Aim To investigate the effects of osthol on bone marrow stromal stem cells in vitro under the con-ditions of the ability to differentiate into osteoblasts and the case of proliferation. Methods The rat bone marrow sample was obtained,and the all bone marrow cell culture methods were used to separate and collect the stratum of mononuclear cells. The cells were cultured in DMEM containing 10% fetal bovine serum. Three days later the culture medium was changed for the first time. Nine days later,serial subcultivation proceeded. The final concentration of osthol was 1 ? 10 -4,1 ? 10 -5,1 ? 10 -6,1 ? 10 -7 mol?L -1 respectively. MTT method was adopted in proliferation analysis. Under the induced condition,the Alkaline phosphatase activity, calcium salt sediment yield and osteocalcin were meas-ured on 4th,8th,12th,16th day. On the fifteenth day,his-tochemistry dyeing proceeded for calcified tubercle. Total RNA was isolated and the gene expression of bFGF, IGF-1,Osterix and Runx-2 was investigated by RT-Real Time PCR. Results The BMSCs proliferation was refrained by osthol dose-dependently. But it evidently led to osteogenesis. The ALP activity,calcium salt sediment yield and osteocalcin were raised,and calcified tubercle amount was increased. Besides,it also could enhance the mRNA level of bFGF,IGF-1,Osterix and Runx-2. Conclusion The osthol with final concentra-tion of 1 ? 10 -5 mol?L -1 can markedly promote BM-SCs differentiation to osteogenesis. which proves osthol is an active constituent of the traditional Chinese medi-cine Common Cnidium Fruit.

Objective To explore the feasibility and significance of detecting Cyclin D1 and bcl-2 with flow cytometry in the diagnosis of B-cell lymphoma.Methods 45 patients patients with confirmed hematologic diseases in final diagnosis were collected and recorded 45 cases in detail included in the study.25 healthy persons showing normal results in routine physical examinations and laboratory tests were also selected and matched according to the age and gender.In addition,according to the age and gender matching principle,selected routine physical examination and all tests were within the normal range in 25 healthy persons as a healthy control group.Select the pathologically confirmed Cyclin D1 or bcl-2 positive cases were selected as the positive control group,select and the negative cases as the negative control group.Four-color monoclonal antibodies directly immunofluorescence monoclonal antibody labelinged with immunofluorescence were used to analyze the surface and cytoplasmic antigens,and multi-parameter flow cytometry was used to investigate the peripheral blood Cyclin D1 and bcl-2 subsets in lymphoma patients.Results The normal values of Cyclin D1 MFI and bcl-2 were obtained from the 25 normal volunteers by analyzing the (x)±s values and 95 ％ confidence interval,thus establishing the diagnostic criteria.Compared to the healthy control (0.356±0.159,1.938±0.324),all B-cell lymphoma patients had increased expression in Cyclin D1 (1.824±0.312)and bcl-2 MFI (4.259±0.541) in their peripheral blood,results showing statistical significance (P ＜ 0.01).Patients with different subtypes of lymphoma expressed differing Cyclin D1 MFI.In patients with Hodgkin' s lymphoma,Cyclin D1 MFI (0.386±0.112) was significantly lower than that in the non-Hodgkin' s lymphoma patients (1.623±1.987) (P ＜ 0.01).Further,in non-Hodgkin' s lymphoma patients,mantle cell lymphoma was 100 ％ positive for Cyclin D1,while the remaining subtypes were negative.Patients with different subtypes of lymphoma also showed differences in bcl-2 expression.In the Hodgkin' s lymphoma,bcl-2 (2.045±0.877) was significantly lower than the non-Hodgkin' s lymphoma (4.045±0.499) (P ＜ 0.01).In non-Hodgkin' s lymphoma,T cell lymphoma expressed the lowest,while MCL and FL showed 100 ％ positivity.In patients with positive Cyclin D1 or bcl-2,the mean fluorescence intensity level of Cyclin D1 (before treatment 3.099±0.349; after treatment 1.008±0.279) or bcl-2 (before treatment 7.814± 1.030,after treatment 3.131±0.522) was significantly lowered after treatment (P ＜ 0.01).Conclusion Using the method of flow cytometry for detecting Cyclin D1 and bcl-2 in lymphoma cells is feasible,and it can be applied clinically to evaluate the treatment.

