【作者】 王晓燕；

【导师】 王志军；

【作者基本信息】 遵义医学院 ， 外科学， 2011， 硕士

【摘要】 目的：探讨异体骨髓间充质干细胞(Bone marrow mesenchymal stem cells, BM-MSCs)在扩张皮肤中发挥的作用,为临床更好的使用皮肤扩张术提供新的方法。方法：无菌条件下解剖乳猪双侧股骨,冲出骨髓后淋巴细胞分离液分离,接种于培养基中。取培养第三至四代的BM-MSCs,分别加入FITC标记的鼠抗猪CD90、CD34、CD45、CD29抗体,PBS重悬后上流式细胞仪检测。16头小型家猪随机分为4组,每组4头。A组：埋植扩张器后,局部皮下注射细胞悬液；B组：埋植扩张器后,耳缘静脉注射细胞悬液；C组：仅埋植扩张器；D组：空白对照组。在猪的脊柱两侧各设计3个皮瓣,皮瓣中心上文出4×4 cm2大小面积。距脊柱2 cm作纵行切口,按扩张器基底部大小在深筋膜浅层分离出4×6 cm2的皮下腔隙,置入80ml矩形单向扩张器。各组每隔三天注水一次,每次注水10ml。扩张后第7,14,28天分别测量各组标记面积,取出扩张器后即时测量标记面积。取各组扩张第28天的皮肤标本,检测4组皮肤血管内皮细胞、增殖细胞核抗原、表皮干细胞的表达量,分别用抗体CD31、PCNA(proliferating cell nuclear antigen)、K19(epidermal stem keratinocytes)进行免疫荧光染色标记。取各组扩张第14天的皮肤组织,实时定量PCR分析目的基因血管内皮细胞生长因子(vascular endothelial cell growth factor, VEGF)、表皮生长因子(epidermal growth factor, EGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)及基质细胞衍生因子-1 (Stromal cell-derived factor-1, SDF-1)相对表达量。结果：体外培养的BM-MSCs呈多角形或梭形,贴壁细胞呈旋涡状、网状、辐射状排列生长。流式细胞检测结果表明BM-MSCs不表达CD34和CD45,但表达CD29和CD90。随着扩张时间延长,各组标记面积增加,A、B、C组扩张明显,与D组相比有统计学意义(P<0.01)。取出扩张器后测量标记面积,A、B、C组均有一定程度回缩,回缩后的剩余面积A、B组仍大于C组,A组与C组比较有统计学意义(P<0.05)。随着扩张时间延长,表皮层明显增厚,细胞层次增多,以A组扩张第28天最明显,扩张后真皮层变薄,胶原纤维排列松散,纤维间隙增加。A组扩张皮肤第7、14、28天均可看到明显的DiI荧光标记细胞分布,主要分布在真皮下层。随着时间延长,B组皮肤中DiI细胞分布增多,主要在真皮层。CD31检测：A.B.C.D四组血管断面密度(条/视野)分别为9.1±2.31、7.85±1.93、6.5±1.64、4.2±1.54。PCNA检测：A.B.C.D四组PCNA阳性细胞(个/视野)分别为163.25±35.4、108.62±8.93、52±8.07、13.63±3.96。K19检测：A.B.C.D四组K19阳性细胞(个/视野)分别为42±5.24、31.75±4.33、20.88±3.27、13.75±2.38,各组之间比较均具有统计学意义(P<0.01)。实时定量PCR检查结果示VEGF、SDF-1、EGF、bFGF的表达差异倍数,A组比B组分别为为5.4、1.07、1.21、6.03倍。结论：1、局部及全身移植BM-MSCs于扩张器模型可以促进扩张皮肤的生物性生长。2、局部移植组效果好于全身移植组,是一种比较好的移植方案。更多还原

【Abstract】 Objective:To investigate the feasibility of transplantation of mesenchymal stem cells into expanion skin.Methods:The femurs of the porket were harvested in asterile fashion and the marrow was flushed with phosphate buffered saline (PBS).The mononuclear cell layer of the density medium’s upper layer was collected to begin the culture. Passage 3 BM-MSCs were resuspended in PBS. Cell aliquots were incubated with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies specific for CD29, CD90, CD34 and CD45.Cells cultured were analyzed by fluorescence-activated cell sorting.Sixteen pigs divided into four groups randomly with four each. In Group A. each flap received suspended stem cells around the wound. In Group B, stem cells in medium were injected into the ear vein. In Group C, each wound received medium and Group D was the blank control group.After hair removal from the dorsal surface and anesthesia, six flaps were marked on either side of the spine midline by injection of ink in dermal and the area was 4cm×4cm.The 80 ml silicone expanders were implanted under the flap as described previously. The animals were injected the normal saline (N. S.) (10 ml every three days until day 28).The marked area was measured at day 7,14,28 after the expansion. After the expanders were removed, the area was measured immediately. Skin tissue specimens taken after 28 days of expansion were tested the content of vascular endothelial cells, proliferating cell nuclear antigen, and the epidermal stem keratinocytes. They were identified with an antibody against CD31、PCNA and K19 respectively, compare the positive cells between four groups.Skin tissue specimens taken after 14 days of expansion were tested the target gene expression of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and stromal cell-derived factor-1 (SDF-1) by real-time PCR.Result:Mesenchymal stem cells appeared morphologically to be ahomogenous population of fibroblast shaped cells. The results showed that the BM-MSCs did not express CD34 nor CD45. However, almost all the cells expressed CD29 and CD90.With the expansion prolonged, the marked area in each group increased. Compared to Group D, the marked areas of Groups A, B and C increased significantly (P<0.01). Measurements of the marked area after removing the expander showed that Groups A, B, and C all had different degrees of retraction, and the remained areas of Group A and Group B after retraction were still larger than that of Group C. Group A’s was statistically larger than Group C’s (P <0.05).BM-MSCs labeled with CM-DiI were observed throughout the subcutaneous fascial layer of flaps on day 7,14, and 28 after the implantation. There was obviously distribution in group A. With the extending of expansion, the cells labeled with CM-DiI increased in group B, which were mainly distributed in the dermis. None CM-DiI-Labeled BM-MSCs were detected in groups C and D. The vascular cross section densities (article/field) of groups A, B, C and D were 9.1±2.31,7.85±1.93,6.5±1.64,4.2±1.54 respectively. PCNA staining showed that positive cells in group A were 5-6 layers under the base of Epidermis, while there were 2-3 layers for group B and there were only scattered single cells for group C. K19 staining showed that under the base of epidermis there were full thickness positive cells for group A and part of the stratified region, while the continuous cells distribution were seen in group B, and there were only scattered cells in group C. Real-time quantitative PCR test results showed the multiples of difference in target gene expression of VEGF, SDF, EGF and bFGF were 5.4,1.07,4.03 and 6.03 times respectively for group A, as compared with group B.Conclusion:1、Transplant of BM-MSCs into local or systemic of expansion model can promote the growth of biological skin.2、local transplantation achieves better results than systemic transplantation.更多还原