氟中毒肾损伤时STC1、UCP2的表达与作用及高钙干预对其影响的机制研究
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摘要
长时间、过量的氟摄入会导致机体组织和脏器的损伤，并特征性的引起氟斑牙、骨关节畸形、骨质疏松、骨关节硬化。该类疾病均由氟中毒导致，并统称为地氟病。地氟病是一种覆盖各大洲，影响数千万人的地球化学性疾病。近15年来，氟中毒对非骨相系统的影响正逐渐成为学界的研究热点，其中又因为肾脏是机体排氟的最主要脏器和其本身就是氟中毒的靶器官，而使肾脏成为了非骨相系统研究中的重点。线粒体是氟离子攻击细胞的核心靶点，肾脏作为富含线粒体的脏器，已在前人的大量研究中证实了氟中毒能使肾小管上皮细胞中线粒体的结构，功能活性受到严重影响，并与染氟剂量存在剂量-程度相关性，但是，氟中毒影响肾小管上皮细胞线粒体的毒理机制尚不明确，因此，本课题选择从线粒体层面对氟中毒肾损伤进行相关机制的研究。
斯钙素蛋白1（Stanniocalcin1, STC1）是一种调节细胞钙离子平衡的糖蛋白激素，在人体的许多脏器都有表达，高表达于肾脏、前列腺、子宫和甲状腺。STC1主要通过自分泌和旁分泌起作用，并作用于细胞的线粒体，调节钙及磷酸盐的代谢。近年来STC1的其他功能逐渐被发现，相关研究显示，STC1与损伤修复、细胞信号转导、血管再生、类固醇生成、肌肉和骨的生长、肿瘤等相关。最新的研究显示，STC1可以通过调节钙离子的含量致使细胞第二信使功能减弱，并通过增高解偶联蛋白2（UCP2）的表达来抑制线粒体膜电位降低和减少超氧化物的产量，这与细胞ATP的生成、NADH和FADH2平衡、ROS产量等息息相关。UCP2定位于线粒体内膜，是一种调控线粒体内外膜电化学梯度的解偶联蛋白，占线粒体膜蛋白含量的0.01-0.1%，在正常情况下，UCP2并没有介导线粒体膜质子漏，亦未对线粒体内外膜的电位差产生过多影响。但在细胞受到攻击或刺激时，特别是对能带来氧化应激、脂质过氧化损伤的刺激，可促使UCP2介导线粒体内外膜间发生质子漏，从而降低线粒体内外膜的膜电位差，使ROS产量减少、超氧化物产量减少等，并以之拮抗相关损伤给细胞、组织带来的损害。UCP2介导线粒体膜电位差改变要借助于[Ca~(2+)]及[Ca~(2+)]相关通道蛋白才能完成，且细胞钙含量的改变也能促使UCP2的表达发生改变，近年来的研究发现，钙离子与UCP2的关系不仅在机体抵御外界刺激时才能发生，而且在各种肿瘤的发生、发展过程中，UCP2可以作为一种肿瘤标志物，通过调节钙离子含量及钙离子相关通道影响肿瘤细胞的各种异质化功能。
本课题从线粒体层面对氟中毒肾损伤的分子机制进行研究，紧扣目前氟中毒肾损伤研究的薄弱点；以肾小管上皮细胞线粒体解偶联蛋白2和斯钙素蛋白1作为研究对象，以钙离子作为干扰条件，探讨氟中毒肾损伤时UCP2、STC1是否参与了氟中毒肾损伤过程，是否在参与过程中受到钙离子的影响，初步研究UCP2、STC1在氟中毒肾损伤机制中的作用；本研究先进行动物实验，观察氟中毒大鼠肾脏中UCP2、STC1的改变，线粒体解偶联功能的改变和氧化应激指标的改变，以及这些改变与肾组织中[Ca~(2+)]含量的关系，再给予大鼠高钙饮食，观察UCP2、STC1表达出现的变化，高钙与该变化的关系；构建重组UCP2真核表达质粒质粒，使肾小管上皮细胞（HK2）高表达UCP2，再以氯化钙侵染细胞培养基，研究不同剂量氟化钠干扰高表达UCP2的肾小管上皮细胞时，细胞生长情况、线粒体解偶联相关指标的变化，此类变化被高钙环境干预后出现的改变，以及UCP2、STC1、[Ca~(2+)]在氟中毒肾损伤中的关系；该实验从亚细胞器水平对氟中毒肾损伤机制进行研究，选择了与[Ca~(2+)]、氧化应激密切相关的UCP2、STC1作为研究靶点，实验设计符合逻辑，该实验的结果可为以后从线粒体水平研究氟中毒肾损伤的分子机制提供理论和实验基础。
目的：构建符合实验需要的氟中毒模型，检测UCP2、STC1及钙、磷离子在氟中毒大鼠肾损伤中的变化，探讨UCP2、STC1在氟中毒肾损伤机制中的作用及与钙离子的关系。
方法：24只雄性SD大鼠按体重平均分为4组：对照组、低氟组、中氟组、高氟组；对照组采用生理盐水注射，实验组按体重比分别给予5mg/kg、10mg/kg、20mg/kg的NAF腹腔注射，每48小时注射1次，蒸馏水及常规鼠饵自由饲食；建模16周后，取左肾包埋固定，行免疫组化检测UCP2、STC1的蛋白定位及表达，右肾组织50mg提取总RNA，采用RT-PCR技术分析UCP2、STC1基因表达水平，并荧光探针检测肾细胞内[Ca~(2+)]i水平。
结果：免疫组化提示UCP2高表达于肾脏皮髓交界的近曲小管，STC1也主要定位于近曲小管，但有少量表达与髓质的远曲小管。免疫组化及RT-PCR结果提示三种染氟大鼠的UCP2、STC1表达量均显著高于对照组，并随着染氟剂量的增大而显著增高（两两比较，P<0.05）；各实验组大鼠肾细胞内钙离子含量显著高于对照组，且随着染氟剂量的增大而增高（两两比较，P<0.05）；
结论：氟中毒大鼠肾损伤中，UCP2、STC1可能参与了损伤机制，而该损伤机制可能是由于钙磷离子代谢失衡造成。
目的：观察UCP2、STC1在氟中毒大鼠肾脏中的变化，及高钙对氟中毒的拮抗作用。探讨UCP2、STC1与高钙的相互联系及在氟中毒肾损伤中的作用。
方法：42只雄性SD大鼠按体重分为对照组、低氟加钙组、中氟加钙组、高氟加钙组；对照组采用生理盐水注射，低、中、高氟加钙组同样按体重比分别给予5mg/kg、10mg/kg、20mg/kg的氟化钠腹腔注射，每48小时注射1次，给予高钙饮食。建模16周后行免疫组化及RT-PCR检测UCP2、STC1的定位及表达，荧光探针检测肾细胞内[Ca~(2+)]i水平，并检测肾组织脂质过氧化产物的活性。
结果：印证了第一节中UCP2、STC1主要定位于肾脏皮髓交界的近曲小管，很少量表达于肾髓质的远曲小管；与对照组相比较，UCP2、STC1在各染氟加钙组的的表达随着染氟浓度的增加而增高，差异有显著性（P<0.05）。横向对比同剂量单纯染氟组，UCP2在高氟加钙组的表达显著高于高氟组（P<0.05），UCP2在低、中氟加钙组对比低、中染氟组虽未出现显著性改变，但在表达上也呈现上升的趋势。STC1在各个染氟加钙组的表达与对应剂量的单纯染氟组均未出现显著性改变（P>0.05），但在表达上也呈现上升的趋势；各实验组大鼠肾细胞[Ca~(2+)]i水平显著高于对照组，且随着染氟剂量的增大而升高，高钙可抑制肾细胞内[Ca~(2+)]i水平。
结论：氟中毒肾损伤时UCP2、STC1可反应性增高，高钙能拮抗氟中毒肾损伤作用，并促进UCP2、STC1的表达，且抑制肾细胞内[Ca~(2+)]水平。UCP2、STC1可能参与了氟中毒大鼠肾损伤机制，且具体的参与过程可能与钙离子相关。
目的：检测染氟大鼠肾组织中线粒体解偶联相关指标及氧自由基的变化，并与UCP2的表达进行比较，观察UCP2的解偶联功能是否参与了氟中毒肾损伤机制。
方法：提取大鼠肾脏线粒体，检测线粒体呼吸功能；利用线粒体膜孔道开放可以使线粒体发生肿胀的原理，使用荧光分光光度计检测线粒体肿胀程度；利用罗丹明123可以在线粒体中迅速发生荧光淬灭的原理，使用荧光分光光度计检测检测氟中毒大鼠肾脏中线粒体的膜电位（△Ψm）；利用来源于线粒体的未成对电子与氧结合生成的超氧阴离子可与四唑盐（NBT）反应，其生成物可以在激发波长为570nm时产生特征性吸收的原理，使用酶标仪检测线粒体超氧化物含量。