Acute myeloid leukemia ( AML) is the most common acute leukemia in adults and has the highest death rate of all leukemias. Compared with other hematologic malignancies , there was only a small increment in the 5-year relative overall survival for patients with AML in the last 40 years, despite the advancement in our understanding of AML .CD123 is an AML-associated antigen that expresses at a high level in leukemic stem cells and leukemic blasts and a low level in normal hematopoiet-ic stem cells.As an attractive surface target for AML therapies , immune-based therapies targeting CD 123 are being developed re-cently, especially chimeric antigen receptor ( CAR) T-cell-based immunotherapy .Preclinical data have demonstrated that CD 123 CAR-T cells exhibit potent antileukemic activity and various im-pacts on normal hematopoiesis .This will probably be a promis-ing treatment for patients with relapsed/refractory AML.

This study is to investigate the effects on the expression of iNOS and production of NO in the osteogenic differentiation process of rat bone marrow stromal cells (rBMSCs) by icariside II. rBMSCs were cultured by adherence screening method. When the culture dishes were covered with 80% cells, the osteogenic induced cultures were adopted. Icariside II was supplemented into the culture at 1 x 10(-5) mol x L(-1). The activity of iNOS, content of NO and osteogenic differentiation markers including alkaline phosphatase (ALP) activity, CFU-FALP and mineralized bone nodules were compared among the icariside II-supplemented group, L-NMAE group, icariside II + L-NAME group and the control. Total RNA was isolated and the gene expression of iNOS, Osterix and Runx-2 was investigated by real-time PCR. Total protein was also isolated and the secretion of iNOS and collagen I was examined by Western blotting. Icariside II can significantly improved ALP activity, CFU-FALP amount and mineralized nodules. Besides, the mRNA level of factors related to the osteogenic differentiation includes Osterix and Runx-2 also enhanced. The secretion of collagen I also promoted significantly. But all of these effects can be inhibited by L-NAME which can specifically inhibit the activity of iNOS. Icariside II enhances the osteogenic differentiation of rBMSCs significantly, but if the activity of iNOS was blocked by L-NAME, the osteogenic differentiation markers decrease accompanied with iNOS and NO decrease, suggesting that icariside II stimulates the osteogenic differentiation via enhancing the activity of iNOS and promoting the generation of NO.

Objective To evaluate the proportion and clinical significance of CD4+CD25+ regulatory T cells in childhood acute lymphocyte leukemia(AEL)during different therapeutic stages.Methods 55 peripheral blood samples from 40 children patients with ALL were detected by muhiparameter flow cytometry with fluoresce-hbeled monoclonal antibody.Results Treg cells phenotypically express not only CD62L but also FoxP3 protein.In patients with ALL standard-risk the proportion of CD4+CD25Hi was(1.04±0.33)% in the first course of induction treatment, (1.60±0.44)% in maintenance treatment groups, and(1.29±0.30)% in complete remission groups respectively,while in patients with ALL the intermediate and high risk during maintenance therapy was(2.24±0.75)%.Conclusion Compared with healthy children,the proportion of Treg ceHs in ALL is significantly higher,and may be related to the effect of chemical treatment and severity of ALL.The elevated proportion of Treg may contribute to disease relapse.

Objective To investigate the expression features and clinical significance of CD4+ CD25+ regulatory T cells in elderly patients with newly diagnosed acute myelocytic leukemia.Methods Totally 65 patients newly diagnosed as acute myeloid leukemia (AML group) and 72 healthy volunteers (control group) were divided into the elderly group (aged over 60 years) and the young group aged (44.6±2.9) years.The expression of CD4+ CD25+ regulatory T cells was detected by CD4,CD25 and CD127 three-color fluorescein-labeled and multiparameter flow cytometry.The expression features and clinical significance of CD4+ CD25+ regulatory T cells in each group were analyzed.Results After being gated on CD4+ lymphocytes,the expression level of CD4+ CD25+regulatory T cells was significantly increased in AML group as compared to control group [(7.06±2.60) ％ vs.(5.61 ± 1.06) ％,t =4.19,P=0.000].The expression level of CD4 + CD25 + regulatory T cells was higher in elderly AML patients than in elderly controls [(7.55 ± 2.78)％ vs.(5.98 ±1.08) ％,t=3.42,P=0.001].The expression of CD4+ CD25+ regulatory T cells was higher in young AML patients than in young controls [(6.09±1.91)％ vs.(5.14±0.82)％,t=2.21,P=0.036].The expression level of CD4+ CD25+ regulatory T cells was higher in elderly AML patients than in young AML patients [(7.55±2.78)％ vs.(6.09±1.91)％,t=2.19,P=0.032].Conclusions The dual roles of immunosenescence and tumor may cause the excessive accumulation of CD4+ CD25+regulatory T cells in elderly patients with newly diagnosed acute myeloid leukemia.

