Differential Gene Expression Analysis and Functional Prediction of Decidual Myeloid-Derived Suppressor Cells in Patients with Unexplained Recurrent Abortion
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摘要:
目的 研究不明原因复发性流产（unexplained recurrent abortion ，URSA）患者蜕膜髓系抑制细胞（myeloid-derived suppressor cells，MDSCs）差异基因表达分析及功能预测。 方法 收集2023年7月至2023年12月在云南省第一人民医院生殖妇科就诊的3例早孕期人工流产孕妇及3例URSA患者的蜕膜组织，利用MACS分选出蜕膜MDSCs细胞，经表达谱基因芯片检测，差异基因表达分析（如DESeq2或edgeR）来识别两组间基因表达的显著变化，并通过P值和多重校正（如FDR）控制假阳性率。富集分析（如GO或KEGG分析）用于评估差异基因在生物通路中的富集情况。应用 SPSS16.0 分析软件进行超几何检验或Fisher精确检验确定其显著性。 结果 与正常妊娠相比，URSA患者蜕膜MDSCs中有163个基因发生显著变化（P < 0.05），其中67个基因上调，96个基因下调。这些基因在细胞成分、生物过程、分子功能、蛋白结合、补体系统信号通路、白细胞相关激活炎症反应通路、蛋白多糖和细胞外基质受体相互作用中富集。PPI网络分析和HUB基因鉴定显示前10的HUB基因中，上调基因只有SPP1、CCL5、C3AR1和TNF，下调基因包括MXRA8、IGFBP5、SPARCL1、SAA1、DCN、COL3A1。HUB基因主要涉及免疫调节、炎症反应及细胞间交互作用。 结论 URSA患者蜕膜中的MDSCs与细胞外基质和其他细胞间的相互作用功能低下，免疫抑制能力下降，促炎反应加强。 Abstract:Objective To investigate the differential gene expression and functional prediction of decidual myeloid‐derived suppressor cells （MDSCs） in patients with unexplained recurrent abortion （URSA）. Methods Decidual tissues were collected from three pregnant women undergoing early pregnancy termination and three URSA patients at the First People's Hospital of Yunnan Province from July 2023 to December 2023. MDSCs were isolated using MACS, followed by gene expression profiling with microarray analysis. Differential gene expression was analyzed using methods such as DESeq2 or edgeR to identify significant changes in gene expression between the two groups, with control of false positive rates through P-values and multiple corrections （e.g., FDR）. Enrichment analysis （e.g., GO or KEGG analysis） was performed to evaluate the enrichment of differential genes in biological pathways. The SPSS 16.0 software was utilized for hypergeometric tests or Fisher's exact tests to determine significance. Results Compared to normal pregnancies, 163 genes showed significant changes in the decidual MDSCs of URSA patients （P < 0.05）, with 67 genes upregulated and 96 genes downregulated. These genes were enriched in cellular components, biological processes, molecular functions, protein binding, complement system signaling pathways, leukocyte-related activation inflammatory response pathways, proteoglycans, and extracellular matrix receptor interactions. PPI network analysis and HUB gene identification