【作者】 李艳平；

【导师】 赵家军；

【作者基本信息】 山东大学 ， 内科学， 2008， 硕士

【摘要】 研究目的免疫因素在肾小管性酸中毒发病中的作用日渐受到关注。已有研究报道肾小管性酸中毒患者血清中存在抗肾小管上皮细胞自身抗体,但这些抗体的具体种类尚未明确。本实验旨在检测肾小管性酸中毒患者血清中是否存在抗v型氢离子腺苷三磷酸酶(v-H~+-ATP酶)B1亚基及B2亚基自身抗体。研究方法研究对象:肾小管性酸中毒患者11例,其中10例合并干燥综合征:单纯干燥综合征患者8例;健康对照8例。所有患者均为未治疗的新发或复发病例。取各研究对象全血利用流式细胞技术进行免疫学检查,部分血清进行生化等指标检测,部分血清用于间接免疫荧光检测自身抗体。先确定患者血清中是否存在抗肾小管上皮细胞抗体,以及观察v-H~+-ATP酶B1亚基和B2亚基在正常人肾脏组织的表达特点。在正常人肾脏组织石蜡切片上,加肾小管性酸中毒患者血清原液、单纯干燥综合征患者血清原液、健康对照血清原液、抗v-H~+-ATP酶B1亚基抗体或抗v-H~+-ATP酶B2亚基抗体作为一抗孵育4℃过夜,以异硫氰酸荧光素(Fluoresceinisothiocynate,FITC,绿色荧光)-山羊抗人免疫球蛋白(Immunoglobulin,IgG)孵育,标记血清中的抗肾小管上皮细胞自身抗体,或四甲基异硫氰酸罗丹明(tetramethylrhodamineisothiocyanate,TRITC,红色荧光)-兔抗山羊IgG孵育,标记v-H~+-ATP酶B1亚基或B2亚基,4,6-联脒-2-苯基吲哚(4,6-diamidino-2-phenyl-indolediacetare,DAPI,蓝色荧光)标记细胞核并封片。结果阳性者,进一步稀释相应的一抗,重复上述实验,直至结果为阴性。并为以下间接免疫荧光双标记实验选取适当的稀释比例。为检测肾小管性酸中毒患者血清中是否存在抗v-H~+-ATP酶B1亚基及B2亚基自身抗体,先用肾小管性酸中毒患者血清原液、单纯干燥综合征患者血清原液或健康对照血清原液与正常人肾脏组织石蜡切片孵育4℃过夜,然后加不同稀释度的山羊抗人v-H~+-ATP酶B1亚基抗体孵育4℃过夜,再先后加TRITC-兔抗山羊IgG及FITC-山羊抗人IgG荧光二抗避光孵育,DAPI标记细胞核并封片。结果阳性者,将血清按不同比例稀释,重复上述实验,直至结果阴性。血清中抗v-H~+-ATP酶B2亚基自身抗体的检测同抗v-H~+-ATP酶B1亚基自身抗体的检测。每次实验均设阴性对照。结果一般临床指标检查结果:与单纯干燥综合征患者和健康对照相比较,肾小管性酸中毒患者的血清氯水平明显升高,血清二氧化碳明显下降,尿pH明显升高,流式细胞学检查、免疫球蛋白及补体检查结果均提示免疫学变化。血清抗肾小管上皮细胞自身抗体检测结果:11例肾小管性酸中毒患者中,有6例患者肾小管上皮细胞内呈明亮的绿色荧光,提示抗肾小管上皮细胞抗体阳性;当把自身抗体阳性的血清逐渐稀释时,其绿色荧光的强度呈不同程度地逐渐减弱,提示随着血清的稀释,自身抗体的浓度逐渐下降;而8例单纯干燥综合征患者及8例健康对照的肾小管上皮细胞内无明显绿色荧光,提示其血清中抗肾小管上皮细胞自身抗体阴性。v-H~+-ATP酶B1亚基及B2亚基在正常肾脏组织的表达结果:标记v-H~+-ATP酶B1亚基的红色荧光在肾脏中呈闰细胞特异性散在分布,标记v-H~+-ATP酶B2亚基的红色荧光在肾小管上皮细胞内沿管腔面呈连续的线状分布。抗v-H~+-ATP酶B1亚基及B2亚基自身抗体检测结果:11例肾小管性酸中毒患者中,有6例患者标记v-H~+-ATP酶B1亚基的红色荧光强度明显减弱,提示其血清对于抗v-H~+-ATP酶B1亚基抗体有明显的阻断作用;当把自身抗体阳性患者的血清逐渐稀释,标记v-H~+-ATP酶B1亚基的红色荧光强度逐渐增强,提示随着血清的稀释,其对于抗v-H~+-ATP酶B1亚基抗体的阻断作用呈不同程度的减弱,提示不同稀释度血清的抗体效价强度不同;而8例单纯干燥综合征患者和8例健康对照血清则无上述阻断作用。抗v-H~+-ATP酶B2亚基自身抗体的检测结果同抗v-H~+-ATP酶B1亚基自身抗体检测结果。以上结果提示肾小管性酸中毒血清中存在抗v-H~+-ATP酶B1亚基及B2亚基自身抗体。结论肾小管酸中毒患者血清中存在抗肾小管上皮细胞自身抗体,阳性率为6/11;肾小管性酸中毒患者血清中存在抗v-H~+-ATP酶B1亚基及B2亚基自身抗体,阳性率均为6/11:肾小管性酸中毒患者血清中的抗v-H~+-ATP酶B1亚基及B2亚基自身抗体是抗肾小管上皮细胞自身抗体中的两种。更多还原

