Preparation of CD52-targeted chimeric antigen receptor-modified T cells and their anti-leukemia effects / 中华血液学杂志

Objective: To construct chimeric antigen receptor (CAR) T cells targeting CD52 (CD52 CAR-T) and validate the effect of CD52 CAR-T cells on CD52-positive leukemia. Methods: A second-generation CD52-targeting CAR bearing 4-1BB costimulatory domain was ligated into a lentiviral vector through molecular cloning. Lentivirus was prepared and packaged by 293 T cells with a four-plasmid system. Fluorescein was used to label cell surface antigens to evaluate the phenotype of CD52 CAR-T cells after infection. Flow cytometry and ELISA were used to evaluate the specific cytotoxicity of CD52 CAR-T cells to CD52-positive cell lines in vitro. Results: ①A pCDH-CD52scFv-CD8α-4-1BB-CD3ζ-GFP expressing plasmid was successfully constructed and used to transduce T cells expressing a novel CD52-targeting CAR. ②On day 6, CD52-positive T cells were almost killed by CD52-targeted CAR-T post lentivirus transduction [CD52 CAR-T (4.48 ± 4.99) %, vs Vector-T (56.58±19.8) %, P=0.011]. ③T cells transduced with the CAR targeting CD52 showed low levels of apoptosis and could be expanded long-term ex vivo. ④The CD52 CAR could promote T cell differentiation into central and effector memory T cells, whereas the proportion of T cells with a CD45RA(+) effector memory phenotype were reduced. ⑤CD52 CAR-T cells could specifically kill CD52-positive HuT78-19t cells but had no killing effect on CD52-negative MOLT4-19t cells. For CD52 CAR-T cells, the percentage of residual of HuT78-19t cells was (2.66±1.60) % at an the E:T ratio of 1∶1 for 24 h, while (56.66±5.74) % of MOLT4-19t cells survived (P<0.001) . ⑥The results of a degranulation experiment confirmed that HuT78-19t cells significantly activated CD52 CAR-T cells but not MOLT4-19t cells[ (57.34±11.25) % vs (13.06± 4.23) %, P<0.001]. ⑦CD52 CAR-T cells released more cytokines when co-cultured with HuT78-19t cells than that of vector-T cells [IFN-γ: (3706±226) pg/ml, P<0.001; TNF-α: (1732±560) pg/ml, P<0.01]. Conclusions: We successfully prepared CD52 CAR-T cells with anti-leukemia effects, which might provide the foundation for further immunotherapy.

Preparation of CD33 targeted bispecific- and trispecific-T cell engagers and their cytotoxicity on leukemia cells / 中华血液学杂志

Objective: To investigate the effect of CD33-targeted bi-specific and tri-specific T-cell engagers on T-cell proliferation and explore their cytotoxicity on leukemia cells. Methods: The CD33-targeted bi-specific T-cell engager (CD33-BiTE) and tri-specific T-cell engager (CD33-TriTE) expression vectors were successfully constructed and expressed through a eukaryotic cell expression system. CD33-BiTE and CD33-TriTE were purified by affinity chromatography. The effects of CD33-BiTE and CD33-TriTE on T cells were analyzed through in vitro experiments. Results: ① CD33-BiTE and CD33-TriTE were successfully constructed and purified and could compete with flow cytometry antibodies for binding to the target cells. ② After 12 days of co-culture with CD33-BiTE and CD33-TriTE, the number of human T cells were expanded to 33.89±19.46 and 81.56±23.62 folds, respectively. CD33-TriTE induced a stronger proliferation of T cells than CD33-BiTE (P<0.05) . ③ Both CD33-BiTE and CD33-TriTE induced specific dose-dependent cytotoxicity on CD33(+) leukemia cells. ④ Compared to CD33-TriTE, leukemia cells were prone to express PD-L1 when co-cultured with T cells and CD33-BiTE. CD33-TriTE induced powerful cytotoxicity on leukemia cells with high PD-L1 expression. Conclusion: CD33-BiTE and CD33-TriTE expression vectors were constructed, and fusion proteins were expressed in eukaryotic cells. Our results support the proliferative and activating effects of BiTE and TriTE on T cells. Compared to that of CD33-BiTE, CD33-TriTE induced a stronger proliferative effect on T cells and a more powerful cytotoxicity on leukemia cells with high PD-L1 expression.

