| In this study, the expression of osteopontin (OPN) and OPN mRNA in purified renal tubular epithelial cells (RTEC) of SD rats in vitro were deter- minated by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) respectively. The change of expression was observed subsequently after renal tubular epithelial cells were interacted by calcium oxalate monohydrate crystals (COM). The study try to explore both effect and mechanism of OPN in the formation of calcium oxalate stone, furthermore, provide theoretical proofs and methods for research in prevention of calcium oxalate stone.In recent years, a variety of renal tubular epithelial cell strains have been cultured successfully in different species, such as HKC and HK2 in human RTECs, RRTEp iC in rat RTECs, VEPT and EPT in rabbit PTC, LLC-PK1 in pig PTC, BS-C-1 in monkey RTECs, and JTC-12 in distal tubular cell line, etc. Although the cell strains have satisfied the demand in researching different renal tubular segment diseases in varying degrees, they are not only expensive but also difficult to be obtained. Meanwhile, for their more passages, they may lose some functions in gene transfection, immortalization of virus induction, and process of passage repeatedly, may occur difference with animal models in vivo and be unsuitable for specific research needs. Comparing with the RTEC strains, the renal tubular epithelial cells which were obtained from primary culture are more advantages in both hereditary capacity of diploid, and reflecting characteristics of growth in vivo. Thus, these cells are more suitable for experimental research in toxicology, drug test, and cytodifferentiation, although they are some disadvantages in technical difficulties of primary culture and finiteness of cell passages. In China, there are some reports that renal tubular epithelial cells in different species have been cultured successfully by either mechanical trituration combined with enzyme digestion, or trypsinization combined with density gradient centrifugation. However, there is great difference not only in primary cells mixed with many kinds of cells, but also in the same spicy. Therefore, it is very important that both the purity and stability of primary cells should be ensured for experimental study in vitro. For primary culture of renal tubular epithelial cells, the experimental animals which are requested to be specified-pathogens free (SPF) are practically healthy without any communicable diseases. After cultivated and grew germfreely, they are usually transferred to the facility with Barrier System to be fledged and multipled subsequently. In principle, pathogenic bacterium are forbidden to be present in the room where SPF animals are living, meanwhile,both drinking water and feeds for animals must be sterilized in megatemperature. Generally, it takes one week for the rats to accommodate completely new environment after they get to the experimental center by long-distance transport, because the animals have to receive medical inspection and eliminate stress reaction, which is one kind of adaptation reaction result from being stimulated by internal and external environment. The stress reaction is caused by many stimulating factors, including intrinsic heredity, procreation, and external environmental. After stimulated by "stressors" come from the stimulating factors, the animals are harmful, appearing orgasm,discomfort,restlessness,apocleisis,anaphylaxis,attenuated anabolism,lower transformation efficiency of feedstuff, imbalance in endocrine system, lower production trait,etc. Obviously, experiment will result in unsuccessful culture, or different consequences without the accommodating process.In this study, the common SD rats are used as laboratory animals, the experimental reagents are inexpensive ordinary products, both of them make it possible for large batches of homogeneous animal experiment to be undertaken simultaneously. In addition, the operation is simple, easy to be repeated and spread. All these advantages provide a great convenience for research concerned with renal tubular epithelial cells. During the study, using primary culture, renal tubular epithelial cells are procured from nephric tubules, which are come from renal cortexes of SD rats by mechanical abstraction and Percoll (colloidal silica suspension, the brand name) density gradient centrifugation. After different concentrations of COM were added to cell culture fluid, it was observed that the expression of OPN is enhancing along with the increasing concentrations of COM from 1 to 5mmol/L. The phenomenon indicates that COM may stimulate the expression of OPN of RTEC in vitro, enhance concentration of OPN in urine to resist effectively the formation of crystals. However, the expression intensity of OPN was weaker in 10mmol / L concentrations of COM than in 5mmol / L, the result may hint that COM with high concentration can bring about toxic effect on damage renal tubular epithelial cells. Although it is reported, in literatures, that oxalic acid can stimulate cells from resting stage into dividing phase, the increasing synthesis of OPN mRNA does not mean the increase of cell's quantity. The reason is that oxalic acid in high concentration bring about toxic effect on cells, leading to increase of membrane permeability, promotion of DNA breakage, and decrease of cell's quantity. In fact, as soon as adhering to renal tubular epithelial cells, COM with high concentration can damage cells, result in decreased OPN expression. The basyl's number of the product fragment which was come from nephridial tissue's mRNA, by RT-PCR and agarose gel electrophoresis(AGE), is the same as which of the mRNA gene marked in the gene bank(U.S. National Library Genebanr gi6981579). In 5 groups, it was also found that the expression of OPN mRNA was enhancing along with the increasing concentrations of COM from 1 to 5mmol / L, while the expression was weaker in 10mmol / L concentrations of COM than in 5mmol / L.In summary, we conclude that the expression of OPN is increased by renal tubular epithelial cells are stimulated by COM, but the expression is decreased in high concentrations of COM. It shows that high concentrations of COM can cause the decreased expression of OPN by damaging RTEC, and then result in the formation of kidney stones. On the achievement and innovation, two papers have been published depend on the study. On the other hand, using animal models with calcium oxalate stones, although Chinese researchers in Beijing University found that kidney stones result in up-regulation in the expression of OPN and OPN mRNA, the result demonstrated merely that OPN plays an important role in the formation of kidney stones. It did not explain enough either promotion or inhibitory action. It is unclear that the phenomenon is result from the reaction caused by either the inhibition to kidney stones or the damage of nephric tubule. Moreover, the results which come from our study in purified RTEC is more reliable than that come from other empirical study in urine and renal epithelial cells, for the reason of that nephric tubules are the main place where calcium oxalate calculus form kidney stones.
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