间浓度依赖性;光学显微镜观察结果表明,在青藤碱作用下,Capan-1细胞皱缩、变圆、脱落增多;DAPI/TUNEL染色结果表明,在青藤碱作用下,Capan-1细胞凋亡增多;流式细胞术Annexin V-FITC/PI结果表明,在青藤碱作用下,Capan-1细胞凋亡率增高;Western blot结果表明,在青藤碱作用下,Capan-1细胞cleaved caspase-3、核内NF-κB表达降低,NF-κB信号通路激活剂TNF-α逆转青藤碱对Capan-1细胞增殖的抑制作用、凋亡率的升高作用和cleaved caspase-3表达的下调作用。结论 青藤碱通过调控NF-κB信号通路抑制胰腺癌Capan-1细胞增殖并诱导细胞凋亡。     

基于人肝癌细胞系SMMC-7721的三维培养条件研究

目的 基于人肝癌细胞系SMMC-7721考察合适的海藻酸钠、氯化钙浓度,形成良好的海藻酸钙凝胶,优选三维条件下肿瘤细胞的培养条件。方法 在96孔板中培养SMMC-7721细胞,基于L₉（3⁴）正交表设计实验,以MTT法检测并计算氯化钙溶液、柠檬酸钠溶液不同浓度与作用时间下的细胞存活率;结合正交优选结果,考察适宜的海藻酸钠浓度,筛选适合的成胶、溶胶条件,并在三维培养模式下进行细胞活力确证。结果 最佳凝胶应用条件为1%氯化钙溶液与1%海藻酸钠溶液作用15 min内用于成胶,10%柠檬酸钠溶液作用凝胶10 min内用于溶胶,MTT法检测细胞存活率为94.97%。该三维培养条件下的肝癌细胞72 h内生长状态良好,存活率可达92.10%,细胞堆积紧密,出现肿瘤细胞球样聚集体。结论 本实验筛选出适合肿瘤细胞三维培养的成胶、溶胶条件。在该条件下培养肝癌细胞SMMC-7721,细胞生长状态良好,且细胞生长与聚集形态与传统二维培养存在差异,更接近细胞体内生存环境。基于该方法进行肿瘤细胞三维培养,可为深入研究肿瘤细胞生长状态,为抗肿瘤药物筛选提供更有利的方式。     

Dicranopteris linearis extract inhibits the proliferation of human breast cancer cell line (MDA-MB-231) via induction of S-phase arrest and apoptosis

Context: Dicranopteris linearis (Burm.f.) Underw. (Gleicheniaceae) has been scientifically proven to exert various pharmacological activities. Nevertheless, its anti-proliferative potential has not been extensively investigated.

Objective: To investigate the anti-proliferative potential of D. linearis leaves and determine possible mechanistic pathways.

Materials and methods: MTT assay was used to determine the cytotoxic effects of D. linearis methanol (MEDL) and petroleum ether (PEEDL) extracts at concentrations of 100, 50, 25, 12.5, 6.25 and 3.125?µg/mL against a panel of cancer cell lines (breast [MCF-7 and MDA-MB-231], cervical [HeLa], colon [HT-29], hepatocellular [HepG2] and lung [A549]), as compared to negative (untreated) and positive [5-fluorouracil (5-FU)-treated] control groups. Mouse fibroblast cells (3T3) were used as normal cells. The mode of cell death was examined using morphological analysis via acridine orange (AO) and propidium iodide (PI) double staining. Cell cycle arrest was determined using flow cytometer, followed by annexin V-PI apoptosis detection kit.

Results: MEDL demonstrated the most significant growth inhibition against MDA-MB-231 cells (IC₅₀ 22.4?µg/mL). PEEDL showed no cytotoxic effect. Induction of apoptosis by MEDL was evidenced via morphological analysis and acridine orange propidium iodide staining. MEDL could induce S phase cell cycle arrest after 72?h of incubation. Early apoptosis induction in MDA-MB-231 cells was confirmed by annexin V-FITC and PI staining. Significant increase in apoptotic cells were detected after 24?h of treatment with 15.07% cells underwent apoptosis, and the amount escalated to 18.24% with prolonged 48?h incubation.

Conclusions: MEDL has potential as a potent cytotoxic agent against MDA-MB-231 adenocarcinoma.     