结果：与对照组相比，线粒体Ⅲ态呼吸率在低氟组出现了轻度回升，在中氟组又趋于下降，但在低、中氟组未见显著性差异（P>0.05）；在高氟组出现了显著性降低（P<0.05）；与对照组相比，Ⅲ态呼吸率在低氟加钙组出现了下降、在中氟加钙组亦出现下降，但差异均无显著性（P>0.05），在高氟加钙组能在一定程度上提升呼吸率。Ⅳ态呼吸率在低、中染氟组出现了升高，在高氟组出现了降低，差异均无显著性（P>0.05）。钙对氟中毒大鼠肾脏中线粒体的Ⅳ态呼吸率产生一定程度的抑制作用，但差异不明显。线粒体呼吸控制率（RCR）随着染氟浓度的增高而逐渐减低，在高氟组出现了明显降低（P<0.05），钙可以拮抗这种变化，虽在高氟加钙组仍然呈现显著性降低，但RCR数据出现了回升，在各染氟加钙组均出现了线粒体呼吸控制率的改善。
结论：氟中毒肾损伤时，线粒体解偶联相关指标线粒体膜电位、线粒体肿胀所反映的线粒体膜通道转运孔开放程度会发生显著性改变，提示解偶联功能可能参与到氟中毒肾损伤机制中；随着染氟浓度的增高，线粒体肿胀程度和氧自由基水平都会提高，高钙可以抑制以上的变化，实验结果印证了前人研究的氟中毒会造成线粒体损伤和氟中毒氧自由基损伤的机制，且高钙能拮抗氟中毒损伤的作用。
目的：检测氟化钠对肾小管上皮细胞（HK2）生长的抑制作用，并检测UCP2、STC1、线粒体解偶联相关指标的表达，观察在细胞层面UCP2、STC1、线粒体解偶联相关指标是否也出现了改变，及相互之间的关系如何。通过使肾小管上皮细胞高表达UCP2，观察UCP2对氟中毒造成的肾小管上皮细胞（HK2）的损伤是否存在拮抗作用，并同样检测STC1的表达，线粒体解偶联相关指标的变化和细胞内钙离子含量，观测指标的变化，探讨UCP2、STC1、钙离子与氟中毒肾损伤的关系。
方法：构建含人UCP2基因片段的真核表达质粒，并转染HK2，给予正常的HK2和转染了UCP2的HK2培养基中侵染低（0.8mmol/L）、中（1.6mmol/L）、高（2.4mmol/L）三个剂量的氟化钠，并使用氯化钙调节培养基中的钙浓度，通过MTT实验检测细胞的增殖情况；通过RT-PCR的方法检测STC1的表达；提取细胞线粒体，检测线粒体肿胀程度、线粒体的膜电位和细胞内钙离子含量。
结果：肾小管上皮细胞的增殖活性随着染氟程度的增加而增大，并且在中氟组、高氟组出现了显著性降低（P<0.05），高钙可以拮抗氟中毒对肾小管上皮细胞的增殖抑制作用；高表达UCP2的肾小管上皮细胞在染氟环境下其增殖活性变化不一，在低氟组和中氟组，转染了UCP2的HK2可以在一定程度上拮抗氟中毒给细胞造成的增殖抑制，虽未见明显差异，但出现了增殖活性升高的趋势。在高氟组，高表达了UCP2的HK2的增殖活性进一步降低，且与高氟组正常HK2比较后，出现了显著的抑制作用（P<0.05）。高钙可以拮抗氟中毒对高表达了UCP2的HK2细胞的增殖抑制作用，但差异不显著（P>0.05）。各剂量染氟组肾小管上皮细胞中STC1的mRNA表达与对照组大鼠比较均有明显提高，差异有显著性（P<0.05）；高钙可促进STC1的表达。高表达UCP2的HK2中STC1的表达均显著高于对应剂量的正常HK2组（P<0.05）。高钙可以使该表达增强，但差异无显著性（P>0.05）；肾小管上皮细胞内钙离子含量在不同剂量染氟组均显著高于对照组，且两两相比，差异有显著性（P<0.05）。当给予高钙处理后，正常肾小管上皮细胞中钙离子含量会出现显著降低，且两两相比，差异有显著性（P<0.05）；细胞内钙含量在转染了UCP2的肾小管上皮细胞高染氟组中较之正常HK2细胞高染氟组出现了显著下降，在低、中染氟组虽未出现显著的变化，但也呈现下降趋势（P>0.05）。在各染氟组给予转染了UCP2的肾小管上皮细胞高钙干预后，对照正常HK2的相应染氟组，细胞内钙含量出现了下降趋势，在中氟加钙、高氟加钙组出现了显著降低（P<0.05）
结论：氟中毒对肾小管上皮细胞的生存率有明显的抑制作用，并能提高UCP2、STC1的表达，这种改变可随着染氟剂量的增高而愈加明显；氟中毒能影响肾小管上皮细胞的线粒体解偶联功能，并且与染氟剂量呈一定的剂量相关性，因此可从细胞层面继续佐证UCP2、STC1和线粒体解偶联功能可能参与了氟中毒肾损伤的机制。高表达UCP2能促进STC1的表达，降低细胞内钙含量，拮抗线粒体解偶联功能的改变，但对细胞增殖活性具有双向性作用。
Fluoride intake for a long time or a lot content can cause damage tobody tissues and organs, and characteristic cause dental fluorosis, bone andjoint deformities, osteoporosis, bone and joint sclerosis, such diseasescaused by fluoride poisoning, and are collectively referred to as endemicfluorosis. Endemic fluorosis cover all continents, and affect millions ofpeople. In recent15years, impacts of fluorosis to non-phrenology systemhas gradually become active field of research. Kidney is the most importantorgan of the body to excrete the fluorine, and is also a poisoning target organby fluoride, so, the kidney is become focus of fluorosis research.Mitochondria is the sensitive target withstand attacking by fluoride, andkidneys is a organ rich in mitochondria. A large number of studies has beenconfirmed that structure and functions of mitochondria in renal tubularepithelial cells can be severely affected by fluorosis. Also, there is adose-degree of correlation of fluorosis renal injury, but the toxicologicalmechanism is not clear. Therefore, this research is work for the fluorosismechanisms of renal injury from the mitochondria level.