Functional investigation of chimeric antigen receptor T cells targeting LMP1 antigen / 中华血液学杂志

Objective: This study aimed to create a type of CAR-T cells that targets LMP1 antigen and study its immunotherapeutic effect on LMP1-positive hematological malignancies. Methods: To generate LMP1 CAR-T cells, a plasmid expressing LMP1 CAR was created using molecular cloning technology, and T cells were infected with LMP1 CAR lentivirus. The effects of LMP1 CAR-T cells on specific cytotoxicity against LMP1-positive tumor cell lines infected with the EB virus had been confirmed. Results: ① LMP1 protein expressing on EB virus-positive lymphoma cells surface was verified. ② The LMP1 CAR-expressing plasmid was created, and LMP1 CAR-T cells were obtained by infecting T cells with a lentivirus packaging system, with an infection efficiency of more than 80% . ③LMP1 CAR-T cells have a 4∶1 effect-to-target ratio in killing LMP1-positive lymphoma cells. The killing effect of LMP1 CAR-T cells on Raji cells was enhanced after 48 h of coculture, but there was no significant killing effect on Ramos, which are LMP1-negative lymphoma cells. ④After coculture with LMP1-positive lymphoma cells at a ratio of 1∶1 for 5 h, the degranulation effect was enhanced. The proportion of CD107a(+) T cells in the LMP1 CAR-T cell treatment group was significantly higher than that in the vector-T cell group [ (13.25±2.94) % vs (1.55±0.05) % , t=3.972, P=0.017]. ⑤After coculture with LMP1-positive lymphoma cells, the proportion of CD69(+) and CD25(+) T cells in the LMP1 CAR-T cell group was significantly higher than that in vector-T cell group [ (7.40±0.41) % vs (3.48±0.47) % , t=6.268, P=0.003; (73.00±4.73) % vs (57.67±2.60) % , t=2.842, P=0.047]. ⑥After coculture with LMP1-positive lymphoma cells, cytokine secretion in the LMP1 CAR-T cell group was higher than that in the vector-T cell group [interferon-gamma: (703±73) ng/L vs (422±87) ng/L, t=2.478, P=0.068; tumor necrosis factor-alpha: (215±35) ng/L vs (125±2) ng/L, t=2.536, P=0.064]. Conclusion: In this study, we found that the LMP1 protein is only found on the surface of the EBV-positive tumor cell. Simultaneously, we created an LMP1 CAR-expressing plasmid and obtained LMP1 CAR-T cells by infecting T cells with a lentivirus packaging system. Furthermore, we demonstrated that LMP1 CAR-T cells could specifically kill LMP1-positive tumor cells in vitro. The degranulation and activation effects of LMP1 CAR-T cells were enhanced after coculture with LMP1-positive tumor cells, indicating a potential clinical application.

Clinical features and prognosis of childhood B-lineage acute lymphoblastic leukemia expressing the PRAME gene / 中国当代儿科杂志

OBJECTIVES@#To study the clinical and prognostic significance of the preferentially expressed antigen of melanoma (PRAME) gene in the absence of specific fusion gene expression in children with B-lineage acute lymphoblastic leukemia (B-ALL).@*METHODS@#A total of 167 children newly diagnosed with B-ALL were enrolled, among whom 70 were positive for the PRAME gene and 97 were negative. None of the children were positive for MLL-r, BCR/ABL, E2A/PBX1, or ETV6/RUNX1. The PRAME positive and negative groups were analyzed in terms of clinical features, prognosis, and related prognostic factors.@*RESULTS@#Compared with the PRAME negative group, the PRAME positive group had a significantly higher proportion of children with the liver extending >6 cm below the costal margin (P<0.05). There was a significant reduction in the PRAME copy number after induction chemotherapy (P<0.05). In the minimal residual disease (MRD) positive group after induction chemotherapy, the PRAME copy number was not correlated with the MRD level (P>0.05). In the MRD negative group, there was also no correlation between them (P>0.05). The PRAME positive group had a significantly higher 4-year event-free survival rate than the PRAME negative group (87.5%±4.6% vs 73.5%±4.6%, P<0.05), while there was no significant difference between the two groups in the 4-year overall survival rate (88.0%±4.4% vs 85.3%±3.8%, P>0.05). The Cox proportional-hazards regression model analysis showed that positive PRAME expression was a protective factor for event-free survival rate in children with B-ALL (P<0.05).@*CONCLUSIONS@#Although the PRAME gene cannot be monitored as MRD, overexpression of PRAME suggests a good prognosis in B-ALL.

