肾小管上皮细胞损伤诱导草酸钙结石形成与大鼠泌尿系数字构建
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摘要
1研究背景及目的
泌尿系结石(Urolithiasis)是泌尿系统的常见疾病,迄今为止,对尿石形成的预防尚无十分理想的方法,80%以上的患者病因不清。在外科治疗方面结石病的治疗主要有经皮肾造瘘,输尿管镜取石等多种微创外科技术与体外碎石(ESWL)治疗,泌尿系结石治疗后复发率为30%-50%。如果能够从结石形成的机制出发,研制出对延缓结石形成的针对性药物,有极其重要的临床价值。结石的形成亦受到流体动力学等因素的影响,用计算机模拟有限元分析的方法来进行研究成为目前的热点,这就涉及到计算机建模的问题。如能利用相对较少的解剖数据,对泌尿系进行参数化构建,不仅可满足泌尿外科实验研究的需求,同时也能为泌尿外科动物实验及虚拟手术提供一个良好的平台。
根据研究目的我们的研究分为四个部分：
①对非洲绿猴肾小管上皮细胞采用体外培养的方法,采用H202对其进行损伤,观察损伤前后细胞成活率,并按损伤时间顺序测定细胞与氧化损伤有关的酶及大分子的表达,细胞微结构的变化以及对草酸钙晶体成核和聚集的影响。
②对人近曲肾小管上皮细胞采用体外培养的方法,采用H202对其进行损伤,观察损伤前后细胞成活率,并按损伤浓度测定细胞与氧化损伤有关的酶及大分子的表达,细胞微结构的变化以及对草酸钙晶体成核和聚集的影响。
③对日产大豆多糖进行提纯,并在体外尿液体系中进行相应检测,在非洲绿猴肾小管上皮细胞损伤模型的基础上,研究提纯前后的大豆多糖对Vero细胞中草酸钙晶体生长的调控作用。
④使用Autodesk3ds Max8SP2软件,对COM及COD晶形及结石进行模拟；根据大鼠实体解剖,综合运用放样、布尔运算、多边形造模、倒角剖面和车削等手段,对肾、肾上腺、血管、膀胱、气管、甲状腺和血管等器官进行真实比例构建,合并到手术场景中,经渲染后获取各角度的三维图像。
目前对实验用肾细胞的分类一般是根据动物来源进行划分的。最早培养的细胞来自于鼠,包括NRK-52E,髓质集合管细胞(pIMCD),大鼠的近端小管细胞(pMPT)等；第二种细胞来源于狗,如Madin-Darby犬齿肾细胞(MDCK);第三种来源于猴,如非州绿猴肾上皮细胞(BSC-1);第四种来源为猪,比如LLC-PK1;第五个来源为人,比如人的肾上皮细胞(HK-2)。在这里我们选取非洲绿猴肾小管上皮细胞(Vero)及人近曲肾小管上皮细胞(HKC)两种细胞株进行了研究。
大豆多糖(SPS)是从大豆分离蛋白、豆腐和腐竹等生产加工的副产物豆渣纤维中提取,经过预处理、酶解(纤维素酶、半纤维素酶、蛋白酶等)、分离、脱色、灭菌、干燥等工艺精制而成,且具有来源广,价格低廉和无副作用等优点。分析表明,SPS主要成分是半乳糖、阿拉伯糖、半乳糖醛酸,并含有鼠李糖、岩藻木糖及葡萄糖等多种成分。其结构是以鼠李半乳糖醛酸和高聚半乳糖酸为主链,半乳聚糖和阿拉伯糖为侧链结合的近似于球状结构体,具有良好的分散性、水溶性和乳化性等特点。在这里我们的研究采用过氧化氢(Sevag)法来降解日产大豆多糖。
3D Studio Max软件是Kinetix(Autodesk公司的多媒体商业机构)推出的一个动画产品。使用它可以在PC机上得到真正的工作站动画软件的性能和图像质量。其经过不断的换代已成为目前世界上应用最广泛的三维建模,动画,渲染的大众化软件。在这里我们使用的是软件Autodesk3ds Max8SP2(美国,Autodesk公司,ID：666-12345678),操作平台为windows XP系统。
2研究方法
2.1VERO细胞损伤模型建立
非洲绿猴肾小管上皮细胞株VERO购自中科院上海细胞库(编号：GN017),用含有10%新生小牛血清的DMEM-F12培养液培养,保持37℃及饱和湿度环境,每隔1天更换1次培养液。根据实验设计加入含有不同浓度H202的无血清培养液,细胞消化采用胰蛋白酶消化法,当细胞达80%-90%融合后,用PBS液洗涤细胞2次,用0.02%EDTA与0.25%Trypsin消化细胞,3-5min后于倒置显微镜下观察消化程度；当大部分细胞变圆,细胞分离散开,表明消化适度,加入含10%新生小牛血清的DMEM-F12培养液终止消化,充分吹打细胞使其形成单细胞悬液。用CCK-8法来检测H202造成细胞损伤的程度,用酶标仪在450nm处测量吸光度(A),计算细胞存活率(%),并绘制存活率-时间曲线和存活率-浓度曲线,细胞存活率(%)=A(实验组)/A(对照组)×100%
