Effects of mitochondrial transplantation on full-thickness skin defects in diabetic rats
-
摘要:
目的 探讨线粒体移植对糖尿病大鼠全层皮肤缺损的影响。 方法 该研究为实验研究。提取6~8周龄雄性SD大鼠（鼠龄及性别下同）肝脏组织中功能完整的线粒体。取小鼠L929细胞和人脐静脉内皮细胞（HUVEC）,用含50 mmol/L葡萄糖的培养基培养24 h对细胞造成高糖损伤,随后按照随机数字表法（分组方法下同）将其分为用常规培养基处理的对照组、用含20 U/mL重组人表皮生长因子（rhEGF）的培养基处理的生长因子组和用含12.5 μg/mL外源性线粒体的培养基处理的线粒体组。采用划痕试验检测划痕后6 h时小鼠L929细胞的迁移率；培养2、6 h后,检测HUVEC的成管长度与交叉点数,样本数均为3。培养24 h后,按照试剂盒说明书检测前述2种细胞中的活性氧水平和线粒体膜电位,样本数均为6。取18只SD大鼠,成功制成1型糖尿病模型后于其背部制作直径1 cm的全层皮肤缺损创面。将大鼠分为对照组、生长因子组和线粒体组（每组6只）,均于伤后0（即刻）、3、6 d,分别在其创面皮下注射生理盐水、创面上喷洒等量的剂量为40 U/cm²的rhEGF溶液、创面皮下注射等量的剂量为5 μg/g的线粒体悬液。计算伤后3、6、12 d大鼠剩余创面面积百分比。伤后12 d,行苏木精-伊红染色和Masson染色分别观察大鼠创面中上皮化和胶原沉积情况,采用免疫荧光法检测大鼠创面中新生血管特异性标志物CD31及神经特异性标志物神经丝蛋白200的表达情况,按照试剂盒说明书检测大鼠创面中活性氧水平、凋亡细胞数、ATP含量,采用酶联免疫吸附测定法检测创面中肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、IL-6水平。 结果 划痕后6 h时,与对照组相比,生长因子组小鼠L929细胞迁移率明显升高（P<0.05）；与生长因子组相比,线粒体组小鼠L929细胞迁移率明显升高（P<0.05）。培养2、6 h后,与对照组相比,生长因子组和线粒体组HUVEC的成管交叉点数均显著升高（P<0.05）,成管长度均显著增长（P<0.05）。与生长因子组相比,线粒体组HUVEC培养2、6 h后的成管交叉点数均显著升高（P<0.05）,成管长度均显著增长（P<0.05）。培养24 h后,与对照组相比,生长因子组和线粒体组小鼠L929细胞和HUVEC中活性氧水平均显著降低（P<0.05）,线粒体膜电位均显著升高（P<0.05）；与生长因子组相比,线粒体组小鼠L929细胞和HUVEC中活性氧水平均显著降低（P<0.05）,线粒体膜电位均显著升高（P<0.05）。伤后3、6、12 d,线粒体组大鼠剩余创面面积百分比分别为（46±3）%、（37±3）%、（18±3）%,均明显低于对照组的（71±4）%、（63±4）%、（47±5）%和生长因子组的（62±3）%、（54±3）%、（33±4）%,P<0.05。伤后12 d,线粒体组大鼠创面中上皮化及胶原沉积情况、血管新生及神经修复情况均优于生长因子组和对照组；与对照组相比,生长因子组和线粒体组大鼠创面中活性氧水平均明显降低（P<0.05）,凋亡细胞数均明显减少（P<0.05）,ATP含量均明显升高（P<0.05）,TNF-α、IL-1β、IL-6水平均显著降低（P<0.05）；与生长因子组相比,线粒体组大鼠创面中活性氧水平明显降低（P<0.05）,凋亡细胞数明显减少（P<0.05）,ATP含量明显升高（P<0.05）,TNF-α、IL-1β、IL-6水平均显著降低（P<0.05）。 结论 线粒体移植能够通过促进高糖损伤细胞中线粒体ATP的产生、减少氧化应激水平,改善小鼠L929细胞的迁移能力和HUVEC的血管形成能力；同时可以促进糖尿病大鼠全层皮肤缺损创面的上皮化和胶原沉积,降低炎症因子水平、抑制细胞凋亡,进而加速创面愈合。 Abstract:Objective To explore the effects of mitochondrial transplantation on full-thickness skin defects in diabetic rats. Methods This study was an experimental study. Functionally intact mitochondria were extracted from the liver tissue of 6-8-week-old male Sprague Dawley rats (the same age and sex below). Mouse L929 cells and human umbilical vein endothelial cells (HUVECs) were cultured in medium containing high-glucose (50 mmol/L) for 24 hours to induce high-glucose injury. According to the random number table method (the same grouping method below), they were then divided into control group (Ctrl group, treated with conventional medium), growth factor group (GF group, treated with medium containing 20 U/mL recombinant human epidermal growth factor), and mitochondrial group (Mito