Department of General Surgery, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, 400042, China.
Int J Colorectal Dis. 2010 May;25(5):557-66. doi: 10.1007/s00384-010-0883-z. Epub 2010 Feb 18.
Interaction of c-Kit and its ligand stem cell factor (SCF) is necessary for appropriate development and survival of interstitial cells of Cajal (ICC) in the intestine. Blockade of c-Kit will cause ICC loss in vivo. Stem cell leukemia (SCL) gene acts as a positive regulator of upstream transcription of c-Kit expression. This study aimed to explore whether the restoration of c-Kit expression promoted by SCL gene transfer could rescue ICC in vivo.
A modified ICC-loss mouse model was created by continual administration of anti-c-Kit antibody (ACK2) to obtain a steady status of ICC loss, and a recombinant adenovirus vector containing SCL gene (Ad-SCL) was designed to rescue ICC in these mice. Western blot analysis and immunofluorescence labeling assays were performed to analyze the SCL and c-Kit expression in vitro and in vivo. The distribution and configuration of ICC were observed with immunohistochemistry and electromicroscope.
Western blot analysis and immunofluorescence labeling assays showed that SCL gene was successfully delivered to cultured HeLa and ICC cells in vitro. Moreover, significantly increased c-Kit expression could be detected in the colon of Ad-SCL-infected ICC-loss mice. Furthermore, rescue of the ICC network and ICC with typical ultrastructural features could be detected in Ad-SCL-infected ICC-loss mice at day 37.
Ad-SCL was able to enhance c-Kit expression, reactivate the c-Kit/SCF pathway, and rescue ICC in ICC-loss mice. Since loss and defects of ICC are associated with many human gut motility disorders, Ad-SCL may be of potential use in gene therapy of these patients.
c-Kit 与其配体干细胞因子（SCF）的相互作用对于肠道间质细胞（ICC）的正常发育和存活是必需的。c-Kit 的阻断会导致体内 ICC 的丧失。干细胞白血病（SCL）基因作为 c-Kit 表达的上游转录的正调控因子。本研究旨在探讨 SCL 基因转染恢复 c-Kit 表达是否能在体内拯救 ICC。
通过持续给予抗 c-Kit 抗体（ACK2）来建立改良的 ICC 缺失小鼠模型，以获得 ICC 稳定缺失的状态，并设计了含有 SCL 基因的重组腺病毒载体（Ad-SCL）来拯救这些小鼠中的 ICC。采用 Western blot 分析和免疫荧光标记检测方法分析体外和体内的 SCL 和 c-Kit 表达。采用免疫组织化学和电镜观察 ICC 的分布和形态。
Western blot 分析和免疫荧光标记检测显示，SCL 基因成功转染至体外培养的 HeLa 和 ICC 细胞。此外，在 Ad-SCL 感染的 ICC 缺失小鼠的结肠中可检测到 c-Kit 表达显著增加。进一步观察到，在 Ad-SCL 感染的 ICC 缺失小鼠中，在第 37 天可检测到 ICC 网络和具有典型超微结构特征的 ICC 的拯救。
Ad-SCL 能够增强 c-Kit 表达，重新激活 c-Kit/SCF 通路，并在 ICC 缺失小鼠中拯救 ICC。由于 ICC 的缺失和缺陷与许多人类肠道运动障碍有关，Ad-SCL 可能在这些患者的基因治疗中有潜在的应用价值。