The Impact of Induction Treatment Response on the Prognosis of Pediatric Core Binding Factor-Acute Myeloid Leukemia Patients / 中国实验血液学杂志

OBJECTIVE@#To explore the impact of induction treatment response on the prognosis of pediatric core binding factor-acute myeloid leukemia (CBF-AML).@*METHODS@#The result of induce reaction and survival data of 157 pediatric CBF-AML patients in our hospital from September 2008 to December 2018 were retrospectively analyzed．The survival rate of the patients with different degrees of morphological remission after induction chemotherapy was comparative analyzed.@*RESULTS@#Among the 157 children with CBF-AML, 113 (72.4%) patients achieved morphologic leukemia-free state (MLFS) after the first course of induction chemotherapy, 153 (98.1%) patients achieved MLFS after the second course of induction chemotherapy. The 5-year event-free survival (EFS) rate and 5-year overall survival (OS) rate of patients with non-remission (NR) status after the first course of induction of chemotherapy was significantly lower than the patients achieved MLFS and the patients achieved partial remission (PR). The 5-year EFS rate and 5-year OS rate of the patients with PR status after the second course of induction chemotherapy were lower than the patients achieved MLFS, but the difference was not statistically significant. Multivariable analyze showed that NR after the first course of induction chemotherapy and myeloid sarcoma were the independent risk factors affecting EFS of the patients. There were six patients with NR status after the first course of induction chemotherapy, in which all of them harbored t(8;21), three of them with sex chromosome deletion, two of them with myeloid sarcoma.@*CONCLUSION@#NR status after the first course of induction chemotherapy was the independent risk factor affecting EFS and OS of CBF-AML patients, it can be taken as an indicator for higher risk stratification. PR status after the first course of induction chemotherapy may not be used as a diagnostic criterion for primary drug resistance.

Performance Verification of SeqType® P52 Human Ancestry Identification SNP Detection Kit / 法医学杂志

Objective To evaluate the discrimination efficiency of the SeqType® P52 Human Ancestry Identification SNP Detection Kit based on a high-throughput sequencing platform in five Chinese ethnic groups. Methods Using the SeqType® P52 Human Ancestry Identification SNP Detection Kit based on a high-throughput sequencing platform, a total of 350 samples from Han, Tibetan, Mongolian, Uygur, and Yi populations in China were detected and population cluster analysis was performed. Results The effective sequencing depth of a single site in a single sample was ≥720×, and the average report rate was 96%. The mean values of allele frequency differences between the Tibetan, Mongolian, Uygur, Yi and Han population were 0.20, 0.05, 0.24 and 0.11, respectively. Using Structure 2.3.4 software under K=5 mode, independent ancestral component in Han, Tibetan and Uygur could be detected, which was consistent with the result observed from the principal component analysis （PCA）. For the Yi population, two thirds of them had relatively independent ancestral component close to the Tibetan population and one third were similar to the Uygur population. The Mongolian population had similar ancestral origin component with Han population. Conclusion The composite detection system with 52 screened ancestry-informative SNP sites has been established in this study, which can effectively analyze the composition and individual genetic components of populations from Han, Tibetan and Uygur. The ability to discriminate among Han, Mongolian and Yi needs to be further improved. The SeqType® P52 Human Ancestry Identification SNP Detection Kit can be used to infer the origin of an individual's ancestors in some forensic DNA cases.

Free anterolateral thigh flap with superficial temporal vessels as recipient vessels for reconstruction of defect after maxillectomy in malignant tumor / 中华耳鼻咽喉头颈外科杂志