Andiroba oil (Carapa guianensis Aubl) shows cytotoxicity but no mutagenicity in the ACPP02 gastric cancer cell line

Andiroba (Carapa guianensis Aubl) is an Amazonian plant whose oil has been widely used in traditional medicine for various purposes, including anti-inflammation. Research reports indicate that the oil can confer antitumor activity due to the presence of fatty acids, which can directly influence cell death mechanisms. Thus, andiroba oil (AO) has gained interest for its potential to be used in antineoplastic therapies. Here, we report an in vitro analysis of the cytotoxic and mutagenic potential of AO in the gastric cancer cell line, ACP02. Cell survival was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, differential staining with ethidium bromide and acridine orange assessed apoptosis-necrosis, and mutagenesis was assessed by the micronucleus test. The apolar oil was first diluted in 0.1% dimethyl sulfoxide (DMSO) and then further diluted to six concentrations (0.01, 0.1, 1, 10 and 100 μg/mL and 1 mg/mL) in RPMI medium. Controls included RPMI alone (negative control) and 0.1% DMSO diluted in medium (vehicle control). The MTT test showed that AO significantly reduced cell viability (P < .05) only when the highest tested concentration was applied for 48 hours. The apoptosis/necrosis test showed that the highest concentration of AO induced cell death by apoptosis at 24 and 48 hours. There was no statistically significant increase in the frequency of micronuclei. The ability of the AO to decrease the viability of ACP02 cells via apoptosis, without exerting mutagenic effects, suggests that the oil could be useful as an alternative therapeutic agent for primary tumors of stomach cancer.     

Asiatic acid exerts anticancer potential in human ovarian cancer cells via suppression of PI3K/Akt/mTOR signalling

Context Asiatic acid, a triterpenoid compound extracted from the tropical medicinal plant Centella asiatica (Family: Apiaceae), has exhibited various biological activities.

Objective This study was performed to investigate the cytotoxic effects of asiatic acid on human ovarian cancer cells.

Materials and methods SKOV3 and OVCAR-3 ovarian cancer cells were exposed to different concentrations of asiatic acid (10–100?μg/mL) for 72 or 48?h. Cell viability, colony formation, cell cycle distribution, apoptotic response were examined. Involvement of the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway was tested.

Results At the concentration of 40?μg/mL, asiatic acid caused about 50% reduction in the viability of ovarian cancer cells, but had little effect on the viability of normal human ovarian epithelial cells. Asiatic acid at 10?μg/mL reduced colony formation of ovarian cancer cells by 25–30%. Asiatic acid-treated cells showed a cell cycle arrest at the G0/G1 phase and 7- to 10-fold increase in apoptosis. The phosphorylation levels of PI3K, Akt and mTOR were remarkably lower in asiatic acid-treated cells. Overexpression of constitutively active Akt partially reversed the cytotoxic effects of asiatic acid, as evidenced by increased cell viability and colony formation. Furthermore, knockdown of Akt mimicked the growth-suppressive activity of asiatic acid.

Discussion and conclusion These results provide first the evidence for the anticancer potential of asiatic acid in ovarian cancer cells, partially via inactivation of the PI3K/Akt/mTOR pathway. Asiatic acid may represent a potential therapeutic agent for ovarian cancer.     

Synthesis and antitumor activity of novel aroylthiourea derivatives of podophyllotoxin

A novel series of 4β-[(4-substituted) aroylthiourea] derivatives of podophyllotoxin were synthesized and their abilities to inhibit the growth of cancer cells were investigated by MTT assay. Compound 4a possessed the highest cytotoxicity on HepG2, A549 and HCT-116 cancer cell lines with the IC₅₀ values of 0.1 μM. Apoptosis in HCT-116 cells induced by compound 4a was observed by Hoechst33342-Propidium iodide (PI) and acridine orange (AO)-ethidium bromide (EB) double staining assays. DNA flow cytometric analysis revealed that 4a induced cell cycle arrest at G2/M phase and kDNA decatenation assay indicated that 4a inhibited topoisomerase IIα-mediated kDNA decatenation. Our results indicated that compound 4a possessed promising antitumor activity, which need to be studied further.     

