课题背景：本课题受广东省自然科学基金资助，基金编号为O5004763。RNA干扰技术是一项高效率高特异性的基因沉默技术，随着siRNA 合成、转染方法的极大改进，RNA干扰快速成为基因功能研究及其治疗应用的强有力工具。但是siRNA不稳定，容易被血清酶降解，难以到达靶器官而发挥治疗作用。国内外广泛展开siRNA载体系统的研究，壳聚糖纳米粒载体系统是热点之一，旨在寻找一种能够应用于临床的治疗系统。

应用要点：①将纳米技术与RNA干扰技术相结合，应用于治疗脑恶性胶质瘤，为脑恶性胶质瘤的综合治疗建立新的技术平台。②通过吐温-80对脑肿瘤的靶向性，制备靶向性纳米粒。③初次将壳聚糖-siRNA纳米粒应用于脑恶性胶质瘤细胞的治疗研究，从方法和技术上进行探索。

同行评价：纳米材料可以作为RNA干扰的载体系统。高级别恶性胶质瘤手术后复发率高，化疗效率低，且高表达端粒反转录酶。所以在恶性胶质瘤中进行RNA干扰具备良好前景。本实验采用RNA干扰技术，针对端粒反转录酶(hTERT)-mRNA构建壳聚糖（CS）-siRNA纳米粒，转导入胶质瘤U251细胞，发现CS-siRNA-hTERT对胶质瘤U251细胞有抑制作用，作用机制可能与促使肿瘤细胞凋亡，干扰细胞进入S期等。本实验对RNA干扰在恶性胶质瘤中应用有一定意义。

摘要

目的：预防胶质瘤术后复发采取化学疗法和放射治疗，因敏感性低及胶质瘤对其产生抵抗，并未提高恶性胶质瘤患者的预后。目前广泛开展的基因治疗研究，有望取得突破。观察靶向端粒反转录酶(human telomerase reverse transcriptase，hTERT)基因的壳聚糖(chitosan,CS)- 小干扰RNA（small interferening RNA,siRNA）纳米粒在体外进行RNA干扰对胶质瘤U251细胞增殖及凋亡的影响。
方法：实验于2006-09/2007-07在广东省神经外科研究所（重点实验室）完成。制备针对hTERT基因的CS-siRNA纳米粒，原子力显微镜观察纳米粒的形态及大小。按转染试剂的不同分为8组。空白对照组(不含转染试剂及siRNA)，阳性对照组(LipofectamineTM 2000转染siRNA)，阴性对照组(含30 nmol/L阴性对照siRNA的纳米粒)，裸siRNA对照组(含30 nmol/L siRNA)，空白CS纳米粒对照组，10，30，90 nmol/L siRNA纳米粒组。取对数生长期的U251细胞转染后，采用CCK-8法绘制细胞增殖曲线并计算抑制率；Hoechst 33342/碘化丙啶双荧光染色后，荧光显微镜下观察细胞核形态；流式细胞仪检测细胞周期；反转录-聚合酶链反应法检测hTERT-mRNA表达变化。
结果：①原子力显微镜下CS-siRNA纳米粒，呈球形，形状一致，大小约87 nm。②阳性对照组、30，90 nmol/L siRNA纳米粒组细胞增殖明显受抑制，与其余各组比较差异有显著性（P < 0.05）。③Hoechst 33342/碘化丙啶双染荧光显微镜下可见空白对照组、阴性对照组、裸siRNA对照组和空白CS纳米粒对照组大多为低蓝色的正常细胞；阳性对照组、10，30，90 nmol/L siRNA纳米粒组以高蓝色的凋亡细胞为主。④阳性对照组、10，30，90 nmol/L siRNA纳米粒组与空白对照组、阴性对照组、裸siRNA对照组、空白CS纳米粒对照组相比，G0/G1期细胞显著增加，S期细胞明显减少，阳性对照组最为明显(P < 0.05)，G2/M期细胞变化不明显(P > 0.05)。⑤阳性对照组、10，30，90 nmol/L siRNA纳米粒组hTERT mRNA表达明显下调，30，90 nmol/L siRNA纳米粒组表达下调较10 nmol/L siRNA纳米粒组更明显。
结论：CS-siRNA在体外明显抑制了胶质瘤U251细胞增殖并促进其凋亡。
关键词：壳聚糖;纳米粒;胶质瘤;端粒酶反转录酶;RNA干扰;生物材料

