Characterization and genetic optimization of the MSL-G-AN pancreatic alpha cell line.

Ditte Møller Jakobsen

Student thesis: Project on Graduate Program

Abstract

Diabetes is a chronic non-communicable disease caused by abnormally high amounts of glucose in the bloodstream. The disease affects millions of people each year and the accompanied complications and medications to halt or treat the disease has an immense annual cost.
The primary focus in science, concerning diabetes, has traditionally been very β-centric and the interaction between pancreatic β- and α-cells, the α-cells role in diabetes and possible treatments methods involving α-cells are therefore sparsely understood1. Hence, there is a real need for a reliably and well-characterized α-cell line in order to have a model system for the investigation of the α-cells role in diabetes.
This thesis therefore characterizes different aspects of the 12C3AN pancreatic α-cell line for further scientific research. The 12C3AN cell lines’ doubling time, conditions for growth in media and the secretion and total content of glucagon, GLP-1 and insulin were investigated with ELISA (Enzyme linked immunosorbent assay) and immunohistochemistry. The mRNA expression of several endocrine marker mRNAs was also investigated with Q-PCR (Quantitive PCR) and knock-down of the PCSK1 gene using the CRISPR/Ca9 method was attempted.
The 12C3AN cell line has a doubling time of ~64,5 hours in either conditioned media from IN-28 or INS-1E. The secretion of glucagon, GLP-1, and insulin is not affected by the glucose concentration. Not all cells express glucagon or GLP-1, and 75% of the 12C3AN population express none of the two hormones. The cells express mRNA for proglucagon, nuropeptide Y (NPY), somatostatin, CART (Cocaine- and Amphetamine-Regulated Transcript), PCSK1 and PCSK2. The knock-down of PCSK1 did not succeed.
This thesis shows that the 12C3AN cell line is not suitable as a representative α-cell line. The 12C3AN cells glucagon secretion is not influenced by glucose and the cell line is still multihormonal producing GLP-1 in addition to glucagon.

EducationsPharmaceutical Biology, (Bachelor/Graduate Programme) Graduate
LanguageEnglish
Publication date1 Jun 2018
Number of pages103
SupervisorsLouise Torp Dalgaard