【作者】 温学辉；

【导师】 郭振荣； 付小兵；

【作者基本信息】 中国人民解放军军医进修学院 ， 外科烧伤， 2007， 博士

【摘要】 目的：①探寻烧伤后创面下脂肪组织微循环血流变化规律及烧伤后不同时间切痂对创面下脂肪组织血流量的影响；②研究猪烧伤后创面下脂肪组织与创面愈合时间、创面收缩率及创面细菌量变化的关系，探讨烧伤后不同时间切痂对创面下脂肪组织功能、创面基底血流变化以及与愈合指标的关系；③与传统深筋膜层面植皮术式对比，探寻猪创面下脂肪组织与烧伤后切痂植皮成活率、移植皮片成活质量的关系；④通过观察脂肪匀浆液对猪骨髓间充质干细胞分化的影响来分析猪脂肪组织提高创面愈合质量的机理；⑤通过对人类不同厚度皮下组织层面上愈合的瘢痕组织的研究，阐明人类烧伤后创面下脂肪组织的存在对烧伤创面愈合质量的影响。方法：小型香猪背部制作皮肤全层烧伤模型。①用激光多普勒血流监测仪检测烧伤后不同时相点创面下脂肪组织（AUW）和深筋膜组织（DUW）微循环血流量。②伤后24小时切除痂皮，保留不同厚度的脂肪组织。伤后不同时间用薄膜法测量计算创面收缩率、用HE、VG染色、免疫组化染色、Western-bloting、ELISA、PCR、电镜观察、细菌培养等观察创面组织中-SMA、F-Ⅷ、PCNA、bFGF及其受体、Leptin及其受体、TGF-β1，TGF-β3、超微结构、细菌增殖量等指标的变化。③烧伤24小时切痂后以不同厚度的脂肪组织为基底行自体皮移植。通过大体观察、常规组织病理观察、免疫组化染色、电镜观察等方法对比各组植皮创面收缩率变化、植皮组织中bFGF蛋白表达、F-Ⅷ的表达情况。④用脂肪组织匀浆对分离培养的MSC细胞进行干预，绘制细胞生长曲线、计算细胞增殖率及使用流式细胞仪检测细胞生长周期、DNA含量和凋亡率。⑤烧伤后瘢痕患者，按照切除瘢痕时的基底组织情况将所取瘢痕组织分为深筋膜层愈合瘢痕组（A组，n=15）、脂肪组织层愈合瘢痕（B组，n=11）和正常对照组（C组，n=26）。进行组织学观察和Western-bloting、PCR等方法测量组织中TGF-β1，TGF-β3含量变化。结果：①烧伤前皮下脂肪组织VM和深筋膜组织VM比较无显著差别；烧伤后14天内AUW和DUW的VM均明显减少，伤后1～60小时AUW的VM减少更明显。60小时以后两组差別消失；切痂后60小时内，E72h组AUW的VM比E24h组明显降低。E7d AUW的VM一直到切痂后14天都少于E24h和E72h。②三组创面愈合时间无统计学差别。A组创面收缩最早发生，收缩率大且创面收缩率与脂肪保留厚度相关。切痂后第7天和第14天时B组、C组创面总细胞数明显多于A组。创面愈合后的3个月时A组总细胞数明显多于B、C组。A组创面MFB出现早，组织胶原束较厚，弹性纤维增生较重，肉芽组织中-SMA阳性表达细胞较B、C组明显增多。第14天时电镜下仍见MFB多且功能活跃。伤后7天B、C组肉芽组织形成最为丰富，血管密度最大，F-Ⅷ染色阳性的血管内皮细胞数最多，细胞增殖活跃，肉芽组织中PCNA阳性细胞表达明显高于A组（P＜0.05），伤后7天C组胶原纤维较其他两组明显变粗变长，排列趋向于规则。切痂后21天B、C组瘢痕组织较A组相比较结构更趋向于成熟。切痂后3天到7天C组bFGF阳性表达细胞最多。RT-PCR结果显示切痂后3天时三组创面组织中bFGF表达升高并不明显。7天、14天时三组均明显升高，且B组、C组高于A组。切痂后3个月B组、C组bFGF水平小幅度升高，而A组变化不明显。各组切痂后3天bFGF-R的表达均升高，7天时达最高水平，C组≈B组＞A组，切痂后21天bFGF-R的表达降至正常值以下，三月时低于正常组织。切痂后创面Lep水平快速升高，至切痂后21天C、B组水下一直比A组高。切痂后3天创面局部Lep-R的表达即升高，7天时达最高峰，后明显下降。7天时B组＞C组≈A组。切痂后3天B、C组脂肪组织中ADRP水平明显升高，7天时B组＞C组。伤后3个月时三组瘢痕组织中TGF-β1，TGF-β3水平均较正常组织升高，TGF-β3升得最明显。三组间TGF-β水平没有显著差异，B组、C组TGF-β3水平明显高于A组（P＜0.05）。E24h较E72hbFGF阳性表达出现早且持续时间长。不同时间切痂后创面下脂肪组织中Leptin、Leptin-R及ADRP均较正常脂肪组织明显升高，E24h明显高于E72h。烧伤后3天时创面新生血管内皮细胞密度与其基底组织内皮细胞密度密切相关，而与基底VM相关性不明显。三组组织细菌含量的动态变化基本一致，E24h组切痂后各个时间点三组创面细菌含量无差别。E72h切痂后C组≈B组＞A组；E7d切痂后C组1天、3天、7天三组创面组织细菌量均显著高于A组；各组创面中，切痂时间越晚，创面在切痂后不同时间点的细菌含量越高。③各组皮片均成活，A组皮片收缩幅度明显大于B、C组。植皮后各时相点创面愈合质量C组优于B组，B组优于A组。A组毛细血管增生、改建到恢复至正常密度和胶原的沉积、改建所用的时间都比B、C两组要滞后，改建后结构的重构性也比其他两组差。早期移植床组织bFGF表达B组≈C组＞A组。术后7d，PCNA阳性表达量C组＞B组＞A组。成纤维细胞超微结构变化示A组成纤维细胞增生、功能增强到细胞凋亡、功能衰减的过程较B、C组明显滞后，胶原纤维成熟度差，肌成纤维细胞存在时间长、功能衰减慢。④脂肪匀浆培养基诱导后细胞分裂增殖明显加快、对数生长期群体倍增时间缩短、平台期的细胞总数明显增加、G₀/G₁期细胞比例明显降低、S期细胞的比例明显增高、细胞中DNA含量均明显增高于。而且烧伤创面下脂肪匀浆的作用要强于正常脂肪组织匀浆。烧伤创面下脂肪匀浆还可使MSC细胞的凋亡率明显升高。⑤与正常组织相比A组瘢痕的结构更紊乱、层次模糊，组织中细胞成分比B组明显多。外观比较，A组发生增生性瘢痕的几率比B组高出很多，分别为83.7％和62.4％。A、B两组TGF-β1和TGF-β3蛋白水平和基因表达均明显高于正常组，而且A、B两组的TGF-β1水平没有差别，但TGF-β3水平A组＞B组。结论：①脂肪组织的VM不比深筋膜组织的少。烧伤后早期AUW的VM降低明显，60小时后与DUW的持平。早切痂可以促进AUW微循环血量的恢复。②烧伤后猪创面下脂肪组织能够加速创面愈合、减少创面收缩率、提高愈合质量，早期切痂能够促进脂肪组织功能的恢复，并降低脂肪创面感染的机率。烧伤后早期血管内皮细胞的增殖乃至血管的形成主要与其基底组织的血管密度相关，并不是直接与基底VM直接相关。③与传统深筋膜层面植皮术式相比，猪创面下脂肪组织层面植皮成活率相当，移植皮片成活质量更高；④脂肪组织尤其是创面下脂肪组织可能通过促进创面组织中MSC细胞分裂增殖来促进创面愈合。⑤人类烧伤后创面下脂肪组织的保留能够提高烧伤创面的愈合质量，加速瘢痕的成熟，减少瘢痕增生的发生。更多还原

