纳米羟基磷灰石对肝癌细胞端粒酶基因表达的影响

研究羟基磷灰石（HAP）纳米粒子对Bel-7402人肝癌细胞端粒酶基因表达的影响。采用均相沉淀法制备出稳定单分散的HAP纳米粒子,应用透射电镜、电位粒度仪对其进行表征。HAP纳米粒子作用Bel-7402肝癌细胞4 h后,采用原位杂交技术检测Bel-7402肝癌细胞的端粒酶基因表达。结果表明HAP纳米粒子作用组的Bel-7402肝癌细胞的端粒酶阳性细胞比例为61.38%,而对照组的端粒酶阳性细胞比例为87.89%,2组有显著性差异（P〈0.01）。HAP纳米粒子可使Bel-7402肝癌细胞的端粒酶基因表达下调。     

羟基磷灰石纳米粒子对肝癌细胞PCNA表达的影响

为了研究羟基磷灰石(HAP)纳米粒子对肝癌细胞增殖细胞核抗原表达的影响,采用均匀沉淀法制备了均匀分散的纳米尺度的HAP纳米粒子。以0.56 mmol/L的HAP纳米粒子与Bel-7402肝癌细胞作用后,采用流式细胞技术检测细胞周期时相的变化,行免疫细胞化学法PCNA染色,形态学观察和定量分析细胞的PCNA表达。结果显示HAP纳米粒子能使Bel-7402细胞的细胞增殖周期阻滞于G1期,PCNA表达降低,与对照组比较有显著性差异(P<0.01)。HAP纳米粒子可能通过抑制PCNA的表达,起到抑制肝癌细胞增殖的作用。     

GSK-3抑制剂增强携带TRAIL的溶瘤腺病毒对肝癌细胞的杀伤作用

研究携带TRAIL基因的溶瘤腺病毒联合化疗药物GSK-3抑制剂(氯化锂和SB-415286)对人肝癌细胞的体外杀伤作用.采用MTT法检测病毒ZD55-TRAIL联合化疗药物氯化锂和SB-415286对肝癌细胞株HepG-2、BEL-7404以及正常细胞株L02、QSG-7701增殖的抑制作用,并通过结晶紫实验检测进一步证明联合用药的杀伤作用和安全性;利用Hoechst33342染色对肝癌细胞株BEL-7404的细胞凋亡进行形态学观察;通过Western blot检测肝癌细胞HepG-2细胞凋亡信号通路的变化.结果表明:低剂量的氯化锂和SB-415286能显著提高ZD55-TRAIL对肝癌细胞株HepG-2、BEL-7404的杀伤作用,对人体肝正常细胞株L02、QSG-7701无明显的抑制作用.说明GSK-3抑制剂能够解除肝癌细胞对TRAIL的抗性,增强携带TRAIL基因的溶瘤腺病毒对肝癌细胞的杀伤.     

基于金纳米粒子修饰的喜树碱纳米药物的构建及其抗癌活性

金纳米粒子具有抗癌及高光热转换效率的潜能,以透明质酸为稳定剂、金纳米粒子为还原及固定层,将化疗与光热疗法两种模式进行组合,合成了金纳米粒子修饰的纳米药物晶体(CPT-HA-AuNPs)。利用DLS、SEM等技术对CPT-HA-AuNPs的理化性质及药物控释行为进行表征,利用MTT、细胞摄取实验及AnnexinV-FITC/PI双染法对其生物活性进行了研究。结果表明,CPT-HA-AuNPs呈杆状纳米晶体结构,粒径(238.60±4.51) nm, Zeta电位(-47.29±0.84) mV,显示出较好的稳定性;具有较高的光热转换效率,并展现出光热及pH双响应性释放行为。与对照组相比,CPT-HA-AuNPs在NIR光激发条件下表现出显著提高的MDA-MB-231细胞摄取率、体外抗癌活性及促凋亡作用。     