showed that among the top 10 HUB genes, only SPP1, CCL5, C3AR1 and TNF were up-regulated, while MXRA8, IGFBP5, SPARCL1, SAA1, DCN and COL3A1 were down-regulated. The HUB gene were mainly involved in immune regulation, inflammatory responses and intercellular interactions. Conclusion MDSCs in the decidual of URSA patients exhibit impaired interactions with the extracellular matrix and other cells, resulting in decreased immunosuppressive capacity and enhanced pro-inflammatory responses. -
冠状动脉旁路移植术（coronary artery bypass graft，CABG）或冠脉搭桥术是治疗冠心病的重要手段，但移植静脉桥再狭窄严重影响了该手术的预后，目前临床针对桥血管再狭窄主要采用“双抗血小板治疗”，但静脉桥再狭窄的风险依然很高[1] ，一项随机临床试验显示 CABG 术后 3 个月，只服用阿司匹林抗血小板治疗的患者有 14.3%的静脉桥闭塞，服用阿司匹林加氯吡格雷“双抗血小板治疗”的患者依然有 8.4%的静脉桥闭塞[2]。究其原因，移植静脉中膜平滑肌细胞的增殖、迁移导致的内膜增生是再狭窄的关键因素[3]。
目前冠脉搭桥患者术后服用的药物对缓解移植静脉的内膜增生作用甚微[4]。因此如何抑制静脉桥血管再狭窄仍然是冠心病外科的重点和难点。笔者前期研究发现，在动物模型上，灯盏乙素可以降低静脉桥再狭窄程度，可能和抑制血管平滑肌细胞增殖有关[5]。经医院伦理委员会批准后，笔者给冠脉搭桥术后患者常规服药外，加用灯盏花素片，临床研究其抑制静脉桥再狭窄的作用。
1. 资料与方法
1.1 临床资料
根据入选和排除标准，2020年7月至2021年6月在昆明医科大学第一附属医院心脏大血管外科行CABG术患者64例，其中男37例，女27例，均签署知情同意书。
1.1.1 纳入标准[6]
纳入标准：（1）年龄≥18岁；（2）首次进行开胸冠脉旁路移植术，使用或不使用体外循环；（3）至少一条移植血管选用大隐静脉。
1.1.2 排除标准：
（1）同期行其它心血管手术，如瓣膜置换、瓣膜修复或射频消融术等；（2）二次搭桥手术（redo CABG）；（3）急诊搭桥手术；（4）使用了吻合器进行血管吻合；（5）行冠脉内膜剥脱；（6）由于室壁瘤形成行左室修补；（7）合并恶性肿瘤或其它严重系统疾病；（8）严重肾功能不全（肌酐 > 200 μmol/L）；（9）不能耐受血小板双抗治疗，如严重的消化道溃疡；（10）参加了其它临床研究。
1.2 分组和用药方法
患者随机平均分成2组，对照组和灯盏乙素组。对照组患者服用CABG术后药物，阿司匹林100 mg，1次/d；氯吡格雷75 mg，1次/d；阿托伐他汀20 mg，1次/d。灯盏乙素组患者除以上常规药物外加服灯盏花素片（德众，国药准字Z44022688），40 mg/次，3次/d。
1.3 评价指标
术后3个月、6个月行多层计算机断层血管造影（multi-slics computed tomography angiography，MSCTA），计算机重建将弯曲的静脉桥“拉直”，选取固定的5个测量点（静脉桥两端吻合处、静脉桥中点，距离吻合口2 cm处）静脉桥的CTA图像，测量血管内径，最后取平均值，见图1。
1.4 统计学处理
用SPSS13.0统计软件进行统计处理。连续性变量以均数±标准差（
$\bar x \pm s$ ）表示，资料符合正态分布者采用t检验，不符合正态分布者采用Wilcoxon秩和检验或t′ 检验；偏态分布资料以“中位数（P25，P75）”表示，采用Wilcoxon秩和检验；分类变量、等级资料以“例数（百分率）”表示，采用χ2检验，以 P < 0.05 为差异有统计学意义。2. 结果
2.1 2组患者基本资料围术期临床资料比较
64例入选的患者中，对照组32例，灯盏乙素组32例。没有术后死亡病例，没有提前退出试验病例。2组患者的基本临床资料比较差异均无统计学意义（P > 0.05），见表1。
表 1 2组患者基本临床资料比较[（$\bar x \pm s$ ）/n（%）]Table 1. Compare clinic data of 2 groups [（$\bar x \pm s$ ）/n（%）]临床资料 对照组
（n = 32）灯盏乙素组
（n = 32）t/χ2 P 年龄（岁） 59.7 ± 4.8 59.5 ± 8.6 0.151 0.861 男性 17（53.1） 19（59.4） 0.011 0.45 身高（cm） 166.5 ± 7.8 166.8 ± 8.3 −0.142 0.875 体重（kg） 71.1 ± 10.9 72.7 ± 11.5 −0.968 0.335 高血压病 21（65.6） 20（62.5） 0.311 0.341 糖尿病 10（31.3） 12（37.5） 0.315 0.347 高脂血症 9（28.1） 11（34.3） 0.434 0.312 吸烟史 15（46.9） 17（53.1） 0.276 0.336 家族史 14（43.8） 15（46.9） 0.013 0.519 左心室射血分数（%） 45.3 ± 8.4 46.4 ± 9.6 −0.812 0.451 心肌梗死 9（28.1） 10（31.2） 0.034 0.511 2.2 静脉桥内径比较
