Exploration of the mechanism of human platelet-rich plasma in regulating and controlling human epidermal stem cells for promoting wound re-epithelialization at transcriptome level
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摘要: 目的 分析人富血小板血浆(PRP)调控人表皮干细胞(ESC)的靶基因。 方法 (1)收集陆军军医大学第一附属医院行泌尿外科手术的6例男性患者术后弃用的包皮组织,患者年龄为5~25岁,既往身体健康,无泌尿系统感染,采用快速贴壁法培养人ESC并进行形态学观察及鉴定。收集解放军南部战区总医院1名29岁女性健康志愿者静脉血40 mL,采用二次离心法提取PRP。(2)将培养成功的原代人ESC按照随机数字表法分为对照组和PRP处理组,每组3孔。对照组细胞不行特殊处理;PRP处理组待细胞贴壁12 h,在培养基中加入PRP,使其最终体积分数为2.5%。提取RNA应用RNA测序技术进行2组人ESC转录组测序和数据分析,以错误发现率<0.05、差异倍数≥4为标准应用Dr. Tom数据挖掘系统筛选差异表达基因,对获得的差异表达基因进行基因本体论(GO)富集分析找出显著富集的GO条目。再用京都基因和基因组(KEGG)信号通路注释分析进一步寻找差异表达基因可能参与的生物学过程或者代谢通路。最后,选取与再上皮化过程相关且差异表达明显的基因,利用实时荧光定量反转录PCR验证基因的差异表达情况。对数据行独立样本
t 检验。 结果 (1)培养细胞呈克隆样生长,形态为铺路石样,CD49f阳性率达95.132%、CD71阳性率为0.006%,证明ESC原代培养成功。(2)质控数据分析显示,选取样本质量较好,序列比对百分比较高,满足测序要求。(3)测序数据显示,2组间共有449个差异表达基因,其中上调基因354个、下调基因95个,进一步聚类分析确定2组间有18个显著上调基因和5个显著下调基因。GO富集分析以及KEGG信号通路注释分析表明,显著差异表达基因主要富集在表皮构建和角化过程,同时可能与白细胞介素17信号通路相关。(4)选取了与再上皮化过程相关且差异表达明显的角蛋白19、角蛋白10以及S100A7基因进行验证。实时荧光定量反转录PCR显示,与对照组比较,PRP处理组细胞角蛋白19 mRNA和S100A7 mRNA表达量明显升高(t =10.270、5.690,P <0.01),角蛋白10 mRNA表达量明显降低(t =7.306,P <0.01),与测序数据结果一致。 结论 PRP调节人ESC功能促进创面再上皮化涉及角蛋白19、角蛋白10以及S100A7等多个基因的转录调控,深入探讨PRP影响人ESC的可能调控网络将为其后续临床治疗提供依据。Abstract: Objective To analyze target genes of human platelet-rich plasma (PRP) in regulating and controlling human epidermal stem cells (ESCs). Methods (1) The discarded foreskin tissues were collected from 6 male patients of the First Affiliated Hospital of Army Medical University after urological surgery. The patients aged 5 to 25 years with good health and without urinary system infection. Human ESCs were cultivated using quick attachment method, and were subjected to morphological observation and identification. Venous blood sample in the volume of 40 mL was collected from a female healthy volunteer (aged 29 years) of General Hospital of Southern Theater Command of PLA, and PRP was extracted by second centrifugation method. (2) The successfully cultured primary human ESCs were divided into control group and PRP group according to the random number table, with 3 wells in each group. The cells in control group were not specially treated. In PRP group, PRP was added to the ESC medium to achieve final volume fraction of 2.5% after the cells were adhered for 12 