中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (20): 3706-3710.doi: 10.3969/j.issn.1673-8225.2011.20.023

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

实时荧光PCR法定量小腿慢性静脉性溃疡表面的细菌分布

袁  方，赵  渝，张  矛   

  1. 重庆医科大学附属第一医院血管外科，重庆市400016
  • 收稿日期:2011-01-24 修回日期:2011-03-28 出版日期:2011-05-14 发布日期:2011-05-14
  • 通讯作者: 赵渝，硕士，教授，重庆医科大学附属第一医院血管外科，重庆市 400016 zhaoyu@cta.cq. cn
  • 作者简介:袁方★，男，1985年生，河南省驻马店市人，汉族，2011年重庆医科大学毕业，硕士，医师，主要从事下肢慢性溃疡研究。yuanfang85612@163.com

Real-time fluorescence quantitative PCR analysis of bacterial microfloras in chronic venous leg ulcers

Yuan Fang, Zhao Yu, Zhang Mao   

  1. Department of Vascular Surgery, the First Affiliated Hospital, Chongqing Medical University, Chongqing  400016, China
  • Received:2011-01-24 Revised:2011-03-28 Online:2011-05-14 Published:2011-05-14
  • Contact: Zhao Yu, Master, Professor, Department of Vascular Surgery, the First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China zhaoyu@cta.cq.cn
  • About author:Yuan Fang★, Master, Physician, Department of Vascular Surgery, the First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China yuanfang85612@ 163.com

PDF

427

被引次数

7

摘要：

背景：小腿慢性静脉性溃疡表面的细菌在溃疡的愈合过程中起重要作用，但细菌的种类、密度与溃疡愈合的关系仍然存在争议。
目的：探讨小腿慢性静脉性溃疡中的常见菌群种属和细菌量与溃疡愈合的关系。
方法：纳入2009-05/2010-09重庆医科大学第一附属医院血管外科诊断为小腿慢性静脉性溃疡患者39例，共42条患肢。所有患者均进行标准的加压疗法治疗，经过6个月的随访以确定溃疡的愈合率，42条患肢中，28条患肢在6个月内完全愈合，为愈合组，14条患肢未完全愈合，为未愈合组。获取治疗前所有溃疡的组织样本，提取样本中的细菌基因组DNA，合成细菌的通用引物及下肢慢性静脉性溃疡中常见细菌种属的特异性引物，运用常规PCR及荧光定量PCR方法比较愈合组与未愈合组溃疡表面的细菌差异。
结果与结论：愈合组和未愈合组标本中都可检测到细菌，最常见的细菌为金黄色葡萄球菌和铜绿假单胞杆菌。荧光定量PCR结果显示未愈合组标本中的细菌总量较愈合组明显增多(P < 0.05)，两组中金黄色葡萄球菌、铜绿假单胞杆菌的含量无明显差异(P  > 0.05)。实验结果中未见任何一种溃疡的常见细菌种属与溃疡的愈合具有直接关系，而溃疡中的细菌总量与溃疡的愈合有较大的关联。结果表明溃疡表面细菌总量的增加引起的皮肤慢性感染可能是导致小腿慢性静脉性溃疡长期不愈合的重要原因。

关键词: 实时定量荧光PCR, 细菌, 种属, 溃疡愈合, 小腿静脉性溃疡

Abstract:

BACKGROUND: The bacterial microfloras in chronic venous leg ulcers (CVLU) play an important role in the healing process, but there is still considerable debate as to the importance of individual species or microbial density in relation to healing.
OBJECTIVE: To explore the relationship between the bacterial groups frequently recovered from chronic venous leg ulcers and ulcer healing.
METHODS: Patients with newly diagnosed chronic venous leg ulcers in the First Affiliated Hospital of Chongqing Medical University from 2009.5 to 2010.9 were recruited in this study.Totally 39 patients with 42 chronic venous leg ulcers included in this study were treated with standard regimen of compression therapy. Patients were followed up for 6 months to determine healing rates of ulcers. Twenty-eight ulcers with 100% closure of the wound and did not reoccur within 1 month of wound closure were included in the healing group. Fourteen legs without complete healing served as non-healing group. Tissue samples from ulcer bed were obtained by biopsy prior to treatment, DNA was extracted directly from the tissue sample. A set of universal primer and group or species-specific primers for the bacterial groups frequently recovered from CVLU were synthesized. The bacterial difference between healing and non-healing chronic venous leg ulcers was compared using PCR and real-time fluorescence quantitative PCR.
RESULTS AND CONCLUSION: Bacteria were detected in all ulcers. In the healing and non-healing groups, the most common bacteria were staphylococcus aureus and pseudomonas aeruginosa. Quantitative RCR showed that the total bacterials load in the non-healing chronic venous leg ulcers was significantly higher than those of healing group (P < 0.05), whereas the density of staphylococcus aureus and pseudomonas aeruginosa did not show significant difference (P > 0.05). In this study, no single bacterial species or group frequently recovered from CVLU was shown to be associated with healing outcome. This study suggests that the increase of total bacterial load play important roles in maintaining a chronic inflammation state that ultimately leads to the failure of chronic venous leg ulcers to heal.

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