二氯乙酸钠对溶瘤腺病毒ZD55-Smac抑制肝癌细胞生长的影响

研究了溶瘤腺病毒ZD55-Smac联合小分子药物二氯乙酸钠(DCA)对人肝癌细胞株的体外抑制效果.首先通过PCR及Western blot鉴定病毒构建正确与否及有无野毒污染,再用MTT 法分别检测DCA、ZD55、ZD55-Smac以及DCA与病毒联合对肝癌细胞株Huh-7、PLC、SMMC-7721及肝正常细胞QSG-7701生长的抑制作用;通过倒置显微镜观察了DCA、ZD55-Smac以及DCA和ZD55-Smac联合使用对肝癌细胞株PLC及肝正常细胞QSG-7701的细胞形态变化的影响;利用Hoechst33342染色观察了所处理细胞的凋亡形态学变化.结果表明DCA联合ZD55对肝癌细胞株Huh-7、PLC和SMMC-7721的抑制效果相比较单药物治疗或单病毒治疗有显著增加,而DCA联合ZD55-Smac对肝癌细胞的抑制效果比单病毒治疗有所减弱.该研究为进一步探究ZD55-Smac联合药物治疗打下基础.     

纳米HAP粒子在水介质中的分散稳定性研究

在水分散体系中采用化学沉淀法制备纳米羟基磷灰石（Hydroxyapatite，HAP）粒子，研究了分散介质pH值、稳定剂或表面改性剂及其分子量等因素对纳米粒子粒径及分散稳定性的影响，重点讨论了阴离子表面活性剂PAA-Na（聚丙烯酸纳）对纳米HAP溶胶分散稳定性的影响。同时对纳米HAP溶胶分散稳定性机理在理论上作了探讨。     

四臂星型聚乙二醇/聚乳酸的合成及其负载羟基喜树碱研究

以四臂聚乙二醇为引发剂,辛酸亚锡为催化剂,通过开环聚合反应制备四臂星型聚乙二醇/聚乳酸共聚物。通过透析法制备了负载抗肿瘤药物羟基喜树碱的四臂星型聚乙二醇/聚乳酸纳米粒子,并对其粒径分布、载药量、稳定性、体外释药以及体外细胞毒性等特性进行了考察。结果表明,载药纳米粒子的粒径小于200 nm,粒径分布均匀,稳定性良好,在体外的药物缓释效果显著。体外细胞毒性实验结果表明,载药纳米粒子表现出优异的肿瘤细胞增殖抑制效果。     

聚碳酸酯纳米药物的制备与性能

采用可生物降解高分子材料链接或包埋抗癌药物制备高分子纳米或微米药物,不仅可以提高药物的抗癌功效而且能减小药物对正常组织的毒副作用.以9 苯基 2,4,8,10 四氧螺\[5,5\]十一烷 3 酮与2,2 二甲基二亚甲基合成的羟基化的碳酸酯共聚物P(PTC co DTC)为载体,将肿瘤靶向基团叶酸通过化学反应键连在聚碳酸酯的侧链,从而制备肿瘤靶向性高分子载体,并进行了傅立叶红外光谱、核磁共振氢谱、紫外 可见光谱等结构表征.再将靶向高分子载体与5 氟尿嘧啶复合,采用高压电场喷雾法与透析法分别制得两种肿瘤靶向聚碳酸酯纳米抗癌药物,并初步研究了纳米抗癌药物的体外药物控制释放性能.研究结果表明碳酸酯纳米药物具有较好的药物释放性能,且高压电场喷雾法制备的纳米药物比透析法制备的纳米药物释放药物速率快.     

双靶向溶瘤腺病毒联合奥沙利铂对肿瘤细胞凋亡的研究

研究化疗药物奥沙利铂(Oxaliplatin)联合AdCN205-IL-24对人结肠癌细胞的体外杀伤作用。实验采用MTT法检测肿瘤特异性增殖腺病毒AdCN205-IL-24联合奥沙利铂对两种肿瘤细胞以及正常细胞增殖的抑制作用;Hoechest33342染色,在荧光显微镜对病毒联合化疗诱导的肿瘤细胞凋亡进行形态学观察。结果表明:AdCN205-IL-24联合奥沙利铂处理4 d后对结肠癌细胞株HT-29和SW620的增殖抑制率达到23.17%和22.98%,并且联合化疗并未增加对人正常细胞株L-02的毒性。     

