Objective:To construct a suppression subtractive library of suppression-related genes from c serotype Streptococcus mutans (S.mutans). Methods:After being isolated from virulent and avirulent strain of S. mutans respectively, the intact and high-pure genomic DNAs were digested with restriction enzyme AluⅠ. The digested DNA of the avirulent strain ligated with an adaptor was used as tester DNA, and that of the virulent strain as driver DNA. Then the suppression subtractive hybridization(SSH) was carried out, the efficiency of ligation and subtraction were detected respectively. The subtracted fragments were inserted into vector pCR2.1 using T/A cloning kit and transformed into E. coli TOP10F' competent cells. The white colonies were selected to construct the suppression subtractive library. Results: Through electrophoresis of AluⅠ-digested DNAs, 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 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 in both tester and driver DNA was later than that in the unsubtracted group by twelve cycles. It suggested that suppression subtractive hybridization happened indeed. Then the subtracted fragments were cloned and the suppression-related gene library between virulent and avirulent strain of S. mutans was constructed. Conclusions:The suppression subtracted library of suppression-related genes has been constructed.

Objective:To explore avirulent strain-specific new genes or new functions of already known genes from Streptococcus mutans of serotype c.Methods:Twenty-six DNA fragments unique to avirulent strain of Streptococcus mutans were sequenced.The sequences of these presumptive avirulence DNA fragments were subjected to homology search through BLASTn and BLASTx in public database,and their putative biological functions were analyzed.Results:The size of the DNA fragments ranged from 113 bp to 776 bp.The average G+C content was 38.27%,similar to that of the gene-codingsequences in Streptococcus mutans strain UA159 whose genome sequences were just completed.Seven clones were picked repeatedly.Of the nineteen DNA fragments,eight potentially represented new DNA fragments were registered in GenBank.The remaining DNA fragments showed high homology to known genes of Streptococcus mutans strain UA159.Conclusions: The gene analysis and identification supply the groundwork for further study of gene functions of the avirulent strain of Streptococcus mutans serotype c.

Objective: To study the mechanism of differentiation of rat ectomesenchymal cells to odontoblasts. Methods: Ectomesenchymal cells were cultured in three-dimension culture model using collagen gel as frame, and the change of phenotype of ectomesenchymal cells were observed and detected by phase-contrast microscopy and immunohistochemistry after the cells had been treated by 10 ng/ml of bFGF or/and 100 ng/ml IGF-1. Results: 4 days after treatment by bFGF and IGF-1, the cells appeared to be odontoblast-like cells aligned parallelly and polarized with long cytoplasmic processes attached to one end of the cell body.The cells were positive for DSP expression. However, the cells were DSP negative and aligned disorderly in other groups. Conclusion: Ectomesenchymal cells can be induced to differentiate to odontoblast-like cells in three-dimension culture model with the treatment by bFGF and IGF-1.

Objective:To express human dentin sialoprotein (hDSP) gene in COS-7 cells. Methods:hDSP gene was subcloned into mammalian expression vector pcDNA3. The recombined plasmids were transfected into COS-7 cells using lipofectamune PLUS TM kit for transient expression. Western blot analysis and immunohistochemical staining were used to examine the gene products. Results:The constructed vectors were confirmed by digestion with restriction enzyme. An immuno-reaction positive band with relative molecular mass of 60 000 was found by Western blot analysis in culture supernatant and cytoplasms of COS-7 cells. Immunohistochemical staining showed strong positive particles in the cytoplasms. Conclution:hDSP gene can be expressed in COS-7 cells.

Objective:To analyse the nucleotide polymorphism of dentin sialoprotein(DSP) coden region in Chinese people. Methods:The DSP segments were amplified by PCR;single-stranded conformation polymorphism(SSCP) and DNA sequencing were employed to detect the nucleotide polymorphisms in DSP coden region. Results:Three single nucleotide polymorphisms(SNP) were found in DSP coden region,two were same sense SNPs resulting in no change of amino acid product,and one was missense SNP resulting in change of asparagine and aspartic acid. Conclusion:There are some SNPs existing in DSP coden region in Chinese people.

Objective To understand the intensity and characteristics of acute toxicity of esculentoside A on mice and measure relevant parameters and observe its diuresis effect on rat. Methods After intraperitoneal injection of different concentrations of esculentoside A to mice, toxic reactions were observed. Rats with water load were intraperitoneally injected with different doses of esculentoside A. Total urine volume in six consecutive hours after the injection was determined. Results The LD50 of esculentoside A calculated by Bliss method was 26. 19 mg · kg-1 , and the 95% confidence interval was 23. 11-29. 85 mg·kg-1 . The mortality and acute toxicity of esculentoside A appeared to be dose-dependent while the blank control group had no abnormal reaction. The urine volume was significantly different between high dose group and the negative control group. No significant difference in urine volume was found between middle and the negative control group, and between low dose group and the negative control group. Conclusion Esculentoside A is poisonous to mice when single dose was intraperitoneally injected, and high dose of esculentoside A has diuresis effect on rat.

