Adult Pig Islet Isolation.

Type 1 diabetes mellitus (T1DM) is caused by autoimmune destruction of pancreatic ß cells, which results in little or no insulin production. Islet transplantation plays an important role in the treatment of T1DM, with the improved glycometabolic control, the reduced progression of complications, the reduction of hypoglycemic episodes when compared with traditional insulin therapy. The results of phase III clinical trial also demonstrated the safety and efficacy of islet allotransplantation in T1DM. However, the shortage of pancreas donors limits its widespread use. Animals as a source of islets such as the pig offer an alternative choice. Because the architecture of the pig pancreas is different from the islets of mice or humans, the pig islet isolation procedure is still challenging. Since the translation of alternative porcine islet sources (xenogeneic) to the clinical setting for treating T1DM through cellular transplantation is of great importance, a cost-effective, standardized, and reproducible protocol for isolating porcine islets is urgently needed. This manuscript describes a simplified and cost-effective method to isolate and purify adult porcine islets based on the previous protocols that have successfully transplanted porcine islets to non-human primates. This will be a beginners guide without the use of specialized equipment such as a COBE 2991 Cell Processor.

MicroRNA-135a expression is upregulated in hepatocellular carcinoma and targets long non-coding RNA TONSL-AS1 to suppress cell proliferation.

Dysregulation of long non-coding RNAs (lncRNAs) results in development of human diseases, including hepatocellular carcinoma (HCC). lncRNA TONSL-AS1 has been reported to act as a tumor suppressor in gastric cancer. The present study aimed to investigate the role of TONSL-AS1 in hepatocellular carcinoma (HCC). Reverse transcription-quantitative PCR analysis was performed to detect the expression levels of TONSL-AS1 and microRNA (miRNA/miR)-135a in HCC tissues and paired adjacent normal tissues. A 5-year follow-up study was performed to determine the prognostic value of TONSL-AS1 in HCC. The association between miR-135a and TONSL-AS1 was assessed via overexpression experiments. The Cell Counting Kit-8 assay was performed to assess cell proliferation. The results demonstrated that TONSL-AS1 expression was downregulated in HCC tissues, which was associated with a lower survival rate in patients with HCC. TONSL-AS1 and miR-135a were predicted to interact with each other, whereby overexpression of miR-135a downregulated TONSL-AS1 expression. The results demonstrated that TONSL-AS1 and miR-135a were inversely correlated with each other. Notably, overexpression of TONSL-AS1 inhibited HCC cell proliferation, while overexpression of miR-135a promoted HCC cell proliferation and decreased the effect of overexpression of TONSL-AS1 on cell proliferation. Taken together, the results of the present study suggest that miR-135a expression is upregulated in HCC and targets lncRNA TONSL-AS1 to suppress cell proliferation.

Analysis of Differentially Expressed Proteins Involved in miR-449a for Hepatocellular Carcinoma by iTRAQ Proteomics.

BACKGROUND: To investigate the relationship between down-regulation of miR-449a and prognosis of hepatocellular carcinoma (HCC) and to elucidate the potential target proteins of miR-449a. MATERIAL AND METHODS: The expression of miR-449a in 142 HCC tissues was detected by RT-PCR. The correlation between down-regulation of miR-449a and prognosis of HCC was statistically analyzed during clinical follow-up. The Bel-7042 HCC cell line in miR-449a-mimic and miR-449a-inhihbitor model was used, and the potential target protein of miR-449a was screened by isobaric tags for relative and absolute quantitation (iTRAQ) technology. RESULTS: miR-449a was significantly down-regulated in HCC tissues, which was significantly associated with post-operative metastasis (p < 0.0001) and recurrence (p < 0.0001). The median overall survival time in the low-expression group of miR-449a was significantly lower than that of the high-expression group (19 months vs. 37 months, p = 0.001). In addition, the tumor-free survival time of the low-expression group was significantly lower than that of the high-expression group (14 months vs. 24 months, p = 0.001). iTRAQ analysis screened out 137 differential proteins, among which 88 were up-regulated and 49 were down-regulated. GO clustering, KEGG pathway, and STRING analysis were performed, suggesting that these differential proteins have complicated functions, such as ATP binding, metal ion binding, RNA binding, human papillomavirus infection, and Epstein-Barr virus infection. CONCLUSIONS: miR-449a was negatively correlated with HCC prognosis. The differential proteins screened by iTRAQ can provide the basis for studying the target proteins regulated by miR-449a and understanding the pathogenesis of HCC.

Potential roles of mesenchymal stromal cells in islet allo- and xenotransplantation for type 1 diabetes mellitus.

Islet transplantation is poised to play an important role in the treatment of type 1 diabetes mellitus (T1DM). However, there are several challenges limiting its widespread use, including the instant blood-mediated inflammatory reaction, hypoxic/ischemic injury, and the immune response. Mesenchymal stem/stromal cells (MSCs) are known to exert regenerative, immunoregulatory, angiogenic, and metabolic properties. Here, we review recent reports on the application of MSCs in islet allo- and xenotransplantation. We also document the clinical trials that have been undertaken or are currently underway, relating to the co-transplantation of islets and MSCs. Increasing evidence indicates that co-transplantation of MSCs prolongs islet graft survival by locally secreted protective factors that reduce immune reactivity and promote vascularization, cell survival, and regeneration. MSC therapy may be a promising option for islet transplantation in patients with T1DM.

Heparin inhibits the inflammatory response induced by LPS and HMGB1 by blocking the binding of HMGB1 to the surface of macrophages.

High mobility group box 1 protein (HMGB1), a nuclear non-histone DNA-binding protein, is secreted extracellularly during inflammation and is a late mediator of inflammatory responses. The pro-inflammatory activity of recombinant HMGB1 proteins is dependent upon the formation of complexes with other mediators, such as lipopolysaccharide (LPS). This study investigated the influence of heparin on LPS+HMGB1-mediated inflammatory responses in cultured macrophages and a murine sepsis model. HMGB1 promoted the phosphorylation of p38 and ERK1/2. HMGB1 enhanced the induction of the pro-inflammatory cytokine, TNF-α, by LPS in macrophages. Heparin blocked the binding of HMGB1 to the surface of macrophages, and suppressed the phosphorylation of p38 and ERK1/2, but not JNK; TNF-α secretion was also decreased. However, heparin alone did not affect LPS-induced production of TNF-α. Heparin reduced lethality in mice exposed to LPS+HMGB1. To conclude, heparin inhibited LPS-induced HMGB1-amplified inflammatory responses by blocking HMGB1 binding to macrophage surfaces. Heparin could be used therapeutically as an effective inhibitor of HMGB1-associated inflammation.