2.2人肾小管上皮细胞损伤模型建立
人肾小管上皮细胞由上海长征医院梅长林教授馈赠。细胞培养的条件同前,细胞采用不同浓度H202进行损伤,细胞种植培养液中含双氧水0.1,0.3,0.5,1.0以及2.0mmol/L H2O2的培养基中1h。对照组培养液不含H202。在指定的时间点,吸出培养液,细胞用PBS洗两次,并加入新鲜的培养基。然后,每孔加入10μ1的CCK-8。孵育4h后,450nm酶标仪检测。
2.3细胞MDA和SOD浓度
采用双抗体夹心法测定细胞中MDA、SOD浓度,包被单抗的微孔中依次加入丙二醛(MDA)／超氧化物歧化酶(SOD),再与HRP标记的MDA/SOD抗体结合,形成抗体-抗原-酶标抗体复合物,经过彻底洗涤后加底物TMB显色。TMB在HRP酶的催化作用下转化成蓝色,并在酸的作用下转化成最终的黄色。颜色深浅和样品中的丙二醛(MDA)／超氧化物歧化酶(SOD)正相关。用酶标仪在450nm波长下测定吸光度(OD值),通过标准曲线计算样品中丙二醛(MDA)／超氧化物歧化酶(SOD)的浓度。
2.4细胞的OPN表达
细胞加入4%多聚甲醛固定10min,再用PBS洗三次,每次3min；加入羊血清封闭20min,滴加入骨桥蛋白一抗(1：100),4℃过夜。PBS洗三次,避光滴加FITC二抗(1：100),37℃孵育30min后,PBS洗三次,最后用DAPI给细胞染色并封片,激光共聚焦显微镜观察荧光。细胞核为蓝色,骨桥蛋白显绿色。
2.5SEM, XRD分析
细胞结晶孵育后,取出盖玻片,用PBS液洗涤细胞2次,加入2.5%戊二醛固定24h,1%OsO4将细胞后固定,再用PBS液洗涤3次,梯度乙醇(30%、50%、70%、90%、100%)脱水,再用乙酸异戊酯固定后CO2临界点干燥,喷金包被。用SEM观察细胞形态和晶体生长情况,同时对盖玻片上的晶体进行XRD检测(5°<20<60°),确定晶体组分。
2.6细胞Zeta电位检测
细胞清洗后,用PBS洗去未粘附的晶体,加入0.25%胰酶-0.02%EDTA消化液,充分吹打细胞,使细胞和晶体脱落,以1,000rpm/s离心收集细胞和晶体,吸除上清液,加入1000μl pH=7.86的PBS缓冲液使细胞和晶体重新悬浮,于样品池中吸取约8001μ1悬浮液,用纳米粒度仪测其Zeta电位。
2.7共培养后测量CaOxa的浓度
细胞于6孔板中培养加入含有亚稳定的CaOxa溶液间质孵育6h。取出6孔板中的载玻片,然后用PBS洗涤以去除没有结合的晶体。
载玻片放于25m1烧杯中。然后,倒入10mlHNO3和0.5ml HClO4,样品在电炉中消化直到清晰的固化形成。样品不断地加热直到HClO4沸腾,冒烟,直至干掉。加热干燥后置于室温冷却。然后,烧杯中加入3.0m1水溶解残留。溶液中Ca2＋浓度用ICP方法测量。CaOxa晶体的数量使用Ca2+浓度来测量,结果用微克／每平方厘米表示。
2.8大豆多糖的提取
采用Sevag法对SPS进行处理以除去SPS中可能存在的少量蛋白。向多糖溶液中加入0.2倍体积的Sevag试剂(V氯仿：V正丁e=5：1),将混合液剧烈振荡15分钟后离心(4000rpm,10min),此时溶液分为两层,上层为多糖溶液,下层为Sevag试剂与变性蛋白生成的凝胶。取上层液,再加入0.2倍体积的Sevag试剂,重复上述操作,共5次,合并水相,从中提取多糖。
利用过氧化氢(H202)对SPS进行降解。称取1.2g多糖,用60m1蒸馏水溶解于100ml烧杯中,70℃条件下水浴搅拌溶解。溶解完毕后,迅速加入15m1体积分数为30%的H202,降解1.5h后,待溶液冷却,用浓度为2mol/L的NaOH溶液调pH值到7。然后将溶液转入圆底烧瓶中,在70℃水浴条件下旋转蒸发,待溶液体积减少至原体积的1／3时停止蒸发,加入60ml的无水乙醇沉淀多糖过夜,将沉淀过滤,干燥之后进行称重。
2.9多糖理化性质测定
采用苯酚-硫酸法测定了SPS中多糖的百分含量；采用粘度法测定降解前后SPS的平均相对分子质量。
2.10泌尿系结石数字构建
