TNFAIP3 Derived from Skeletal Stem Cells Alleviated Rat Osteoarthritis by Inhibiting the Necroptosis of Subchondral Osteoblasts.

Recent investigations have shown that the necroptosis of tissue cells in joints is important in the development of osteoarthritis (OA). This study aimed to investigate the potential effects of exogenous skeletal stem cells (SSCs) on the necroptosis of subchondral osteoblasts in OA. Human SSCs and subchondral osteoblasts isolated from human tibia plateaus were used for Western blotting, real-time PCR, RNA sequencing, gene editing, and necroptosis detection assays. In addition, the rat anterior cruciate ligament transection OA model was used to evaluate the effects of SSCs on osteoblast necroptosis in vivo. The micro-CT and pathological data showed that intra-articular injections of SSCs significantly improved the microarchitecture of subchondral trabecular bones in OA rats. Additionally, SSCs inhibited the necroptosis of subchondral osteoblasts in OA rats and necroptotic cell models. The results of bulk RNA sequencing of SSCs stimulated or not by tumor necrosis factor α suggested a correlation of SSCs-derived tumor necrosis factor α-induced protein 3 (TNFAIP3) and cell necroptosis. Furthermore, TNFAIP3-derived from SSCs contributed to the inhibition of the subchondral osteoblast necroptosis in vivo and in vitro. Moreover, the intra-articular injections of TNFAIP3-overexpressing SSCs further improved the subchondral trabecular bone remodeling of OA rats. Thus, we report that TNFAIP3 from SSCs contributed to the suppression of the subchondral osteoblast necroptosis, which suggests that necroptotic subchondral osteoblasts in joints may be possible targets to treat OA by stem cell therapy.

[Effects of different carbon sources on growth and active component contents in Salvia miltiorrhiza and S. castanea f. tomentosa hairy roots].

Salvia miltiorrhiza(Sm) and Salvia castanea f. tomentosa(Sc) hairy roots were used as experimental materials to study the effects of six different carbon sources, galactose, fructose, lactose, glucose, arabinose and sucrose(control), on fresh weight, dry weight, contents and yields of salvianolic acids and tanshinones. The results showed that galactose was most beneficial to the growth of two kinds of hairy roots, while lactose and arabinose were not conducive to their growth. As for Sm hairy roots, fructose significantly promoted the accumulation of salvianolic acid B, and the content increased by 5.801 times and 10.151 times compared with the control group, respectively. Glucose significantly promoted the accumulation of salvianolic acids. The content and yield of rosmarinic acid were 7.674 times and 9.260 times of that of the control group, and the content and yield of salvianolic acid B were 5.532 times and 6.675 times of the control group. For the hairy roots of Sc, galactose significantly increased the content and yield of rosmarinic acid, reaching 7.820 times and 9.944 times of the control group, respectively. Fructose promoted the increase of the content and yield of cryptotanshinone, reaching 9.242 times and 6.609 times of the control group, respectively. The study confirmed the optimal carbon source for the hairy root culture of Sm and Sc, and provided theoretical guidance for large-scale production of Sm drug-derived components and the utilization of Sc.

Efficacy and safety of mesenchymal stromal cells for the prophylaxis of chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation: a meta-analysis of randomized controlled trials.

A meta-analysis of randomized controlled trials (RCTs) was conducted to evaluate the efficacy and safety of mesenchymal stromal cells (MSCs) for the prophylaxis of chronic graft-versus-host disease (cGVHD) in patients with hematological malignancies undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Six studies involving 365 patients were included. The pooled results showed that MSCs significantly reduced the incidence of cGVHD (risk ratio [RR] 0.63, 95% confidence interval [CI] 0.46 to 0.86, P = 0.004). Favorable prophylactic effects of MSCs on cGVHD were observed with umbilical cord-derived, high-dose, and late-infusion MSCs, while bone marrow-derived, low-dose, and coinfused MSCs did not confer beneficial prophylactic effects. In addition, MSC infusion did not increase the risk of primary disease relapse and infection (RR 1.02, 95% CI 0.70 to 1.50, P = 0.913; RR 0.89, 95% CI 0.44 to 1.81, P = 0.752; respectively). Moreover, there was an apparent trend toward increased overall survival (OS) in the MSC group compared with that in the control group (RR 1.13, 95% CI 0.98 to 1.29, P = 0.084). In conclusion, this meta-analysis demonstrated that MSC infusion is an effective and safe prophylactic strategy for cGVHD in patients with hematological malignancies undergoing allo-HSCT.