Objective: To study the effects of superficial temporal artery and vein as recipient vessels for the free anterolateral thigh flap on the appearance and functions after maxillectomy. Methods: Clinical data of 21 patients with malignant maxillary tumors in Department of Oral and Maxillofacial Surgery, the Second Xiangya Hospital of Central South University from January 2014 to November 2019, who were treated by free anterolateral thigh flap with temporal superficial vessels as the recipient vessels were analyzed retrospectively. There were 18 males and 3 females, with the age ranging from 29 to 73 years old, including 19 cases of squamous carcinoma, 1 case of adenoid cystic carcinoma and 1 case of osteosarcoma. Of those 7 patients underwent primary surgery, 14 patients received resurgery, and 6 patients had a history of postoperative radiotherapy and chemotherapy. Among 14 patients with resurgery, 13 had recurrent ipsilateral second site tumor and 1 had recurrent tumor, and all of them received the maxillectomy and reconstructive surgery with the free anterolateral thigh flap. Patients were evaluated with water swallow test and speech intelligibility score in 1, 3 and 6 months after operation. The data were statistically analyzed with SPSS 22.0 statistical software. Water swallow test results before and after operation were compared using the Wilcoxon rank sum test. The mean speech intelligibility scores before and after operation were compared by the paired t test. Results: Patients were followed up for 10-60 months. All free flaps survived after operation. No diplopia occurred. Breathing, swallowing and speaking functions were normal. No movement disorders caused by the donor of thigh flap. Water swallow test showed no phenomenon of water flowing into the nasal cavity or oral and nasal leakage with level Ⅰ for 4 cases, level Ⅱ for 13 cases, level Ⅲ for 3 cases and level Ⅳ for 1 case. The mean speech intelligibility scores before surgery and 1, 3 and 6 months after surgery were 4.31±0.13, 1.46±0.21, 2.15±0.45 and 2.87±0.76 respectively. There was statistically significant difference in the mean speech intelligibility scores between 1 and 6 months after surgery (F=78.456, P<0.05). Conclusion: It is safe and reliable to use the superficial temporal vessels as recipient vessels for free anterolateral thigh flap in the reconstruction of defect after maxillectomy in malignant tumors, with good outcomes of functions and a satisfactory restoration of outward appearance.

Clinical characteristics and prognostic analysis of pediatric pro-B cell acute lymphoblastic leukemia / 中国当代儿科杂志

OBJECTIVE@#To explore the clinical-biological characteristics and prognosis of pediatric pro-B cell acute lymphoblastic leukemia (pro-B-ALL).@*METHODS@#A total of 64 patients aged less than 18 years old with pro-BALL were enrolled. Clinical characteristics, therapeutic effect and prognostic factors were retrospectively analyzed.@*RESULTS@#Pro-B-ALL occurred in 6.23% (64/1 028) of pediatric ALL. Among the 64 patients, 35 were male and 29 were female. The median age was 7.0 years (range 0.4-16.0 years) at diagnosis, of which 39% and 6% were ≥ 10 years old and < 1 year old respectively. The median WBC count was 25.5×10@*CONCLUSIONS@#Pediatric pro-B ALL is a heterogeneous disease with clinical and biological diversity. Biological characteristics, such as immunological markers, genetic alterations, and MRD at 3 months after chemotherapy may be important factors for the long-term prognosis.

Influence of dasatinib treatment on body height in children with acute myeloid leukemia / 中国当代儿科杂志

OBJECTIVE@#To study the influence of dasatinib treatment on body height in children with acute myeloid leukemia (AML).@*METHODS@#A retrospective analysis was performed for the clinical data of 86 AML children aged <17 years. According to the treatment regimen, these children were divided into a conventional chemotherapy group and a dasatinib chemotherapy group. The 57 children in the conventional chemotherapy group were given conventional chemotherapy drugs without tyrosine kinase inhibitor, and the 29 children in the dasatinib chemotherapy group were given conventional chemotherapy drugs and dasatinib. The two groups were compared in terms of height standard deviation score (HtSDS) at the beginning of treatment and after treatment, as well as the change in HtSDS after 1 and 2 years of treatment.@*RESULTS@#There was no significant difference in HtSDS between the conventional and dasatinib chemotherapy groups before treatment. Within the first two years of treatment, the dasatinib chemotherapy group had a similar change trend of HtSDS as the conventional chemotherapy group. Four children in the dasatinib chemotherapy group reached the final adult height during follow-up, which was significantly lower than the target height (P=0.044). In the conventional chemotherapy group, there was no significant difference between final adult height and target height. In the dasatinib chemotherapy group, the children in adolescence had a significant change in HtSDS after treatment (P=0.032).@*CONCLUSIONS@#Dasatinib treatment may affect the final height of children with AML, and the use of dasatinib after the beginning of adolescence may lead to growth disorder, but dasatinib treatment has little effect on body height in the short-term treatment.

Oxidative Damage of Bone Marrow Stromal Cells Caused by Chemotherapy Drugs / 中国实验血液学杂志

