应用凝集素芯片检测细胞膜表面糖链种属特异性

摘要

目的 应用凝集素芯片检测小鼠和兔不同组织细胞膜表面的糖链类型,探求不同物种间相同组织细胞膜表面糖复合物在种群免疫和细胞功能中的作用.方法 选择22种凝集素,利用生物芯片点样仪制各芯片,选用牛血清白蛋白(BSA)为阴性质控,马铃薯凝集素(STL)为阳性质控.分别用胶原酶Ⅰ、Ⅱ、Ⅳ消化小鼠和兔的肾、脾、肝组织,制备细胞悬液,并加入吖啶橙荧光标记细胞.将细胞悬液分别滴加至凝集素芯片中,利用凝集素与糖链的特异亲和性捕获细胞,激光扫描仪检测芯片中各凝集素位点的荧光信号强度,行常规HE染色后于荧光显微镜下观察各凝集素位点所捕获的细胞形态,并分析不同胶原酶消化对检测结果的影响.结果 小鼠和兔相同组织的细胞膜表面糖链类型基本相同,仅个别位点略有不同.在GNA、LTL、HHL,、NPL位点,兔脾细胞均为阴性,小鼠脾细胞均为阳性.在BPL、WFA位点,兔肾细胞均为阴性,小鼠肾细胞均为阳性;在MAL-I位点,兔肾细胞为阳性,小鼠肾细胞为阴性.MPL、WBA、LTL、SBA、BPL、WFA、MAL-I、AAL、BCL位点,兔肝细胞均为阳性,小鼠肝细胞均为阴性;在NPL位点,小鼠肝细胞为阳性,兔肝细胞为阴性.DSL、STL位点,小鼠和兔各组织细胞检测结果均为阳性;在EEL,、DBA位点,小鼠和兔各组织细胞检测结果均为阴性.在GNA位点,兔脾组织经胶原酶Ⅱ消化后可见极少量细胞,经其他2种胶原酶消化后则未见捕获细胞.兔肾组织以及小鼠的脾和肾组织经3种胶原酶消化后,检测结果无明显差异.在BCL位点,兔肝组织经胶原酶Ⅳ消化后捕获细胞最多,其次为胶原酶Ⅱ,胶原酶Ⅰ最少.结论 通过制备的凝集素芯片成功检测了小鼠和兔不同组织细胞膜表面的糖链特异性.小鼠和兔相同组织的细胞膜表面具有相同类型糖链,但不同组织之间细胞膜糖链类型也存在其特异性,这可能与组织分化及其功能密切相关.%Objective To detect the types of glycoprofiles on the different tissues cells surface of miceand rabbits by lectin microarray, in order to explore the roles in the population immunity and cell function of glycoprofiles on the same tissue cells surface between different species.Methods 22 lectins were printed on array using microarray spotting instrument to make chips, choosing the bovine serum albumin (BSA) as the negative control and STL as the positive control.The cells were extracted from the kidney, liver and spleen tissues of mice and rabbits with collagenase Ⅰ, Ⅱ, Ⅳ digests respectively, prepared the cells suspension and labeled the cells by AO.Dropping the cells suspension on the lectin microarray, the cells were caught by lectins though the distinctive binding specificities with glycoprofiles.The fluorescence signal intensities on the chips were detected by laser scanner.After conventional HE staining, the morphology of cells caught at various lectin points were observed by fluorescence microscope, and analyzed the effects of different collagenase digestion on test results.Results The types of glycoprofiles on the same tissues cells surface of mice and rabbits were basically the same, except particular points were slightly different.The test results were negative in rabbits spleen cells and positive in mice spleen cells at the points of GNA, LTL, HHL, NPL.That were negative in rabbits kidney cells and positive in mice kidney cells at the points of BPL, WFA, but positive in rabbits kidney cells and negative in mice kidney cells at the point of MAL-Ⅰ.Those were positive in rabbits liver cells and negative in mice liver cells at the points of MPL, WBA, LTL, SBA, BPL, WFA, MAL-Ⅰ, AAL, BCL, but positive in mice liver cells and negative in rabbits liver cells at the point of NPL.Those were all positive in rabbits and mice cells at the points of DSL, STL, but all negative in rabbits and mice cells at the points of EEL, DBA.At the point of GNA, very few rabbits spleen cells were detected by collagenase Ⅱ digesting, while none was detected by other two collagenases.There was no significant difference in rabbits kidney cells and mice spleen and kidney cells by there kinds of collagenases digesting.At the point of BCL, the cells number was the largest in rabbits liver tissues by collagenase Ⅳ digesting, the collagenase Ⅱ was the next and the collagenase Ⅰ was the least.Conclusions The specificities of glycoprofiles on the different tissues cells surface of mice and rabbits were successfully detected by lectin microarray.The types of glycoprofiles on the same tissues cells surface of mice and rabbits were basically the same, but there were also the specificities, that may be related to the differentiation and function of the tissues.