[Establishment and Evaluation Strategy of an in vitro Cell Model of Bone Marrow Microenvironment Injury in Mouse Acute Graft-Versus-Host Disease].

OBJECTIVE: To establish a mesenchymal stem cell（MSC）-based in vitro cell model for the evaluation of mouse bone marrow acute graft-versus-host disease (aGVHD). METHODS: Female C57BL/6N mice aged 6-8 weeks were used as bone marrow and lymphocyte donors, and female BALB/c mice aged 6-8 weeks were used as aGVHD recipients. The recipient mouse received a lethal dose (8.0 Gy,72.76 cGy/min) of total body γ irradiation, and injected with donor mouse derived bone marrow cells (1×107/mouse) in 6-8 hours post irradiation to establish a bone marrow transplantation (BMT) mouse model (n=20). In addition, the recipient mice received a lethal dose (8.0 Gy,72.76 cGy/min) of total body γ irradiation, and injected with donor mouse derived bone marrow cells (1×107/mouse) and spleen lymphocytes (2×106/mouse) in 6-8 hours post irradiation to establish a mouse aGVHD model (n=20). On the day 7 after modeling, the recipient mice were anesthetized and the blood was harvested post eyeball enucleation. The serum was collected by centrifugation. Mouse MSCs were isolated and cultured with the addition of 2%, 5%, and 10% recipient serum from BMT group or aGVHD group respectively. The colony-forming unit-fibroblast（CFU-F) experiment was performed to evaluate the potential effects of serums on the self-renewal ability of MSC. The expression of CD29 and CD105 of MSC was evaluated by immunofluorescence staining. In addition, the expression of self-renewal-related genes including Oct-4, Sox-2, and Nanog in MSC was detected by real-time fluorescence quantitative PCR（RT-qPCR). RESULTS: We successfully established an in vitro cell model that could mimic the bone marrow microenvironment damage of the mouse with aGVHD. CFU-F assay showed that, on day 7 after the culture, compared with the BMT group, MSC colony formation ability of aGVHD serum concentrations groups of 2% and 5% was significantly reduced （P < 0．05）; after the culture, at day 14, compared with the BMT group, MSC colony formation ability in different aGVHD serum concentration was significantly reduced （P < 0．05）. The immunofluorescence staining showed that, compared with the BMT group, the proportion of MSC surface molecules CD29+ and CD105+ cells was significantly dereased in the aGVHD serum concentration group (P < 0.05), the most significant difference was at a serum concentration of 10% （P < 0．001, P < 0.01）. The results of RT-qPCR detection showed that the expression of the MSC self-renewal-related genes Oct-4, Sox-2, and Nanog was decreased, the most significant difference was observed at an aGVHD serum concentration of 10% （P < 0.01,P < 0．001,P < 0．001）. CONCLUSION: By co-culturing different concentrations of mouse aGVHD serum and mouse MSC, we found that the addition of mouse aGVHD serum at different concentrations impaired the MSC self-renewal ability, which providing a new tool for the field of aGVHD bone marrow microenvironment damage.

[Establishment and Evaluation of Intestinal Injury Model of Mouse Acute Graft Versus Host Disease Based on An Organoid Technology].

