CAJ | 학술논문

目的：探讨不同浓度改性甲壳素对体外培养人成纤维细胞的影响。方法使用含0、10、100、500、1000、2000、5000 mg/L改性甲壳素的培养基体外培养人成纤维细胞株HFF，培养0、1、2、4、8、14 d后进行相应检测。采用MTT法评价改性甲壳素的细胞毒性，酶联免疫吸附法（ELISA）检测细胞培养上清Ⅰ型胶原和Ⅲ型胶原含量，反转录（RT）?PCR、免疫印迹法检测Ⅰ型胶原、增殖细胞核抗原（PCNA）mRNA和蛋白的表达。结果培养0、1、2、4、8、14 d MTT法检测显示，与对照组（0 mg/L改性甲壳素）比较，浓度为10、100 mg/L的改性甲壳素对HFF细胞无明显细胞增殖抑制作用；而浓度为500、1000、2000、5000 mg/L的改性甲壳素对HFF细胞增殖具有明显抑制作用，且具有时间和浓度依赖性（均P<0.05）。ELISA检测显示，500、1000、2000、5000 mg/L的改性甲壳素处理HFF细胞2 d，各组细胞分泌Ⅰ型胶原和Ⅲ型胶原含量均显著减少，与对照组比较差异均有统计学意义（均P<0.05）。RT?PCR和免疫印迹检测显示，500 mg/L改性甲壳素处理HFF细胞0~14 d，随着培养时间延长Ⅰ型胶原、PCNA mRNA和蛋白表达均逐渐降低（均P<0.05）。结论改性甲壳素可明显抑制成纤维细胞增殖及Ⅰ型胶原、PCNA表达，有望在疤痕的防治中发挥作用。
목적：탐토불동농도개성갑각소대체외배양인성섬유세포적영향。방법사용함0、10、100、500、1000、2000、5000 mg/L개성갑각소적배양기체외배양인성섬유세포주HFF，배양0、1、2、4、8、14 d후진행상응검측。채용MTT법평개개성갑각소적세포독성，매련면역흡부법（ELISA）검측세포배양상청Ⅰ형효원화Ⅲ형효원함량，반전록（RT）?PCR、면역인적법검측Ⅰ형효원、증식세포핵항원（PCNA）mRNA화단백적표체。결과배양0、1、2、4、8、14 d MTT법검측현시，여대조조（0 mg/L개성갑각소）비교，농도위10、100 mg/L적개성갑각소대HFF세포무명현세포증식억제작용；이농도위500、1000、2000、5000 mg/L적개성갑각소대HFF세포증식구유명현억제작용，차구유시간화농도의뢰성（균P<0.05）。ELISA검측현시，500、1000、2000、5000 mg/L적개성갑각소처리HFF세포2 d，각조세포분비Ⅰ형효원화Ⅲ형효원함량균현저감소，여대조조비교차이균유통계학의의（균P<0.05）。RT?PCR화면역인적검측현시，500 mg/L개성갑각소처리HFF세포0~14 d，수착배양시간연장Ⅰ형효원、PCNA mRNA화단백표체균축점강저（균P<0.05）。결론개성갑각소가명현억제성섬유세포증식급Ⅰ형효원、PCNA표체，유망재파흔적방치중발휘작용。
Objective To investigate the effects of different concentrations of modified chitin on human fibroblasts cultured in?vitro. Methods Human fibroblast cell line HFF was cultured in medium containing 0,10,100,500,1 000,2 000,5 000 mg/L modified chitin,and was evaluated at 0,1,2,4,8 and 14 days of the culture. MTT method was used to evaluation the cytotoxicity of modified chitin,enzyme?linked immunosorbent assay(ELISA)was used to detect the levels of types Ⅰ andⅢ collagen in the cell culture supernatant,and the reverse transcription(RT)?PCR and Western blot was used to measure the mRNA and protein expressions of typeⅠcollagen and proliferating cell nuclear antigen(PCNA). Results MTT assay at days 0,1,2,4,8 and 14 of culture showed that modified chitin at the concentrations of 10,100 mg/L did not exhibit any inhibitory effects on proliferation of HFF cells compared with the control group (0 mg/L modified chitin),while modified chitin at the concentrations of 500,1 000,2 000 and 5 000 mg/L significantly inhibited the HFF cell proliferation in a time? and concentration?dependent manner (all P<0.05). As shown by ELISA assay,treatment of HFF cells with 500,1 000,2 000 and 5 000 mg/L modified chitin for 2 d resulted in significantly reduced production of typesⅠandⅢcollagen in each group of cells compared with the control group(all P<0.05). RT?PCR and Western blot analysis showed that mRNA and protein expression levels of typeⅠcollagen and PCNA gradually decreased over the 14?day treatment of HFF cells with 500 mg/L modified chitin (all P<0.05). Conclusion Modified chitin can inhibit fibroblast proliferation and expression of type Ⅰ collagen and PCNA in the fibroblast,which may be useful in the prevention and treatment of scarring.