Abstract
Background & Aims: Chronic pancreatitis (CP) is characterized by chronic inflammation and progressive pancreatic fibrosis leading eventually to the loss of exocrine and endocrine pancreas functions. The loss-of-function mutations of serine protease inhibitor Kazal type 1 (SPINK1) gene are associated with various forms of human CP. We previously showed that deletion of Spink3, the mouse homologue of SPINK1, causes pancreatitis-like changes in the mouse. Spink3-/mice die within 2 weeks after birth, making it impossible to monitor long-term effects of Spink3 deficiency. The aim of this study was to rescue the Spink3-/phenotype by generating Spink3-/mice with knocked-in SPINK1, and to characterize timedependent changes in the pancreas in this geneticmodel. Methods:We placedCAG promoterSPINK1 minigene (CAG-SP1) into diaphanous homolog 2 (Diap2) locus, which is located on X chromosome, using the Cre-Lox technology. X-inactivation is a process whereby one of the two copies of the X chromosome present in female mammals is inactivated. By utilizing X-inactivation, we were able to create mice in which SPINK level was partially, but not completely, reduced compared with the wild type. The CAG-SP1 knock-in mice were crossed to Spink3+/mice, thus generating Spink3-/-; CAG-SP1 knock-in mice. Mice were followed up for up to 6 months. Time-dependent changes in pancreatic histology, autophagy, inflammatory infiltration, acinar cell death, stellate cell activation, fibrosis, as well as trypsinogen activation and serum amylase were measured. Results: Spink3-/-;YXCAG-SP1, as well as Spink3-/-;XCAG-SP1/CAG-SP1 mice in which CAG-SP1 was present on both X chromosomes, showed no abnormalities in pancreas or any other organ during the 6 months of observation, indicating that human Spink1 rescues Spink3 deficiency in mice. Spink3-/-; XCAG-SP1/wild mice in which CAG-SP1 is present on only one of the two X chromosomes, showed slight growth retardation but were healthy and fertile. Pancreas of Spink3-/-; XCAGSP1/wildmice at birth contained both normal and degenerated acinar cells, with accumulation of autophagic vacuoles. The Spink3-/-;XCAG-SP1/wild mice time-dependently developed pathologic features of CP, including loss of acinar cells, intralobular fibrosis with activated stellate cells, and neutrophilic infiltration. In the interlobular areas, fibrosis was not evident, but instead prominent lipomatosis was observed. Interlobular ducts were dilated and contained protein plugs, which resembled CP in human. Older mice displayed acinar-ductal metaplasia and prominent expression of proto-oncogenes Egfr, Her2, and Ras, but did not develop pancreatic intraepithelial neoplasia. Conclusions: The results re-inforce the role of SPINK1/Spink3 deficiency in the development of CP and indicate that CP conditions trigger factors promoting pancreatic cancer development.
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