COM与COD单晶模型COM按六方体建模,先按比例建成圆柱形,边数设为6,双底面各顶点采用多边形编辑命令调整顶点位置。COD按四角双锥建模,先半高构建四棱锥,再用镜相命令于Z轴复制另一半,两个四棱锥成组。选用透明玻璃材质球,调整参数为颜色白色,透明度40,半透明80,折射率1.5,亮度200,赋予晶体材质。
结石模型使用圆球体,保持表面光滑。先用FFD4×4×4圆柱体调整表面,再利用噪波分别于X、Y、z轴设置0-100的数值,材料球填加合适贴图。尿路采用管状体造模,FFD4×4×4圆柱体调整表面,移至与结石相适应的部位。分别渲染生成图片。
2.11大鼠泌尿系数字构建
使用Autodesk3ds Max8SP2软件运用放样、布尔运算、多边形造模、倒角剖面和车削等手段,对肾、肾上腺、血管、膀胱、气管、甲状腺和血管等器官进行真实比例构建,合并到腹部及颈部手术场景中,并安装自由摄像头经渲染后获取各角度的三维图像。
2.12统计学分析
统计学分析使用SPSS13.0软件。数据表达采用均数±标准差来表示(χ-±s)。细胞存活率检测采用One-Way ANOVA或Two-Way ANOVA方法,MDA、SOD, OPN表达,结晶体大小,数目等采用One-Way ANOVA方法检验,先进行Levene检验方差齐性,若P0.05,选择基于方差齐性的方差分析结果；若P<0.05选择基于方差不齐的方差分析结果(Welch法)。若方差分析显著后,则进一步多重比较,方差齐者采用LSD法比较,方差不齐者采用Games-howell比较法。降解前、后SPS的含糖量检测采用配对样本t检验。P<0.05认为是差异具有统计学意义。
3结果
1浓度为0.15mmol/LH2O2可以明显的损伤Vero细胞并在0.5h-2h内按时间相关地减小细胞存活率；
2细胞损伤后,MDA浓度和OPN表达上升,而SOD水平减低；
3损伤的Vero细胞容易导致草酸钙晶体的成核及聚集,诱导更多的COM结晶形成；
4损伤Vero细胞表面的Zeta变化比正常细胞更为剧烈,几乎呈线性上升。
5H202会明显的损伤HKC细胞,且在0.1～2mmol/L浓度范围内随着剂量的增加会使细胞存活率下降。
6在细胞损伤后,MDA浓度以及OPN表达会升高,而SOD水平下降,这就会引起损伤细胞诱导的草酸钙晶体数目的增多。
7正常细胞的表面初始Zeta电位高于损伤细胞,损伤细胞在c(H2O2)=0～1.5mmol/L的范围内,随其浓度的升高其细胞表面的Zeta电位呈一直下降的趋势,而加入晶体后,Zeta电位会先下降再上升的再趋于平缓。说明细胞表面Zeta电位的下降可以为CaOxa晶体粘附提供更多的位点。
8通过双氧水对水溶性的大豆多糖进行降解,降解后大豆多糖去除了蛋白成分,含糖量显著的减少,且分子量由98,000降低到28,379；
9降解前、后的大豆多糖均能有效的抑制COM晶体的形成,但降解后的小分子量大豆多糖体现出更强的分子活性,对草酸钙晶体的调控作用进一步的增强。
10SPS可以提高损伤Vero细胞的线粒体的膜电位。
11Vero细胞经SPS作用后,细胞的形貌能逐渐恢复到正常状态,且其能诱导更多COD晶体的生成。
12用降解后小分子量SPS对损伤Vero细胞修复后,其诱导了尺寸较小的草酸钙聚集体。
13利用3D Studio Max软件,构建出结石单晶及泌尿系结石三维模型。
14采取相对简单的方法和较少的数据在个人PC机上成功构建出大鼠泌尿系参数化模型。
15在手术场景中进行合并,模拟出大鼠原位,髂位及颈部肾移植情况。
4讨论
本研究采用化学及生物学分析的研究方法,建立了稳定的肾小管上皮细胞损伤模型,摸索出了一套细胞体外化学测试方法。这可以为生物矿化与临床诊断相结合提供了依据,利用生物统计学的方法设计实验及分析数据,使得出的结论更为客观、准确。同时利用相对较少的解剖数据,使用3D Studio Max软件模仿结石的晶体及全貌,并对大鼠的泌尿系统进行参数化构建,模拟出大鼠原位及异位肾移植场景,以满足泌尿外科动物解剖实验的需求,同时也为泌尿系流体分析及虚拟手术提供一个良好的平台。
1Background and purpose
Urolithiasis 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 Methods
2.1Established Damage Model of Vero Cell Line