group, treated with medium containing 12.5 μg/mL exogenous mitochondria). The scratch assay was performed to evaluate the migration rate of mouse L929 cells at 6 hours after scratching (n=3). The length and number of branch nodes of tube formed by HUVECs were measured after 2 and 6 hours of culture (n=3). After 24 hours of culture, the reactive oxygen species (ROS) levels and mitochondrial membrane potential in aforemetioned two types of cells were detected according to the kit instructions (n=6). Eighteen Sprague Dawley rats were selected and a type 1 diabetic rat model was successfully established. Then, the full-thickness skin defects with a diameter of 1 cm were created on their backs. The rats were divided into Ctrl group, GF group, and Mito group (with each group of 6 rats). At post-injury days (PID) 0 (immediately), 3, and 6, the wounds were subcutaneously injected with normal saline (Ctrl group), topically sprayed with an equal amount of rhEGF solution at a dose of 40 U/cm² (GF group), or subcutaneously injected with equal amount of mitochondrial suspension at a dose of 5 μg/g (Mito group), respectively. The percentage of remaining wound area of rats was calculated at PID 3, 6, and 12. At PID 12, the epithelialization and collagen deposition in the wound of rats were detected by hematoxylin and eosin staining and Masson's staining, respectively. Immunofluorescence staining was used to detect the expression of CD31 (a marker for neovascularization) and neurofilament 200 (a marker for nerves) in the wound of rats. The ROS levels, number of apoptotic cells, ATP content in the wound of rats were detected according to the kit instructions. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 in the wound of rats were detected by enzyme-linked immunosorbent assay. Results At 6 hours after scratching, compared with that in Ctrl group, the migration rate of mouse L929 cells in GF group was significantly increased (P<0.05). compared with that in GF group, the migration rate of mouse L929 cells in Mito group was significantly increased (P<0.05); After 2 and 6 hours of culture, compared with those in Ctrl group, the numbers of branch nodes and the lengths of tube formed by HUVECs in both GF group and Mito group were significantly increased (P<0.05). Compared with those in GF group, the numbers of branch nodes and the lengths of tube formation of HUVECs after 2 and 6 hours of culture in Mito group were significantly increased (P<0.05). After 24 hours of culture, compared