【Abstract】 Objective: ① To investigate the regularity of microcirculiation change of adipoise tissue under the burned wound(ATUW), and to detect the effect of the interval between thermal injury and escharectomy on ATUW; ② To explore the relationship between adipose tissue preservation and healing speed, wound construction rate, some kinds of healing-related index respectively; the effect of adipose tissue preservation on bacterium content in the wounds; the effect of the interval between thermal injury and escharectomy on the function of ATUW; as well as the relationship between the change of microcirculiation change of ATUW and the changes of healing-related index; ③ Compared with the traditional skingrafting technique on deep fascia of wound, to evaluate the survival rate and the survival quality of skingrafting on ATUW; ④ To explore a part of mechanism of the capability of adipose tissue on improving wound healing by observing the effect of the homogenate of adipose tissue on the biological properties and generation ability of porcine MSCs in vitro; ⑤ To elucidate the effect of adipose tissue preservation on maturity and proliferation of burn scar by studying on the reparation and regeneration quality of human scars which had been healed on various base structure of wound.Methods: Seventy-two full thickness skin thermal wounds were produced on the back in 6 male minipigs. ① LDF was used to detect the regulation of microcirculation changes of both deep fascia and adipose tissue under the wounds. ② we preserved various thickness of adipose tissue in escharectomy which undertook 24 hours after the burn injury. On day 3, 7, 14 and 21 after injury, we compare the healing speeds and the rate of wound contraction in wounds on the whole subcutaneous fat (n = 24), half subcutaneous fat (n = 24) and deep fascia (n = 24) in burned pigs after excision of eschar by using sketchy observation . light microscope , electron microscope , HE staining ,VG staining and immunohistochemistry, RT-PCR. ELISA. Western blotting methods and bacterial culture were performed to detect thedifference of genes or proteins of -SMA, F-VDIRA, PCNA, bFGF, bFGf-R, TGF-β1, TGF-β3, Lep, Lep-R, ADRP, and the changes of bacteria content in wounds. ③ 24 wounds on minipig were undertook excision of eschar and autografting on various thickness of adipose tissue 24 hours after burn injury, We compare the survival rate and survival quality of grafting on the whole subcutaneous fat (n = 8), half subcutaneous fat (n = 8) and deep fascia (n = 8) by using sketchy observation, light microscope, electron microscope and immunohistochemistry methods to detect the difference of proteins of F-ⅧRA and bFGF. ④ We use homogenate of both normal adipose tissue and adipose tissue under the wounds to intervene the biological behavior and generation ability of MSCs. Grouth curve of MSCs was made, the population doubling time of MSCs was culculated and cell cycle, DNA content ,apoptosis rate of MSCs was detected by Flow Cytometry. ⑤ 26 scar samples obtained from 18 patients who had suffered burn injury and the wounds had healed with scar. All scar samples were divided into 3 groups according to the thickness of adipose tissue under the scar: Scar healed on deep fascia group(n=15), Scar healed on adipose tissue group(n=ll) and normal tissue control group(n=26).We use histological examination, Western-bloting, PCR method to detect protein level changes of TGF-β1,TGF-β3 in samples.Results: ①The