本文關(guān)鍵詞： 人腎小球系膜細(xì)胞 免疫球蛋白A 蛋白質(zhì)譜 DNA測(cè)序　出處：《北京大學(xué)學(xué)報(bào)(醫(yī)學(xué)版)》2017年06期 　論文類型：期刊論文

【摘要】：目的:探討人腎小球系膜細(xì)胞(human mesangial cells,HMCs)中是否表達(dá)免疫球蛋白A(immunoglobulin A,Ig A)。方法:培養(yǎng)人HMCs細(xì)胞系,以免疫熒光染色檢測(cè)Igα、Igκ、Igλ在系膜細(xì)胞中的表達(dá);以反轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(reverse transcription polymerase chain reaction,RT-PCR)和DNA測(cè)序檢測(cè)HMCs中Igα、Igκ、Igλ恒定區(qū)轉(zhuǎn)錄本表達(dá);以Western blot方法檢測(cè)細(xì)胞裂解液及培養(yǎng)上清液中Igα、Igκ、Igλ蛋白水平表達(dá);以jacalin為配體的親和層析純化上清液蛋白質(zhì),以蛋白質(zhì)譜檢測(cè)純化的蛋白氨基酸序列。結(jié)果:免疫熒光染色檢測(cè)到HMCs細(xì)胞漿Igα、Igκ、Igλ的陽性表達(dá);RT-PCR檢測(cè)到細(xì)胞內(nèi)Igα1、Igα2重鏈、Igκ輕鏈、Igλ輕鏈恒定區(qū)轉(zhuǎn)錄本表達(dá),且其核苷酸序列與美國國立生物技術(shù)信息中心(National Center of Biotechnology Information,NCBI)數(shù)據(jù)庫相應(yīng)的mRNA序列比對(duì),同源性分別達(dá)到99%、97%、98%和97%;Western blot檢測(cè)到細(xì)胞內(nèi)Igα1、Igα2和Igλ蛋白水平的表達(dá),Igκ目前檢測(cè)為陰性;細(xì)胞上清液中可見完整Ig A分子表達(dá);以jacallin為配體親和層析純化的培養(yǎng)上清液中的Ig A,蛋白質(zhì)譜測(cè)序所得的氨基酸片段序列與NCBI數(shù)據(jù)庫比對(duì),顯示HMCs分泌的蛋白質(zhì)與B細(xì)胞表達(dá)的Igα1恒定區(qū)52~104之間53個(gè)氨基酸、154~221之間68個(gè)氨基酸、以及276~327之間52個(gè)氨基酸的序列完全相同,與Igα2重鏈恒定區(qū)52~113之間62個(gè)氨基酸、151~204之間54個(gè)氨基酸、以及251~314之間64個(gè)氨基酸的序列完全相同,提示系膜細(xì)胞可以將合成的Ig A分泌到胞外。結(jié)論:HMCs可能合成并分泌Ig A。
[Abstract]:Objective: to investigate the expression of immunoglobulin A immunoglobulin A (Ig A) in human Mesangial cells (mesangial cells). Methods: human HMCs cell line was cultured and the expression of Ig 偽 Ig 魏 Ig 位 in Mesangial cells was detected by immunofluorescence staining. Reverse transcription polymerase chain reactionation (RT-PCR) and DNA sequencing were used to detect the expression of Ig 偽 transcription 魏 Ig 位 constant region in HMCs, and the expression of Ig 偽 Ig 魏 Ig 位 protein in cell lysate and culture supernatant was detected by Western blot method. Supernatant proteins were purified by affinity chromatography with jacalin as ligand. Results: the positive expression of Ig 偽 + Ig 魏 Ig 位 in the cytoplasm of HMCs cells was detected by immunofluorescence staining. RT-PCR was used to detect the expression of Ig 偽 1, Ig 偽 2 heavy chain and Ig 魏 light chain constant region transcripts of Ig 位 in HMCs cells. The nucleotide sequence was aligned with the corresponding mRNA sequence in the National Center of Biotechnology Information (NCBI) database of the National Biotechnology Information Center, and the homology reached 990.97% and 97% respectively. The expression of Ig 偽 2 and Ig 位 protein in the cells was negative at present. The complete expression of IgA was found in the supernatant, and the sequence of the amino acid fragment was compared with that of the NCBI database, which was purified by affinity chromatography with jacallin as the ligand, and the sequence of the amino acid fragment of the supernatant was compared with that of the NCBI database. The results showed that the protein secreted by HMCs was identical to that of Ig 偽 1 expressed in B cells. The sequence of 53 amino acids between 53 amino acids and 158 amino acids in the constant region of Ig 偽 1, and between 2766 and 327 amino acids, was the same as that in B cells. The sequence of 64 amino acids and 64 amino acids between 62amino acid and 151C204 in the constant region of heavy chain of Ig 偽 2 is identical to that in the region of Ig 偽 2, suggesting that the synthesized IgA can be secreted into extracellular by Mesangial cells. Conclusion\\\;
【作者單位】： 北京大學(xué)第三醫(yī)院腎內(nèi)科;北京大學(xué)基礎(chǔ)醫(yī)學(xué)院免疫學(xué)系;
【分類號(hào)】：R446.6

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