Repeated Gene Transfection Impairs the Engraftment of Transplanted Porcine Neonatal Pancreatic Cells

BACKGROUND: Previously, we reported that neonatal porcine pancreatic cells transfected with hepatocyte growth factor (HGF) gene in an Epstein-Barr virus (EBV)-based plasmid (pEBVHGF) showed improved proliferation and differentiation compared to those of the control. In this study, we examined if pancreatic cells transfected repeatedly with pEBVHGF can be successfully grafted to control blood glucose in a diabetes mouse model. METHODS: Neonatal porcine pancreatic cells were cultured as a monolayer and were transfected with pEBVHGF every other day for a total of three transfections. The transfected pancreatic cells were re-aggregated and transplanted into kidney capsules of diabetic nude mice or normal nude mice. Blood glucose level and body weight were measured every other day after transplantation. The engraftment of the transplanted cells and differentiation into beta cells were assessed using immunohistochemistry. RESULTS: Re-aggregation of the pancreatic cells before transplantation improved engraftment of the cells and facilitated neovascularization of the graft. Right before transplantation, pancreatic cells that were transfected with pEBVHGF and then re-aggregated showed ductal cell marker expression. However, ductal cells disappeared and the cells underwent fibrosis in a diabetes mouse model two to five weeks after transplantation; these mice also did not show controlled blood glucose levels. Furthermore, pancreatic cells transplanted into nude mice with normal blood glucose showed poor graft survival regardless of the type of transfected plasmid (pCEP4, pHGF, or pEBVHGF). CONCLUSION: For clinical application of transfected neonatal porcine pancreatic cells, further studies are required to develop methods of overcoming the damage for the cells caused by repeated transfection and to re-aggregate them into islet-like structures.

Inhibition of hyperplasia of vascular smooth muscle cells in rat by the plasmid containing the short hairpin RNA of angiotensin II type 1 receptor / 中华外科杂志

<p><b>OBJECTIVE</b>RNA interference is a new technology that inhibit effectively the expression the specific genes. The current study was designed to investigate whether the plasmid containing the short hairpin RNA (shRNA) of angiotensin II type 1 receptor (AT(1)R) can inhibit the hyperplasia of vascular smooth muscle cells in rat.</p><p><b>METHODS</b>The plasmids containing the shRNA of AT(1)R were constructed, and transfected vascular smooth muscle cell (VSMC) to detect the effect on the AT(1)R expression by RT-PCR and Western blot, observe the shape of VSMCs by the inverted phase contrast microscope, and detect the hyperplasia of VSMCs by trypan blues staining and MTT.</p><p><b>RESULTS</b>The plasmids was certified to be in the right rank. After transfecting cells, there was significant difference (P < 0.01) in the expression of AT(1)R mRNA between the plasmid transfected group (pAT(1)R-shRNA(1) 1.37 +/- 0.15; pAT(1)R-shRNA(2) 1.45 +/- 0.12) and the control group (2.09 +/- 0.26), and there was significant difference (P < 0.01) in the expression of AT(1)R protein between the gene transfected group (pAT(1)R-shRNA1 1.12 +/- 0.04; pAT(1)R-shRNA2 1.20 +/- 0.07) and the control group (3.17 +/- 0.21). It is shown that pAT(1)R-shRNA can decrease the expression of AT(1)R mRNA and protein. There was significant difference (P < 0.01) in the Cell number between the plasmid transfected adding AngII group (pAT(1)R-shRNA1 5.48 +/- 0.44; pAT(1)R-shRNA2 5.55 +/- 0.45) and the AngII control group (8.13 +/- 0.41); there was significant difference (P < 0.01) in the Ratio of light density by MTT between the plasmid transfected adding AngII group (pAT(1)R-shRNA1 0.365 +/- 0.024; pAT(1)R-shRNA2 0.307 +/- 0.025) and the control group (0.485 +/- 0.011); It is shown that that pAT(1)R-shRNA can inhibit the hyperplasia of VSMCs, and matching the result of morphology observation.</p><p><b>CONCLUSIONS</b>The plasmids containing the shRNA of AT(1)R can inhibit the expression of AT(1)R mRNA and protein in VSMCs, and inhibit the hyperplasia of VSMCs induced by AngII in rat.</p>

Effect on hyperplasia of VSMCS in rat by the plasmid containing the short hairpin RNA of AT1R / 中国应用生理学杂志

<p><b>AIM</b>RNA interference is a new technology that inhibit effectively of the expression the specific genes. The present study was designed to investigate whether the plasmid containing the short hairpin RNA (shRNA) of angiotensin II type 1 receptor (AT1R) can inhibit the hyperplasia of VSMCs in rat.</p><p><b>METHODS</b>The plasmids containing the shRNA of AT1R were constructed, and transfected vascular smooth muscle cell (VSMC) to detect the effect on the AT1R expression by RT-PCR and Western blot, and detect the hyperplasia of VSMCs by trypan blues training and MTT.</p><p><b>RESULTS</b>The plasmids was certified to be in the right rank, and there was significant difference (P < 0.01) in the expression of AT1R mRNA and protein between the plasmid transfected group and the control group. There was significant difference (P < 0.01) in the hyperplasia of VSMCs between the plasmid transfected adding Ang II group and the control group.</p><p><b>CONCLUSION</b>The plasmids containing the shRNA of AT1R have the effect of RNAi, and inhibit the hyperplasia of VSMCs induced by Ang II in rat.</p>