Objective To investigate the effects of c-myc promoter binding protein(MBP-1)gene expression silencing on the pro-liferation in vitro in human gastric cancer cell line SGC-7901.Methods The cells divided into three groups:blank control group (cells without transfecting gastric cancer cell),negative control group(cells transfecting missense sequence)and experimental group (cells transfecting MBP-1 shRNA).Two MBP-1 shRNA sequences and one negative control shRNA sequence were designed,syn-thesized and cloned into pSIREN-retroQ plasma.Then the recombinant plasmids were constructed and transfected into human gas-tric cancer SGC-7901 cells by Lipofectamine 2000.After selection of cells with puromycin for two weeks,stable cell line SGC-7901 expressing gene of MBP-1 specific shRNA was obtained.The expressions of MBP-1 mRNA and protein in SGC-7901 were deter-mined by the real time PCR and Western blot,respectively.The effects of altered expression of MBP-1 on the cell proliferation were measured by MTT cell proliferation assay.Results PCR and sequencing indicated that the recombinant plasmids pSIREN-retroQ was constructed.Then the recombinant plasmids were transfected into human gastric cancer SGC-7901 cells by Lipofectamine 2000. After selection of cells with puromycin for two weeks,stable cell line SGC-7901 expressing gene of MBP-1 specific shRNA was ob-tained.The relative expression level MBP-1 mRNA in the MBP-1 siRNA transfection group was significantly decreased compared with the blank control group(P <0.05).Compared with the blank group,the expression levels of MBP-1 protein in the experimental group also significantly decreased.The proliferation abilities of SGC-7901 cells at 48,72,96,120 h after MBP-1 siRNA transfection were significantly increased compared with the blank control group (P < 0.05 ).Conclusion Down-regulating the expression of MBP-1 can obviously promote the proliferation of human gastric cancer cell line SGC-7901.MBP-1 gene may become the new target of gene therapy for gastric cancer.

OBJECTIVE To clone the MSI-78 gene for the purpose of providing evidence for further studies in prokaryotic expression and activities of antimicrobial peptides. METHODS According to the amino acid sequences of MSI-78,the MSI-78 gene was designed favorable for the Escherichia coli codons. After EcoRⅠand PstⅠ disgestion,cohesive ends were added to both ends respectively and the MSI-78 gene was synthesized by chemical methods. Then,the MSI-78 gene was ligated with pUC-18,transformed into the E. coli DH5?. Through filtration of ? complementary screening,the positive recombinant was finally identified by enzyme digestion of ECORⅠand ECORⅠ/PstⅠ and by PCR. RESULTS The MSI-78 gene was ligated with pUC-18 and transformed into the E. coli DH5?. As a result,MSI-78 gene was cloned in E. coli DH5? successfully. CONCLUSIONS The cloning of the MSI-78 gene provides evidence for further studies of its prokaryotic expression and activities of antimicrobial peptides.

Objective To master the technique of mouse embryonic stem (ES) cells differentiate into endothelial cells,which would be a new therapeutic approach for cardiovascular disease.Methods Expression of selfrenewal marker genes in E 14 cells was assessed.Expression of vascular endothelial growth factor receptor 2 (Flk 1) in monolayer differentiation on day 4 and vascular endothelial cadherin (VE-cadherin) on day 8 were detected.On day 8,differentiation cells were also observed under phase contrast microscopy (PCM) and transmission electron microscope (TEM).ES cells and endothelial-specific molecular markers were assessed by RT-PCR at different time-points.Results As self-renewal marker genes were expressed in E14 cells,E14 cells was identified to maintain their selfrenewal pluripotency.The marker gene of letarl,Flk1 was expressed on differentiation day 4.On differentiation day 8 the marker gene VE-cadherin was expressed and as observed under PCM endothelial cells with spindle shape and TEM with Weibel-Palade body,thus were the major populations generated after VEGF induction,and E14 cells were confirmed differentiated into mature endothelial cells.The expressions of genes octamer binding transcription factor 4 (Oct4),Flk1 and VE-cadherin were detected on differentiation day 2,4,6,8 and 10.Conclusions As VE-cadherin gene was expressed in monolayer on differentiation day 8,E14 cells were confirmed differentiated into endothelial cells,which would be a new therapeutic approach for cardiovascular disease.

