摘要
目的探讨神经生长因子(NGF)对H9c2心肌细胞缺氧/复氧(H/R)后凋亡的保护作用及其机制。方法将培养的H9c2心肌细胞随机分为5组:正常对照组(C组)、缺氧/复氧组(H/R组)、NGF组(N组)、NGF+PI3k受体特异性阻断剂LY294002组(N+L组)和LY294002组(L组)。C组用正常培养液在通有95%空气+5%CO2的37℃培养箱内正常培养8 h;H/R组用预先经95%N2+5%CO2的混合气饱和1 h的模拟缺氧液置换正常培养液后,放入通有95%N2+5%CO2的37℃培养箱缺氧培养4 h,然后将模拟缺氧液换用预先经95%空气+5%CO2饱和的正常培养液,在正常条件下复氧4 h;N组、N+L组和L组先缺氧培养4 h(缺氧条件同H/R组),再分别用预先经95%空气+5%CO2饱和的含NGF、NGF+LY294002和LY294002的正常培养液置换模拟缺氧液,在正常条件下复氧4 h。NGF和LY294002的终浓度分别为0.05 mg·L-1和3 mg·L-1。CCK-8比色法测定各组细胞的存活率;应用碘化丙啶(PI)染色流式细胞术检测细胞凋亡率;Western blot法检测胞内Akt磷酸化水平及内质网应激蛋白Caspase-12蛋白的表达。结果 N组与N+L组或H/R组或L组相比能明显提高H9c2心肌细胞经H/R损伤后的存活率,减少细胞凋亡率,上调Akt磷酸化水平,降低内质网应激蛋白Caspase-12表达;LY294002明显抑制了NGF对Akt磷酸化水平的上调作用。结论 NGF对经H/R损伤的H9c2心肌细胞有抗凋亡作用,其机制可能与PI3k-Akt通路有关。
Aim To investigate the anti-apoptotic effect of NGF on H9c2 cardiac myocytes in a hypoxia / reox- ygenation injury model and its mechanism. Methods The H9c2 cardiac myocytes were randomly divided into five groups： control group （C group）, hypoxia/reoxy- genation group （H/R group）, NGF group （N group）, NGF ＋ LY294002 group （ N ＋ L group） and LY294002 group（ L group）. Each group received the correspond- ing treatment. Cell survival rate was tested by cell counter kit-8 methods. Apoptotic rate was evaluated by propidium iodide （PI） staining and flow cytometry （FCM）. The levels of Caspase-12, p-Akt/Akt were e- valuated by Western blot. Results The NGF group could significantly protect the H9c2 cardiac myocytes under the hypoxia / reoxygenation injury with increased cell survival rate. It also decreased the apoptotic per- centage, upregulated the level of p-Akt/Akt and inhib- ited the expression of Caspase-12. As the specific in- hibitor of PI3k receptor, LY294002 decreased the level of p-Akt. Conclusion NGF has the effect of anti-ap- optosis on H9c2 cardiac myocytes exposed to hypoxia / reoxygenation injury via PI3k-Akt signal pathway.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2014年第4期506-510,共5页
Chinese Pharmacological Bulletin
基金
国家自然科学基金青年基金项目(No 81000066)
卫生部国家临床重点专科[No财社〔2011〕170号]
重庆市医学重点学科建设项目[No渝卫科教〔2007〕2号]
作者简介
谢飞（1988一），男，硕士生，研究方向：围术期内重要脏器保护，E-mail：xiefei_8896@126．com；
魏珂（1976一），男，博士生，研究方向：围术期内重要脏器保护，E-mail：962881848@qq．com；
闵苏（1960一），女，教授，主任医师，博士生导师，研究方向：围术期内重要脏器保护，通讯作者，Tel：023．89011068，E—mail：ms89011068@163．com
