Child ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell

Objective To explore the impact factors for post-thaw embryo survival rate and clinical pregnancy rate in frozen-thawed embryo transfer program. Methods The clinical data of 573 cycles of frozen-thawed embryo transfers were retrospectively analysed. Groups were divided according to the pre-freeze embryo quality, pre-freeze embryonic developmental stage, frozen-thawed embryo quality and cryopreservation technique, respectively, and post-thaw embryo survival rates and/or clinical pregnancy rates were compared among groups. Results The clinical pregnancy rate of high quality pre-freeze embryo was significantly higher than that of low quality pre-freeze embryo (31.8% vs 20.0%) (P< 0.05). There was no significant difference in the post-thaw survival rates and clinical pregnancy rates between embryos frozen at day 2 of ferrtilization and those frozen at day 3 of ferrtilization(79. 1% vs 82.9% and 25.5% vs 31.2%, respectively) (P>0.05). The clinical pregnancy rates of the transfer cycles only with fully intact embryos and with mixed embryos were significantly higher than that only with partially damaged embryos(36.7% vs 24.1% and 29.2% vs 24.1%, respectively)(P<0.05). The post-thaw survival rate and post-thaw high-quality embryo rate were significantly higher in those processed with modified cryopreservation technique than in those processed with original cryopreservation technique (82.0% vs 66.3% and 50.0% vs 27.5%, respectively)(P<0.05). Conclusion Pre-freeze embryo quality, post-thaw embryo survival rate and post-thaw embryo quality have a positive correlation to subsequent clinical pregnancy rate. Favorable cryopreservation technique may ensure the success of post-thaw embryo recovery and transfer.

OBJECTIVETo investigate the methylation Oct4 in orientation induced differentiation in bone marrow mesenchymal stem cells

METHODSMice BMSCs were isolated and purified from bone marrow by adherent culture,and then identified by morphology and immunocytochemistry.Mouse osteoblastic cells were cultured by bone fragments inoculation,and then identified by alkaline phosphatase(AKP)staining and alizarin red staining.BMSCs were induced to differentiate into osteoblasts in vitro. Indirect immunofluorescence staining and reverse transcription polymerase chain reaction(RT PCR)were used to detect the expressions of Oct4 in BMSCs before and after induction.The methylation status of Oct4 gene in mouse BMSCs was explored by a methylation specific PCR before and after induction

RESULTSThe isolated mice BMSCs massively proliferated in vitro and formed cell colones with uniform morphology.Positive expressions of CD29,cKit,and CD44 and negative expression of CD34 were found in the isolated cells.After 10 days[DK]'[DK] induction,both AKP and the alizarin red were positive in cells and osteoblastic cells isolated from mice skull bones.The indirect immunoinfluorescence staining and RT-PCR also showed that the Oct4 expression in the directed differentiation of mouse BMSCs was down-regulated.The CpG island of Otc4 gene promoter in mouse BMSCs became methylated during the induced differentiation.

CONCLUSIONSMice BMSCs and osteoblasts were successfully cultured in vitro in this studyOct4 may be involved in the maintenance of adult stem cell pluripotency.The down regulated expression of Oct4 gene in mouse BMSCs during the directed differentiation may contribute to the methylation of CpG island in Otc4 gene promoter.

Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cells, Cultured ; CpG Islands ; DNA Methylation ; Mesenchymal Stromal Cells ; cytology ; Mice ; Octamer Transcription Factor-3 ; metabolism ; Osteoblasts ; cytology ; Promoter Regions, Genetic

Collagen is the most abundant protein in human body and a periodic helix, i. e. , triple helix, fibrous protein, which provides the scaffold structures for the cell adhesion and macromolecule aggregation, etc. With the development of gene engineering and biomaterial technologies, and the incessant studies on the technique to obtain the proteins with special functions, the collagen protein has been one of the third generation biomaterials that attract more attention than others. In this paper, we reviewed the recent structure-based design and biosynthesis of collagen.
Animals ; Biotechnology ; methods ; Collagen ; biosynthesis ; chemistry ; genetics ; Humans ; Models, Biological ; Models, Molecular ; Protein Conformation ; Protein Stability ; Protein Structure, Secondary ; Temperature

OBJECTIVETo in vestigate the chemical constituents of Sarcandra glabra and obtain a more comprehensive understanding on its effective components.

