摘要
目的观察没药提取物对人皮肤Fh生物学特性的影响，探讨其促进创面愈合的可能机制。 方法从人正常包皮组织中分离并培养Fb，取第3-5代细胞用于实验。（1）将Fh接种至96孔板，按随机数字表法分为对照组，1×10-4、1×10 1、1×10-3、1×10-21、1、10、1×102 g/L没药水提取物组以及上述7种浓度的没药醇提取物组。对照组用含体积分数0. 25%小牛血清的DMEM培养液（简称低浓度血清培养液）培养，各浓度没药水及没药醇提取物组分别用含相应终浓度2种没药提取物的低浓度血清培养液培养。培养48 h，用倒置相差显微镜观察细胞形态，噻唑蓝法测定各组Fh增殖活性（以吸光度值表示）。（2）将Fh分别接种于培养瓶和培养皿中，均按随机数字表法分为2组：1 g/L没药水提取物组，用含终浓度1 g/L没药水提取物的低浓度血清培养液培养；对照组，采用低浓度血清培养液培养。培养72 h，分别采用流式细胞仪、实时荧光定量PCR法检测Fb周期与I、Ⅲ型胶原mRNA的表达。对数据进行LSD-t检验。 结果 （1）各组细胞均呈长梭形生长，1 g/L没药水提取物组细胞融合较对照组更显著。1×10-3、1×10-2、1×10-11，1、10 g/L没药水提取物组Fb吸光度值分别为0. 378±0.032、0.402±0.007、0.390±0.038、0.453±0.036、0.390±0.037，均高于对照组的0. 332±0.044，t值分别为2.24、2.93、2.69、5.73、2.71，P值均小于0.05。1×10-3、1×10-2 g/L没药水提取物组吸光度值分别为0.312±0.048、0.154±0.009，前者与对照组比较，差异
无统计学意义（t=2.84，P〉0.05）；后者显著低于对照组（t=7. 17，P〈0.05）。1×10。3、1×10。1、1、10、1×102 g/L没药醇提取物组Fh吸光度值显著低于对照组（z值为2.30 24. 79，P值均小于0. 05）。（2）1 g/L没药水提取物组CO/G1期细胞百分比为（74.3±6.3）%，明显少于对照组的（82.2 ±7.9）010，z=6.77，P〈0.05；S期及G2/M期细胞百分比分别为（16.6±3.4）%、（9.1±1.6）%，明显多于对照组的（13.3±2.3）%、（4.5±0.8）%，t值分别为7. 53、6.34，P值均小于0.05。1g/L没荮水提取物组Fb中I型胶原mRNA相对表达量（0.89 ±0.08）与对照组（1.00 ±0.06）比较，差异无统计学意义（t=1.17，P〉0.05）；Ⅲ型胶原mRNA相对表达量（1.38±0.12）显著高于对照组（1. 00±0.05，t=3.81，P〈0.01）。 结论 没药水提取物能显著促进Fb增殖，加快Fb细胞周期进程，上调Fb中Ⅲ型胶原mRNA表达，可能与其促进创面愈合的机制相关。
Objective To observe the effects of myrrh extract on biological characteristics of human dermal fibroblasts （Fb） , and to explore its possible mechanisms in promoting wound healing. Methods Normal Fb was isolated from human foreskin tissue and cuhured in vitro. The third to fifth passages of Fb were used in the experiment. （ 1 ） Fb were planted onto 96-well plate and divided into control group, and 1 x10-, 1 x10 3, 1 ×10^-2, 1 ×10^ -1, 1, 10, 1 ×10^2 g/L myrrh water extract groups and myrrh ethanol extract groups according to the random number table. Fb in control group were cultured with DMEM medium containing 0.25% calf serum （briefly called low-concentration serum medium）, and those in various concentrations of myrrh water extract and myrrh ethanol extract groups respectively with low-concentration serum medium containing corresponding concentration of 2 kinds of myrrh extract. After being cultured for 48 h, cell morphology was observed with inverted-phase contrast microscope, and Fb proliferation activity （ denoted as absorbance value） was determined with MTT method. （2） Fb were respectively planted into flasks and dishes and divided into two groups according to the random number table. Fb in control group were cultured with low-concentration serum medium, and that in 1 g/L myrrh water extract group with low-concentration serum medium containing 1 g/L myrrh water extract. After being cultured for 72 h, Fb cell cycle and the type Ⅰ and Ⅲ collagen mRNA expression were respectively determined by flow cytometry and real-time fluorescent quantitative PCR. Data were processed with LSD- t test. Results （ 1 ） Fb in all groups grew in long-spindle shape, but the cell fusion was much obvious in 1 g/L myrrh water extract group than in control group. Fb absorbanee value in 1 ×10-3, 1 ×10-2, 1 ×10-1, 1, 10 g/L myrrh water extract groups was respectively 0. 378 ± 0. 032, 0. 402 ± 0. 007, 0. 390 ± 0. 038, 0. 453 ± 0. 036, 0. 390 ± 0. 037, all higher than that in control group （0. 332 ± 0. 044, with t value respectively 2.24, 2.93, 2.69, 5.73, 2.71, P values all below 0. 05 ）. Compared with that in control group, Fb absorbance value in 1 × 10 -4 g/L myrrh water extract group was not statistically different （0. 312 ±0. 048, t =2.84, P 〉 0.05） , while that in 1 ×102 g/L myrrh water extract group was significantly lower （0. 154 ± 0. 009, t = 7. 17, P 〈 0. 05）. Fb absorbance values in 1 ×10-3, 1× 10-1 , 1, 10, 1 ×102 g/L myrrh ethanol extract groups were significantly lower than that in control group （ with t values from 2.30 to 24.79, P values all below 0.05 ）. （2） Compared with those in control group [（82.2 ±7.9）% and （13.3 ±2.3）%, （4.5 ±0.8）%1, the percentage of ceils in G0/G1 phase in 1 g/L myrrh water extract group was obviously decreased [ （74.3 ± 6.3） % , t = 6.77, P 〈 0.05], while those in S and G2/M phases increased [（16.6 ±3.4）%, （9.1 ±1.6）%, withtvalue respectively 7.53, 6.34, P values below 0.05 ]. Compared with those in control group （ 1.00 ± 0.05, 1.00 ±0. 06） , the mRNA level of collagen Ⅲ in 1 g/L myrrh water extract group was significantly up-regulated （ 1.38 ± 0.12, t = 3.81, P 〈 0.01 ）, while that of collagen Ⅰ was not statistically different （0.89 ±0.08, t = 1.17, P 〉 0.05）. Conclusions Myrrh water extract can notably promote the proliferation of Fb, accelerate the cell cycle of Fb, and up-regulate the mRNA expression of type Ⅲ collagen in Fb, which may be related to its mechanisms in promoting wound healing.
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2012年第2期130-133,共4页
Chinese Journal of Burns
基金
基金项目：陕西省“13115”科技创新工程重大科技专项（2009ZDKG-87）
关键词
没药属
成纤维细胞
细胞增殖
胶原
伤口愈合
Commiphora
Fibroblasts
Cell proliferation
Collagen
Wound healing
