老年髋部骨折患者骨髓单个核细胞的单细胞转录组测序分析研究

Single cell RNA sequencing of bone marrow mononuclear cells from elderly patients with hip fracture

目的探讨2例老年髋部骨折患者骨髓单个核细胞(BMMNCs)中是否存在与调节免疫炎症相关的亚群,比较其BMMNCs亚群差异。方法选取2例老年髋部骨折患者作为研究对象,记为A和B。检测患者外周静脉血补体(C)3、C4、白细胞介素(IL)-2、IL-6、IL-10和淋巴细胞亚群。对2例患者的BMMNCs进行单细胞转录组测序分析,将细胞聚类分群,对每个亚群差异表达倍数前20的基因进行基因本体论(GO)及京都基因和基因组百科全书(KEGG)分析,判断每个亚群的主要功能,找出调节免疫炎症的相关亚群及基因。比较2例患者的预后、BMMNCs亚群种类及对应细胞比例差异。结果A患者外周血指标仅IL-10稍偏高。B患者C3偏低,C4接近正常值下限,IL-2、IL-6、IL-10明显偏高,CD8+T细胞百分比偏低,CD4+/CD8+偏高,其余指标未见明显异常。经单细胞转录组测序分析,将2例患者的BMMNCs分成相同的5个亚群,其中亚群2和亚群4的功能与调节免疫炎症相关,亚群2中的CCL4、CCL5、LTB、CXCR4及亚群4中的C1QA、C1QB、CD14、SPP1可能是关键基因。A患者最终预后较好,其BMMNCs中构成亚群2和亚群4的细胞比例均高于B患者[47.00%(1431/3045)vs.29.28%(882/3012),5.88%(179/3045)vs.3.85%(116/3012)]。结论运用单细胞转录组测序技术能将老年髋部骨折患者的BMMNCs分成不同亚群,其中有与调节免疫炎症相关的亚群,其可能影响患者伤后免疫炎症状态及预后。

Objective To determine whether subpopulations may exist which are related to regulation of immunization or inflammation in bone marrow mononuclear cells(BMMNCs)from 2 elderly patients with hip fracture and whether there might be any difference in the subpopulations between them.Methods Two elderly patients with hip fracture were enrolled in this study.Their venous blood was harvested to determine subpopulations of complement(C)3,C4,interleukins(IL)-2,IL-6,IL-10,and lymphocytes.Single cell RNA sequencing(scRNA-Seq)was used to group their BMMNCs.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses were performed of the top 20 differentially expressed genes for each subpopulation to judge the main function of each subpopulation.The subpopulations and the key genes related to regulation of immunization or inflammation were found out.The relationships were explored between subpopulations and prognosis of the patients.Results By the venous blood indexes,IL-10 was slightly high in patient A;C3 below normal,C4 close to the lower normal limit,IL-2,IL-6 and IL-10 were significantly high,CD8+T%was low,and CD4+/CD8+high in patient B.After scRNA-Seq and bioinformatics analyses,the BMMNCs in the 2 patients were divided into 5 subpopulations.GO and KEGG enrichment analyses showed that the functions of subpopulation 2 and subpopulation 4 were related to immunization or inflammation.CCL4,CCL5,LTB and CXCR4 in subpopulation 2 and C1QA,C1QB,CD14 and SPP1 in subpopulation 4 were related to the regulation of immunization or inflammation.The final prognosis of patient A was much better.The proportions of BMMNCs involved in subpopulation 2 and subpopulation 4 from patient A were higher than those from patient B[47.00%(1,431/3,045)versus 29.28%(882/3,012);5.88%(179/3,045)versus 3.85%(116/3,012)].Conclusions The BMMNCs from elderly patients with hip fracture can be divided into subpopulations by scRNA-Seq.Some of the subpopulations may be related to regulation of immunization or inflammation,which may affect the post-injury immune inflammatory state and prognosis of the patients.