A highly sensitive and specific Homo1-based real-time qPCR method for quantification of human umbilical cord mesenchymal stem cells in rats.

BACKGROUND: Owing to the characteristics of easier access in vitro, low immunogenicity, and high plasticity, human umbilical cord-derived mesenchymal stem cells (UC-MSCs) are considered as a promising cell-based drugs for clinical application. No internationally recognized technology exists to evaluate the pharmacokinetics and distribution of cell-based drugs in vivo. METHODS: We determined the human-specific gene sequence, Homo1, from differential fragments Homo sapiens mitochondrion and Rattus norvegicus mitochondrion. The expression of Homo1 was utilized to determine the distribution of UC-MSCs in the normal and diabetic nephropathy (DN) rats. RESULTS: We observed a significant correlation between the number of UC-MSCs and the expression level of Homo1. Following intravenous transplantation, the blood levels of UC-MSCs peaked at 30 min. A large amount of intravenously injected MSCs were trapped in the lungs, but the number of them decreased rapidly after 24 h. Additionally, the distribution of UC-MSCs in the kidneys of DN rats was significantly higher than that of normal rats. CONCLUSIONS: In this study, we establish a highly sensitive and specific Homo1-based real-time quantitative PCR method to quantify the distribution of human UC-MSCs in rats. The method provides guidelines for the safety research of cells in preclinical stages.

The Murine Reg3a Stimulated by Lactobacillus casei Promotes Intestinal Cell Proliferation and Inhibits the Multiplication of Porcine Diarrhea Causative Agent in vitro.

Lactobacillus casei (L. casei), a normal resident of the gastrointestinal tract of mammals, has been extensively studied over the past few decades for its probiotic properties in clinical and animal models. Some studies have shown that some bacterium of Lactobacillus stimulate the production of antimicrobial peptides in intestinal cells to clear enteric pathogens, however, which antimicrobial peptides are produced by L. casei stimulation and its function are still not completely understood. In this study, we investigated the changes of antimicrobial peptides' expression after intragastric administration of L. casei to mice. The bioinformatics analysis revealed there were nine genes strongly associated with up-regulated DEGs. But, of these, only the antimicrobial peptide mReg3a gene was continuously up-regulated, which was also confirmed by qRT-PCR. We found out the mReg3a expressed in engineering E.coli promoted cell proliferation and wound healing proved by CCK-8 assay and wound healing assay. Moreover, the tight junction proteins ZO-1 and E-cadherin in mReg3a treatment group were significantly higher than that in the control group under the final concentration of 0.2 mg/ml both in Porcine intestinal epithelial cells (IPEC-J2) and Mouse intestinal epithelial cells (IEC-6) (p < 0.05). Surprisingly, the recombinant mReg3a not only inhibited Enterotoxigenic Escherichia coli (ETEC), but also reduced the copy number of the piglet diarrheal viruses, porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine rotavirus (PoRV), indicating the antimicrobial peptides mReg3a may be feed additives to resist the potential of the intestinal bacterial and viral diarrhea disease.

Umbilical Cord-Derived Mesenchymal Stem Cells Ameliorate Nephrocyte Injury and Proteinuria in a Diabetic Nephropathy Rat Model.