with those in Ctrl group, the ROS levels in both mouse L929 cells and HUVECs in GF group and Mito group were significantly decreased (P<0.05), while the mitochondrial membrane potentials were significantly increased (P<0.05); compared with those in GF group, the ROS levels in both mouse L929 cells and HUVECs in Mito group were significantly decreased (P<0.05), while the mitochondrial membrane potentials were significantly increased (P<0.05). At PID 3, 6, and 12, the percentages of remaining wound area of rats in Mito group ((46±3)%, (37±3)%, (18±3)%) were significantly lower than those in Ctrl group ((71±4)%, (63±4)%, (47±5)%) and GF group((62±3)%, (54±3)%, (33±4)%), P<0.05. At PID 12, in the wounds of rats in Mito group, the status of epithelialization and collagen deposition, as well as the conditions of angiogenesis and nerve repair, were superior to those in growth factor and control groups. Compared with those in Ctrl group, the ROS levels and number of apoptotic cells in the wounds of rats in GF group and Mito group were significantly decreased (P<0.05), the ATP content significantly increased (P<0.05), and the levels of TNF-α, IL-1β, and IL-6 were all significantly reduced (P<0.05). Compared with those in GF group, the ROS levels and number of apoptotic cells in the wounds of rats in Mito group were significantly decreased (P<0.05), the ATP content significantly increased (P<0.05), and the levels of TNF-α, IL-1β, and IL-6 were all significantly reduced (P<0.05). Conclusions Mitochondrial transplantation enhances mitochondrial ATP production and reduces the level of oxidative stress in cells under high-glucose injury, which improves the migration capacity of mouse L929 cells and the angiogenesis capacity of HUVECs. Simultaneously, it facilitates epithelialization and collagen deposition in full-thickness skin defect wounds of diabetic rats, reduces the levels of inflammatory cytokines, and inhibits cell apoptosis, thereby accelerating wound healing. -
参考文献
(42) [1] XiongY, LinZ, BuP, et al. A whole-course-repair system based on neurogenesis-angiogenesis crosstalk and macrophage reprogramming promotes diabetic wound healing[J]. Adv Mater, 2023,35(19):e2212300. DOI: 10.1002/adma.202212300. [2] ZhaoH, LiuY. Neutrophil extracellular traps induce fibroblast ferroptosis via IRE1α/XBP1-mediated ER stress to impair diabetic wound healing[J]. Free Radic Biol Med, 2025,236:17-27. DOI: 10.1016/j.freeradbiomed.2025.05.391. [3] HuangX, ZhengL, ZhouY, et al. Controllable adaptive molybdate-oligosaccharide nanoparticles regulate M2 macrophage mitochondrial function and promote angiogenesis via PI3K/HIF-1α/VEGF pathway to accelerate diabetic wound healing[J]. Adv Healthc Mater, 2024,13(3):e2302256. DOI: 10.1002/adhm.202302256. [4] 徐旭英. 