Expression of CHMP4A and TSPYL-2 in early esophageal cancer and their significance / 临床与实验病理学杂志

Purpose To investigate the role of CHMP4A and TSPYL-2 in early pathogenesis of esophageal cancer.Methods Through comparison of the four subtractive libraries,early esophageal squamous cell carcinoma genes CHMP4A and TSPYL-2 were chosen for further study.Through RT-PCR and immunohistochemistry methods,CHMP4A and TSPYL-2's expression was detected in esophageal squamous cell carcinoma tissue,cancerous tissue and normal esophageal mucosa.Results CHMP4A and TSPYL-2 expression between esophageal squamous cell carcinoma and normal esophageal epithelium tissue had significant differences (P ＜ 0.05),and the CHMP4A gene expression in esophageal mucosa,field cancerization areas,esophageal squamous cell carcinoma tissue increased,while TSPYL-2 gene expression in esophageal mucosa,field cancerization areas,esophageal squamous cell carcinoma tissue decreased,which were consistent with the protein expression of CHMP4A and TSPYL-2.Conclusion CHMP4A and TSPYL-2 genes are differentially expressed in esophageal squamous cell carcinoma,which can be used as alternative genetic markers for further research.

Multidrug resistance associated genes of leukemia separated by suppression subtractive hybridization / 国际检验医学杂志

Objective To isolate and identify differential expression genes associated with multidrug resistance of leukemia . Methods Differential expression genes between leukemia cell line K 562 and resistant cell lines K562/DOX were isolated by using suppression subtractive hybridization (SSH) technique .Total RNA were extracted .cDNA were synthesized and digested by restric-tion enzyme Rsa Ⅰ ,then connected with adopter1 and adopter2R ,and linked with pMD19-T vector .Constructed vectors were trans-ferred into E .coli .Subtracted cDNA library was constructed ,and the positive clones were screened according to base sequences and homologous sequences .The differential expression genes were indentified by comparison analysis of Gene Bank database .Results A total of 220 differential expression genes were sequenced ,including hemoglobin ,ribosomes and mitochondria related genes ,and heat shock factor binding protein 1 (HSPB1) gene and other genes .Conclusion SSH method and molecular cloning technique could be used to construct subtracted cDNA library of differential expression genes between drug resistant and not -resistant leukemia cells , which might be useful for further screening and cloning of differential expression genes of multidrug resistant tumor cells .

Subtractive Hybridization Identifies Stem Cell-Associated Genes in an Acute Myeloid Leukemia with Poor Prognosis

Introduction: Current prognostic markers have improved survival prediction, however, it has not advanced treatment strategies. Gene expression profiling may identify biological markers suitable as therapeutic targets. Leukaemia stem cell is associated with adverse outcome, however, its biological characteristics are still being investigated. We observed higher in vitro cell viability in acute myeloid leukaemia (AML) samples with poor prognosis, which may be stem cell related. Objective: The objective of this study was to profile highly expressed genes in an AML sample of poor prognosis/high viability and compare with a sample of good prognosis/low viability. Method: Subtractive hybridization was performed on two AML samples with high blast counts (>80%), a poor prognosis, PP (disease free survival, DFS12 months) sample. The PP sample had higher CD34+ counts (73% vs 46%) and higher cell viability than the GP sample. cDNA libraries were subsequently cloned and sequenced. Results: cDNA subtracted from the PP samples was identified as genes active during fetal/embryonic development (LCOR, CNOT1, ORMDL1), HOX- related genes (HOXA3, PBX3, SF3B1), hematopoiesis (SELL, IL-3RA) and aerobic glycolysis/hypoxia (PGK1, HIGD1A) -associated genes. Majority of GP clones isolated contained genes involved in oxidative phosphorylation, OXPHOS (COXs, ATPs, MTND4 and MTRNR2), protein synthesis (including ribosomal proteins, initiating and elongation factors), chromatin remodeling (H2AFZ, PTMA), cell motility (MALAT1, CALM2, TMSB4X), and mitochondria (HSPA9, MPO) genes. Conclusion: Thus, the PP sample exhibited stem cell-like features while the GP sample showed cells at a high level of cell activity. These genes are potential prognostic markers and targets for therapy.

