目的 比较冷冻前和复苏后脐带血样本的造血功能,探讨冷冻袋附属小管是否可用于脐带血质量控制及移植供体发放前复核。方法 选取2009年3月—2021年2月在广州脐血库冻存的53份脐带血为研究对象,常规复温后,从冷冻袋和附属小管抽取样本,分为冷冻前、小管复苏和大袋复苏组。评价各组的总有核细胞(total nucleated cells,TNCs)数、细胞活率、CD34阳性细胞数量、粒-巨噬细胞集落(colony-forming units-granulocyte/macrophages, CFU-GMs)数量、祖细胞集落(colony-forming units, CFUs)数量等质量参数。结果 小管复苏后TNCs数量、细胞活率、CFU-GMs数量及CFUs数量较冷冻前均显著减少,差异有统计学意义(P＜0.05),而二者的CD34阳性细胞数量差异无统计学意义(P＞ 0.05);大袋复苏后TNCs数量、细胞活率及CFUs数量较冷冻前各相应值比较,差异有统计学意义(P＜0.05),而二者的CD34阳性细胞数量及CFU-GMs数量差异无统计学意义(P＞ 0.05);小管复苏后的TNCs数量、细胞活率、CD34阳性细胞活率、CFU-GMs数量及CFUs数量较大袋复苏后的相应值均减少,差异有统计学意义(P＜0.05),而二者的CD34阳性细胞数量差异无统计学意义(P＞ 0.05)。小管复苏与大袋复苏后的各项参数存在高度相关性。结论 附属小管在一定程度上可作为脐血库质量控制和产品发放前复核的取材。

Objective To compare the hematopoietic function of cord blood before and after cryopreservation, and to explore whether sampling from the attached segment can be used for quality control of cord blood units and confirmatory testing before transplantation. Methods A total of 53 cryopreserved cord blood units from March 2009 to February 2021 in Guangzhou Cord Blood Bank were enrolled in this study. After routine thawing, samples were taken from main bag and its attached segment. They were divided into pre-freezing, post-thawing segment and post-thawing main bag groups. Total nucleated cells (TNCs), cell viability, CD34 positive cells, colony-forming units-granulocyte/macrophages(CFU-GMs)and colony-forming units (CFUs) were examined. Results The number of TNCs [(1.12±0.38)×10⁹], cell viability [(79.11±7.71)%], the number of CFU-GMs [(5.55±4.58)×10⁵] and the number of CFUs [(15.11±9.68)×10⁵] were significantly decreased in samples from post-thawing attached segments compared with those pre-freezing(P＜0.05), but there was no significant difference in the number of CD34 positive cells between two groups (P＞0.05). The number of TNCs [(1.25±0.40)×10⁹], cell viability [(84.75±5.89)%] and CFUs [(18.61±6.42)×10⁵] in samples from post-thawing main bags were significantly different from those pre-freezing (P＜0.05), but there were no significant differences in the number of CD34 positive cells and CFU-GMs between them (P＞0.05). The number of TNCs, cell viability, CD34 positive cell viability, the numbers of CFU-GMs and CFUs were significantly decreased in post-thawing segments compared with those in main bags after thawing (P＜0.05), but there was no significant difference in the number of CD34 positive cells between the two groups (P＞0.05). Spearman correlation analysis showed that the parameters of samples from main bag and its attached segment were highly correlated after thawing. Conclusion Cord blood from attached segment could be quality control sample for cord blood bank and confirmatory testing before product release in some degree.

目的 构建靶向CXCR7基因的CRISPR/Cas9基因编辑系统,并应用于HEK 293T细胞系。方法 设计两对靶向CXCR7基因的sgRNAs,分别插入PX458载体中,并转化DH5α大肠埃希菌。经菌液PCR和测序验证,挑选序列正确的sgRNA-CXCR7-PX458质粒,转染HEK 293T细胞,用流式分选转染阳性细胞,提取其DNA,PCR扩增后测序验证。结果 经测序验证,成功构建了靶向CXCR7基因的CRISPR/Cas9系统,转染HEK 293T细胞后,测序鉴定发现成功编辑CXCR7基因。结论 成功构建了靶向CXCR7的sgRNA-CXCR7-PX458质粒,可在HEK 293T上成功编辑CXCR7基因,为进一步的功能研究奠定基础。

