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.

OBJECTIVETo compare the immunophenotypic and clinical characteristics between NPM1 mutated acute myeloid leukemia (AML) (NPM1m(+)AML) and unmutated AML(NPM1m(-)AML) not otherwise characterized (NOS) under similar FAB subtypes constituent ratio.

METHODSImmunophenotyping and NPM1 gene mutation type-A, B and D and other leukemic related fusion genes were detected by multiparameter flow cytometry and real time RT-PCR or PCR, respectively. 104 AML patients with NPM1m(+)AML and performed immunophenotyping assay were included, 97 with NPM1m(-)AML.

RESULTSThere were significant difference between the two groups at presentation in terms of sex, white blood count(WBC), platelet counts (PLT), blast ratio, normal karyotype ratio, WT1 expression level, FLT3-ITD mutation positive rate and remission rate of first course of induction therapy (P < 0.05). On the immunophenotype, the expression of early differentiation antigens (CD34, HLA-DR, CD117, CD38), lymphocytic antigens (CD7, CD4, CD19, CD2), myeloid and monocytic differentiation-associated antigens (CD13, CD14, CD15) were lower, and that of CD33 as well as CD123 were higher in NPM1m(+)AML patients. Among them, only CD34, HLA-DR, CD7, and CD4 positive cases were significantly lower in NPM1m(+)AML group than in NPM1m(-)AML group (P < 0.05), the rest of them had significant difference in the number of positive cells (P < 0.05). Above features were further analyzed between the M1/M2 and M4/M5 subgroups. M1/M2 cases retained the women prominent and had a higher WT1 expression level (P < 0.05). The expression of monocytic differentiation-associated antigens including HLA-DR and lymphocytic antigens were higher and that of CD117 were lower in M4/M5 subtype (P < 0.05). Among them, the positive rates of HLA-DR, CD64, CD11b, CD10, CD15, and CD4 were significantly higher in M4/M5 than in M1/M2 in NPM1m(+)AML group (P < 0.05).

CONCLUSIONThe most clinical characteristics in NPM1m(+)AML patients are consistent with reports, but some immunophenotype are different to the previous reports under similar FAB subtypes constituent ratio. The major immunophenotypic features of NPM1m(+)AML patients are lower expression of progenitor, myeloid and lymphoid lineage antigens. Monocytic differentiation-associated antigens are only higher expression in M4/M5 cases when comparison with M1/M2 cases within NPM1m(+)AML group.

Adolescent ; Adult ; Aged ; Antigens, CD ; metabolism ; Child ; Child, Preschool ; Female ; HLA-DR Antigens ; immunology ; Humans ; Immunophenotyping ; Leukemia, Myeloid, Acute ; diagnosis ; genetics ; immunology ; Male ; Middle Aged ; Mutation ; Nuclear Proteins ; genetics ; Young Adult

The expression levels of programmed cell death 5 (PDCD5) are down-regulated in many malignancies. SG611-pdcd5, a recombinant conditionally replicative adenovirus carrying pdcd5 gene expression cassette, can evidently kill the leukemic cells and protect selectively the normal cells. The purpose of this study was to investigate the synergistic killing effect of SG611-pdcd5 and low-dose etoposide (VP-16) on K562 cells. K562 cells were treated with different concentrations of VP-16 or different multiplicities of infection (MOI) of SG611-pdcd5. After 48 hours of incubation the cell viability was determined by using MTT assay. The results showed that the cell viability of SG611-pdcd5 (MOI = 40) plus VP-16 (0.5 µg/ml) group significantly decreased as compared with single SG611-pdcd5 (MOI = 40) treatment group or single VP-16(0.5 µg/ml) treatment group (42.00 ± 5.75% vs 59.45 ± 4.12%; 42.00 ± 5.75% vs 82.91 ± 3.41%, respectively, both p < 0.05). The synergistic killing effect of SG611-pdcd5 plus VP-16 was higher than that of PDCD5 protein plus VP-16 or that of non-replicating adenovirus carrying pdcd5 (Ad-pdcd5) plus VP-16 (both p < 0.05). The cell viability of VP-16 (4.0 µg/ml) plus SG611-pdcd5 (MOI = 40) group, VP-16 (4.0 µg/ml) plus proPDCD5 (40 µg/ml) group and VP-16 (4.0 µg/ml) plus Ad-pdcd5 (MOI = 80) group was 37.09 ± 1.89%, 52.36 ± 1.64% and 73.64 ± 4.33%, respectively. It is concluded that SG611-pdcd5 can promote K562 cell death induced by low-dose VP-16. The combination of SG611-pdcd5 and VP-16 can enhance the killing effect on leukemic cells.
Adenoviridae ; genetics ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; Apoptosis Regulatory Proteins ; genetics ; Cell Survival ; Etoposide ; pharmacology ; Genetic Vectors ; Humans ; K562 Cells ; Neoplasm Proteins ; genetics