《中国糖尿病足防治指南2019》解读[J].中国临床医生杂志,2023,51(4):394-397. [5] WongA, OngB, LeeA, et al. Topical biological agents as adjuncts to improve wound healing in chronic diabetic wounds: a systematic review of clinical evidence and future directions[J]. Cureus, 2022,14(7):e27180. DOI: 10.7759/cureus.27180. [6] ChiuA, SharmaD, ZhaoF. Tissue engineering-based strategies for diabetic foot ulcer management[J]. Adv Wound Care (New Rochelle), 2023,12(3):145-167. DOI: 10.1089/wound.2021.0081. [7] ZhaoX, ZhangY, HuangZ, et al. Innovative therapies for diabetic foot ulcers: application and prospects of smart dressings[J]. Biomed Pharmacother, 2025,191:118498. DOI: 10.1016/j.biopha.2025.118498. [8] JiangG, JiangT, ChenJ, et al. Mitochondrial dysfunction and oxidative stress in diabetic wound[J]. J Biochem Mol Toxicol, 2023,37(7):e23407. DOI: 10.1002/jbt.23407. [9] PrabhakaranHS, HuD, HeW, et al. Mitochondrial dysfunction and mitophagy: crucial players in burn trauma and wound healing[J/OL]. Burns Trauma, 2023,11:tkad029[2025-07-21].https://pubmed.ncbi.nlm.nih.gov/37465279/. DOI: 10.1093/burnst/tkad029. [10] 王齐, 朱冠娅, 谢挺, 等. ATP代谢及嘌呤信号受体在糖尿病创面愈合炎症反应阶段的变化[J].上海交通大学学报(医学版),2020,40(1):10-17. DOI: 10.3969/j.issn.1674-8115.2020.01.002. [11] ZhangZ, HuangQ, ZhaoD, et al. The impact of oxidative stress-induced mitochondrial dysfunction on diabetic microvascular complications[J]. Front Endocrinol (Lausanne), 2023,14:1112363. DOI: 10.3389/fendo.2023.1112363. [12] DengL, DuC, SongP, et al. The role of oxidative stress and antioxidants in diabetic wound healing[J]. Oxid Med Cell Longev, 2021,2021:8852759. DOI: 10.1155/2021/8852759. [13] 徐一凡, 张雨建, 郑嘉溢, 等. 靶向线粒体的糖尿病干预策略及相关药物研究进展[J].药学进展,2025(3):219-227. DOI: 10.20053/j.issn1001-5094.20250009. [14] PantT, UcheN, JuricM, et al. Clinical relevance of lncRNA and mitochondrial targeted antioxidants as therapeutic options in regulating oxidative stress and mitochondrial function in vascular complications of diabetes[J]. Antioxidants (Basel), 2023, 12(4):898. DOI: 10.3390/antiox12040898. [15] Krako JakovljevicN, PavlovicK, JoticA, et al. Targeting mitochondria in diabetes[J]. Int J Mol Sci, 2021,22 (12):6642. DOI: 10.3390/ijms22126642. [16] McCullyJD, CowanDB, PacakCA, et al. Injection of isolated