【作者】 吴晓霞；

【导师】 刘淑杰；

【作者基本信息】 中国医科大学 ， 口腔临床医学， 2002， 硕士

【摘要】 前言 分泌型免疫球蛋白A(secretory immunoglobulin A，SIgA)自然存在于唾液中，也可由特异性抗原刺激产生，可干预微生物对口腔表面的附着，改变细菌的表面状态，阻碍其在牙面的粘附和定居，还可抑制细菌合成多糖，从而减少变形链球菌(mutans streptococci，简称MS)致龋的机会和可能。唾液过氧化物酶(salivary peroxidase，简称Spx)，在过氧化氢存在的条件下，能催化硫氰酸根离子(SCN~-)发生氧化反应，中间产物均是强氧化剂，进入菌体后，能阻断糖代谢通路，从而抑制细菌增殖，因而也有一定的防龋作用，目前对于SIgA、Spx和龋病的关系的研究尚有争议，原因之一是实验方法的不准确性和不一致性，本实验应用先进、敏感的非竞争性生物素-抗生物素酶联免疫吸附法(non-competitive avidin-biotin immuno-enzymatic assay，简称NABA法)测定唾液中SIgA浓度，用Nb-SCN~-法测定唾液Spx浓度，并研究它们和龋病的关系。同时在国内首次对多龋组、无龋组儿童唾液中SIgA、Spx的相关关系进行了研究，为唾液抗菌蛋白和龋病关系的研究提供了新线索。 实验方法 随机选取1个幼儿园中4-5岁多龋儿童20名(dmft≥5)及无龋儿童20名。采样均在上午9-10时，由同一检查者完成。用吐唾法取非刺激性全唾液2ml，同时记录dmft。取1ml样本离心(18，000g，15min，4℃)，上清液-20℃保存待测Spx。其余1ml唾液-20℃保存待测SIgA。 NABA法测定唾液SIgA浓度：向兔抗人 IgA（a链）包被的96IL酶标板中依次加人稀释唾液（标准品X生物素标记羊抗人 IgA…链X亲和素－辣根过氧化物酶、酶底物，最后以硫酸终止反映，405urn处读取 OD值，以 IgA浓度的对数为横坐标，以 OD值对数为纵坐标，绘制标准曲线，计算样本SIgA浓度。 Nb－SCN－法测定唾液 SpX浓度：混合物为 1．5 Inl NbS，1．3 rnl0．0092M KSCN，100gi标准品（唾液广向96孔酶标板的每孔中加入 200 pl混合物。加人过氧化氢溶液使反应开始。记录第 1分钟内405 urn处OD值的变化，按Mansson＿Rahem扣ua的公式： 活性＝AA／6t xl八 x反应混合物总体俐加人样本体积 来计算酶活性，以LP浓度为横坐标，酶活性为纵坐标，绘制标准曲线，计算样本SPX浓度。 实验结果 多龋组儿童唾液SIgA浓度为52．44 L 13．23pg／Inl，无龋组为乃．73。22．匕冲d，前者低于后者，二者有显著性差异（P＜0·of人多龋组儿童唾液SPX浓度为5．865 tZ，297pg／Inl，无龋组为7．om土3．228 p才M，二者无显著性差异（P＞0．05）。 无龋组儿童唾液SIgA／Px浓度呈显著正相关，相关系数r值为 0．72（P＜0．of），多龋组儿童唾液 SIgA＊px相关系数为口．38，无统计学意义汀＞o．05八 讨 论 早在60年代，Keyes就已论证，龋病是一种细菌感染性疾病，MS是人类的主要致龋菌，SIgA可通过干扰变链对牙面的粘着，或通过凝集细菌，使其便于从口腔中清除，以及抑制多糖合成，减少 ·2·菌斑量等作用来抑制龋病发生。对于SIgA和龋病的关系，国内外均作了大量研究，但结果很不一致。Benderli研究了26名肾移植病人唾液SIgA浓度和龋齿的关系，发现肾移植病人唾液SIgA浓度显著低于对照组，术后 12月病人的龋齿数明显增加；tehtonen发现多龋者唾液SIgA浓度高于低龋者；张志平测定了60名龋患者和 60名健康者唾液 SIgA含量，前者显著低于后者涨君平测定了 3－4岁 39名无龋儿童和 64名多龋儿童唾液中 SIgA浓度，未发现二者的差别。对于研究结果有差异的原因，Rudney认为首先是实验方法的不准确性和不一致性。目前常用于SIgA浓度测定的方法有单向免疫扩散法、免疫比浊法、放射免疫分析法及酶联免疫吸附法（enZym 一linked i。unosorbent ass印，ELISA法），前两者准确性差，人工测量误差大，而放射免疫分析需要较长时间，还有要求使用放射性同位素的缺点，普通ELISA法和NABA法相比也不够敏感。非竞争性生物素－抗生物素酶联免疫吸附实验hon－competitive avldln－blotin。uno－e啊m叨c ass盯s，简称 NABA法）是用生物素标记抗体取代普通ELISA法中的酶标抗体，由于生物素－亲和素之间的亲和常数高，每个抗体可结合10个左右生物素分子，而只能结合1个酶分子，因而具有更高的灵敏度；此外，由于抗体可高度稀释，减少了背景着色的可能性，使结果更准确。国外 NABA法已开始用于唾液 SIgA浓度的测定，而国内尚无有关报道。我们通过实验认为，NABA法操作简便快速，结果准确，是一种适用于检测唾液SIgA浓度的先迸方法。本研究应用先进敏感快速的 NABA法研究幼儿非刺激性全唾液 SIgA浓度和龋病的关系，结果显示多龋组儿童非刺激性全唾液SIgA浓度为52．44 Z13．23p矿Inl，无龋组为75．73。22．15pyrnl，前者显著低于后者，有统计学意义，与大多数学者的研究结果一致，认为唾液 SIgA对于预防龋齿发生有一定作用。 唾液SpX能催更多还原

