BACKGROUND:Bone tissue transplantation or osteogenic material fil ing is after used for bone defect repair. To remove autologous bone tissues can lead to additional damage and secondary deformity, therefore, it is extremely urgent to search for a new osteogenic material. OBJECTIVE:To construct the porousβ-tricalcium phosphate (β-TCP)/col agen scaffold modified with human bone morphogenetic protein 2 (hBMP2) gene, and to observe its effects on differentiation of MC3T3-E1 cel lines. METHODS:The porousβ-TCP/col agen scaffold modified with hBMP2 gene was prepared. Then in vitro culture system of MC3T3-E1 cel lines with composite scaffold was established. There were scaffold and plate groups, and each group was divided into two subgroups according to the different concentrations of plasmid. Samples were col ected and observed morphological y by scanning electron microscope and light microscope after complex culture. After 1, 3, 7 and 14 days of induction, calcium nodules were observed through alizarin red staining, the cel cycle was detected by real-time PCR, and expressions ofαI-chain col agen type I gene, Osterix and bone sialoprotein were observed. RESULTS AND CONCLUSION:The number of cel s adhered, differentated and distributed on the composite scaffold was significantly higher than that of the single scaffold (P<0.05). Alizarin red staining and real-time PCR detection showed that the osteogenesis ability of MC3T3-E1 cel lines in the scaffold group was stronger than that in the plate group. To conclude, the porousβ-TCP/col agen scaffold modified with hBMP2 gene is an appropriate candidate for bone defect repair.

BACKGROUND:Ligustrazine has been shown to restore the function of the femoral headviathe revascularization, increased blood flow, theabsorption ofnecroticbone, and bone regeneration. OBJECTIVE:To study the effects of ligustrazine on remodeling of periodontal tissues and the expression of osteoprotegerin in the maintenance phase in rats with orthodontic tooth movement. METHODS:Thirty-two healthy male Wistar rats were included and equaly randomized into four groups. Maxilary left first molar mesialization was performed through traction of 50 g force for 21 days to establish the rat model of tooth movement. 5, 10, 15 mg/L ligustrazine (50 μL) were localy injected into the first molar periosteum in model rats on the day before removing the orthodontic forcing device. Same volume of saline was injected in the control group. The injection was administered every other day. At 1 and 4 weeks after injection, the distance of tooth movement, the recurrence distances and percentage were determined and calculated. The pathological changes in periodontal tissues were observed by immunohistochemistry and hematoxylin-eosin staining. The width ofthe parodontium and number of osteoblasts were observed under an optical microscope. RESULTS AND CONCLUSION:The recurrence distance inthecontrol group was increased compared withtheexperimental group, while the number of osteoblasts and osteoprotegerin immunoreactivity were decreased. Good width of the parodontium and smal recurrence trend were found in 10mg/L ligustrazine group. These findings indicate that ligustrazine promotes the proliferation of osteoblasts and enhances the expression of osteoprotegerin, which is beneficial to the retention of teeth after orthodontic surgery.

Objective To evaluate the effects of the chronic Keshan disease's self-management in Liangshan Prefecture of Sichuan Province. Methods According to "The Serf Management Program of Chronic Keshan Disease in Liangshen Prefecture of Sichuan Province", 56 chronic Keshan disease patients were selected in the personalized self-manngemant evaluation under the instructions by endemic disease specialists and the rural doctors. Evaluation was based on changes of indexes such as the clinical symptoms, general health conditions, the electrocardiogram, X-ray, the heart function, etc, before treatment and 3 and 6 months following the treatment. Results Clinical signs and symptoms of the patients were significantly improved 3 and 6 months after the treatment, and the improvement was more obvious 6 months than 3 months following the treatment(P<0.05 or< 0.01). After treatment for 3 months, the patients'electrocardingram and heart function did not show obvious change (X2=0.05,039, P0.05); hut obvious improvements Eexcept X ray results(X2=0.61 ,P0.05)] were seen 6 months after treatment (X2=4.36,16.84, P<0.05 or<0.01). Altogether, among the 56 patients evaluated after treatment for 6 months, none achieved the clinical cure standard, 26 cases(46.3%) showed significant improvement, 17 cases (30.4%) were stable, 5 cases (8.9%) were aggravated,one case (1.8%) lost contact, and 6 cases (10.6%) died. Conclusion The project of the chronic Keshan disease's self-management is suitable for the present situati,on of the endemic regions and can he introduced to many places in our country.