mitochondria during early reperfusion for cardioprotection[J]. Am J Physiol Heart Circ Physiol, 2009,296(1):H94-H105. DOI: 10.1152/ajpheart.00567.2008. [17] HayashidaK, TakegawaR, ShoaibM, et al. Mitochondrial transplantation therapy for ischemia reperfusion injury: a systematic review of animal and human studies[J]. J Transl Med, 2021,19(1):214. DOI: 10.1186/s12967-021-02878-3. [18] LiZ, CaoX, LiuZ, et al. Therapeutic effect of mitochondrial transplantation on burn injury[J]. Free Radic Biol Med, 2024,215:2-13. DOI: 10.1016/j.freeradbiomed.2024.02.019. [19] JiaoQ, XiangL, ChenY. Mitochondrial transplantation: a promising therapy for mitochondrial disorders[J]. Int J Pharm, 2024,658:124194. DOI: 10.1016/j.ijpharm.2024.124194. [20] LiX, GuanY, LiC, et al. Recent advances in mitochondrial transplantation to treat disease[J]. Biomater Transl, 2025,6(1):4-23. DOI: 10.12336/biomatertransl.2025.01.002. [21] LuW, LiX, WangZ, et al. Mesenchymal stem cell-derived extracellular vesicles accelerate diabetic wound healing by inhibiting NET-induced ferroptosis of endothelial cells[J]. Int J Biol Sci, 2024,20(9):3515-3529. DOI: 10.7150/ijbs.97150. [22] ZhouS, WanL, LiuX, et al. Diminished Schwann cell repair responses play a role in delayed diabetes-associated wound healing[J]. Front Physiol, 2022,13:814754. DOI: 10.3389/fphys.2022.814754. [23] WangW, BaiD, WuC, et al. A protocol for constructing a rat wound model of type 1 diabetes[J]. J Vis Exp, 2023(192): e64914. DOI: 10.3791/64914. [24] ZhaoM, WangY, LiL, et al. Mitochondrial ROS promote mitochondrial dysfunction and inflammation in ischemic acute kidney injury by disrupting TFAM-mediated mtDNA maintenance[J]. Theranostics, 2021,11(4):1845-1863. DOI: 10.7150/thno.50905. [25] YaoWD, ZhouJN, TangC, et al. Hydrogel microneedle patches loaded with stem cell mitochondria-enriched microvesicles boost the chronic wound healing[J]. ACS Nano, 2024,18(39):26733-26750. DOI: 10.1021/acsnano.4c06921. [26] McDermottK, FangM, BoultonAJM, et al. Etiology, epidemiology, and disparities in the burden of diabetic foot ulcers[J]. Diabetes Care, 2023,46(1):209-221. DOI: 10.2337/dci22-0043. [27] XuS, LiS, BjorklundM, et al. Mitochondrial fragmentation and ROS signaling in wound response and repair[J]. Cell Regen, 2022,11(1):38. DOI: 10.1186/s13619-022-00141-8. [28] ClarkMA, ShayJW. Mitochondrial transformation of mammalian cells[J]. Nature, 1982,295(5850):605-607. DOI: 10.1038/295605a0. [29] KingMP, AttardiG. Injection of mitochondria into