图1 

注：A～C，Y27632组、Fasudil组和对照组小鼠术后5 d的皮瓣形态（皮瓣手术前分别腹腔注射Y27632溶液、Fasudil溶液和等体积生理盐水3 d）。D，统计学分析结果显示Fasudil组的皮瓣坏死率明显低于对照组和Y27632组，Y27632组的皮瓣坏死率也明显低于对照组。每组6只小鼠，^*** P<0.001。

Figure 1 

Note： A-C， Flap morphology of the Y27632， Fasudil， and control groups on day 5 after surgery， respectively （mice were intraperitoneally injected with Y27632 solution， Fasudil solution， and an equal volume of normal saline for 3 days before surgery）. D， Statistical analysis showed that the flap necrosis rate in the Fasudil group was significantly lower than that in the Y27632 group and the control group， and the necrosis rate in the Y27632 group was also significantly lower than that in the control group. n=6 mice per group，^*** P<0.001.

图2 

注：A，激光散斑衬比成像系统展示的皮瓣切取后即刻（记为第0天）和第5天各组小鼠皮瓣血流灌注情况（术前连续3 d分别腹腔注射Y27632溶液、Fasudil溶液和等体积生理盐水）；B，各组小鼠术后第0天的蒂部髂腰动脉管径、皮瓣近端血流灌注量和远端血流灌注量对比；C为各组小鼠术后第5天的蒂部髂腰动脉管径、皮瓣近端血流灌注量和远端血流灌注量对比。PU，灌注单位。每组6只小鼠。^ns P>0.05，^** P<0.01，^*** P<0.001。

Figure 2 

Note： A， Laser speckle contrast imaging showing flap blood perfusion immediately after flap harvesting （recorded as day 0）and on day 5 in each group （mice were intraperitoneally injected with Y27632 solution， Fasudil solution and an equal volume of normal saline， respectively， for 3 consecutive days before surgery）. B， Quantitative analysis of the pedicle iliolumbar artery diameter， proximal flap perfusion （PU， perfusion unit）， and distal flap perfusion at day 0 after surgery. C， Quantitative analysis of the corresponding indicators at day 5 after surgery. PU， perfusion units. n = 6 mice per group. ^ns P > 0.05， ^** P < 0.01， ^*** P < 0.001.

图3 

注：A～C，CD31免疫荧光标记的Y27632组、Fasudil组和对照组小鼠皮瓣远端存活组织内微血管情况（皮瓣手术前分别腹腔注射Y27632溶液、Fasudil溶液和等体积生理盐水3 d），标尺为250 μm。D，各组小鼠皮瓣远端存活组织中平均每个视野的微血管数定量分析结果，每组6只小鼠，^* P<0.05，^*** P<0.001。

Figure 3 

Note： A–C， CD31 immunofluorescence staining of microvessels in the distal surviving tissue of the flaps in the Y27632， Fasudil， and control groups， respectively （mice were intraperitoneally injected with Y27632 solution， Fasudil solution， and an equal volume of normal saline for 3 days before surgery）. Scale bar = 250 μm. D， Quantitative analysis of the mean microvessel number per field in the distal surviving tissue of the flap in each group. n=6 in each group， ^* P<0.05， ^*** P<0.001.

图4 

注：A，EdU增殖实验的代表性荧光图片（标尺为250 μm）；B，定量分析3组细胞中EdU阳性细胞所占比例差异（^ns P>0.05，^*** P<0.001）。Y27632、Fasudil组及对照组细胞分别用Y27632溶液（10 μmol/L）、Fasudil（10 μmol/L）及等体积DMSO处理6 h，并用10 μmol/L的EdU试剂标记，每组3孔。EdU，5-乙炔基-2'-脱氧尿苷；DAPI，4'，6-二脒基-2-苯基吲哚。

Figure 4 

Note： A， Representative fluorescence images of the EdU proliferation assay （scale bar=250 μm）； B， Quantitative analysis of the proportion of EdU-positive cells among the three groups （^ns P>0.05， ^*** P<0.001）. Cells in the Y27632， Fasudil， and control groups were treated with Y27632 solution （10 μmol/L）， Fasudil solution （10 μmol/L）， and an equal volume of DMSO for 6 h， respectively， and labeled with 10 μmol/L EdU reagent， with 3 wells per group. EdU， 5-ethynyl-2'-deoxyuridine；DAPI， 4'，6-diamidino-2-phenylindole.

图5 

注：A～B，平板划痕实验和 Transwell实验分别测定体外细胞横向及纵向迁移能力（标尺均为100 μm）；C～D，细胞迁移率（即划痕愈合面积占比）和穿透聚碳酸酯膜的细胞数统计（^ns P>0.05，^*** P<0.001）。Y27632组、Fasudil组及对照组细胞分别用Y27632（10 μmol/L）、Fasudil溶液（10 μmol/L）及等体积DMSO处理，每组3孔。

Figure 5 

Note： A-B， Horizontal and vertical migration abilities of HUVECs measured by the plate scratch assay and Transwell assay respectively （the scale bar is 100 μm）； C-D， Statistics of cell migration rate （i.e.， the proportion of scratch healing area） and the number of cells penetrating the polycarbonate membrane respectively （^ns P>0.05， ^*** P<0.001）. Cells in the Y27632， Fasudil， and control groups were treated with Y27632 solution （10 μmol/L）， Fasudil solution （10 μmol/L）， and an equal volume of DMSO， respectively， with 3 wells per group.

图6 

注：A，基质胶成管实验检测各组HUVECs在体外的小管形成能力（标尺为100 μm）；B～E，定量分析成管实验显示总主段血管的长度、主段血管的数量、管状结构中两个或多个管状段交汇点的数量，以及血管网络形成数量。Y27632、Fasudil组及对照组细胞分别经Y27632溶液（10 μmol/L）、Fasudil溶液（10 μmol/L）及等体积DMSO处理，每组3孔，^*** P<0.001。

Figure 6 

Note： A， Matrigel tube formation assay was used to detect the in vitro tube-forming ability of HUVECs in each group （scale bar = 100 μm）； B-E， Quantitative analysis of the tube formation assay showing the total length of master segments， the number of master segments， the number of master junctions where two or more tubular segments intersect in the tubular structure， and the number of master meshes. The Y27632， Fasudil， and control groups were treated with Y27632 solution （10 μmol/L）， Fasudil solution （10 μmol/L）， and an equal volume of DMSO， respectively， with 3 wells per group， ^*** P<0.001.