论文标题:HAP纳米粒子对肝癌细胞增殖及内质网影响的研究
Research on Effect of HAP Nano-particles on Proliferation and Endoplastimic Reticulum of Hepatocellular Carcinoma Cells
论文作者 张然
论文导师 李世普,论文学位 硕士,论文专业 生物医学工程
论文单位 武汉理工大学,点击次数 79,论文页数 66页File Size12030k
2005-05-01论文网 http://www.lw23.com/lunwen_33414602/ 纳米粒子;细胞集落;内质网;肝癌细胞;激光共聚焦扫描显微镜
Nano-particles; Cell Clong; Endoplastimic Reticulu;Hepatocellular carcinoma Cells; Laser Scanning Confocal Microscope
纳米粒子对肿瘤细胞增殖的抑制作用是其本身的一种纳米生物学效应,也是生物中心多年来的研究课题。本论文围绕这一课题,利用均相沉淀法合成了羟基磷灰石(HAP)、掺锶磷灰石(Sr-HAP_1)、锶钙磷灰石(Sr-HAP_2)、二氧化钛(TiO_2)四种不同成分的纳米粒子,还控制试验条件制备了四种不同粒径的羟基磷灰石(HAP)粒子,即HAP_1、HAP_2、HAP_3和HAP_4。利用MTT实验法筛选出了对Bel-7402肝癌细胞增殖抑制明显而对L-02肝细胞增殖抑制不明显,分散均匀,生物相容性好,可以在体内代谢的粒径在100nm以内的HAP_1纳米粒子作为后期实验的材料基础。运用生长曲线法和集落生成率法检测三种不同浓度的HAP_1纳米粒子对Bel-7402肝癌细胞增殖的抑制作用和对细胞克隆原的抑制作用;借助倒置显微镜观察HAP_1纳米粒子作用后Bel-7402肝癌细胞发生的形态变化;利用激光共聚焦扫描显微镜,通过荧光标记的手段,定性、定量的研究了HAP_1纳米粒子作用前后Bel-7402肝癌细胞中内质网的变化。最后得出结论:1.HAP_1纳米粒子对Bel-7402肝癌细胞的增殖具有抑制作用,且这种抑制作用有剂量依赖和时间依赖性。2.不同浓度的HAP_1纳米粒子作用后,Bel-7402肝癌细胞形成集落的能力明显下降,对照组的集落形成率为97.78±1.50%,而HAP_(1-1)组、HAP_(1-2)组、HAP_(1-3)组的集落形成率分别为73.11±1.67%、40.89±2.16%、15.44±2.50%,HAP_(1-2)组和HAP_(1-3)组与对照组相比有显著差异(p<0.05),形成集落的直径和面积上较对照组明显减小(p<0.01)。3.不同浓度的HAP_1纳米粒子作用后,Bel-7402肝癌细胞内出现了多少不等的空泡,空泡间互相融合,最终导致细胞死亡。4.HAP_1纳米粒子作用后,Bel-7402肝癌细胞中内质网的数量与分布发生变化,内质网的荧光强度值和平均荧光值都较对照组明显减少,推测HAP-1纳米粒子通过影响Bel-7402肝癌细胞内质网结构和功能对细胞的转录产生影响,抑制蛋白质的合成,致使肿瘤细胞的代谢受阻,从而改变细胞的分化过程,影响肿瘤细胞的增殖或生存。
Inhibition effects on proliferation of cancer cells is a kind of biological effect of nano-particles himself, and it had been studies by the Biomedical Materials and Engineering Research Center of Wuhan University of Technology for many years. In this study, different component nano-particles, HAP, Sr-HAP_1, Sr-HAP_2 and TiO_2, were prepared by the homogeneous precipitation, and different size HAP particles, HAP_1, HAP_2, HAP_3 and HAP_4, were prepared by different experiment condition controlled. According to the character of nano-particles that was distinct inhibition on proliferation of Bel-7402 hepatocellular carcinoma cells and mild inhibition on L-02 hepatocytes, HAP_1 nano-particles which average size was less-than 100nm and were well distributed, and could be metabolized in vivo, were selected as the material basis for future research. Method of growth curve and cell clone were used to detected the inhibition on proliferation and rate of cell clone formed of Bel-7402 hepatocellular carcinoma cells treated with different concentration HAP_1 nano-particles. Morphology change was observed through microscope after treated with different concentration HAP_1 nano-particles. Laser scanning confocal microscope (LSCM) was used to research endoplastimic reticulum (ER) in Bel-7402 hepatocellular carcinoma cells qualitatively and quantitatively after treated with different concentration HAP_1 nano-particles. The conclusions are received. 1. Proliferation of Bel-7402 hepatocellular carcinoma cells is inhibited treated with HAP_1 nano-particles, and the inhibition is dose-dependent and time-dependent in range of concentration. 2. Rate of cell clone formed is descent after treated with HAP_1 nano-particles, rate of cell clong formed of control group is 97.78 ± 1.50%, and the rate of different concentration HAP_1 nano-particles, HAP_(1-1) group, HAP_(1-2) group and HAP_(1-3) group, are 73.11 ± 1.67%, 40.89±2.16% (p<0.01, compared with control group) and 15.44±2.50% (p<0.01, compared with control group) respectively. Radiu and area of cell clone are distinct decreased (p<0.01) compared with control group. 3. Vaculoes are observed in cells after treated with different concentration HAP_1 nano-particles. Then many vacuoles are fused, which perhaps causes Bel-7402 hepatocellular carcinoma cellsdeath. 4. Quantity and distribution of ER in Bel-7402 hepatocellular carcinoma cells are changed treated with different concentration HAP_1 nano-particles. Number of fluorescence intension and average fluorescence of treated groups are distinct decreased compared with control group. Formation and function of ER are varied after treated with HAP_1 nano-particles, which affect cell transcription, inhibit protein synthesis, block cell metabolism, change the process of cell differentiation and made cells death.
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