真皮组织微观化重建的探索性研究

Exploratory study on the micro-remodeling of dermal tissue

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×3μm、间距6μm，16μm×3μm、间距6μm，16μ×5μm、间距8μm，20μm×3μ间距2μm）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Fh，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Fh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。

Objective To explore the effect of three-dimensional structure of dermal matrix on biological 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 array 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 ×3 μm, 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 seeretion were detected with immunohistochemistry, fluorescent immunohistochemistry, MTT test and the hydroxyproline content 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, O. 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 c^-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 template to call. Different three-dimensional circumstances for dermal tissue can result in different template effect and wound healing condition.