The Stanniocalcin-1(STC1) is a glycoprotein hormone which regulate the balance of cellular calcium, is expressed in many organs of humanbody.STC1is high expression in the kidney, prostate, uterus, and thyroid.STC1is secreted by autocrine and paracrine,effects on mitochondria of cells,regulate the metabolism of calcium and phosphate. In recent years, someother functions of the the STC1were found, which relate to the damagerepair, cell signal transduction, angiogenesis, steroidogenesis, muscle andbone growth, tumor and so on. The latest researches have shown that STC1reduce the level of Ca~(2+)which is an important second messager, binging onthe level of uncouple protein2(UCP2) is highly increased, resulting theinhibition to the mitochondrial membrane potential reduction and reduce theproduction of superoxide. This process is closely relate to the generation ofcellular ATP, NADH and FADH2balance and ROS production. UCP2wasfound on the inner mitochondrial membrane,which is a kind of uncouplingprotein that regulating the mitochondrial outer and inner membraneelectrochemical gradient. It is accounted for0.01-0.1%of the mitochondrialmembrane protein content. UCP2neither mediate mitochondrial membraneproton leak, nor produce too much impact on the mitochondrial membrane’sEMF. But when the cell is stimulated, especially the stimulation resultingoxidative stress or stimulation of lipid oxidative damage, it can contribute tothe UCP2-mediated proton leak in mitochondrial membrane, therebyreducing the EMF of mitochondrial inner/outer membrane, the membranepotential of reduce ROS production and production of superoxide which can damage to cells and tissue. UCP2-mediated proton leak is complete with theaid of [Ca~(2+)] and of [Ca~(2+)] related channel proteins, and the changes of Ca~(2+)content can induce UCP2expression changes. Recent studies show thatcalcium and UCP2relations not only in the body to defend stimulation, butalso in the processes of a variety of tumor occurrence and development.UCP2as a tumor marker, by regulating the calcium content and calciumcorrelation channels affect tumor cell heterogeneity functions.
Our subject sduties the molecular mechanisms as a breakthrough offluorosis renal injury, closely catch the weak point of fluorosis renal injury;We study on the mitochondrial uncoupling protein2and Stanniocalcinprotein in renal tubular epithelial cells as a research object, while the calciumis defined as the interference conditions to explore if UCP2and STC1isinvolved in the process of fluorosis renal injury. Find out the approximaterole of UCP2and STC1in the fluorosis kidney damage mechanism. In thisstudy, we conducted animal experiments firstly, to observe changes influorosis rat kidney of UCP2, STC1, mitochondrial solution couplingfunction changes and the level of oxidative stress parameters. And then ourteam give the rats high-calcium diet, observe if the expression of UCP2,STC1changed,dissuse the relationship between high-calcium and thechange; Constructing the recombinant the UCP2eukaryotic expressionplasmid vector, make sure the tubular epithelial cells (HK2) high expressionof UCP2, and then inject CaCl2to the cell cultures to study the results of different level of sodium fluoride effect on the cells, such the state,mitochondrial solution uncoupling index changes. Find out such changes ifhave relationship with the high calcium environment intervention, as well asthe relationship of UCP2, STC1[Ca~(2+)] in the of fluorosis kidney injury; Thisexperiment studies the mechanisms of fluoride poisoning kidney damage insub-cells level, choosing the Ca2+, UCP2and STC1as a research point. Theexperimental design is logical, and the results can provide a basis for thestudy the molecular mechanisms of fluorosis kidney damage from themitochondrial level in the future.
Objective: Establish fluorosis model and detect changes of UCP2,STC1and Ca~(2+), explore the role of UCP2and STC1in mechanisms offluorosis kidney damage and the their ralationships with calcium.
Methods: According to the weight24male SD rats divided averagelyinto4groups, such as control group, low dose group, the middle fluoridegroup and the high fluoride group; The control group injected physiologicalsaline, and the experimental group was given the NAF intraperitonealinjection5mg/kg body weight,10mg/kg,20mg/kg respectively. We inject1time every48hours, and fed distilled water and regular rat feedingstufffreely; After establishing modle16weeks, took out and immobilize the leftkidney. Detecte the UCP2, STC1protein’s localization and expression byimmunohistochemistry. Extract50mg right kidney organization’s totalRNA,and detecte expression levels of UCP2, STC1by RT-PCR. and finallydetect content of renal cells calcium.
Results: immunohistochemistry prompted UCP2highly expressed inkidney skin medullary junction of the proximal convoluted tubule.STC1ismainly localized in the proximal tubule and a small amount of expression inthe medulla of the distal convoluted tubule. Immunohistochemistry andRT-PCR’s results suggest that the UCP2, STC1expression in fluorosis rats were significantly higher than the control group. And the Ca~(2+)is increased in3groups while the fluoride increased(pairwise comparisons, P<0.05); ratsrenal cells calcium content in each experimental group were significantlyhigher than the control group and it increase while the fluoride increased(pairwise comparisons, P <0.05).
Conclusion: UCP2and STC1maybe involved in mechanism offluorosis injury of rat kidney in which calcium imbalance also play animportant part.
Objective: To observe of UCP2and STC1changes in the kidneys ofrats with fluoride with high calcium against rats renal injury, and explorethe relationship of UCP2, STC1, and high calcium in the renal injury offluorosis rats.