Objective To observe the therapeutic effects of Liuwei Shunji Capsule on survival state, visceral hypersensitivity, the content of 5-HT of model rats with liver depression and spleen deficiency-type irritable bowel syndrome (IBS), and explore its mechanism.Methods SD rats were randomly divided into normal group, model group, pinaverium bromide group and Liuwei Shunji Capsule of high, medium and low dose groups. Senna with restraint stress was used in duplicating liver depression and spleen deficiency-type IBS model. From the second day after the model was established, each drug treatment group was administered with corresponding drugs once a day for two weeks. The effects of Liuwei Shunji Capsule on survival state, visceral hypersensitivity, the content of 5-HT in serum and hypothalamus were observed.Results Compared with model group, Liuwei Shunji Capsule could raise weight growth rate of model rats with liver depression and spleen deficiency-type IBS (P<0.05), improve their diarrhea, fatigue demeanor, fur quality, irritability and other symptoms, significantly decrease their visceral hypersensitivity, the content of 5-HT in serum and hypothalamus (P<0.01).Conclusion Liuwei Shunji Capsule can effectively improve survival state and intestinal disorders of rats with liver depression and spleen deficiency-type IBS, and the mechanism of treatment could be realized through regulating 5-HT level.

AIM:Toobservetheeffectsofmicroparticlesderivedfrombonemarrowmesenchymalstemcells ( MSC-MPs) on angiogenesis and cardiac function in a rat myocardial infarction model .METHODS:MSCs were obtained from Sprague-Dawley rats.MSCs were treated under serum-free condition in hypoxia for 72 h, and the microparticles were isolated from the supernatants .The phenotypic profile of MSC-MPs was determined by bead-based flow cytometry and the morphology was observed under a transmission electron microscope .The rat myocardial infarction model was established . The cardiac function was evaluated by echocardiography after the intramyocardial injection of MSC -MPs.The myocardial in-farct size was observed by Masson staining .The blood vessel density in the peri-infarcted area was measured using immuno-histochemical staining for von Willebrand factor and α-smooth muscle actin.The expression of vascular endothelial growth factor ( VEGF) was analyzed by real-time PCR.RESULTS: Apoptotic MSCs released a large quantity of microparticles which were phenotypically similar to the parent MSCs and 100~1 000 nm in diameter.The cardiac functions of myocardial infarction rat model were improved at 7 d and 28 d after intramyocardial injection of MSC-MPs compared with control group . The myocardial infarct size was reduced and angiogenesis was promoted significantly in the infarcted heart injected with MSC-MPs 28 d after treatment .MSC-MPs treatment also increased the expression level of VEGF within 7 d.CONCLU-SION:MSC-MPs protect cardiac tissue from ischemic injury and improve cardiac function by promoting angiogenesis after myocardial infarction .

Objective To investigate the molecular mechanisms of synergistic effects of BFA and CDDP on human lung cancer GLC-82 cells, and to test the levels of PERK-ATF4 pathway. Methods GLC-82 cells were incubated with 50 ng/mL of BFA or/and 2 μg/mL of CDDP for 24 or 48 hours. The levels of PERK, p-PERK and ATF4 in GLC-82 were analyzed by real-time PCRand/or Western Blot. Results The levels of PERK were lowest in CDDP group, but higher in BFA group (P < 0.05), the highest in group of BFA+CDDP (P < 0.05 or P < 0.01). The p-PERK level decreased in group of BFA+CDDP (P < 0.05 or P < 0.01). There was no significant change of ATF4 expression in CDDP group, but ATF4 expression increased slightly in BFA group, and increased further in group of BFA+CDDP (P < 0.05 or P < 0.01)which was also higher than that in BFA group or CDDP group (P < 0.05 or P < 0.01). Conclusions The upregulated levels of PERK and ATF4 by the combination of BFA and CDDP may be one of the mechanisms of synergistic anti-cancer effect of BFA and CDDP on GLC-82 cells.

Objective To investigate the effects of c?myc promoter binding protein 1(MBP?1)gene on the proliferation of human Saos?2 osteo?sarcoma cells in vitro. Methods Saos?2 cells were divided into three groups:blank control group(untransfected cells),negative group(cells transfected with missense sequence)and experimental group(cells transfected with MBP?1 shRNA). Two MBP?1 shRNA sequences and one neg?ative control shRNA sequence were designed ,synthesized and cloned into pSIREN?retroQ plasma. Then the recombinant plasmids were construct?ed and transfected into human Saos?2 osteosarcoma cells by Lipofectamine 2000. The expressions of MBP?1 mRNA and protein in Saos?2 cells were detected by real?time PCR and Western blot ,respectively. The effects of altered expression of MBP?1 on cell proliferation were measured by CCK?8 cell proliferation assay. The expressions of cyclin D1 and cyclin E in Saos?2 were determined by Western blot. Results PCR and sequenc?ing results indicated that the recombinant plasmids pSIREN?retroQ was constructed. The relative expression level of MBP?1 mRNA in the MBP?1 siRNA transfection group was significantly decreased than that in blank control group(P<0.05). Compared with the blank control group,the ex?pression levels of MBP?1 protein in the experimental group also significantly decreased. The proliferation abilities of Saos?2 cells at 48,72,and 96 hours after MBP?1 siRNA transfection were significantly increased than those in the blank control group(P<0.05). Compared with the blank con?trol group,the expression levels of cyclin D1 and cyclin E protein in the experimental group also significantly increased(P<0.05). Conclusion Knockdown of the expression of MBP?1 gene promotes the proliferation of human Saos?2 osteosarcoma cells. MBP?1 gene may become the new tar?get of gene therapy for osteosarcoma.

