Acquired aplastic anemia(AA) is a disease of bone marrow failure mediated by autoimmune T cells. CD4+CD25+ regulatory T cells, which have immunosuppressive and anergic, belong to a subpopulation of T cells specialized for immune regulation and play important roles in the development of autoimmune T cells. Foxp3 plays a significant role in the development and function of regulatory T cells which have been confirmed by many investigations. In recent years, the relationship between CD4 +CD25 +Foxp3 + regulatory T cells and AA has become a hot spot of research. What’s more, its clinical significance is becoming more and more obvious in AA.

Objective To study the expression of PI3K and Akt mRNA in acute leukemia(AL) and chronic myelocytic leukemia (CML) and try to find out a relationship between the prognostic significance of acute leukemia and the expression level of PI3K and Akt. Methods The samples were collected from 63 ALpatients, 7 relapsed AL patients, 14 CML patients and 10 AL patients in complete remission(CR) patients, 11samples of normal controls(NC). The expression of PI3K, Akt mRNA were measured by semi-quantity reverse transcription polymerase chain reaction(RT-PCR). Results The expression of PI3K mRNA in AL and relapse group were significantly higher than that in normal control. In CML, the P13K mRNA expression level was higher than that in NC and CR. The similar results of the expression of Akt mRNA were observed in CML, NC and CR. At the same time, the expression level of Akt mRNA in relapse group was significantly higher than that in NC. The complete remission rate in PI3K (+) was lower than that in PI3K (-). Similar result was obtained in Akt (+) and Akt (-). Conclusion The PI3K/Akt pathway may contribute to the occurrence of leukemia. The patients with positive expression of the members of this pathway had a lower remission rate.

BACKGROUND: In the past, guided tissue regeneration materials were produced by 13-tricalcium phosphate (β-TCP) and gelatin (Gel), but the production of fiber membrane by electro-spinning technique was reported less.OBJECTIVE: To prepare a new kind of Gel with β-TCP hybrid nanofibrous membrane and testing its biocompatibility compared with polylactic acid (PLLA) and poly(lactide-co-glycolide) (PLGA) membrane.METHODS: The β-TCP/Gel guided tissue regeneration membrane was made by electro-spinning technique.Scanning electron microscopy (SEM) was employed to observe membrane surface.MTT test was performed to compare toxicity among β-TCP/Gel,PLLA, and PLGA membranes.RESULTS AND CONCLUSION: The β-TCP/Gel membrane was porous, and β-TCP granules were nodosity-adhered on surface of Gel.The average diameter of fiber was 500-600 nm.The distribution of fiber ranged from 200 to 500 nm.There was no significant difference in toxicity among PLLA, PLGA, and negative control group (P > 0.05).The results suggested that the β-TCP /Gel membrane was low cytotoxicity and suitable for tissue engineering.It would be a promising material for periodontal tissue regeneration.

Objective To investigate the proliferation inhibition and the apoptosis induction effect of brucine on human chronic myeloid leukemia cell line HL-60 cells.Methods HL-60 cells were exposed to various dosages of brucine 24,48,72 h respectively,MTT method was used to assay the growth inhibition effect of brucine on HL-60 cells and the IC50 of brucine was evaluated at the same time.The morphology was observed by AO/EB stains.The cell apoptosis and cell cycle was tested by flow cytometry with Annexin V-FITC/PI double staining and PI labeling respectively.Results The results indicated that the brucine displayed significant anti-proliferative effect on HL-60 cells in a dose-and time-dependent manner,with IC50 value of 211.8 μg/ml(24 h),107 μg/ml(48 h),83 μg/ml(72 h)respectively.The most significant inhibition was observed at 320 μg/ml for 48 h.In this condition,apoptosis morphology was induced by brucine with nuclear chromatin condensation,most of the nuclears were orange stained and condensation-like or bead-like,which was consistent with the Annexin V-FITC/PI results.The cell apoptosis rates were(2.1±1.1)％,(21.3±1.2)％,(38.6±1.3)％,(58.5±4.1)％,(75.3±0.87)％and(66.2±0.75)％in different dose of brucine,respectively.At the same time,the cell cycle analysis results showed that the cell ratio in G0/G1 phase was decreased while in G2/M and sub-G0 phases was increased,comparing with blank control group.Conclusion Brucine can inhibit cell growth dramatically,which may be related to the cell apoptosis and the cell cycle arrest.