METHODThe constituents were isolated by various column chromatographic method and their structures were elucidated by physico-chemical properties and spectroscopic analysis.

RESULTFive flavonoid glycosides were isolated and identified as kaempferol-3-O-beta-D-glucuronide (1), quercetin-3-O-alpha-D-glucuronide (2), quercetin-3-O-beta-D-glucuronopyranoside methyl ester (3), 5, 7, 4'-trihydroxy-8-C-beta-D-glucopyranosyl flavanone (4), neoastilbin (5), 5-O-caffeoylquinic acid methyl ester (6), 3, 4-dihydroxybenzoic acid (7), isofraxidin (8).

CONCLUSIONCompounds 1-6 were isolated from the genus Sarcandra for the first time. The glucuroide compounds compounds 1-3, were first isolated from the genus Sarcandra.

Caffeic Acids ; chemistry ; Coumarins ; chemistry ; Drugs, Chinese Herbal ; chemistry ; Flavonoids ; chemistry ; Glucuronides ; chemistry ; Glycosides ; chemistry ; Magnetic Resonance Spectroscopy ; Magnoliopsida ; chemistry ; Spectrometry, Mass, Electrospray Ionization

OBJECTIVETo investigate the role of tissue factor (TF) expression in the invasive and metastatic ability of colorectal carcinoma and explore the influence of TF on the invasive ability of HT-29 cells.

METHODSTF expression of specimens from 85 colorectal carcinomas and 6 colorectal adenomas was observed by immunohistochemistry. The role of TF expression in prognosis and tumor invasion and metastasis was analyzed. The plasmids pcDNA3.1/Zeo bearing either sense or antisense-TFcDNA were transfected into HT-29 cells by the way of Lipofectamine 2000. TF proteins in transfected and untransfected HT-29cells were detected by Western blot. In vitro Matrigel invasion assays were performed to show the invasive ability of those cells.

RESULTSTF expression was positive in 40 (47.1%) of 85 colorectal carcinoma specimens, but negative in normal mucosa and adenoma specimens. TF expression showed significant correlation with tumor invasive depth (r = 0.895, P < 0.01). TF expression showed significant correlation with synchronous and metachronous hepatic metastasis (r = 0.974, P < 0.01 and r = 0.963, P < 0.01 respectively). TF expression was a significant risk factor for hepatic metastasis (P < 0.01) and prognosis (P < 0.01). TF expression in HT-29 cells with sense/antisense-TFcDNA transfection was more/less than that of the cells without transfection. The invasive ability of HT-29 cells with sense-TFcDNA transfection was increased in vitro compared with the untransfected cells, but HT-29 cells with antisense-TFcDNA transfection got the contrary change.

CONCLUSIONSTF may take part in the invasive and metastatic process of primary colorectal carcinoma, and TF expression may be an indicator of hepatic metastasis and prognosis for colorectal carcinoma patients. TF expression may increase the invasive ability of HT-29 cell in vitro.

Blotting, Western ; Cell Movement ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; HT29 Cells ; Humans ; Immunohistochemistry ; Logistic Models ; Multivariate Analysis ; Thromboplastin ; analysis ; genetics

OBJECTIVETo investigate whether insertion of TC motif into hepatitis B virus (HBV) core protein c/e1 site affects the expression of S and e antigen.

METHODSDifferent oligonucleotides encoding TC motif were inserted into the c/e1 site of the core gene of a 1.3 copy wild-type HBV genome vector. HepG2 cells were divided into several groups of cells to transiently transfect with the wild-type and mutant HBV vectors, respectively. In each group, the expression level of core protein inside cells was detected by western blotting, and the levels of S and e antigen in culture medium were analyzed by ELISA assay.