African 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 Line
The 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 concentration
Determinated 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 cells
4%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 analysis
After 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 measurement
Washed 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-cultured
The 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 polysaccharides
Sevag 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 nature
Used 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 building
COM 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 tubular
Used 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 analysis
Statistical 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.
3Results
1H2O2with 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.
4Discussion
In 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.
泌尿系结石(Urolithiasis)是泌尿系统的常见疾病,迄今为止,对尿石形成的预防尚无十分理想的方法,80%以上的患者病因不清。在外科治疗方面结石病的治疗主要有经皮肾造瘘,输尿管镜取石等多种微创外科技术与体外碎石(ESWL)治疗,泌尿系结石治疗后复发率为30%-50%。如果能够从结石形成的机制出发,研制出对延缓结石形成的针对性药物,有极其重要的临床价值。结石的形成亦受到流体动力学等因素的影响,用计算机模拟有限元分析的方法来进行研究成为目前的热点,这就涉及到计算机建模的问题。如能利用相对较少的解剖数据,对泌尿系进行参数化构建,不仅可满足泌尿外科实验研究的需求,同时也能为泌尿外科动物实验及虚拟手术提供一个良好的平台。
根据研究目的我们的研究分为四个部分：
①对非洲绿猴肾小管上皮细胞采用体外培养的方法,采用H202对其进行损伤,观察损伤前后细胞成活率,并按损伤时间顺序测定细胞与氧化损伤有关的酶及大分子的表达,细胞微结构的变化以及对草酸钙晶体成核和聚集的影响。
②对人近曲肾小管上皮细胞采用体外培养的方法,采用H202对其进行损伤,观察损伤前后细胞成活率,并按损伤浓度测定细胞与氧化损伤有关的酶及大分子的表达,细胞微结构的变化以及对草酸钙晶体成核和聚集的影响。
③对日产大豆多糖进行提纯,并在体外尿液体系中进行相应检测,在非洲绿猴肾小管上皮细胞损伤模型的基础上,研究提纯前后的大豆多糖对Vero细胞中草酸钙晶体生长的调控作用。
④使用Autodesk3ds Max8SP2软件,对COM及COD晶形及结石进行模拟；根据大鼠实体解剖,综合运用放样、布尔运算、多边形造模、倒角剖面和车削等手段,对肾、肾上腺、血管、膀胱、气管、甲状腺和血管等器官进行真实比例构建,合并到手术场景中,经渲染后获取各角度的三维图像。
目前对实验用肾细胞的分类一般是根据动物来源进行划分的。最早培养的细胞来自于鼠,包括NRK-52E,髓质集合管细胞(pIMCD),大鼠的近端小管细胞(pMPT)等；第二种细胞来源于狗,如Madin-Darby犬齿肾细胞(MDCK);第三种来源于猴,如非州绿猴肾上皮细胞(BSC-1);第四种来源为猪,比如LLC-PK1;第五个来源为人,比如人的肾上皮细胞(HK-2)。在这里我们选取非洲绿猴肾小管上皮细胞(Vero)及人近曲肾小管上皮细胞(HKC)两种细胞株进行了研究。
