Use of EIF-5A1 SIRNA to protect islets cells from apoptosis and to preserve their functionality
A islet cell and functional technology, applied in the direction of DNA/RNA fragments, pancreatic cells, cell culture active agents, etc., can solve problems such as damage to islet viability, increased risk of hyperlipidemia and hypertension
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Embodiment 1
[0061] Example 1: Portal vein perfusion
[0062] Mouse islets express eIF-5A. Total RNA was extracted from isolated mouse islets, and RT-PCR was performed for β-actin and eIF-5A1 ( figure 1 ). Resting unstimulated islets showed positive levels of eIF-5A1-mRNA.
[0063] In eIF5A1-siRNA delivery: eIF5A1-mRNA levels decreased after slow portal vein perfusion. Introduce 1ml siRNA (CT (control) sequence or eIF-5A1, 5 μ g) or saline to mice by slow retrograde portal vein perfusion, n=2 for each group ( figure 2 ). The digested pancreas was digested by collagenase flushing of pancreatic ducts, and islets were isolated as described by Lewi et al., Proc. Islets (50 per mouse) were incubated for 16 hours. Total RNA was then extracted for RT-PCR against β-actin and eIF-5A1 ( image 3 ). The mRNA ratios of eIF-5A1 / β-actin were 5.24 (CT-siRNA) and 3.01 (eIF-5A1-siRNA). image 3 showed that the mRNA level of eIF-5A1 was reduced in those cells treated with siRNA. The experiment wa...
Embodiment 2
[0069] Example 2: Intraperitoneal injection
[0070] Antibodies and siRNA: Raise a mouse monoclonal antibody against eIF-5A1. Anti-actin monoclonal antibody (clone C4, #69100) was purchased from MP Biomedicals. In Western blots, secondary antibodies were labeled with near-infrared fluorophores from Li-Cor (IRDye 800 and IRDye 700). siRNA specific for eIF-5A1 was directed against the following sequence of eIF-5A1: 5'-AAAGGAATGACTTCCAGCTGA-3'. Whereas the control siRNA was the transcribed sequence 5'-AAAGTCGACCTTCAGTAAGGA-3', which was previously shown not to induce knockdown of any known protein. See co-pending applications U.S. 11 / 725,470 and PCT / US07 / 64424, which are hereby incorporated by reference in their entirety.
[0071] IP (intraperitoneal) injection of siRNA into mice: Twelve 8- to 10-week-old C57BL / 6 male mice were purchased from Charles River. Male mice were chosen to avoid the metabolic changes that accompany the female estrous cycle. Mice were randomly assign...
Embodiment 3
[0076] Example 3: Application of siRNA against Pdx-1 in vitro and in vivo
[0077] βTC3 cells were transfected with two different Pdx-1 siRNA constructs (constructs A and B) or controls and immunoblotted for the proteins Pdx-1, GAPDH and β-actin. see Figure 17A . C57B 1 / 6 mice were injected intraperitoneally with the same siRNAs once daily for 3 days, and islets were isolated and purified by collagenase digestion and differential gradient centrifugation. Islets were lysed in 2% SDS and subjected to immunoblotting. see Figure 17B . Isolated islets were loaded with Fura2 for 30 min before imaging in 3 mM D-glucose. Islets were stimulated with 11 mM D-glucose for approximately 300 s, and Fura2 ratios were continuously monitored by fluorescence microscopy. see Figure 17C . Glucose-stimulated insulin secretion (GSIS) was performed using 50 islets from each treatment group. see Figure 17D .
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