Objective To explore clinical characteristics and diagnosis of aggressive natural killercell leukemia (ANKL),and evaluate the value of flow cytometry (FCM) in diagnosing it.Methods A case of ANKL was reported and literatures were reviewed.Results The patient presented with persistent high fever,progressive pancytopenia and hepatosplenomegaly.The untypical cells could be seen by morphology.By FCM,NK cells consisted 83.3 ％ of total lymphocytes in bone marrow and immunophenotypes were CD34-,CD2+,CD7+,CD3-,CyCD3+,CD5-,CD16+,CD56+,CD30-,CD4-,CD8-,CD117-,CD11c,CD19-,CD45++,SSC+-++.T-cell receptor (TCR) and IgH gene rearrangement were negative and chromosome was normal.The patient was diagonised ANKL eventually.Conclusions ANKL is a quite rare disease with highly aggressive,poor prognosis and could be misdiagnosed easily.FCM combined with morphology is a convenient,handy,practicable and less invasive device for the diagnosis,and can be a preferred detection technique in some cases.

Purpose To probe into the value of M-mode echocardiography and spectral Doppler echocardiography in diagnosing fetal atrial premature beats.Materials and Methods Echocardiography examinations were given to the seventy-three fetuses around 16-40 gestational weeks which were found suffering from fetal arrhythmia in the clinical examinations by using M-mode echocardiography and spectral Doppler. Thirty-two of them were screened out with fetal atrial premature beats, and their ultrasonic cardiograms were analyzed. The follow-up visits were later conducted.Results Among the thirty-two cases with fetal atrial premature beats, fourteen were attacked frequently, and the other eighteen were attacked accidentally. Two cases were documented with muscular ventricular septal defect. Apart from two missing cases, the rest thirty cases were found to be recovered from arrhythmia before or after birth in the follow-up visits. The disappearance rate of atrialpremature beats in the fetuses attacked frequently by the disease before birth was clearly lower than that in those attacked accidentally (P<0.05). Muscular ventricular septal defect in the two cases were found closed after birth. Conclusion M-mode echocardiography and spectral Doppler echocardiography, as non-invasive imagining techniques to take antenatal examination of fetal arrhythmia, have advantages such as reliable, direct, convenient and can be used repetitively, therefore can provide important information for clinical treatment and prognosis.

ObjectiveTo explore clinical characteristics and diagnosis of patients with granulocytic sarcoma (GS),and to evaluate the value of FCM in diagnosing it.MethodsClinical data of one patient with GS was reviewed and related literature was reviewed. ResultsThe patient was diagnosed as AML-M2,chromosomal karyotype was 46.XY, t (8;21)(q22;q22)and the AML/ETO gene was positive. Systemic chemotherapy with daunorubicin plus cytarabine was given and complete remission was received. Then a nodular in medial angle of right eye was found. Result of CT indicated the possibility of leukemia infiltration.Needle aspiration cytology was conducted and many blast cells were found by microscope.CD34+ CD117+ CD13+ CD33+CD45dim SSC+ can be found by FCM. The cytology was complete remission and minimal residual disease was negative. Finally diagnosis was GS, relapse of AML. After a systemic chemotherapy with large dose of cytarabine plus teniposide (cytarabine 6.0 g/d, d1-3;teniposide 50 mg/d, d1-3), the mass could not be touched and the follow-up was continued.Conclusion Although relapse of AML often occurs in the testicle or the central nervous system,it pay attention to the possibility of relapse of AML presenting as GS.Fine needle aspiration cytology(FNAC)combined with FCM can provide an convenient, handy, practicable and less invasive way of the diagnosis and can be a preferred detection technique.

Objective To investigate the proportion of CD_4~+ CD_(25)~+ Tregs in the peripheral blood of the patients suffering from acute myeloid leukemia (AML) with or without chemotherapy and clinical significance.Methods The flow cytometry was used to evaluate the proportion of CD_4~+ CD_(25)~+ CD_(127)~(low) T cells in the peripheral blood of the patients with primary AML group (group A), AML who achieved complete remission over 3 years (group C) or less than 3 years(group B) and the healthy donors. Results The percentages of CD_4~+ CD_(25)~+ Tregs of group A and B was significantly higher than that of control group (P <0.05); but not significantly defferent among the group C and control group (P >0.05). The percentages of CD_4~+ CD_(25)~+ Tregs of group A was significantly higher than that of group B and C. Conclusion These results suggested that CD_4~+ CD_(25)~+ Tregs played an important role in acute myeloid leukemia.