摘要
目的探讨富血小板血浆(platelet rich plasma,PRP)与人毛囊干细胞(hair follicle stem cells,HFSCs)共移植对大鼠深Ⅱ度烧伤创面愈合的作用及其机制。方法通过细胞计数试剂盒(cell counting kit 8,CCK-8)细胞增殖实验、细胞划痕实验探究PRP在体外培养条件下对人HFSCs增殖、迁移的影响。使用28只SD大鼠建立深Ⅱ度烧伤模型,随机取4只造模后大鼠的烧伤创面组织和正常皮肤组织,经苏木精-伊红(hematoxylin and eosin,HE)染色后观察鉴定模型建立是否成功。将造模成功的24只大鼠按随机数字表法分为对照组[在烧伤创面组织下多点注射500μl磷酸盐缓冲液(phosphate buffered saline,PBS)]、HFSCs组(在烧伤创面组织下多点注射500μl PBS混匀的人HFSCs悬液)、PRP组(在烧伤创面组织下多点注射500μl PRP)、PRP+HFSCs组(在烧伤创面组织下多点注射PRP混匀的人HFSCs悬液),每组6只。各组每周给予2次相应干预。干预第7、14、20天计算各组大鼠烧伤创面的愈合率;第20天取各组大鼠烧伤创面组织,通过免疫组织化学染色技术检测CD31阳性血管数;通过Masson染色观察烧伤创面组织胶原纤维再生情况;通过实时荧光逆转录聚合酶链反应(reverse transcription quantitative polymerase chain reaction,RT-PCR)检测烧伤创面组织中白细胞介素1β(interleukin 1 beta,IL-1β)、超氧化物歧化酶1(superoxide dismutase 1,SOD1)、B淋巴细胞瘤2基因(B-cell lymphoma 2,Bcl-2)、转化生长因子β(transforming growth factor beta,TGF-β)和基质金属蛋白酶9(matrix metalloproteinase 9,MMP-9)基因的mRNA相对表达量;通过Western blot法检测烧伤创面组织中IL-1β、SOD1和Bcl-2蛋白的相对表达量。结果在培养基中使用10%PRP能显著增强HFSCs的增殖与迁移能力。与对照组、HFSCs组、PRP组比较,在干预第7、14、20天,PRP+HFSCs组大鼠创面组织愈合率最高(P均<0.05);第20天,PRP+HFSCs组CD31阳性血管数最多(P<0.05),IL-1β、TGF-β、MMP-9的mRNA表达水平最低,Bcl-2、SOD1的mRNA与蛋白表达水平最高(P均<0.05),IL-1β蛋白表达水平最低(P均<0.05)。此外,与对照组、HFSCs组、PRP组比较,PRP+HFSCs组大鼠烧伤创面组织胶原纤维最致密有序,无过度胶原纤维沉积。结论在体外培养条件下,PRP可促进人HFSCs增殖与迁移。PRP与人HFSCs共移植可以控制烧伤部位炎症反应与氧化应激,促进新生血管形成,从而加快创面愈合。
Objective To investigate the effects and mechanism of co-transplantation of platelet rich plasma(PRP)and human hair follicle stem cells(HFSCs)on deep second-degree burn wounds in rats.Methods Cell counting kit 8(CCK-8)cell proliferation experiment and cell scratch experiment were used to explore the effect of PRP on the proliferation and migration of human HFSCs under in vitro culture conditions.Twenty-eight SD rats were used to establish deep second-degree burn wound model,the burn wound tissues and normal skin tissues of 4 rats after modeling were randomly selected to observe and identify whether the model was successfully established by hematoxylin and eosin(HE)staining.Twenty-four rats with successful modeling were randomly divided into control group[injection of 500μl phosphate buffered saline(PBS)under burn wound tissues at multiple point injections],HFSCs group(the human HFSCs suspension mixed with 500μl PBS was injected at multiple points under the burn wound tissues),PRP group(500μl PRP was injected at multiple points under the burn wound tissues)and PRP+HFSCs group(the HFSCs suspension mixed with PRP was injected at multiple points under the burn wound tissues)by random number table method,with 6 rats in each.Injections were administered twice weekly around the burn area.Burn wound healing rates were calculated on the 7th,14th and 20th days of the interventions;on the 20th day of the interventions,burn wound tissues was harvested for further analysis,immunohistochemical staining was conducted to quantify number of CD31 positive vessels,and Masson staining was performed to observe collagen fiber regeneration in burn wound tissues;reverse transcription quantitative polymerase chain reaction(RT-PCR)was used to assess the mRNA relative expression levels of interleukin 1 beta(IL-1β),superoxide dismutase 1(SOD1),B-cell lymphoma 2(Bcl-2),transforming growth factor beta(TGF-β)and matrix metalloproteinase 9(MMP-9)in burn wound tissues;Western blot was conducted to evaluate the protein relative expression levels of IL-1β,SOD1 and Bcl-2 in burn wound tissues.Results The 10%PRP in the medium enhanced the proliferation and migration of HFSCs.Compared with the control group,HFSCs group and PRP group,the PRP+HFSCs group demonstrated the highest wound healing rates on the 7th,14th and 20th days of the interventions(all P<0.05);on the 20th day of the interventions,the PRP+HFSCs group exhibited the highest number of CD31 positive vessels(P<0.05),the mRNA and protein expression levels of pro-inflammatory factor IL-1β,TGF-βand MMP-9 were the lowest,and the mRNA expression levels of Bcl-2 and SOD1 were the highest(all P<0.05),the protein expression level of IL-1βwas the lowest(all P<0.05).Furthermore,compared with the control,HFSCs and PRP groups,the burn wound tissues of rats in PRP+HFSCs group exhibited the most compact and organized collagen fibers without excessive collagen deposition.Conclusion PRP can promote the proliferation and migration of human HFSCs in vitro.The co-transplantation of PRP and human HFSCs can control the inflammatory response and oxidative stress at the burn site,promote the formation of new blood vessels,thereby accelerating the wound healing.
作者
徐林胜子
邱伟明
王钰
刘跃平
刁波
XU Linshengzi;QIU Weiming;WANG Yu;LIU Yueping;DIAO Bo(School of Medicine,Wuhan University of Science and Technology,Wuhan Hubei 430062,China)
出处
《联勤军事医学》
2025年第5期378-385,402,共9页
Military Medicine of Joint Logistics
基金
湖北省卫生健康委员会面上项目(WJ2019M263)。
关键词
深Ⅱ度烧伤
富血小板血浆
毛囊干细胞移植
血管生成
创面愈合
瘢痕
Deep second-degree burn
Platelet rich plasma
Hair follicle stem cells transplantation
Angiogenesis
Wound healing
Scar