姚鹏飞，柯以铨，王建奇，许刚，周振军. 壳聚糖-小干扰RNA纳米粒对U251细胞增殖及凋亡的影响[J].中国组织工程研究与临床康复，2008，12(14):2640-2644 [www.zglckf.com/zglckf/ejournal/upfiles/08-14/14k-2640(ps).pdf]

Proliferation and apoptosis of U251 cells induced by chitosan-small interference RNA nanoparticle

Abstract

AIM: Current chemotherapy and radiotherapy are doomed to prevent glioblastoma recurrence, but don't improve the prognosis of patients with malignant gliomas due to low sensitivity and easy induction for drug resistance in glioma cells. However, the widespread gene therapy may bring the breakthrough in this field. This study was designed to investigate cell proliferation and apoptosis of glioblastoma U251 by RNA interference with chitosan (CS)-small interference RNA (siRNA) nanoparticle targeted human telomerase reverse transcriptase (hTERT) gene in vitro.
METHODS: The experiment was carried out in the Neurosurgery Institute of Guangdong Province (Key Laboratory) between September 2006 and July 2007. CS-siRNA nanoparticle targeting hTERT gene was prepared. The morphology and size of the nanoparticle were observed with atomic force microscope. The transfected neurogliocytoma cells U251 were divided into 8 groups: blank control group (without transfection reagents or siRNA), positive control group (LipofectamineTM 2000 transfected siRNA), negative control group (30 nmol/L controlled siRNA nanoparticle), bare siRNA control group (30 nmol/L siRNA), blank CS nanoparticle control group, siRNA nanoparticle groups (10, 30, 90 nmol/L). U251 cells at logarithmic growth phase were detected to draw the curve of cell proliferation and calculate inhibition ratio by means of CCK-8 method. The morphology of the nucleus was observed by Hoechst 33342/propidium iodide double staining under fluorescence microscope. Flow cytometry results showed the cell cycle. Reverse transcription-polymerase chain reaction was used to detect the expression changes of hTERT-mRNA.
RESULTS: ①Under atomic force microscope, CS-siRNA nanoparticles were spherical and uniform, and the mean particle diameters were 87 nm.②The cell proliferation was obviously inhibited in positive control group and siRNA nanoparticle groups (30 and 90 nmol/L), with significant differences compared with other groups (P < 0.05).③The results of Hoechst33342/propidium iodide double staining under fluorescence microscope revealed that, the blank control group, negative control group, bare siRNA control group and blank CS nanoparticle group contained low blue normal cells, while positive control group and siRNA nanoparticle groups (10, 30, 90 nmol/L) represented high blue apoptotic cells.④Compared with blank control group, negative control group, bare siRNA control group and blank CS nanoparticle group, the cells at S phase remarkably decreased, while those at G0/G1 phase increased in siRNA nanoparticle groups (10, 30, 90 nmol/L). And the changes were the most obvious in positive control group (P < 0.05). Cells at G2/M phase were not changed (P > 0.05).⑤The mRNA level of hTERT was obviously decreased in positive control group and siRNA nanoparticle groups, especially in 30 and 90 nmol/L siRNA nanoparticle groups.
CONCLUSION: CS-siRNA effectively inhibits cell proliferation and promotes apoptosis of U251 in vitro.

Yao PF, Ke YQ, Wang JQ, Xu G, Zhou ZJ. Proliferation and apoptosis of U251 cells induced by chitosan-small interference RNA nanoparticle.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(14):2640-2644(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-14/14k-2640(ps).pdf]