大豆多糖(SPS)是从大豆分离蛋白、豆腐和腐竹等生产加工的副产物豆渣纤维中提取,经过预处理、酶解(纤维素酶、半纤维素酶、蛋白酶等)、分离、脱色、灭菌、干燥等工艺精制而成,且具有来源广,价格低廉和无副作用等优点。分析表明,SPS主要成分是半乳糖、阿拉伯糖、半乳糖醛酸,并含有鼠李糖、岩藻木糖及葡萄糖等多种成分。其结构是以鼠李半乳糖醛酸和高聚半乳糖酸为主链,半乳聚糖和阿拉伯糖为侧链结合的近似于球状结构体,具有良好的分散性、水溶性和乳化性等特点。在这里我们的研究采用过氧化氢(Sevag)法来降解日产大豆多糖。
3D Studio Max软件是Kinetix(Autodesk公司的多媒体商业机构)推出的一个动画产品。使用它可以在PC机上得到真正的工作站动画软件的性能和图像质量。其经过不断的换代已成为目前世界上应用最广泛的三维建模,动画,渲染的大众化软件。在这里我们使用的是软件Autodesk3ds Max8SP2(美国,Autodesk公司,ID：666-12345678),操作平台为windows XP系统。
2研究方法
2.1VERO细胞损伤模型建立
非洲绿猴肾小管上皮细胞株VERO购自中科院上海细胞库(编号：GN017),用含有10%新生小牛血清的DMEM-F12培养液培养,保持37℃及饱和湿度环境,每隔1天更换1次培养液。根据实验设计加入含有不同浓度H202的无血清培养液,细胞消化采用胰蛋白酶消化法,当细胞达80%-90%融合后,用PBS液洗涤细胞2次,用0.02%EDTA与0.25%Trypsin消化细胞,3-5min后于倒置显微镜下观察消化程度；当大部分细胞变圆,细胞分离散开,表明消化适度,加入含10%新生小牛血清的DMEM-F12培养液终止消化,充分吹打细胞使其形成单细胞悬液。用CCK-8法来检测H202造成细胞损伤的程度,用酶标仪在450nm处测量吸光度(A),计算细胞存活率(%),并绘制存活率-时间曲线和存活率-浓度曲线,细胞存活率(%)=A(实验组)/A(对照组)×100%
2.2人肾小管上皮细胞损伤模型建立
人肾小管上皮细胞由上海长征医院梅长林教授馈赠。细胞培养的条件同前,细胞采用不同浓度H202进行损伤,细胞种植培养液中含双氧水0.1,0.3,0.5,1.0以及2.0mmol/L H2O2的培养基中1h。对照组培养液不含H202。在指定的时间点,吸出培养液,细胞用PBS洗两次,并加入新鲜的培养基。然后,每孔加入10μ1的CCK-8。孵育4h后,450nm酶标仪检测。
2.3细胞MDA和SOD浓度
采用双抗体夹心法测定细胞中MDA、SOD浓度,包被单抗的微孔中依次加入丙二醛(MDA)／超氧化物歧化酶(SOD),再与HRP标记的MDA/SOD抗体结合,形成抗体-抗原-酶标抗体复合物,经过彻底洗涤后加底物TMB显色。TMB在HRP酶的催化作用下转化成蓝色,并在酸的作用下转化成最终的黄色。颜色深浅和样品中的丙二醛(MDA)／超氧化物歧化酶(SOD)正相关。用酶标仪在450nm波长下测定吸光度(OD值),通过标准曲线计算样品中丙二醛(MDA)／超氧化物歧化酶(SOD)的浓度。
2.4细胞的OPN表达
细胞加入4%多聚甲醛固定10min,再用PBS洗三次,每次3min；加入羊血清封闭20min,滴加入骨桥蛋白一抗(1：100),4℃过夜。PBS洗三次,避光滴加FITC二抗(1：100),37℃孵育30min后,PBS洗三次,最后用DAPI给细胞染色并封片,激光共聚焦显微镜观察荧光。细胞核为蓝色,骨桥蛋白显绿色。
2.5SEM, XRD分析
细胞结晶孵育后,取出盖玻片,用PBS液洗涤细胞2次,加入2.5%戊二醛固定24h,1%OsO4将细胞后固定,再用PBS液洗涤3次,梯度乙醇(30%、50%、70%、90%、100%)脱水,再用乙酸异戊酯固定后CO2临界点干燥,喷金包被。用SEM观察细胞形态和晶体生长情况,同时对盖玻片上的晶体进行XRD检测(5°<20<60°),确定晶体组分。
2.6细胞Zeta电位检测
细胞清洗后,用PBS洗去未粘附的晶体,加入0.25%胰酶-0.02%EDTA消化液,充分吹打细胞,使细胞和晶体脱落,以1,000rpm/s离心收集细胞和晶体,吸除上清液,加入1000μl pH=7.86的PBS缓冲液使细胞和晶体重新悬浮,于样品池中吸取约8001μ1悬浮液,用纳米粒度仪测其Zeta电位。