Objective To construct the CRISPR/Cas9 gene editing system targeting C-X-C chemokine receptor 7 (CXCR7) gene and to edit CXCR7 gene in 293T cell line. Methods Two pairs of small guide RNAs (sgRNAs) targeting CXCR7 gene were designed and inserted into PX458 vector, which were transformed into host bacterium Escherichia coliDH5α. The correct sgRNA-CXCR7-PX458 plasmids were selected by PCR and further Sanger sequencing verification. HEK 293T cell line was transfected by DNA of sgRNA-CXCR7-PX458 plasmid. After 72 hours,GFP-positive cells were sorted by flow cytometry. We did DNA extraction of the GFP-positive cells and amplified the CXCR7 gene corresponding fragment by PCR and investigated the CXCR7 gene editing results by Sanger sequencing. Results The CRISPR/Cas9 system targeting CXCR7 gene was successfully constructed. After 293T cells were transfected, the CXCR7 gene was edited in HEK 293T cells successfully. Conclusion The sgRNA-CXCR7-PX458 plasmid targeting CXCR7 gene was successfully constructed. The CRISPR/Cas9 gene editing system targeting CXCR7 gene were used on the HEK 293T cell line, which lays a foundation for further study of BCOR function.

目的 采用生物信息学方法预测低氧预处理人胎盘绒毛膜间充质干细胞环状RNAs相对应的miRNA及其靶基因,并分析靶基因所参与的生物学过程和信号通路。方法 用Arraystar公司的商业软件为环状RNAs预测其相对应的miRNAs,分别用targetScan7.1和mirdbV5数据库预测miRNAs的靶基因,并取两个预测结果的合集,应用在线网站http://www.geneontology.org和http://www.genome.ad.jp/kegg对靶基因进行功能富集分析和信号通路富集分析。结果 功能富集分析表明,circRNAs的靶基因主要涉及到细胞发育、细胞分化和细胞发育调控。东京基因和基因组百科全书信号通路富集分析表明肿瘤中转录失控和有丝分裂原激活蛋白激酶(MAPK)信号通路最有意义,而且分析发现MAPK信号通路为核心通路。本研究表明,低氧预处理使得间充质干细胞中部分circRNAs的表达量发生差异性变化。结论 低氧预处理人胎盘绒毛膜间充质干细胞环状RNAs同低氧预处理间充质干细胞的生物学特性变化密切有关,为了解低氧预处理影响间充质干细胞特性发生变化的分子机制提供新思路。

Objective To predict the miRNA and its target genes of circular RNAs in hypoxia- preconditioned human palcenta chorionic mesenchymal stem cells using bioinformatics, and analyze the biological process and signaling pathway. Methods Arraystar's commercial software was used to predict the corresponding miRNAs of circular RNAs. The target genes of miRNAs were predicted by targetScan7.1 and mirdbV5 databases respectively, and an intersection of two prediction results was obtained. The online databases http://www. geneontology.org and http://www.genome.ad.jp/kegg performed functional enrichment analysis and signal pathway enrichment analysis of target genes. Results Functional enrichment analysis indicated that the target genes of circRNAs mainly involved cell development, cell differentiation and cell development regulation. The signal enrichment analysis of the Tokyo Gene and Genome Encyclopedia indicates that transcriptional misregulation in cancer and mitogen-activated protein kinase (MAPK) signaling pathway are most meaningful, and the MAPK signaling pathway is found to be the core pathway. This study showed that hypoxic preconditioning caused significant changes in the expression of mesenchymal stem cell circRNAs. Conclusion The changes of circular RNAs in hypoxia-preconditioned human placental chorionic mesenchymal stem cell is closely related to the biological characteristics of hypoxia-preconditioned mesenchymal stem cells. This study provides a new idea for understanding the molecular mechanism of hypoxic preconditioning affecting the changes of biological characteristics in mesenchymal stem cells.