Construction of targeted plasmid vector pcDNA3.1-Egr.1p-p16 and its expression in pancreatic cancer JF305 cells induced by radiation in vitro

AIM: To construct pcDNA3.1-Egr.1p-p16 recombinant plasmid and investigate the expression of p16 in pancreatic cancer JF305 cells induced by radiation and the feasibility of gene radiotherapy for pancreatic carcinoma. METHODS: Human p16 cDNA was ligated to the downstream of Egr-1 promotor to construct pcDNA3.1-Egr.1p-p16 plasmid by restriction enzyme digested. The recombined plasmids were transfected into pancreatic cancer JF305 cells with lipofectamine. p16 mRNA level was detected by RT-PCR. The expression of p16 after different doses of X-ray radiation was detected by Western blot technique. Cell survival was assessed by clonogenic assays and cell viability was analysed by trypen blue exclusion. Flow cytometry was performed to study the apoptosis of JF305 cells. RESULTS: Restriction enzyme digestion showed the correctly constructed pcDNA3.1-Egr.1p-p16. The p16 expression in cells transfected with pcDNA3.1-Egr.1p-p16 induced by different doses of radiation was higher than that in the control group (P < 0.05). Eight hours after 2 Gy X-ray radiation, the expression reached its peak(87.00 ng/L), and was signifi cantly higher than that in the control group (P < 0.0.5). Clonogenic analysis and trypan blue extraction test showed that the pcDNA3.1-Egr.1p-p16 transfer enhanced radiation-induced cell killing in p16-null JF305 cell lines. The induction of apoptosis was lower in combined transfection and irradiation group than that in irradiation alone. CONCLUSION: X-ray can induce the recombinant plasmid pcDNA3.1-Egr.1p-p16 expression in JF305 cells. The detection of dose and time provides an experimental basis for in vivo study in future.

Embryonic Stem Cells-loaded Gelatin Microcryogels Slow Progression of Chronic Kidney Disease

Background： Chronic kidney disease （CKD） has become a public health problem.New interventions to slow or prevent disease progression are urgently needed.In this setting, cell therapies associated with regenerative effects are attracting increasing interest.We evaluated the effect of embryonic stem cells （ESCs） on the progression of CKD.Methods： Adult male Sprague-Dawley rats were subjected to 5/6 nephrectomy.We used pedicled greater omentum flaps packing ESC-loaded gelatin microcryogets （GMs） on the 5/6 nephrectomized kidney.The viability of ESCs within the GMs was detected using in vio two-photon fluorescence confocal imaging.Rats were sacrificed after 12 weeks.Renal injury was evaluated using serum creatinine, urea nitrogen, 24 h protein, renal pathology, and tubular injury score results.Structural damage was evaluated by periodic acid-Schiff and Masson trichrome staining.Results： In vitro, ESCs could be automatically loaded into the GMs.Uniform cell distribution, good cell attachment, and viability were achieved from day 1 to 7 in vitro.After 12 weeks, in the pedicled greater omentum flaps packing ESC-loaded GMs on 5/6 nephrectomized rats group, the plasma urea nitrogen levels were 26% lower than in the right nephrectomy group, glomerulosclerosis index was 62% lower and tubular injury index was 40% lower than in the 5/6 nephrectomized rats group without GMs.Conclusions： In a rat model of established CKD, we demonstrated that the pedicled greater omentum flaps packing ESC-loaded GMs on the 5/6 nephrectomized kidney have a long-lasting therapeutic rescue function, as shown by the decreased progression of CKD and reduced glomerular injury.