OBJECTIVE: To establish an intestinal organoid model that mimic acute graft versus host disease (aGVHD) caused intestinal injuries by using aGVHD murine model serum and organoid culture system, and explore the changes of aGVHD intestine in vitro by advantage of organoid technology. METHODS: 20-22 g female C57BL/6 mice and 20-22 g female BALB/c mice were used as donors and recipients for bone marrow transplantation, respectively. Within 4-6 h after receiving a lethal dose (8.0 Gy) of γ ray total body irradiation, a total of 0.25 ml of murine derived bone marrow cells (1×107/mice, n=20) and spleen nucleated cells (5×106/mice, n=20) was infused to establish a mouse model of aGVHD (n=20). The aGVHD mice were anesthetized at the 7th day after transplantation, and the veinal blood was harvested by removing the eyeballs, and the serum was collected by centrifugation. The small intestinal crypts of healthy C57BL/6 mice were harvested and cultivated in 3D culture system that maintaining the growth and proliferation of intestinal stem cells in vitro. In our experiment, 5%, 10%, 20% proportions of aGVHD serum were respectively added into the organoid culture system for 3 days. The formation of small intestinal organoids were observed under an inverted microscope and the morphological characteristics of intestinal organoids in each groups were analyzed. For further evaluation, the aGVHD intestinal organoids were harvested and their pathological changes were observed. Combined with HE staining, intestinal organ morphology evaluation was performed. Combined with Alcian Blue staining, the secretion function of aGVHD intestinal organoids was observed. The distribution and changes of Lgr5+ and Clu+ intestinal stem cells in intestinal organoids were analyzed under the conditions of 5%, 10% and 20% serum concentrations by immunohistochemical stainings. RESULTS: The results of HE staining showed that the integrity of intestinal organoids in the 5% concentration serum group was better than that in the 10% and 20% groups. The 5% concentration serum group showed the highest number of organoids, the highest germination rate and the lowest pathological score among experimental groups, while the 20% group exhibited severe morphological destruction and almost no germination was observed, and the pathological score was the highest among all groups(t=3.668, 4.334,5.309,P<0.05). The results of Alican blue staining showed that the secretion function of intestinal organoids in serum culture of aGVHD in the 20% group was weaker than that of the 5% group and 10% of the organoids, and there was almost no goblet cells, and mucus was stainned in the 20% aGVHD serum group. The immunohistochemical results showed that the number of Lgr5+ cells of intestinal organoids in the 5% group was more than that of the intestinal organoids in the 10% aGVHD serum group and 20% aGVHD serum group. Almost no Clu+ cells were observed in the 5% group. The Lgr5+ cells in the 20% group were seriously injuried and can not be observed. The proportion of Clu+ cells in the 20% group significantly increased. CONCLUSION: The concentration of aGVHD serum in the culture system can affect the number and secretion function of intestinal organoids as well as the number of intestinal stem cells in organoids. The higher the serum concentration, the greater the risk of organoid injury, which reveal the characteristics of the formation and functional change of aGVHD intestinal organoids, and provide a novel tool for the study of intestinal injury in aGVHD.

Single-cell transcriptomic analysis identifies a highly replicating Cd168+ skeletal stem/progenitor cell population in mouse long bones.

Skeletal stem/progenitor cells (SSPCs) are tissue-specific stem/progenitor cells localized within skeletons and contribute to bone development, homeostasis, and regeneration. However, the heterogeneity of SSPC populations in mouse long bones and their respective regenerative capacity remain to be further clarified. In this study, we perform integrated analysis using single-cell RNA sequencing (scRNA-seq) datasets of mouse hindlimb buds, postnatal long bones, and fractured long bones. Our analyses reveal the heterogeneity of osteochondrogenic lineage cells and recapitulate the developmental trajectories during mouse long bone growth. In addition, we identify a novel Cd168+ SSPC population with highly replicating capacity and osteochondrogenic potential in embryonic and postnatal long bones. Moreover, the Cd168+ SSPCs can contribute to newly formed skeletal tissues during fracture healing. Furthermore, the results of multicolor immunofluorescence show that Cd168+ SSPCs reside in the superficial zone of articular cartilage as well as in growth plates of postnatal mouse long bones. In summary, we identify a novel Cd168+ SSPC population with regenerative potential in mouse long bones, which adds to the knowledge of the tissue-specific stem cells in skeletons.

Human osteoarthritic articular cartilage stem cells suppress osteoclasts and improve subchondral bone remodeling in experimental knee osteoarthritis partially by releasing TNFAIP3.