Methods:24male SD rats were divided into four groups according tobody weight, control group low fluoride plus calcium group, middle fluorideplus calcium group, high fluoride plus calcium group. The control groupwith normal saline injection, low, middle and high fluoride plus calciumgroups were given5mg/kg,10mg/kg and20mg/kg of sodium fluoridesolution by intraperitoneal injection according to body weight ratios,1injection every48hours. Low, meddle and high fluoride plus calcium groupswere given high calcium diet for16weeks. Ways of immunohistochemistryand RT-PCR were used to detect location and expression of STC1and UCP2.Content of renal cell [Ca2+]i was detect by fluorescent probes, and we alsodetected the lipid peroxides activity of rats renal tissue.
Results: Confirmed that UCP2/STC1was mainly localized in renalcortex and a very small amount expressed in the renal medulla distalconvoluted tubule. Expression of UCP2and STC1was increasing with the contents of fluoride compared with the control group and high calciumcould increase the expression of UCP2and STC1, the difference wassignificant (P<0.05). Lateral compared with the dose simply exposed tofluoride group of UCP2expression in the high fluoride plus calcium groupwas significantly higher than the high fluoride group (P<0.05). UCP2in thelow, low fluoride plus calcium group compared exposed to fluoride grouphad significantly changes in expression also showed an upward trend. STC1in the expression of each exposed to fluoride plus calcium group and thecorresponding dose simply exposed to fluoride group were not significantlychanged (P>0.05), but expression also showed an upward trend; in eachexperimental group of rat kidney cells of [Ca~(2+)] i levels were significantlyhigher than that control group and exposed to fluoride dose increaseselevated, high calcium and inhibit renal cell [Ca~(2+)]i level.
Conclusion: The UCP2/STC1of fluorosis renal injury can beincreased reactivity, high calcium can antagonize the the fluorosis renalinjury, and promoting of UCP2, STC1expression and inhibition of renal cell[Ca~(2+)] level. UCP2, STC1may be involved in fluorosis rat renal injurymechanism, and the participation process may be related to the calcium ion.
Objective: To detect the changes of mitochondrial uncoupling-relatedindicators and oxygen free radical in the tissue of SD rats and compare withexpression of UCP2. Explore whether the uncoupling function of UCP2isinvolved in mechanism of fluorosis kidney injury.
Method: Extract of rat kidney mitochondria and detect mitochondrialrespiratory function. By the principle that mitochondrial membrane poreopening make the mitochondria swelling, detect the swelling level ofmitochondria by fluorescence spectrophotometer. By the principle thatrhodamine123will fluorescence quench in mitochondria rapidly, detectmitochondrial membrane EMF by fluorescence spectrophotometer (△Ψm).Measure mitochondrial superoxide content by coelosphere reader becauseunpaired electrons and superoxide anion from mitochondria react withtetrazolium salt (NBT) and the production will be waken in570nmlight-wave.
Results: Compared with the control group, mitochondrial III staterespiration rate raise mildly in the low fluoride group. Tended to decline inthe medium fluoride group, but low fluoride group was no significantdifference with medium group (P>0.05); significantly decreased in the highfluoride group (P<0.05); Compared with the control group, the respiratory state III rate decrease in the low fluoride plus calcium group and the mediumfluoride plus calcium group, but no significant differences (P>0.05)In thehigh fluoride plus calcium group, a certain extent enhance of the rate isfound. The Ⅳ state respiration rate increases in the low fluoride pluscalcium group and the medium fluoride plus calcium group and the highfluoride group’s decrease, but there was no significant difference (P>0.05).Calcium to produce a certain degree of inhibition of state IV respiration rateof fluorosis in the rat kidney mitochondria, but the difference is not obvious.Mitochondrial respiratory control ratio (RCR) as exposed to fluorideincreased gradually to reduce and the high fluoride group was significantlylower (P<0.05). Although a significant reduction was found in the highfluoride plus calcium group, but the RCR recovery,because calcium canantagonize this change. Mitochondrial respiratory control ratio raise influoride plus calcium groups.
Conclusion: When fluorosis kidney injury happened, themitochondrial uncoupling-related indicators, EMF, mitochondrial swellinglevel changing significantly, suggest that in the mitochondrial membranechannel transition pore opening degree of a significant change. Thesesuggest that the uncoupling function maybe involve in the mechanism offluorosis renal injury. With fluoride concentration increased, the degree ofswelling of mitochondria and the level and oxygen free radicals content willincrease, but high calcium can inhibit this change. The experimental results confirmed previous studies that mitochondrial damage and fluorosis oxygenfree radical damage caused by fluorosis, and high calcium can antagonizethis damage.
Objective: To determine sodium fluoride effect on the renal tubularepithelial cells (HK2), and detect UCP2, STC1, mitochondrialuncoupling-related indicators changes and their relation. Make HK2highlyexpress UCP2, and observe UCP2, STC1, uncoupling indicators’ changesand calcium content. Explore if UCP2have antagonistic effect to fluorosis.Discuss the relation between UCP2, STC1, Ca~(2+) and calcium.
Method: To construct plasmid containing human UCP2genefragments, transfect HK2. Infect the normal HK2, transfected UCP2,transfected HK2low(0.8mmol/L), medium(1.6mmol/L), high (2.4mmol/L),three levels of sodium fluoride. Use calcium chloride to adjust the calciumconcentration in the medium. Detect cell proliferation by the MTT assay;Detect the expression of STC1by RT-PCR. Extract mitochondria,detectmitochondrial swelling level, mitochondrial membrane EMF andintracellular calcium ion content.
Results: The proliferative activity of renal tubular epithelial cells withthe dye fluorine increase in the degree of increase and in the fluoride group,the high fluoride group was significantly decreased (P<0.05). High calciumcontent can antagonize the negative effect of fluorosis to HK2’s proliferation. HK2’s change is different in three groups which UCP2is highly expressed.Although there is no significant different, in the low fluoride group and themedium fluoride group, the HK2transfected with UCP2’s proliferation israise. Compare the high fluoride group with HK2high expression of UCP2,the transfected HK2’s proliferation activity was further reduced.(P<0.05).High calcium can antagonize the effect of fluorosis to the HK2celltransfected but the difference is not significant (P>0.05). STC1mRNAexpression in renal tubular epithelial cells of fluoride groups is significantlydifferent with the control group (P<0.05); High calcium content can promotethe expression of STC1. Transfected HK2’s STC1expression is significantlyhigher than the normal of HK2group (P<0.05). The high calcium contentcan enhance the expression of STC1, but the difference is no significant(P>0.05). The fluoride groups’ calcium content is higher than the controlgroup(p<0.05). Calcium content in HK2cells from fluoride groups issignificantly higher than control groups, by pairwise compared (P<0.05).When given high calcium handling, calcium content in normal renal tubularepithelial cells appear significantly reduced, and pairwise compared, thedifference was significant (P<0.05); Intracellular calcium content intransfected HK2cells from the high fluoride group is less than the normalHK2cells while the low fluoride group and the medium group have nosignificant change (P>0.05). Give transfected HK2cells high calciumhandling the calcium content becoming lower than the normal HK2cells. The high fluoride group and the medium fluoride group is reducedsignificantly.(P<0.05).
