Objective:To investigate the effect of gemcitabine on myeloid derived suppressor cells (MDSC) in the spleen of B lymphoma cell-bearing mice, and the therapeutic effect of gemcitabine combined with intratumoral injection of dendritic cells (DCs) in treatment of large B lymphoma. Methods: BALB/c mice were inoculated subcutaneously with B lymphoma A20 cells; large tumors were formed 30 d after inoculation. Gr-1~+ CD11b~+ MDSC proportion in the spleen was analyzed by flow cytometry before and after gemcitabine treatment. Splenic MDSC sorted by immunomagnetic beads was further treated with gemcitabine, and then the apoptosis of MDSC was examined by Annexin-V/PI staining. Tumor growth and survival time of A20 tumor-bearing mice were observed after treatment with gemcitabine and intratumoral injection of DCs. Results: Splenic Gr-1~+ CD11b~+ MDSC ratio in A20 cell-bearing mice was 10 times higher than that in the normal mice. Gemcitabine induced apoptosis and necrosis of purified MDSC in vitro in a time-dependent manner. The percentage of MDSC in the spleen of A20 tumor-bearing mice was decreased after injection of a single dose of gemcitabine. Gemcit-abine or intratumoral injection of DCs alone inhibited growth of tumor to a certain degree, with the mean survival periods of mice in the gemcitabine, DCs, and untreated groups being (48.8±3.6) d, (47.2±7.4) d, and (38.8±2.2) d, respectively. Gemcitabine chemotherapy combined with intratumoral DC injection resulted in continuous shrink of the tumors, and 60% of the mice survived for more than 90 d. Conclusion: Gemcitabine can effectively eliminate splenic MDSC in tumor-bearing mice. Gemcitabine chemotherapy and DCs immunotherapy can work synergistically in the treat-ment of huge lymphoma. These results provide an experimental basis for the comprehensive chemotherapy and immunotber-apy of relapsed or refractory lymphoma.

Objective:To investigate the tumor-specific neoantigen for primary plasma cell leukemia (PCL) using gene sequencing technology combined with bioinformatic analysis. Methods: Peripheral blood samples of one patient with primary PCL during relapse and remission periods were collected. HLA molecular typing was performed using polymerase chain reaction with sequencing-based typing; whole-exome and transcriptome were sequenced by next-generation sequencing method; and bioinformatics software NetMHCpan was used to predict neoantigens. Results: Six tumor-specific missense mutations were found in the patient's peripheral blood during relapse period, located in genes FRG1, MLL3, SVIL, MYOM1, ZDHHC11 and RFPL4A.Considering patient's HLA sub-types, 43 neoantigens were predicted via bioinformatics. Considering that FRG1 and MLL3 had relatively high gene expression levels, 20 neoantigens derived from mutations of the two genes were preferentially selected, among which four neoantigens had high affinity with the patient's HLA molecules and thus had potential clinical application value. Conclusion: The study has completed a tumor neoantigen screen and prediction for primary PCL. This practice demonstrates that predicting neoantigen based on tumor-specific somatic mutation is feasible for primary PCL.

