生物人工肾小管上皮种子细胞的建立

Construction of seeding cells for bioartificial kidney tubule

目的利用人Nanog基因转染生物人工肾的种子细胞（肾近曲小管上皮细胞HKC），提高其增殖能力，在较短时间内获得大量种子细胞，为克服肾脏组织工程种子细胞来源的匮乏及生物人工肾的快速构建提供新途径。方法制备含有人Nanog基因的复制缺陷型重组腺相关病毒血清2型病毒颗粒rAAV2-hNanog，用rAAV2-hNanog转染肾小管上皮细胞，然后采用RT-PCR检测hNanog基因在转染的肾小管上皮细胞中的表达，再用MTT、免疫荧光及激光共聚焦技术检测hNanog基因对肾小管细胞HKC生长的影响。结果rAAV2-hNanog转染肾小管上皮细胞后，RT-PCR检测表明hNanog基因能在肾小管上皮细胞中稳定表达；同时，小管上皮细胞在转染后的增殖能力也显著增强（P〈0．05）。光镜下观察发现，转染的小管上皮细胞的形态无明显改变。结论利用hNanog基因提高生物人工肾小管种子细胞的增殖能力，可为生物人工肾的快速构建及应用提供一种新的方法。

Objective To promote the proliferation of seeding cells, obtain plenty of engineering cells for bioartificial kidney （BAK） in relatively short time and facilitate the construction of BAK system. Methods Recombinant adenovirus-associated virus Ⅱ type harboring human Nanog gene （rAAV2- hNanog） was prepared, then rAAV2-hNanog was transfected into seeding cell HKC. The expression of human Nanog in seeding cells was detected by RT-PCR after transfection. The effects of human Nanog on HKC cell line were examined by tetrazolium salt colorimetry （MTr）, immunofluorescence and confocal laser scanning microscope（ CLSM ）. Results The human Nanog could be expressed in engineering cells. The proliferation activity of transfected seeding cell was significantly improved（P〈0.05 ）. Meanwhile, no obvious morphology changes could be found under inverted microscope in the seeding cells transfected by rAAV2-hNanog compared with control cells. Conclusions The proliferation of seeding cells modified by human Nanog gene is strikingly accelerated. This new approach will overcome the insufficiency of seeding cells for BAK system.