中国实验血液学杂志最新文献

Objective: To prepare mesenchymal stem cells with antioxidant capacity (AO-MSC) from human umbilical cords and evaluate its cell biological properties.

Methods: In control group, mesenchymal stem cells (MSC) were isolated by digesting human umbilical cord Wharton's Jelly tissues with 0.2% collagenase II, and the released cells were collected and cultured in an animal serum-free culture medium. In AO-MSC group, incompletely collagenase II-digested tissue debris were allowed to adhere to flusk flat bottoms and the AO-MSC was harvested by adherent culture. The conventional digestion and culture method was used as control. MSC colony forming ability was evaluated by fibroblast colony forming assay (CFU-F). MSC proliferative capacity was evaluated by CCK-8 assay. The MSC surface markers were detected by using flow cytometry and immunofluorescence staining. The adipogenic and osteogenic capacity of MSC was evaluated by multi-differentiation in vitro, and the mRNA expression of genes that control adipogenic and osteogenic differentiation was detected by real-time fluorescence quantitative PCR (RT-qPCR); Moreover, the mRNA expression of antioxidant substances such as SOD-1, GSH, GAT, and NQO1 in MSC was also evaluated by RT-qPCR.

Results: The AO-MSC isolated by this strategy reached a confluence of 80%-90% at around 18 days and grew in a swirling pattern. Flow cytometry and immunofluorescence staining assays showed that CD73, CD29, CD105, CD90 were highly expressed and CD31, CD45, HLA-DR were scarcely expressed in AO-MSC. AO-MSC exhibited stronger self-renewal and differentiation ability compared to MSC. However, the in vitro adipogenic-osteogenic capacity of MSC in the control group was stronger than that of AO-MSC. RT-qPCR assay showed that AO-MSC expressed higher mRNA levels of antioxidant substances compared to MSC.

Conclusion: Human AO-MSC is successfully prepared from human umbilical cord without animal serum.

Objective: To investigate the influence of Ku80 inhibition on the chemotherapeutic sensitivity of the T-acute lymphoblastic leukemia(T-ALL) cell line Jurkat, and to explore the potential mechanism.

Methods: The transcription and expression level of Ku80 in 6 hematological malignant cell lines were detected by RT-qPCR and Western blot, respectively. The expression of Ku80 in Jurkat cells was detected by Western blot after transfection with the recombinant shKu80 lentiviral vector. The proliferation capacity of Jurkat cells was explored by CCK-8 after Ku80 inhibition. The colony formation ability, apoptosis, and γH2AX(a protein marker of DNA damage) expression in Jurkat cells were investigated after Ku80 silencing and co-treated with etoposide(VP16) for 4 hours through soft agar assay, flow cytometry and Western blot, respectively.

Results: The mRNA level and protein expression of Ku80 were both highest in Jurkat among 6 hematological malignant cell lines. Ku80 expression was successfully down regulated in Jurkat cells after relative plasmid transfected. The proliferative ability of cells was significantly decreased after Ku80 inhibition(P < 0.05). The colony formation capacity of Jurkat cells was obviously reduced and the cells apoptosis and γH2AX expression were increased after Ku80 inhibition, with or without VP16 incubation.

Conclusion: Targeted silencing of Ku80 could enhance the sensitivity of VP16 in Jurkat cells, which might be associated with the elevated level of DNA damage accumulation.

Objective: To investigate the effect of different isoforms of RBBP6 on the proliferation of multiple myeloma (MM) cells after alternative splicing mediated by splicing factor SRSF1 .

Methods: RT-PCR was used to detect the expression levels of RBBP6 mRNA splicing isoforms regulated by SRSF1 . The GEO database was used to analyze the changes of RBBP6 isoform 1 in the progression of plasma cell disease, and survival analysis was used to evaluate the value of this gene in the prognosis of MM patients. in vitro loss-of-function and gain-of-function experiments were conducted by transfecting control siRNA, RBBP6 isoform 1 siRNA, empty vector (EV), OE-RBBP6 isoform 3 into MM.1S cells, then the expression levels of RBBP6 isoform 1 and RBBP6 isoform 3 were detected by real-time PCR and Western blot. CCK-8 assay was performed to detect the cell proliferation ability.