BACKGROUND: Though articular cartilage stem cell (ACSC)-based therapies have been demonstrated to be a promising option in the treatment of diseased joints, the wide variety of cell isolation, the unknown therapeutic targets, and the incomplete understanding of the interactions of ACSCs with diseased microenvironments have limited the applications of ACSCs. METHODS: In this study, the human ACSCs have been isolated from osteoarthritic articular cartilage by advantage of selection of anatomical location, the migratory property of the cells, and the combination of traumatic injury, mechanical stimuli and enzymatic digestion. The protective effects of ACSC infusion into osteoarthritis (OA) rat knees on osteochondral tissues were evaluated using micro-CT and pathological analyses. Moreover, the regulation of ACSCs on osteoarthritic osteoclasts and the underlying mechanisms in vivo and in vitro were explored by RNA-sequencing, pathological analyses and functional gain and loss experiments. The one-way ANOVA was used in multiple group data analysis. RESULTS: The ACSCs showed typical stem cell-like characteristics including colony formation and committed osteo-chondrogenic capacity. In addition, intra-articular injection into knee joints yielded significant improvement on the abnormal subchondral bone remodeling of osteoarthritic rats. Bioinformatic and functional analysis showed that ACSCs suppressed osteoarthritic osteoclasts formation, and inflammatory joint microenvironment augmented the inhibitory effects. Further explorations demonstrated that ACSC-derived tumor necrosis factor alpha-induced protein 3 (TNFAIP3) remarkably contributed to the inhibition on osteoarhtritic osteoclasts and the improvement of abnormal subchondral bone remodeling. CONCLUSION: In summary, we have reported an easy and reproducible human ACSC isolation strategy and revealed their effects on subchondral bone remodeling in OA rats by releasing TNFAIP3 and suppressing osteoclasts in a diseased microenvironment responsive manner.

Landscape of Immune Microenvironment in Epithelial Ovarian Cancer and Establishing Risk Model by Machine Learning.

BACKGROUND: Epithelial ovarian cancer (EOC) is an extremely lethal gynecological malignancy and has the potential to benefit from the immune checkpoint blockade (ICB) therapy, whose efficacy highly depends on the complex tumor microenvironment (TME). METHOD AND RESULT: We comprehensively analyze the landscape of TME and its prognostic value through immune infiltration analysis, somatic mutation analysis, and survival analysis. The results showed that high infiltration of immune cells predicts favorable clinical outcomes in EOC. Then, the detailed TME landscape of the EOC had been investigated through "xCell" algorithm, Gene set variation analysis (GSVA), cytokines expression analysis, and correlation analysis. It is observed that EOC patients with high infiltrating immune cells have an antitumor phenotype and are highly correlated with immune checkpoints. We further found that dendritic cells (DCs) may play a dominant role in promoting the infiltration of immune cells into TME and forming an antitumor immune phenotype. Finally, we conducted machine-learning Lasso regression, support vector machines (SVMs), and random forest, identifying six DC-related prognostic genes (CXCL9, VSIG4, ALOX5AP, TGFBI, UBD, and CXCL11). And DC-related risk stratify model had been well established and validated. CONCLUSION: High infiltration of immune cells predicted a better outcome and an antitumor phenotype in EOC, and the DCs might play a dominant role in the initiation of antitumor immune cells. The well-established risk model can be used for prognostic prediction in EOC.

Emerging role of long non-coding RNAs in normal and malignant hematopoiesis.

Long noncoding RNAs (lncRNAs) have recently been discovered and are increasingly recognized as vital components of modern molecular biology. Accumulating evidence shows that lncRNAs have emerged as important mediators in diverse biological processes such as cell differentiation, pluripotency, and tumorigenesis, while the function of lncRNAs in the field of normal and malignant hematopoiesis remains to be further elucidated. Here, we widely reviewed recent advances and summarize the characteristics and basic mechanisms of lncRNAs and keep abreast of developments of lncRNAs within the field of normal and malignant hematopoiesis. Based on gene regulatory networks at different levels of lncRNAs participation, lncRNAs have been shown to regulate gene expression from epigenetics, transcription and post transcription. The expression of lncRNAs is highly cell-specific and critical for the development and activation of hematopoiesis. Moreover, we also summarized the role of lncRNAs involved in hematological malignancies in recent years. LncRNAs have been found to play an emerging role in normal and malignant hematopoiesis, which may provide novel ideas for the diagnosis and therapeutic targets of hematological diseases in the foreseeable future.

