中华皮肤科杂志
中華皮膚科雜誌
중화피부과잡지
Chinese Journal of Dermatology
2015年
11期
801-806
,共6页
康玉英%孙彩虹%鞠梅%陈崑%顾恒
康玉英%孫綵虹%鞠梅%陳崑%顧恆
강옥영%손채홍%국매%진곤%고항
白细胞,单核%成纤维细胞%基质金属蛋白酶类%细胞增殖%植物血凝素类
白細胞,單覈%成纖維細胞%基質金屬蛋白酶類%細胞增殖%植物血凝素類
백세포,단핵%성섬유세포%기질금속단백매류%세포증식%식물혈응소류
Leukocytes,mononuclear%Fibroblasts%Matrix metalloproteinases%Cell proliferation%Phytohemagglutinins
目的 研究不同活化剂对外周血单一核细胞(PBMC)产生基质金属蛋白酶(MMP)的影响以及PBMC活化后培养上清液[称为条件培养基(CM)]对培养的皮肤成纤维细胞增殖活性、产生MMP的影响.方法 抽取、分离健康人PBMC,分为植物血凝素(PHA)组、双抗体组、对照组,分别用活化剂PHA、CD3和CD28双抗体、含10％胎牛血清的RPMI 1640培养基处理细胞,72 h后收集三组CM,并将获得的CM按不同比例稀释后作用于培养的成纤维细胞,以不加CM作为对照组,培养48或24 h.采用噻唑蓝法检测细胞增殖能力,半定量反转录(RT)-PCR法检测细胞MMP-1、MMP-3、MMP-9 mRNA表达水平,酶联免疫吸附实验(ELISA)检测上清液中白介素(IL)-6、MMP-1、MMP-3、MMP-9蛋白含量.采用单因素方差分析、Tukey HSD检验、Games-Howell检验等方法进行统计学分析.结果 与对照组相比,PHA组PBMC增殖活性及分泌IL-6、MMP-3水平明显增加,差异均有统计学意义(P＜0.05);3组活化后上清液中均未检测到MMP-1、MMP-9蛋白.对照组、双抗体组、PHA组PBMC中MMP-1 mRNA相对表达水平(以靶基因与β肌动蛋白mRNA之比表示)0.083±0.016、0.188±0.030、0.714土0.104,差异有统计学意义(P＜0.05),MMP-3、MMP-9 mRNA均无表达.不同稀释度CM刺激成纤维细胞后上清液中可检测到MMP-3蛋白,MMP-3含量以原液CM中最高,1/10 CM刺激时最低;在相同的稀释度刺激时,上清液中MMP-3含量以PHA刺激组最高,对照刺激组最低;不同CM刺激成纤维细胞后上清液中均未检测到MMP-1、MMP-9蛋白.不同处理组成纤维细胞的增殖能力及MMP-1、MMP-3 mRNA表达差异均无统计学意义(P＞0.05),MMP-9 mRNA无表达.结论 PBMC活化后可表达MMP-1 mRNA并分泌MMP-3蛋白,PBMC活化后上清液不能刺激成纤维细胞MMP-1、MMP-3、MMP-9 mRNA及蛋白表达,提示炎症细胞可能通过自身产生MMP而发挥作用.
目的 研究不同活化劑對外週血單一覈細胞(PBMC)產生基質金屬蛋白酶(MMP)的影響以及PBMC活化後培養上清液[稱為條件培養基(CM)]對培養的皮膚成纖維細胞增殖活性、產生MMP的影響.方法 抽取、分離健康人PBMC,分為植物血凝素(PHA)組、雙抗體組、對照組,分彆用活化劑PHA、CD3和CD28雙抗體、含10％胎牛血清的RPMI 1640培養基處理細胞,72 h後收集三組CM,併將穫得的CM按不同比例稀釋後作用于培養的成纖維細胞,以不加CM作為對照組,培養48或24 h.採用噻唑藍法檢測細胞增殖能力,半定量反轉錄(RT)-PCR法檢測細胞MMP-1、MMP-3、MMP-9 mRNA錶達水平,酶聯免疫吸附實驗(ELISA)檢測上清液中白介素(IL)-6、MMP-1、MMP-3、MMP-9蛋白含量.採用單因素方差分析、Tukey HSD檢驗、Games-Howell檢驗等方法進行統計學分析.結果 與對照組相比,PHA組PBMC增殖活性及分泌IL-6、MMP-3水平明顯增加,差異均有統計學意義(P＜0.05);3組活化後上清液中均未檢測到MMP-1、MMP-9蛋白.對照組、雙抗體組、PHA組PBMC中MMP-1 mRNA相對錶達水平(以靶基因與β肌動蛋白mRNA之比錶示)0.083±0.016、0.188±0.030、0.714土0.104,差異有統計學意義(P＜0.05),MMP-3、MMP-9 mRNA均無錶達.不同稀釋度CM刺激成纖維細胞後上清液中可檢測到MMP-3蛋白,MMP-3含量以原液CM中最高,1/10 CM刺激時最低;在相同的稀釋度刺激時,上清液中MMP-3含量以PHA刺激組最高,對照刺激組最低;不同CM刺激成纖維細胞後上清液中均未檢測到MMP-1、MMP-9蛋白.不同處理組成纖維細胞的增殖能力及MMP-1、MMP-3 mRNA錶達差異均無統計學意義(P＞0.05),MMP-9 mRNA無錶達.結論 PBMC活化後可錶達MMP-1 mRNA併分泌MMP-3蛋白,PBMC活化後上清液不能刺激成纖維細胞MMP-1、MMP-3、MMP-9 mRNA及蛋白錶達,提示炎癥細胞可能通過自身產生MMP而髮揮作用.
