| 1Background and purposeUrolithiasis is a normal disease in urinary system, and up to now, there is no ideal method for conquer it. The origin of this disease still remains unclear for over80persent of patients. The main surgical therapies on urolithiasis are minimally invasive surgical techniques such as percutaneous nephrostomy, ureteroscopic stone removal and ESWL with the following30%-50%recurrent rate. If we can develop some effective medicine for preventing the formation of stone from the mechanism, that would be a very important contribution for clinic of urology. Stone formation are also subject to the impact of factors such as fluid dynamics, computer simulation and finite element analysis has become the current hot spots, which relates to computer modeling building. If we can uild parameterized urinary tract model with a relatively smaller number of anatomical data, it will not only to meet the demand for urology experimental studies, but also can provide a good platform for of urology animal experiments and virtual surgery.This study includes four parts according to our purpose:①Cultivated African green monkey renal tubular epithelial cells in vitro, then used H2O2to hurt the cells, tested cell viability, enzymes and macromolecules express involved in oxagen injury in each time point. Observed the changes of cells microstructure and the effection of CaOxa crystals nucleation and aggregation.②Cultivated human renal proximal tubular epithelial cells in vitro, then used H2O2to hurt the cells, tested cell viability, enzymes and macromolecules express involved in oxagen injury in each C(H2O2). Observed the changes of cells microstructure and the effection of CaOxa crystals nucleation and aggregation.③Degradated of Japanese SPS, then tested in urine system in vitro. Studied the crystals growing regular effection of raw and degradated SPS on Vero cells Based on the African green monkey renal epithelial cells model.④Used software of Autodesk3ds Max8SP2, simulated crystals shapes of COM, COD and calcium oxalate calculus. Based on the anatomic stucture of rats, builded organs such as kidney, adrenal gland, blood vessel, bladder airtub and gladula thyreoidea model using the commands of lofte, boolean controler, poly select, bevel profile, lethe et al. Merged those organ into surgical scenes, and got multi angle3D images after rendering.At present, cultured epithelial cells fall into several categories based on their origin. The first includes cell cultures derived from mouse, such as normal NRK-52E, primary inner medullary collecting duct cells (pIMCD), and primary mouse proximal tubule cells (pMPT); the second are those derived from dogs, such as Madin-Darby canine kidney cells (MDCK); the third are those derived from monkeys, such as African green monkey kidney epithelial cells (BSC-1); the fourth are those derived from pigs, such as LLC-PK1;and the fifth are the cell cultures derived from human beings, such as human renal tubular epithelial cells (HK-2). Here, we chose African green monkey renal tubular epithelial and human renal tubular epithelial cell lines for study.Soybean polycose (SPS) is made of soy asymphytous protein, soybean dregs from the by-product of soybean curd and bean-curd stick, through procedures such as fore treatment, enzymolysis (cellulase, hemicellulase, protease et al.), disassociation, bleaching, degerming. The advantages of it are wide source of low price and no side effects. Analysis showed that the main components of the SPS are galactose, arabinose, galacturonicacid, rhamnose, fucose xylose, glucose et al. The structure of it is rhamnose semi-galacturonic acid and high poly galactono-based chain, galactan and arabinose side chains combined with the structure similar to globular, with good dispersion, water solubility and emulsifying properties features. Here we study the hydrogen peroxide (Sevag) method to degradate Japanese soybean polysaccharides.3D Studio Max software is the world first-class3D modeling and animation system developed by Kinetix (Autodesk, Inc., Multimedia commercial organizations), You can get real image quality of the animation on a PC workstation. It was updated continuously and become the world most widely used3D modeling, animation, rendering popular software. Here we used software of Autodesk3ds Max8SP2(U.S., Autodesk, ID:666-12345678), operating platform was windows XP system.2Study Methods2.1Established Damage Model of Vero Cell LineAfrican green monkey renal tubular epithelial cell line, VERO was purchased from Chinese Academy of Sciences Shanghai Cell Bank(Num:GN017), cultured with DMEM-F12culture medium containing10%newborn calf serum, maintained in a37℃and saturated humidity environment, replaced medium every other day. added serum-free medium with different H2O2concentrations according to the experimental design. When the cells reached80%-90%confluence, washed them with PBS2times. Then used0.25%trypsin with0.02%EDTA to digest cells.3-5min after digestion, observed under inverted microscope; When the majority of the cells became round and separated spread, that indicated that the digestion suitble. Joined with10%newborn calf serum, DMEM-F12medium to terminate the digestion, full wind and percussion cells to form a single cell suspension. Applied CCK-8assay to detect the extent of H2O2resulting in cell damage, measuring the absorbance (A) at450nm with a microplate reader, and calculate the cell varbility rate (%), and draw survival-time curve and the survival rate-concentration curve,The cell survival