Deficiency of rpoS is the major factor leading to attenuation of Salmonella enterica serovar Choleraesuis vaccine strain C500 / 中国人兽共患病学报

Salmonella enterica serovar Choleraesuis strain C500 is a live attenuated vaccine that has been widely used in Chi‐na for over 50 years to prevent piglet paratyphoid .However ,as C500 is obtained by chemical methods ,the genetic background of this strain remained unclear .In this study ,we compared the genomic differences between the virulent reference strain C 78‐2 and C500 by suppression subtractive hybridization combined with the mirror orientation selection method (MOS‐SSH ) .Six genes (asr ,ydgF ,ydgD ,ydgE ,rpoS ,and ptsG) were lost in C500 strain .Using real‐time PCR analysis ,we demonstrated that the genes regulated by rpoS ,a vital transcriptional regulator playing an important role in Salmonella infection ,were downregulated in C500 .Additionally ,the virulence of the rpoS mutant strain C78‐2ΔrpoS was 100 000 times lower than the parental strain in BALB/c mice .So loss of rpoS gene is the major factor leading to the attenuation of C500 strain .

Identification of differentially expressed genes in Oncomelania hupensis liver induced by sanguinarine / 中草药

Objective: To identify the differentially expressed genes in Oncomelania hupensis liver induced by sanguinarine. Methods: The median lethal concentration (LC50) of sanguinarine for O. hupensis was determined by concentration gradient method. Livers were isolated from live O. hupensis after being treated by 80% LC50 of sanguinarine or clean water. Then the mRNA of livers was purified and used for RT-PCR. The differentially expressed genes were acquired by suppression subtractive hybridization (SSH) and amplified by nested PCR before cloned into pMD-18T vecter. Positive clones were identified using PCR and DNA sequencing analyses. Then the further study was accomplished by bioinformatic analysis with blastx in NCBI. Results: The LC50 of sanguinarine for O. hupensis was 7.5 mg/L. The differentially expressed sequences were between 150 and 450 bp. After blastx homological analysis, the up-regulated genes were obtained as collagen α-4 (VI) chain, collagen α-5 (VI) chain, 40S ribosomal protein S19, hypothetical protein, kelch-like protein, and granulin-like protein. The down-regulated genes were obtained as β-tubulin, α-amylase 1, large subunit ribosomal protein L23e, chitotriosidase-1, Chit3 protein, hypothetical protein, mitochondrial aldehyde dehydrogenase, peptidoglycan recognition protein, eukaryotic translation elongation factor 1A, ferritin, disintegrin, and metalloproteinase with thrombospondin motifs 3, lysozyme 1, endo-1, 2 (4)-β-glucanase 1, and hemocyanin α-4D-subunit. The functions of these genes encoding proteins were related to material transportation, protein synthesis, proliferation induction, nutrition, anti-oxidation, anti-inflamation, respiration, signal transduction, and so on. Conclusion: Sanguinarine could cause the significant changes of gene expression in O. hupensis liver.

Analysis of gene expression in Kalanchoe daigremontiana leaves during plantlet formation under drought stress

Background: Kalanchoe daigremontiana is an attractive model system for the study of the molecular mechanisms of somatic embryogenesis and organogenesis competence due to its formation of plantlets with adventitious roots on the leaf margins that are derived from somatic embryos. The suppression subtractive hybridization technique was used to investigate gene expression during asexual reproduction. Leaves from plants subjected to drought stress provided the source of ‘Tester’ DNA, and leaves from plants grown under normal conditions provided the ‘Driver’ DNA for subtractive hybridization. Results: A total of 481 high quality ESTs were generated, which clustered into 390 unigenes. Of these unigenes, 132 grouped into 12 functional categories, suggesting that asexual reproduction is a complicated process involving a large number of genes. The expression characteristics of selected genes from the SSH library were determined by real-time PCR and were classified into five groups, suggesting that gene expression patterns during asexual reproduction are complex. Up-regulation of S-adenosylhomocysteine hydrolase suggested that a decrease in cytokinin levels promotes the initiation of plantlet formation. Many other genes, such as inorganic pyrophosphatase and glutamate decarboxylase, play important roles in gene regulation during asexual reproduction. Conclusion: Our results provide a framework and unified platform on which future research on asexual reproduction in K. daigremontiana can be based. This represents the first genome-wide study of asexual reproduction in K. daigremontiana.

Construction of an SSH cDNA library from symbiotic germinated seeds of Dendrobium officinale and sequence analysis / 中国药学杂志

OBJECTIVE: To investigate differential gene expression profiling of symbiotic germinated seeds of Dendrobium officinale. METHODS: cDNAs from 5-week symbiotic germinated seeds and 5-week aseptic cultivated seeds, taken as the tester and driver respectively, were used to construct a suppressive subtractive hybridization (SSH) cDNA library. RESULTS: By sequencing positive clones and BLASTx analysis against GenBank database, 100 expressed sequence tags (EST) homologous to plant known genes were obtained. Functional annotation revealed that they were grouped into serials of cellular processes including cell and chromosome structure, RNA synthesis, signal transduction, energy metabolism, protein synthesis and degradation, and cell defense, etc. Real-time quantitative RT-PCR analyses showed that the five randomly selected genes were all up-regulated in symbiotic germinated seeds. CONCLUSION: The symbiotic seed germination of D. officinale is involved in multiple pathways, and the results of this study will lay a foundation for further molecular elucidation of seed germination in Orchidaceae.