OBJECTIVE@#To explore the oxidative damage of OP9 cells induced by daunorubicin (DNR) treatment.@*METHODS@#The TMRM probe was used to detect mitochondrial membrane potential by flow cytometry; the reactive oxygen species (ROS) was determined by flow cytometry DCFDA probe; the real-time PCR was used to detect the molecular expression of antioxidant enzyme，glutathione peroxidase (GPX) in OP9 cells; the expression of γ-H2AX was determined by flow cytometry.@*RESULTS@#Compared with normal OP9 cells, the positive rate of TMRM in DNR-treated OP9 cells decreased by 56.7% (P＜0.05); the positive rate of DCFDA in DNR-treated OP9 cells increased by 3.52 times (P＜0.01). Compared with normal OP9 cells, DNR-treated OP9 cells showed a decrease in the expression of GPX4 by 44.22% (P＜0.001); the expression of GPX7 decreased by 65.7% (P＜0.001); the expression of GPX8 decreased by 24.7% (P＜0.001); the positive rate of γ-H2AX in DNR-treated OP9 cells increased (P＜0.05).@*CONCLUSION@#After DNR treatment, mitochondrial membrane potential of OP9 cells decreases; the level of reactive oxygen species increases; the expression of glutathione peroxidase (GPX) molecules decreases significantly; genomic instability increases obviously; the oxidative damage of cells increased.

Effectiveness analysis of intensive management of dyslipidemia in community based on family doctor contracting service / 中华健康管理学杂志

Objective@#To analysis of the effect of strengthening management of dyslipidemia in community based on family doctor contracting service.@*Methods@#In December 2014, 1267 cases patients with dyslipidemia aged over 18 years were selected from three community health service centers in Hangzhou, including 645 in intervention group (311 males and 334 females) and 622 in control group (307 males and 315 females). In the intervention group, the management of dyslipidemia in community was strengthened by graded follow-up and personalized diagnosis and treatment based on the contracted services of family doctors, while the control group adopted the general management of dyslipidemia in the community. After 12 months of intervention, the changes of lifestyle (smoking, drinking, body mass index, waist circumference), regularly taking lipid-regulating drugs, blood lipid, blood pressure, blood sugar levels and their control rates were compared by χ2 test or t test before and after intervention between the intervention group and the control group. Non-conditional logistic regression analysis was used to analyze the influencing factors of blood lipid attainment.@*Results@#Before intervention, there were no significant differences in gender, age, cardiovascular risk stratification, the levels of lipid and other metabolic indicators, lipid compliance rate between intervention group and control group (P> 0.05). After intervention, the intervention group improved in drinking, overweight,obesity, abdominal obesity, and the rate of regularly taking lipid-regulating drugs increased,compared with the control group, the difference was statistically significant (χ2=5.923,4.765,8.587,5.341, 5.654; all P<0.05). The levels of total cholesterol, triglyceride, low density lipoprotein cholesterol, body mass index, waist circumference, systolic blood pressure, diastolic blood pressure, fasting blood glucose, glycosylated hemoglobinin the intervention group were lower than those in the control group, the differences were statistically significant (t=-4.987,-3.207, -6.280, -3.339, -2.466, -4.052, -5.012, -2.865, -2.450; all P<0.05), while the HDL-C level in the intervention group was higher than that in the control group (t=2.294; P<0.05). The control rate of blood lipids, the control rate of blood pressure, and the combined control rates of blood lipid, blood pressure and blood sugar in the intervention group were higher than those in the control group, the differences were all significant (χ2=31.262,4.818,17.245; all P<0.05). Unconditional logistic regression analysis showed that family doctor contracted services (OR=1.961, 95%CI: 1.485-2.589), gender (OR=0.662, 95%CI: 0.471-0.930), smoking (OR=0.498, 95%CI: 0.332-0.745), obesity (OR=0.570, 95%CI: 0.359-0.904), hypertension (OR=0.353, 95%CI: 0.259-0.480), diabetes mellitus (OR=0.340, 95%CI: 0.239-0.483) was the influencing factor of blood lipid reaching the target (all P<0.05).@*Conclusion@#Intensive management of dyslipidemia in community based on family doctor's contracting service is helpful to improve the management effect of dyslipidemia.