human cells leads to a rapid replacement of the endogenous mitochondrial DNA[J]. Cell, 1988,52(6):811-819. DOI: 10.1016/0092-8674(88)90423-0. [30] WuHC, FanX, HuCH, et al. Comparison of mitochondrial transplantation by using a stamp-type multineedle injector and platelet-rich plasma therapy for hair aging in naturally aging mice[J]. Biomed Pharmacother, 2020,130:110520. DOI: 10.1016/j.biopha.2020.110520. [31] CaiW, ZhangJ, YuY, et al. Mitochondrial transfer regulates cell fate through metabolic remodeling in osteoporosis[J]. Adv Sci (Weinh), 2023,10(4):e2204871. DOI: 10.1002/advs.202204871. [32] MoskowitzovaK, OrfanyA, LiuK, et al. Mitochondrial transplantation enhances murine lung viability and recovery after ischemia-reperfusion injury[J]. Am J Physiol Lung Cell Mol Physiol, 2020,318(1):L78-L88. DOI: 10.1152/ajplung.00221.2019. [33] JavaniG, BabriS, FarajdokhtF, et al. Mitochondrial transplantation improves anxiety- and depression-like behaviors in aged stress-exposed rats[J]. Mech Ageing Dev, 2022,202:111632. DOI: 10.1016/j.mad.2022.111632. [34] ZhaoZ, HouY, ZhouW, et al. Mitochondrial transplantation therapy inhibit carbon tetrachloride-induced liver injury through scavenging free radicals and protecting hepatocytes[J]. Bioeng Transl Med, 2021,6(2):e10209. DOI: 10.1002/btm2.10209. [35] YuZ, HouY, ZhouW, et al. The effect of mitochondrial transplantation therapy from different gender on inhibiting cell proliferation of malignant melanoma[J]. Int J Biol Sci, 2021,17(8):2021-2033. DOI: 10.7150/ijbs.59581. [36] ParkKH, HanSH, HongJP, et al. Topical epidermal growth factor spray for the treatment of chronic diabetic foot ulcers: a phase Ⅲ multicenter, double-blind, randomized, placebo-controlled trial[J]. Diabetes Res Clin Pract, 2018,142:335-344. DOI: 10.1016/j.diabres.2018.06.002. [37] ZhaoDY, SuYN, LiYH, et al. Efficacy and safety of recombinant human epidermal growth factor for diabetic foot ulcers: a systematic review and meta-analysis of randomised controlled trials[J]. Int Wound J, 2020,17(4):1062-1073. DOI: 10.1111/iwj.13377. [38] RibeiroFM, VolpatoH, Lazarin-BidóiaD, et al. The extended production of UV-induced reactive oxygen species in L929 fibroblasts is attenuated by posttreatment with Arrabidaea chica through scavenging mechanisms[J]. J Photochem Photobiol B, 2018,178:175-181. DOI: 10.1016/j.jphotobiol.2017.11.002. [39] DuH, LiS, LuJ, et al. Single-cell RNA-seq and bulk-seq identify RAB17 as a potential regulator of angiogenesis by human dermal microvascular