CAJ | 학술논문

目的：已有的研究表明包括糖尿病肾病在内的多种肾病患者肾小管上皮细胞存在C3a／C3aR轴过度活化现象，但有关C3 a／C3 aR轴过度活化在肾小管上皮细胞中的病理意义未知。文中构建分泌性过表达C3 a的肾小肾小管上皮细胞株，为探讨各种病理情况下C3a／C3aR轴过度活化的病理意义提供细胞模型。方法设计合成人C3a 分泌性表达单元，将其克隆到慢病毒表达载体pLenti6．3-MCS-IRES2-EGFP的多克隆位点，构建成C3a分泌性表达载体pLenti6．3-C3a-IRES2-EGFP；将pLenti6．3-C3a-IRES2-EGFP和包装质粒共转染293 T细胞，包装成C3a表达重组慢病毒LV-C3a；以LV-C3a感染人肾小管上皮细胞系HK2，根据LV-C3a上带有杀稻瘟菌素抗性基因的特点，以杀稻瘟菌素筛选稳定转染细胞克隆；利用荧光定量PCR和ELISA方法对稳定转染细胞克隆的C3a表达水平和分泌情况进行分析，从中鉴定出稳定分泌性过表达C3a的人肾小管上皮细胞株。结果①成功构建了序列完全正确的C3a分泌性表达载体pLenti6．3-C3a-IRES2-EGFP；②成功进行了重组慢病毒包装，得到了高滴度（5×108个／mL）的C3a表达重组慢病毒LV-C3a；③成功进行了LV-C3a对HK2细胞的转染，得到了稳定转染LV-C3a的HK2细胞株；基于荧光定量PCR和ELISA的分析证实，与HK2细胞比较，HK2-C3a细胞株中C3a mRNA表达水平显著升高[（1．0±0．5） vs （1321．0±18．0）， P＜0．01]，分泌的C3a水平显著升高[（0．3±0．2）ng／mL vs （249．0±37．0） ng／mL， P＜0．01]。结论成功构建的C3a分泌性慢病毒表达载体和分泌性过表达C3a的人肾小管上皮细胞株为进一步研究各种病理情景中C3a／C3aR过度活化在人肾小管上皮细胞中的病理意义提供了很好的细胞模型，也为进一步开展C3a／C3aR过度活化在其他细胞中的病理意义创造了条件。
목적：이유적연구표명포괄당뇨병신병재내적다충신병환자신소관상피세포존재C3a／C3aR축과도활화현상，단유관C3 a／C3 aR축과도활화재신소관상피세포중적병리의의미지。문중구건분비성과표체C3 a적신소신소관상피세포주，위탐토각충병리정황하C3a／C3aR축과도활화적병리의의제공세포모형。방법설계합성인C3a 분비성표체단원，장기극륭도만병독표체재체pLenti6．3-MCS-IRES2-EGFP적다극륭위점，구건성C3a분비성표체재체pLenti6．3-C3a-IRES2-EGFP；장pLenti6．3-C3a-IRES2-EGFP화포장질립공전염293 T세포，포장성C3a표체중조만병독LV-C3a；이LV-C3a감염인신소관상피세포계HK2，근거LV-C3a상대유살도온균소항성기인적특점，이살도온균소사선은정전염세포극륭；이용형광정량PCR화ELISA방법대은정전염세포극륭적C3a표체수평화분비정황진행분석，종중감정출은정분비성과표체C3a적인신소관상피세포주。결과①성공구건료서렬완전정학적C3a분비성표체재체pLenti6．3-C3a-IRES2-EGFP；②성공진행료중조만병독포장，득도료고적도（5×108개／mL）적C3a표체중조만병독LV-C3a；③성공진행료LV-C3a대HK2세포적전염，득도료은정전염LV-C3a적HK2세포주；기우형광정량PCR화ELISA적분석증실，여HK2세포비교，HK2-C3a세포주중C3a mRNA표체수평현저승고[（1．0±0．5） vs （1321．0±18．0）， P＜0．01]，분비적C3a수평현저승고[（0．3±0．2）ng／mL vs （249．0±37．0） ng／mL， P＜0．01]。결론성공구건적C3a분비성만병독표체재체화분비성과표체C3a적인신소관상피세포주위진일보연구각충병리정경중C3a／C3aR과도활화재인신소관상피세포중적병리의의제공료흔호적세포모형，야위진일보개전C3a／C3aR과도활화재기타세포중적병리의의창조료조건。
[Abstract ] Objective Evidence from previous studies indicated that over-activation of C3a/C3aR axis existed in the renal tubular epithelial cells of patients with renal diseases including diabetic nephropathy .However , the pathological significance of over-ac-tivating C3a/C3aR axis still remains to be elucidated .In this study, we constructed a renal tubular epithelial cell line over-expressing C3a in a secretory manner in order to provide a cell model to investigate the pathological significance of over -activating C3a/C3aR axis under various pathological scenes . Methods We designed a synthesized C3a secretory expression unit and cloned it into the multi-clonal site of lentivirus expression vector pLenti 6.3-MCS-IRES2-EGFP.After identification by sequencing , recombinant lentivirus was packaged by using pLenti 6.3-C3a-IRES2-EGFP and packaging plas-mid in 293T cells.Then, the recombinant lentivirus was used to in-fect HK2, a cell line of human renal tubular epithelial cells .After screening in medium with blasticidin , blasticidin resistant cell clones were obtained .Real-time PCR and ELISA method were applied to analyze the expression and secretion of stable transfected cells cloned C3a and identify renal tubular epithelial cell lines with stable over-activating C3a. Results ①C3a secretory expression unit was suc-cessfully synthesized and correctly cloned into the multi-clonal site of pLenti6.3-IRES2-EGFP; ②C3a secrectory expression recombi-nant lentivirus LV-C3a was successfully packaged with a high titer of 5 ×108/mL;③HK2 Cell clones resistant for blasticidin were ob-tained;according to the analysis of Real-time PCR and ELISA, the C3a mRNA level in HK2-C3a cell lines was significantly higher than that of HK2 cells(1.0 ±0.5 vs 1321.0 ±18.0, P<0.01) and the secreted C3a level increased significantly ([0.3 ±0.2]ng/mL vs [249.0 ±37.0] ng/mL, P<0.01). Conclusion The present study successfully constructed C 3a secretory expression vector pLenti6.3-C3a-IRES2-EGFP and C3a over-expression renal tubular epithelial cell line HK 2-C3a, which is very useful in further study of the function and significance of C 3a/C3aR axis not only in renal tubular epithelial cells but also in other cell types .