【Abstract】 ObjectiveAutoimmunity plays an important role in renal tubular acidosis(RTA).The presence of autoantibodies against renal tubular epithelial cells has been found in patients with RTA,but the detailed description of these autoantibodies is current lacking.Our aims of this study was to detect a presence or absence of autoantibodies against B1 and/or B2 subunits of vacuolar H~+-adenosine triphosphatease(v-H~+-ATPase) in sera of patients diagnosed with RTA.MethodsEleven untreated patients diagnosed with RTA(10 of these cases were associated with Sjogren syndrome),eight untreated patients with simple Sjogren syndrome(sSS)and eight healthy controls(HC)were enrolled in the study.Part of the fasting whole blood was taken out for measuring immuno-function by flow cytometry, and other serum was for detecting serum biochemistry,and autoantibodies presence or absence by indirect immunofluorescence assay.Firstly,to detect a presence or absence of serum autoantibodies against renal tubular epithelial cells and observe the expression of B 1 subunit and B2 subunits of v-H~+-ATPase in normal renal tissue of human,paraffin sections of normal human renal tissue were incubated with sera of RTA,sSS,HC or anti- B1 or B2 subunits of v-H~+-ATPase antibodies overnight at 4℃,then autoantibodies against renal tubular epithelial cells in sera were conjugated with fluorescein isothiocyanate(FITC,green color)-labeled goat-anti-human immunoglobulin G(IgG),while B1 or B2 subunits of V-H~+-ATPase were conjugated with tetramethylrhodamine isothiocyanate(TRITC, red color)-labeled rabbit-anti-goat IgG.Nuclei were stained with 4,6-diamino-2-phenyl indole(DAPI,blue color).For each positive sample,the sera or anti-B1 or B2 subunits of V-H~+-ATPase antibodies were diluted to repeat the trial until negative results were obtained.To confirm a presence or absence of autoantibodies against B1 and B2 subunits of v-H~+-ATPase in sera of RTA,paraffin sections of normal human renal tissue were firstly incubated with sera of RTA,sSS or HC overnight at 4℃,and with goat-anti-v-H~+-ATPase B1 subunit antibodies followed later overnight at 4℃.Then,the sections were incubated with TRITC-labeled rabbit-anti-goat secondary antibodies for B subunit of v-H~+- ATPase assay,and with FITC-labeled goat-anti-human secondary antibodies for autoantibodies against renal tubular epithelial cells.Finally,nuclei were stained with DAPI.For each positive sample,we diluted the sera and repeated the trial until negative.Similarly,detection of autoantibodies against B2 subunit of v-H~+-ATPase was performed by the same protocol as that against B1 subunit of v-H~+-ATPase assay described previously.ResultsResults of general indicators:Compared with patients with sSS and HC,results in sera of patients with RTA showed that serum chloride increased,CO2 decreased and urine pH increased.And results of flow cytometry,IG and complements implied immune disorders.Results of autoantibodies against renal tubular epithelial cells:The sera of 6 cases of all RTA patients showed green fluorescence staining in renal tubular epithelial cells,additional experiments showed that the staining intensity was also reduced following the sera of patients with RTA were diluted.Moreover,none of those sera from patients with either sSS or HC showed green fluorescence in renal tubular epithelial cells,which indicated no autoantibodies against renal tubular epithelial cells in sera of patients with sSS and HC.The results indicated the presence of autoantibodies against renal tubular epithelial cells in sera of patients with RTA.Results of distribution characteristics of B1 and B2 subunits of v-H~+-ATPase in normal human renal tissue:Red fluorescence representing B1 subunit of v-H~+-ATPase distributed specifically in intercalated cells,while B2 subunit distributed continuously in the cavosurface of renal tubular epithelial cells.Assay results of autoantibodies against B1 and B2 subunits of v-H~+-ATPase: when sera were incubated prior to anti-B1 subunit antibodies,the red fluorescence intensity showed a decrease in 6 cases of the 11 patients with RTA,which implied that blocking effect of the sera of patients with RTA to anti-B1 subunit of v-H~+-ATPase antibodies was occurred.The sera-induced blocking effects were reduced following the sera of patients with RTA diluted.While none of sera from these patients with either sSS or HC showed the blocking effect like that.The assay results of autoantibodies against B2 subunit of v-H~+-ATPase were as the same as that of B1 subunit.ConclusionThere are autoantibodies against renal tubular epithelial cells in sera of patients with RTA,and the positive ratio is 6/11.There are autoantibodies against B1 and B2 subunits of v-H~+-ATPase in sera of patients with RTA,and the positive ratio are both 6/11.Autoantibodies against B1 and B2 subunits of v-H~+-ATPase are two kinds of autoantibodies against renal tubular epithelial cells in sera of patients with RTA.更多还原