Mesenchymal stem cells (MSCs) are shown to alleviate renal injury of diabetic nephropathy (DN) in rats. However, the underlying mechanism of this beneficial effect is not fully understood. The aims of this study are to evaluate effects of umbilical cord-derived mesenchymal stem cells (UC-MSCs) on renal cell apoptosis in streptozotocin- (STZ-) induced diabetic rats and explore the underlying mechanisms. Characteristics of UC-MSCs were identified by flow cytometry and differentiation capability. Six weeks after DN induction by STZ injection in Sprague-Dawley rats, the DN rats received UC-MSCs once a week for consecutive two weeks. DN-related physical and biochemical parameters were measured at 2 weeks after UC-MSC infusion. Renal histological changes were also assessed. Moreover, the apoptosis of renal cells and expression of apoptosis-related proteins were evaluated. Compared with DN rats, rats treated with UC-MSCs showed suppressed increase in 24-hour urinary total protein, urinary albumin to creatinine ratio, serum creatinine, and blood urea nitrogen. UC-MSC treatment ameliorated pathological abnormalities in the kidney of DN rats as evidenced by H&E, PAS, and Masson Trichrome staining. Furthermore, UC-MSC treatment reduced apoptosis of renal cells in DN rats. UC-MSCs promoted expression of antiapoptosis protein Bcl-xl and suppressed expression of high mobility group protein B1 (HMGB1) in the kidney of DN rats. Most importantly, UC-MSCs suppressed upregulation of thioredoxin-interacting protein (TXNIP), downregulation of thioredoxin 1 (TRX1), and activation of apoptosis signal-regulating kinase 1 (ASK1) and P38 MAPK in the kidney of DN rats. Our results suggest that UC-MSCs could alleviate nephrocyte injury and albuminuria of DN rats through their antiapoptotic property. The protective effects of UC-MSCs may be mediated by inhibiting TXNIP upregulation in part.

Multifunctional TK-VLPs nanocarrier for tumor-targeted delivery.

Virus-like particles (VLPs) have been exploited for various biomedical applications, such as the monitoring, prevention, diagnosis and therapy of disease. In this study, a novel multifunctional VLPs nanocarrier (TK-VLPs) was prepared and used for tumor-targeted delivery. The SPR and cell uptake results indicated that the TK peptide is a "bi-functional ligand" with high affinity for Caco-2, HRT-18 and HUVEC cells through the integrin α6ß1 and integrin αvß3 receptors. The results of the direct immunofluorescence, SDS-PAGE and western blot assays demonstrated that the TK-VLPs were successfully prepared using the baculovirus expression system. Confocal laser scanning microscopy and the flow cytometry analysis validated that the TK-VLPs could target to Caco-2, HRT-18 and HUVEC cells. An in vivo study further confirmed that the TK-VLPs could target and efficiently deliver fluorescein to tumor cells and the tumor vasculature in mice bearing subcutaneous tumors. TK-VLPs-DOX displayed a uniform, spherical shape and an average size of approximately 28nm. The results of the cell uptake and cytotoxicity assays indicated that TK-VLPs-DOX could enhance the selectivity for colorectal cancer cells. Together, our studies provide strong evidence that TK-VLPs could target colon tumor cells and tumor angiogenesis with enhanced permeability and retention effects, suggesting that the TK-VLPs are a multifunctional nanocarrier with potential applications in a colon tumor-targeted drug delivery system.

Prokaryotic diversity in continuous cropping and rotational cropping soybean soil.

In spite of the techniques based on the amplification of 16S rRNA genes (16S rDNA) to compare bacterial communities that are now widely in use in microbial ecology, little is known about the composition of the soybean continuous cropping (CC) and rotational cropping (RC) soil microbial community. To address this, we compared the levels of bacterial community diversity in RC and 5-year CC rhizosphere soil samples. We selected 407 clones in RC and 490 clones in CC for restriction fragment length polymorphism analysis. A total of 123 phylotypes were identified among the 16S rDNA clones, while 78 unique and 21 common phylotypes were identified among the CC soil isolates. Analysis of sequences from a subset of the phylotypes showed that at least 11 bacterial divisions were represented in the clone libraries. The phylotype richness, frequency distribution (evenness), and composition of the two clone libraries were investigated using a variety of diversity indices. Although the analysis of diversity indices and LIBSHUFF comparisons revealed that the compared libraries were not significantly different (P=0.05) between the RC vs. CC soils, some differences could be observed in terms of specific phyla and groups. We concluded that the group variance was not determined immediately by the cropping system's induction, but was a long-term and slow process.