volume of microcirculation(VM)in adipose tissue and in deep fascia tissue had no statistic difference before injury; But the VM both in AUW and DUW reduced rapidly after burn injury, the VM in AUW decreased more than that of DUW 1 ～60 hours after the thermal injury. the AUW’s VM in E72h were lower than that in E24h within 60 hours after escharectomy,and the AUW’s VM in E7d remained lower than that in E24h and in E72h 14 days after escharectomy. ② The healing time of the three groups had no difference. The wounds’ construction occurred earlier and the wounds’ construction rate (WCR) which had relationship with the thickness of preserved adipose tissue in A group remains more than that in other groups to the end .The totle amount of cytes in wounds of B and C group were more than that of A group 7 days and 14 days after escharectomy, But when 3 months passed , the changes of totle amount of cytes in wounds was reverse,the group A contained morecytes component than the other two groups. The MFB appeared ealier and remained more functional for longer time in group A than that in the other groups. The a-SMA positive cytes in group A were more than that in B and C group. The granulation tissue in B and C gtoup generate earlier and become more abundant, cell multiplication was more active in earlier phase of healing than that in group A, which was manifested by more F-ⅧRA positive cytes and more PCNA positive cytes in wounds of B and C group. And the array of collagen fibers trend to be thick, long and regulative earlier in group B and C than that in group A. The overall degree of maturity of repairing tissue in group B and C was higher than that in group A 21 days after escharectomy. The immunohistochemistry staining showed that the bFGF positive cytes increased just after the inury and incraesed more in group C and B than that in group A,but the results of RT-PCR showed that the gene expression of bFGF was not increase until 7 days after the injury, and got to the top at 14 days after injury, the gene expression of bFGF in group B and C was higher than that in group A. And moreover, bFGF level in group C and B increase in small extent 3 months after the escharectomy after a stable decrease, and this phenomena didn’t appear in group A. The protein expression of bFGF-R, Lep, Lep-R and ADRP all increased in various time points , and in group B and C these levels were higher than those in group A. 3 months after injury , the TGF-(31, TGF-β3 protein level especially TGF-β3 protein level in the three groups were higher than normal tissue, there was no difference amang the three groups in TGF-β1 level, but there was obvious difference in TGF-β3 level: group C≈group B>group A. The bFGF positive cytes appeared earlier and persist longer in E24h than that in E72h. The Lep,Lep-R,and ADRP level in adipose tissue under the wound were higher than those in normal adipose tissue, and the interval time between escharectomy and injury may affect the Lep,Lep-R,and ADRP levelin wound:the earlier escharectomy was undertaken, the higher level of these indix may present. The cell density of neogenesis vascular endothelial cell were closely correlated with the