Objective To study the Janus Kinase 2 V617F (JAK2V617F) point mutation in bcr-abl-negative myeloproliferative disorders (MPD) and explore its clinical significances. Methods Genomic DNA was isolated from bone marrow or peripheral-blood granulocytes. Allelespecific-polymerase chain reactions (AS-PCR), restriction enzyme digestion in combination with PCR product sequencing were performed to detect the mutation in genomic DNA. 110 patients were detected, including 41 with bcr-ablnegative MPD, 25 with bcr-abl-positive chronic myelogenous leukemia (CML), and 44 with acute leukemia.Results JAK2V617F was presented in 11 cases(91.7 %) of 12 polycythemia vera (PV), 8 cases(53.3 %) of 15 essential thrombocythemia(ET), 4 cases (57.1%) of 7 idiopathic myelofibrosis (IMF), while in other patients including 7 hypereosinophilic syndrome (HES), 25 bcr-abl-positive CML, 24 acute myelocytic leukemia (AML), 18 acute lymphoblastic leukemia(ALL), and 2 acute mixed lineage leukemia, JAK2V617F can not be detected. All positive samples and 10 negative samples identified by AS-PCR and restriction enzyme digestion were confirmed further by DNA sequencing. Conclusion The frequency of JAK2V617F mutation was more than 90 % among patients with PV, more than 50 % among patients with ET and IMF. The detection of JAK2V617F mutation will be of great significanees in the diagnosis and differential diagnosis of MPD. This mutation can be a molecular marker of MPD and might be a treatment target in the future.

Objective: To investigate the value of NPM1 and FLT3 gene mutation combined with bone marrow imaging detection in the prognosis judgement of initial treatment cytogenetically normal acute myeloid leukemia (CN-AML). Methods: The clinical data of 100 patients (non-M₃ type) with primary and initial treatment CN-AML from January 2010 to January 2014 in the Peace Hospital Affiliated of Changzhi Medical College were retrospectively analyzed. All patients were enrolled in the bone marrow imaging examination on the end day of induction treatment or the first day after the end of induction treatment (T time point). Univariate and multivariate prognostic analyses were performed on AML patients according to FLT3 and NPM1 gene status,bone marrow juvenile cell ratio at T time point. Results: A total of 100 patients included 36 cases with FLT3 gene mutation and 44 cases with NPM1 gene mutation. The complete remission (CR) rate of CN-AML patients was 13.9% (5/36) and 71.9% (46/64), respectively (P < 0.01), 2-year recurrence-free survival (RFS) rate was 5.6% and 59.8%, respectively (P < 0.01), 2-year overall survival (OS) rate was 15.6% and 66.2%, respectively in FLT3⁺ group and FLT3^- group (P < 0.01). The median RFS and median OS time in FLT3⁺ group was 6.9 months and 9.4 months, respectively, and the median RFS and median OS time in FLT3^- group had not yet reached. There were no significant differences of all the indexes between the two groups (all P < 0.01). And there were no significant differences in CR rate, 2-year RFS rate and 2-year OS rate between NPM1⁺ group and NPM1^- group (all P > 0.05). The CR rate, 2-year RFS rate and 2-year OS rate in NPM1⁺ FLT3^- group were better than those in NPM1^- FLT3^-, NPM1^- FLT3⁺ and NPM1⁺ FLT3⁺ groups; NPM1^- FLT3⁺ and NPM1⁺ FLT3⁺ groups had the worst prognosis, and there were no statistical differences in the CR rate, RFS and OS time between the two groups (all P > 0.05). The prognosis of the patients in bone marrow juvenile cell ratio < 0.05 group at T time point was better than that in ratio ≥0.05 group (all P < 0.05). CN-AML patients were classified into the good prognosis group (NPM1^- FLT3^-), the medium prognosis group (NPM1⁺ FLT3^-), and the poor prognosis group (NPM1^- FLT3⁺ and NPM1⁺ FLT3⁺) according to the FLT3 and NPM1 genes. The good prognosis group+ the ratio of bone marrow juvenile cells at T time point < 0.05 group, the good prognosis group+ the ratio of bone marrow juvenile cells at T time point ≥0.05 group, the medium prognosis group+ the ratio of bone marrow juvenile cells at T time point < 0.05 group had no statistical differences in CR rate, 2-year RFS rate and 2-year OS rate (all P > 0.05). The medium prognosis group+ the ratio of bone marrow juvenile cells at T time point ≥0.05 group, the poor prognosis group+ the ratio of bone marrow juvenile cells at T time point < 0.05 group had the equivalent prognosis, and the average prognosis was moderate; the poor prognosis group+ the ratio of bone marrow juvenile cells at T time point ≥ 0.05 group had the worst prognosis. According to Cox multivariate regression analysis, FLT3 gene mutation and the ratio of bone marrow juvenile cells at T time point were independent influencing factors for RFS and OS in CN-AML patients (all P < 0.05). NPM1 was an independent prognosis factor affecting RFS and OS of FLT3^- patients (all P < 0.05). Conclusions After induction chemotherapy, the responsiveness and sensitivity of AML patients to chemotherapy regimen can be assessed early and objectively according to molecular genetics and the ratio of bone marrow juvenile cells at T time point, which has a certain value in the prognosis judgement.