[Clinical Significance of Serum Immunoglobulin Level after Peri- pheral Blood Haploidentical Hematopoietic Stem Cell Transplantation in Patients with Hematologic Malignancies].

OBJECTIVE: To investigate the clinical significance of post-transplantation serum immunoglobulin level in the outcome of patients with hemalologic malignancies treated by haploidentical peripheral hematopoietic stem cell transplanta-tion(Haplo-HSCT). METHODS: The clinical data of 157 patients treated by haplo-HSCT were analyzed retrospectively. The overall survival rate (OS), graft versus host disease (GVHD) incidence, infection incidence, serum immunoglobulin level, the relationship of immunoglobulin levels with OS and transplant complications were analyzed. RESULTS: The 2-year OS rate was 59.2%(95%CI:51.6%-66.9%), 2-year relapse mortality was 11.5%(95%CI: 6.4%-16.6%), and non-relapse mortality was 29.3%(95%CI:21.7%-36.9%). The cumulative incidence of III-IV aGVHD was 16.6%(95%CI:10.8%-22.9%); the cumulative incidence of extensive cGVHD was 21.7%(95%CI:15.3%-28.6%); the cumulative incidence of severe bacterial infection within 1 year was 59.2%(95%CI:51.6%-66.2%); the cumulative incidence of invasive fungal infection was 47.1%(95%CI:38.9%-54.8%). The occurrence of extensive cGVHD after haplo-HSCT related with the gender match of donor-recipient and bacterial infection. The levels of IgG in patients with 0-II aGVHD and patients with III-IV aGVHD for 1 month after haplo-HSCT were (6.96±2.47) and (4.27±2.42) g/L （P=0.003）, IgG levels at 3 months afte haplo-HSCT were (8.71±4.47) and (6.65±2.95) g/L (P=0.038); IgG levels at 1 month after haplo-HSCT showed predictive value for III-IV aGVHD susceptibility(P=0.003); for patients with IgG<4 g/L at any time after haplo-HSCT, the incidence of extensive cGVHD was significantly increased (35.5% vs 18.3%) (P=0.037), the incidence of fungal infection within 1 year after haplo-HSCT was significantly increased(71.0% vs 41.3%) (P=0.003), and the 2-year survival rate was reduced significantly (P=0.035). CONCLUSION: Haplo-HSCT is effective for the treatment of hematologic malignancies. Patients with lower IgG at 1 month after haplo-HSCT are more likely to develop III-IV aGVHD, and IgG levels at 1 month after haplo-HSCT can predict its susceptibility to a certain extent. Patients with severe hypoimmunoglobulinemia (IgG<4 g/L) after haplo-HSCT are more likely to develop extensive cGVHD, fungal infection and show worse survival prognosis.

[Analysis of Clinical Pathological Features and Prognosis in Young Patients with Diffuse Large B Cell Lymphoma].

OBJECTIVE: To explore the clinical pathological features of the patients with diffuse large B cell lymphoma (DLBCL) and their prognostic factors. METHODS: The prognosis of the clinical pathological features and their influence on prognosis of 177 patients diagnosed as DLBCL at the first visit from January 2013 to May 2017 in our hospital were analyzed retrospectively. RESULTS: The univariate analysis showed that overall survival (OS) and progression-free survival (PFS) were associated with later Ann Arbor stage (Ⅲ-Ⅳ) ( P＜0.01, P＜0.05), high performance status (ECOG score 2-4) (P＜0.01, P＜0.05), extranodal involvement ＞1 (P＜0.01, P＜0.05), elevated LDH level (P＜0.01, P＜0.05). B symptom (P＜0.05) and elevated ß2-MG level (P＜0.05) also influenced OS. COX multivariate analysis showed that the elevated ß2-MG level (P＜0.05) and later stage (Ⅲ-Ⅳ) (P＜0.05) have an independent influence on OS, later stage (Ⅲ-Ⅳ) (P＜0.05) also independently influenced PFS. The patients with high aaIPI score (2-3) and bone marrow involvement before treatment had poor OS (P＜0.01, P＜0.01) and PFS (P＜0.05, P＜0.01). CONCLUSION: Elevated ß2-MG level can independently influence OS, and later stage (Ⅲ-Ⅳ) can independently influence both OS and PFS. High aaIPI score (2-3) and bone marrow involvement before treatment have an inferior influence on OS and PFS.

