氧濃度及 Notch 通路對大鼠纖維環(huán)細胞增殖和細胞周期的影響

·基礎研究·

氧濃度及Notch通路對大鼠纖維環(huán)細胞增殖和細胞周期的影響

馬俊，張穎，陳元元，石長貴，袁文

作者單位：200003上海，第二軍醫(yī)大學附屬長征醫(yī)院骨科

通信作者：袁文yuanwenspine@163.com

【摘要】目的檢測不同氧濃度條件下椎間盤纖維環(huán)細胞Notch信號通路相關分子的表達變化，明確參與調(diào)節(jié)纖維環(huán)細胞增殖的相關靶基因。方法體外分離、培養(yǎng)大鼠纖維環(huán)細胞，運用CCK-8細胞活力檢測試劑盒、流式細胞儀檢測不同氧濃度培養(yǎng)條件下Notch信號通路抑制劑L685458(2 μmol/L、4 μmol/L、8 μmol/L)對纖維環(huán)細胞增殖及細胞周期的影響；熒光定量RT-PCR檢測不同氧濃度培養(yǎng)條件下纖維環(huán)細胞Notch信號表達水平變化。結(jié)果CCK-8結(jié)果顯示與常氧條件相比，低氧培養(yǎng)纖維環(huán)細胞時所測吸光度值明顯升高；無論常氧或低氧條件，加入Notch抑制劑干預后所測吸光度值明顯降低；流式細胞儀結(jié)果顯示與常氧條件相比，低氧培養(yǎng)纖維環(huán)細胞處于S/G2期細胞所占百分率明顯升高，加入Notch抑制劑干預后處于S/G2期細胞所占百分率明顯降低。低氧干預8～24 h后，Notch3、Notch4、Delta-like1、Delta-like3、Hes1、Hes5、Hes7 mRNA都有不同水平上調(diào)，Hey2 mRNA表達水平下調(diào)。結(jié)論低氧可以通過上調(diào)Notch信號通路的表達水平促進纖維環(huán)細胞增殖，Notch信號可能作為一個研究靶點用于延緩椎間盤退變的過程。

【關鍵詞】大鼠; 椎間盤退行性變; 細胞增殖; 受體，Notch; 細胞低氧

基金項目：上海市自然科學基金青年項目(12ZR1454900)

作者簡介：馬俊(1989— )，碩士，醫(yī)師

【中圖分類號】R 349.53 【文獻標志碼】 A

DOI【】

收稿日期：(2014-03-05)

Influences of oxygen tension and Notch signaling pathway on proliferation and cell cycle of rat annulus fibrosus cells

MAJun,ZHANGYing,CHENYuan-yuan,SHIChang-gui,YUANWen.DepartmentofOrthopaedics,ChangzhengHospital,SecondaryMilitaryMedicalUniversity，Shanghai200003，China

Abstract【】ObjectiveTo observe the influences of O2 concentration on the expression levels of Notch signaling components in rat annulus fibrosus cells(AFCs), and to explore the target gene participates in controlling the proliferation process of AFCs. MethodsRat annulus fibrosus cells were harvested and cultured in vitro under hypoxia or normoxia condition. AFCs were administrated to different concontrations of Notch signal inhibitor, L685458(2 μmol/L， 4 μmol/L， 8 μmol/L)， for 8-24 h, the proliferation and cell cycle of AFCs were examined by CCK-8 assay and flow cytometry. The expression level of Notch signaling components were tested by quantitative real-time polymerase chain reaction (qRT-PCR). Results CCK-8 assay indicated that the optical density value of AFCs under hypoxia was increased when compared with under normoxia, while decreased with different concontrations of L685458 whether under hypoxia or normoxia. Flow cytometry indicated that the viability and percentage of S/G2 phase cells of AFCs under hypoxia was increased when compared with under normoxia, while decreased with different concontrations of L685458 whether under hypoxia or normoxia. When AFCs were cultured under hypoxia for 8-24 h, the expression of Notch3， Notch4， Delta-like1， Delta-like3， Hes1， Hes5， Hes7 were increased and Hey2 was decreased at mRNA level compared with under normoxia . ConclusionHypoxia promotes the proliferation process of AFCs through up-regulation of Notch signaling components, and Notch signaling may become a therapeutic target for retarding the process of disc degeneration.

【Key words】Rats; Intervertebral disc degeneration; Cell proliferation; Receptors, notch; Cell hypoxia

J Spinal Surg, 2015,13(1):50-55

椎間盤退變是臨床上引起頸腰痛的最主要原因之一。以椎間盤自身細胞為基礎的細胞治療技術在延緩椎間盤退變的進程中可能具有重要的意義[1]?！?br>