【作者】 吴黎明；

【导师】 郭定宗；

【作者基本信息】 华中农业大学 ， 临床兽医学， 2014， 博士

【摘要】 斯钙素1（Stanniocalcin1, STC-1）是一种最初在硬骨鱼类发现的低钙性激素,在鱼类它是由紧邻肾脏而且在整个肾脏中均有分布的特殊器官—斯坦尼氏小体所分泌,该激素主要通过调节腮、小肠和肾脏Ca2+和无机磷酸盐（Inorganic phosphate, Pi）的转运来发挥其抗高钙效应。哺乳动物体内,STC-1在多种器官中均有表达,包括Ca2+吸收上皮如小肠、回肠、肾脏、胎盘以及其它血管化组织。哺乳动物STC-1是一多功能效应蛋白,如它能调节肾脏和肠道Ca2+/Pi吸收,小鼠中其超表达可致高血Pi、侏儒症、代谢率增加等效应,近来发现它和癌症也有一定的联系。STC-1的表达受1,25（OH）2D3等理化因素调节。然而,STC-1在哺乳动物中的多种生理作用还未完全清楚,还有待进一步研究。1.原核表达牛源STC-1重组蛋白本试验以奶牛肾脏组织提取的总RNA合成cDNA,然后使用PCR技术扩增出除信号肽序列外的奶牛STC-1全长结构基因。利用T-A克隆原理将扩增产物克隆到pMD-18T载体,转化至DH5α感受态细胞,经过夜培养后挑取单菌落进行扩大培养,然后将菌液进行测序。结果表明该扩增产物和GenBank登录号BC105435.1奶牛斯STC-1基因序列完全符合。然后将测序正确的扩增产物进行双酶切并亚克隆到pET-32a+表达载体,转化到感受态细胞Rosetta（DE3）中,经0.6mM IPTG诱导3h后,成功表达出奶牛STC-1融合蛋白。2.STC-1蛋白在奶牛胃肠道差异性表达情况本实验分别采用半定量实时荧光PCR和western blotting技术来分析STC-1基因和蛋白相对表达量。STC-1基因表达情况以表达率形式予以展示：胃和肠道的相对表达值除以肾脏的相对表达值的百分数。结果显示,在胃中STC-1基因和蛋白表达量最高是在皱胃；而在肠道中十二指肠和结肠的基因表达量最高,蛋白表达量最高却是在十二指肠和空肠。肾脏中STC-1基因和蛋白的表达量均高于胃肠道中的表达量。另外,本实验发现肠道斯钙素1在30bp处出现免疫条带,说明该STC-1可能分子量约为50kD （STC50）,而非更大分子量（bigSTC）。从结果可以看出STC-1表达最丰富的部位正好是胃肠道消化和吸收最活跃的部位,表明STC-1可能参与了奶牛消化与吸收过程。3.新生牛小肠上皮细胞培养方法的建立本实验建立了一种从新生犊牛小肠组织获取小肠上皮细胞的方法。0.25%胰蛋白酶、0.1%胶原酶Ⅰ以及胶原酶Ⅺ/中性蛋白Ⅰ混合物等方法用于从小肠组织分离小肠绒毛和隐窝。然后利用小肠上皮细胞和成纤维细胞的贴壁能力和对胰蛋白酶消化耐受性之间的差异来纯化小肠上皮细胞。使用上皮细胞特异性角蛋白抗体进行免疫细胞化学染色的方法来区别培养物中上皮细胞和成纤维细胞。光学和电子显微镜用于细胞形态学观察。结果表明所有分离方法均能分离一定量隐窝和/或肠绒毛,但胶原酶Ⅰ分离效率更高、所得消化物结构更完整,但需要消化很长时间。原代培养物能在孵育3-7d内贴壁,其间夹杂着一定量成纤维细胞和平滑肌样细胞。经多种方法纯化后,能得到较高纯度的上皮样细胞,该培养物能存活1.5月并传代5-7次。从培养细胞中能克隆到蔗糖酶、钙结合蛋白和上皮钙离子通道,说明该培养物保留了部分小肠上皮细胞生物活性。根据以上特点说明该培养物能用于小肠上皮细胞功能的研究。4.STC-1对过氧化氢诱导的奶牛肠道上皮细胞损伤的作用研究该研究利用H₂O₂处理原代小肠上皮细胞以模拟慢性肠炎造成的细胞损伤。上皮细胞经重组质粒超表达STC-1后,再由200μM H2O2处理不同时间,以研究STC-1对氧化损伤的作用。AO/EB双染色和台盼蓝排除法用于鉴定细胞的损伤情况,STC-1和凋亡相关蛋白表达情况由Real-time PCR和western blotting方法检测。结果表明STC-1基因和蛋白表达均随H2O2诱导时间延长而增加,H₂O₂诱导的细胞损伤也具有时间依赖而加剧的趋势,该趋势能为STC-1过表达而逆转。此外,细胞过表达STC-1也能上调凋亡相关蛋白Bcl-2的表达,同时轻微下调caspase-3的表达。本实验表明STC-1保护细胞免受氧化损伤的可能机制之一是它具有抗氧化和抗凋亡作用,因此,STC-1是奶牛慢性肠炎的一个潜在诊断或治疗指标。5.STC-1对Caco2细胞钙离子转运蛋白调节作用研究本实验的目的是初步探索STC-1影响Ca2+吸收的可能分子机制,多种新型Ca2+通道和维生素D受体是本研究的对象。该实验采用外源导入真核表达载体的方式增强Caco2细胞STC-1的表达,导入特异性siRNA的方式抑制该激素的表达,两方面分别观察TRPV5、TRPV6、PMCA1b、NCX1和VDR等钙离子转运蛋白表达水平结果表明,增强STC-1表达后TRPV5、TRPV6和VDR mRNA和蛋白的表达水平均有所下调,而抑制了该激素的表达后TRPV5和TRPV6的表达有所上升,尤以TRPV6表达明显,当添加外源重组STC-1后,其表达又下调了。但增强和抑制该激素的表达都对PMCA1b和NCX1表达没有显著影响。因此,斯钙素1影响上皮细胞Ca2+摄取可能是通过抑制了Ca2+进入细胞的通道而实现的。更多还原

【Abstract】 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.更多还原