CAJ | 학술논문

目的探讨4种炎性因子在真菌性小鼠角膜炎发病中的表达和作用.方法实验研究.240只BALB/c小鼠采用随机数字表法分为4组,分别为茄病镰刀菌感染组、烟曲霉菌感染组、白色念珠菌感染组及损伤对照组,每组60只.建立小鼠真菌性角膜炎模型.分别于术后1、3、5及7 d,于裂隙灯显微镜下观察角膜疾病的发展,病理学检查病变角膜的炎症反应和菌丝生长情况,反转录聚合酶链反应(RT-PCR)和酶联免疫吸附试验(ELISA)检测巨噬细胞炎性蛋白2(MIP-2)、细胞因子诱生性中性粒细胞趋化物(KC)、白细胞介素(IL)1β和IL-6炎性因子的mRNA和蛋白质表达水平.对临床评分、RT-PCR和ELISA结果比较应用参数检验中方差分析(one-way ANOVA),进一步两两比较采用LSD检验法.结果随时间变化各感染组小鼠角膜病变和炎症反应出现先增强到术后7 d开始减弱的趋势.RT-PCR结果显示,MIP-2和IL-1β的表达在烟曲霉菌感染组最强,KC在白色念珠菌感染组最强,IL-6在茄病镰刀菌感染组最强.各感染组中IL-1β和KC的表达强于IL-6和MIP-2.MIP-2、KC及IL-1β在茄病镰刀菌感染组、烟曲霉菌感染组及白色念珠菌感染组的表达高峰分别为术后5、1及3 d,IL-6在茄病镰刀菌和烟曲霉菌感染组的高峰为术后1和7 d,在白色念珠菌感染组的高峰为术后1和5 d.对各实验组同一因子mRNA的表达强度的相对值进行相互比较分析显示,各组间差异均有统计学意义(MIP-2:F=1675.339,P<0.01;KC:F=730.267,P<0.01;IL-1β:F=297.106,P<0.01;IL-6:F=174.513,P<0.01).ELISA结果显示,各感染组的4种因子蛋白表达量从高到低依次为IL-1β、MIP-2、KC及IL-6.不同实验组间和相同实验组中每种因子的蛋白表达随时间变化规律与4种因子的mRNA表达规律吻合.对各实验组同一因子的蛋白表达水平进行相互比较分析表明,各组间差异均有统计学意义(MIP-2:F=2871.736,P<0.01;KC:F=886.598,P<0.01;IL-1β:F=3595.488,P<0.01;IL-6:F=89.225,P<0.01).结论 MIP-2、KC和IL-1β的mRNA和蛋白表达水平与真菌性角膜炎的病变程度和炎症反应密切相关,其中MIP-2和IL-1β的持续高水平表达可能在真菌性角膜炎病变损害中起着重要的作用. 目的探討4種炎性因子在真菌性小鼠角膜炎髮病中的錶達和作用.方法實驗研究.240隻BALB/c小鼠採用隨機數字錶法分為4組,分彆為茄病鐮刀菌感染組、煙麯黴菌感染組、白色唸珠菌感染組及損傷對照組,每組60隻.建立小鼠真菌性角膜炎模型.分彆于術後1、3、5及7 d,于裂隙燈顯微鏡下觀察角膜疾病的髮展,病理學檢查病變角膜的炎癥反應和菌絲生長情況,反轉錄聚閤酶鏈反應(RT-PCR)和酶聯免疫吸附試驗(ELISA)檢測巨噬細胞炎性蛋白2(MIP-2)、細胞因子誘生性中性粒細胞趨化物(KC)、白細胞介素(IL)1β和IL-6炎性因子的mRNA和蛋白質錶達水平.對臨床評分、RT-PCR和ELISA結果比較應用參數檢驗中方差分析(one-way ANOVA),進一步兩兩比較採用LSD檢驗法.結果隨時間變化各感染組小鼠角膜病變和炎癥反應齣現先增彊到術後7 d開始減弱的趨勢.RT-PCR結果顯示,MIP-2和IL-1β的錶達在煙麯黴菌感染組最彊,KC在白色唸珠菌感染組最彊,IL-6在茄病鐮刀菌感染組最彊.各感染組中IL-1β和KC的錶達彊于IL-6和MIP-2.MIP-2、KC及IL-1β在茄病鐮刀菌感染組、煙麯黴菌感染組及白色唸珠菌感染組的錶達高峰分彆為術後5、1及3 d,IL-6在茄病鐮刀菌和煙麯黴菌感染組的高峰為術後1和7 d,在白色唸珠菌感染組的高峰為術後1和5 d.對各實驗組同一因子mRNA的錶達彊度的相對值進行相互比較分析顯示,各組間差異均有統計學意義(MIP-2:F=1675.339,P<0.01;KC:F=730.267,P<0.01;IL-1β:F=297.106,P<0.01;IL-6:F=174.513,P<0.01).ELISA結果顯示,各感染組的4種因子蛋白錶達量從高到低依次為IL-1β、MIP-2、KC及IL-6.不同實驗組間和相同實驗組中每種因子的蛋白錶達隨時間變化規律與4種因子的mRNA錶達規律吻閤.對各實驗組同一因子的蛋白錶達水平進行相互比較分析錶明,各組間差異均有統計學意義(MIP-2:F=2871.736,P<0.01;KC:F=886.598,P<0.01;IL-1β:F=3595.488,P<0.01;IL-6:F=89.225,P<0.01).結論 MIP-2、KC和IL-1β的mRNA和蛋白錶達水平與真菌性角膜炎的病變程度和炎癥反應密切相關,其中MIP-2和IL-1β的持續高水平錶達可能在真菌性角膜炎病變損害中起著重要的作用.