hours. RNA was extracted, and transcriptome sequencing and data analysis of human ESCs of two groups were performed using RNA sequencing technology. Using the false discovery rate less than 0.05 and the fold change more than or equal to 4 as the standard, the differentially expressed genes were screened by Dr. Tom data mining system. Gene ontology (GO) enrichment analysis was performed on the obtained differentially expressed genes to find out the GO entries with significant enrichment. Then Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation analysis was used to further analyze the biological processes or metabolic pathways in which differentially expressed genes might be involved. Finally, the genes related to re-epithelialization and significantly differentially expressed were selected, and the differential expression of genes was verified by real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR). Data were statistically analyzed with independent-samplest test. Results (1) The cultured cells were cloned with a paving stone-like shape and positive rate of CD49f of 95.132% and CD71 of 0.006%, which proved that the primary culture of ESCs was successful. (2) The quality control data analysis showed that the selected samples had better quality and higher sequence alignment rate, which met the requirements of sequencing. (3) Sequencing data showed that there were a total of 449 differentially expressed genes between the two groups, including 354 up-regulated genes and 95 down-regulated genes. Further cluster analysis determined that there were 18 significantly up-regulated genes and 5 significantly down-regulated genes between the two groups. GO enrichment analysis and KEGG pathway annotation analysis showed that the significantly differentially expressed genes were mainly enriched in the epidermis construction and keratinization process, which also might be related to interleukin 17 signaling pathway. (4) Keratin 19, keratin 10, and S100A7 genes which were related to the process of re-epithelialization and significantly differentially expressed were selected for verification. Real-time fluorescent quantitative RT-PCR showed that compared with those of control group, the mRNA expressions of keratin 19 and S100A7 of cells in PRP group were significantly increased (t =10.270, 5.690,P <0.01), while the mRNA expression of keratin 10 was significantly decreased (t =7.306,P <0.01), which was consistent with the result of sequencing data. Conclusions PRP regulates function of human ESCs and promotes wound re-epithelialization involving transcriptional regulation of multiple genes, including keratin 19, keratin 10, and S100A7. In-depth exploration of the possible regulatory network of PRP affecting human ESCs will provide the basis for its subsequent clinical application. -
(1)采用数字减影血管造影技术定位分析旋股外侧动脉横支,直观了解了横支的出现情况和发出位置、共干情况、走行情况等形态学特点。
以旋股外侧动脉不同分支为蒂的股前外侧皮瓣被广泛应用于四肢软组织损伤的修复 [ 1, 2, 3] 。在旋股外侧动脉各分支中,横支具有解剖学位置较高、供区隐蔽、供皮量大等特点,可为股前外侧皮瓣设计方案提供更多选择 [ 4, 5, 6] 。同时,在旋股外侧动脉的降支与斜支变异或缺如时,可将横支用作股前外侧皮瓣的血管蒂 [ 7, 8] 。然而,目前横支的形态学特点,包括其与旋股外侧动脉其他分支尤其是斜支的相对关系仍需系统性阐明。为明确横支的形态学特征,从而为股前外侧皮瓣设计提供依据,本课题组对四肢软组织损伤患者行股前外侧皮瓣修复术前数字减影血管造影(DSA),对成像资料中旋股外侧动脉横支的形态学特点进行分析并讨论其临床意义。
1. 对象与方法
本回顾性观察性研究符合《赫尔辛基宣言》的基本原则。
1.1 入选标准
纳入标准:采用股前外侧皮瓣修复四肢软组织缺损创面,且术前行下肢DSA定位。排除标准:造影剂充盈不良,血管显影不佳;合并脉管炎、下肢动脉闭塞、动脉瘤等动脉性疾病。
1.2 临床资料
2020年10月—2021年5月,苏州瑞华骨科医院收治62例符合入选标准的四肢软组织损伤患者,其中男40例、女22例,年龄20~72岁(平均50岁),包括机器绞伤者12例、机器压伤者27例、交通伤者23例,均以股前外侧皮瓣修复四肢软组织缺损,术前行下肢DSA检查,均签署特殊检查知情同意书。
1.3 检查方法及相关处理
患者取仰卧位(术中保持体位不变),术前以安尔碘皮肤消毒剂对双侧腹股沟区进行消毒,铺无菌洞巾。于皮瓣供区对侧腹股沟区股动脉浅表搏动点下方2~3 cm处选择穿刺点,常规进行局部浸润麻醉。以髂前上棘内侧水平10 cm为起点,与髂前上棘和髌骨外侧缘的连线平行固定显影标尺。使用改良Seldinger法 [ 9] 置入外径4 F血管穿刺鞘,在直径0.035 mm泥鳅导丝导引下置入外径4 F的Cobra导管或单弯导管,将导管置入股深动脉开口处后,用造影剂注射器(压力2.07 MPa)注入碘克沙醇(流速4 mL/s、总量12 mL)进行首次造影,采集图像,初步观察旋股外侧动脉类型。随后,在泥鳅导丝辅助下推进Cobra导管或单弯导管至旋股外侧动脉处,拔除导丝,行第2次造影(压力1.38 MPa、流速3 mL/s、总量8 mL)并采集图像。拔除导管及血管穿刺鞘,由医师按压穿刺点15 min,密切关注患者出血情况,按压后以无菌纱布覆盖并予弹力胶布固定。返回病房后予沙袋压迫穿刺点4 h、心电监护2 h,按期常规复查肝肾功能。
1.4 观察指标与方法
使用TView 6.0.1.2104图像分析软件(上海卫宁健康科技集团股份有限公司)结合显影标尺对造影采集的图像进行测量分析,观测下肢血管大体情况以及旋股外侧动脉横支的出现情况(横支细小或横支缺如均判定为无横支,计算出现率)、源动脉、发出点位置、走行方向与皮穿支穿出点位置,另进行横支形态学特点分类。
2. 结果
2.1 下肢血管大体情况
DSA检查显示,62例患者股动脉、股深动脉及旋股外侧动脉各分支均清晰可辨。
2.2 旋股外侧动脉横支大体情况
在59例患者中观察到旋股外侧动脉横支,其中52例为单横支、7例为双横支,横支出现率为95.2%(59/62)。共观察到66条旋股外侧动脉横支,其中3条发自股深动脉、63条发自旋股外侧动脉。旋股外侧动脉横支发出点多位于髂前上棘与髌骨外侧缘连线近端,距髂前上棘6.5~12.7 cm,平均9.7 cm,见 图1、 2。旋股外侧动脉横支约与身体长轴垂直向外侧发出,在旋股外侧动脉升支与斜支之间走行,沿途发出分支,主干走行至大转子下方。旋股外侧动脉横支皮穿支穿出点距髂前上棘8.0~18.0 cm,平均13.8 cm。
2.3 旋股外侧动脉横支形态学特点分类
旋股外侧动脉横支形态学特点分类中,以与1条旋股外侧动脉其他分支共干发出者最为常见,占50.0%(31/62);其次为自旋股外侧动脉(12例)或股深动脉(3例)单干发出者,占24.2%(15/62);特殊类型者占21.0%(13/62),包括双横支者7例、与多条旋股外侧动脉其他分支共干者6例;横支细小/缺如者仅占4.8%(3/62)。在前述双支共干关系中,以升支与横支共干者最为常见,占77.4%(24/31);横支与斜支共干者(5例)、横支与降支共干者(2例)共占22.6%(7/31)。见 图3。
