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Objective: In order to conduct high-throughput genome-wide translocation sequencing based on CRISPR/Cas9, Nalm6-cas9 monoclonal cell line expressing Cas9 protein was constructed by lentivirus transduction., Methods: Lentiviral vectors LentiCas9-Blast, pSPAX2, and pMD2.G were used to co-transfect HEK293T cells to obtain recombinant lentivirus. After Nalm6 cells were infected with the recombinant lentivirus, the cells were screened by Blasticidin, and multiple monoclonal cell lines expressing Cas9 protein were obtained by limited dilution. Western blot was used to detect the expression level of Cas9 protein in monoclonal cell lines, and cell count analysis was used to detect the proliferation activity of monoclonal cell lines. LentiCRISPRV2GFP-Δcas9, LentiCRISPRV2GFP-Δcas9-AF4, LentiCRISPRV2GFP-Δ cas9-MLL plasmids were constructed, and transfected with pSPAX2 and pMD2.G, respectively. T vector cloning was used to detect the function of Cas9 protein in Nalm6-Cas9 monoclonal cell line infected with virus., Results: Western blot showed that Nalm6-Cas9_1-6 monoclonal cell line had high expression of Cas9 protein. Cell count analysis showed that high expression of Cas9 protein in Nalm6-Cas9_1-6 monoclonal cell line did not affect cell proliferation activity. The Nalm6-Cas9_1-6 monoclonal cell line had high cleavage activity, and the editing efficiency of AF4 and MLL genes was more than 90% which was determined by T vector cloning., Conclusion: Nalm6-Cas9_1-6 monoclonal cell line stably expressing highly active Cas9 protein was obtained, which provided a basis for exploring the translocation of MLL in therapy-related leukemias based on CRISPR/Cas9 genome-wide high-throughput genome-wide translocation sequencing.

Objective: To study the expression of B lymphocyte-induced mature protein-1 (BLIMP-1) in regulatory T cells (Tregs) of children with aplastic anemia (AA), and analyze its correlation with the number of Tregs and the levels of inhibitory cytokines interleukin (IL)-10 and transforming growth factor (TGF)-β in plasma., Methods: The peripheral blood samples of 10 newly diagnosed AA children and 10 healthy children were collected for experiment. qPCR was used to detect FOXP3 and PRDM1 mRNA expression levels. Flow cytometry was used to detect the proportion of Tregs, the expression of BLIMP-1 in Tregs, and the levels of cytokines such as IL-2, IL-17A, IL-6, interferon (IFN)-γ, IL-10 and TGF-β in plasma. Pearson correlation model was used to evaluate the relationship between the expression of BLIMP-1 in Treg and the number of Tregs, as well as the levels of IL-10 and TGF-β in plasma., Results: Compared with control group, the proportion of Tregs in peripheral blood of AA children was decreased significantly (P<0.001); The plasma levels of proinflammatory cytokines IL-2, IL-6 and IFN-γ in AA children were increased significantly (P=0.033, P=0.031, P=0.006), and IL-17A also was increased but the difference was not statistically significant (P=0.052), while anti-inflammatory cytokines IL-10 and TGF-β were significantly reduced (P=0.048, P=0.002). The relative expressions level of FOXP3 and PRDM1 mRNA in AA children were significantly lower than those in control group (P=0.037, P=0.016). The expression of BLIMP-1 protein in Tregs of AA children was significantly lower than that in control group (P<0.001). The expression level of BLIMP-1 protein in Tregs was positively correlated with the percentage of Tregs in lymphocytes (r=0.671, P=0.001), and was also positively correlated with the levels of IL-10 and TGF-β in plasma (r=0.500, P=0.029; r=0.486, P=0.030)., Conclusion: The expression of BLIMP-1 in Tregs of AA children is impaired, and the low expression of BLIMP-1 is related to the decrease of the number in Tregs and IL-10 and TGF-β expressions.

Objective: To analyze retrospectively the postoperative complications of acromioclavicular joint dislocation (Tossy III) and explore the preventative methods for the complications., Methods: Forty-eight cases of acromioclavicular joint dislocation (Tossy III) were reviewed, 14 cases treated with open reduction and Kirschner wire combined with steel wire fixation (group A), 11 cases treated with open reduction and lag screw or steel wires fixation (group B), 23 cases treated with open reduction and AO clavicular hook plate fixation (group C). The acromioclavicular ligament, articular capsule, coracoclavicular ligament and coracoacromial ligament were repaired in all patients. The reasons of postoperative complications were analyzed., Results: Forty-eight patients got average follow-up of 18 months. In group A, 8 patients obtained excellent results, 4 good and 2 poor; in group B, 7, 3 and 1, respectively; in group C, 21, 1 and 1, respectively. The excellence rate showed statistical difference between group A and C (P < 0.05). In group A, 4 cases with postoperative pain, 3 cases with periarthritis of shoulder, 3 cases with arthritides of acromioclavicular joint, 4 cases with internal fixation failure and 2 cases with recurrence of joint dislocation; in group B, 3, 2, 1, 3 and 1, respectively; in group C, 22, 2, 1, 2 and 1, respectively. There was no significant difference in postoperative complications in three groups (P > 0.05)., Conclusion: Selection of the suitable internal fixation and reconstruction of coracoclavicular and coracoacromia ligaments is the basic operation. Acromioclavicular space debridement, ligamentous reconstruction, rigid internal fixation are effective methods to reduce postoperative complications for the treatment of acromioclavicular joint dislocation.