cell density of endothelial cell in the base tissue of wound, but not the volune of microcirculation in the base tissue of wound 3 days after injury. The bacteria content of wounds were seriously affected by the interval time betweenescharectomy and injury: The earlier escharectomy was made, the fewer bacteria may existed and the preservation of adipose tissue under the scar which was remained longer than 3 days may cause more bacteria content in wounds. ③ The skin graft in the three groups all survived, but the skin grafts in group A contract more than that in the other groups. The healing quality of graft in group C was superior to group B and group A. The interval time for blood vessel hyperplasia and rebuilting and for collagenic deposition and restructure in group A was longer than that in group B and C, and the maturation degree of rebuilt tissue in group A was worse than that in the other groups.And in group A, the expression of bFGF and PCNA were lower than that in group B and C, and the ultramicrostructure change of fibroblast showed that the process of proliferation, function enhancement,and apoptosis, function decline were delayed than group B and C, the maturate degree of collagenous fibers were poor, myofibroblast exist longer time and its founction attenuated much slower. ⑤Compare with normal tissue , in group A,the constructure of scar was more disorder, layer of tissue was blur, the cell component was more, and the occurence of Hyperplastic scar was more frequent than those in group B. the frequent of group A and B was 83.7% and 62.4%,respectively. The TGF-β1 and TGF-β3 protein level in both groups were higher than that of normal tissue, there was no difference in TGF-β1 between A and B group , but the TGF-β3 level in group A was higher than that in group B. Conclusion: ① Adipose tissue is not a kind tissue lacking of blood supply compare with deep fascia tissue.The microcirculation of AUW was obviously decreased,and reach the same level with DUW 60 hours after injury .Early escarectomy may improve the recover of microcirculation . ② Preservation of adipose tissue in burned wound may accelerate healing process, reduce wound contraction rate, enhance healing quality, escharectomy in early phase may promote the recover of adipose tissue’ s function, and reduced the infected chance of adipose tissue remained wounds. In early phase of healing process, the generation of vascular endothelial cell and Angiopoiesis were closely correlated with the cell density of endothelial cell in the base tissue of wound, but not the volune of microcirculation in the base tissue ofwound. ③Grafting on adipose tissue may obtain the same survival rate and better survival quality, compared with the traditional grafting technique on deep fascia; ④The perserved adipose tissue in the wound improve the healing quality and promote the grafting survival rate, which may be attributed to the ability of fat to promote the stem cells’ generation and differentiation. ⑤The perserved adipose tissue in human burned wound had apotential to improve the quality of healing, to accelerate the maturation of scar, to reduce the occur of hyperplastic scar.更多还原