Effect of Chemotherapeutic Drug-Induced Damage of Bone Marrow Stroma Cells on Normal Hematopoietic Cells / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of damage of bone marrow stroma cells induced by chemotherapeutic drug on the function of normal hematopoitic cells.@*METHODS@#Senescence cells were detected by flow cytometry after SA-β-gal staining; real-time PCR was used to detect the expression of a serial molecules in bone marrow stromal cell line OP9 cells; the expression of γ-H2AX was determined by flow cytometry after histone γ-H2AX staining; the colony forming ability of hematopoietic cells was tested by colony formation assay.@*RESULTS@#The percentage of senescence cells in OP9 cells after DNR treatment was 2.24 times as much as that in untreated OP9 cells (P<0.05). Compared with normal OP9 cells, the expression levels of IL-6 and TNF-alpha in DNR-treated OP9 cells increased by 2.73 times (P<0.01) and 0.56 times (P<0.01), and the expression levels of N-cadherin, alpha smooth muscle actin (alpha-SMA), angiopoietin1 (Angpt1) and osteopontin (OPN) decreased by 69.54%（P<0.01），63.90%（P<0.01），87.41%（P<0.01）and 42.78%（P<0.01）respectively. After the co-culture with DNR-treated OP9 cells, the colony formation of normal hematopoietic cells decreased by 47.10% than that co-cultured with untreated OP9 cells (P< 0.05), meanwhile, the percentage of γ-H2AX+ cells in normal hematopoietic cells increased by 2.19 times (P<0.05).@*CONCLUSION@#After treatment with DNR, the senescence cell number of OP9 cells sgnificantly increases; the expression of TNF-α and IL-6 is up-regulated, while the expression of α-SMA, Angpt-1 and OPN is down-regulated as compared with normal OP9 cells. In addition, after co-culture of DNR-treated OP9 cells with normal hematopoietic cells, the colony formation ability of hematopoietic cells decreases and the genome instability of hematopoietic cells increases as compared with normal hematopoietic cells.

Exploration of Mechanism for Meisoindigo-Inducing K562 Cell Apoptosis by Using Quantitative Proteomic Analysis / 中国实验血液学杂志

OBJECTIVE@#To screen the differentially expressed proteins at the early stage of K562 cells treated with meisoindigo by using tandem mass tags (TMT)-based proteomics technology, and to explore the mechanism for meisoindigo-inducing apoptosis.@*METHODS@#The half inhibitory concentration (IC) of mesoindigo on K562 cells was determined by CCK8. The flow cytometry was used to assay the apoptosis of K562 cells treated by meisoindigo or DMSO. Total proteins were extracted from the cells treated with 0.2% DMSO (control) or 20 μmol/L meisoindigo (Test) for 2 hours. Then, the TMT-labeling HPLC-MS/MS was used to identify and quantify the peptides and their abundance, all the tests were repeated for 3 times. The Mascot software was used to identify the proteins; the GO annotations, enrichment and cluster analysis were used to analyze the differentially expressed proteins.@*RESULTS@#Meisoindigo-induced K562 cell apoptosis in a dose-dependent manner (r=0.98), 5 544 proteins were identified, 4792 of which were quantified. The protein with expression difference>1.5-folds in Test group accoanted for 8, out of which the expression of 4 proteins were up-regulated and 4 were down-regulated. The differentially expressed proteins mainly associated with reactive oxygen species (ROS).@*CONCLUSION@#Several proteins including DDIT4 were found to have dramatic changes in the early stage of K562 cells treated with meisoindigo by using quantitative proteomics technology. The ROS metabolic process may play important roles in meisoindigo-inducing apoptosis of K562 cells.

Clinical Study of Futongning Granules in Treating Functional Abdominal Pain in Children / 广州中医药大学学报

Objective To observe the clinical effect of Futongning Granules for the treatment of functional abdominal pain in children. Methods A total of 187 children with functional abdominal pain were randomized into treatment group(N = 122)and control group(N = 65). The treatment group was treated with Futongning Granules and the control group was treated with Compound Pepsin Powder. The gastrointestinal syndrome scores of the two groups were observed, and the short-term and long-term clinical efficacy of the two groups was evaluated after treatment. Results (1)The total effective rate and the effective rate for syndromes of abdominal internal cold, spleen-stomach deficiency cold, indigestion induced by milk and food stagnation, liver-Qi stagnation, blood collateral obstruction, gastrointestinal heat stagnation in the treatment group were 92.3％, 92.9％, 95.0％, 95.2％, 90.3％, 85.7％, 90.0％, and those in the control group were 71.0％, 69.2％, 69.2％, 86.7％, 66.7％, 50.0％, 60.0％, respectively. The total effective rate and the effective rate for various syndromes in the treatment group were superior to those of the control group (P < 0.05). (2) After treatment, gastrointestinal syndrome scores of various syndromes and overall scores in the two groups were obviously decreased(P<0.05 or P<0.01 compared with those before treatment), and the decrease in the treatment group was superior to that in the control group (P < 0.05). (3) The results of 6-month follow-up showed that the clinical efficacy during the follow-up in the treatment group was better than that in the control group(P < 0.05), and the difference was statistically significant(P<0.05). Conclusion Futongning Granules exert certain effect for the treatment of functional abdominal pain in children, and are effective for significantly relieving clinical symptoms and reducing the recurrence of disease.