OBJECTIVETo evaluate levels of common specific fusion transcripts M-bcr-abl, m-bcr-abl, TEL-AML1, AML1-ETO, PML-RAR alpha, CBF beta-MYH11 in untreated leukemia patients.

METHODSSpecific fusion transcript levels were detected by TaqMan-based real-time quantitative RT-PCR technique in a total of 208 samples, including 195 bone marrow samples from 50 M-bcr-abl(+) chronic phase-chronic myeloid leukemia (CML-CP), 10 M-bcr-abl(+) acute lymphoblastic leukemia (ALL), 19 m-bcr-abl(+) ALL, 11 TEL-AML1(+) ALL, 30 AML1-ETO(+) acute myeloid leukemia (AML), 58 PML-RAR alpha(+) acute promyelocytic leukemia (APL) and 17 CBF beta-MYH11(+) AML patients and 13 peripheral blood samples from 13 M-bcr-abl(+) CML-CP patients. abl was chosen as internal control gene. Fusion transcript level was calculated as fusion transcript copies/abl transcript copies in percentage.

RESULTSBone marrow and peripheral blood samples of CML-CP patients had similar M-bcr-abl fusion transcript levels (median 30% vs 35%, P > 0.05). M- and m-bcr-abl (median 64% vs 54%) levels were similar in ALL patients (P > 0.05), M-bcr-abl level was significantly higher in ALL than CML-CP patients(P < 0.001). Median TEL-AML1 level was 228% in ALL patients. Among AML patients, AML1-ETO level was significantly higher than CBF beta-MYH11 and PML-RAR alpha levels (median 388% vs 145%, 388% vs 47%, all P < 0.001), CBF beta-MYH11 level was significantly higher than PML-RAR alpha level (P < 0.001). Fusion transcript levels of L-, V- and S-type PML-RAR alpha were 45%, 44% and 55%, respectively. L-type was significantly lower than S-type (P = 0.04).

CONCLUSIONSFusion transcript levels in untreated leukemia patients were different and patient-to-patient variations did exist. Detection of fusion transcript levels in untreated leukemia patients not only provides baseline for minimal residual disease monitoring and treatment evaluation but also enable the comparison in inter-laboratory data.

Adolescent ; Adult ; Bone Marrow Cells ; metabolism ; Child ; Child, Preschool ; Core Binding Factor Alpha 2 Subunit ; genetics ; Female ; Fusion Proteins, bcr-abl ; genetics ; Humans ; Leukemia ; genetics ; Male ; Middle Aged ; Oncogene Proteins, Fusion ; genetics ; RUNX1 Translocation Partner 1 Protein ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Transcription, Genetic

