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。     

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.     

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。     

Selective Anti-Hepatoma Treated with Titanium Oxide Nanoparticles in vitro

1　ＩｎｔｒｏｄｕｃｔｉｏｎＳｏｍｅｉｎｏｒｇａｎｉｃｎａｎｏｐａｒｔｉｃｌｅｓｅｘｈｉｂｉｔａｎａｎｏｂｉｏｌｏｇｉ ｃａｌｅｆｆｅｃｔ[1,2 ] ,ｆｏｒｅｘａｍｐｌｅ ,ｗｉｔｈｉｎａｃｅｒｔａｉｎｓｉｚｅ ,ｐａｒｔｉｃｌｅｓｃａｎａｆｆｅｃｔｃａｎｃｅｒｃｅｌｌ’ｓｂｉｏｌｏｇｉｃａｌｃｈａｒａｃｔｅｒｉｓｔｉｃｓｓｕｃｈａｓｇｒｏｗｔｈａｎｄｐｒｏｌｉｆｅｒａｔｉｏｎ[3] .Ｓｏｆａｒ ,ｔｈｅｅｆｆｅｃｔｏｆＴｉＯ2ｎａｎｏｐａｒｔｉｃｌｅｏｎｈｅｐａｔｏｍａｃｅｌｌｓｈａｓｎｏｔｂｅｅｎｒｅｐｏｒ…     

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.     

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.     

Selective anti-hepatoma treated with titanium oxide nanoparticles in vitro

Effects of titanium oxide (TiO₂) nanoparticles on Bel-7402 human hepatoma hepatoma cells and L-02 human hepatocytes at different times were observed. Using cell culture, cell growth curves of Bel-7402 cells and L-02 cells treated with TiO₂ nanoparticles were examined by MTT assay, and the cellular ultrastructure was observed by an analytical transmission electron microscope (ATEM). It is found that OD value of Bel-7402 cell treated with TiO₂ nanoparticles for 48–144h is obviously lower than that of control group (p<0.01). However the growth curve of L-02 cells is almost not affected by TiO₂ nanoparticles. ATEM and energy dispersive X-ray (EDX) analyses show that there are obvious vacuoles increased heterolysosome, and particles with high electron density which are confirmed to be TiO₂ nanoparticles in Bel-7402 cytoplasm. More interestingly, it is alse found that TiO₂ nanoparticle obviously inhibits the proliferation of hepatoma cells by altering lysosome activity and destroying cytoplasm structure. The inhibition on proliferation of hepatocytes by TiO₂ nanoparticles is much slighter. The results demonstrate that TiO₂ nanoparticle has different killing effects on cancer cell and normal cell. CAO Xian-ying: Born in 1962 Funded by the National Natural Science Foundation of China (No:39770225)     

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.     

Ultrastructure Changes and Mechanism of Cancer Cells Induced by Inorganic Crystal Nanoparticles

1Introduction Itwasreportedthatnanoparticlescankillcancer cell[1],andTiO2nanoparticlescaninhibittheproliferation oftumorcells[2,3].Hydroxyapatite(HAP)nanoparticles wasreportedthatcouldinducethedeathofhepatoma cells[4,5].Buthowthecancercelldiedisnotstillun…     

Change in Shape and Crystal Structure of HAP Nanoparticles during Absorption into Cell

The change of hydroxyapatite （HAP） nanoparticles in shape and crystal structure after endocytosis into cancer cells was studied. BEL7402 cells were incubated with HAP nanoparticles for 2 hour, 8 hours, 20 hours, respectively. Then, the cells were collected and viewed under a transmission electronic microscope （TEM）. Electronic diffraction （ED） attached to TEM was used to detect the properties of the particles. The results show that HAP particles in the cytoplasm can be degraded in cytoplasm. The degradation process is prolonged by more than 20 hours. Thus, it is concluded that HAP nanoparticles would be degraded after kill cells or delivery gene.     

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.     

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.     

HAP纳米粒子与传统抗癌药物的抗癌效果比较

采用MTT法检测HAP纳米粒子及5种传统化疗药物对体外培养肝癌Bel-7402细胞及正常对照细胞的敏感性，研究对比HAP纳米粒子及传统抗癌药物对体外肝癌细胞的作用特点。在相同的作用时间内，不同浓度的常用抗癌药物大部分表现出良好的抑癌效果，而HAP纳米粒子较大部分化疗药物对肝癌细胞抑制作用低，但对于正常对照细胞无影响，结果HAP纳米粒子能够作为一种抗癌药物，与传统抗癌药物相比，它存在着优越性。     

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.     