Optimization of cryopreservation procedures for rat and human hepatocytes

1. Rat hepatocytes were cryopreserved using a number of procedures and the viability, attachment, and metabolic activity of the cryopreserved cells were compared to freshly isolated hepatocytes. Several cryopreservation agents (dimethylsulphoxide [DMSO], glycerol, polyvinylpyrrolidone [PVP], dextrans), and combinations of these agents, were examined. Other variables tested included the freezing rate, thawing rate, and the concentration of serum in the freezing medium. 2. Recovery of viable attached cells was optimal using DMSO at concentrations of 10% or higher, a slow stepwise cooling procedure, and a quick thaw. The concentration of serum in the freezing medium (0% to 90%) did not affect cryopreservation results. Using this procedure the recovery of viable hepatocytes was 70%. 3. Levels of hepatocyte ethoxycoumarin-O-deethylase (ECOD) activity did not change following cryopreservation. The rate of decline of ECOD activity with time in culture was similar in freshly isolated and cryopreserved hepatocytes. 4. Hepatocytes isolated from three human livers were cryopreserved and recovered with viabilities similar to those obtained with the rat. A preliminary experiment also showed no loss of metabolic activity in human hepatocytes following cryopreservation.     

Investigation of freezing- and thawing-induced biological, chemical, and physical changes to enoxaparin solution

This study investigated the effect of freezing and thawing on the biological, physical, and chemical properties of enoxaparin solution. Solutions were frozen and thawed under different conditions, in the presence or absence of dimethyl sulfoxide (DMSO) or 1,2-propanediol (1,2-PD), and the antifactor Xa (AFXa) activity was determined. Enoxaparin solution lost more than 60% of its AFXa activity when thawed rapidly after freezing at -196 degrees C. The loss of AFXa activity was less with higher freezing temperatures and increased with the number of freeze/thaw cycles, but was independent of the duration of freezing. Slow freezing to -196 degrees C with rapid thawing, or rapid freezing with slow thawing, resulted in negligible loss of AFXa activity. The loss of AFXa activity did not involve the loss of N-sulfate groups, the breakdown of glycosidic bonds or the glassy state transition. Controlling the freezing or thawing conditions, dilution with water or addition of a small percentage of DMSO ameliorated the loss of enoxaparin AFXa activity. The loss in AFXa activity was found by size exclusion chromatography to be primarily due to aggregation and was reversed by sonication in the presence of DMSO. These results may provide insight into solutions for the long-term storage of concentrated or diluted enoxaparin.     

吲哚-1,3,4-噁二唑类衍生物的合成及其体外抗肿瘤活性研究

目的 合成吲哚-1,3,4-噁二唑类衍生物,并进行体外抗肿瘤活性研究。方法 以吲哚-3-甲酰肼为起始原料,通过[4+1]环加成反应、水解反应、缩合反应得到目标化合物。采用MTT法测试目标化合物对HeLa、SCG-7901和MDA-MB-231细胞的体外抗肿瘤活性。采用克隆形成实验考察化合物6f对SCG-7901细胞增殖的影响。采用Annexin V-APC/PI双染法检测化合物6f对SCG-7901细胞凋亡和坏死的影响。采用DAPI染色法检测化合物6f对SCG-7901细胞凋亡核染色质形态学的影响。采用DCFH-DA染色法检测化合物6f对SCG-7901细胞中活性氧含量的影响。结果 合成了15个吲哚-1,3,4-噁二唑类衍生物,其结构经¹H-NMR、¹³C-NMR和HR-ESI-MS确证。部分化合物表现出良好的抗肿瘤活性,其中化合物6f对HeLa、SCG-7901和MDA-MB-231 3种肿瘤细胞株均表现出明显的抗增殖作用,且具有一定的选择性。进一步研究表明,化合物6f以浓度相关的方式促进细胞产生活性氧,抑制细胞增殖,诱导细胞凋亡。结论 部分吲哚-1,3,4-噁二唑类衍生物表现出良好的抗肿瘤活性,为该类化合物的进一步抗肿瘤活性研究提供思路。     

细胞因子诱导的杀伤细胞对Raji细胞的杀伤效应及其机制的实验研究

目的探讨细胞因子诱导的杀伤（CIK）细胞对Raji细胞的杀伤效应及其作用机制。方法采用MTT法测定CIK细胞对Raji细胞的杀伤效应。用免疫组化测定bcl-2蛋白的表达。结果随着CIK细胞和Raji细胞效靶比例增加及时间的延长，CIK细胞明显增强其对Raji细胞的杀伤活性，与对照组相比差异有显著性（P〈0．01），bcl-2蛋白的表达随时间的延长而下降，与对照组相比有显著性差异（P〈0．01）。结论CIK细胞对Raji细胞有较强的杀伤活性。杀伤机制可能与下调bcl-2蛋白的表达有关。