Conclusions: Fluorosis have negative effect on the proliferation ofHK2cells and it can enhance the expression of UCP2and STC1.the changeabove is more significant with the fluoride content. Fluorosis affect theuncouple function of HK2cells’ mitochondria and the result have some doserelation with fluoride content. So the result prove that UCP2, STC1and theuncouple function of mitochondria involve the mechanism of kidneyfluorosis. The present findings provided that UCP2is a factor to stimulateSTC1expression and reduce the calcium content of UCP2, that have effectson cell in many side.
斯钙素蛋白1（Stanniocalcin1, STC1）是一种调节细胞钙离子平衡的糖蛋白激素，在人体的许多脏器都有表达，高表达于肾脏、前列腺、子宫和甲状腺。STC1主要通过自分泌和旁分泌起作用，并作用于细胞的线粒体，调节钙及磷酸盐的代谢。近年来STC1的其他功能逐渐被发现，相关研究显示，STC1与损伤修复、细胞信号转导、血管再生、类固醇生成、肌肉和骨的生长、肿瘤等相关。最新的研究显示，STC1可以通过调节钙离子的含量致使细胞第二信使功能减弱，并通过增高解偶联蛋白2（UCP2）的表达来抑制线粒体膜电位降低和减少超氧化物的产量，这与细胞ATP的生成、NADH和FADH2平衡、ROS产量等息息相关。UCP2定位于线粒体内膜，是一种调控线粒体内外膜电化学梯度的解偶联蛋白，占线粒体膜蛋白含量的0.01-0.1%，在正常情况下，UCP2并没有介导线粒体膜质子漏，亦未对线粒体内外膜的电位差产生过多影响。但在细胞受到攻击或刺激时，特别是对能带来氧化应激、脂质过氧化损伤的刺激，可促使UCP2介导线粒体内外膜间发生质子漏，从而降低线粒体内外膜的膜电位差，使ROS产量减少、超氧化物产量减少等，并以之拮抗相关损伤给细胞、组织带来的损害。UCP2介导线粒体膜电位差改变要借助于[Ca~(2+)]及[Ca~(2+)]相关通道蛋白才能完成，且细胞钙含量的改变也能促使UCP2的表达发生改变，近年来的研究发现，钙离子与UCP2的关系不仅在机体抵御外界刺激时才能发生，而且在各种肿瘤的发生、发展过程中，UCP2可以作为一种肿瘤标志物，通过调节钙离子含量及钙离子相关通道影响肿瘤细胞的各种异质化功能。
本课题从线粒体层面对氟中毒肾损伤的分子机制进行研究，紧扣目前氟中毒肾损伤研究的薄弱点；以肾小管上皮细胞线粒体解偶联蛋白2和斯钙素蛋白1作为研究对象，以钙离子作为干扰条件，探讨氟中毒肾损伤时UCP2、STC1是否参与了氟中毒肾损伤过程，是否在参与过程中受到钙离子的影响，初步研究UCP2、STC1在氟中毒肾损伤机制中的作用；本研究先进行动物实验，观察氟中毒大鼠肾脏中UCP2、STC1的改变，线粒体解偶联功能的改变和氧化应激指标的改变，以及这些改变与肾组织中[Ca~(2+)]含量的关系，再给予大鼠高钙饮食，观察UCP2、STC1表达出现的变化，高钙与该变化的关系；构建重组UCP2真核表达质粒质粒，使肾小管上皮细胞（HK2）高表达UCP2，再以氯化钙侵染细胞培养基，研究不同剂量氟化钠干扰高表达UCP2的肾小管上皮细胞时，细胞生长情况、线粒体解偶联相关指标的变化，此类变化被高钙环境干预后出现的改变，以及UCP2、STC1、[Ca~(2+)]在氟中毒肾损伤中的关系；该实验从亚细胞器水平对氟中毒肾损伤机制进行研究，选择了与[Ca~(2+)]、氧化应激密切相关的UCP2、STC1作为研究靶点，实验设计符合逻辑，该实验的结果可为以后从线粒体水平研究氟中毒肾损伤的分子机制提供理论和实验基础。
目的：构建符合实验需要的氟中毒模型，检测UCP2、STC1及钙、磷离子在氟中毒大鼠肾损伤中的变化，探讨UCP2、STC1在氟中毒肾损伤机制中的作用及与钙离子的关系。
方法：24只雄性SD大鼠按体重平均分为4组：对照组、低氟组、中氟组、高氟组；对照组采用生理盐水注射，实验组按体重比分别给予5mg/kg、10mg/kg、20mg/kg的NAF腹腔注射，每48小时注射1次，蒸馏水及常规鼠饵自由饲食；建模16周后，取左肾包埋固定，行免疫组化检测UCP2、STC1的蛋白定位及表达，右肾组织50mg提取总RNA，采用RT-PCR技术分析UCP2、STC1基因表达水平，并荧光探针检测肾细胞内[Ca~(2+)]i水平。
结果：免疫组化提示UCP2高表达于肾脏皮髓交界的近曲小管，STC1也主要定位于近曲小管，但有少量表达与髓质的远曲小管。免疫组化及RT-PCR结果提示三种染氟大鼠的UCP2、STC1表达量均显著高于对照组，并随着染氟剂量的增大而显著增高（两两比较，P<0.05）；各实验组大鼠肾细胞内钙离子含量显著高于对照组，且随着染氟剂量的增大而增高（两两比较，P<0.05）；
结论：氟中毒大鼠肾损伤中，UCP2、STC1可能参与了损伤机制，而该损伤机制可能是由于钙磷离子代谢失衡造成。
目的：观察UCP2、STC1在氟中毒大鼠肾脏中的变化，及高钙对氟中毒的拮抗作用。探讨UCP2、STC1与高钙的相互联系及在氟中毒肾损伤中的作用。
方法：42只雄性SD大鼠按体重分为对照组、低氟加钙组、中氟加钙组、高氟加钙组；对照组采用生理盐水注射，低、中、高氟加钙组同样按体重比分别给予5mg/kg、10mg/kg、20mg/kg的氟化钠腹腔注射，每48小时注射1次，给予高钙饮食。建模16周后行免疫组化及RT-PCR检测UCP2、STC1的定位及表达，荧光探针检测肾细胞内[Ca~(2+)]i水平，并检测肾组织脂质过氧化产物的活性。
结果：印证了第一节中UCP2、STC1主要定位于肾脏皮髓交界的近曲小管，很少量表达于肾髓质的远曲小管；与对照组相比较，UCP2、STC1在各染氟加钙组的的表达随着染氟浓度的增加而增高，差异有显著性（P<0.05）。横向对比同剂量单纯染氟组，UCP2在高氟加钙组的表达显著高于高氟组（P<0.05），UCP2在低、中氟加钙组对比低、中染氟组虽未出现显著性改变，但在表达上也呈现上升的趋势。STC1在各个染氟加钙组的表达与对应剂量的单纯染氟组均未出现显著性改变（P>0.05），但在表达上也呈现上升的趋势；各实验组大鼠肾细胞[Ca~(2+)]i水平显著高于对照组，且随着染氟剂量的增大而升高，高钙可抑制肾细胞内[Ca~(2+)]i水平。
结论：氟中毒肾损伤时UCP2、STC1可反应性增高，高钙能拮抗氟中毒肾损伤作用，并促进UCP2、STC1的表达，且抑制肾细胞内[Ca~(2+)]水平。UCP2、STC1可能参与了氟中毒大鼠肾损伤机制，且具体的参与过程可能与钙离子相关。
目的：检测染氟大鼠肾组织中线粒体解偶联相关指标及氧自由基的变化，并与UCP2的表达进行比较，观察UCP2的解偶联功能是否参与了氟中毒肾损伤机制。
