真皮组织微观化重建的探索性研究

目的 从微观角度探讨真皮基质三维结构对Fb生物学行为的影响.方法 借鉴平面几何、三角函数分析真皮组织三维结构,按照真皮组织由黏附成分和非黏附成分组成的原理,通过计算机辅助设计不同参数具有细胞黏着作用的点状结构阵列;运用微图案印刷和分子自组装法,建立4种(8μm×3 μm、间距6 μm,16 μm×3μm、间距6 μm,16 μm×5μm、间距8 μm,20 μm×3 μm、间距2 μm)具有细胞黏附点的桥墩样结构阵列细胞培养基质(MPGCC)培养人Fb,以无MPGCC培养的Fb为对照.利用免疫组织化学、荧光免疫组织化学、噻唑蓝法、羟脯氨酸含量测定法,检测培养Fb中骨架蛋白α-平滑肌肌动蛋白(α-SMA)表达、细胞活力和细胞分泌情况.结果 数学推导结果提示,真皮组织三维结构可用MPGCC进行模拟.用上述4种规格MPGCC培养的Fb其α-SMA表达百分率依次为(49±3)%、(61±3)%、(47±4)%、(51±3)%,与对照组(12±3)%比较,差异有统计学意义(P<0.05);Fb活力依次为0.12±0.03、0.13±0.04、0.14±0.03、0.19±0.03,与对照组0.35±0.04比较显著下降(P<0.05);羟脯氨酸含量依次为(0.95±0.04)、(0.87±0.03)、(0.81±0.03)、(0.77±0.03)μg/mg,与对照组(0.53±0.03)μg/mg比较显著上升(P<0.05).通过调整桥墩角度和桥墩间阵列参数,4组阵列相互对比,α-SMA表达、细胞活力和羟脯氨酸含量差异均有统计学意义(P<0.05).结论 MPGCC可能是真皮模板的基本功能单位或称为真皮模板单元,不同的真皮组织三维环境可产生不同的模板效应和创面愈合结局.

Exploratory study on the micro-remodeling of dermal tissue

Objective To explore the effect of three-dimensional structure of dermal matrix on bio-logical behavior of fibroblasts (Fb) in the microcosmic perspective. Methods The three-dimensional structure of dermal tissue was analyzed by plane geometric and trigonometric function. Microdots structure ar-ray with cell adhesion effect was designed by computer-assisted design software according to the adhesive and non-adhesive components of dermal tissue. Four sizes (8 μm × 3 μm, space 6 μm ;16 μm× 3μm, space 6 μm;16 μm×5 μm, space 8 μm;20 μm × 3 μm, space 2 μm) of micropier grid used for cell culture (MPGCC) with cell-adhesive microdots, built up with micro-pattern printing and molecule self-assembly method were used to culture dermal Fb. Fb cultured with cell culture matrix without micropier grid was set up as control. The expression of skeleton protein (α-SMA) of Fb, cell viability and cell secretion were de-tected with immunohistochemistry, fluorescent immunohistochemistry, MTT test and the hydroxyproline con-tent assay. Results The three-dimensional structure of dermal tissue could be simulated by MPGCC as shown in arithmetic analysis. Compared with those of control group [(12 ± 3)% and (0.53 ± 0.03) μg/mg, (0.35 ±0.04)] , the expression of α-SMA[(49±3)% , (61 ±3)% , (47±4) % , (51±3)%] and the content of hydroxyproline [(0.95±0.04), (0.87±0.03), (0.81±0.03), (0.77±0.03) μg/mg] were increased significantly (P <0.05), the cell viability of Fb (0. 12±0.03, 0. 13±0.04, 0.14± 0.03, 0.19± 0.03) cultured in MPGCC was decreased significantly (P < 0.05). When the parameters of micropier grid were changed, the expression of α-SMA, the cell viability and the content of hydroxyproline of Fb cultured in four sizes of MPGCC were also significantly changed as compared with one another (P < 0.05). Conclusions MPGCC may be the basic functional unit of dermal template, or unit of dermal tem-plate to call. Different three-dimensional circumstances for dermal tissue can result in different template effect and wound healing condition.