ObjectiveTo investigate the effect of Qiling Baitouweng Tang （QLBTWT） on proliferation and apoptosis， Janus kinase 2 （JAK2）/signal transducer and activator of transcription 3 （STAT3） signaling pathway and interleukin-10 （IL-10） in diffuse large B-cell lymphoma （DLBCL）. MethodWith human DLBCL cells OCI-LY10 and U2932 as research objects， cell proliferation was detected by cell counting kit-8 （CCK-8） assay. After treatment with 0， 4.6， 9.3， 18.7， 37.5， 75， 150 mg·L^-1 QLBTWT for 24 h， the half-inhibitory concentration （IC₅₀） of OCL-LY10 and U2932 cells was calculated to be 9.33， 16.13 mg·L^-1， respectively， based on which， 9.5， 19， 38 mg·L^-1 QLBTWT were selected for subsequent experiments. After 0， 9.5， 19， 38 mg·L^-1 QLBTWT treatment for 24 h， the zymogen activities of Caspase-3， Caspase-8 and Caspase-9 in OCI-LY10 and U2932 cells were detected using corresponding activity assay kits （colorimetric）， and the IL-10 expression was detected by enzyme-linked immuno sorbent assay （ELISA）. The apoptosis rate and cell cycle of OCI-LY10 and U2932 cells treated with different concentrations of QLBTWT for 24 h were detected by flow cytometry. The expressions of apoptosis-related proteins ［B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， cleaved poly adenosine diphosphate ribose polymerase （cleaved PARP）， cleaved Caspase-3］， JAK2， STAT3， phospho-JAK2 （p-JAK2）， phospho-STAT3 （p-STAT3） pathway proteins， and c-Myc protein in OCL-LY10 and U2932 cells after 24 h treatment with 0， 9.5， 19， 38 mg·L^-1 QLBTWT were all tested by Western blot. ResultAfter QLBTWT treatment on OCI-LY10 and U2932 cells for 24 h， cell proliferation was inhibited in each QLBTWT group compared with that in the control group （P<0.05， P<0.01）. The zymogens of Caspase-3， Caspase-8 and Caspase-9 were activated （P<0.01）， and there was an increase in cell apoptosis （P<0.05， P<0.01） and cell cycle arrest at Gap phase1 （G₁） phase in 9.5， 19 and 38 mg·L^-1 QLBTWT group （P<0.05， P<0.01）. After 9.5， 19 and 38 mg·L^-1 QLBTWT treatment on OCI-LY10 and U2932 cells for 24 h， the expressions of Bcl-2， p-JAK2 and p-STAT3 proteins were decreased （P<0.01）， and the expressions of Bax， cleaved PARP and cleaved Caspase-3 proteins were increased （P<0.01）， but no significant change was observed in the expressions of JAK2 and STAT3 proteins. Compared with the conditions in the control group， the expressions of c-Myc， p-JAK2， and p-STAT3 proteins were down-regulated in 19 mg·L^-1 QLBTWT group and 19 mg·L^-1 QLBTWT+10 μg·L^-1 IL-10 group （P<0.05， P<0.01）， and up-regulated in 10 μg·L^-1 IL-10 group （P<0.05， P<0.01）， while there was no difference in JAK2/STAT3 proteins. ConclusionQLBTWT can inhibit proliferation and induce apoptosis of human DLBCL cells OCI-LY10 and U2932， and the potential mechanism may be related to the regulation of JAK2/STAT3 signaling pathway.

ObjectiveTo investigate the efficacy and safety of cinobufagin tablets combined with thalidomide/dexamethasone （TD） regimen in the treatment of newly diagnosed multiple myeloma （NDMM） with phlegm and stasis obstruction. MethodsThe clinical data of 50 patients with NDMM of phlegm and stasis obstruction who were hospitalized at the Jiangsu Province Hospital of Chinese Medicine from June 1st， 2015 to July 31th， 2019 were retrospectively analyzed， and they were divided into a control group （bortezomib/dexamethasone-containing regimen， 27 cases） and an observation group （cinobufagin tablets combined with TD regimen， 23 cases）. The clinical efficacy and safety were compared between the two groups after two or three courses of treatment. The primary outcomes were clinical remission rate including overall response rate and deep remission rate， one-year and two-year overall survival rate， and adverse effects. The secondary outcomes were the proportion of plasma cells in bone marrow， hemoglobin， β2-microglobulin， lactate dehydrogenase， serum creatinine， blood urea nitrogen， bone pain score， and KPS functional status score （KPS score） before and after treatment. ResultsIn terms of clinical efficacy， there was no statistically significant difference （P＞0.05） in the overall response rate ［the observation group 69.57%（16/23） vs the control group 70.37% （19/27）］ and deep remission rate ［the observation group 56.52% （13/23） vs the control group 55.56% （15/27）］ between groups after the treatment. The one-year overall survival rates of the observation group and the control group were 90.9% and 92.4%， and the two-year overall survival rates were 81.8% and 80.9% respectively， with no statistically significant differences between groups （P＞0.05）. During the treatment， no renal function injury occurred in both groups. The incidence of peripheral nerve injury in the observation group was 8.70%， which was lower than 48.15% in the control group （P＜0.01）. After the treatment， the proportion of myeloma plasma cells， β2-microglobulin， serum creatinine level， and bone pain score decreased， while the hemoglobin level and KPS score increased in both groups （P＜0.05 or P＜0.01）. Compared between groups after treatment， the bone pain score of the observation group was lower than that of the control group， while the KPS score was higher than that of the control group （P＜0.05）. ConclusionThe clinical efficacy of cinobufagin tablets combined with TD in the treatment of NDMM is equivalent to bortezomib/dexamethasone-containing regimen， but the former is more helpful in relieving the pain and improving the quality of life， and has better safety.