2.7共培养后测量CaOxa的浓度
细胞于6孔板中培养加入含有亚稳定的CaOxa溶液间质孵育6h。取出6孔板中的载玻片,然后用PBS洗涤以去除没有结合的晶体。
载玻片放于25m1烧杯中。然后,倒入10mlHNO3和0.5ml HClO4,样品在电炉中消化直到清晰的固化形成。样品不断地加热直到HClO4沸腾,冒烟,直至干掉。加热干燥后置于室温冷却。然后,烧杯中加入3.0m1水溶解残留。溶液中Ca2＋浓度用ICP方法测量。CaOxa晶体的数量使用Ca2+浓度来测量,结果用微克／每平方厘米表示。
2.8大豆多糖的提取
采用Sevag法对SPS进行处理以除去SPS中可能存在的少量蛋白。向多糖溶液中加入0.2倍体积的Sevag试剂(V氯仿：V正丁e=5：1),将混合液剧烈振荡15分钟后离心(4000rpm,10min),此时溶液分为两层,上层为多糖溶液,下层为Sevag试剂与变性蛋白生成的凝胶。取上层液,再加入0.2倍体积的Sevag试剂,重复上述操作,共5次,合并水相,从中提取多糖。
利用过氧化氢(H202)对SPS进行降解。称取1.2g多糖,用60m1蒸馏水溶解于100ml烧杯中,70℃条件下水浴搅拌溶解。溶解完毕后,迅速加入15m1体积分数为30%的H202,降解1.5h后,待溶液冷却,用浓度为2mol/L的NaOH溶液调pH值到7。然后将溶液转入圆底烧瓶中,在70℃水浴条件下旋转蒸发,待溶液体积减少至原体积的1／3时停止蒸发,加入60ml的无水乙醇沉淀多糖过夜,将沉淀过滤,干燥之后进行称重。
2.9多糖理化性质测定
采用苯酚-硫酸法测定了SPS中多糖的百分含量；采用粘度法测定降解前后SPS的平均相对分子质量。
2.10泌尿系结石数字构建
COM与COD单晶模型COM按六方体建模,先按比例建成圆柱形,边数设为6,双底面各顶点采用多边形编辑命令调整顶点位置。COD按四角双锥建模,先半高构建四棱锥,再用镜相命令于Z轴复制另一半,两个四棱锥成组。选用透明玻璃材质球,调整参数为颜色白色,透明度40,半透明80,折射率1.5,亮度200,赋予晶体材质。
结石模型使用圆球体,保持表面光滑。先用FFD4×4×4圆柱体调整表面,再利用噪波分别于X、Y、z轴设置0-100的数值,材料球填加合适贴图。尿路采用管状体造模,FFD4×4×4圆柱体调整表面,移至与结石相适应的部位。分别渲染生成图片。
2.11大鼠泌尿系数字构建
使用Autodesk3ds Max8SP2软件运用放样、布尔运算、多边形造模、倒角剖面和车削等手段,对肾、肾上腺、血管、膀胱、气管、甲状腺和血管等器官进行真实比例构建,合并到腹部及颈部手术场景中,并安装自由摄像头经渲染后获取各角度的三维图像。
2.12统计学分析
统计学分析使用SPSS13.0软件。数据表达采用均数±标准差来表示(χ-±s)。细胞存活率检测采用One-Way ANOVA或Two-Way ANOVA方法,MDA、SOD, OPN表达,结晶体大小,数目等采用One-Way ANOVA方法检验,先进行Levene检验方差齐性,若P0.05,选择基于方差齐性的方差分析结果；若P<0.05选择基于方差不齐的方差分析结果(Welch法)。若方差分析显著后,则进一步多重比较,方差齐者采用LSD法比较,方差不齐者采用Games-howell比较法。降解前、后SPS的含糖量检测采用配对样本t检验。P<0.05认为是差异具有统计学意义。
3结果
1浓度为0.15mmol/LH2O2可以明显的损伤Vero细胞并在0.5h-2h内按时间相关地减小细胞存活率；
2细胞损伤后,MDA浓度和OPN表达上升,而SOD水平减低；
3损伤的Vero细胞容易导致草酸钙晶体的成核及聚集,诱导更多的COM结晶形成；
4损伤Vero细胞表面的Zeta变化比正常细胞更为剧烈,几乎呈线性上升。
5H202会明显的损伤HKC细胞,且在0.1～2mmol/L浓度范围内随着剂量的增加会使细胞存活率下降。
6在细胞损伤后,MDA浓度以及OPN表达会升高,而SOD水平下降,这就会引起损伤细胞诱导的草酸钙晶体数目的增多。
