High temperature reduces the proliferation of and occludin expression in rat Sertoli cells in vitro / 中华男科学杂志

<p><b>OBJECTIVE</b>To explore the action mechanisms of temperature in male infertility or subfertility by observing the effects of different temperatures on the proliferation of and occludin (OCLN) expression in rat Sertoli cells in vitro.</p><p><b>METHODS</b>We isolated Sertoli cells from the testis of male Wistar rats, and performed oil red O staining and immunohistochemistry to identify their FasL. We cultured the Sertoli cells at 34 degrees C (control group) and at 35, 36, 37, 38 and 39 degrees C (experimental groups) for 4 days. Then we measured their proliferation by CCK-8 assay, observed their morphology and structure by hematoxylin-eosin staining, and determined their OCLN expression level by Western blotting and immunofluorescence.</p><p><b>RESULTS</b>The purity of the isolated Sertoli cells was (96.20 +/- 1.95)%. CCK-8 assay indicated that the proliferation of the Sertoli cells was increased between 34 and 36 degrees C, and decreased at 36-39 degrees C. The pyknotic nuclei and fragmentation of the Sertoli cells were more obvious at > 36 degrees C. Western blot and immunofluorescence showed the highest level of OCLN expression at 36 degrees C, which, however, decreased while the temperature rose above 36 degrees C (P < 0. 01).</p><p><b>CONCLUSION</b>High temperature (> 36 degrees C) inhibited the proliferation of rat Sertoli cells in vitro, and decreased the expression of OCLN, which suggests that a higher temperature above 36 degrees C may reduce male fertility by affecting the proliferation of Sertoli cells and integrity of the tight junction among Sertoli cells or Sertoli cells and other cells.</p>

Construction of lentivirus vector containing human homeobox gene HOXB4 and its expression in human umbilical cord mesenchymal stem cells / 中国实验血液学杂志

This study was purposed to construct lentivirus vector containing human homeobox gene HOXB4 and explore changes of human umbilical cord mesenchymal stem cells (HUCMSC) after infected with HOXB4 mediated by lentivirus. PCR amplification was performed to obtain HOXB4, which was cloned in lenti-shuttle vector. Four-plasmid lentivirus packaging system was used to transfect HEK293T cells. After 48 h, lentivirus Lenti-HOXB4 was harvested and lentivirus titer was determined. Lenti-HOXB4 was used to infect HUCMSC. The infected cells were observed under inverted fluorescence microscope to determine the optimal multiplicity of infection (MOI). Meanwhile, RT-PCR, immune fluorescence staining, CCK-8 and flow cytometry (FCM) were used to determine the expression of HOXB4 and its effect on cell growth. The results indicated that lenti-HOXB4 was successfully obtained by co-transfecting the 293T cells with four plasmids. The determined virus titer was 3×10(8) TU/ml; when MOI was 20. Lenti-HOXB4 had a high transfection rate in HUCMSC, over 80%. In HUCMSC infected with lenti-HOXB4, the expression of target gene could be detected both at mRNA and protein levels. It could promote the proliferation of HUCMSC. FCM results indicated HOXB4 gene did not significantly influence the surface marker of HUCMSC. It is concluded that HOXB4 gene can promote the high proliferation of HUCMSC and does not significantly influence the expression of the surface marker of HUCMSC.

Construction and identification of a eukaryotic expression vector containing human homeobox gene / 中国实验血液学杂志

In order to investigate the special role of HOXB4 in expansion and self renewal of hematopoietic stem cells, the cDNA of HOXB4 was extracted and cloned from umbilical cord blood mononuclear cells by using RT-PCR. Then the eukaryotic expression bicistronic plasmid vector pIRES2-EGFP/HOXB4 was designed and constructed after cutting HOXB4 and pIRES2-EGFP respectively by restriction enzyme EcoRI and BamHI. The recombinant plasmid was delivered into competent cells of Escherichia coli. The successful construction of plasmid was confirmed by the identification of endonuclease cutting and sequencing. The results showed that the HOXB4 cDNA was cloned successfully from umbilical cord blood mononuclear cells and the recombinant eukaryotic expression bicistronic plasmid vector was constructed, and then introduced it into 293T cells successfully. It is concluded that a pIRES2-EGFP/HoxB1 eukaryotic expression bicistronic plasmid vector has been constructed successfully, which results provide a useful material basis for exploration of HoxB4 function in the proliferation and differentiation of hematopoietic cells.