Outcome of Rituximab-Based Treatment for Post-Transplant Lymphoproliferative Disorder After Allogeneic Hematopoietic Stem Cell Transplantation: A Single-Center Experience.

BACKGROUND Post-transplant lymphoproliferative disorder (PTLD) is a rare complication following solid organ transplantation and allogeneic hematopoietic stem cell transplantation (Allo-HSCT), which gives rise to high mortality rates. MATERIAL AND METHODS This was a single-center retrospective analysis based on 27 patients who were diagnosed with PTLD following Allo-HSCT between January 1, 2007 and June 2018 at the Chinese PLA General Hospital. The purpose of this analysis was to investigate responses and prognostic factors of rituximab-based treatment. RESULTS Twenty-seven patients were treated with rituximab. Among them, 20 of 27 patients (74.07%) had a complete response, 2 of 27 patients (7.41%) had a partial response, 5 of 27 patients (18.52%) had no response, and 22 of 27 patients (81.48%) cleared Epstein-Barr virus (EBV) copies. There were no obvious side effects. The 1-year overall survival (OS) estimate was 46.8% (95% CI, 23.1-65.5%). Univariate analysis revealed that lower OS was correlated with Eastern Cooperative Oncology Group (ECOG) score standard (3-4), Epstein-Barr virus (EBV) viral load (≥106 copies/mL), bacteria or fungal infection, and EBV reactivation were positive after treatment with 1 or 2 doses of rituximab (P<0.05). Multivariate analysis showed that each of the following were independently associated with lower OS (P<0.05): female, ECOG score standard (3-4), and EBV reactivation were positive after treatment with 1 or 2 doses of rituximab. CONCLUSIONS Our results demonstrated that rituximab-based treatment was a safe and effective strategy for patients who were diagnosed with PTLD following Allo-HSCT. The identified prognostic factors may help to detect which PTLD patients are at a higher risk of mortality.

Single- Versus Double-Unit Umbilical Cord Blood Transplantation for Hematologic Diseases: A Systematic Review.

Controversial results exist regarding the clinical benefits of single- vs double-unit umbilical cord blood transplantation (UCBT) in patients with hematologic diseases. A systematic review was conducted to evaluate this issue. The PubMed, Embase, and Cochrane Library databases were searched up to May 2018. A total of 25 studies including 6571 recipients were identified. Although double-unit UCB contained higher doses of total nucleated cells and CD34+ cells, it offered no advantages over single-unit UCB in terms of hematologic recovery, including the rate and speed of neutrophil and platelet engraftment. Double-unit UCBT was associated with higher incidences of grades II-IV acute and extensive chronic graft-vs-host disease, accompanied by a lower relapse incidence, which may be attributed to a graft-vs-graft effect induced by double-unit UCB. However, transplant-related mortality, disease-free survival, and overall survival were comparable between single- and double-unit UCBT. Although double-unit UCBT confers no clinical advantages over single-unit UCBT, certain patients, such as those at high risk of relapse, might benefit from double-unit UCBT, a possibility that needs to be clarified in future randomized trials.

Outcome and Prognostic Factors of High-Risk Acute Myeloid Leukemia After Allogeneic Hematopoietic Stem Cell Transplantation.

