摘要
目的构建基质金属蛋白酶-12(MMP-12)基因小干扰RNA片段(siRNA)表达质粒,探讨MMP-12基因在矽肺纤维化发展过程中的功能。方法利用RNA干扰技术,将构建的MMP-12-siRNA表达质粒转染大鼠肺成纤维细胞(FB),筛选出最佳干扰基因序列。实验分为3组:对照组(C),加未经SiO_2粉尘刺激的肺泡巨噬细胞(AM)培养上清液;SiO_2刺激组(S),加经SiO_2粉尘刺激的AM培养上清液;转染组(T),首先将筛选出的含最佳干扰基因序列的质粒转染FB,基因沉默后,再加经SiO_2粉尘刺激的AM培养上清液。运用免疫细胞化学法检测各组中I型及Ⅲ型胶原蛋白的表达情况。结果经条件上清培养液刺激后,S组FB中各时段I型胶原及Ⅲ型胶原表达均比C组增加,S组I型胶原蛋白为0.360 8±0.006 9~0.694 3±0.007 3,C组为0.240 9±0.005 8~0.257 8±0.009 0,差异均有统计学意义;Ⅲ型胶原蛋白为0.198 9±0.027 8~0.412 9±0.010 8,差异亦均有统计学意义(P<0.05或P<0.01);T组FB中I型胶原及Ⅲ型胶原的表达均比S组明显减少,差异有统计学意义(P<0.01)。结论利用RNA干扰技术将MMP-12-siRNA表达质粒转染FB,可沉默MMP-12基因的表达,并可减少I及Ⅲ型胶原蛋白的沉积,证实MMP-12基因在矽肺纤维化的发展中起重要作用。
[Objective]To construct small interfering RNA（si RNA）expression plasmids of matrix matalloproteinases-12（MMP-12）gene,explore the functions of MMP-12 in development process of pulmonary fibrosis in silicosis.[Methods] MMP-12-si RNA expressing plasmid were constructed and stably transfected into rat lung fibroblast（FB） by RNA interfering（RNAi） technology,to find out the si RNA which had the best inhibitory effect. The experiment was divided into three groups. Control group（C）：alveolar macrophage（AM） supernatant of the C group was without SiO_2 dust conditions to stimulate incubated with rat FB. SiO_2group（S）： AM supernatant was with SiO_2 dust conditions to stimulate incubated with rat FB. Transfection group（T）： the plasmid which had the best inhibitory effect was transfected into FB,then added to AM supernatant stimulated by SiO_2 dust after Gene silencing. The expressions of collagen typeⅠand collagen type Ⅲ were detected by immunocytochemistry.[Results]After the stimulation by supernatant,the expressions of collagen type I and collagen type Ⅲ in S group were higher than those in C group.The expression of collagen type I in S group and C group was 0.360 8 ±0.006 9-0.694 3±0.007 3 and 0.240 9±0.005 8-0.257 8±0.009 0 respectively,which the difference was statistically significant,while the expression of collagen type Ⅲ was 0.198 9±0.027 8-0.412 9±0.010 8,and the difference was statistically significant（P〈0.05 or P〈0.01）. The expressions of collagen type I and collagen type Ⅲ in T group were lower than those in S group,and the difference was statistically significant（P 0.01）.[Conclusion]MMP-12-siRNA expressing plasmid transfected into FB can make MMP-12 gene silencing by RNAi technology and reduce the ex-pression of collagen type I and collagen type Ⅲ,indicating that MMP-12 plays an important role in the pulmonary fibrosis process of silicosis.
作者
尹丹丹
唐军栋
高路杨
景友玲
马小兵
张云香
王献华
YIN Dan-dan TANG Jun-dong GAO Lu-yang JING You-ling MA Xiao-bing ZHANG Yun-xiang WANG Xian-hua(Pathology Department, Welfang People ＇s Hospital, Weifang Shandong,261041, China Ji Tang College College of Basic Medicine ,North China University of Science and Technology, Tangshan Hebei, 063000, China)
出处
《职业与健康》
CAS
2017年第16期2193-2197,共5页
Occupation and Health
基金
河北省自然科学基金资助项目(C2008001007)
河北省科学技术研究与发展计划项目(09276199D)
关键词
矽肺纤维化
成纤维细胞
基质金属蛋白酶-12
RNA干扰技术
胶原
Pulmonary fibrosis of silicosis
Fibroblast
Matrix matalloproteinases-12
RNA interfering technology
Collagen
作者简介
尹丹丹，女，在读硕士研究生，研究方向为矽肺纤维化分子病理学。
通讯作者：王献华，教授，E-mail：wangxianhua0921@163．com