2组静脉桥内径比较，术后3个月和术后6个月灯盏乙素组高于对照组，差异有统计学意义（P < 0.05），见表2。
表 2 静脉桥内径比较（$ \bar x \pm s$ ）Table 2. Compare the inner diameter of graft vein （$\bar x \pm s $ ）组别 术后3月（mm） 术后6月（mm） 对照组 5.0 ± 0.5 4.6 ± 0.4 灯盏乙素组 5.1 ± 0.6* 4.9 ± 0.6* 与对照组比较，*P < 0.05。 3. 讨论
冠心病是全球首位死亡原因。临床上通过移植自体血管恢复冠心病患者心肌灌注（再血管化）的冠脉搭桥术是临床上广泛应用的治疗手段[7]。大隐静脉由于长度长、解剖位置表浅等优点成为80% 的CABG再血管化中选用的桥血管[8]。但静脉桥再狭窄使患者需要二次手术，严重制约了CABG的疗效[9-10]。
静脉桥再狭窄，主要因为静脉移植到动脉环境后发生“动脉”粥样硬化。正常静脉不会发生粥样硬化，但移植静脉血流力学环境改变后，移植静脉为了适应新环境发生“动脉化”，血管平滑肌细胞的增殖、迁移引起内膜增厚是后期发生粥样硬化的细胞学基础[11]。内膜增生是启动静脉桥粥样硬化的早期改变，因此抑制内膜增生是防止静脉桥再狭窄的关键[12]。内膜增生是一个复杂的过程，尚不清楚确切分子机制，研究显示内膜中过度的平滑肌细胞增殖和迁移是一个重要的因素[13-14]。目前对于冠脉搭桥术患者，术后常期服用阿司匹林、氯吡格雷和阿托伐他汀钙来减缓静脉桥再狭窄。阿司匹林和氯吡格雷均是抑制血小板功能的药物，阿托伐他汀钙是降脂药，主要起到稳定钙化斑块的作用，它们对平滑肌细胞增殖和迁移无效。
灯盏乙素是从彝药灯盏花中提取的单体，结构式鉴定为4，5，6-三羟基黄酮-7葡萄糖醛酸苷。灯盏花始载于《滇南本草》，现收载于《中国药典》2005年版第一部[15]，又名灯盏细辛，为菊科植物短葶飞蓬的全草，主要产于云南、贵州、四川等省，云南省能入药的灯盏花资源得天独厚，约占全国资源的95%以上。20世纪70年代云南省药物研究所首次从灯盏花中分离得到灯盏乙素，从此灯盏乙素被制成各种剂型应用于临床，灯盏乙素对心血管具有保护作用，其机制包括调节血管内皮功能、缓解血管痉挛、减轻炎性反应、抗自由基损伤、抑制血小板凝集等[16-18]。临床上主要用于心、脑血管疾病的治疗，如 ：高血压、冠心病等的治疗。随着研究不断的深入，灯盏乙素应用范围不断拓宽，研究显示灯盏乙素可以抑制高糖诱导的鼠血管平滑肌细胞的增殖和迁移[19]。笔者前期研究发现，在CABG动物模型上，灯盏乙素可以降低静脉桥再狭窄程度，其机制与抑制血管平滑肌细胞增殖有关。但结果限于动物实验。
为此，经医院伦理委员会批准，本研究将冠脉搭桥术后患者随机分组，对照研究，观察临床已使用多年的灯盏花素片（其中活性成分为灯盏乙素）对冠脉搭桥术后患者静脉桥再狭窄的作用，采用计算机断层血管造影测量冠脉搭桥术后患者的静脉桥内径，通过统计分析，得出结论，口服含有灯盏乙素的灯盏花素片对减缓静脉桥再狭窄可能有一定作用，但要到达最后的结论还需要多中心、大样本的研究结果支持。
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图 1 蜕膜MDSCs细胞形态学及分子机制图
注：蜕膜MDSCs分为两种类型M-MDSCs和PMN-MDSCs，在蜕膜界面分别趋化不同的免疫分子。
Figure 1. Cellular morphology and molecular mechanism of MDSCs in uterine decidua
图 3 差异表达基因火山图
注：每个点代表1个基因，共
28264 基因，红／绿色虚线分别代表 P < 0.05且倍数筛选阈值|Fold change| ≥1。蓝色的点代表显著变化基因，其中左侧代表表达下调的基因，右侧代表表达上调的基因。黑色的点表示表达量没有明显变化的基因。Figure 3. Volcano plot of differential gene expression
图 4 差异表达基因热图
注：线的长度则代表距离指标，同簇样本或基因的相似性高（模式相近）。每1行为1张芯片，每1列为1个基因探针，共163个差异表达基因，红色代表上调基因，蓝色代表下调基因。
Figure 4. Heatmap of differentially expressed genes
图 5 差异表达基因GO富集分析柱状图
A：细胞组成富集分析；B：分子功能富集分析；C：生物学过程富集分析；横轴表示功能富集显著性水平（P-Value）的-Log10P 值，纵轴表示 GO term；CC: cellular component; MF：molecular function；BP: biological process。
Figure 5. Histogram of differentially expressed gene GO enrichment analysis
图 6 差异表达基因KEGG富集分析
Figure 6. KEGG enrichment analysis of differentially expressed genes
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