双基因溶瘤腺病毒联合5-FU抑制肺癌细胞增殖的研究

研究携带TRAIL和Smac的双基因溶瘤腺病毒（ZD55-TRAIL-IETD-Smac）联合化疗药物5-氟尿嘧啶（5-FU）对肺癌细胞的体外杀伤作用。通过MTT法检测细胞抑制率,流式细胞术检测细胞凋亡率,Western Blot检测杀伤性基因表达与凋亡相关蛋白表达。结果显示ZD55-TRAIL-IETD-Smac与5-FU联合应用能有效地抑制肺癌细胞的增殖,并通过激活Caspase通路诱导肺癌细胞产生凋亡,表明,5-FU能够显著增强携带TRAIL和Smac的双基因溶瘤腺病毒对肺癌细胞的杀伤作用,为肺癌的临床应用提供了参考。     

Cytotoxinic Mechanism of Hydroxyapatite Nanoparticles on Human Hepatoma Cell Lines

1　IntroductionWiththedevelopmentofthenanometerscienceandtechnology ,theuseandstudyofthenano biomaterialsonmedicalsciencebegintoshowitsbrillianttalents .Itwasfoundthatsomeinorganicnanoparticles[1] havethenano biologicaleffectascomparedwithnon nanometer[2 ] .Whenthoseinorganicnanoparticlesaresmalltothenano level,theycaninhibittheproliferationofcancercells ,andatthesametimetheyaffectedthenormalcellslittle .Tofur therconfirmthebiologicalcharacterofthenanoparticle ,thefollowingexperimentshavebeenp…     

Apoptosis of Cancer Cells Induced by HAP Nanoparticles

To confirm apoptosis is one of the hepatoma cells death pathways after HAP nanoparticles absorption,hepatoma cells were collected for ultrathin sections preparation and examined under a transmission electron microscope（TEM）after 1 h incubation with HAP nanoparticle.Apoptosis was detected by TUNEL technique.After absorption.some vacuoles with membrane containing HAP nanoparticles were found in cytoplasma.The nuclear enrelope shrinked.and some area pullulated from nucleus.The karyotin became pycnosis and assembled at the edge.An apoptosis body was found.and the data of IOD and numbers of the positive apoptosic signals in nuclear area of slides could illustrate much more apoptosis in the HAP group than those in the control group（P〈0.001）.The experimental results indicate that the HAP nanoparticles can induce cancer cells apoptosis.     

Effects of the interaction between hydroxyapatite nanoparticles and hepatoma cells

To gain a better understanding of the anticancer effects of hydroxyapatite(HAP) nanoparticles in vivo and in vitro, the effects of the interaction of HAP nanoparticles with hepatoma cells were explored. HAP nanoparticles were prepared by homogeneous precipitation and characterized by laser particle analysis and transmission electron microscopy(TEM). HAP nanoparticles were observed to be uniformly distributed, with rod-like shapes and diameters in the range of 42.1-87.1 nm. Overnight attached, suspended, and proliferating Bel-7402 cells were incubated with HAP nanoparticles. Inverted microscopy observation revealed that HAP nanoparticles with a cell membrane showed good adsorption. TEM demonstrated that HAP nanoparticles were present on the surface of cells, continuously taken up by cells through endocytosis, and transported in vesicles close to the nucleus. Fluorescence microscopy showed that the concentrations of intracellular Ca2+ labeled with Fluo-3 calcium fluorescent probe were significantly enhanced. In addition, inverted microscopy observation revealed that suspended cells treated with HAP nanoparticles did not adhere to the culture bottle, resulting in cell death. After the overnight attached cells were treated with HAP nanoparticles for 96 h with increasing doses of HAP nanoparticles, inverted microscopy observation revealed that cell proliferation was slowed and cell–cell adhesion was weakened. Feulgen staining and image analysis indicated that the nuclear DNA content of the cells was markedly reduced, and argyrophilic nucleolar organizer region(AgNOR) staining and image analysis indicated that the number of AgNORs was signifi cantly decreased. Therefore, hepatoma cells brought about the adsorption, uptake, transport and degradation of HAP nanoparticles. In addition, HAP nanoparticles affected hepatoma cells with regard to cell–cell adhesion, cell and extracellular matrix adhesion, and DNA and protein synthesis; thus inhibiting cell proliferation. This understanding of the effects of interaction between HAP nanoparticles and hepatoma cells is useful for further study of the anticancer mechanisms of HAP nanoparticles.     