endothelial cells in diabetic foot ulcers[J/OL]. Burns Trauma, 2023,11:tkad020[2025-07-21]. https://pubmed.ncbi.nlm.nih.gov/37605780/. DOI: 10.1093/burnst/tkad020. [40] ShepherdJ, SarkerP, RimmerS, et al. Hyperbranched poly(NIPAM) polymers modified with antibiotics for the reduction of bacterial burden in infected human tissue engineered skin[J]. Biomaterials, 2011,32(1):258-267. DOI: 10.1016/j.biomaterials.2010.08.084. [41] McCullyJD, LevitskyS, Del NidoPJ, et al. Mitochondrial transplantation for therapeutic use[J]. Clin Transl Med, 2016,5(1):16. DOI: 10.1186/s40169-016-0095-4. [42] ShanmughapriyaS, LangfordD, NatarajaseenivasanK. Inter and intracellular mitochondrial trafficking in health and disease[J]. Ageing Res Rev, 2020,62:101128. DOI: 10.1016/j.arr.2020.101128. -
图 1 从大鼠肝脏中提取的线粒体及其结构完整性与质量验证。1A.线粒体（阳性染色为红色）被成功提取 线粒体红色荧光探针×10；1B.可见线粒体内、外膜连续,嵴结构清晰 透射电子显微镜×140 000；1C、1D.分别为提取的线粒体呈红色荧光（正常线粒体）和绿色荧光（受损或膜电位下降的线粒体）显色情况 JC-1×20
图 2 共孵育3 h后外源性线粒体与小鼠L929细胞和HUVEC的共定位情况 4′,6-二脒基-2-苯基吲哚-线粒体绿色荧光探针-线粒体红色荧光探针×60。2A、2B.分别为小鼠L929细胞和HUVEC,2种细胞的胞质内均可见外源性线粒体和内源性线粒体共存
注：细胞核阳性染色为蓝色,细胞内线粒体阳性染色为绿色,外源性线粒体阳性染色为红色,图片显示颜色为3种颜色的复合色；HUVEC为人脐静脉内皮细胞
图 3 划痕后即刻及6 h时3组小鼠L929细胞迁移情况 倒置荧光显微镜×10。3A、3B、3C.分别为对照组、生长因子组、线粒体组细胞划痕后即刻的划痕面积,基本相近；3D、3E、3F.分别为对照组、生长因子组、线粒体组细胞划痕后6 h时划痕面积,依次减小
注：对照组、生长因子组、线粒体组细胞分别于高糖损伤24 h后加入常规培养基及含重组人表皮生长因子的培养基和含外源性线粒体的培养基进行培养
图 4 培养2 h及6 h后3组人脐静脉内皮细胞的成管情况 倒置荧光显微镜×10。4A、4B、4C.分别为对照组、生长因子组、线粒体组细胞培养2 h后的成管情况,图4C成管交叉点数明显多于图4A、4B,图4C成管长度明显长于图4A、4B；4D、4E、4F.分别为对照组、生长因子组、线粒体组细胞培养6 h后的成管情况,图4D中的成管交叉点数最少,成管长度最短,图4F中的成管长度最长,成管交叉点数最多
注：对照组、生长因子组、线粒体组细胞分别于高糖损伤24 h后加入常规培养基及含重组人表皮生长因子的培养基和含外源性线粒体的培养基进行培养
图 5 培养24 h后3组小鼠L929细胞和HUVEC中活性氧水平 4′,6-二脒基-2-苯基吲哚-2',7'-二氯二氢荧光素二乙酸酯×10。5A、5B、5C.分别为对照组、生长因子组、线粒体组小鼠L929细胞,活性氧水平依次降低；5D、5E、5F.分别为对照组、生长因子组、线粒体组HUVEC,活性氧水平依次降低
注：对照组、生长因子组、线粒体组细胞分别于高糖损伤24 h后加入常规培养基及含重组人表皮生长因子的培养基和含外源性线粒体的培养基进行培养；细胞核阳性染色为蓝色,细胞中活性氧阳性染色为绿色；HUVEC为人脐静脉内皮细胞
图 6 培养24 h后3组小鼠L929细胞和HUVEC中线粒体膜电位情况 4′,6-二脒基- 2-苯基吲哚-JC-1×20。6A、6B、6C.分别为对照组、生长因子组、线粒体组小鼠L929细胞,图6A较图6B和6C中的线粒体膜电位下降明显,图6C中正常线粒体的红色荧光明显强于图6A和6B；6D、6E、6F.分别为对照组、生长因子组、线粒体组HUVEC,图6D较图6E和6F中的线粒体膜电位下降明显,图6F中正常线粒体的红色荧光明显强于图6D和6E
注：对照组、生长因子组、线粒体组细胞分别于高糖损伤24 h后加入常规培养基及含重组人表皮生长因子的培养基和含外源性线粒体的培养基进行培养；细胞核阳性染色为蓝色,受损或膜电位下降的线粒体阳性染色为绿色,正常线粒体阳性染色为红色,图片显示颜色为3种颜色的复合色；HUVEC为人脐静脉内皮细胞
图 7 伤后各时间点3组糖尿病大鼠全层皮肤缺损创面的愈合情况。7A、7B、7C.分别为对照组大鼠伤后0（即刻）6、12 d创面情况,呈逐渐愈合趋势；7D、7E、7F.分别为生长因子组大鼠伤后0、6、12 d创面情况,图7E、7F创面面积分别明显小于图7B、7C；7G、7H、7I.分别为线粒体组大鼠伤后0、6、12 d创面情况,图7H、7I创面面积分别明显小于图7E、7F
注：对照组、生长因子组、线粒体组大鼠创面分别采用生理盐水、重组人表皮生长因子和含提取的外源性线粒体溶液处理；白色橡胶圈内、外圈直径分别为16、20 mm
