CAJ | 학술논문

目的观察不同条件下骨髓间充质干细胞(MSC)体外诱导分化为肾小管上皮样细胞的差异.方法抽取SD大鼠的骨髓,经密度梯度离心分离,联合贴壁筛选法获取纯化的MSC.以流式细胞仪鉴定间充质干细胞表面标志.取扩增3代的MSC分组培养:(1)对照组:用含胎牛血清培养基;(2)全反式维甲酸(ATRA)组:胎牛血清+缺血再灌注肾脏匀浆上清+ATRA;(3)联合诱导组:胎牛血清+缺血再灌注肾脏匀浆上清+ATRA+表皮生长因子(EGF)+骨形成蛋白(BMP-7).诱导7 d后,倒置显微镜下观察细胞形态变化;化学染色检测细胞碱性磷酸酶表达;免疫细胞化学法检测细胞角蛋白18(cytokeratin-18)、E钙黏蛋白(E-cadherin)的表达.结果流式细胞仪显示,体外分离培养的第3代MSC,CD44阳性细胞表达率为97.8%±0.9%;CD90阳性细胞表达率为96.8%±1.4%;CD29阳性细胞表达率为97.6%±2.4%;而CD11b/c阳性细胞表达率为13.2%±0.6%;CD34阳性细胞表达率为1.2%±0.5%.诱导7 d后,与对照组长梭形细胞相比,ATRA组部分细胞为圆形、短梭形单层排列;联合诱导组的大部分细胞为圆形、短梭形,细胞密集处呈鹅卵石样排列.碱性磷酸酶染色显示,对照组细胞为阴性;ATRA组部分细胞阳性;联合诱导组阳性细胞数明显增多.免疫细胞化学显示,ATRA组和联合诱导组细胞cytokeratin-18阳性表达率分别为29.47%±1.08%和47.52%±2.13%,显著高于对照组(P<0.05);E-cadherin阳性表达率分别为14.88%±2.46%和36.15%±1.13%,也显著高于对照组(P<0.05).结论在体外模拟的急性肾衰竭微环境中加入ATRA可诱导MSC部分分化为肾小管上皮样细胞.联合EGF、BMP-7共同诱导能进一步促进MSC向肾小管上皮样细胞分化.
목적 관찰불동조건하골수간충질간세포(MSC)체외유도분화위신소관상피양세포적차이.방법 추취SD대서적골수,경밀도제도리심분리,연합첩벽사선법획취순화적MSC.이류식세포의감정간충질간세포표면표지.취확증3대적MSC분조배양:(1)대조조:용함태우혈청배양기;(2)전반식유갑산(ATRA)조:태우혈청+결혈재관주신장균장상청+ATRA;(3)연합유도조:태우혈청+결혈재관주신장균장상청+ATRA+표피생장인자(EGF)+골형성단백(BMP-7).유도7 d후,도치현미경하관찰세포형태변화;화학염색검측세포감성린산매표체;면역세포화학법검측세포각단백18(cytokeratin-18)、E개점단백(E-cadherin)적표체.결과 류식세포의현시,체외분리배양적제3대MSC,CD44양성세포표체솔위97.8%±0.9%;CD90양성세포표체솔위96.8%±1.4%;CD29양성세포표체솔위97.6%±2.4%;이CD11b/c양성세포표체솔위13.2%±0.6%;CD34양성세포표체솔위1.2%±0.5%.유도7 d후,여대조조장사형세포상비,ATRA조부분세포위원형、단사형단층배렬;연합유도조적대부분세포위원형、단사형,세포밀집처정아란석양배렬.감성린산매염색현시,대조조세포위음성;ATRA조부분세포양성;연합유도조양성세포수명현증다.면역세포화학현시,ATRA조화연합유도조세포cytokeratin-18양성표체솔분별위29.47%±1.08%화47.52%±2.13%,현저고우대조조(P<0.05);E-cadherin양성표체솔분별위14.88%±2.46%화36.15%±1.13%,야현저고우대조조(P<0.05).결론 재체외모의적급성신쇠갈미배경중가입ATRA가유도MSC부분분화위신소관상피양세포.연합EGF、BMP-7공동유도능진일보촉진MSC향신소관상피양세포분화.
Objective To investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to renal tubular epithelial-like cells under different conditions. Methods MSCs were obtained from rat marrow. MSCs were isolated by gradient density centrifugation and plastic adherence and then purified. Surface markers were identified with flow cytometry after amplification in vitro. The purified MSCs of the third passage were cultured respectively as follows: (1) control group: DMEM medium with fetal bovine serum(FBS). (2) all-trans retinoic acid (ATRA) group: DMEM medium with FBS, ATRA and ischemic reperfusion-injured kidney tissue homogenate. (3)combination group: DMEM medium with FBS, ATRA, ischemic reperfusion-injured kidney tissue homogenate, epidermal growth factor (EGF) and bone morphogenetic protein 7 (BMP-7). After 7 days, the MSCs were collected for alkaline phosphatase (AKP) staining, cytokeratin-18 and E-cadherin immunocytochemical analysis. Results The positive rates of the third passage MSCs in CD44, CD90 and CD29 were 97.8%±0.9%, 96.8%±1.4% and 97.6%±2.4%,respectively, but in CD11b/c and CD34 were only 13.2%±0.6% and 1.2%±0.5%. The MSCs in control group were spindle. The MSCs in ATRA group were round and elliptic. The MSCs in combination group became cobblestone-like cells after 7 days. AKP staining showed that tubular epithelial-like cells from MSCs in control group were negative, some above cells in ATRA group were positive and number of above cells increased in combination group. Compared with negative control group, the ratios of cytokeratin-18 positive cells in ATRA group and combination group were respectively increassed by 29.47%±1.08% and 47.52%±2.13% (all P<0.05), the ratios of E-cadherin positive cells in ATRA group and combination group were respectively increased by 14.88%±2.46% and 36.15%±1.13% (all P<0.05). Conclusion MSCs may differentiate by renal tubular epithelial-like cells under the induction of ischemic reperfusion-injured kidney tissue homogenate and ATRA in vitro, which are further differentiated under the combined induction of EGF and BMP-7.