The set-up of an in vitro model for stable knockdown of MyD88 by lentivirus-based RNAi in IEC-6 cell line and the study on its early apoptosis.
- Author:
Pingqian BAO
1
;
Yang LI
;
Keling CHEN
;
Bin ZHOU
;
Bin LIU
;
Yuan LI
;
Zongguang ZHOU
Author Information
1. Department of Pediatric Surgery, West China Hospital, Sichuan University, Chendu 610041, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Apoptosis;
genetics;
Cell Line;
Epithelial Cells;
cytology;
Gene Knockdown Techniques;
Humans;
Inflammatory Bowel Diseases;
physiopathology;
Intestines;
cytology;
Lentivirus;
genetics;
metabolism;
Myeloid Differentiation Factor 88;
genetics;
RNA Interference;
RNA, Messenger;
genetics;
RNA, Small Interfering;
genetics;
Rats;
Toll-Like Receptor 4;
metabolism
- From:
Journal of Biomedical Engineering
2012;29(6):1138-1149
- CountryChina
- Language:Chinese
-
Abstract:
Intestinal inflammatory disease is a kind of non-specific disease with morbidity increasing yearly. It has been proved that the Toll like receptor 4 (TLR4) signaling pathways are closely related to intestinal inflammatory diseases. Myeloid differentiation protein 88 (Myd88) is a critical adaptor protein of TLR4 signaling and critical for the study of intestinal inflammatory disease, but stable Myd88 knockdown in vitro models of cell line are still very few. In the present study, an HIV-1-based lentivirus three-plamid packaging system was used for the construction of a lentivirual vector mediating RNA interference (RNAi) against Myd88 in intestinal fossae epithelial cell line-6 (IEC-6). Real-time PCR and Western blot were used to detect Myd88 expression. Annexin V staining and flowcytometry (FLM) were applied to detect and evaluate the early apoptosis. The results showed that lentiviral vectors containing the shRNA expression cassette specifically targeting Myd88 were constructed and efficiently stably knocked down Myd88 expression in IEC-6 cell line. Early apoptosis was significantly decreased after Myd88 knockdown. This study successfully constructed a lentivirus-based RNAi for Myd88 and detailed the key technique combined with characteristics of the early apoptosis after the Myd88 knockdown, provided a novel, stable and repeatable in vitro model for the pathogenesis, targeting therapeutic study for the intestinal inflammatory diseases.