OBJECTIVETo detect the activity of autophagy and explore the impact on survival and proliferation of HEL cells and hematopoietic cells of polycythemia vera （PV） patients with JAK2 V617F mutation.

METHODSFlow cytometry, AO staining and Western blot methods were used to detect the autophagy activity and the expression of LC3-Ⅱ protein of JAK2 V617F+ HEL cells and hematopoietic cells of 12 newly diagnosed PV patients with JAK2 V617F mutation. HEL cells and bone marrow cells of 3 PV patients were treated with rapamycin or 3-MA to induce and inhibit autophagy, respectively. CellTiter Glo(R) method was used to detect the proliferation activity of cells.

RESULTSThere was higher level of mean LC3-Ⅱ fluorescence intensity in HEL cells (159 389 ± 29 001) than that in K562 cells (96 047 ± 24 134) (P=0.044). The formation of autophagosome in HEL cells is more than that in K562 cells detected by microscope. What's more, the level of mean LC3-Ⅱ fluorescence intensity in 12 PV patients' myeloid cells (92 842 ± 4 250) was higher than that of 15 healthy volunteers (86 633 ± 2 504) (P=0.001). The expression of LC3-Ⅱ protein was higher in PV patients' peripheral blood cells than that in healthy volunteers detected by Western blot. After treated with rapamycin 12, 24, 48 h, the activity of autophagy in HEL cells and bone marrow cells of 3 PV patients were increased and the proliferation activity was higher than the control group, the proliferation activity at 48 h were (101 413 ± 3 720), (18 744 ± 1 015), respectively. However, after treated with 3-MA 12, 24, 48 h, the activity of autophagy was decreased and the proliferation activity was lower than the control group, the proliferation activity at 48 h were (5 732 ± 166), (5 371 ± 56), respectively.

CONCLUSIONThere is high basical activity of autophagy in JAK2 V617F+ HEL cells and hematopoietic cells of PV patients with JAK2 V617F mutation. Up-regulated autophagy promotes proliferation of JAK2 V617F⁺ HEL cells and bone marrow cells of PV patients with JAK2 V617F mutation. Decreased autophagy inhibits proliferation of JAK2 V617F+ HEL cells and bone marrow cells of PV patients with JAK2 V617F mutation.