Results: Knockdown of SRSF1 increased the expression of RBBP6 isoform 3 and decreased the expression of RBBP6 isoform 1. RBBP6 isoform 1 was closely related to the progression of plasma cell disease, and the high expression of RBBP6 isoform 1 was associated with poor prognosis in patients with MM. Downregulation of RBBP6 isoform 1 expression and overexpression of RBBP6 isoform 3 both reduced the proliferation ability of MM cells. And after downregulating the expression of RBBP6 isoform 1, the level of p53 protein in MM cells was significantly increased (P < 0.05).

Conclusion: In MM, splicing factor SRSF1 can cause alternative splicing abnormalities in RBBP6 . The RBBP6 isoform 1 promotes MM cell proliferation, while the RBBP6 isoform 3 inhibits MM cell proliferation, and the mechanism may be related to regulating the p53 pathway.

Objective: To investigate the clinical phenotype and molecular pathogenic mechanism of a hereditary coagulation factor V deficiency (FⅤD) family.

Methods: A phase I assay was used to measure coagulation factors II, V, VII, VIII, IX, X, Ⅺ, Ⅻ (FⅡ∶C, FⅤ∶C, FⅦ∶C, FⅧ∶C, FⅨ∶C, FⅩ∶C, FⅪ∶C, FⅫ∶C), activated partial thromboplastin time (APTT) and prothrombin time (PT) to determine the clinical phenotype and molecular pathogenesis of F VD. Prothrombin time (PT) were used for phenotypic identification; high-throughput exome sequencing was applied to screen the whole gene variants, and Sanger sequencing was used to verify the suspected variants in F5 gene; MutationTaster, PolyPhen-2 bioinformatics software was used to predict the pathogenicity of the variants, ClustalX software was used to analyze the amino acid conservatism, and PyMol software was used to simulate the model of the mutant protein.

Results: The pre-documented patient had significantly prolonged PT and APTT, FⅤ∶C was only 5.45%, and there was no significant abnormality in TT, FIB and the rest of the coagulation factors. The mother, father and sister of the proband had prolonged PT and APTT, and FⅤ∶C was reduced to different degrees. Genetic testing revealed the presence of a c.286G>C (p.Asp96His) pure missense variant in exon 3 of F5 in the prior witness, and a c.286G>C (p.Asp96His) heterozygous missense variant in father, mother, and sister of the proband. Bioinformatics analysis suggested that p.Asp96His was a pathogenic variant, and the associated amino acid site was highly conserved among 10 species. Protein simulation showed that the mutation of Asp96 to His96 could lead to the disappearance of the original hydrogen bond and the change of the distance, destroying the original hydrogen bond interaction force and affecting the stability of the protein structure.

Conclusion: The F5 exon 3 c.286G>C (p.Asp96His) missense variant may have contributed to the reduction of FⅤ∶C in the preexisting individual and family members, as well as being the genetic etiology of coagulation factor V deficiency.

Objective: To investigate the clinical features, gene mutation profile, efficacy and prognostic factors of primary extranodal diffuse large B-cell lymphoma(EN-DLBCL).

Methods: A retrospective analysis was performed for 382 patients with primary EN-DLBCL with complete clinical data who were treated in West China Hospital from January 2013 to January 2023, and their clinical characteristics,gene mutation profile, efficacy and prognostic factors were analyzed.

Results: The median age of the 382 patients with EN-DLBCL was 56(18-89) years old. The male-to-female ratio was 1.12∶1, and the most common primary sites were gastrointestinal tract (31.7%), Wechsler ring (19.1%) and breast gland (7.1%). A total of 51 gene mutations were fund, and the most common frequencies of gene mutations were TP53 (32.5%), MYD88 (32.5%), and CD79B (30.0%). The median follow-up was 63 months, and the 5-year progression-free survival (PFS) rate was 74.5% and the 5-year overall survival (OS) rate was 89.6%. The adverse factors on PFS were as follows: >1 extranodal sites involvement ( P <0.001), P53≥50% ( P <0.001), hyper double expression(hDEL) of C-myc >50%/Bcl-2>70% ( P <0.001). The adverse factors affecting the OS of patients were as follows: >1 extranodal sites involvement ( P <0.001), P53≥50% (P < 0.001), hDEL( P <0.001). Chemotherapy combined with local radiotherapy could improve PFS (P =0.041) and OS (P =0.003), while R-CHOP+X (molecule agents as BTKi、HDACi、Lenalidomide) failed to show a significant difference in PFS (P =0.075) and OS (P =0.767). Among the 40 patients who underwent next-generation sequencing at high risk, there was no significant in PFS (P =0.849) and OS (P =0.500) of patients with positive MYD88 and/or CD79B mutations (MCD subtype) treated with BTKi and patients with negative MYD88 and CD79B mutations.