ObjectiveTo investigate the effect of pulsatilla saponin A （PSA） on proliferation and apoptosis of human Burkitt lymphoma （BL） cell line Raji cells and expression of related pathway proteins. MethodWith Raji cells as the research object， the cell proliferation was detected by cell counting kit-8 （CCK-8） method， and the half-maximal inhibitory concentration （IC₅₀） values of 24 h， 48 h and 72 h were calculated to be 19.77， 18.31， 16.70 μmol·L^-1， respectively. In subsequent related experiments， 0， 8， 16， 32 μmol·L^-1 PSA were selected according to the IC₅₀ value of Raji cells treated with PAS for 72 h. After 0， 8， 16， 32 μmol·L^-1 PSA acted on Raji cells for 24， 48， 72 h， the optical density values of cell growth curve were detected by CCK-8 method. The zymogen activities of cysteine aspartate-specific protease （Caspase）-3， Caspase-8 and Caspase-9 in Raji cells treated with 0， 8， 16 and 32 μmol·L^-1 PSA for 24 h were measured by Caspase-3， Caspase-8 and Caspase-9 colorimetric assay kit. The apoptosis rate and cell cycle of Raji cells treated with different concentrations of PSA after 24 h were detected by flow cytometry. The expression of B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， cleaved poly（ADP-ribose） polymerase （cleaved PARP）， cleaved cysteinyl aspartate-specific protease-3 （cleaved Caspase-3） apoptosis related protein and Janus kinase 2 （JAK2）， signal transducer and activator of transcription 3 （STAT3）， phosphorylated-JAK2 （p-JAK2）， and phosphorylated- STAT3 （p-STAT3） pathway proteins in Raji cells after 24 h of treatment with 0， 8， 16 and 32 μmol·L^-1 PSA were tested by Western blot. ResultCompared with control group， decreased cell survival rate， inhibited cell proliferation， activated zymogens of Caspase-3， Caspase-8 and Caspase-9 （P<0.01）， increased apoptosis （P<0.05， P<0.01）， and enhanced cell cycle arrest in Gap phase 2 （G₂） were observed in 8， 16 and 32 μmol·L^-1 PSA groups（P<0.05， P<0.01）. Compared with control group， cells treated with 8， 16 and 32 μmol·L^-1 PSA had lower expression of Bcl-2， p-JAK2， p-STAT3 proteins （P<0.05， P<0.01）， and higher expression of Bax， cleaved PARP and cleaved Caspase-3 protein （P<0.01）， while no significant change was found in the expression of JAK2 and STAT3 proteins. ConclusionPSA could inhibit proliferation and induce apoptosis of Raji cells， and its potential mechanism might be related to the regulation of JAK2/STAT3 signaling pathway.

[Abstract] Objective: To establish a chimeric antigen receptor（CAR）modified T cells specifically targeting CD19 molecule (CD19CAR-T cells) and to testify their in vitro killing effect on target cells. Methods: CD19-CAR fragments yielded by PCR were constructed into pCDH-GFP lentiviral vectors by molecular cloning technology. The packaged lentiviral particles were transducted into CD3+ T cells of donors. Transduction efficiency was measured by flow cytometry and PCR. The in vitro cytotoxicity of obtained CD19CAR-T cells against CD19+ Ramos cells was tested by 7-AAD staining. Results: The amplification folds of CD3+ T cells increased to (78.8± 23.2) folds after in vitro culture for 10 days, and about (58.3±5.4)% cells expressing GFP.About (57.4±9.3)% CD19+Ramos cells were specifically killed by the CD19-CAR-T cells in vitro at the E∶T ratio of 5∶1. Conclusion: This study successfully established an effective method for constructing and amplifying CD19-CAR-T cells in vitro, which showed profound efficiency and specific cytotoxity against CD19+ Ramos cells.And this report might provide an experimental evidence for clinical treatment of CD19+ B cell neoplasmas.