Division of Hand, Plastic and Aesthetic Surgery, University Hospital, LMU, Munich, Germany.
Department of Dermatology and Allergy, University Hospital of Munich, LMU, Munich, Germany.
Lett Appl Microbiol. 2022 Aug;75(2):401-409. doi: 10.1111/lam.13740. Epub 2022 May 31.
Dermal replacement materials bioactivated with cyanobacteria have shown promising potential for wound regeneration. To date, extraction of cyanobacteria RNA from seeded scaffolds has not been described. The aim of this study was to develop a method to isolate total RNA from bioactivated scaffolds and to propose a new approach in determining living bacteria based on real-time PCR. Transgenic Synechococcus sp. PCC 7002 (tSyn7002) were seeded in liquid cultures or scaffolds for dermal regeneration in vitro and in vivo for 7 days. RNA was extracted with a 260/280 ratio of ≥2. The small subunit of the 30S ribosome in prokaryotes (16S) and RNAse P protein (rnpA) were validated as reference transcripts for PCR analysis. Gene expression patterns differed in vitro and in vivo. Expression of 16S was significantly upregulated in scaffolds in vitro, as compared to liquid cultures, whilst rnpA expression was comparable. In vivo, both 16S and rnpA showed reduced expression compared to in vitro (16S: in vivo Ct value 13.21 ± 0.32, in vitro 12.44 ± 0.42; rnpA in vivo Ct value 19.87 ± 0.41, in vitro 17.75 ± 1.41). Overall, the results demonstrate rnpA and 16S expression after 7 days of implantation in vitro and in vivo, proving the presence of living bacteria embedded in scaffolds using qPCR.
经蓝藻生物激活的皮肤替代材料在伤口再生方面显示出了有前景的潜力。迄今为止，尚未有关于从接种支架中提取蓝藻 RNA 的报道。本研究旨在开发一种从生物激活支架中分离总 RNA 的方法，并提出一种基于实时 PCR 确定活细菌的新方法。转基因聚球藻 sp. PCC 7002（tSyn7002）被播种在液体培养物或用于体外和体内皮肤再生的支架中，持续 7 天。用 260/280 比值≥2 的方法提取 RNA。原核生物的 30S 核糖体小亚基（16S）和 RNAse P 蛋白（rnpA）被验证为 PCR 分析的参考转录物。体外和体内的基因表达模式不同。与液体培养物相比，支架中 16S 的表达显著上调，而 rnpA 的表达则相当。体内，与体外相比，16S 和 rnpA 的表达均降低（16S：体内 Ct 值 13.21±0.32，体外 12.44±0.42；rnpA 体内 Ct 值 19.87±0.41，体外 17.75±1.41）。总的来说，这些结果表明 rnpA 和 16S 在体外和体内植入 7 天后的表达情况，使用 qPCR 证明了支架中存在活细菌。
