Changes in gene expression in beta cells after islet isolation and transplantation using laser-capture microdissection.
OBJECTIVE
The process of islet isolation can cause chemical and mechanical injury to beta cells. In addition, hyperglycaemia after islet transplantation can compromise beta cell function. The aim of this experiment was to evaluate changes in gene expression in endogenous islets using laser-capture microdissection (LCM).


METHODS
Islets from B6AF1 mice were studied in situ in the pancreas as well as those freshly isolated or cultured for 24 h. Fresh islets were transplanted under the kidney capsule of syngeneic diabetic (streptozocin-induced) and non-diabetic mice. Frozen sections from all the samples were prepared for LCM to obtain beta cell-enriched tissue; RNA was extracted and amplified using T7 polymerase. RT-PCR was used to assess expression of selected genes critical for beta cell function (Ins, Ipf1 [previously known as Pdx1], Slc2a2 [previously known as GLUT2] and Ldha) and the stress response (Hmox1 [previously known as HO-1], Gpx1, Tnfaip3 [previously known as A20] and Fas). Immunostaining was also performed.


RESULTS
In freshly isolated and cultured islets, insulin and Ipf1 mRNA levels were decreased by 40% (compared with islets in situ), while stress genes were upregulated. Comparison between in situ pancreatic islets and engrafted beta cells of cured mice showed declines in Ipf1 expression.


CONCLUSIONS
Our experiment, the first report to investigate changes in gene expression in endogenous islets using LCM, indicate that beta cells following islet isolation and residing in a foreign graft environment have decreased expression of genes involved in insulin production and increased expression of stress genes. Our data suggest that an islet graft, even in successful transplantation, may be different from endogenous islets in gene expression.
Ahn
Y B
YB
Section on Islet Transplantation and Cell Biology, Research Division, Joslin Diabetes Center and the Department of Medicine, Harvard Medical School, Boston, MA 02215, USA.
Xu
G
G
Marselli
L
L
Toschi
E
E
Sharma
A
A
Bonner-Weir
S
S
Sgroi
D C
DC
Weir
G C
GC
D000818:Animals; D001786:Blood Glucose / Q000378:metabolism; D001835:Body Weight; D002469:Cell Separation / Q000379:methods; D017931:DNA Primers; D005786:Gene Expression Regulation; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000502:physiology* / Q000637:transplantation; D007515:Islets of Langerhans / Q000166:cytology* / Q000502:physiology; D016381:Islets of Langerhans Transplantation / Q000502:physiology*; D007834:Lasers; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D042282:Microdissection / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics
D001786:Blood Glucose; D017931:DNA Primers; D012333:RNA, Messenger; D034741:RNA, Small Interfering