恒定及波动高糖下牛视网膜血管周细胞凋亡的线粒体机制

目的 研究恒定及波动高糖培养对牛视网膜血管周细胞凋亡的影响,并探讨其线粒体调控机制.方法 体外培养接近融合的视网膜血管周细胞在恒定及波动高糖中孵育6 d后,采用电镜观察周细胞超微结构改变;TUNEL法检测周细胞凋亡;激光共聚焦显微镜检测周细胞线粒体跨膜电位的改变;分光光度计检测周细胞胞质细胞色素c的变化;免疫组化和RT-PCR测定Bax、Bel-2基因的表达.结果 (1)恒定及波动高糖卜周细胞呈现出典型的细胞凋亡改,变,恒定高糖作用强于波动高糖组;(2)恒定及波动高糖培养使用细胞线粒体跨膜电位降低,周细胞线粒体跨膜电位与周细胞凋亡率负相关(r=-0.89,P<0.01);(3)恒定及波动高糖促使周细胞线粒体细胞色素c转移到胞质,胞质细胞色素c浓度增加,与细胞凋亡率正相关(P<0.01);(4)恒定及波动高糖能诱导凋亡基因Bax表达增加,抑制Bcl-2的表达,Bax/Bcl-2比值增加.Bax/Bcl-2比值与线粒体跨膜电位负相关,与胞质细胞色素c浓度正相关,与周细胞凋亡率正相关(均P<0.01).结论 恒定及波动高糖均可诱导培养的周细胞线粒体跨膜电位降低,细胞色素c释放,细胞发生凋亡,且恒定高糖作用强于波动高糖;线粒体凋亡途径在周细胞凋亡中起重要作用,凋亡基因Bax、Bcl-2可能参与其调控.

The role of mitochondrial apoptotic pathway in the apoptosis of cultured bovine retinal capillary pericytes induced by constant and intermittent high glucose

Objective To study the effects of constant and intermittent high glucose on the apoptosis of cultured bovine retinal capillary pericytes (BRPs), and to investigate the role of mitochondrial apoptotic pathway in the apoptosis of BRPs. Methods After being cultured under glucose with different concentrations tot 6 days, the change of uhrastructure of BRPs was observed under electronmieroscope, the apoptosis of pericytes was detected by TUNEL method, the mitochondrial inner transmembrane potential (△Ψm) was detected by laser scanning confocal microscopy, the change of cytochrome c (cyt-c) was assayed by spectrophotometer and the expression of apoptotic genes was detected by immunohistochemical method and RT-PCR. Results (1) BRPs showed typical changes of apoptosis in constant and intermittent high glucose concentrations. The apoptosis induced by constant high glucose concentration was more obvious than that by intermittent high glucose. (2) Constant and intermittent high glucose concentrations obviously decreased △Ψm compared with control group. The △Ψm of BRPs was correlated negatively with the apoptotie rate of BRPs (r = - 0.89, P < 0.01) ; (3) Constant and intermittent high glucose concentrations increased the release of cyt-c from mitochondria to cytoplasm, and the concentration of cyt-c in the cytoplasm was correlated positively with the apoptotic rate of BRPs (P < 0.01) ; (4) Constant and intermittent high glucose concentrations increased the expression of proapoptotie gene Bax and decreased the expression of prosurvival gene Bcl-2, resulting in increased Bax/Bcl-2 ratio. Bax/Bcl-2 ratio was negative correlatied with the △Ψm of BRPs, and positively eorrclaticd with the concentration of cyt-c in cytoplasm and apoptotic rate (both P < 0.01). Conclusion Constant and intermittent high glucose concentrations could decrease △Ψm, increase the release of cyt-c and induce the apoptosis of BRPs, the effects being stronger with constant high glucose concentration. The mitochondrial apoptotic pathway plays an important role in the apoptosis of BRPs, in which Bax and Bcl-2 are involved.