纳米金载体输送基于“甲硫氨酸应激”疗法的rMETase基因靶向性抑制胃癌干细胞能量代谢以治疗胃癌

Elimination or inhibition of gastric cancer stem cells is the key to prevent the recurrence of gastric cancer. At present there is no satisfactory clinical method of removal of gastric cancer stem cell. Our previous study showed that p38 protein is activated and the expression of antioxidase PRDX6 protein is downregulated in gastric cancer cells in methionine stress. The new study find that disturbance of energy metabolism can lead to change of biological characteristics of stem cells. We hypothesize that methionine stress can kill gastric cancer stem cell when methionine enzyme gene may be transducted into gastric cancer stem cells with dynamic METase. As methionine stress may inhibit glycolysis of gastric cancer cells through activating p38 protein. As p38 protein can block the mTOR signaling pathway. At the same time, methionine stress may cut off the sources of energy of gastric cancer stem cells through injurying mitochondria with the oxygen free radical. Based on this new idea , we construct reduction response type gold nanoparticles carried rMETase gene using the layer by layer method. Effect of the non viral delivery carrier specific Killing gastric cancer stem cells is be detected.And then, the change and molecular mechanism of mitochondria and glycolytic of gastric cancer stem cell in methionine stress are studied. The results will provide a new way of targeted therapy of gastric cancer.

胃癌无法根治的原因之一是由于胃癌干细胞的存在，目前临床上尚没有满意的清除胃癌干细胞的方法。我们前期研究结果提示：甲硫氨酸应激可以激活胃癌细胞p38蛋白、下调抗氧化酶PRDX6蛋白表达。最新研究发现，干扰能量代谢可以导致干细胞生物学特性改变。为此，我们提出假说：如果能将重组甲硫氨酸酶（rMETase）基因特异性导入胃癌干细胞内并合成有活性的rMETase，就可以在胃癌干细胞内造成甲硫氨酸应激，从而激活p38蛋白阻断mTOR信号通路来抑制糖酵解，同时又能通过增加氧自由基来损伤线粒体功能，达到全面切断胃癌干细胞能量供应的目的，进而杀灭胃癌干细胞。基于此新设想，本项目在前期制备纳米载体的基础上，构建装载rMETase基因的仿生型纳米金给药载体，检测其对胃癌干细胞特异性杀伤作用；同时研究甲硫氨酸应激对胃癌干细胞线粒体和糖酵解两个主要能量代谢途径的影响和调控的机制。所得结果将为胃癌靶向治疗提供新思路。

有效清除胃癌干细胞对于获得理想的抗癌疗效是十分必要的。研究发现，线粒体结构和功能的改变会干扰肿瘤细胞的生长、能量代谢、增殖和凋亡，还能干扰肿瘤细胞的分化、自我更新等特性；肿瘤细胞在有氧条件下，仍主要依靠糖酵解产能，增强的糖酵解能促进肿瘤细胞快速增殖，增强其扩散、侵袭能力。胃癌细胞具有甲硫氨酸依赖性，结合前期工作，我们推测：甲硫氨酸饥饿可以干扰胃癌干细胞的糖酵解及线粒体功能、有效杀死胃癌干细胞。本课题利用肿瘤球培养法和免疫磁珠法分别从胃癌细胞株和胃癌病人血清中分离癌干细胞；制备靶向性HA-G5 PAMAM-Au-METase基因纳米复合物，对其杀灭胃癌干细胞的效应进行体内外生物学评价；研究甲硫氨酸饥饿对胃癌细胞能量代谢的影响及其机制。研究结果如下: （1）从胃癌细胞株中分离、鉴定胃癌干细胞，利用免疫磁珠成功从胃癌病人的外周血中分离得到CD133+/CD44+细胞。(2）制备HA-G5 PAMAM-Au-METase基因纳米复合物，该基因纳米复合物能够抑制CD44+胃癌干细胞的增殖和线粒体功能，HA-G5-PAMAM-AU- METase转染胃癌干细胞后，线粒体释放的Cyt C水平和胞质内c-caspase 3的表达显著增强，细胞中ROS水平明显升高。将HA-G5 PAMAM-Au-METase注射到胃癌裸鼠模型中，肿瘤组织生长明显受到抑制，CD44表达水平也相应降低，Cyt C水平和c-caspase 3表达有明显的增强。（3） rMETase能够明显降低胃癌细胞糖酵解产物乳酸和ATP产生，抑制有氧糖酵解；rMETase可以通过抑制PI3K/Akt信号通路调控GLUT-1来抑制有氧糖酵解。本课题构建HA-G5 PAMAM-Au-METase基因纳米复合物，将其导入胃癌干细胞内，研究甲硫氨酸饥饿对糖酵解及线粒体功能的影响，为开发高效、低毒的靶向性药物提供了理论依据。