Yukawa Yasutsugu, Lou Jueren, Fukui Naoshi, Lenke Lawrence G
Department of Orthopaedic Surgery, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Neurotrauma. 2002 Sep;19(9):1091-103. doi: 10.1089/089771502760341992.
Although Bcl-2 gene transfer can rescue cells from neuronal apoptosis, the temporal relationship between treatment initiation time and effectiveness is unknown. The purpose of present study is to investigate the optimal treatment timing of Bcl-2 gene transfer in saving cells after neural insults. Bcl-2 gene transfer was mediated by recombinant adenovirus carrying human bcl-2 oncogene (Adv-Bcl-2). Adenovirus carrying beta-galactosidase gene (Adv-Bgal) served as a control. A serum withdrawal model of NSC-19 cell culture was used to induce apoptosis in vitro. At various time points before or after serum withdrawal, the motor neuron cells (NSC-19 cells) were infected with either Adv-Bcl-2 or Adv-Bgal. At 72 h after serum withdrawal, the number of apoptotic cells and DNA fragmentation were examined to evaluate the effect of Bcl-2 gene transfer. A weight-drop spinal cord injury model in rats was used as in vivo model. At various time points before or after experimental spinal injury, virus solution, including Adv-Bcl-2 or Adv-Bgal, was injected at the spinal cord in injured rats. The degree of cord injury was measured at 72 h after injury. TUNEL staining was performed to count cells that have undergone DNA damage in sections. Bcl-2 protein overexpression was confirmed by immunostaining both in vitro and in vivo model. In vitro, Adv-Bcl-2 infection produced a less prominent DNA laddering pattern. Adv-Bcl-2 infection between 24 h before and 4 h after serum withdrawal significantly reduced the apoptotic cell death. In vivo Adv-Bcl-2 injection immediately after injury effectively suppressed the injury lesion by blocking DNA fragmentation and irreversible cellular injury. Our data demonstrate that earlier initiation of Bcl-2 gene transfer can produce improved neural cell rescue following neural insults. These results stress important temporal considerations in future gene therapy strategies for spinal cord injury.
尽管Bcl-2基因转移可使细胞免于神经元凋亡，但其治疗开始时间与疗效之间的时间关系尚不清楚。本研究的目的是探讨Bcl-2基因转移在神经损伤后挽救细胞的最佳治疗时机。Bcl-2基因转移由携带人bcl-2癌基因的重组腺病毒（Adv-Bcl-2）介导。携带β-半乳糖苷酶基因的腺病毒（Adv-Bgal）用作对照。采用NSC-19细胞培养的血清剥夺模型在体外诱导凋亡。在血清剥夺之前或之后的不同时间点，运动神经元细胞（NSC-19细胞）用Adv-Bcl-2或Adv-Bgal感染。血清剥夺后72小时，检测凋亡细胞数量和DNA片段化情况，以评估Bcl-2基因转移的效果。大鼠重物坠落性脊髓损伤模型用作体内模型。在实验性脊髓损伤之前或之后的不同时间点，将包括Adv-Bcl-2或Adv-Bgal的病毒溶液注射到受伤大鼠的脊髓中。在损伤后72小时测量脊髓损伤程度。进行TUNEL染色以计数切片中发生DNA损伤的细胞。通过体外和体内模型的免疫染色证实了Bcl-2蛋白的过度表达。在体外，Adv-Bcl-2感染产生的DNA梯状条带模式不太明显。在血清剥夺前24小时至后4小时之间进行Adv-Bcl-感染可显著减少凋亡细胞死亡。在体内，损伤后立即注射Adv-Bcl-2可通过阻断DNA片段化和不可逆的细胞损伤有效抑制损伤病变。我们的数据表明，更早开始Bcl-2基因转移可在神经损伤后更好地挽救神经细胞。这些结果强调了在未来脊髓损伤基因治疗策略中重要的时间因素。