Clinical observation of influences of donor HBV infection on allogeneic hematopoietic stem cell transplantation recipients / 西安交通大学学报(医学版)

Objective To investigate the influences of donor HBV infection on allogeneic hematopoietic stem cell transplantation recipients.Methods We made a retrospective analysis of data of four patients without HBV infection who underwent allogeneic hematopoietic stem cell transplantation from January 2015 to December 2016. Among them donors of these patients all had HBV infection.We then observed the influences of HBV infection on hematopoietic reconstruction,hepatic vein occlusive disease and HBV infection.Results HBV serological conditions of two donors were HbsAb,HbeAb and HbcAb positive,and quantitative of HBV-DNA was negative;the donor and the recipient did not use anti-HBV drugs.One donor was HbsAg,HbeAb and HbcAb positive,and the quantitative of HBV-DNA was also positive.Another donor was HbsAg and HbcAb positive,and the quantitative of HBV-DNA was also positive.These two donors received oral nucleoside therapy one month before stem cell collection and the recipients of these two donors also took nucleoside drugs one week before the conditioning.Hepatitis B immune globulin was given after transfusion of stem cells and the third day and seventh day after transplantation.Quantitative of HbsAb was detected each month and if it was less than 150 IU,hepatitis B immune globulin would be given.All the recipients had hematopoietic reconstruction and no VOD or hepatitis B virus infection occurred.Conclusion Oral administration of nucleoside drugs combined with hepatitis B immunoglobulin can effectively prevent HBV infection in recipients with HBV infection donors.

Relationship between the Therapeutic Dose of Arsenic Trioxide and Relapse in Acute Promyelocytic Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyze the correlation of ATO therapeutic dose with the relapse of patients with acute promyelocytic leukemia (APL) and to investigate the optimal dose and courses of ATO.</p><p><b>METHODS</b>The clinical data of 102 patients with APL from January 2008 to June 2015 were analyzed retrospectively. The clinical characteristics of APL patients in relapsed group and maintained remission group were compared. According to ATO dose in 2 years recommended in chinese guideline as criteria of grouping, the patients were divided into ATO high and low dose groups, then the relapse rate in groups was compared. The cut-off value of ATO dose was analyzed by ROC curve.</p><p><b>RESULTS</b>Univariate analysis showed that the relapse rate in high ATO and low ATO groups on 2 year treatment was 2.5% and 17.7% respectively (P<0.05); multiple variate analysis demonstrated that the ATO dose>22.4 mg/kg on 2 year treatment was independent preventive factor for the relapse of APL (OR=0.119, P<0.05). The ROC curve showed that the cut-off value of ATO dose on 2 year treatment was 8.765 mg/kg. The relapse rate of APL in group of ATO dose >8.765 mg/kg group was significantly lower than that in group of ATO dose <8.765 mg/kg.</p><p><b>CONCLUSION</b>The relapse of APL relates with used ATO dose, sufficient use of ATO dose can decrease the relapse rate of APL.</p>

Expression and Asymmetric Division of Numb in Leukemia Cell K562 Line / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the role of asymmetric division in leukemia cells through detection of expression and asymmetric division of Numb in differentiated and undifferentiated K562 cells.</p><p><b>METHODS</b>Firstly, Hemin was used to induce K562 cell differentiation, and the expression of Numb was detected by the real-time quantitative RT-PCR and flow cytometry. After K562 cells were synchronized by nocodazole, the Numb protein was labeled by immunohistochemical staining, followed by the determination of the terminally differentiated cells through confocal microscopy. The fluorescence intensity was calculated by Image J software, and the cell division pattern was analyzed on the basis of the fluorescence intensities of Numb in 2 divided daughter cells.</p><p><b>RESULTS</b>Compared with the undifferentiated K562 cells, the level of Numb mRNA expression increased 2.3 times (P<0.001). The ratio of Numb positive cells was(67.37±5.01)% in differentiated K562 cells, while that was (43.97±5.72)% in undifferentiated K562 cells (P<0.01). Compared with undifferentiated K562 cells, the ratio of cells with asymmetric division in differentiated K562 cells increased 18.3%, the percentage of cells with symmetry self-renewal reduced 49.7%(P<0.001) and that with symmetry differentiation increased 32%(P<0.001).</p><p><b>CONCLUSION</b>In differentiated K562 cells, expression of Numb and proportion of cells with asymmetric division were higher than that in undifferentiated cells. With the differentiation of leukemia cells, the proportion of cells with asymmetrical division increases, and the proportion of cells with symmetrical self-renewal decreases. The stemness of leukemia cells is maintained mainly through the symmetrical self-renewal.</p>