Conclusion: Primary EN-DLBCL can involve multiple organs or tissue sites. TP53 , MYD88 , and CD79B are the most common gene mutations. The efficacy of BTKi in patients with positive MCD subtypes at intermediate and high risk is not inferior to that in MCD-negative control patients.

Objective: To explore the changes in number and immune function of mucosal-associated invariant T (MAIT) cells in peripheral blood of patients with newly diagnosed acute myeloid leukemia (AML), and its correlation with the occurrence and development of AML.

Methods: Seventy-five clinical samples of patients with newly diagnosed AML and 48 healthy control samples in our hospital from January 2022 to February 2023 were included. Multiparametric flow cytometry was used to detect the number of MAIT cells, membrane surface markers, effector phenotypes and functional indicators in the samples.

Results: Compared with healthy controls, the percentage of MAIT cells in CD3⁺ T cells in peripheral blood of newly diagnosed AML patients was significantly reduced ( P < 0.001). The percentage of MAIT cells in all CD3⁺ T cells in bone marrow of AML patients was similar to that in peripheral blood (P >0.05). Most of MAIT cells in peripheral blood of AML patients were effector memory T cells. Compared with healthy controls, the proportion of effector memory MAIT cells decreased ( P < 0.05), while the proportion of terminally differentiated effector memory MAIT cells and PD-1⁺MAIT cells increased significantly (both P < 0.05). AML patients' peripheral blood MAIT cells expressed significantly higher levels of granzyme B and perforin than healthy controls (both P < 0.05), and secreted significantly lower levels of cytokines such as gamma interferon and tumor necrosis factor α than healthy controls (both P < 0.001).

Conclusion: Compared with healthy controls, the proportion of MAIT cells in AML patients is reduced and the expression of functional markers is abnormal, suggesting that their function is impaired and may be involved in the occurrence and development of AML.

Objective: To evaluate the gene mutation profile and prognostic significance of adult cytogenetically normal acute myeloid leukemia (CN-AML) with CEBPA-bZIP mutation.

Methods: Targeted sequencing was implemented on the diagnostic bone marrow DNA samples of 141 adult CN-AML subjects with CEBPA-bZIP mutation. The nomogram model for leukemia-free survival (LFS) rate was generated by combining genetic abnormalities and clinical data. Risk stratification was conducted based on prognostic variables and the effect of risk-adjusted consolidation therapy was investigated by Kaplan-Meier method.

Results: Four variables were finally included in our nomogram model after multivariate Cox analysis, and an equation for risk score calculation was obtained, risk score=1.300 2×white blood cell (WBC) (≥18.77×10⁹/L)+1.406 5×CSF3R mutation positive+2.648 9× KMT2A mutation positive+1.012 8×DNA methylation-related genes mutation positive. According to the nomogram model, patients were further divided into low-risk group (score=0, n=46) and high-risk group (score>0, n=95). Prognostic analysis showed that the 5-year LFS rate, 5-year overall survival (OS) rate, and 5-year cumulative incidence of relapse (CIR) of patients who received allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the high-risk group were 93.5%, 97.1%, and 3.5%, while those in patients who received maintenance chemotherapy were 32.9%, 70.5%, and 63.4%, respectively. The differences were statistically significant (all P < 0.05). Allo-HSCT could significantly improve the prognosis of patients in high-risk group. However, no corresponding benefit was observed in the low-risk group.

Conclusion: Adult CN-AML with CEBPA-bZIP mutation has a complex co-mutation pattern. The nomogram model based on mutations of CFS3R, KMT2A and DNA methylation-related genes together with WBC count can further divide this subset of patients into a relatively low-risk group and a relatively high-risk group. For individuals in the high-risk group, allo-HSCT is proposed as post-remission therapy. The above data will benefit the prognosis estimation and treatment decision for adult CN-AML with CEBPA-bZIP mutation.

Objective: To investigate the expression of serum histone deacetylase 2 (HDAC2) in patients with acute myeloid leukemia (AML) and its clinical significance.

