Expression Of Stanniocalcin1in The Gastrointestinal Tract Of Neonatal Calf And The Effects On Oxidative Stress And Novel Calcium Channels

Stanniocalcin1(STC-1) is hypocalcemic hormone that is originally identified in fish where it is released by specialized organs, the corpuscles of Stannius that are located adjacent to the kidney and scattered throughout the kidney and exerts antihypercalcemic effect by regulating Ca2+and inorganic phosphate (Pi) transport in the gill, intestine, and kidney. This hormone has also been recently identified in mammals where it is expressed in multiple organs including Ca2+-transporting epithelia like intestine, colon, kidney, placenta and other vascularized tissues. STC-1acts as local mediator, such as modulating gut/renal Ca2+/Pi excretion, over-expressing in mice results in high serum phosphate, dwarfism, and increased metabolic rate, and being liked to cancer in recent reports. STC-1was modulated by1,25(OH)2D3and other physical and chemical factors treatment. Nevertheless, the exact physiological effects of STC-1in mammals are less defined, and further studies are needed to establish its functional importance.1. The prokaryotic expression of cow’s STC-1fusion proteinThe cDNA was synthesized from the total RNA obtained from calf kidney tissue, and the full-length of CDS of STC-1was then amplified using PCR. The PCR amplicon was cloned into a pMD-18T vector, transformed into DH5a, cultured in large scale and then sequenced. The amplicon with completely correct sequence was subcloned into the pET-32a+vector and transformed into Rosetta (DE3) competent cells. After a3-h induction by0.6mM IPTG, we successfully expressed the fusion protein of STC-1.2. The differential expression of STC-1in bovine gastrointestinal tractReal-time PCR and western blotting assays were used to analyze the relative expression levels of STC-1gene and protein, respectively. The data of relative gene expression levels were shown as the expression ratio:the relative value of stomach and intestinal tract to the standard sample in kidney. Quantitative analyses indicated that the highest levels of both STC-1mRNA and protein are expressed in the kidney. In contrast, the highest STC-1mRNA and protein levels in the stomach are expressed in the abomasum. In addition, the duodenum and colon exhibited the highest STC-1mRNA relative expression ratios among the intestines, whereas the highest protein expression levels were found in the duodenum and jejunum. Furthermore, the result showed that the STC-1detected in this experiment may be a STC50rather than a big STC. Thus, STC-1 3. The development of a method for preparation of bovine intestinal epithelial cell (IEC) primary cultures0.25%trypsin,0.1%(w/v) collagenase I, and collagenaseXI/dispase I digestion were used to isolate the villi and crypts, Purification of the IEC was achieved using the difference of adherence and tolerance of trypsin digestion between epithelial cells and fibroblast. The characterization test for IEC was performed by an immunocytochemistry staining for cytokeratins that unique to epithelium. Optical and electron microscopy assay were used to investigate the cellular morphology. The results showed that the optimal primary cultures were achieved by using a collagenase I digestion singly.7-12days post-incubation, the primary cultures reached confluence and consisted of epithelial colonies together with varying amounts of fibroblasts and smooth-muscle-like cells. Combination of various strategies for purification could obtain the epithelial cells with a higher percentage of purification. In addition, the sucrase, Calbindin D9k and Ca2+channels genes were cloned from the cultures also suggests that the physiological potential of IEC is still maintained in vitro. The primary cultures could be maintained for a maximum period of1.5month and be sub-cultured5-7times. Thus, these cultured cells displayed important functional properties that could be utilized in future studies of primary IEC.4. STC-1protects bovine intestinal epithelial cells from hydrogen peroxide-induced damageThis study utilized the primary intestinal epithelial cells (IEC) exposed to hydrogen peroxide (H2O2) for different time intervals to mimic the chronic enteritis induced cellular damage. Prior to treatment with200μM H2O2, the cells were transfected with recombinant plasmid for48h to over-express STC-1. AO/EB double staining and trypan blue exclusion assays were then utilized to detect the viability and death rate of the cells, respectively. The expressions of STC-1and apoptosis-related proteins in the cells were detected by real-time PCR and western blotting. The results indicated that both of the STC-1mRNA and protein expression levels positively correlated with the duration of H2O2treatment. The cellular damage induced by H2O2in bovine IEC in a time-dependent manner, and this effect could be attenuated by the over-expression of STC-1. Furthermore, the over-expression of STC-1can up-regulate Bcl-2protein expression, and down-regulate caspase-3expression slightly in damaged cells. This study suggested that STC-1may play a protective role in intestinal cells through its activity of anti-oxidative stress. Thus, STC-1may be a therapeutic target for chronic enteritis.5. Regulation of intestinal epithelial calcium transport proteins by STC-1in Caco2cellsThis study has examined the expression levels of the calcium transport proteins involved in transcellular transport across intestinal epithelia in Caco2cells following over-expression or inhibition of STC-1. These proteins include the transient receptor potential vanilloid members (TRPV)5and6, the plasma membrane calcium ATPase lb (PMCAlb), the sodium/calcium exchanger (NCX1), and the vita min D receptor (VDR). Both gene and protein expressions of TRPV5and6were attenuated in response to over-expression of STC-1, and the opposite trend was observed in cells treated with siRNASTC-1. To further investigate the ability of STC-1to influence TRPV6expression, cells were treated with100ng/mL recombinant human STC-1(rhSTC-1) for4h following pre-transfection with siRNASTC-1for48h. Intriguingly, the increase in the expression of TRPV6resulting from siRNASTC-1was reversed by treatment with rhSTC-1. No significant effect of STC-1on the expression of PMCAlb, NCX1or VDR was observed in this study. We therefore conclude that the inhibition of STC-1on calcium transport in intestinal epithelia is due at least in part to its negative regulation of expression of the epithelial channels TRPV5/6that mediate calcium influx.