图 8 伤后12 d的3组糖尿病大鼠全层皮肤缺损创面中上皮化和胶原沉积情况。8A、8B、8C.分别为对照组、生长因子组和线粒体组上皮化情况,图8C的上皮化情况优于图8A、8B 苏木精-伊红×40；8D、8E、8F.分别为对照组、生长因子组和线粒体组胶原沉积情况,图8F较图8D、8E胶原沉积更明显,分布更加有序和紧实 Masson×40
注：对照组、生长因子组、线粒体组大鼠创面分别采用生理盐水、重组人表皮生长因子和含提取的外源性线粒体溶液处理；图8A、8B、8C中的双向箭头之间的距离指示新生上皮长度
图 9 伤后12 d的3组糖尿病大鼠全层皮肤缺损创面中血管再生和神经修复情况。9A、9B、9C.分别为对照组、生长因子组和线粒体组血管再生情况,图9C中血管再生情况优于图9A、9B 4′,6-二脒基-2-苯基吲哚-Alexa Fluor 594×10；9D、9E、9F.分别为对照组、生长因子组和线粒体组神经修复情况,图9D、9E、9F中的再生神经数目依次增多 4′,6-二脒基-2-苯基吲哚-Alexa Fluor 488×10
注：对照组、生长因子组、线粒体组大鼠创面分别采用生理盐水、重组人表皮生长因子和含提取的外源性线粒体溶液处理；细胞核阳性染色为蓝色,新生血管特异性标志物CD31阳性染色为红色,神经特异性标志物神经丝蛋白200阳性染色为绿色,箭头指示阳性染色区域
图 10 伤后12 d的3组糖尿病大鼠全层皮肤缺损创面中活性氧水平和细胞凋亡情况。10A、10B、10C.分别为对照组、生长因子组和线粒体组创面中活性氧水平,图10C中活性氧水平显著低于图10A、10B 4′,6-二脒基-2-苯基吲哚-2',7'-二氯二氢荧光素二乙酸酯×10；10D、10E、10F.分别为对照组、生长因子组和线粒体组细胞凋亡情况,图10D中凋亡细胞数明显多于图10E、10F,图10F中凋亡细胞最少 4′,6-二脒基-2-苯基吲哚-异硫氰酸荧光素×10
注：对照组、生长因子组、线粒体组大鼠创面分别采用生理盐水、重组人表皮生长因子和含提取的外源性线粒体溶液处理；细胞核阳性染色为蓝色,细胞内活性氧、凋亡细胞阳性染色均为绿色
Table 1. 培养2 h及6 h后3组人脐静脉内皮细胞的成管交叉点数及长度比较（
组别 样本数 成管交叉点数（个） 成管长度（μm） 2 h 6 h 2 h 6 h 对照组 3 10.3±1.2 23.7±3.3 417±107 1650±167 生长因子组 3 30.3±3.9a 50.7±3.3a 3171±817a 4175±711a 线粒体组 3 41.0±2.2ab 65.3±2.9ab 4904±223ab 6518±2981ab F值 68.88 89.34 41.63 59.41 P值 <0.001 <0.001 <0.001 <0.001 注：对照组、生长因子组、线粒体组细胞分别于高糖损伤24 h后加入常规培养基及含重组人表皮生长因子的培养基和含外源性线粒体的培养基进行培养；成管交叉点数及长度的处理因素主效应,F值分别为158.10、98.76,P值均<0.001；时间因素主效应,F值分别为131.60、22.20,P值均<0.001；二者交互作用,F值分别为3.64、0.43,P值分别为0.058、0.663；与对照组比较,aP<0.05；与生长因子组比较,bP<0.05 
下载: 导出CSV
Table 2. 伤后各时间点3组糖尿病大鼠剩余全层皮肤缺损创面面积百分比比较（%,
组别 样本数 3 d 6 d 12 d 对照组 3 71±4 63±4 47±5 生长因子组 3 62±3 54±3 33±4a 线粒体组 3 46±3ab 37±3ab 18±3ab F值 28.28 26.74 25.82 P值 <0.001 0.001 0.001 注：对照组、生长因子组、线粒体组大鼠创面分别采用生理盐水、重组人表皮生长因子和含提取的外源性线粒体溶液处理；处理因素主效应,F=154.60,P<0.001；时间因素主效应,F=386.20,P<0.001；二者交互作用,F=7.44,P<0.001；与对照组比较,aP<0.05；与生长因子组比较,bP<0.05
下载: 导出CSV
Table 3. 伤后12 d的3组糖尿病大鼠全层皮肤缺损创面中各指标比较（
组别 样本数 活性氧水平 凋亡细胞数（个） ATP含量 对照组 6 72±21 39.4±13.3 3.04±0.27 生长因子组 6 46±17a 15.4±5.5a 3.83±0.32a 线粒体组 6 19±6ab 8.6±2.1ab 4.63±0.68ab F值 15.19 18.50 14.85 P值 <0.001 <0.001 <0.001 注：对照组、生长因子组、线粒体组大鼠创面分别采用生理盐水、重组人表皮生长因子和含提取的外源性线粒体溶液处理；与对照组比较,aP<0.05；与生长因子组比较,bP<0.05
下载: 导出CSV
Table 4. 伤后12 d的3组糖尿病大鼠全层皮肤缺损创面中炎症因子水平比较（pg/mL,
组别 样本数 TNF-α IL-1β IL-6 对照组 6 309±25 160±29 240±31 生长因子组 6 256±40a 111±30a 182±29a 线粒体组 6 172±25ab 64±21ab 122±35ab F值 25.37 15.74 17.23 P值 <0.001 <0.001 <0.001 注：对照组、生长因子组、线粒体组大鼠创面分别采用生理盐水、重组人表皮生长因子和含提取的外源性线粒体溶液处理；TNF为肿瘤坏死因子,IL为白细胞介素；与对照组比较,aP<0.05；与生长因子组比较,bP<0.05
下载: 导出CSV
-
李玉骞.mp4
-
下载:
图(11) / 表(4)
计量
- 文章访问数: 190
- HTML全文浏览量: 34
- PDF下载量: 22
- 被引次数: 0
