Study On The Bone Marrow Mononuclear Cells Of Elderly Hip Fracture Patients With Different Prognosis Based On Single Cell RNA Sequencing

ObjectiveTo perform a subpopulation analysis of elderly hip fracture patients' bone marrow mononuclear cells(BMMNCs)and explore the relationship between it and patients' prognosis by single cell RNA sequencing(scRNA-Seq).MethodsProspective analysis on the clinical data of the elderly hip fracture patients who treated in our hospital from January 2018 to December 2018.Use Nottingham Hip Fracture Score(NHFS)to evaluate clinical prognosis.The score less than 6 was defined as a good prognosis,while a score greater than or equal to 6 was defined as a poor prognosis.The patient with good prognosis was recorded as A,and the patient with poor prognosis was recorded as B.Two patients were followed up one month after surgery.Drew the patients' venous blood to test immunoglobulins(IgA,IgG,IgM),complements(C3,C4),lymphocyte subpopulation,interleukins(IL-2,IL-6,IL-10,TNF-?)on the morning of surgery.Drew the patients' bone marrow of fractured femur about 10ml during operation,extract and prepare bone marrow mononuclear cell suspension and test sample quality.We used scRNA-Seq to group the cells.Perform GO and KEGG analyses on the top20 differentially expressed genes of each subpopulation and judge the main function of each subpopulation.Search for genetic information through NCBI database and find out the key genes.Finally,explore the relationship between different subpopulations and patients'prognosis.ResultsThe patient(A)with a good prognosis was determined to be a woman whose NHFS was 4.And the patient(B)with a poor prognosis was determined to be a woman whose NHFS was 7.The patient(A)had no complications such as pulmonary infection,cardiovascular and cerebrovascular accidents within 1 month after surgery;The patient(B)was readmitted 19 days after operation due to acute myocardial infarction and eventually died of cardiogenic shock.The venous blood indexes of the patient(A)showed only a slight high in IL-10,and no obvious abnormalities were found in other indexes;AS for patient(B),her C3 was below normal,C4 was close to the normal lower limit,IL-2,IL-6 and IL-10 were significantly high,the CD8+T%was low,and CD4+/CD8+ was high.High activity BMMNCs were obtained from bone marrow of tw-o patients by density gradient centrifugation.After scRNA-Seq and bioinformatics analysis,the BMMNCs of two patients were divided into five subpopulations.GO and KEGG enrichment analyses showed that the functions of subpopulation 2 and subpopulation 4 were related to inflammatory immune response.Search for genetic information of subpopulation 2 and subpopulation 4 through NCBI database,we found that CCL4,CCL5,LTB,CXCR4 in subpopulation 2 and C1QA,C1QB,CD14,SPP1 in subpopulation 4 were related to the regulation of immunization or inflammation.47%and 5.88%of the BMMNCs from patient(A)were involved in subpopulation 2 and subpopulation 4 respectively.And the ratio was 29.28%and 3.85%in patient(B).ConclusionBMMNCs with high activity can be isolated from femoral bone marrow of elderly hip fracture patients by density gradient centrifugation.BMMNCs of elderly hip fracture patients can be divided into subpopulations with different functions,and there are subpopulations related to the regulation of immunization or inflammation.CCL4,CCL5,LTB,CXCR4,C1QA,C1QB,CD14 and SPP1 may be the key genes.Patients with different prognosis have the same BMMNCs subpopulations but different proportion of corresponding cells.The patients with high proportion of corresponding cells of subpopulations related to the regulation of immunization or inflammation may have a good prognosis.