Ishiwata Toshiyuki, Fujii Takenori, Ishiwata Shunji, Ikegawa Shigeo, Naito Zenya
Department of Pathology II, Nippon Medical School, Tokyo, Japan.
Pathol Int. 2004 Feb;54(2):77-81. doi: 10.1111/j.1440-1827.2004.01593.x.
Lumican is a member of the small-leucine-rich proteoglycan (SLRP) family and is overexpressed during wound healing of the cornea, in ischemic and reperfused heart, and in several cancer tissues. Lumican is considered to regulate the collagen fibril diameter and interfibrillar spacing. However, the effect of lumican on cell growth has not been adequately examined. In the present study, we attempted to clarify whether lumican contributes to human embryonic kidney (HEK) 293 cell growth, using the morpholino antisense oligonucleotide (m-anti oligo) against lumican mRNA. M-anti oligo is a novel oligonucleotide and exhibits a higher antisense activity, higher water solubility, and greater resistance to nucleases in target cells than phosphorothioate types of oligonucleotide. After delivery of m-anti oligo against lumican mRNA, the fluorescein 5-isothiocyanate (FITC) conjugated oligonucleotides were observed in the cytoplasm and nucleus of HEK 293 cells at 24 h by confocal laser microscopy. M-anti oligo for lumican mRNA strongly inhibited the synthesis of lumican protein in the HEK 293 cells, and the HEK cell growth rate was higher than those in the control groups. These findings may indicate that lumican protein has an inhibitory effect on HEK 293 cell growth in vitro.
层黏蛋白聚糖是富含亮氨酸的小分子蛋白聚糖（SLRP）家族的成员，在角膜伤口愈合过程中、缺血再灌注的心脏以及几种癌组织中过度表达。层黏蛋白聚糖被认为可调节胶原纤维直径和纤维间间距。然而，层黏蛋白聚糖对细胞生长的影响尚未得到充分研究。在本研究中，我们试图使用针对层黏蛋白聚糖mRNA的吗啉代反义寡核苷酸（m-anti oligo）来阐明层黏蛋白聚糖是否有助于人胚肾（HEK）293细胞的生长。M-anti oligo是一种新型寡核苷酸，与硫代磷酸酯类寡核苷酸相比，在靶细胞中表现出更高的反义活性、更高的水溶性和更强的核酸酶抗性。在递送针对层黏蛋白聚糖mRNA的m-anti oligo后，通过共聚焦激光显微镜在24小时时观察到异硫氰酸荧光素（FITC）偶联的寡核苷酸存在于HEK 293细胞的细胞质和细胞核中。针对层黏蛋白聚糖mRNA的M-anti oligo强烈抑制HEK 293细胞中层黏蛋白聚糖蛋白的合成，并且HEK细胞的生长速率高于对照组。这些发现可能表明层黏蛋白聚糖蛋白在体外对HEK 293细胞生长具有抑制作用。
