Development of Gene Delivery Systems to Intestinal Epithelial Cells as a Target

以肠上皮细胞为靶标的基因传递系统的开发

• 批准号: 09672324
• 负责人: TAKAKURA Yoshinobu
• 金额: $ 2.43万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09672324/
• 关键词: gene transfer; epithelial cells; Caco-2 cells; Pam-T cells; MDCK cells; LLC-PK_1 cells; interferon; secretion polarity; 遺伝子治療; Caco-2; 分泌タンパク; カチオン性リポソーム

In the present study, the secretion polarity of interferon (IFN)-beta exogenously expressed following transfection of mouse or human IFN-beta expression plasmid in four kinds of epithelial cells was studied using a bicameral culture system : human colorectal carcinoma Caco-2, mouse squamous cell carcinoma Pam-T, Mardin-Darby canine kidney MDCK, and porcine tubular epithelial cell LLC-PK1 I_n the former three kinds, transiently expressed IFN-beta was predominantly secreted from the cell membrane side to which the transfection was carried out ; if the plasmid was applied to the upper or lower compartment, IFN-beta was secreted from the apical or basolateral membrane, respectively. Interestingly, simultaneous transfection from both sides, apically with mouse IFN-beta gene and basally with human LFN-betag ene, resulted in mouse IFN-beta secretion into upper compartments and human IFN-beta secretion into lower compartments, and vice versa. LLC-PK_1 cells showed non-polarized transient secretion. Meanwhile, stable secretion from the transformants, which is established from either cell line transfected with IFN-beta expression plasmid was non-polarized. Taken together these results suggest that epithelial cells have various protein sorting-secretion pathways for the same protein. The sorting manner seems to be depended on how the protein is expressed, constitutive oremergent. These findings would be useful to construct the strategy for gene transfer into intestinal epithelial cells and other polarized cells.

在本研究中，使用双室培养系统研究了在四种上皮细胞中转染小鼠或人IFN-β表达质粒后外源表达的IFN-β的分泌极性：人大肠癌Caco-2、小鼠鳞状细胞癌Pam-T、犬肾MDCK、猪肾小管上皮细胞LLC-PK 1在前三种中，瞬时表达的IFN-β主要从进行转染的细胞膜侧分泌;如果将质粒应用于上室或下室，则IFN-β分别从顶膜或基底外侧膜分泌。有趣的是，从两侧同时转染，顶部用小鼠IFN-β基因和底部用人IFN-β基因，导致小鼠IFN-β分泌到上部隔室中，而人IFN-β分泌到下部隔室中，反之亦然。LLC-PK_1细胞呈非极性瞬时分泌。同时，从用IFN-β表达质粒转染的任一细胞系建立的转化体的稳定分泌是非极化的。综合这些结果表明，上皮细胞有不同的蛋白分选分泌途径相同的蛋白质。分选方式似乎取决于蛋白质的表达方式，组成型或突现型。这些结果将有助于构建基因转移到肠上皮细胞和其他极化细胞的策略。

项目成果

Yoshinobu, Takakura: "Cellular uptake properties of oligonucleotides in LLC-PK_1 renal epithelial cells." Antisense and Nucleic Acid Drug Development. 8. 67-73 (1998)

Yoshinobu, Takakura：“LLC-PK_1 肾上皮细胞中寡核苷酸的细胞摄取特性。”

Sachiko, Okamoto: "Stimulation side-dependent asymmetrical secretion of poly C-induced interferon-β from polarized epithelial cell lines" Biochemical and Biophysical Research Communications. 254. 5-9 (1999)

Sachiko，Okamoto：“从极化上皮细胞系刺激多聚 C 诱导的干扰素-β 的侧依赖性不对称分泌”《生物化学和生物物理研究通讯》254. 5-9 (1999)。

Taro Kanamaru: "Biological effects and cellular uptake c-myc antisense oligonucleotides and their cationic liposome complexes." Jourmal of Drug Targeting. 5(4). 235-246 (1998)

Taro Kanamaru：“c-myc 反义寡核苷酸及其阳离子脂质体复合物的生物效应和细胞摄取。”

Taro, Kanamaru: "Biological effects and cellular uptake of c-myc antisense oligonucleotides and their cationic liposome complexes." Journal of Drug Targeting. 5(4). 235-246 (1998)

Taro, Kanamaru：“c-myc 反义寡核苷酸及其阳离子脂质体复合物的生物效应和细胞摄取。”

Yoshinobu Takakura: "Cellular uptake properties of oligonucleotides in LLC-PK_1 renal epithelial cells." Antisense and Nucleic Acid Drug Development. (in press).

Yoshinobu Takakura：“LLC-PK_1 肾上皮细胞中寡核苷酸的细胞摄取特性。”