Methods: The 150 AML patients who received treatment in our hospital from April 2017 to April 2019 were included as AML group, and further divided into survival group (108 cases) and death group (42 cases) according to their survival status. In addition, 100 health individuals undergoing health examination in the same period were included as control group. The expression of HDAC2 in serum was detected by real-time quantitative PCR. Cox regression was used to analyze the influencing factors of adverse prognosis. The predictive effect of serum HDAC2 for the adverse prognosis of AML patients was evaluated by the receiver operating characteristic (ROC) curve.

Results: Compared with the control group, the expression of serum HDAC2, interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1β and C-reactive protein (CRP) in AML group was obviously increased (all P <0.05). Compared with the survival group, the expression of serum HDAC2 in the death group was also increased (P <0.05). Serum HDAC2 was positively correlated with IL-6, TNF-α, IL-1β and CRP in AML patients (r =0.567, 0.559, 0.623, 0.537). According to Cox regression analysis, the IL-6, TNF-α, IL-1β and HDAC2 were independent risk factors affecting the prognosis of AML patients (all P <0.05). ROC analysis showed that the AUC of serum HDAC2 level in predicting the adverse prognosis of AML was 0.862.

Conclusion: The expression level of serum HDAC2 in AML patients is increased, which has positive correlations with IL-6, TNF-α, IL-1β and CRP. HDAC2 is an independent risk factor for the poor prognosis of AML patients, and has a high predictive value.

Objective: To analyze the clinical characteristics and prognosis of children with hypodiploid B-cell precursor acute lymphoblastic leukemia (BCP-ALL).

Methods: The clinical data of 1 287 children with BCP-ALL admitted to five hospital in Fujian province from April 2011 to December 2020 were retrospectively analyzed. According to the results of chromosome karyotype, all the patients were grouped into hypodiploid subgroup and non-hypodiploid subgroup. The clinical characteristics, early treatment response ［minimal residual disease (MRD) on middle stage of induction chemotherapy and end of induction chemotherapy］ and long-term efficacy ［overall survival (OS) and event-free survival (EFS)］ were compared. The prognostic factors of hypodiploid BCP-ALL were further explored.

Results: Among 1 287 BCP-ALL patients， 28 patients (2.2%) were hypodiploid BCP-ALL. The proportion of patients with white blood cell count （WBC）≥50×10⁹/L in the hypodiploid subgroup was significantly higher than that in the non-hypodiploid subgroup (P =0.004), while there was no statistically significant difference in gender ratio, age group at initial diagnosis, and early treatment response between the two groups (all P >0.05). The 5-year EFS and OS rate of the hypodiploid subgroup were 75.0%(95%CI ：66.8%-83.2%) and 77.8%(95%CI ：69.8%-85.8%), respectively, which were lower than those of non-hypodiploid subgroup ［EFS: 79.6%(95%CI ：78.4%-80.8%); OS: 86.4%(95%CI ：85.4%-87.5%)］, but the difference was not statistically significant (all P >0.05). Further subgroup analysis by risk stratification showed that the 5-year EFS and OS rates of the hypodiploid subgroup were significantly lower than those in the low-risk (LR) group ［LR group EFS: 91.4% (95%CI ：88.4%-93.6% ), P < 0.001; OS: 94.7% (95%CI ：92.1%-96.4%), P < 0.001］ ; it was similar to that of BCP-ALL children stratified into intermediate-risk (IR) excluding hypodiploid ［IR group EFS: 79.4%(95%CI ：74.9%-83.2%), P =0.343; OS: 87.3%(95%CI ：83.6%-90.2%), P =0.111］; while was higher than that of EFS in HR group, but the difference was not statistically significant ［HR group EFS: 58.7%(95%CI ：52.6%-64.8%), P =0.178. OS: 69.9%(95%CI ：63.5%-75.4%), P =0.417］. Univariate analysis showed that gender, age, white blood cell count, and MRD on middle stage of induction chemotherapy had no significant impact on OS and EFS; chromosome count< 40 was a risk factor for lower OS (P =0.026), but has no significant effect on EFS; MRD≥0.01% after induction therapy was a risk factor for lower OS and EFS (P =0.002, and 0.001, respectively).

Conclusion: Children with hypodiploid BCP-ALL have an intermediate prognosis, and MRD ≥0.01% after induction chemotherapy may be a risk factors for poor prognosis.