Experiment on inducing human dental pulp stem cells into neural-like cells / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To explore the multi-differentiated capability of human dental pulp stem cells (hDPSCs) obtained by cell-clone culture approach and to determine the appropriate induced medium.</p><p><b>METHODS</b>The cloned isolation and expansion of hDPSCs were preinduced for 24 h, and were subsequently replaced with neural-inductive medium containing certain concentration of dimethylsulfoxide (DMSO), butylated hydroxyanisode (BHA), forskolin, P-mercaptoethanol (p-ME) and hydrocortisone for 4 days. Then induced cells were analyzed by morphological observation, immnocytochemical staining for non-specific esterase (NSE) and glial fibrillary acidic protein (GFAP) expression, RT-PCR for GFAP mRNA. Meanwhile, the uninduced hDPSCs were used as negative control.</p><p><b>RESULTS</b>The morphology of induced cells changed at the initial 12 h, and displayed a typical neuron-like cells at 24 h. There was a gradual increase in the number of these neuronal differentiated cells with continuous induction. Furthermore, immnocytochemical staining showed that the induced cell expressed NSE and GFAP, two marked enzymes of neuron cell. The GFAP mRNA was also detected in induced cells by RT-PCR assay. In contrast, the uninduced cells maintained its original appearance and had no expression on them.</p><p><b>CONCLUSION</b>hDPSCs may possess potential of multiple-differentiation and may differentiate into neuron-like cells on certain inductive condition.</p>

Sequencing and bioinformatical analysis of virulent strain-specific DNA fragments from Streptococcus mutans / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To search the DNA sequences specific to virulent strain of Streptococcus mutans in the public database and explore new genes or new functions of already known genes from Streptococcus mutans of serotype c and suppose their functions.</p><p><b>METHODS</b>Thirty-one DNA fragments unique to virulent strain of Streptococcus mutans were sequenced. The sequences of these presumptive virulence DNA fragments were subjected to search through software BLASTn and BLASTx in public database, and their putative biological functions were analyzed. RESULTS Two clones were picked repeatedly. The size of the remaining DNA fragments ranged from 113 bp to 776 bp. The average G+C content was 38.59%, similar to that of the gene-coding sequences in Streptococcus mutans strain UA159 whose genome sequences were just complete. Of the twenty-nine DNA fragments, five potentially represented new DNA fragments in Streptococcus mutans, thus registered and obtained their gene's accession number in GenBank. The remaining DNA fragments showed high homology to known genes of Streptococcus mutans strain UA159. Their predicted functions of these fragments were associated to bacterial signal transduction, transcriptional regulation, stress-damage repair, biochemical metabolism, outer membrane protein synthesis, adhesion on tooth surface and hypothetical proteins.</p><p><b>CONCLUSION</b>The gene analysis, identification and functional forecasting were carried out through bioinformatics associated software and database to find out new genes and new functions of known genes, and to supply the groundwork for researches in gene functions.</p>

Construction of a virulence-related gene library of Streptococcus mutans by suppression subtractive hybridization / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To construct a suppression subtractive library of virulence-related genes from c serotype Streptococcus mutans (S. mutans), and lay foundations for screening the virulent genes.</p><p><b>METHODS</b>After being isolated from virulent and avirulent strain of S. mutans respectively, the intact and high-pure genomic DNA was digested with three appropriate four-base-cutting restriction endonueleases to produce fragments of optimal length. The digested DNA of the virulent strain ligated with adaptor was used as tester DNA, and that of the avirulent strain as driver DNA. Then the suppression subtractive hybridization was carried out, and the efficiency of ligation and subtraction detected respectively. The subtracted fragments were inserted into vector pCR2. 1 using T/A cloning kit, and transformed into E. coli TOP10F' competent cells. Those white colonies were selected to construct the suppression subtractive library.</p><p><b>RESULTS</b>Alu I chosen from three restriction endonucleases was verified to be suitable for preparing restriction fragments from S. mutans genomic DNA. Through electrophoresis of Alu I -digested DNA, a smear ranged from 0.1 to 2.0 kb was observed. The ligation efficiency of tester DNA with adaptor was at least higher than 25 percent. The subtraction efficiency of suppression subtractive hybridization confirmed the success in enrichment of differential genes between virulent and avirulent strain of S. mutans. In the subtracted group, the appearance time of the 23S rRNA gene both in tester and driver DNA was later than that in the unsubtracted group by six cycles. It suggested that suppression subtractive hybridization happened indeed. After the subtracted fragments were cloned, 96 colonies were picked up for constructing the suppression subtractive library of virulence-related genes of S. mutans.</p><p><b>CONCLUSION</b>Suppression subtractive hybridization allows rapid and easy construction of virulence-related gene library of S. mutans.</p>

Tissue engineering of dentin-pulp complex-like structures by human dental mesenchymal cells / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To establish three-dimensional culture model of human dental mesenchymal cells and bioengineer in vivo with ceramic bovine bone (CBB) and Collagraft as scaffolds.</p><p><b>METHODS</b>Human dental mesenchymal cells induced upon stimulation of bFGF and IGF-1 or TGF-beta(1) were implanted onto CBB and Collagraft containing the same kinds of growth factors respectively. Then cell/scaffold constructs were transplanted into nude mice to establish in vivo culture model of dental mesenchymal cells. Control groups were set up at the same time. After 4 weeks or 10 weeks, the implants were taken out for histological and immunohistochemical analysis.</p><p><b>RESULTS</b>Within 10-week implant tissues, typical dentin-pulp complex-like structures were generated in scaffolds containing growth factors. Human dentin sialoprotein (DSP) was expressed in the newly formed dentin. This phenomenon wasn't observed in control groups and 4-week implants.</p><p><b>CONCLUSIONS</b>Dentin-pulp complex-like structures could be bioengineered successfully with human dental mesenchymal cells and CBB or Collagrafts containing growth factors in nude mice.</p>

The positive effect of transforming growth factor beta on ectomesenchymal stem cells of embryonic facial processes differentiating to smooth muscle cells / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of transforming growth factor beta (TGF-beta) on ectomesenchymal stem cells differentiating to smooth muscle cells.</p><p><b>METHODS</b>60 pmol/L TGF-beta was added to the ectomesenchymal stem cells of embryonic facial processes. Immunohistochemistry assay and image analysis were used to value the expression extent of a smooth muscle actin (alpha-SMA) and quantitative RT-PCR was used to value the quantity of alpha-SMA.</p><p><b>RESULTS</b>2 days later, about 95% cells in TGF-beta group and 65% cells in control group without differentiation inhibitor expressed alpha-SMA. Expression of alpha-SMA in TGF-beta group was stronger than that of control group after one and two days. Quantitative RT-PCR showed the quantity of alpha-SMA mRNA in treated group cells was more than that of in control group.</p><p><b>CONCLUSION</b>Quantity of alpha-SMA in TGF-beta group is more than that of spontaneous differentiation group. TGF-beta has positive effect on ectomesenchymal stem cells differentiating to smooth muscle cells.</p>