Effect of Amino Acid Motifs in Integrin β3 Cytoplasmic Tail on αⅡbβ3-Mediated Cell function in 293T cell models / 中国实验血液学杂志

OBJECTIVE@#To establish 293T cell lines stably expressing Calpain-cleavage related α3 cytoplasmic tail mutants, and to explore the effect of amino acid motifs in integrin β3 cytoplasmic tail on αⅡbβ3-mediated cell function.@*METHODS@#293T cell lines stably co-expressing human wild type integrin αⅡb and full length β3 or mutant β3, including β3-ΔNITY (β3 cytoplasmic tail NITY motif deleted), β3-Δ754 (β3 cytoplasmic tail TNITYRGT motif deleted) and β3-Δ759 (β3 cytoplasmic tail RGT motif deleted) were established. Spreading and adhesion of these stable cell lines on immobilized fibrinogen were tested.@*RESULTS@#293T-αⅡbβ3ΔNITY, 293T-αⅡbβ3Δ754, 293T-αⅡbβ3Δ759 and 293T-αⅡbβ3 cell lines were successfully established. Compared with the 293T cells, 293T-αⅡbβ3 cells which expressed full β3, possessed well adhesion and spread ability on immobilized fibrinogen, suggesting it can be as a surrogate for platelet. Compared with 293T-αⅡbβ3 cells, the 293T-αⅡbβ3ΔNITY cells showed a partial impairment of adhesion and spreadability on immobilized fibrinogen. while the 293T-αⅡbβ3Δ754 cells and 293T-αⅡbβ3Δ759 cells failed to adhere or spread on immobilized fibrinogen.@*CONCLUSION@#To the cell spreading function mediated by integrin β3, RGT motif is vital, while NITY can be dispensable. These established 293T cell lines stably expressing different β3 mutants provide a solid basis for a further analysis of mass spectrometry.

Establishment of an internal control for directed differentiation using pluripotent stem cell lines derived from heterozygotic twins / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To reprogram amniotic fluid cells into pluripotent stem cells in order to create an optimal internal control model for directed cell differentiation.</p><p><b>METHODS</b>Human amniotic fluid-derived cells (hAFDCs) from heterozygotic twin fetuses were induced by retroviral vectors encoding Oct4, Sox2, c-Myc and Klf4. In vivo pluripotency, differentiation capacity and karyotype of hAFDCs induced pluripotent stem cells (hAFDCs-iPSCs) were determined.</p><p><b>RESULTS</b>hAFDC-iPSCs derived from heterozygotic twins have maintained self renewal, with expression of high pluripotency marker gene detected at both mRNA and protein levels. The cells have maintained their differentiation capacity both in vitro and vivo, and showed normal karyotypes after long-term culturing in vitro.</p><p><b>CONCLUSION</b>hAFDCs-iPSCs derived from heterozygotic twins have good consistency in terms of genetic background, and can provide a good internal control for directed differentiation of iPSCs, and may be used an ideal source for autologous cell replacement therapy in the later life of the fetus.</p>

Clinical analysis of 19 cases of adult primary mediastinal liposarcoma / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the pathoclinical features of adult primary mediastinal liposarcoma and their correlation with the prognosis.</p><p><b>METHODS</b>The clinical data of 19 patients with adult primary mediastinal liposarcoma who were treated in our hospital between 1970 and 2011 were retrospectively analyzed. Eighteen patients underwent open thoracic tumor excisions for at least one time, and the remaining one patient only received biopsy surgery. Histopathological results after surgery revealed that 6 well-differentiated type tumors, 6 myxoid type tumors, 3 pleomorphic type tumors, 2 mixed type tumors, and 2 un-classified tumors.</p><p><b>RESULTS</b>Among 6 patients with well-differentiated type tumors, 1 died after having been survived for 10 years; 5 were still alive, with a mean duration of 126.2 months,the 5-year survival rate was 100%. Among 6 patients with myxoid type tumors, 5 patients had follow-up data, with a mean survival of 26.2 months. Among 3 patients with pleomorphic type tumors, only one patient had follow-up data: the patient finally died, with a survival of 34 months. Of 2 patients with mixed type tumors, only one patient had follow-up data: the patient survived 8 months and died. Of 2 patients with un-classified type tumors, one had follow-up data: the patient lived for 24 months and died.</p><p><b>CONCLUSIONS</b>Mediastinal liposarcoma is a rare disease. Surgery is the primary therapeutic modality. Different pathological subtypes have different epidemiological features, biological behaviors, and malignant potentials. Pathological subtype is an important prognosis factor. Patients with well-differentiated tumors have much better prognosis than those with other subtypes.</p>

Cloning of hsa-miR-148a and construction of its retroviral expression vector / 南方医科大学学报

<p><b>OBJECTIVE</b>To clone hsa-miR-148a and construct its retroviral expression vector.</p><p><b>METHODS</b>The pre-miR-148a amplified by PCR was inserted to pMSCV to construct the recombinant retroviral expression plasmid pMSCV-miR-148a, which was confirmed by restriction endonuclease analysis and DNA sequencing. The retroviral expression vector pMSCV-miR-148a and PIK packaging plasmid were cotransfected into 293FT packaging cells by calcium phosphate-mediated transfection to produce the retrovirus, and the retrovirus titer was measured by infection of NIH3T3 cells.</p><p><b>RESULTS</b>Restriction enzyme digestion and DNA sequencing demonstrated that the retroviral vector pMSCV-miR-148a was constructed successfully, and the virus titer was 5x10(8) CFU/ml after infection of NIH3T3 cells.</p><p><b>CONCLUSION</b>The successful construction of the retroviral expression vector MSCV-miR-148a allows the production of high-titer retrovirus to facilitate further study of the molecular functions of miR-148a.</p>

Sperm acrosin activity helps predict IVF-ET outcome / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the effect of sperm acrosin activity on the IVF-ET outcome.</p><p><b>METHODS</b>We analyzed sperm parameters, morphology and acrosin activity for 909 infertile husbands by computer-assisted self-assessment (CASA), modified Papanicolaou staining and N-alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA), respectively, and detected the rates of fertilization, cleavage, quality embryos, embryo cryopreservation, implantation, clinical pregnancy and abortion. The wives were identified as normal or with mere oviduct problems.</p><p><b>RESULTS</b>The rate of normal sperm morphology and sperm motility, vitality, rapid progressive velocity and concentration were significantly lower in the abnormal acrosin activity group than in the normal one (P < 0.01). Significant positive correlations were observed between acrosin activity and the above-mentioned semen parameters (P < 0.01). There were no significant differences in the number of retrieved eggs, the rates of cleavage, quality embryos, embryo cryopreservation, non-embryo transfer cycles and miscarriages, and the number of transferred embryos between the two groups (P > 0.05). The fertilization rate, the percentage of transfer cycles with only 1 embryo and the rate of implantation and clinical pregnancy were notably higher in the normal acrosin activity group than in the abnormal one (P < 0.01).</p><p><b>CONCLUSION</b>Sperm acrosin activity is closely related with semen parameters, and it helps to predict the sperm fertilizing capacity and IVF-ET outcome.</p>