方法：提取大鼠肾脏线粒体，检测线粒体呼吸功能；利用线粒体膜孔道开放可以使线粒体发生肿胀的原理，使用荧光分光光度计检测线粒体肿胀程度；利用罗丹明123可以在线粒体中迅速发生荧光淬灭的原理，使用荧光分光光度计检测检测氟中毒大鼠肾脏中线粒体的膜电位（△Ψm）；利用来源于线粒体的未成对电子与氧结合生成的超氧阴离子可与四唑盐（NBT）反应，其生成物可以在激发波长为570nm时产生特征性吸收的原理，使用酶标仪检测线粒体超氧化物含量。
结果：与对照组相比，线粒体Ⅲ态呼吸率在低氟组出现了轻度回升，在中氟组又趋于下降，但在低、中氟组未见显著性差异（P>0.05）；在高氟组出现了显著性降低（P<0.05）；与对照组相比，Ⅲ态呼吸率在低氟加钙组出现了下降、在中氟加钙组亦出现下降，但差异均无显著性（P>0.05），在高氟加钙组能在一定程度上提升呼吸率。Ⅳ态呼吸率在低、中染氟组出现了升高，在高氟组出现了降低，差异均无显著性（P>0.05）。钙对氟中毒大鼠肾脏中线粒体的Ⅳ态呼吸率产生一定程度的抑制作用，但差异不明显。线粒体呼吸控制率（RCR）随着染氟浓度的增高而逐渐减低，在高氟组出现了明显降低（P<0.05），钙可以拮抗这种变化，虽在高氟加钙组仍然呈现显著性降低，但RCR数据出现了回升，在各染氟加钙组均出现了线粒体呼吸控制率的改善。
结论：氟中毒肾损伤时，线粒体解偶联相关指标线粒体膜电位、线粒体肿胀所反映的线粒体膜通道转运孔开放程度会发生显著性改变，提示解偶联功能可能参与到氟中毒肾损伤机制中；随着染氟浓度的增高，线粒体肿胀程度和氧自由基水平都会提高，高钙可以抑制以上的变化，实验结果印证了前人研究的氟中毒会造成线粒体损伤和氟中毒氧自由基损伤的机制，且高钙能拮抗氟中毒损伤的作用。
目的：检测氟化钠对肾小管上皮细胞（HK2）生长的抑制作用，并检测UCP2、STC1、线粒体解偶联相关指标的表达，观察在细胞层面UCP2、STC1、线粒体解偶联相关指标是否也出现了改变，及相互之间的关系如何。通过使肾小管上皮细胞高表达UCP2，观察UCP2对氟中毒造成的肾小管上皮细胞（HK2）的损伤是否存在拮抗作用，并同样检测STC1的表达，线粒体解偶联相关指标的变化和细胞内钙离子含量，观测指标的变化，探讨UCP2、STC1、钙离子与氟中毒肾损伤的关系。
方法：构建含人UCP2基因片段的真核表达质粒，并转染HK2，给予正常的HK2和转染了UCP2的HK2培养基中侵染低（0.8mmol/L）、中（1.6mmol/L）、高（2.4mmol/L）三个剂量的氟化钠，并使用氯化钙调节培养基中的钙浓度，通过MTT实验检测细胞的增殖情况；通过RT-PCR的方法检测STC1的表达；提取细胞线粒体，检测线粒体肿胀程度、线粒体的膜电位和细胞内钙离子含量。
结果：肾小管上皮细胞的增殖活性随着染氟程度的增加而增大，并且在中氟组、高氟组出现了显著性降低（P<0.05），高钙可以拮抗氟中毒对肾小管上皮细胞的增殖抑制作用；高表达UCP2的肾小管上皮细胞在染氟环境下其增殖活性变化不一，在低氟组和中氟组，转染了UCP2的HK2可以在一定程度上拮抗氟中毒给细胞造成的增殖抑制，虽未见明显差异，但出现了增殖活性升高的趋势。在高氟组，高表达了UCP2的HK2的增殖活性进一步降低，且与高氟组正常HK2比较后，出现了显著的抑制作用（P<0.05）。高钙可以拮抗氟中毒对高表达了UCP2的HK2细胞的增殖抑制作用，但差异不显著（P>0.05）。各剂量染氟组肾小管上皮细胞中STC1的mRNA表达与对照组大鼠比较均有明显提高，差异有显著性（P<0.05）；高钙可促进STC1的表达。高表达UCP2的HK2中STC1的表达均显著高于对应剂量的正常HK2组（P<0.05）。高钙可以使该表达增强，但差异无显著性（P>0.05）；肾小管上皮细胞内钙离子含量在不同剂量染氟组均显著高于对照组，且两两相比，差异有显著性（P<0.05）。当给予高钙处理后，正常肾小管上皮细胞中钙离子含量会出现显著降低，且两两相比，差异有显著性（P<0.05）；细胞内钙含量在转染了UCP2的肾小管上皮细胞高染氟组中较之正常HK2细胞高染氟组出现了显著下降，在低、中染氟组虽未出现显著的变化，但也呈现下降趋势（P>0.05）。在各染氟组给予转染了UCP2的肾小管上皮细胞高钙干预后，对照正常HK2的相应染氟组，细胞内钙含量出现了下降趋势，在中氟加钙、高氟加钙组出现了显著降低（P<0.05）
结论：氟中毒对肾小管上皮细胞的生存率有明显的抑制作用，并能提高UCP2、STC1的表达，这种改变可随着染氟剂量的增高而愈加明显；氟中毒能影响肾小管上皮细胞的线粒体解偶联功能，并且与染氟剂量呈一定的剂量相关性，因此可从细胞层面继续佐证UCP2、STC1和线粒体解偶联功能可能参与了氟中毒肾损伤的机制。高表达UCP2能促进STC1的表达，降低细胞内钙含量，拮抗线粒体解偶联功能的改变，但对细胞增殖活性具有双向性作用。
Fluoride intake for a long time or a lot content can cause damage tobody tissues and organs, and characteristic cause dental fluorosis, bone andjoint deformities, osteoporosis, bone and joint sclerosis, such diseasescaused by fluoride poisoning, and are collectively referred to as endemicfluorosis. Endemic fluorosis cover all continents, and affect millions ofpeople. In recent15years, impacts of fluorosis to non-phrenology systemhas gradually become active field of research. Kidney is the most importantorgan of the body to excrete the fluorine, and is also a poisoning target organby fluoride, so, the kidney is become focus of fluorosis research.Mitochondria is the sensitive target withstand attacking by fluoride, andkidneys is a organ rich in mitochondria. A large number of studies has beenconfirmed that structure and functions of mitochondria in renal tubularepithelial cells can be severely affected by fluorosis. Also, there is adose-degree of correlation of fluorosis renal injury, but the toxicologicalmechanism is not clear. Therefore, this research is work for the fluorosismechanisms of renal injury from the mitochondria level.