RESULTSWestern blotting showed that these TC-tagged core proteins were expressed at similar level of wild-type one. ELISA assay indicated that the level of S and e antigen in culture medium of different groups were not significantly different.

CONCLUSIONInsertion of TC motif into HBV core protein c/e1 site does not interference with the expression of viral protein encoded by HBV genome.

Amino Acid Sequence ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Genetic Vectors ; genetics ; metabolism ; Hep G2 Cells ; Hepatitis B Core Antigens ; genetics ; metabolism ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; Humans ; Mutagenesis, Insertional ; Recombinant Proteins ; genetics ; metabolism ; Transfection ; Viral Core Proteins ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism ; Virus Replication

OBJECTIVEFMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is constitutively activated in (70-90)% pediatric patients with acute myeloid leukemia (AML) and appears to confer an adverse prognosis. Although several FLT3-selective small molecule inhibitors and antibodies were developed with varied degrees of success, to address the specificity and resistance, new approaches for specifically targeted FLT3 are needed and RNA interference is a promising choice. The aim of the present study was to investigate the efficacy of suppression of FLT3 induced by small hairpin interfering RNA (shRNA) on myeloproliferation and apoptosis in an acute monocytic leukemia (AMOL) cell line THP-1.

METHODSFLT3-targeted small hairpin interfering RNA (FLT3-shRNA) was designed and synthesized by transcription system in vitro was transfected into THP-1 cells. Firstly FLT3 mRNA level was detected by semi-quantitative RT-PCR and FLT3 protein level was detected by flow cytometry (FCM) to verify the efficacy on FLT3-shRNA interference at 48 h after transfection. Cell growth viability was measured at 24 h, 48 h and 72 h after treatment with CCK-8. The distribution of cell cycle was assayed by FCM, and apoptosis was analyzed by DNA Ladder and Annexin V-FITC Staining at 48 h.

RESULTSFLT3 targeted shRNAs was synthesized successfully and the concentration of 15 nmol/L for 48 h could obtain desirable downregulation of FLT3 expression, the inhibitory percentages of FLT3 mRNA and protein were (72.95 +/- 2.07)% and (65.39 +/- 5.57)%, respectively. The suppression of FLT3 induced by FLT3-shRNA resulted in marked inhibition of cell growth and the inhibitory percentages were (36.66 +/- 3.67)% at 48 h, (35.56 +/- 0.73)% at 72 h. FLT3-shRNA induced the inhibition of cell cycle from G(0)/G(1) phase to S phase, the percentage of sub-G(0)/G(1) phase (65.71 +/- 4.47)% was higher than those in the PBS-control group (52.23 +/- 2.98)%, NC-shRNA control group (51.81 +/- 1.44)%, P < 0.01; the percentage of S phase (25.11 +/- 2.70)% was lower than those in the PBS-control group (34.41 +/- 4.07)% and NC-shRNA control group (32.50 +/- 1.46)%, P < 0.05. Furthermore treatment with FLT3-shRNA for 48 h resulted in clear apoptosis ladder, the percentage of early apoptosis detected by Annexin V-FITC was (18.59 +/- 2.07)% which was significantly higher than that in the PBS-control group (4.00 +/- 0.50)% and the NC-shRNA control group (6.06 +/- 0.70)%, P < 0.001.

CONCLUSIONThe suppression of FLT3 induced by the shRNA can effectively inhibit cell proliferation, and apoptosis induction on THP-1 cells, which indicates that this approach may bear the therapeutic potential on childhood AMOL.

