EA 3102, Hôpital Robert Debré, AP-HP, Paris, France.
J Gene Med. 2010 May;12(5):413-22. doi: 10.1002/jgm.1451.
In utero tracheal occlusion (TO) has been developed to improve the lung hypoplasia associated with congenital diaphragmatic hernia (CDH). However, although TO stimulates fetal lung growth, it results in a decrease of alveolar type II cells (ATII) and surfactant production. Because keratinocyte growth factor (KGF) is a potent stimulus of ATII proliferation and maturation, we evaluated, in a fetal lamb model of CDH, a gene therapy strategy combining TO and ovine KGF transfection into the fetal airways using bisguanidinium-tren-cholesterol/dioleoyl-phosphatidylethanolamine (BGTC/DOPE) cationic liposomes.
Three groups of sheep fetuses with CDH and a group of normal fetuses were studied. The fetuses of the three groups with CDH (KGF, Medium and Hernia groups) underwent surgery at 85 days of gestation to create a diaphragmatic hernia. The KGF and medium group fetuses underwent a second surgery step at day 125 to perform TO associated with injection of the KGF transfection mixture (KGF group) or control medium (Medium group), whereas the fetuses of the Hernia group were left untreated. Normal fetuses were used as a control (Normal group). All fetuses were euthanized at 132 days of gestation and various analytical studies [lung weight, radial alveolar count (RAC), KGF and surfactant protein B (SPB) expression, number of ATII cells] were performed to assess the efficiency of KGF transfection and its effects on fetal lung development.
TO was associated with lung hyperplasia and increased RAC in the Medium and KGF groups versus the Hernia group. Expression of KGF was increased in the KGF group compared to all other groups and was associated with an increased synthesis of SPB by alveolar cells and an ectopic synthesis of SPB by bronchiolar cells compared to TO treatment alone.
Thus, BGTC/DOPE liposomes can mediate efficient KGF transfection into the airways in a fetal sheep model of CDH. Furthermore, combining KGF transfection and TO resulted not only (as did TO alone) in the correction of the CDH-associated lung hypoplasia and decreased RAC, but also in increased SPB synthesis, suggesting a better maturation of the re-growing lung (compared to TO alone). Additional studies are required to further explore the therapeutic potential of such a combined strategy; in particular, studies evaluating the lung function of in utero-treated CDH lamb newborns.
在子宫内进行气管结扎（TO）已被开发用于改善与先天性膈疝（CDH）相关的肺发育不良。然而，尽管 TO 刺激胎儿肺生长，但它会导致肺泡 II 型细胞（ATII）和表面活性剂产生减少。由于角质细胞生长因子（KGF）是刺激 ATII 增殖和成熟的有效刺激物，我们在 CDH 的胎羊模型中评估了一种基因治疗策略，该策略使用双胍基-tren-胆固醇/二油酰基磷脂酰乙醇胺（BGTC/DOPE）阳离子脂质体将 TO 和绵羊 KGF 转染到胎儿气道中。
研究了三组患有 CDH 的绵羊胎儿和一组正常胎儿。三组患有 CDH 的胎儿（KGF、中值和疝组）在妊娠 85 天时接受手术以创建膈疝。KGF 和中值组胎儿在第 125 天进行第二次手术步骤，进行 TO 并注射 KGF 转染混合物（KGF 组）或对照培养基（中值组），而疝组胎儿则未接受治疗。正常胎儿用作对照（正常组）。所有胎儿均在妊娠 132 天时安乐死，并进行各种分析研究[肺重、肺泡计数（RAC）、KGF 和表面蛋白 B（SPB）表达、ATII 细胞数量]，以评估 KGF 转染的效率及其对胎儿肺发育的影响。
TO 与中值和 KGF 组与疝组相比，与肺过度增生和 RAC 增加有关。与所有其他组相比，KGF 组的 KGF 表达增加，并且与肺泡细胞中 SPB 的合成增加以及单独 TO 治疗时支气管细胞中 SPB 的异位合成有关。
因此，BGTC/DOPE 脂质体可以介导在 CDH 的胎羊模型中有效转染气道中的 KGF。此外，与单独的 TO 治疗相比，将 KGF 转染与 TO 结合不仅（如 TO 单独治疗一样）纠正了与 CDH 相关的肺发育不良和减少 RAC，而且还增加了 SPB 的合成，表明重新生长的肺成熟度更高（与单独的 TO 相比）。需要进一步的研究来进一步探索这种联合策略的治疗潜力；特别是，评估宫内治疗 CDH 羔羊新生儿的肺功能的研究。