The objective of this study was to estimate a novel apoptosis-promoting molecule PDCD5 expression in the bone marrow cells from adult acute myeloid leukemia (AML) for investigation of its significance in the pathogenesis of AML. Flow cytometry assay was used for detection of PDCD5 expression in the different groups of cells from bone marrow of AML patients and normal controls by using 21 monoclonal antibodies with different fluorescent markers. The PDCD5 expressions in bone marrow cells from some AML patients and normal controls were also detected by Western blot. The results showed that the mean PDCD5 fluorescence intensity in bone marrow nucleated cells (MNC) from the bone marrow of 36 untreated AML patients was significantly lower than that from the bone marrow of 30 normal controls (3059 +/- 1392) vs (7432 +/- 1261) (P < 0.01). The mean PDCD5 fluorescence intensity was lower in the marrow granulocytes, monocytes, blast cells, and lymphocytes from untreated AML patients than that from normal (3939 +/- 2121) vs (8367 +/- 1045); (3156 +/- 1635) vs (5917 +/- 2329); (2824 +/- 1592) vs (3998 +/- 2106); (1474 +/- 816) vs (3355 +/- 2042) respectively, (all P < 0.01). Western blot analysis demonstrated that PDCD5 expression was significantly decreased in the AML cells, as compared with normal cells. It is concluded that PDCD5 expression in MNC in untreated AML patients is lower than that in the normal. PDCD5 expression in the marrow granulocytes, monocytes, blast cells, and lymphocytes of untreated AML patients is significantly lower than that in the normal. It suggests that the abnormally low expression of PDCD5 may be involved in the pathogenesis of AML.
Apoptosis ; physiology ; Apoptosis Regulatory Proteins ; metabolism ; Bone Marrow Cells ; metabolism ; Humans ; Leukemia, Myeloid, Acute ; metabolism ; pathology ; Neoplasm Proteins ; metabolism

OBJECTIVETo explore the specificity of anti-phosphotyrosine monoclonal antibody PY20 in bcr-abl+ cells and its possible clinical applications.

METHODSBcr-abl cell lines( K562, MEG-01) and bcr-abl- cells lines( Jurkat, MCF-7 )were stained with PY20. Phosphotyrosine protein of K562 and MEG-01 cells was detected by flow cytometry before and after treatment with imatinib. Phosphotyrosine protein in bone marrow cells from 49 patients with chronic myeloid leukemia (CML), Ph+ acute lymphoblastic leukemia(Ph(+) -ALL), Ph- ALL, acute myeloid leukemia (AML-M1, M2, M3, M5, FAB classification), chronic lymphocytic leukemia (CML) and 3 normal donor. Positive cells over 5% of total cells was considered positive cases for phosphotyrosine protein. The level of tyrosine phosphorylation was determined by median fluorescence intensity (MFI).

RESULTSBcr-abl cell lines and marrow cells from 10 CML patients and 8 ALL patients were all PY20-positive, while bcr-abl- cell lines and marrow cells from 18 leukemia patients and 3 normal donor were all PY20-negative. MFI decreased remarkably after blocked by imatinib in K562 cells and MEG-01 cells. The positive cell percent of marrow cells from 10 newly diagnosed CML patients and 9 imatinib-sensitive CML patients was (54.20 +/- 19.82)% and (14.84 +/- 6.17)% (P < 0.05), while that of 2 cases of imatinib-resistant was 64.3% and 57.2%. There was significant difference of MFI between imatinib-resistant patients and imatinib-sensitive patients (99.42 +/- 4.87 vs 46.41 +/- 4.67, P < 0.01).

CONCLUSIONPY20 monoclonal antibody is highly specific for bcr-abl+ cells. It might be useful in rapid detection of bcr-abl+ cells and sensitivity to imatinib of CML patients.

Antibodies, Monoclonal ; analysis ; Bone Marrow Cells ; metabolism ; Flow Cytometry ; Fusion Proteins, bcr-abl ; analysis ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism ; Leukemia, Myeloid, Acute ; metabolism ; Phosphotyrosine ; analysis ; immunology

OBJECTIVETo evaluate the significance of quantification of WT1 mRNA for monitoring minimal residual disease (MRD) in patients with acute myeloid leukemia (AML).

METHODSWT1 mRNA level was detected with real-time quantitative RT-PCR (RQ-PCR) technique in bone marrow samples from 15 normal subjects (NBM) and 123 AML patients. Sixty-two AML samples were also detected AML1-ETO mRNA expression by RQ-PCR. Simultaneously follow-up of WT1 and AML1-ETO levels were carried out in 50 samples from 8 AML patients. WT1 and AML1-ETO levels were normalized by internal control ABL gene.