7正常细胞的表面初始Zeta电位高于损伤细胞,损伤细胞在c(H2O2)=0～1.5mmol/L的范围内,随其浓度的升高其细胞表面的Zeta电位呈一直下降的趋势,而加入晶体后,Zeta电位会先下降再上升的再趋于平缓。说明细胞表面Zeta电位的下降可以为CaOxa晶体粘附提供更多的位点。
8通过双氧水对水溶性的大豆多糖进行降解,降解后大豆多糖去除了蛋白成分,含糖量显著的减少,且分子量由98,000降低到28,379；
9降解前、后的大豆多糖均能有效的抑制COM晶体的形成,但降解后的小分子量大豆多糖体现出更强的分子活性,对草酸钙晶体的调控作用进一步的增强。
10SPS可以提高损伤Vero细胞的线粒体的膜电位。
11Vero细胞经SPS作用后,细胞的形貌能逐渐恢复到正常状态,且其能诱导更多COD晶体的生成。
12用降解后小分子量SPS对损伤Vero细胞修复后,其诱导了尺寸较小的草酸钙聚集体。
13利用3D Studio Max软件,构建出结石单晶及泌尿系结石三维模型。
14采取相对简单的方法和较少的数据在个人PC机上成功构建出大鼠泌尿系参数化模型。
15在手术场景中进行合并,模拟出大鼠原位,髂位及颈部肾移植情况。
4讨论
本研究采用化学及生物学分析的研究方法,建立了稳定的肾小管上皮细胞损伤模型,摸索出了一套细胞体外化学测试方法。这可以为生物矿化与临床诊断相结合提供了依据,利用生物统计学的方法设计实验及分析数据,使得出的结论更为客观、准确。同时利用相对较少的解剖数据,使用3D Studio Max软件模仿结石的晶体及全貌,并对大鼠的泌尿系统进行参数化构建,模拟出大鼠原位及异位肾移植场景,以满足泌尿外科动物解剖实验的需求,同时也为泌尿系流体分析及虚拟手术提供一个良好的平台。
1Background and purpose
Urolithiasis 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 Methods
2.1Established Damage Model of Vero Cell Line
African 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 Line
The 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 concentration
Determinated 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 cells
4%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 analysis
After 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 measurement
Washed 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-cultured
The 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 polysaccharides
Sevag 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 nature
Used 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 building
COM 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 tubular
Used 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 analysis
Statistical 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.
3Results
1H2O2with 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.
4Discussion
In 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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