The purpose of this study was to construct a recombinant conditionally replicating adenovirus (CRAd) expressing programmed cell death 5 (pdcd5). Pdcd5 gene was inserted in the E3 region of SG600-a CRAd in which the key genes for virus replication E1a and E1b were controlled under the human telomerase reverse transcriptase promoter (hTERTp) and the hypoxia response element (HRE) respectively, and with a deletion of 24 nucleotides within CR2 region of E1a. The insertion and orientation of all recombined plasmids were confirmed by restriction enzyme digestion and polymerase chain reaction (PCR). The infection efficiencies of a recombined virus carrying enhanced green fluorescent protein (EGFP) in leukemic cell lines were observed by using fluorescence microscope. The relative pdcd5 expression levels of K562 after being infected with SG611-pdcd5 were detected by real-time quantitative PCR. The results showed that the construction of SG611-pdcd5 was completed and confirmed. Pdcd5, hTERTp, HRE, skeleton and fiber11 of recombinant adenovirus SG611-pdcd5 were successfully amplified. The infection efficiencies of SG611-EGFP were all above 70% in both leukemic K562 and MEG-01 cell lines. SG611-pdcd5 expressed pdcd5 with high efficiency in leukemic cells as compared with Ad-pdcd5 or SG611 (p < 0.001). The expression level of pdcd5 increased gradually along with the increase of MOI. It is concluded that the triple-regulated adenovirus of SG611-pdcd5 containing the pro-apopro-tic gene pdcd5 has been successfully established with high pdcd5 expression level in leukemic cells, indicating that the recombinant adenovirus, SG611-pdcd5, promises further development of targeted tumor gene therapy.
Adenoviridae ; genetics ; Apoptosis Regulatory Proteins ; genetics ; Genetic Therapy ; methods ; Neoplasm Proteins ; genetics ; Oncolytic Viruses ; genetics ; Promoter Regions, Genetic ; Telomerase ; genetics

OBJECTIVETo investigate the unusual bcr/abl fusion gene structures of two Ph chromosome positive chronic myelogenous leukemia (CML) patients in chronic phase (CP).

METHODSBy using general M- and micro -bcr/abl specific primers respectively, bcr/abl fusion transcripts were detected by reverse transcription-polymerase chain reaction (RT-PCR). The RT-PCR products sequencing was performed, the DNA sequences were analyzed in Genebank and the bcr and abl sequences at the fusion site were identified. DNA was amplified by PCR using a set of primers designed according to the sequencing result of RT-PCR products.

RESULTSTwo patients showed typical manifestations of CML-CP. Their RT-PCR products were different from usual M- or micro -type; one was longer than M-bcr/abl but shorter than micro -bcr/abl, the other one was shorter than M-bcr/abl. The RT-PCR products sequencing showed that both products contained bcr and abl gene sequences. The first patient's bcr gene was broken within exon 18, and fused to abl gene exon 2(a2), and a 40 bp of partial abl intron 1b fragment was inserted between them, resulting in a novel in-frame bcr/abl fusion transcript-e18-int-a2 which has not been reported in the literature so far. In the second patient, deletion of abl exon2(a2) led to exon 13(b2) of bcr gene fusing with abl exon 3(a3).

CONCLUSIONUncommon bcr/abl fusion gene may occur in typical Ph(+) CML patient.

Adult ; Fusion Proteins, bcr-abl ; genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Male ; Molecular Sequence Data ; Philadelphia Chromosome ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA

This study was purposed to investigate the effect of adenovirus-mediated transfer of PDCD5 gene on apoptosis of K562 cells induced by etoposide. Recombinant adenovirus PDCD5 (Ad-PDCD5), control vectors Ad-null and Ad-eGFP were constructed by AdMax vector system respectively. After K562 cells were transfected by Ad-PDCD5, Ad-null or Ad-eGFP with different multiplicity of infection (MOI), the expression level of the PDCD5 gene was examined by RQ-RT-PCR assay. The effects of etoposide in combination with Ad-PDCD5 on the proliferation and apoptosis of K562 cells were measured by using MTT assay and flow cytometry with Annexin-V-FITC/PI dual labeling technique, respectively. The results showed that the transfection efficiencies of Ad-eGFP in K562, Jurkat and CEM cells ranged from 60% to 86%. Expression level of PDCD5 gene in K562 cells was evidently increased following transfection with Ad-PDCD5. The Ad-PDCD5 synergistically enhanced the apoptotic percentage of K562 cells induced by VP-16, as compared with that of Ad-null + VP16 and VP-16 alone respectively. It is concluded that Ad-PDCD5 may be a potential agent for enhancing the chemotherapy effect.
Adenoviridae ; genetics ; metabolism ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Etoposide ; pharmacology ; Humans ; K562 Cells ; Neoplasm Proteins ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Transfection

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