无机纳米粒子进入后肝癌细胞超微结构的变化

将生物性状较为稳定的二氧化钛（TiO2）和生物相容性较好的羟基磷灰石（HAP）纳米粒子,与肝癌细胞共同孵育1 h和8 h后,收集细胞并制样,用透射电镜观察肝癌细胞超微结构的变化.结果发现肝癌细胞质内有纳米粒子的团块,周围的细胞质有自行溶解现象,内质网过度肿胀,线粒体肿胀崩解、嵴结构紊乱,核膜周间隙扩大、核固缩等死亡现象.     

Investigation of HAP Nanoparticles Absorbed by Hepatoma Cells in vitro

Many particles are found in the cytoplasm area after the mixture of hydroxyapatite(HAP) nanoparticles and cultured cancer cells.The purpose of this study was to confirm whether these particles in cytoplasm are HAP nanoparticles exactly.BEL7402 cells were incubated in HAP sol for 8 hours.Then,the cells were collected for specimen preparation.Transmission electron microscope(TEM),energy dispersing spectrum (EDS)and electronic diffraction(ED)attached to TEM were used to detect the properties of the particles.It is found that many particles similar to HAP in shape are in the cytoplasm under TEM.By EDS analysis,they are the particles containing calcium(Ca)and phosphorus(P).The classic rings of HAP crystal appear in the ED pictures of these particles.So the particles are confirmed as HAP nanoparticles.Thus,it is concluded that HAP nanoparticles as the crystal particles can be absorbed by hepatoma cells.     

Investigation of HAP Nanopartieles Absorbed by Hepatoma Cells in vitro

Many particles are found in the cytoplasm area after the mixture of hydroxyapatite （HAP） nanoparticles and cultured cancer cells. The purpose of this study was to confirm whether these particles in cytoplasm are HAP nanoparticles exactly. BEL7402 cells were incubated in HAP sol for 8 hours. Then, the cells were collected for specimen preparation. Transmission electron microscope （TEM）, energy dispersing spectrum （EDS） and electronic diffraction （ED） attached to TEM were used to detect the properties of the particles. It is found that many particles similar to HAP in shape are in the cytoplasm under TEM. By EDS analysis, they are the particles containing calcium （Ca） and phosphorus （P）. The classic rings of HAP crystal appear in the ED pictures of these particles. So the particles are confirmed as HAP nanoparticles. Thus, it is concluded that HAP nanoparticles as the crystal particles can be absorbed by hepatoma cells.     

Effect of hydroxyapatite nanoparticles on K562 cells in vitro

Stable and single-dispersed hydroxyapatite （HAP） nanoparticles were synthesized with ultrasonic-assisted method. HAP nanoparticles were characterized by dynamic light scattering, XRD （X-ray diffraction） and TEM （Transmission Electron Microscopy）. The effect of HAP nanoparticles on the K562 human myelogenous leukemia cell line was investigated by MTT assay and cell count test, and the mechanism was studied through the changes of cell cycle and ultrastructure. The results showed that HAP nanoparticles inhibited the proliferation of K562 cells dramatically in vitro. HAP nanoparticles entered the cytoplasm of K562 cells and the cells were arrested at G/M phase, thus, the cells died directly.     

Effects of Antihepatocarcinoma with Apatite Nanoparticles in vivo

The inhibition effect of hydroxyapatite （ HAP ） nanoparticles on hepatocarcinoma was investigated in vivo. The human hepatocarcinoma cell line Bel- 7402 was transplanted subcutaneously into nude mice. Hydroxyapatite nanoparticles suspension at a dose of 0. 2 mL was injected into the transplanted tumors every day for 2 weeks, and saline was used us control. The efficacy of hydroxyapatite nanoparticles on this carcinoma was surveyed and morphological changes of tissue and cells were observed by light microscopy and transmission electron microscopy （TEM）. Experimental results show that hydroxyapatite nanoparticles have a visible destructive effect on the structures of hepatocarcinoma cells and tissue. The inhibition rates of tumor growth were 77.21% and 51. 32% after intra-tumor injection of hydroxyapatite nanoparticles for 1 week and 2 weeks, respectively. Compared with the control group, hydroxyapatite nanoparticles can also prolong the survival time of the nude mice bearing this cancer significantly. This indicates that hydroxyapatite nanoparticles have the therapeutic potential on hepatoma in vivo.