The Stanniocalcin-1(STC1) is a glycoprotein hormone which regulate the balance of cellular calcium, is expressed in many organs of humanbody.STC1is high expression in the kidney, prostate, uterus, and thyroid.STC1is secreted by autocrine and paracrine,effects on mitochondria of cells,regulate the metabolism of calcium and phosphate. In recent years, someother functions of the the STC1were found, which relate to the damagerepair, cell signal transduction, angiogenesis, steroidogenesis, muscle andbone growth, tumor and so on. The latest researches have shown that STC1reduce the level of Ca~(2+)which is an important second messager, binging onthe level of uncouple protein2(UCP2) is highly increased, resulting theinhibition to the mitochondrial membrane potential reduction and reduce theproduction of superoxide. This process is closely relate to the generation ofcellular ATP, NADH and FADH2balance and ROS production. UCP2wasfound on the inner mitochondrial membrane,which is a kind of uncouplingprotein that regulating the mitochondrial outer and inner membraneelectrochemical gradient. It is accounted for0.01-0.1%of the mitochondrialmembrane protein content. UCP2neither mediate mitochondrial membraneproton leak, nor produce too much impact on the mitochondrial membrane’sEMF. But when the cell is stimulated, especially the stimulation resultingoxidative stress or stimulation of lipid oxidative damage, it can contribute tothe UCP2-mediated proton leak in mitochondrial membrane, therebyreducing the EMF of mitochondrial inner/outer membrane, the membranepotential of reduce ROS production and production of superoxide which can damage to cells and tissue. UCP2-mediated proton leak is complete with theaid of [Ca~(2+)] and of [Ca~(2+)] related channel proteins, and the changes of Ca~(2+)content can induce UCP2expression changes. Recent studies show thatcalcium and UCP2relations not only in the body to defend stimulation, butalso in the processes of a variety of tumor occurrence and development.UCP2as a tumor marker, by regulating the calcium content and calciumcorrelation channels affect tumor cell heterogeneity functions.
Our subject sduties the molecular mechanisms as a breakthrough offluorosis renal injury, closely catch the weak point of fluorosis renal injury;We study on the mitochondrial uncoupling protein2and Stanniocalcinprotein in renal tubular epithelial cells as a research object, while the calciumis defined as the interference conditions to explore if UCP2and STC1isinvolved in the process of fluorosis renal injury. Find out the approximaterole of UCP2and STC1in the fluorosis kidney damage mechanism. In thisstudy, we conducted animal experiments firstly, to observe changes influorosis rat kidney of UCP2, STC1, mitochondrial solution couplingfunction changes and the level of oxidative stress parameters. And then ourteam give the rats high-calcium diet, observe if the expression of UCP2,STC1changed,dissuse the relationship between high-calcium and thechange; Constructing the recombinant the UCP2eukaryotic expressionplasmid vector, make sure the tubular epithelial cells (HK2) high expressionof UCP2, and then inject CaCl2to the cell cultures to study the results of different level of sodium fluoride effect on the cells, such the state,mitochondrial solution uncoupling index changes. Find out such changes ifhave relationship with the high calcium environment intervention, as well asthe relationship of UCP2, STC1[Ca~(2+)] in the of fluorosis kidney injury; Thisexperiment studies the mechanisms of fluoride poisoning kidney damage insub-cells level, choosing the Ca2+, UCP2and STC1as a research point. Theexperimental design is logical, and the results can provide a basis for thestudy the molecular mechanisms of fluorosis kidney damage from themitochondrial level in the future.
Objective: Establish fluorosis model and detect changes of UCP2,STC1and Ca~(2+), explore the role of UCP2and STC1in mechanisms offluorosis kidney damage and the their ralationships with calcium.
Methods: According to the weight24male SD rats divided averagelyinto4groups, such as control group, low dose group, the middle fluoridegroup and the high fluoride group; The control group injected physiologicalsaline, and the experimental group was given the NAF intraperitonealinjection5mg/kg body weight,10mg/kg,20mg/kg respectively. We inject1time every48hours, and fed distilled water and regular rat feedingstufffreely; After establishing modle16weeks, took out and immobilize the leftkidney. Detecte the UCP2, STC1protein’s localization and expression byimmunohistochemistry. Extract50mg right kidney organization’s totalRNA,and detecte expression levels of UCP2, STC1by RT-PCR. and finallydetect content of renal cells calcium.
Results: immunohistochemistry prompted UCP2highly expressed inkidney skin medullary junction of the proximal convoluted tubule.STC1ismainly localized in the proximal tubule and a small amount of expression inthe medulla of the distal convoluted tubule. Immunohistochemistry andRT-PCR’s results suggest that the UCP2, STC1expression in fluorosis rats were significantly higher than the control group. And the Ca~(2+)is increased in3groups while the fluoride increased(pairwise comparisons, P<0.05); ratsrenal cells calcium content in each experimental group were significantlyhigher than the control group and it increase while the fluoride increased(pairwise comparisons, P <0.05).
Conclusion: UCP2and STC1maybe involved in mechanism offluorosis injury of rat kidney in which calcium imbalance also play animportant part.
Objective: To observe of UCP2and STC1changes in the kidneys ofrats with fluoride with high calcium against rats renal injury, and explorethe relationship of UCP2, STC1, and high calcium in the renal injury offluorosis rats.
Methods:24male SD rats were divided into four groups according tobody weight, control group low fluoride plus calcium group, middle fluorideplus calcium group, high fluoride plus calcium group. The control groupwith normal saline injection, low, middle and high fluoride plus calciumgroups were given5mg/kg,10mg/kg and20mg/kg of sodium fluoridesolution by intraperitoneal injection according to body weight ratios,1injection every48hours. Low, meddle and high fluoride plus calcium groupswere given high calcium diet for16weeks. Ways of immunohistochemistryand RT-PCR were used to detect location and expression of STC1and UCP2.Content of renal cell [Ca2+]i was detect by fluorescent probes, and we alsodetected the lipid peroxides activity of rats renal tissue.