Study on the Variation of Calcium and Phosphorus in Nano Hydroxyapatie Treated Cancer Cells through Synchrotron Radiation XRF

1Introduction Synchrotronradiation(SR)isanexcellentsource withhighbrightnessforX rayfluorescence(XRF).Syn chrotronradiationX rayisappliedaslightresourceofSR XRF.TheSR XRFisgeneratedbyhighenergyelectron accelerator.Itoffershighsensitivityandspatialresoluti…     

Inhibition of the growth of human hepatoma cell line both in vitro and in vivo by transducing CKI gene p21WAF-1 with GE7 targeting gene delivery system

The EGF receptor-mediated targeting gene delivery system GE7 was used to transduce exogenous gene pCEP-p21WAF-1 into human hepatocellular carcinoma cell both in vitro and in vivo. After in vitro transduction of the exogenous gene, the growth of the cell lines SMMC-7721 and BEL-7402 was significantly inhibited compared with the control. On day 8 the inhibition rates of the above cell lines reached 56.0% and 66.7%, respectively. The in vivo experiment showed that the growth of human hepatoma transplanted in nude mice injected with GE7 gene delivery system subcutaneously once a week for 3 weeks was remarkably inhibited compared with that of untransfected control. The average tumor weight of the experiment group was (0.083 ± 0.043) g, while that of the control group was (0.281± 0.173) g. The difference is significant (P<0.05). It was indicated that GE7 gene delivery system could efficiently transduce exogenous gene pCEP-p21WAF-1 into hepatoma cell with high EGF receptor expression, and inhibit the cell growth with high efficacy both in vivo and in vitro.     

Titanium Dioxide Nanoparticle Absorbed by Hepatoma Cells in Vitro

It is reported thut nanoporticles can be applied as carriers and anti-cancer medicines. But the interaction of nanoparticles and cells is unclear. The purpose of this study was to discuss whether inorganic crystal nanoparticles can get through cells with intact crystal. BEL7402 heputoma cells and titanium dioxide （ TiO2 ） nanoporticles were selected and incubated together in vitro. All specimens were prepared and observed under a traasmission electron mieroscope （TEM）. TiO2 nanopartieles were found not in the nuclear area but in the cytoplasma. TiO2 nanoponieles maintained the plate-like shape during absorbing. The result shows that hepatoma cells can endocytose the intact TiO2 crystal nanoparticles. It implies that novel nano-effect plays an important role in the biomedicinal application of inorganic crystal nanopartieles.     

Titanium dioxide nanoparticle absorbed by hepatoma cells in vitro

It is reported that nanoparticles can be applied as carriers and anti-cancer medicines. But the interaction of nanoparticles and cells is unclear. The purpose of this study was to discuss whether inorganic crystal nanoparticles can get through cells with intact crystal. BEL7402 hepatoma cells and titanium dioxide (TiO₂) nanoparticles were selected and incubated together in vitro. All specimens were prepared and observed under a transmission electron microscope (TEM). TiO₂ nanoparticles were found not in the nuclear area but in the cytoplasma. TiO₂ nanoparticles maintained the plate-like shape during absorbing. The result shows that hepatoma cells can endocytose the intact TiO₂ crystal nanoparticles. It implies that novel nano-effect plays an important role in the biomedicinal application of inorganic crystal nanoparticles. Funded by National Natural Science Foundation of China (No. 50472040)     

Preparation of Ca_9Sr(PO_4)_6(OH)_2 Nanoparticles and Effects on Proliferation of Bel-7402 and L-02 Cells in vitro

1Introduction Hydroxyapatite(Ca10(PO4)6(OH)2,HAP)isthe maincomponentofinorganicmineralinanimalsclerous tissue.ArtificialHAPiswidelyusedassubstituteorfill ingmaterialforscleroustissuesbecauseofitsgoodbio compatibilityandosteoconductivity[1,2].Itisalsousedi…     

Size Depending Separation of HAP:Eu Nanoparticles in Dispersed Sol

1Introduction Hydroxyapatite(Ca10(PO4)6(OH)2)isanimportant biomaterialthatiswidelyusedassubstituteorfillingma terialforscleroustissuesduetoitsgoodbiocompatibility andosteoconductivity[1,2].Inordertoinvestigatethein teractionsbetweenHAPnanoparticleswithana…