BACKGROUND Allogeneic transplantation remains one of the best therapies for high-risk acute myeloid leukemia (HR-AML). MATERIAL AND METHODS This study retrospectively analyzed 126 patients with HR-AML after allogeneic hematopoietic stem cell transplantation (allo-HCST). RESULTS The disease-free survival (DFS) rates of 1 year and 3 years were 58.83% (95%CI: 50.75-68.20%) and 53.09% (95%CI: 44.59-63.22%) respectively. The cumulative relapse rates of 1 year and 3 years were 21.1% (95%CI: 14.4-28.8%) and 25.9% (95%CI: 18.1-34.5%) respectively. The cumulative incidences of III to IV acute graft-versus-host disease (aGVHD) for 100 days was 8.70% (95%CI: 4.6-14.5%). The cumulative rate of extensive chronic graft-versus-host disease (cGVHD) for 1-year was 4.1% (95%CI: 1.5-8.7%). The cumulative transplantation related mortality rate of 1 year and 3 years were 20.1% (95%CI: 13.6-27.6%) and 21.0% (95%CI: 14.3-28.6%) respectively. Univariate analysis revealed that lower overall survival was correlated with age, bacterial or fungal infection, disease status at transplantation, III-IV aGVHD, post-transplantation lymphoproliferative disorders (PTLD), white blood cell engraftment, and extramedullary involvement (P<0.05). The results of multivariate analysis were that the aforementioned factors were also related to lower overall survival except for PTLD (P<0.05). The results of univariate and multivariate analysis were that extramedullary involvement, III-IV aGVHD, and status pre-transplantation influenced DFS (P<0.05). The risk factors for relapse were status pre-transplantation and extramedullary involvement by univariate and multivariate analysis (P<0.05). CONCLUSIONS HR-AML has inferior prognosis. Our study indicated the necessity of achieving remission status prior to hematopoietic stem cell transplantation, and administration of preventive treatments on high-risk patients after hematopoietic stem cell transplantation. In addition, adequate prevention and treatment of complications are needed.

[Establishment of A20 Murine B Lymphoma Transplantation Model Labeled with Luciferase].

OBJECTIVE: To establish the animal model of luciferase-transfected A20 murine B cell lymphoma, so as to provide experimental tools to explore the effect of graft versus tumor. METHODS: Luciferase- labeled A20 cells were cloned with puromycin selection. Transfected A20 cells and C57BL/6 bone marrow were inoculated into the irradiated BALB/c mice by injection in tailvein to establish the transplantation model. The bioluminescent imaging technique was used to monitor the tumor growth, and then the survival, body weight, tumor formation and pathological characteristics of target organs were observed. RESULTS: A20 cell line stably expressing luciferase gene was successfully obtained. The the bioluminicent imaging found that the tumor luminescence could be observed on day 8 of A20 cell inoculation, and the mean fluorescent intensity was increased along with the tumor growth. Compared with the BMT group, the survival rate and body weight of BMT+A20-Luc+ mice were decreased significantly. General anatomy showed the tumor mainly formed in the liver and spleen. CONCLUSION: A mouse transplantation model with luciferase- transfected A20 cells has been successfully established, thus laying a foundation for investigation of graft-versus-tumor.

[Application of Chimeric Antigen Receptor-Modified NK Cells in Multiple Myeloma].

OBJECTIVE: To explore the killing effect of CAR (CD138-CD28-CD3ζ)-NK cells on myeloma cells through construction of CAR(CD138-CD28-CD3)-NK cells. METHODS: The antiCD138scFv-CD28-CD3 zeta plasmid pcDNA3.1 was constructed, which then together with 3 plasmid lentiviral packaging system were transfected into 293T cells, the virus was collected. Furthermore, in order to get the stably transfected cell line, the NK92MI cell line was infected by the virus, then the positive cells were screened by puromycin. The expression of the CARNK cells were verified by RT-PCR and Western blot. At last the ability of secreting cytokine CD107a was detected by flow cytometry, and the statistical analysis was carried out to verify the anti-myeloma effect of CAR-NK cells. RESULTS: Gene fragment of the CAR(antiCD138scFv-CD28-CD3ζ) was constructed successfully by gene engineering technique in vitro, and the gene sequence was verified to be correct by sequencing. By virus packaging technology, the virus expressing the protein of the CAR was obtained. PCR and Western blot verified the expression of CAR fusion protein on the sufurce of NK cells. The cell killing experiment confirmed that the CAR-NK cells possessed the ability to secrete cytokine CD107a superior to control cells and showed the obvious killing effect on multiple myeloma cells. CONCLUSION: The CAR can be constructed in vitro, and express on NK92 cells. The CAR-NK cells can kill the multiple myeloma cells expressing CD138 antigen, thereby plays an antimyeloma effect.