Identification of chilling-responsive transcripts in peanut (Arachis hypogaea L. )

To isolate differentially expressed peanut genes responsive to chilling, a suppression subtractive hybridization (SSH) cDNA library was constructed for a chilling tolerant peanut cultivar A4 with mRNAs extracted from the seeds imbibed at 2ºC and 15ºC, respectively, for 24 hrs. A total of 466 cDNA clones were sequenced, from which 193 unique transcripts (73 contigs and 120 singlets) were assembled. Of these unique transcripts, 132 (68.4 percent) were significantly similar to the sequences in GenBank non-redundant (nr) protein database, which belonged to diverse functional categories including metabolism, signal transduction, stress response, cell defense and transcriptional regulation. The remaining 61 (31.6 percent) showed no similarity to either hypothetical or known proteins. Six differentially expressed transcripts were further confirmed with real-time quantitative PCR (RT-qPCR).

Construction of subtracted cDNA library for differentially expressed genes associated with chronic optic nerve injury / 第二军医大学学报

Objective: To construct subtractive cDNA libraries of differentially expressed genes associated with chronic optic nerve injury in cats. Methods: Fifteen adult cats were randomly divided into 3 groups (n=5): control group, 4-w compression group and 8-w compression group. The chronic optic nerve injury was produced by an inflatable balloon implanted under the optic chiasm. The total RNA was prepared from optic nerves of each group by TRIzol method. Double-stranded cDNA was produced by SMART PCR cDNA synthesis protocol. Suppression subtractive hybridization (SSH) was used to isolate the cDNA fragments of differentially expressed genes in the optic nerves after 4-w and 8-w compression. The cDNA fragments were directly inserted into T/A cloning vector to establish the subtractive library,followed by amplification of the libraries through E. coli transformation with calcium chloride and screening of blue and white clones. Three hundred positive bacterial clones were randomly picked in each library and identified by colony PCR. Results: Analysis of the white clones by PCR showed that 80% clones contained 200-800 bp inserts in each library. Conclusion: Four subtractive cDNA libraries of differentially expressed genes associated with chronic optic nerve injury have been successfully constructed by SSH and T/A cloning techniques,which lays a solid foundation for screening and cloning specific differentially expressed genes associated with chronic optic nerve injury.

Screening for clear cell renal cell carcinoma metastasis-associated methylations by genome subtractive hybridization / 第二军医大学学报

Objective: To screen for methylated biomarkers for the early diagnosis and prediction of clear cell renal cell ancer (ccRCC) metastasis. Methods: Highly and lowly metastatic ccRCC cell lines,which were established using fresh surgical specimens in our laboratory,were used in this study. Genomic DNA was extracted after pathological identification. Methylated genomic fragments were enriched by 3 cycles of Not I genomic DNA subtractive hybridization and were linked to vectors,which was then used for transformation, followed by α selection. Forty positive clones were randomly selected for DNA sequencing. Bioinformatics analysis was used to identify methylation of CpG islands and to predict the function of unknown genes. Results: DNA sequencing revealed 27 independent clones with different methylations between the highly and lowly metastatic ccRCC. Five of the 27 clones contained CpG islands,and 2 of the 5 fragments contained CpG islands in the promoter regions of genes MYADM and LOC646024. MYADM was associated with maturation of hematopoietic cells and regeneration of stem cells. LOC646024 shared 85% homology with UL16 binding protein 1; the latter was related to tumor killing function of NK cells. Conclusion: Novel methylated sequences have been discovered from Chinese ccRCC cells with different potentials for metastasis. Methylation of 2 candidate genes,MYADM and LOC646024,is indicated to be involved in ccRCC metastasis. Our findings are valuable for the biomarker exploration to predict metastasis as well as molecular epidemiological research.

