Effects of osteogenic growth peptide to the proliferation and differentiation of bone marrow stromal cells / 中国骨伤

<p><b>OBJECTIVE</b>To investigate the effects of osteogenic growth peptide (OGP) to the proliferation and differentiation of cultured bone marrow stromal cells (BMSCs) of rats.</p><p><b>METHODS</b>The SD rats (age 6 weeks) were sacrified, and the bone marrow stromal cells as the adherent stromal cell population were separated from the bone marrow culcure. The proliferation curves of bone marrow stromal cells which were conditioned cultured with four kind of different concentrations of osteogenic growth peptide were measured with the MTT method, and the osteogenesis markers including alkaline phosphatase and calcic deposition detected by histochemical staining.</p><p><b>RESULTS</b>Osteogenic growth peptide at the concentration of 10(-10), 10(-11) mol/L promoted the proliferation of bone marrow stromal cells, while at the concentrations of 10(-8), 10(-9) mol/L suppressed the proliferation of bone marrow stromal cells. However,osteogenic growth peptide at the concentration of 10(-9), 10(-8) mol/L advanced the ratio of positive cells in alkaline phosphatase histochemical staining.</p><p><b>CONCLUSION</b>Osteogenic growth peptide shows distinct effects on the proliferation and differentiation of bone marrow stromal cells depending on its concentration. Osteogenic growth peptide at the concentration of 10(-8), 10(-9) mol/L can promote bone marrow stromal cell differentiation to the osteogenic in vitro.</p>

Reconstruction of embryo using an improved nuclear transfer method / 生物工程学报

Previous methods used for nuclear transplantation were further investigated to develop a method that was both easy to carryout and did not require any special apparatus, such as Piezoimpact or Spindle-View. Following the puncture of zona pellucida with two holes by injection pipette that contained donor nuclei or cells, the injection pipette was pulled back to the perivitelline space while the negative pressure was increased in the holding pipette until the polar body and karyoplasm were wiped off completely. Then a reconstructed embryo was completed by the direct injection of the donor nucleus or cell without pulling out the injection pipette. 200 oocytes were manipulated using this method and it cost about 40 seconds with nucleus injection and about 30 seconds with cell injection to complete a reconstructed embryo. The success rates were 62.6% and 86. 0%, respectively, and enucleation rate was about 73.3% validated by Hoechst 33342. Using this method, the nucleus was completely eliminated and another was injected using the microscope and micromanipulator. Moreover, the efficiency of nuclear transplantation and survival rate of reconstructed embryos were greatly improved. Furthermore, it is very easy to manipulate and popularize in practice.

Inhibitory role of transcription factor COUP-TFII in expression of hTERT in HeLa cells / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To clone and identify the proteins involved in regulating the transcription of hTERT and study the role of genes in both hTERT transcription and telomerase activity.</p><p><b>METHODS</b>The full cDNA of COUP-TFII was cloned from HeLa cDNA library by hTERT promoter-based yeast one-hybrid assay and then in-frame inserted into His-tag fusion expression vector pEK318. The His-tag COUP-TFII fusion proteins were purified by Ni-NTA chromatography. The interaction of COUP-TFII with hTERT promoter in vitro was identified by electrophoretic mobility shift assay and Footprint. The role of COUP-TFII in both hTERT transcription and telomerase activity were probed through Luciferase reporter assay, Northern blot, and TRAP-PCR ELISA.</p><p><b>RESULTS</b>COUP-TFII could firmly bind to the downstream E-box and the other two binding sites in hTERT promoter. Luciferase reporter assay indicated COUP-TFII could suppress hTERT promoter activity and stable introduction of COUP-TFII into HeLa cells also decreased both endogenous hTERT transcription and telomerase activity.</p><p><b>CONCLUSION</b>The human COUP-TFII can firmly bind to hTERT promoter, and inhibit telomerase activity through decreasing hTERT transcription. It will greatly facilitate understanding of telomerase regulation in normal and cancer cells.</p>