【Abstract】 PrefaceSecretory immunoglobulin A ( SIgA) not only is a naturally occurred antimicrobial factor in saliva, but also can be induced by specific antigens. It can interfere with attachment of microorganisms on the oral tissure, aggregate bacteria to facilitate their clearance by secretions , and inhibit glucans synthesis so as to lessen the cariogenieity of the mutans streptococcus ( MS). Salivary peroxidase (Spx)can catalyze oxidization of SCN- , all of the product are strong oxidizing a-gents, which can block carbohydrate metabolism after entering bacterial cell, so it also has some inhibition effect on caries. Now there is controversy about the relationship between SIgA ,Spx and caries. One of the reasons is the difficulty in standardization of the measurement method. In this experiment, we used the sensitive, accurate non -competitive avidin - biotion immuno - enzymatic assay ( NAB A) to determine the SIgA concentration , Nb - SCN- method to determine the Spx concentration, at the same time we studied the difference of SIgA, Spx concentration between the caries - susceptible children and caries - resistent ones, as well as their correlation in this two groups. The object of this study is to find some new information about the rela-tionship between antimicrobiol proteins and caries.MethodsTo choose randomly twenty 4 - Syr - old children with caries (dmft ≥5 ) , and twenty caries - free children as control. From the children, 2 ml unstimulated saliva were collected, and their dmft values were recorded. The SIgA concentration were assayed by NABA method in saliva that had been stored at -20℃. Microplates coated with and -human IgA (a chain) were incubated overnight at 37Tl. Then saliva (diluted buffer, standard) , biotinylated anti -human IgA ( a chain) , avidin - peroxidase, enzyme substrate were added successively, at last H2SO4 were added to stop the reaction. Absorbances were read at 405 nm. Calculate SIgA concentration according to the standard curve.The spx concentration were measured by Nb - SCN- method in saliva that had been centrifugated(18,000g, 15min, 4℃)and stored at -20℃. The reaction mixture was made up of 1. 5ml Nbs, 1. 3ml KSCN, 100(0,1 saliva (standard). 200ul mixture were added to the microplates, then 100ul H2O2 were added to make the reaction begin. Absorbances were read at 405 nm for the first minute. Bovine peroxidase was used as standard. The Spx concentration were calculated according to the standard curve.Results1. The SIgA concentration in unstimulated saliva of caries - re-sistent children was 75. 73 ±2. 1μxg/ml, in saliva of caries - sus-ceptible children is 52.44± 13.23u,g/ml. The former was significantly higher than the latter ( P < 0.01) .2. The Spx concentration in unstimulated saliva of caries - resis-tent children was 7.007 ± 3.228μg/ml, in saliva of caries - susceptible children was 5. 865± 2. 297μLg/ml. There was no significant difference between them ( P >0.05).3. SIgA and Spx correlated significantly in caries - resistant children, correlation coefficient was 0. 72(P <0.01) , but there was no significant correlation for them in caries - susceptible children, correlation coefficient was 0. 38 ( P >0.05 ).DiscussionEarly in 1960s, Keyes had proved caries is a kind of infectious disease. MS is the major cariogenic bacteria. Both SIgA and Spx are important antimicrobial proteins in saliva, which can disturb the action of bacteria through several mechanisms. But studies aimed at correlating SIgA and Spx with resistance or susceptibility to dental caries have given variable and conflicting results over the years for several reasons. One of the most important reasons is the difficulty of the standardization of the measurement method. The usually used single immunodiffusion is too crude; radioimmuno - assay has the shortcoming of applying radioisotope. Enzyme - linked immunosorbent assay (ELISA)is also less sensitive than NAB A. Over the past few years, NAB A had been applied to determine SIgA concentration overseas. Th更多还原