rate (%)=A (experimental group)/A (control group)×100%.2.2Established Damage Model of HK Cell LineThe human renal tubular epithelial cells line was presented by Pro. Mei CL of the Shanghai Changzheng Hospital. Cell culture conditions were the same with the former, the cells were damaged with different concentrations of H2O2, cell culture medium containing hydrogen peroxide,0.1,0.3,0.5,1.0and2.0mmol/L for1h. The control group applied H2O2-free medium. At the specified point of time, suck out the medium, washed the cells twice with PBS then add fresh media. Added10μl of CCK-8to each well. Readed the absorbance through microplate reader under450nm wavelength after incubated for4h.2.3Cells SOD and MDA concentrationDeterminated MDA and SOD by double antibody sandwich method. Added malondialdehyde (MDA)/superoxide dismutase (SOD) into coated monoclonal antibody micropores, and then binded with HRP-labeled MDA/SOD antibody. Here the antibody-antigen-enzyme-antibody complex formed. After a thorough washing, added substrate TMB for coloration. TMB changed into blue catalysed by HRP enzyme, and final yellow by acid. The shades of color and malondialdehyde (MDA)/superoxide dismutase (SOD) level in samples is a positive correlation. Measured absorbance (OD) in450nm wavelength with a microplate reader, calculated the concentration of malondialdehyde (MDA)/superoxide dismutase (SOD) in the sample by standard curve.2.4Osteopontin expression in cells4%formaldehyde fixed cells for10min, then washed cells by PBS three times,3min once;Joined the sheep serum blocked for20min, then joined osteopontin primary antibody (1:100) at4℃overnight. Washed cells by PBS three times, dark dropped FITC secondary antibody (1:100) in darkroom, incubated at37℃for30min, PBS washed three times, and finally stained cells with DAPI and mounted, then ovserved fluorescence under laser scanning confocal microscopy. Nuclei in blue, and osteopontin in green.2.5SEM, XRD analysisAfter the cells incubated with crystal, remove the coverslip, washed the cells twice with PBS, added2.5%glutaraldehyde,1%OsO4fixed24h, and then washed with PBS three times, gradient ethanol (30%,50%,70%,90%,100%) dehydrated, then fixed samples with isoamyl acetate, dryed them with CO2at critical point, sprayed gold package. Observed cell morphology and crystal growth by SEM, at the same time detected the crystals on coverslips by XRD (5°<20<60°), to determine the crystal composition.2.6Zeta potential in cells membrane measurementWashed Cells with PBS to remove non-adhesion of the crystals, added0.25%trypsin-0.02%EDTA digestive juice, full winded and percused cells so that the cells and crystals shedding, collected cells and crystals after centrifuged(1,000rpm/s), cleared supernatant and re-suspended cells and crystals with1000μl PBS (pH=7.86), sucked about800μl suspension in the sample pool, measured Zeta potential with nano-particle size analyzer.2.7CaOxa concentration measurement after co-culturedThe cells were cultured in6-well plates, joined metastable CaOxa incubated for6h. Picked up slides from the6-well plates, and then washed slides with PBS to remove unbound crystals.The slides were put into a25ml beaker. Then, poured10ml HNO3and0.5ml HClO4into the beaker. The samples were digested in the furnace until clear curing formation. Samples were continuously heated until HClO4boiling, smoke untill dryed. After heating and drying the samples cooled at room temperature. And then dropped3.0ml water in the beaker to dissolve residue. Ca2+concentration in solution was measured by ICP method. The number of CaOxa crystals was measured the through Ca2+concentration, results in mg/cm2.2.8The extraction of soybean polysaccharidesSevag method was used in SPS extraction processing for removing slight protein. Sevag reagent (Vchloroform:Vn_butanol=5:1, Volumn0.2×) was added into the polysaccharide solution, shaked the mixture up and down vigorously for15minutes, then centrifuged (4000rpm,10min). After that the solution was divided into two layers, the upper was the polysaccharide solution, the lower is the gel generated by the Sevag reagent and denatured protein. Take the upper liquid, then add0.2times the volume of Sevag reagent, repeated the above operation for five times. Combined aqueous phase from which we can extract polysaccharides. Applied hydrogen peroxide (H2O2) to degradate SPS. Weighed1.2g polysaccharide and dissolved it in a100ml beaker with60ml distilled water. Stirring dissolve was at70℃water bath. After that, quickly added the15ml H2O2(volume fraction was30%). Degradated for1.5h till the solution was cooled the adjusted pH to7with NaOH (2mol/L). Transferred the liquid into a round bottom flask, rotary evaporated it at70℃water bath till the solution volume reduced to1/3of original volume. Added60ml of anhydrous ethanol, precipitated polysaccharides out overnight, filtered the precipitate, dried and weighed.2.9Determinate the polysaccharides physical and chemical natureUsed the phenol-sulfuric acid method for the determination of the percentage content of polysaccharides in the SPS; viscosity method for the determination of the average relative molecular mass before and after degradation of SPS.2.10Urinary calculi digital buildingCOM and COD monocrystalline model:COM model was based on six-party body, according to the proportion of built cylindrical, the number of edges is set to6, the vertices of double-dip surface applied polygon editing commands to adjust the vertex position. COD was base on double cone model with four corners, first