Results: Confirmed that UCP2/STC1was mainly localized in renalcortex and a very small amount expressed in the renal medulla distalconvoluted tubule. Expression of UCP2and STC1was increasing with the contents of fluoride compared with the control group and high calciumcould increase the expression of UCP2and STC1, the difference wassignificant (P<0.05). Lateral compared with the dose simply exposed tofluoride group of UCP2expression in the high fluoride plus calcium groupwas significantly higher than the high fluoride group (P<0.05). UCP2in thelow, low fluoride plus calcium group compared exposed to fluoride grouphad significantly changes in expression also showed an upward trend. STC1in the expression of each exposed to fluoride plus calcium group and thecorresponding dose simply exposed to fluoride group were not significantlychanged (P>0.05), but expression also showed an upward trend; in eachexperimental group of rat kidney cells of [Ca~(2+)] i levels were significantlyhigher than that control group and exposed to fluoride dose increaseselevated, high calcium and inhibit renal cell [Ca~(2+)]i level.
Conclusion: The UCP2/STC1of fluorosis renal injury can beincreased reactivity, high calcium can antagonize the the fluorosis renalinjury, and promoting of UCP2, STC1expression and inhibition of renal cell[Ca~(2+)] level. UCP2, STC1may be involved in fluorosis rat renal injurymechanism, and the participation process may be related to the calcium ion.
Objective: To detect the changes of mitochondrial uncoupling-relatedindicators and oxygen free radical in the tissue of SD rats and compare withexpression of UCP2. Explore whether the uncoupling function of UCP2isinvolved in mechanism of fluorosis kidney injury.
Method: Extract of rat kidney mitochondria and detect mitochondrialrespiratory function. By the principle that mitochondrial membrane poreopening make the mitochondria swelling, detect the swelling level ofmitochondria by fluorescence spectrophotometer. By the principle thatrhodamine123will fluorescence quench in mitochondria rapidly, detectmitochondrial membrane EMF by fluorescence spectrophotometer (△Ψm).Measure mitochondrial superoxide content by coelosphere reader becauseunpaired electrons and superoxide anion from mitochondria react withtetrazolium salt (NBT) and the production will be waken in570nmlight-wave.
Results: Compared with the control group, mitochondrial III staterespiration rate raise mildly in the low fluoride group. Tended to decline inthe medium fluoride group, but low fluoride group was no significantdifference with medium group (P>0.05); significantly decreased in the highfluoride group (P<0.05); Compared with the control group, the respiratory state III rate decrease in the low fluoride plus calcium group and the mediumfluoride plus calcium group, but no significant differences (P>0.05)In thehigh fluoride plus calcium group, a certain extent enhance of the rate isfound. The Ⅳ state respiration rate increases in the low fluoride pluscalcium group and the medium fluoride plus calcium group and the highfluoride group’s decrease, but there was no significant difference (P>0.05).Calcium to produce a certain degree of inhibition of state IV respiration rateof fluorosis in the rat kidney mitochondria, but the difference is not obvious.Mitochondrial respiratory control ratio (RCR) as exposed to fluorideincreased gradually to reduce and the high fluoride group was significantlylower (P<0.05). Although a significant reduction was found in the highfluoride plus calcium group, but the RCR recovery,because calcium canantagonize this change. Mitochondrial respiratory control ratio raise influoride plus calcium groups.
Conclusion: When fluorosis kidney injury happened, themitochondrial uncoupling-related indicators, EMF, mitochondrial swellinglevel changing significantly, suggest that in the mitochondrial membranechannel transition pore opening degree of a significant change. Thesesuggest that the uncoupling function maybe involve in the mechanism offluorosis renal injury. With fluoride concentration increased, the degree ofswelling of mitochondria and the level and oxygen free radicals content willincrease, but high calcium can inhibit this change. The experimental results confirmed previous studies that mitochondrial damage and fluorosis oxygenfree radical damage caused by fluorosis, and high calcium can antagonizethis damage.
Objective: To determine sodium fluoride effect on the renal tubularepithelial cells (HK2), and detect UCP2, STC1, mitochondrialuncoupling-related indicators changes and their relation. Make HK2highlyexpress UCP2, and observe UCP2, STC1, uncoupling indicators’ changesand calcium content. Explore if UCP2have antagonistic effect to fluorosis.Discuss the relation between UCP2, STC1, Ca~(2+) and calcium.
Method: To construct plasmid containing human UCP2genefragments, transfect HK2. Infect the normal HK2, transfected UCP2,transfected HK2low(0.8mmol/L), medium(1.6mmol/L), high (2.4mmol/L),three levels of sodium fluoride. Use calcium chloride to adjust the calciumconcentration in the medium. Detect cell proliferation by the MTT assay;Detect the expression of STC1by RT-PCR. Extract mitochondria,detectmitochondrial swelling level, mitochondrial membrane EMF andintracellular calcium ion content.
Results: The proliferative activity of renal tubular epithelial cells withthe dye fluorine increase in the degree of increase and in the fluoride group,the high fluoride group was significantly decreased (P<0.05). High calciumcontent can antagonize the negative effect of fluorosis to HK2’s proliferation. HK2’s change is different in three groups which UCP2is highly expressed.Although there is no significant different, in the low fluoride group and themedium fluoride group, the HK2transfected with UCP2’s proliferation israise. Compare the high fluoride group with HK2high expression of UCP2,the transfected HK2’s proliferation activity was further reduced.(P<0.05).High calcium can antagonize the effect of fluorosis to the HK2celltransfected but the difference is not significant (P>0.05). STC1mRNAexpression in renal tubular epithelial cells of fluoride groups is significantlydifferent with the control group (P<0.05); High calcium content can promotethe expression of STC1. Transfected HK2’s STC1expression is significantlyhigher than the normal of HK2group (P<0.05). The high calcium contentcan enhance the expression of STC1, but the difference is no significant(P>0.05). The fluoride groups’ calcium content is higher than the controlgroup(p<0.05). Calcium content in HK2cells from fluoride groups issignificantly higher than control groups, by pairwise compared (P<0.05).When given high calcium handling, calcium content in normal renal tubularepithelial cells appear significantly reduced, and pairwise compared, thedifference was significant (P<0.05); Intracellular calcium content intransfected HK2cells from the high fluoride group is less than the normalHK2cells while the low fluoride group and the medium group have nosignificant change (P>0.05). Give transfected HK2cells high calciumhandling the calcium content becoming lower than the normal HK2cells. The high fluoride group and the medium fluoride group is reducedsignificantly.(P<0.05).
Conclusions: Fluorosis have negative effect on the proliferation ofHK2cells and it can enhance the expression of UCP2and STC1.the changeabove is more significant with the fluoride content. Fluorosis affect theuncouple function of HK2cells’ mitochondria and the result have some doserelation with fluoride content. So the result prove that UCP2, STC1and theuncouple function of mitochondria involve the mechanism of kidneyfluorosis. The present findings provided that UCP2is a factor to stimulateSTC1expression and reduce the calcium content of UCP2, that have effectson cell in many side.
引文
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