Apoptosis ; drug effects ; genetics ; Cell Proliferation ; drug effects ; Child ; Humans ; Leukemia, Monocytic, Acute ; enzymology ; pathology ; Protein-Tyrosine Kinases ; metabolism ; RNA Interference ; physiology ; RNA, Small Interfering ; pharmacology ; Receptor Protein-Tyrosine Kinases ; metabolism ; fms-Like Tyrosine Kinase 3 ; metabolism

OBJECTIVETo investigate the association of childhood hemophagocytic syndrome (HPS) with human parvovirus B19 (HPVB19) infection, and to analyze the clinical features of this disease.

METHODSELISA and quantitative real-time PCR were used to detect HPVB19-IgM, HPVB19-IgG and HPVB19-DNA in 65 children with HPS (HPS group) and 65 healthy children (control group). The HPS group was divided into HPVB19-infected (n=14) and non-infected (n=51) groups according to the detection results of HPVB19-DNA. The clinical data of two groups were compared.

RESULTSThe positive rate of HPVB19-IgM in the HPS group (26%, 17/65) was significantly higher than that in the control group (9%, 6/65) (P=0.011), and there was no significant difference in the positive rate of HPVB19-IgG between the HPS (38%, 25/65) and control groups (29%, 19/65) (P=0.266). The infection rate of HPVB19 in the HPS group (22%, 14/65) was significantly higher than that in the control group (3%, 2/65) (P=0.001). Compared with the non-infected group, the HPVB19-infected group had significantly lower platelet count and hemoglobin level on admission, significantly more severe liver function damage, a significantly earlier onset time, and a significantly longer course of disease (P<0.05).

CONCLUSIONSThe pathogenesis of HPS may be associated with HPVBl9 infection. HPVBl9-infected children with HPS have more acute onset, more severe clinical manifestations, and a longer disease duration.

Adolescent ; Antibodies, Viral ; analysis ; Child ; Child, Preschool ; DNA, Viral ; analysis ; Female ; Humans ; Infant ; Lymphohistiocytosis, Hemophagocytic ; etiology ; Male ; Parvoviridae Infections ; complications ; Parvovirus B19, Human

This study was aimed to investigate the incidence of thrombosis in patients with primary thrombocytosis (PT) and its correlation with function changes of platelets, and to explore the effect of thromboxane A2 (TXA2) inhibitor-ozagrel sodium on platelet activity and its efficacy for prevention and treatment of thrombosis. The CD62P and PAC-1 levels on platelet surface were detected by flow cytometry; the levels of TXB2 (metabolic product of TXA2) and 6-keto-PGFIalpha (metabolic product of prostacyclin) were detected by FLISA. The function change of platelets and its correlation with thrombosis were observed and compared in PT patients with and without thrombosis. The results indicated that the TXB2, PAC-1 and CD62P level, and TXB2/6-keto-PGF1alpha ratio in PT patients with thrombosis were higher than those in PT patients without thrombosis before treatment with ozagrel sodium (p<0.01). After treatment with ozagrel sodium, the function indexes of platelets such as CD62P, PAC-1, TXB2 and TXB2/6-keto-PGF1alpha except 6-keto-PGF1alpha in PT patients with and without thrombosis decreased obviously (p<0.01), but there was no significant difference in TXB2, 6-keto-PGF1alpha and TXB2/6-keto-PGF1alpha levels between PT patients with and without thrombosis except CD62P and PAC-1. It is concluded that the multi-index of platelets in PT patients with thrombosis are higher than that in PT patients without thrombosis, the activation of platelet function is a high risk factor for thrombosis of PT patients. The ozagrel sodium can obviously reduce the platelet activation, decrease the production of TXA2 and ameliorate the TXB2/6-keto-PGF1alpha ratio. The ozagrel sodium not only possesses therapeutic effect, but also preventive efficacy for thrombosis.
Adolescent ; Adult ; Aged ; Female ; Fibrinolytic Agents ; therapeutic use ; Humans ; Male ; Methacrylates ; therapeutic use ; Middle Aged ; Thrombocythemia, Essential ; complications ; drug therapy ; Thrombosis ; complications ; drug therapy ; Young Adult