目的 研究1例RhD阴性产妇所育新生儿因D抗原遮蔽现象造成血清学假阴性的特点,及分析新生儿发生溶血病多次配血不合的原因,探讨提高抗体检出率的方法,及时解决新生儿HDFN的输血救治问题.方法 采用盐水法和微柱凝集法结合吸收放散实验鉴定产妇及新生儿的抗体,RhCE exon1-10外显子测序鉴定新生儿血型,找到相匹配的血液给新生儿换血治疗.结果 新生儿血型初次鉴定为AB、ccdEe,患儿母亲血型为AB、ccdee;患儿红细胞经ZZAP处理及基因鉴定后Rh分型为ccDEe;母亲、新生儿游离血清及新生儿红细胞放散液中检测出抗-D+抗-G.测得产妇血清中抗-D效价为1024、抗-G效价为8;患儿抗-D与抗-G效价分别为128、4.从血库中筛选出与新生儿血清交叉配血相匹配的0型洗涤红细胞.结论 产妇抗-D可导致新生儿D抗原完全遮蔽,在临床检测过程中需采用多种血清学方法结合基因方法鉴定;对RhD阴性的孕妇要进行产前抗体筛查和鉴定,新生儿注射免疫球蛋白后交叉配血要格外关注,及时为新生儿发生HDFN的换血输血提供了相匹配血液具有十分重要的意义.

Objective To construct the Hut78 cell line with EZH2 gene knocked into by CRISPR/Cas9 system. Methods The EZH2 expression vector pMD-18T-EZH2 with homologous arm and the sgRNA expression vector pSpCas9 (BB)-2A-Puro-sgRNA, which could cut the double stranded genomic DNA, were constructed, and the two vectors were co-transfected into Hut78 cells. Then the expression of EZH2 mRNA was detected by qPCR, and the expressions of EZH2 and H3K27me3 proteins were detected by Western blot assay. Results The pMD-18T-EZH2 and pSpCas9(BB)-2A-Puro-sgRNA recombinant vectors were confirmed by DNA sequencing. When Hut78 cells were transfected with the two recombinant plasmid, qPCR results showed that the expression of EZH2 mRNA was significantly increased, and Western blot analysis showed that the expressions of EZH2 and H3K27me3 proteins were significantly increased. Conclusion EZH2 gene is successfully knocked into Hut78 cells by CRISPR/Cas9 system.

Objective To constructe the rat-specific EZH2 overexpression plasmid and the EZH2 catalytic subunit SET domain deletion plasmid,as well as assess their levels of expression in 293T cells.Methods The RNA extracted from rat cardiac tissue was reverse-transcribed into cDNA,the cDNA of catalytic subunit SET domain deleted EZH2 was amplified by overlap PCR,then the PCR amplification was achieved using the EZH2 gene as template.Products through PCR amplification and vector were cut with double enzyme,and then ligated the target fragments,purified by gel extraction,using T4 ligase.The ligated products were transferred into competent cells.After plate coating,monoclonal picking,shaking cultivation and sequencing procedures,the plasmids were extracted and transfected into 293T cells using liposome-mediated method.Results Western Blot and RT-PCR assays showed recombinant plasmids DNA can successfully overexpress EZH2 gene in 293T cells.Conclusions Two plasmids were successfully constructed.This study laid the foundation for the further study on the relationship between EZH2 and myocardial hypertrophy,and the function of EZH2 catalytic subunit.