目    录
中文摘要 (1)
英文摘要 (3)
英文缩写 (5)
研究论文低水平铅通过AMPK/mTOR/FoxO3a信号通路诱导VSMCs 增殖、迁移
前言 (6)
材料与方法 (8)
结果 (18)
讨论 (28)
结论 (34)
参考文献 (34)
综述重金属铅对心血管系统的影响及机制研究进展 (40)
致谢 (52)
个人简历 (53)
proliferation低水平铅通过AMPK/mTOR/FoxO3a信号通路
诱导VSMCs增殖、迁移
摘    要
目的：本研究通过建立体外染毒大鼠血管平滑肌细胞模型，观察低浓度铅对血管平滑肌细胞增殖和迁移的影响，并探索AMPK/mTOR/FoxO3a 信号通路在其中的作用。
方法：选取体重100-120g的雄性SD大鼠，在无菌环境下分离其腹主动脉，采用组织贴块法培养原代血管平滑肌细胞。本实验分为0（对照组）、0.1组（铅染毒低剂量0.1μM）、1.0组（铅染毒高剂量1.0μM）、0.1 +R（雷帕霉素）组、R组、0.1+CC（AMPK抑制剂）组、CC组。通过c ck8细胞增殖实验和划痕实验检测血管平滑肌细胞增殖、迁移的能力；使用荧光显微镜检测FoxO3a在细胞核内的定位及细胞骨架的形态；采用W estern Blot法检测细胞中PCNA、MMP-2、MMP-9、mTOR、p-mTOR、A
MPK、p-AMPK蛋白的表达水平；采用SPSS 21.0软件对数据进行统计分析，P<0.05为差异有统计学意义。
结果：
2. Western Blot法检测增殖迁移相关蛋白PCNA、MMP-2、MMP-9，发现低剂量组蛋白含量均显著升高；
3.免疫荧光结果显示低剂量铅可使F-actin聚集、增粗；
4.免疫荧光结果显示低剂量铅可使FoxO3a发生核转位，由细胞质转移到细胞核中；
5.加入雷帕霉素后FoxO3a定位于细胞质中，核转位被抑制，F-actin 排列松散、肌丝细小，PCNA、MMP-2、MMP-9蛋白表达含量明显降低，细胞增殖、迁移被抑制；
6.加入AMPK抑制剂后p-mTOR含量升高，FoxO3a发生核转位，定位于细胞核中，F-actin呈束状排列，荧光强度增高，PCNA和MMP-9蛋白含量升高，细胞发生增殖、迁移。
结论：低剂量铅可诱导血管平滑肌细胞发生增殖和迁移，可能与铅抑制AMPK磷酸化有关。铅抑制AMPK磷酸化使p-mTOR的表达增加，调控下游蛋白FoxO3a发生核转位，从而使细胞骨架应力纤维增粗，同时细胞增殖的标志性蛋白PCNA和迁移标志蛋白MMP-2、MMP-9表达含量增高，细胞发生增殖、迁移。综上，铅通过AMPK/mTOR/FoxO3a信号通路诱导血管平滑肌细胞增殖、迁移。
关键词：铅，血管平滑肌细胞，增殖，迁移
Low Level Lead Regulates the Vascular Smooth Muscle Cells
Proliferation and Migration via
AMPK/mTOR/FoxO3a Signaling Pathway
ABSTRACT
Objective：We have established an in vitro model which to evaluate the role of low level lead on VSMC proliferation、migration and the underlying mechanism. We aimed at clarifying the contribution of the AMPK/mTOR/FoxO3a signaling pathway.
Methods：Male SD rats weighing 100-120 g were selected to isolate their abdominal aortas in a steri
le environment, and primary VSMCs were cultured by tissue patch method. The experiment was divided into control group、low dose group (0.1 mmol/L)、high dose group (1.0mmol/L)、low dose + rapamycin group、rapamycin group, low dose + AMPK inhibitor group and AMPK inhibitor group. The ability of VSMCs to proliferate and migrate was detected by CCK8 test and scratch test; the localization of FoxO3a in the nucleus and the morphology of cytoskeleton were detected by fluorescence microscopy; the expression levels of PCNA、MMP-2、MMP-9、mTOR、
p-mTOR、AMPK and p-AMPK proteins in VSMCs were detected by Western Blot method; the data were statistically analyzed by SPSS 21.0 software, P<0.05 was statistically significant.
Result:
1. CCK8 and scratch test showed that lead could induce VSMCs to proliferate and migrate, and the proliferation and migration were most obvious in the low dose group;
2. Western Blot assay was used to detect PCNA, MMP-2 and MMP-9, which showed that the content of low-dose histone was significantly increased.
3. Immunofluorescence results showed that low-dose lead could agglomerate and thicken F-actin;
4. Immunofluorescence results showed that low-dose lead could cause nuclear translocation of FoxO3a from cytoplasm to nucleus;
5. After the addition of rapamycin, FoxO3a was localized in the cytoplasm, nuclear translocation was inhibited, F-actin arranged loosely, myofilament was fine, no phenotypic transformation occurred, PCNA, MMP-2, MMP-9 protein expression content was significantly reduced, cell proliferation and migration were inhibited;
6. After adding AMPK inhibitor, the content of p-mTOR increased, FoxO3a translocated into the nucleus, F-actin arranged in bundles, fluorescence intensity increased, PCNA and MMP-9 protein content increased, and cell proliferation and migration occurred.
Conclusion: Low level lead induced cell proliferation and migration，the molecular regulation mechanism of this phenomenon may correlate with a reduced phosphorylation of AMPK. Lead inhibits AMPK phosphorylation and promote mTOR phosphorylation，and promoted the nuclear translocation of FoxO3a，induces redistribution of the actin cytoskeleton and formation of stress fibers，transformation occurs in cells，VSMCs switch from a contractile to a proliferative. The expression of the PCNA、MMP-2、MMP-9 protein was significantly elevated，this implies cell prolif
eration and migration. In summary,we demonstrated that lead induces the proliferation and metastasis of VSMCs via AMPK/mTOR/FoxO3a signaling pathway.
Keywords: Lead, VSMCs, Proliferation, Migration

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