Relationship between Peripheral Blood White Blood Cell Count and Early Death Rate in Patients with Acute Promyelocytic Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between peripheral white blood cell count and early death rate of the patients with acute promyelocytic leukemia (APL).</p><p><b>METHODS</b>Through retrospective study, the relationship of early death rate in 116 cases newly diagnosed APL patients with maximum of peripheral blood white blood cell count should be analyzed before and after induction therapy as well as in the whole course of disease during the past 8 years.</p><p><b>RESULTS</b>There was a close relationship between the peripheral white blood cell count and the early death rate in APL patients. Peripheral blood white blood cell count in the early died patients was significantly higher than that of the survival patients (P<0.05). ROC analysis showed that the highest risk threshold of peripheral white cell count was 70×10/L (P<0.05) before treatment, while the highest risk threshold after treatment and in the whole course of disease were 96.4×10/L(P<0.05) and 91.5×10/L(P<0.01) respectively. The dealth rate of patients with highest risk threshold was significantly increased (P<0.05).</p><p><b>CONCLUSION</b>The highest peripheral blood white blood cell count closely relates with the early death rate of patients at different time points in the whole course of disease. Control of peripheral white blood cell count may effectively reduce the early death rate of APL patients.</p>

Risk Factors and Therapeutic Outcome of Intestinal Acute Graft Versus Host Disease in Acute Leukemia Patients after Haploidentical Peripheral Hematopoietic Stem Cell Transplantation / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the risk factors and therapeutic outcome of acute graft versus host disease (aGVHD) in patients with acute leukemia after haploidentical peripheral hematopoietic stem cell transplantation.</p><p><b>METHODS</b>The clinical data of 19 cases of acute leukemia underwent haploidentical hematopoietic stem cell transplanttion during January 2010 and December 2010 were retrospectively analyzed. The effects of patients sex, donor-recipient sex difference, donor age, conditioning regimen, dosage of anti-thymocyte globulin(ATG), mononuclear cell and CD34cell counts on the intestinal aGVHD were analyzed by Logistic regression.</p><p><b>RESULTS</b>Intestinal aGVHD occurred in 5 cases with 1 case at stage II 3 cases at stage III and 1 case at stage IV on the 7th, 22th, 27th, 70th and 154th day after transplantation, respectively. Single factor analysis showed that the patient's sex, donor-recipient sex difference, donor age, dosage of ATG, mononuclear cell and CD34cell counts were not related with the occurrence of the intestinal aGVHD, and the conditoning regimen was the risk factor for the intestinal aGVHD. 2 cases among 5 cases with intestinal aGVHD were treated with methylprednisolone at dosage of 1 mg/kg per day, 1 case was treated with methylprednisolone therapy combined with tacrolimus. 2 cases of methylprednisolone-resistance were treated with CD25 monoclonal antibody. Intestinal aGVHD of all patients was improved after the above-mentioned treatment.</p><p><b>CONCLUSION</b>Conditioning regimen of haploidentical peipheral hematopoieitc stem cell transplantaion has effects on the intestinal aGVHD, which needs to be confirmed by further research.</p>

Relationship between High Expression of c-FLIP and Drug Resistance of Leukemia Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the effect of c-FLIP expression on drug resistance of Kasumi-1 leukemia cells and its mechanisms.</p><p><b>METHODS</b>Tet-on inducible system was used to construct the conditional expression vector of c-FLIP by cloning the c-FLIP gene into lentivirus vector pLVX-Tight-Puro, then the Kasumi-1 cells were transfected with lentivirus pLVX-Tight-Puro-c-FLIP. The expression of c-FLIP was induced by doxycycline(Dox) for different time and doses, and verified by qRT-PCR and Western blot. On the basis of the overexpression of c-FLIP, the Kasumi-1-c-FLIP cells were treated with CH11 and PB in order to induce apoptosis, and the Giemsa staining was used to show the apoptotic cell morphology.</p><p><b>RESULTS</b>qRT-PCR and Western blot showed the overexpression of c-FLIP, the CH11 and PB can induce Kasumi-1 cell apoptosis, while the c-FLIP overexpression weakened this effects. Western blot showed that the c-FLIP blocked the caspase-8 activation.</p><p><b>CONCLUSION</b>The overexpression of c-FLIP inhibits the apoptosis caused by CH11 and PB, and leads to drug resistance in leukemia cells.</p>