半胱氨酸蛋白酶3、bax和bcl-xl在N-甲基-N亚硝酸脲诱导的大鼠视网膜损伤后的表达

目的 观察N-甲基-N亚硝酸脲（MNU）诱导的大鼠视网膜损伤过程中凋亡相关分子半胱氨酸蛋白酶3（caspase-3）、bcl-2相关X蛋白（bax）和B细胞淋巴瘤/白血病-xl（bcl-xl）在视网膜中的时空表达，探讨其在MNU诱导的视网膜损伤中的作用。方法 将鼠龄50 d的30只SPF级雌性SD大鼠随机分为MNU模型组（24只大鼠）和正常对照组（6只大鼠）。模型组按40 mg/kg的剂量腹腔注射MNU建立MNU模型；正常对照组大鼠腹腔注射生理盐水5ml/kg。模型组大鼠根据MNU注射后不同处死时间再分为1、3、7、10 d组，每小组各6只大鼠。正常对照组大鼠注射后3 d处死。逆转录-聚合酶链反应（RT-PCR）检测各组视网膜中caspase-3、bax和bcl-xl的基因表达情况；免疫荧光技术检测其在视网膜中的表达以及分布的变化，并用末端脱氧核苷酸转移酶（TdT）介导dUTP 缺口末段标记（TUNEL）法检测视网膜细胞凋亡的变化。结果经病理学检测确定造模成功。RT-PCR检测结果表明，［与正常对照组相比（caspase-3和bax的相对吸光度（RA）值为62.45±7.65、46.53±4.41］，MNU模型1 d 组caspase-3（83.23±8.11，P=0.009）和bax（72.73±9.46，P=0.004）的表达均增强，3 d 组二者表达水平均最高 （caspase-3 RA =140.48±18.40，P=0.000；bax- RA=102.36±13.97，P=0.001），10 d 组caspase-3的表达水平（RA =47.32±5.37，P=0.027）低于对照组，而10 d bax的表达 （RA=48.27±4.40，P=0.997）基本恢复至对照组水平；bcl-xl在4个造模组中的表达水平（1 d RA =63.29±4.29，P=0.000；3d RA =79.83±5.58，P=0.000；7 d RA=70.19±4.42，P=0.000；10 d RA =66.05±4.97，P=0.000）均高于正常对照组（RA =45.98±3.06），3 d 组的表达水平最高。MNU诱导后1、3、7 d 组的bax/bcl-xl比值（1 d为1.15±0.14，P=0.143；3 d为1.28±0.16，P=0.001；7 d为1.17±0.08，P=0.079）大于对照组（1.01±0.09），其中3 d组的比值最大，但10 d 组bax / bcl-xl比值（0.73±0.07，P=0.001）小于对照组。免疫荧光检测结果显示，caspase-3、bax和bcl-xl的表达变化分别与其RT-PCR检测结果一致；正常对照组在视网膜各层均未发现凋亡细胞，MNU 1 d 组的外核层可检测到大量凋亡细胞核［凋亡率（AI）为34.96±3.44，P=0.000］，3 d 组的外核层检测的凋亡细胞最多（AI=76.97±5.83，P=0.000），10 d 组未检测到凋亡细胞。结论 MNU诱导的视网膜光感受器细胞凋亡可能是通过上调caspase-3及引起bax/bcl-xl表达失衡并明显倾向促进细胞凋亡造成的。

Temporal and spatial expressions of caspase-3, bax and bcl-xl in rat retina with MNU-induced photoreceptor damages

Objective To investigate the temporal and spatial expression pattern of Caspase-3、 Bax and Bcl-xl in N-methyl-N-nitrosourea (MNU) damaged rat retina. Methods Twenty-four 50-day-old female Sprague-Dawley rats (n=24) received single intraperitoneal injection of MNU 40 mg/kg and were examined at 1, 3, 7 and 10 days after MNU treatment (6 rats sacrificed at each timepoint). As control, six rats were injected with saline (5 ml/kg) and sacrificed 3d after injection. Expressions of Caspase-3 and bax and bcl-xl were detected by RT-PCR and immunofluorescence assays, photoreceptor cell apoptosis was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling (TUNEL). Results Animal models were successful established and confirmed by pathological studies. RT-PCR results indicated that caspase-3 and bax up-regulated at 1 d (caspase-3 RA=83.23±8.11, P=0.009;bax RA=72.73±9.46,P=0.004) and peaked at 3 d (caspase-3 RA=140.48±18.40, P=0.000;bax RA=102.36±13.97, P=0.001) compared with control (caspase-3 RA=62.45±7. 65;bax RA46.53±4.41). Bcl-xl expression increased and peaked at 3d (3d RA=79.83±65.58, P=0.000 vs control 45.98±3.06). It was noted that the ratios of bax / bcl-xl expression at 1 d, 3 d and 7 d after MNU injection were enhanced (1 d 1.15±0.14, P=0.143;3 d 1.28±0.16, P=0.001;7 d 1.17±0.08, P=0. 079, vs control 1.01±0.09), and at 3 d the ratio reached the peak, whereas at10 d bax / bcl-xl ratio (0.73±0.07, P= 0.001) was decreased compared with the control. Immunofluoreseence assays demonstrated that the changes of bax, bcl-xl and caspases-3 protein expressions coincided with their RT-PCR results respectively. The Bax positive cells were detected in the outer nuclear layer;while caspase-3 and bcl-xl positive cells emerged in several layers of retina included the pigment epithelium layer, the photoreceptor cell inner segments, the outer nuclear layer, the outer plexiform layer, the inner plexiform layer and the ganglion cell layer. Photoreceptor cell apoptosis was only detected in the outer nuclear layer and peaked at 3 d in MNU treated rats (AI=76. 97±5.83, P= 0. 000 vs control 0.00±0.00). Conclusions These data suggest that bax and bcl-xl and caspases-3 may involve in the MNU-induced rat photoreceptor cell apoptosis.