Present study investigated the effect of endotoxin from Bacteroides melaninogenicus ATCC 25845 on release of colony-stimulating factor (CSF)in mice. The bone marrow cells were cultured in semisolid agar medium,the number of colonies was as a level index of CSF. The results showed that as much as 0.1?g endotoxin could induce the release of CSF,moreover, The level of CSF increased with dose of endotoxin untill 50 ?g. The colony-stimulatin factor level of B. melaninogenicus endotoxin was 66.6?8.5(CFU-C). This endotoxin showed significant effect on bone marrow cells of mice.

We discussed the function of four pairs of genes in the toxin-antitoxin system of Mycobacterium tuberculosis,providing theoretical foundation and scientific basis for studying the transmission mechanism of Mycobacterium tuberculosis.Four pairs of genes which belong to VapBC family,including four VapC genes (Rv1720c,Rv2103c,Rv2494,Rv3408) and four VapB genes (Rv1721c,Rv2104c,Rv2493,Rv3407) were chosen.We constructed a serial of arabinose-induced hybrid plasmid system in Escherichia coli and a serial of acetamide-induced hybrid plasmid system in Mycobacterium smegmatis respectively,in order to observe the potential inhibition effect of VapC and the release inhibition of homologous VapB.Results showed that only one toxin gene(Rv2103c) showed the function of bacteriostasis in both E.coli and M.smegmatis and the homologous antitoxin gene(Rv2104c) could release the inhibition of growth.We built the inducible systems of VapBC family in both E.coli and M.smegmatis respectively and found only a pair of toxin and antitoxin genes(Rv2103c,Rv2104c) had the function of inhibition and release for the growth of bacteria.And two pairs of toxin genes(Rv1720c,Rv2494) did not have the function of inhibition for the growth of both E.coli and M.smegmatis.Whereas,another toxin gene VapC47(Rv3408) also did not have the bacteriostastic activity,only this result was not consistent with the existing literature.We speculated that the reason for this kind of difference may be the different inducible systems we used.Cause the other three results were consistent with all existing literature and the doubtful result also appeared in other reports,so our protocol could be confirmed as reliable,and we would use it to build inducible systems and make further functional identification of certain toxin and antitoxin genes that we are interested in.

This study was aimed to analyze changes of content and quantity of essential oil of processing drugs of Rhizome Atractylodes and to achieve the impact of pyrolysis characteristics for using excipients, in order to offer evi-dences for further research and its processing technology. Steam distillation was used to extract essential oil in the Rhizome Atractylodes. Infrared spectroscopy and gas chromatography were used in the qualitative and quantitative analysis on constituents of essential oil of processing products of Rhizome Atractylodes. Thermogravimetric analysis was used in the comparison of pyrolysis characteristics between Rhizome Atractylodes and its excipients. The results showed that the content of essential oil was declined after processing. However, after being processed, the content of atractylodin was increased at different degrees compared to crude product. The change of atractylodin showed differ-ent tendency in different processing drugs. The atractylodin content from high to low was in the order of products stir-baked to yellowish, products roasted by bran, products prepared with rice water, crude drug. At the temperature of more than 220oC, excipients had major impact for the pyrolysis characteristics of Rhizome Atractylodes. It was concluded that the essential oil declined, but atractylodin increased after Rhizome Atractylodes being processed. It also provided experimental basis for further research on processing technology, ormulation of quality standard and improvement of processing mechanism.

This study was aimed to reveal current achievements and the future trend about Chinese materia medica (CMM) processing, which pointed future research direction for scholars.“CMM processing” was used as the subject term in document retrieval to the Web of Science (WoS) and CNKI. The CitespaceII and Ucinet & Netdraw were used to build mapping knowledge domains to acceptable both English and Chinese literature data. The results showed that the mapping knowledge for“CMM processing” for co-citation, source journal, author, key words and so on were established. It was concluded that the rapid present period of Chinese literature on CMM processing was earlier for around 15 years than English literature. And the total amount of English documents was much less than Chinese documents. Combing the developing process of CMM, it revealed its current achievements and future trend, which pointed future research direction for scholars, and moreover it put forward new thinking and method to summarize or expand traditional medicine and related research fields.