Suppression subtraction hybridization in screening of differentially expressed genes in radiation-induced malignant transformation cellular model / 第二军医大学学报

Objective: To screen for the differentially expressed genes during irradiation-induced malignant transformation of human bronchus epithelium cells (BEAS-2B). Methods: Suppression subtraction hybridization (SSH) was used to construct a subtracted cDNA library of differentially expressed genes during irradiation-induced malignant transformation of BEAS-2B cells. Then the subtracted library was screened by PCR and the differential fragments were sequenced and analyzed with BLAST. Fluorescent real-time quantitative PCR was used to investigate some of the differentially expressed genes. The new EST was registered in GenBank. Results: Then 40 clones were chosen to be sequenced from the library of increased expression and decreased expression respectively according to the length of insertion element. Totally 73 sequences were obtained from the 80 sequenced clones. Forty-one sequences were decreased in the transformed cells; BLAST analysis indicated that there were 6 known sequences, 20 unknown sequences, 7 void sequences and 8 repeated sequences. Thirty-two sequences were increased in the transformed cells; Blast analysis indicated that there were 14 known sequences, 9 unknown sequences, and 9 repeated sequences. Fluorescent quantitative PCR revealed that, compared with control group, the expression of MY06, HACE1, ZNF143, and HNRPH1 were significantly increased in the radiation transforming group, with their mRNAs increased by 3.49, 29.38, 12.99 and 5.00 folds, respectively. Compared with control group, the expression of PCBP2, RPL15, and TCERG1 in the radiation transforming group was significantly decreased, with their mRNAs decreased by 1.89,48.77 and 11.95 folds, respectively. The 29 unknown sequences were registered in the GenBank (ID: EB643220-EB643248). Conclusion: The cDNA library has been successfully established for malignant transformation cellular model by suppression subtractive hybridization; the library includes a number of unknown genes. The increased gene ZNF143 is associated with cell proliferation and cell division. TCERG1, as an assistant transcription activation factor, plays an important role in the mRNA transcription and later modification. PCBP2, a Polyc connection protein, plays a modulating role in protein translation. These genes have not been reported in the radiation carcinogenicity.

Screening for rat vascular calcification related genes using suppression subtractive hybridization / 中华肾脏病杂志

Objective To determine the differentially expressed genes in the development of vascular medium calcification in rats using the suppression subtractive hybridization (SSH). Methods Twenty-four 6-week old SD rats of specific pathogen free grade were recruited and randomly allocated into calcified group (n=12) and control group (n=12). Rats were made for vascular calcification model in calcified group (vitamin D3 plus nicotine, VDN). All rats were sacrificed to measure concentration of calcium in the arterial tissue and examine the pathological lesion changes. RNA in rat aortic tunica tissue was extracted and reverse transcripted into cDNA. cDNA fragments which highly expressed calcification were isolated in calcified group using the SSH. Differentially expressed genes with cDNA fragment were inserted into PMD18-T plasmid vector and transformed to competent DH-5α by means of heating transfer. cDNA libraries of differentially expressed gene between calcified group and control group were successfully constructed. Recombinant vectors were analyzed by colony PCR. Positive genes were randomly selected for sequencing and analyzed by BLAST. Six genes, for example, were randomly selected for RT-PCR certification. Results (1) The pathological examination results demonstrated that in calcified group there were obvious calcium diposits and media squirm in tunica media of rat aortic wall, while in control group no calcium diposit was found. (2) There was statistical significance in calcium concentration in vascular tissue between calcified group[(15.34 ± 2.51)mg/g] and control group [(5.20 ± 0.75) mg/g] (P＜0.01). (3) Subtracted libraries in vascular calcification was successfully established. Ninety-two positive clones in positive library and 18 positive clones in reverse library were obtained after the colony PCR identification. The length of insertion fragments was concentrated between 150 bp and 400 bp. Calcification-related 43 up-regulated genes and 11 down-regulated genes were obtained through sequencing and BLAST analysis in positive clones. RT-PCR validation indicated that the expressions of 5 genes such as CytoP450 and Nell1 had greater increase in calcified group than those in control group, the average fold change was 1.71.Conclusions Model of vascular calcification induced by vitamin D3 plus nicotine is successfully constructed. Related gene expression spectrum is changed in the process of vascular calcification.Some ossification genes and genes associated with apoptosis, oxidation, inflammation and cytokines are up-regulated. At the same time, some genes which possibly inhibit vascular calcification are down -regulated.