목적 연구불동활화제대외주혈단일핵세포(PBMC)산생기질금속단백매(MMP)적영향이급PBMC활화후배양상청액[칭위조건배양기(CM)]대배양적피부성섬유세포증식활성、산생MMP적영향.방법 추취、분리건강인PBMC,분위식물혈응소(PHA)조、쌍항체조、대조조,분별용활화제PHA、CD3화CD28쌍항체、함10％태우혈청적RPMI 1640배양기처리세포,72 h후수집삼조CM,병장획득적CM안불동비례희석후작용우배양적성섬유세포,이불가CM작위대조조,배양48혹24 h.채용새서람법검측세포증식능력,반정량반전록(RT)-PCR법검측세포MMP-1、MMP-3、MMP-9 mRNA표체수평,매련면역흡부실험(ELISA)검측상청액중백개소(IL)-6、MMP-1、MMP-3、MMP-9단백함량.채용단인소방차분석、Tukey HSD검험、Games-Howell검험등방법진행통계학분석.결과 여대조조상비,PHA조PBMC증식활성급분비IL-6、MMP-3수평명현증가,차이균유통계학의의(P＜0.05);3조활화후상청액중균미검측도MMP-1、MMP-9단백.대조조、쌍항체조、PHA조PBMC중MMP-1 mRNA상대표체수평(이파기인여β기동단백mRNA지비표시)0.083±0.016、0.188±0.030、0.714토0.104,차이유통계학의의(P＜0.05),MMP-3、MMP-9 mRNA균무표체.불동희석도CM자격성섬유세포후상청액중가검측도MMP-3단백,MMP-3함량이원액CM중최고,1/10 CM자격시최저;재상동적희석도자격시,상청액중MMP-3함량이PHA자격조최고,대조자격조최저;불동CM자격성섬유세포후상청액중균미검측도MMP-1、MMP-9단백.불동처리조성섬유세포적증식능력급MMP-1、MMP-3 mRNA표체차이균무통계학의의(P＞0.05),MMP-9 mRNA무표체.결론 PBMC활화후가표체MMP-1 mRNA병분비MMP-3단백,PBMC활화후상청액불능자격성섬유세포MMP-1、MMP-3、MMP-9 mRNA급단백표체,제시염증세포가능통과자신산생MMP이발휘작용.
Objective To study the effects of different stimulators on the production of matrix metalloproteinases (MMPs) by peripheral blood mononuclear cells (PBMCs),and to evaluate the effects of the culture supernatant of activated PBMCs,named conditioned media (CM),on the proliferation of and production of MMPs by cultured human fibroblasts.Methods PBMCs were isolated from the venous blood samples of healthy volunteers and divided into three groups to be stimulated by phytohemagglutinin (PHA group),the combination of antibodies against CD3 and CD28 (double-antibody group),or the RPMI 1640 medium containing 10％ fetal calf serum (control group).After 72-hour stimulation,CM was collected from all the three groups,diluted to several different degrees.Cultured human fibroblasts were classified into several groups to be treated with different dilutions of CM from the three groups for 48 or 24 hours,with the fibroblasts untreated with CM serving as the control group.Methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate cellular proliferative activity,semi-quantitative reverse transcription (RT)-PCR to detect the expressions of MMP-1,MMP-3 and MMP-9 mRNAs in cells,and enzyme-linked immunosorbent assay (ELISA) to measure the levels of interleukin (IL)-6,MMP-1,MMP-3 and MMP-9 proteins in the culture supernatant of cells.Statistical analysis was carried out mainly by using one-way analysis of variance (ANOVA),Tukey HSD test,and GamesHowell test.Results Compared with the control group,the PHA group showed increased cellular proliferative activity,IL-6 and MMP-3 protein levels in the culture supernatant of activated PBMCs (all P ＜ 0.05).Significant differences were observed among the PHA group,double-antibody group and control group in the relative mRNA expression level (expressed as the ratio of target mRNA to β-actin mRNA) of MMP-1 in activated PBMCs (0.083 ± 0.016 vs.0.188 ± 0.030 vs.0.714 ± 0.104,F =85.905,P ＜ 0.05),but neither MMP-3 nor MMP-9 mRNA was expressed by activated PBMCs.MMP-3 protein was detectable in the culture supernatant of fibroblasts after the treatment with CM,and the level of MMP-3 protein was highest in that of fibroblasts treated with undiluted CM,and lowest with 1/10 diluted CM;at the same dilutions,the level of MMP-3 protein was highest in the culture supernatant of fibroblasts treated with CM from the PHA group,but lowest with that from the control group.Neither MMP-1 nor MMP-9 protein was detected in the culture supernatant of activated PBMCs or treated fibroblasts.There were no significant differences in cellular proliferative activity of and mRNA expressions of MMP-1 or MMP-3 in fibroblasts among these groups (all P ＞ 0.05),and MMP-9 mRNA expression was undetected in the treated fibroblasts.Conclusions PBMCs can be induced to express MMP-1 mRNA and secret MMP-3 protein after activation.However,the culture supernatant of activated PBMCs has no capacity to stimulate the expressions of MMP-1,MMP-3 and MMP-9 mRNAs or proteins by fibroblasts,suggesting that inflammatory cells may function through self-production of MMPs.
