[Construction of Nalm6-Cas9 Cell Line for Genome-Wide Translocation Sequencing].

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.

Presence of Tertiary Lymphoid Organ in Nasal Inverted Papilloma Is Correlated with Eosinophil Infiltration and Local Immunoglobulin Production.

INTRODUCTION: Nasal inverted papilloma (NIP) is a benign tumour with multiple inflammatory cell infiltration. Tertiary lymphoid organs (TLOs) support local antibody production and play important roles in airway inflammation. However, the evidence of TLOs and local immunoglobulins in NIP has not been reported yet. We investigated the presence of TLOs and immunoglobulins in NIP tissues and their association with the clinical-pathological characteristics of NIPs. METHODS: We analyzed the occurrence and composition of TLOs and local immunoglobulins by immunohistochemistry and evaluated the lymph organogenesis associated genes and cytokines by quantitative qPCR and Luminex assays, respectively, in papilloma tissues from 84 NIP cases. RESULTS: TLOs were present in 54% (45/84) of the NIP patients but not in control subjects. TLOs were composed of T cells, B cells, follicular dendritic cells, macrophages, and natural killer cells. Compared to NIP tissues without TLOs, tissues with TLOs showed significantly higher eosinophil infiltration levels (3.5-fold), elevation of lymphorganogenic genes (CXCL12, CXCL13, CCL20, CCL21, CD21L, and lymphotoxin alpha and beta), and increased Th17 (IL-21, IL-22, and GM-CSF) and Th2 (IL-5 and IL-13) cytokine production. Moreover, NIP with TLOs demonstrated a higher number of follicular T helper cells and immunoglobulin-producing plasma cells (CD138+ IgA+, CD138+ IgM+, CD138+ IgE+, and CD138+ IgG+) than those without TLOs, and these antibody-producing cells were positively correlated with the eosinophil number. CONCLUSION: The high frequency of TLOs and excess local immunoglobulin production are associated with an eosinophilic and Th2 skew microenvironment in the NIP mucosa, which would contribute to an important immunopathogenic response during NIP pathogenesis.

[The influence of different treating agents on the shear bond strength between zirconia and orthodontic brackets].

PURPOSE: To compare the effect of different treating agents on the shear bond strength (SBS) between zirconia and orthodontic brackets. METHODS: Fifty zirconia specimens were randomly divided into 5 groups(n=10), and treated with sandblasted and 37% phosphoric acid.Group A was control sample without any treatment; Group B was treated with silane coupling agent; Group C was treated with silane coupling agent and SE Bond; Group D were treated with Z-Prime(TM) Plus; Group E were treated with Z-Prime(TM) Plus+SE Bond. The upper incisor brackets were bonded to each specimen using 3M Transbond light-cured resin adhesive. Each specimen was put into 37°C water, and SBS tests were performed after 24 hours. Then statistical analysis was carried out regarding to the adhesive residual index(ARI) of the zirconia oxide surface after removing the brackets. The surface of zirconia in each group was scanned with scanning electron microscope after surface treatment, and the surface morphology changes were observed, infrared spectra analysis was conducted on zirconia and treating agents. SPSS 17.0 software package was utilized for statistical analysis. RESULTS: The mean SBS in each group was as follows: group A (3.12±0.84) MPa; group B (1.92±0.83) MPa; group C (5.26±0.80) MPa; group D (6.54±0.98) MPa; group E (9.47±2.11) MPa. Significant difference existed between each group (P<0.05), group A and B had lower ARI, while ARI in group E was highest. Group D and E achieved effective bond strength. CONCLUSIONS: Using Z-Prime(TM) Plus can achieve effective bond strength, which is lower than Z-Prime(TM) Plus combined with SE Bond. It's suggested to use Z-Prime(TM) Plus combined with SE Bond to achieve higher SBS for orthodontic treatment.