3. 讨论
经典的股前外侧皮瓣以旋股外侧动脉降支为蒂,在修复较大面积的软组织缺损时,单穿支有时难以满足血供需求;且降支多为肌皮穿支,解剖游离时对供区肌肉损伤较大;当降支发出穿支细小时,不利于血管吻合 [ 10, 11, 12, 13, 14, 15] 。为确保皮瓣血供、减少供区肌肉损伤带来的并发症,高位穿支已成为股前外侧皮瓣血管蒂的重要选择 [ 16] 。由于术中视野的限制,按照传统方式设计股前外侧皮瓣难以解剖到高位穿支,对横支的形态学认识尚有不足。因此,本课题组术前对股前外侧区进行DSA定位寻找合适穿支,通过探讨横支的形态学特点以指导临床应用。
穿支常用影像学定位技术有超声、CT血管造影(CTA)、磁共振血管造影(MRA)和DSA等,后三者均基于造影技术,穿支检出率高 [ 17, 18, 19, 20] 。与MRA、CTA相比,DSA属于有创操作,费用较高 [ 21] 。CTA及MRA可以通过三维技术对目标血管进行多角度观察,但是其准确度受患者身体质量指数、仪器设备要求等因素影响较大 [ 22, 23, 24, 25] 。与CTA和MRA相比,DSA直接置管于目标血管,局部对比剂浓度高,管腔充盈效果好,局部血管对比度、锐利度高 [ 26] 。此外,DSA图像质量稍优于CTA和MRA。本研究中,患者均采用DSA定位股前外侧区血管,各动脉分支清晰可辨。
横支是旋股外侧动脉的主要分支,本研究观察到横支出现率为95.2%。横支多位于升支的下方和斜支的外侧,在与身体水平轴成角约15°的范围内走行,可略向下或向上。横支与其他分支间存在一定的联系 [ 27] ,其常与升支、斜支、降支共干发出(其中与升支共干最为多见),多表现为旋股外侧动脉主干自发出后,向外下走行一段距离后分为上下2支,上支向外上走行,沿途发出横支与升支;下支向外下走行,发出斜支与降支 [ 27, 28] 。本研究显示,旋股外侧动脉横支的发出点距髂前上棘平均距离为9.7 cm,而横支皮穿支穿出点距髂前上棘平均距离为13.8 cm,解剖学位置偏高,提示若以横支为蒂设计皮瓣,具有位置隐蔽、供皮面积较大等优点。
本课题组根据本组患者股前外侧区旋股外侧动脉横支形态学特点,将其分为4类:(1)横支直接由旋股外侧动脉或股深动脉发出,与其他穿支无任何共干关系,即单干发出。(2)横支与1条旋股外侧动脉其他分支由同一源自旋股外侧动脉或股深动脉的短干发出,包括与升支共干、与斜支共干、与降支共干,即共干发出。(3)旋股外侧动脉或股深动脉发出2条横支,或横支与多条旋股外侧动脉其他分支共干,即特殊类型。(4)无横支或横支口径细小,DSA无法探查。以上横支形态学特点分类,可为临床实践提供下述参考:(1)单干发出的横支,血管解剖较为简单,是减轻供区肌肉损伤,增加手术成功率的合适选择。(2)针对共干发出的横支,可联合共干穿支设计多叶皮瓣、嵌合皮瓣、血流桥接皮瓣,为修复合并血管、肌肉等复合组织损伤或大面积软组织缺损提供设计方案。(3)源动脉发出双横支或横支与多条源动脉其他分支共干时,大腿近端血管丰富,横支血管走行较为复杂,需格外谨慎,尽可能采取逆行解剖法,追溯源动脉,避免损伤血管。(4)横支细小/缺如时,不宜选取横支设计股前外侧皮瓣,以避免手术失败。
基于DSA进行血管定位有以下优点:(1)可进行动态的旋股外侧动脉分支成像研究,以更全面地了解血管的形态与分布,有助于制订较具体的手术方案,便于术前模拟设计双叶皮瓣、嵌合皮瓣等 [ 29] 。(2)术前可以准确掌握血管走行分布,明确穿支与源动脉之间关系,避免术中盲目分离,为皮瓣的设计及切取提供依据,减少血管损伤。(3)结合显影标尺,明确穿支血管长度及供血范围,为皮瓣切取范围定位提供参考依据。(4)定位时可使指定穿支管腔充盈,便于术前模拟与受区血管吻合,对于避免吻合口堵塞、降低动脉危象发生概率具有重要意义 [ 30] 。但基于DSA进行血管定位也存在以下不足:(1)以碘克沙醇为造影剂,尽管不良反应较少,但仍有可能引发过敏反应、碘中毒、神经系统并发症 [ 31, 32, 33] 。(2)DSA是一种有创操作,存在X线辐射影响 [ 34] 。(3)DSA造影图像为二维图像,存在空间重叠可能,可造成将深部血管误判为表浅穿支的情况。
在DSA定位及参考定位结果进行手术时,需要注意以下几点:(1)行股前外侧区DSA时,常于腹股沟区股动脉浅表处寻找穿刺点,以便穿刺。但是肥胖患者股动脉位置较深,穿刺难度较大,反复穿刺可能造成穿刺点血肿或假性动脉瘤 [ 35] ,需密切关注患者出血情况,出现穿刺部位出血或血肿时,利用1 kg沙袋持续压迫穿刺点4~6 h,可换对侧继续穿刺造影。(2)一旦造影过程中观察到过敏反应,应立即停止造影,积极抢救;对于甲亢患者或有甲亢病史的患者,严禁进行DSA。(3)定位过程中需要患者保持特定体位,避免血管偏移正常位置,术后需复查肝肾功能。(4)术者熟练程度是重要因素,可缩短操作时间,避免反复穿刺,降低辐射影响。(5)DSA图像空间显影能力不佳,皮瓣切取过程中应以术中所见为准。
综上所述,DSA可在一定程度上反映旋股外侧动脉横支形态学特点,结合横支形态学特点,可为以旋股外侧动脉横支为蒂的股前外侧皮瓣设计提供一定思路。然而DSA造影不能显示旋股外侧动脉横支尤其是穿支走行的层次,这仍是目前横支切取过程中的难题,需要临床及解剖的进一步研究。
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