RESULTSAll correlation co-efficiencies were over 0.99 for WT1, AML1-ETO and ABL standard curves. Co-efficiencies of both interassay and intraassay variation were below 4%. The WT1 expression levels in NBM were 0.001 to 0.019 with a median level of 0.008. Higher levels of WT1 expression were found in 61 of 67 (91%) newly diagnosed AML patients compared with NBMs and 37 of the 67 (55.2%) showed 100-fold higher WT1 levels than that in NBMs. WT1 mRNA levels were highest in M(4EO) and M(3) and lowest in M(1) and M(5) patients. There was an excellent correlation between WT1 and AML1-ETO gene expression levels (r = 0.88, P < 0.001). WT1 expression levels in three patients who were in continuous complete hematological remission (CHR) were within normal range. In three of four relapsing patients, WT1 expression levels increased 31.4, 11.4 and 4.0 fold respectively one month before hematological relapse.

CONCLUSIONSQuantification of WT1 expression level by RQ-PCR may be used to monitor MRD for most AML patients, but it is less sensitive than fusion gene. Continuous or significant increase of WT1 expression in CHR patients predicts an impending relapse.

Adolescent ; Adult ; Aged ; Bone Marrow ; metabolism ; Child ; Child, Preschool ; Core Binding Factor Alpha 2 Subunit ; genetics ; metabolism ; Female ; Humans ; Leukemia, Myeloid, Acute ; diagnosis ; metabolism ; pathology ; Male ; Middle Aged ; Neoplasm, Residual ; diagnosis ; metabolism ; pathology ; Oncogene Proteins, Fusion ; genetics ; metabolism ; RNA, Messenger ; genetics ; RUNX1 Translocation Partner 1 Protein ; Recurrence ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; WT1 Proteins ; genetics ; metabolism

Vascular endothelial growth factor (VEGF) is a specific mitogen for vascular endothelial cells. It has been associated with angiogenesis, growth, metastasis and poor prognosis in solid tumors. Lately, it has been known that VEGF expression is higher in bone marrow from chronic myeloid leukemia (CML) patients than that in normal subjects. However, it is not clarified that the effect of VEGF on the abnormal proliferation of CML cells. In order to explore the effect of autocrine VEGF on CML cells, K562 cells were transfected with the VEGF(121) cDNA sense vector (K562/S) or with the VEGF(121) cDNA antisense vector (K562/As). K562 cells were transfected with the pcDNA(3) vector (K562/V) as the control. Cell proliferation was determined by MTT and colony forming assay in vitro. Flow cytometric Annexin-V-FITC/PI dual labeling technique was performed to observe the effect of VEGF(121) cDNA transfection on apoptosis of K562 cells. Results indicated that K562/S transfectants exhibited a 3-fold increase in VEGF secretion, and K562/As transfectants exhibited a 49% reduction in VEGF secretion. K562/As showed a reduced growth rate and colony forming efficiency as compared to K562/V. K562/S showed an increasing growth rate and colony forming efficiency as compared to K562/V. K562/As had more apoptotic cells than K562/V and K562/S in the same culture condition. These data suggest that VEGF plays an important role in the abnormal proliferation and apoptosis in CML cells through an autocrine mechanism.

OBJECTIVETo investigate the clinical characteristics of newly diagnosed acute myeloid leukemia (AML) with NPM1 mutation.

METHODSNPM1 mutation (including A, B, D mutation type) was detected in 206 patients with newly diagnosed AML by real-time quantitative RT-PCR.

RESULTSThe incidence of NPM1 mutation was 15.5% in total AML patients and 32.5% in normal karyotypes AML patients. The characteristics of 174 NPM1 wild type patients v.s. that of 32 NPM1 mutation patients was as follow, median age (46 vs 35 years old, P < 0.01), WBC counts (27 × 10(9)/L vs 8 × 10(9)/L, P < 0.01), BPC (82 × 10(9)/L vs 36 × 10(9)/L, P < 0.01), proportion of AML-M(5) (31.2% vs 5.8%, P = 0.01), incidence of normal karyotypes (92.6% vs 40.8%, P < 0.01), incidence of FLT3-ITD-positive (25.0% vs 7.5%, P < 0.01), CD34-positvie (23.3% vs 69.5%, P < 0.01), cases with fusion gene (0 vs 47.1%, P < 0.01). No statistic difference was found in sex, percentage of blasts in bone marrow, complete remission rate, overall survival between the two groups. Relapse-free survival in AML patients with NPM1-mutation and FLT3-ITD-negative tended to be higher than in those with NPM1-mutation and FLT3-ITD-positive.