we built pyramid of half-height, and then use the mirror relative command to copy the other half of model along Z axis, grouped the two Pyramid. Used transparent glass material ball, adjusted the parameters to white color, transparency40, translucent80, refractive index of1.5, and the brightness of200, gifted the material situations to the crystals.Stone model:based on a sphere, keeping the surface smooth. Used FFD4×4×4cylinder to adjust the surface, and then use the noise, set up0-100parameters along X, Y, and Z axis respectively, material ball plus stone alike picture. Urinary tract tubular applied tube body modeling, Used FFD4×4×4cylinder to adjust the surface, moved it to the site compatible with the stones, rendered and generated picture.2.11Digital building of rat urinary renal tubularUsed software of Autodesk3ds Max8SP2, based on the anatomic stucture of rats, builded organs such as kidney, adrenal gland, blood vessel, bladder airtub and gladula thyreoidea model using the commands of lofte, boolean controler, poly select, bevel profile, lethe et al. Merged those organ into the abdomen and neck scenes, installed free camera to get multi angle3D images after rendering.2.12Statistical analysisStatistical analysis used SPSS13.0software. Data expresses as mean±standard deviation (x±s). The cell viability was detected by One-Way ANOVA or Two-Way, the ANOVA method; MDA, SOD, OPN expression, crystals size and number were detected by One-Way ANOVA method of testing. First,Levene’s test was performed for testing variance homogeneity of, when P≥0.05, selected the analysis results based on homogeneity of variance, of variance; when P<0.05, selected the analysis results based on heterogeneity(Welch method). When analysis of variance was significant, further multiple comparisons, homogeneity of variance used LSD method, heterogeneity of variance used Games-howell method to compare. The sugar content of SPS before and after degradation used paired samples t-test. P<0.05was considered as there are statistically significant different between groups.3Results1H2O2with the concentration of0.15mmol/L of can significant damage to Vero cells and time-dependent decrease of cell viability within0.5h-2h;2With the extent of cell injury increased, MDA level and the expression of OPN would be increase, and SOD level is in the contrary;3Damaged Vero cells prone to induce calcium oxalate crystal nucleation and aggregation, at the same time induce the formation of COM crystals;4Damaged Vero cell surface zeta potential changes more intense than normal cells, almost linear rise.5H2O2would obvious damage HKC cells, and in0.1～2mmol/L concentration range, the cell viability decreased with the increase of dose.6After HK cells were damaged, the concentration of MDA and OPN expression were both increased, and SOD levels decrease, which gives chances for the increase number of calcium oxalate crystals induced by damaged cells.7The initial Zeta potential in the surface of normal cells was higher than that of damaged cells, damaged cells cell surface zeta potential was on the downward within the range of C(H2O2)=0～1.5mmol/L with increasing concentration of H2O2. After incubated with CaOxa, the Zeta potential first decreased and then rising again to flatten. Cell surface zeta potential can decrease CaOxa crystal adhesion by provide more sites.8Degradated water-soluble soybean polysaccharide by hydrogen peroxide, the protein composition in soybean polysaccharide was removal, and the sugar content was significant decreased. The molecular weight of soybean polysaccharide was decent from98,000to28,379;9Both raw and degradated soybean polysaccharide can effectively inhibit the formation of COM crystals, but the degradation of the small component reflect molecular activity for regulation of calcium oxalate crystals.10SPS can increase the mitochondrial membrane potential of injured Vero cells.11Injured Vero cells can gradually restored to its normal state by SPS in cell morphology, and SPScan induce the generation of more COD crystals.12Injured Vero cells can induce smaller aggregates after exposed to degradated water-soluble soybean polysaccharide.13Used3D Studio Max software, built monocrystalline of COM and COD model and three-dimensional model of urinary stones.14Successfully built a parametric model of the rat urinary system on a personal with a relatively smaller number of anatomical data.15Mergered simulation into surgical scenes, simulated the kidney transplant scenes in situ, right iliac fossa as well as neck front.4DiscussionIn this study, we used chemical and biological analysis methods, established a stable renal tubular epithelial cell injury model, and got a series methods for cell detecting in vitro, and for urine chemical analysis. This can provide models and platforms for combining the study of biological mineralization and clinical examination. At the same time we use statistic method for experiement design and analysis data analysis to get result more objective and accurate. At the same time we used3D Studio Max software to mimic the crystals and the whole picture of the stones, and built parameter model of rats urinary system, simulated the kidney transplant scenes in situ, right iliac fossa as well as neck front based on a relatively smaller number of anatomical data. Thus we can not only meet the urology animal anatomy experimental needs, but also provide a good platform for fluid analysis and virtual surgery of the urinary system. |
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