Effects of template molecules on the structures and luminescence intensities of a series of porous Tb-MOFs based on the 2-nitroterephthalate ligand.

Four novel porous Tb(iii) metal-organic frameworks (Tb-MOFs) have been designed and prepared hydrothermally from 2-nitroterephthalate (2-H2ntp), namely {[Tb(2-ntp)1.5(H2O)]·H2O} n (1), {[Tb(2-ntp)2(H2O)]·4,4'-Hbipy} n (2), {[Tb(2-ntp)2(H2O)]·2,4-Hbipy} n (3), and {[Tb(2-ntp)2(H2O)]·(1,4-H2bbi)0.5} n (4) (4,4'-bipy = 4,4'-bipyridine; 2,4-bipy = 2,4-bipyridine; 1,4-bbi = 1,4-bisbenzimidazole). X-ray diffraction structural analyses show these Tb-MOFs are porous and are based on Tb3+ ions and 2-nitroterephthalate, in which water molecules (1) or protonated N-donor ligands (2-4) exist as templates. The fluorescence properties of complexes 1-4 could be associated with the characteristic peaks of Tb(iii) ions, and the existence of different guest molecules affects the intensities of peaks, which means that these could be potential fluorescence materials, with intensities adjusted using guests.

[Advances of Diagnostic Biomarkers on Acute Graft Versus Host Disease -Review].

Acute graft-versus-host disease (aGVHD) is one of the main complications after allogeneic hematopoietic stem cell transplantation (HCT), resulting in considerable morbidity and mortality. Diagnosis of aGVHD is typically based on clinical presentation and confirmed by biopsy. In recent years, aGVHD researches have been focused on diagnostic biomarkers, and several microRNAs, cytokines and immunoregulated cells have been dicovered currently. This review summarizes the advances on aGVHD biomarkers.

[Treatment of Chronic Graft versus Host Disease -Review].

Chronic graft versus host disease (cGVHD) is the major reason of late non-relapse related death after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The therapeutic regimen based on glucocorticoids remains as the standard initial treatment of cGVHD. But due to the significant side effects and unsatisfactory outcomes, particularly for patients with steroid-refractory disease, the more effective and less toxic therapies should be needed. Abnormal regulation for the stable state of T lymphocytes and B lymphocytes for allo-HSCT patients is the current focus of studying cGVHD pathogenesis. This article summarizes the treatment of cGVHD targeted to pathogenic factors and signaling pathways in pathogenetic process.

Discovery of 5,6-diaryl-1,2,4-triazines hybrids as potential apoptosis inducers.

A series of 5,6-diaryl-1,2,4-triazines hybrids bearing a 1,2,3-triazole linker were synthesized by molecular hybridization strategy and evaluated for antiproliferative activity against three selected cancer cell lines (MGC-803, EC-109 and PC-3). The first structure-activity relationship (SAR) for these 5,6-diaryl-1,2,4-triazines is explored in this report with evaluation of 15 variants of the structural class. Among these chemical derivatives, 3-(((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-5,6-diphenyl-1,2,4-triazine (11E) showed the more potent inhibitory effect against three cell lines than 5-Fu. Cellular mechanism studies in MGC-803 cells elucidated 11E inhibited colony formation and arrested cell cycle at G2/M phase. Furthermore, compound 11E caused morphological changes, decreased mitochondrial membrane potential, and induced apoptosis through the apoptosis-related proteins in MGC-803 cells. It was the first time, to our knowledge, that 5,6-diaryl-1,2,4-triazines bearing a 1,2,3-triazole linker were used as potential apoptosis inducers.