목적 탐토4충염성인자재진균성소서각막염발병중적표체화작용.방법 실험연구.240지BALB/c소서채용수궤수자표법분위4조,분별위가병렴도균감염조、연곡매균감염조、백색념주균감염조급손상대조조,매조60지.건립소서진균성각막염모형.분별우술후1、3、5급7 d,우렬극등현미경하관찰각막질병적발전,병이학검사병변각막적염증반응화균사생장정황,반전록취합매련반응(RT-PCR)화매련면역흡부시험(ELISA)검측거서세포염성단백2(MIP-2)、세포인자유생성중성립세포추화물(KC)、백세포개소(IL)1β화IL-6염성인자적mRNA화단백질표체수평.대림상평분、RT-PCR화ELISA결과비교응용삼수검험중방차분석(one-way ANOVA),진일보량량비교채용LSD검험법.결과 수시간변화각감염조소서각막병변화염증반응출현선증강도술후7 d개시감약적추세.RT-PCR결과현시,MIP-2화IL-1β적표체재연곡매균감염조최강,KC재백색념주균감염조최강,IL-6재가병렴도균감염조최강.각감염조중IL-1β화KC적표체강우IL-6화MIP-2.MIP-2、KC급IL-1β재가병렴도균감염조、연곡매균감염조급백색념주균감염조적표체고봉분별위술후5、1급3 d,IL-6재가병렴도균화연곡매균감염조적고봉위술후1화7 d,재백색념주균감염조적고봉위술후1화5 d.대각실험조동일인자mRNA적표체강도적상대치진행상호비교분석현시,각조간차이균유통계학의의(MIP-2:F=1675.339,P<0.01;KC:F=730.267,P<0.01;IL-1β:F=297.106,P<0.01;IL-6:F=174.513,P<0.01).ELISA결과현시,각감염조적4충인자단백표체량종고도저의차위IL-1β、MIP-2、KC급IL-6.불동실험조간화상동실험조중매충인자적단백표체수시간변화규률여4충인자적mRNA표체규률문합.대각실험조동일인자적단백표체수평진행상호비교분석표명,각조간차이균유통계학의의(MIP-2:F=2871.736,P<0.01;KC:F=886.598,P<0.01;IL-1β:F=3595.488,P<0.01;IL-6:F=89.225,P<0.01).결론 MIP-2、KC화IL-1β적mRNA화단백표체수평여진균성각막염적병변정도화염증반응밀절상관,기중MIP-2화IL-1β적지속고수평표체가능재진균성각막염병변손해중기착중요적작용.
Objective To investigate the expression, contribution and regulation of predominant inflammatory cytokines in the inflammation process of in a mouse model of keratomycosis. Methods It was an exprimental study. Two hundred forty inbred BALB/c mice were randomly divided into four groups (injury control group, F. solani infected group, A. fumigatus infected group and C. albicans infected group) and establishment fungal keratitis was performed. At 1, 3, 5, and 7 days after infection, the photographs of their corneas were taken using the slit lamp and the digital camera system. The presence of inflammatory cells and fungal hyphae were observed by HE and PAS staining. Four inflammatory cytokines in infectious corneas, including MIP-2, KC, IL-1β, and IL-6, were detected respectively for expression of mRNA and protein levels, using RT-PCR and ELISA. One-way ANOVA was used to test the comparisons of the clinical scoring, RT-PCR, and ELISA, and LSD was used to test multiple comparisons. Results The course and inflammatory reaction of corneal tissue in all infected group increased at day 3 and 5 and gradually decreased after day 7 with time. The RT-PCR results showed, the mRNA expression levels of MIP-2 and IL-1β were the highest in A. fumigatus infected group, and the expression levels of KC was the highest in C. albicans infected group. Additionally, the expression levels of IL-6 were found to be the highest in F.solani infected group. The mRNA expression levels of IL-1β and KC in each experimental group were higher than those of IL-6 and MIP-2. The peak expression levels of MIP-2, KC, and IL-1β occurred at day 5, day 1, and day 3 in F. solani infected group, A. fumigatus infected group and C. albicans infected group,respectively. However, the peak expression level of IL-6 occurred at day 1 and 7, day 1 and 7, and day 1 and 5 in the F. solani, A. fumigatus, and C. albicans groups, respectively. The difference of mRNA expression of each cytokine had statistically significant between each groups(MIP-2: F = 1675.339, P <0.01; KC: F=730.267, P<0.01; IL-1β: F =297. 106, P<0.01; IL-6: F=174.513, P<0.01). The protein expression levels of the four cytokines in the infected groups from high to low in turn were IL-1β,MIP-2, KC and IL-6. The trend of protein expression of the four cytokines at each time point was the same as the cytokine mRNA expression in different experimental groups as well as within the same group. The difference of protein expression of each cytokine had statistically significant between each groups ( MIP-2:F=2871.736, P<0.01; KC:F=886.598, P<0.01; IL-Iβ: F=3595.488, P<0.01; IL-6: F=89.225, P < 0.01 ). Conclusions The expression levels of MIP-2, KC and IL-1β are closely related to the performance of the disease severity and inflammatory reaction of FK. The expression of MIP-2 and IL-1β may play important role in the pathogenesis of FK.