Objective To investigate the expression and clinical significance of P-selectin (CD62P) in bone marrow hematopoietic stem cells of patients with acute leukemia (AL). Methods The CD62P expression in bone marrow mononuclear cells of 15 healthy donors and 56 untreated patients with AL, were examined by flow cytometry. Results The average rate of CD62P expression was (6.72±7.64) % in hematopoietic stem cells (CD+45 CD+34 CD-38) of the 38 patients with acute myeloid leukemia (AML), was (3.46±2.51) % in hematopoietic stem cells (CD+45 CD+34 CD+19) of the 12 patients with B-acute lymphoblastic leukemia (B-ALL), and was (6.23±4.95) % in hematopoietic stem cells (CD+45 CD+34 CD+7) of 6 patients with T-acute lymphoblastic leukemia (T-ALL). The expression rates in those AL patients were higher than that in the healthy controls (1.04 ±1.23) % (t = 2.847, 3.284, 3.091, respectively, P ＜0.01), while there was no difference between the control group and the group who reach CR after routine treatment (t =1.932, P ＞0.05). Furthermore, the leukocyte,hemoglobin and platelet count in CD+62P patients with AML and T-ALL were significantly higher than CD-62P ones (t =4.153, 8.095, 8.289, 7.235, 8.692, 9.832, respectively, P ＜0.05), but there was no significant difference between CD+62P and CD-62P patients with B-ALL (t =0.340, 1.142, 0.019, respectively, P ＞0.05).Conclusion The CD62P is one of the markers of platelet activation, and its expression varies in different types of AL. The CD62P in hematopoietic stem cells of AL could be regarded as a new sign for the leukemic stem cells, as well as a helpful prognostic indicator in treatment response assessment.

Nowadays the role of genetic findings in determining the diagnosis, therapy and prognosis of acute myeloid leukemia (AML) has become more valuable. To improve and validate the detection of clonal chromosomal aberrations in leukemia, we designed a combined application of karyotyping with multiplex reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH), and addressed the expression and distribution of fusion genes among the subtypes of Chinese adult patients with de novo AML. Multiplex RT-PCR assays were performed on 477 samples from newly diagnosed AML patients, and cytogenetic data were obtained from 373 of them by R or G banding techniques and those in some cases were confirmed by FISH. The PCR products in some suspected cases were tested by two-directional sequencing. The results showed that except unqualified samples, fusion genes were detected by multiplex RT-PCR in 211 of 474 patients (44.51%), including AML1-ETO, CBFβ-MYH11, PML-RARα, PLZF-RARα, NPM-RARα, MLL rearrangements, BCR-ABL, DEK-CAN, SET-CAN, TEL-PDGFR, TLS-ERG, AML1-MDS1 (EVI-1). In 373 patients, who took both multiplex RT-PCR and karyotype analysis, the detection rate of chromosomal aberrations by using multiplex RT-PCR and karyotyping was 160/373 (42.89%) and 179/373 (47.98%) respectively, and the combination could optimize the detection rate of clonal genetic abnormalities to 216/373 (57.90%). The PCR results from 11 cases "normal" in karyotyping but abnormal in RT-PCR for MLL rearrangements were confirmed by two-directional sequencing. It is concluded that karyotype studies remain the cornerstone for genetic testing; conventional cytogenetics and molecular-based methods are complementary tests for the detection of clonal genetic aberrations in AML, especially for the cryptic or submicroscopic aberrations. Once a genetic marker has been identified by combined analysis, it could be used to monitor residual disease during/after chemotherapy, by quantitative RT-PCR and/or FISH.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive leukemia. However the poor prognosis and low morbidity restrict further analysis of the disease. Therefore there is an increasing demand to develop animal models for identifying novel therapeutic approaches. In this study, we inoculated the anti-mouse CD122 monoclonal antibody conditioned NOD/SCID mice with the leukemia cells from 9 T-ALL patients and 1 cell line via the tail vein. Four of the 9 patients and the cell line were successfully engrafted. Flow cytometry detected high percentage of human CD45(+) cells in recipient mice. Immunohistochemistry showed infiltration of human CD45(+) cells in different organs. Serial transplantation was also achieved. In vivo drug treatment showed that dexamethasone could extend survival, which was consistent with clinical observation. These results demonstrated that we successfully established 5 xenotransplantation models of T-ALL in anti-mCD122 mAb conditioned NOD/SCID mice, which recapitulated the characteristics of original disease.