CONCLUSIONIt is necessary to detect NPM1 mutation and FLT3-ITD in newly diagnosed AML patients, especially in patients with high WBC and BPC, CD34-negative, normal karyotype, which might help to molecular classification and treatment.

Humans ; Leukemia, Myeloid, Acute ; genetics ; Mutation ; Nuclear Proteins ; genetics ; Prognosis ; fms-Like Tyrosine Kinase 3 ; genetics

OBJECTIVETo investigate the relationship between three types of bcr/abl fusion transcripts and clinical manifestation in chronic myeloid leukemia (CML).

METHODM-, m- and micro -bcr/abl fusion transcripts were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) technique in 537 fresh bone marrow samples of patients suspected CML clinically.

RESULTSOf 573 patients, 479 expressed M-bcr/abl transcripts, among whom 370 were in chronic phase (CP), and 109 in accelerated (AP)/blastic phase (BP). The percentages of patients with b2a2 transcripts in CP and AP/BP were 32.4% (120/370) and 36.7% (40/109) (P > 0.05). The b2a2 transcript patients in blastic crisis were 52.6% (10/19) for lymphoblastic and 33.3% (30/90) for myeloblastic (P > 0.05). The platelet count of untreated patients with b3a2 isoform [(485.9 +/- 333.8) x 10(9)/L, n = 125] was distinctly higher than those with b2a2 isoform [(380.5 +/- 321.9) x 10(9)/L, n = 62] (P < 0.05). 66.0% (31/47) and 64.4% (29/45) of the patients in CP and AP/BP respectively co-expressed M- and m-bcr/abl transcripts (P > 0.05). One patient expressed only m-bcr/abl transcript was of typical acute myeloblastic leukemia (AML). Both two micro -bcr/abl(+) patients were of typical CML.

CONCLUSIONSAlmost all typical CML patients express M-bcr/abl transcripts, most of them coexpress M-bcr/abl and m-bcr/abl transcripts, a few possesses only micro -bcr/abl fusion gene. m-bcr/abl(+) are usually associated with AML or CML in myeloblastic crisis besides acute lymphoblastic leukemia (ALL). Patients with b3a2 isoform are prone to higher platelet count before treatment.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; Child, Preschool ; Female ; Fusion Proteins, bcr-abl ; genetics ; Genotype ; Humans ; Infant ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Male ; Middle Aged ; Reverse Transcriptase Polymerase Chain Reaction ; Transcription Factors ; genetics

Vascular endothelial growth factor (VEGF) is one of the main angiogenic cytokines and plays an important role in the development of human solid tumors. However, it is not clarified whether VEGF governs the progress of the chronic myelogenous leukemia (CML). This study is to estimate VEGF expression in the bone marrow cells from normal and adult CML patients and various leukemic cell lines. Reverse transcription-polymerase chain reaction (RT-PCR) was used for detection of VEGF mRNA. VEGF concentrations in the cell cultural supernatant and the plasma from normal and CML patient bone marrows were determined by enzyme linked immunosorbent assay (ELISA). VEGF mRNA was positive in 67 of 72 cases of bcr/abl(+) CML patient bone marrow cells (93.1%), in 5 of 10 CML patients post Allo-BMT bone marrow cells (50%), and in 6 of 10 normal bone marrow cells (60%), the expression rate of VEGF mRNA in CML patients bone marrow cells was higher than that in CML patients post Allo-BMT and normal bone marrow cells. VEGF mRNA also expressed in the HL-60, K562, CEM, KG1a, NB4, and Nalm6 cells, but not in the Jurkat cells. The mean VEGF concentration in the plasma (380.6 pg/ml) from 22 untreated CML patients was 9 folds higher than that from 9 CML patients post Allo-BMT (38.0 pg/ml). The mean VEGF concentration in the cultural supernatant (499.8 pg/ml) of 17 newly diagnosed CML bone marrows was 2.5-folds higher than that in 11 normal donors (141.3 pg/ml). The CML marrow cells secrete more VEGF than normal marrow cells do. Our results suggest that the abnormality of VEGF transcription and translation expression may play an important role in the development of CML.