CAJ | 학술논문

目的探讨肠三叶因子(ITF)保护甲氨喋呤(MTX)所致肠黏膜损伤的可能机制.方法 IEC-6细胞分为仅予培养液的空白对照组、经MTX处理的MTX对照组、仅予ITF预处理的ITF对照组和经不同浓度ITF预处理后再予MTX的ITF实验组.RT-PCR法检测E-cadherinmRNA表达变化.明胶酶谱法检测基质金属蛋白酶(MMP)-2和MMP-9活性.分光光度计法检测Caspase-3活性.细胞计数试剂盒-8法检测细胞增殖情况.改良的Boyden趋化小室法观测细胞迁移情况.结果 0.1 mg/mlITF实验组、1 mg/ml ITF实验组E-cadherin mRNA的相对表达量(分别为0.538±0.109和0.528±0.132)与MTX对照组(0.763±0.139)比较差异有统计学意义(P值分别=0.021和0.025).各组间MMP-2和MMP-9活性差异均无统计学意义(P值均＞0.05).0.1 mg/ml ITF实验组和1.0 mg/ml ITF实验组Caspase-3活性分别为0.077±0.009和0.044±0.009,较MTX对照组(0.090±0.011)下降(P值分别=0.032和0.005).各ITF实验组的细胞增殖情况与MTX对照组比较差异均无统计学意义(P值分别=0.132、0.150、0.114、0.367).1.0 mg/ml ITF实验组、0.1 mg/mlITF实验组Boyden小室穿膜细胞数[分别为(224.33±34.67)和(143.56±25.23)个]与MTX对照组[(50.11±7.77)个]比较差异均有统计学意义(P值均＜0.05),两实验组间亦差异有统计学意义(P＜0.05).结论 ITF的保护作用与其促细胞迁移和抗细胞凋亡功能有关,且不会导致明显的细胞增殖.其促细胞迁移功能可能与下调E-cadherin基因转录水平有关.
목적 탐토장삼협인자(ITF)보호갑안첩령(MTX)소치장점막손상적가능궤제.방법 IEC-6세포분위부여배양액적공백대조조、경MTX처리적MTX대조조、부여ITF예처리적ITF대조조화경불동농도ITF예처리후재여MTX적ITF실험조.RT-PCR법검측E-cadherinmRNA표체변화.명효매보법검측기질금속단백매(MMP)-2화MMP-9활성.분광광도계법검측Caspase-3활성.세포계수시제합-8법검측세포증식정황.개량적Boyden추화소실법관측세포천이정황.결과 0.1 mg/mlITF실험조、1 mg/ml ITF실험조E-cadherin mRNA적상대표체량(분별위0.538±0.109화0.528±0.132)여MTX대조조(0.763±0.139)비교차이유통계학의의(P치분별=0.021화0.025).각조간MMP-2화MMP-9활성차이균무통계학의의(P치균＞0.05).0.1 mg/ml ITF실험조화1.0 mg/ml ITF실험조Caspase-3활성분별위0.077±0.009화0.044±0.009,교MTX대조조(0.090±0.011)하강(P치분별=0.032화0.005).각ITF실험조적세포증식정황여MTX대조조비교차이균무통계학의의(P치분별=0.132、0.150、0.114、0.367).1.0 mg/ml ITF실험조、0.1 mg/mlITF실험조Boyden소실천막세포수[분별위(224.33±34.67)화(143.56±25.23)개]여MTX대조조[(50.11±7.77)개]비교차이균유통계학의의(P치균＜0.05),량실험조간역차이유통계학의의(P＜0.05).결론 ITF적보호작용여기촉세포천이화항세포조망공능유관,차불회도치명현적세포증식.기촉세포천이공능가능여하조E-cadherin기인전록수평유관.
Objective To investigate the potential mechanism of intestinal trefoil factor(ITF)against methotrexate (MTX)- induced injury in intestinal mucosa. Methods Cultured IEC-6 cells were divided into groups as follows: blank group, MTX treated group, ITF treated group and experimental group treated with gradient concentrations of ITF plus MTX. Expression of E-cadherin mRNA was determined by Real-Time polymerase chain reaciton (RT- PCR). The activity of matrix metalloproteinase(MMP)-2 and MMP-9 was measured by gelatin zymogramphy. Caspases-3 activity was measured by colorimetric assay. Cell proliferation was assessed by cell counting kit-8 (CCK-8)assay. Migration of IEC-6 in vitro was observed using modified Boyden chamber assay. Results The expression of E-cadherin mRNA in experimental group (treated with 0.1 mg/ml or 1 mg/ml of ITF) was significantly down-regulated (0. 538±0. 109 or 0. 528±0. 132, respectively) in comparison with MTX treated group (0. 763±0. 139) with significant difference (P=0. 021 or P=0. 025, respectively). There was no significant difference in activity of MMP-2 and MMP-9 among groups (P＞0. 05). When compared with MTX treated group (0. 090 ±0. 011 ), the activity of Caspase3 in experimental group (treated with 0. 1 mg/ml or 1 mg/ml of ITF) was significantly decreased (0. 077±0. 009, P=0. 032 or 0. 044±0. 009,P=0. 005, respectively). There was no statistical difference in cell proliferation between experimental group (treated with 1 μg/ml, 0.01 mg/ml, 0. 1 mg/ml or 1.0 mg/ml of ITF) and MTX treated group (P=0. 132,0. 150,0. 114 or 0. 367, respectivley). More migratory cells attached to the bottom surface of the membrane in experiment group (treated with 0. 1 mg/ml or 1 mg/ml of ITF) in comparison with MTX treated group (P ＜0. 001 ). Moreover, more migratory cells were found in experimental group treated with 1.0 mg/ml of ITF than those in group treated with 0. 1 mg/ml of ITF (P＜0. 001). Conclusions Without cell proliferation, the protective effect of ITF is related to its functions of promoting cell migration and inhibiting cell apoptosis, which may down-regulate expression of E-cadherin mRNA.