| Background and objective: Tissue inhibitor of metalloproteinase-l(TIMP-l), a major member of TIMPs family, is a multifacial protein which is involved in both pathological and physiological processes. Besides its MMP-inhibiting activity, TIMP-1 also exhibits other multifunctional activities, such as cell growth-promoting, anti-apoptotic, steroidogenic and anti-angiogenic activities. Recent study suggested that TIMP-1 could prevent pancreatic p-cell from cytokine-mediated (IL-1β, TNF-α, IFN-γ) apoptosis in vitro, and that the apoptosis of β-cell was critical to the etiology of diabetes. Therefore, our hypothesis was that TIMP-1 may delay the onset of diabetes induced by streptozotocin (STZ) by preventing pancreatic p-cell from apoptosis. There have been no studies on the protective effects of TIMP-1 on pancreatic P-cell injury in vivo. The aim of present study was to establish a homozygous hTIMP-1 trangenic mouse strain, and to explore the possible protective effects and its mechanism on pancreatic p-cell injury induced by STZ.Methods: (1) The hTIMP-1 transgenic founder mice was obtained by the technique of microinjecting foreign DNA into the pronuclear of fertilized eggs in our previous study. Testing breeding and fluorescence in situ hybridization (FISH) were used to screen homozygote, and to establish homozygous transgenic mouse strain. The integration pattern of foreign gene in transgenic mice genome was identified by the combination of Southern blot and inverse PCR. The tissue expression profile of foreign gene hTIMP-1 in transgenic mice was studied by Northern blot. (2) The effects of different doses of STZ on blood glucose and survival rate in homozygous transgenic mice were observed. TUNEL staining was used to determine the peak of apoptosis in pancreatic islet cells after a single dose of STZ (100mg/kg) stimulation in normal model group mice. The peak time point of apoptosis was chosen for comparison of p-cell apoptosis between normal model group and transgenic model group by means of double staining of TUNELand insulin. The expression of Bcl-2, Bcl-XL, Bax, caspase-3 and caspase-9 were assesed with RT-PCR and Western blot at the peak time point of apoptosis.Results: (1) The homozygous hTIMP-1 trangenic mouse strain was established successfully. The results confirmed that the foreign gene hTIMP-1 was integrated into the transgenic mice Chrl7 El.3 in the form of a single copy and single locus, indicating that foreign gene hTIMP-1 was integrated stably into the chromosomes of homozygous transgenic mice, and could be transmitted to offsprings. Foreign gene hTIMP-1 was expressed mainly in kidney, liver and pancreas. (2) hTIMP-1 transgenic model group mice developed delayed hyperglycemia in comparison to normal model group induced with different doses of STZ. 12h time point was found to be the peak of apoptosis in pancreatic islet cells after stimulation with a single dose of STZ (lOOmg/kg). When compared with normal model group at the 12h time point, the transgenic model group showed a much lower apoptotic index [(2.5±0.6)% vs (12.7±2.6)%, P = 0.01] of (3-cell which showed the positive double staining of TUNEL and insulin; The results of analysis of covariance of the mRNA and protein levels of apoptosis-related factors showed: CD There was no significant difference in the pancreatic expression levels of Bcl-2 and Bax between normal group and transgenic group (P > 0.05), which were both upregulated markly after STZ stimulation in two groups, but there was no significant difference in the response trend to STZ stimulation between the two groups (P > 0.05), suggesting that the expression levels of Bcl-2 and Bax weren't affected by the foreign gene hTIMP-1. (2) when compared with normal group, the pancreatic expression level of BcI-Xl was higher in transgenic group(P < 0.05), and was upregulated significantly after STZ stimulation in the two groups, but there was no significant difference in the response trend to STZ stimulation between two groups(P > 0.05), suggesting that foreign gene hTIMP-1 may upregulate the expression of BcI-Xl. (D There was no significant difference in the pancreatic expression levels of caspase-3 and caspase-9 between normal and transgenic groups (P > 0.05). They were both upregulated markly in normal model group, but only slightly upregulated in transgenic model group after STZ stimulation. And there was significant difference in the response trend to STZ stimulation between the two groups (P <...
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