CAJ | 학술논문

目的:研究pcDU6载体质粒介导的转化生长因子β(TGF-β1)短发夹RNA(pcDU6-A1-A2和pcDU6-B1-B2)对人血清白蛋白(HAS)致人肾小管上皮细胞(HK2细胞)增殖,TGF-β1、结缔组织生长因子(CTGF)和纤连蛋白(FN)表达的影响,并探讨HAS刺激HK2细胞增殖及CTGF和FN基因过表达是否通过TGF-β1介导.方法:构建TGF-β1短发夹RNA的pcDU6载体质粒,体外培养HK2细胞株.采用脂质体转染将表达TGF-β1 shRNA的pcDU6质粒载体(pcDU6-A1-A2和pcDU6-B1-B2)分别导入试验组细胞.使用HAS(5 g/L)刺激HK2细胞12 h或24 h.用四甲基偶氮唑盐比色法测定细胞增殖水平;逆转录多聚酶链反应半定量分析HK2细胞中TGF-β1,CTGF和FN mRNA的表达水平;双抗夹心酶联免疫吸附法检测HK2细胞培养液中TGF-β1及FN蛋白质水平.结果:HAS对HK2细胞增殖在5 g/L作用24 h最明显;HK2细胞在HAS刺激下可明显上调TGF-β1,CTGF及FN mRNA的表达,培养液中TGF-β1和FN的蛋白质含量亦明显升高 (P＜0.05).与pcDU6空载体组比较,pcDU6载体质粒介导的TGF-β1 shRNA干扰组TGF-β1, CTGF及FN mRNA的表达明显下调(P＜0.05).TGF-β1 shRNA转染HK2细胞后12 h或24 h,细胞培养液中TGF-β1和FN蛋白质含量明显下降,HK2细胞增殖被部分抑制(P＜0.05).TGF-β1shRNA干扰组组间比较以及pcDU6空载体转染组与HAS刺激组比较,差异均无统计学意义(P＞0.05).结论:pcDU6载体质粒介导的TGF-β1shRNA能够明显抑制HAS刺激下HK2细胞增殖以及TGF-β1,CTGF和FN基因的表达,HAS刺激HK2细胞增殖及CTGF和FN基因的过表达可能通过TGF-β1介导.
목적:연구pcDU6재체질립개도적전화생장인자β(TGF-β1)단발협RNA(pcDU6-A1-A2화pcDU6-B1-B2)대인혈청백단백(HAS)치인신소관상피세포(HK2세포)증식,TGF-β1、결체조직생장인자(CTGF)화섬련단백(FN)표체적영향,병탐토HAS자격HK2세포증식급CTGF화FN기인과표체시부통과TGF-β1개도.방법:구건TGF-β1단발협RNA적pcDU6재체질립,체외배양HK2세포주.채용지질체전염장표체TGF-β1 shRNA적pcDU6질립재체(pcDU6-A1-A2화pcDU6-B1-B2)분별도입시험조세포.사용HAS(5 g/L)자격HK2세포12 h혹24 h.용사갑기우담서염비색법측정세포증식수평;역전록다취매련반응반정량분석HK2세포중TGF-β1,CTGF화FN mRNA적표체수평;쌍항협심매련면역흡부법검측HK2세포배양액중TGF-β1급FN단백질수평.결과:HAS대HK2세포증식재5 g/L작용24 h최명현;HK2세포재HAS자격하가명현상조TGF-β1,CTGF급FN mRNA적표체,배양액중TGF-β1화FN적단백질함량역명현승고 (P＜0.05).여pcDU6공재체조비교,pcDU6재체질립개도적TGF-β1 shRNA간우조TGF-β1, CTGF급FN mRNA적표체명현하조(P＜0.05).TGF-β1 shRNA전염HK2세포후12 h혹24 h,세포배양액중TGF-β1화FN단백질함량명현하강,HK2세포증식피부분억제(P＜0.05).TGF-β1shRNA간우조조간비교이급pcDU6공재체전염조여HAS자격조비교,차이균무통계학의의(P＞0.05).결론:pcDU6재체질립개도적TGF-β1shRNA능구명현억제HAS자격하HK2세포증식이급TGF-β1,CTGF화FN기인적표체,HAS자격HK2세포증식급CTGF화FN기인적과표체가능통과TGF-β1개도.
Objective To determine the effect of 2 transforming growth factor β1 (TGF-β1) short hairpin RNA (shRNA) expression plasmids (pcDU6-A1-A2 and pcDU6-B1-B2) on proliferation, TGF-β1, connective tissue growth factor (CTGF), and fibronectin (FN) expression induced by human serum albumin (HAS) in HK2 cells. Methods A vector plasmid containing the TGF-β1 shRNA was generated. An HK2 cell line was used in the study. The 2 TGF-β1 shRNA expression plasmids were transfected into cultured HK2 cells by lipofectamine 2000. Cellular proliferation was assessed by tetrazolium salt colorimetry. The semi-quantitative reverse transcriptive PCR was performed to detect the expression of TGF-β1,CTGF, and FN mRNA. Levels of TGF-β1 and FN protein were measured with a sandwich enzyme-linked immunosorbent assay. Results After treating with 5 g/L HAS for 24 hours in HK2 cells, cellular proliferating capacity increased significantly (P＜0.05). The expression levels of TGF-β1, CTGF, and FN mRNA were upregulated in HK2 cells stimulated by 5 g/L HAS, and levels of TGF-β1 and FN protein in the culture supernatant increased (P＜0.05). The introduction of pcDU6-A1-A2 and pcDU6-B1-B2 resulted in significant reduction of cellular proliferation activity, and the expression levels of TGF-β1, CTGF, and FN mRNA were downregulated (P＜0.05). Levels of TGF-β1 and FN protein in the culture supernatant decreased (P＜0.05) after 12 or 24 hours of TGF-β1 shRNA transfection into HK2 cells There was no significant difference in the expression levels of TGF-β1, CTGF, and FN mRNA between the 2 pcDU6 vector plasmid mediated TGF-β1 shRNA groups (P＞0.05). Conclusion pcDU6 vector plasmid mediated TGF-β1 shRNAs could obviously inhibit the expression levels of TGF-β1, CTGF, FN and cellular proliferation stimulated by HAS in HK2 cells, which may be related to the mediation of TGF-β1.