烧伤创面水疱液对人骨髓间充质干细胞体外培养及表型转化的影响

Effect of burn blister fluid on human mesenchymal stem cell （MSC） in vitro and its phenotypic modulation in culture

目的了解烧伤水疱液对人骨髓间充质干细胞（MSC）体外培养及表型转化的影响。方法体外分离、培养、扩增、鉴定人骨髓MSC，收集烧伤患者伤后12、24、48h水疱液，分别加入MSC培养液中，分为水疱液1组：MSC培养液中加入伤后12h水疱液；水疱液2组：MSC培养液中加入伤后24h水疱液；水疱液3组：MSC培养液中加入伤后48h水疱液；对照组：常规培养。各组所加水疱液体积分数均为20％。观察水疱液微生物生长情况及各组MSC生长情况。流式细胞仪检测培养8d时各组MSC中CD44、细胞角蛋白7（CK7）阳性表达率。结果15个水疱液标本细菌、真菌培养均为阴性；各组MSC细胞形态无明显变化，但各水疱液组细胞数量均少于对照组，水疱液3组细胞数量下降最明显。水疱液1、2、3组CD44阳性表达率分别为（83．0±3．1）％、（77．2±2．9）％、（65．1±2．3）％，均低于对照组[（89．5±3．2）％，P〈0．01]；水疱液1、2、3组CK7阳性表达率分别为（24．06±0．11）％、（16．41±0．09）％、（4．48±0．07）％，明显高于对照组[（3．87±0．04）％，P〈0．01]。结论烧伤创面水疱液对MSC体外培养有明显的抑制作用，并具有一定的促进MSC表型转化作用。

Objective To study the effect of blister fluid obtained from burn patient on human MSCs in vitro and its phenotypic modulation in culture. Methods Blister fluid from burn patients was collected at 12, 24, 48 post burn hour （PBH）. The human MSCs were isolated, cultured, amplified and identified in vitro, then were divided into A （culture with 20% blister fluid collected at 12 PBH） , B （culture with 20% blister fluid collected at 24 PBH） , C （culture with 20% blister fluid collected at 48 PBH） , N （with ordinary culture medium） groups. The growth of MSCs and micro-organisms in blister fluid were observed. Positive expression rates of CD44 and CK7 were detected by flow eytometry after culture for 8 days. Results Bacterial and fungal growths were absent in 15 blister fluid samples. There was no obvious change in MSC morphology in each group. Compared with that of N group, the number of MSCs in A, B, C groups was decreased, especially in C group. CD44 positive expression rate in A, B, C groups was （83. 0 ± 3. 1）%, （77.2±2.9）% and （65. 1 ±2.3）%, respectively, which was obviously lower than that in N group [（89.5± 3. 2）%, P 〈0.01]. CK7 positive expression rate in A, B, C groups was （24.06± 0. 11）%, （16.41 ±0.09）% and （4.48±0.07）%, respectively, which was obviously higher than that in N group [ （3. 87 ± 0.04） % , P 〈 0. 01 ]. Conclusions Burn blister fluid can obviously inhibit the growth of human MSC cultured in vitro, and may promote modulation of its phenotype to certain extent.