小鼠皮肤损伤修复过程中白细胞介素1α和白细胞介素1β变化与损伤时间的关系★
李 树1，王 杰2
课题背景：成功应用成纤维细胞生长因子2基因治疗的技术关键步骤是成纤维细胞生长因子2基因的克隆及其真核表达载体的构建。国内外数据库中关于大鼠成纤维细胞生长因子2分子的克隆和及其真核表达载体的构建的研究很少。
应用要点：在克隆和扩增大鼠成纤维细胞生长因子的过程中，由于此基因本身结构的特异性，无法扩增出与Genebank中百分之百吻合的基因片段，最终扩增出的基因序列有一个无意突变，不影响其在体内的表达。
同行评价：从文章叙述看，研究者有较多自己的经验，应该有过多次的探索试验，为文章最后的结果奠定了基础。具有一定的新颖性。从研究者描述的操作方法看，其过程正确，结果应该能够重复，实验过程及结果对其他研究者均具有借鉴作用。
摘要
目的：研究表明，细胞因子在各种炎症反应、损伤修复及细胞突变过程中发挥着重要作用，并且有望成为推断损伤时间新的有效指标。实验拟验证皮肤损伤后损伤区组织内不同时间白细胞介素1α和白细胞介素1β基因表达的变化及其与损伤时间的关系。
方法：实验于2006-03/2007-12在中国刑警学院法医系实验室完成。①实验材料：清洁级BALB/c纯系雄性小白鼠35只，体质量25~27 g。②实验方法：建立动物皮肤损伤模型，在脊柱两侧1.0 cm 处，左右对称各做一处皮肤缺损损伤，损伤不伤及肌肉，每只鼠做3对损伤。损伤后分别在0，3，6，12 h，1，3，6 d（0 h 作为对照）取5只动物麻醉断头处死。③实验评估：免疫组织化学染色后，显微镜下观察不同时间点白细胞的变化，实时定量聚合酶链反应检测白细胞介素1α和白细胞介素1β两种炎症因子的基因表达。
结果：纳入BALB/c纯系雄性小白鼠35只，均进入结果分析。①伤后0~5 h，皮肤创缘呈弱阳性反应；伤后6~24 h，表皮阳性反应减弱，创伤局部以巨噬细胞为主的阳性细胞逐渐增多。伤后1 d 可见嗜中性白细胞大量浸润及少量单核细胞；伤后2~4 d，单核细胞数目逐渐增加，3 d 达高峰。对照组皮肤表皮、毛囊、皮脂腺和汗腺均呈弱阳性反应。②皮肤损伤后在早期可诱导白细胞介素1α和白细胞介素1β的基因表达，6 h 达第一次高峰，24 h 下降，第3天再次出现高峰，第6天下降至正常水平。
结论：损伤组织中白细胞介素1α和白细胞介素1β的基因表达随伤后不同时间呈现一定规律性变化，可为法医学案例早期损伤时间的推断提供参考。
关键词：损伤时间；细胞因子；皮肤损伤；基因表达；实时定量聚合酶链反应；组织构建
李树，王杰. 小鼠皮肤损伤修复过程中白细胞介素1α和白细胞介素1β变化与损伤时间的关系 [J].中国组织工程研究与临床康复，2008，12(15):2879-2882 [www.zglckf.com/zglckf/ejournal/upfiles/08-15/15k-2879(ps).pdf]
1中国刑事警察学院法医学系，辽宁省沈阳市 110035；2沈阳医学院基础医学部生物化学与分子生物学教研室，辽宁省沈阳市
110034
李 树★，女，1964年生，吉林省辽源市人，汉族，1989年中国刑警学院法医专业双学位毕业，硕士，副教授，主要从事DNA多态性分析的研究。
lishu19852006@126.com
通讯作者：王 杰，教授，硕士生导师，沈阳医学院基础医学院副院长，沈阳医学院生物化学及分子生物学教研室，辽宁省沈阳市
110034
wangjie19932002@
163.com
中图分类号:R392.114
文献标识码:B
文章编号:1673-8225
(2008)15-02879-04
收稿日期：2007-12-07 修回日期：2008-02-12 (07-50-12-6803/WL·Y)
Time-course analysis of interleukin 1 alpha and 1 beta gene expression during the skin wound healing in mice
Abstract
AIM: Studies reveal that, cytokines play an important role in the process of various inflammatory reaction, wound repair and cell mutation, also, cytokines are expected to be a novel indicator of ratiocinating wound time. This study was designed to explore the changes in gene expression of interlukin-1α (IL-1α) and interlukin-1β (IL-1β) during the skin wound healing process in mice at different phases.
METHODS: The experiment was carried out in the laboratory of Forensic Medicine Department of China Criminal Police University from March 2006 to December 2007. ①Thirty-five BALB/c inbred male white mice of cleaning grade, aged 25-27 g, were used in this study. ②Skin wound model was induced by injuries in the area of 1.0 cm away from two sides of spinal column, without affection on muscles. Three pairs of injuries were established in each mouse. Five animals were decapitated under anesthesia at 0, 3, 6, 12 hours and 1, 3, 6 days, respectively, whereas those at 0 hour were taken as controls. ③The white blood cells at different phases were observed under microscope after immunochemical staining. Real-time quantitative polymerase chain reaction method was applied to detect gene expression of IL-1α and IL-1β.
RESULTS: Thirty-five BALB-c inbred male white mice were all involved in the result analysis. ①Skin wound was weakly positive during 0-5 hours after injury; the positive reaction was attenuated during 6-24 hours, and positive cells increased, of which macrophages were dominant. Infiltration of massive leucocyte neutrophils and a small quantity of mononuclear cells were detectable at 1 day; mononuclear cells increased during 2-4 days, and reached a peak at 3 days. In control group, the epidermis, folliculus pili, glandulae sebaceae and coil gland were all weakly positive. ②IL-1α and IL-1β were expressed at early phase after injury, and two expressing peaks were found at 6 and 72 hours, then decreased to normalized levels at 6 days after injury.
CONCLUSION: Genet expression of IL-1α and IL-1β in damaged tissues has a regularity with wound time, and can be used for reference to estimate early injury age in forensic cases.
Li S, Wang J. Time-course analysis of interleukin 1 alpha and 1 beta gene expression during the skin wound healing in mice.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(15):2879-2882(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-15/15k-2879(ps).pdf]
1Department of Forensic Medicine, China Criminal Police University, Shenyang 110035, Liaoning Province, China; 2Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Shenyang Medical College, Shenyang 110034, Liaoning Province, China
Li Shu★, Double bachelor, Master, Associate professor, Department of Forensic Medicine, China Criminal Police University, Shenyang 110035, Liaoning Province, China
lishu19852006@126.com
Correspondence to: Wang Jie, Professor, Tutor of master, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Shenyang Medical College, Shenyang 110034, Liaoning Province, China
wangjie19932002@
163.com
Received: 2007-12-07
Accepted: 2008-02-12
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