Comparison of detection sensitivity of RDA and SHDD methods in cloning differential genes / 肿瘤研究与临床

Objective To establish matched-pairs of DNA samples with copy-number controlled differential target genes, and to compare the detection sensitivity of typical Representational Difference Analysis (RDA) method and Subtractive Hybridization Difference Display (SHDD) method in isolating differential genes.Methods Two gene fragments (376 bp and 869 bp in length respectively) cloned by PCR using Human Papilloma Virus (HPV) DNA as template were used as differential target genes, and mixed with human genome DNA. Five matched, pairs of human genome DNA samples with gradually increased difference in copy numbers of target genes were established and RDA and SHDD methods were performed to clone differential target genes and compared their detection sensitivity. Results By using RDA method, 376 bp fragment with 6-fold difference and 869 bp fragment with 8-fold difference were cloned.However, both of these two target fragments with 4-fold difference were isolated using SHDD method.Conclusion The SHDD method adopts balanced bi-directional subtractive hybridization between two sample difference products and avoids loss of differential target genes caused by unbalanced subtractive hybridization of RDA method, and thus outweighs RDA method in isolating target genes, especially long-length target fragments, with small difference.

The differential expression of the anti-oxidation related genes in Echinococcus granulosus by real-time PCR / 中国人兽共患病学报

To isolate the specific genes in protoscoleses (PSC) of Echinococcus granulosus under oxidative stresses from the SSH library constructed in the previous study, the gene expression in PSC under oxidative stresses was studied by using real-time PCR. The previously amplified library was sequenced and analyzed in GenBank with Blast research. Sequence analysis indicated that all clones in the SSH library contained the coding sequences, of which some clones showed homology in the GenBank and others were unknown. Differential expression of 4 genes randomly selected and the TPx gene in this library were studied with real-time PCR. It was demonstrated that the gene expression of S88 and H32-1 in oxidative tissues was 2.0 and 2.3 times higher than the un-oxidative stresses respectively. The TPx gene was up-regulated when PSC was induced with H_2H_2 of more than 0.8 mmol/L. These results implies that the up-regulated expression of the above-mentioned genes may be related with the related functions of anti-oxidative process in PSC and they may be used as the candidate genes for the study of anti-oxidation of E.granulosus.

Construction of subtracted cDNA library by suppression subtracted hybridization for differentially expressed genes in Mycobacterium tuberculosis / 中国免疫学杂志

Objective: To isolate Mycobacterium tuberculosis H37Rv and H37Ra genes especially,and to construct two cDNA libraries by using suppression subtractive hybridization (SSH).Methods: We used suppression subtractive hybridization (SSH) to clone the differential genomic genes between Mycobacterium tuberculosis virulence strain H37Rv and attenuated strain H37Ra.After two times of subtractive hybridization and two times of PCR,the products of last PCR amplification were inserted into pGEM-T Easy vector and be transformed into the E.coli DH5α and screened of blue and white clones of the transformants.The subtracted cDNA library of differentially expressed genes were identified by RT-PCR.Results:High or especially expressed genes as tester had been obtained by SSH in correctitude reaction (H37Rv as tester) and reverse reaction (H37Ra as tester),the cDNA libraries of A and B of Mycobacterium tuberculosis H37Rv and H37Ra were successfully constructed.90% of the colonies were white clones,the single band of the colonies was 75% and 80%.Conchision:The cDNA libraries of Mycobacterium tuberculosis H37Rv and H37Ra were successfully constructed using SSH technique,which lay a solid foundation for screening and cloning new specific differentially expressed genes in them.

Effects of the recipe for activating blood circulation and nourishing qi on expression of mitochondrial ribosomal protein L51 in CVB3 infection model in rat cardiac myocytes / 国际中医中药杂志

Objective To study the effects of the recipe for activating blood circulation and nourishing qi on expression of mitochondrial ribosomal protein L51 (MRPL51) in CVB3 infection model in rat cardiac myocytes, to reveal the pathogenesis of CVB3 myocarditis in the genetic level, to explore the therapeutic mechanism of the recipe for activating blood circulation and nourishing qi on CVB3 myocarditis, and to confirm the validity of the recipe for activating blood circulation and nourishing qi on CVB3 myocarditis. Methods After establishing CVB3 infection model and treatment model with recipe for activating blood circulation and nourishing qi by culturing neonatal rat myocardial cells, a modified suppression subtractive hybridization (SSH) was used to isolate differentially expressed genes between two model groups. These results were further verified by fluorescence RT-PCR. Results The results of SSH showed that gene expression of the treatment group was lower than that of the CVB3 infection group. The results of fluorescent RT-PCR which agreed with that of SSH displayed the threshold cycle number (Ct) in the treatment group was higher than the virus group. Conclusion Up-regulation of MRPL51 might be one of the pathogenesis of CVB3 myocarditis. The recipe for activating blood circulation and nourishing qi could treat viral myocarditis by regulating the expression of MRPL51.

Screening of Mycobacterium tuberculosis distinctive genes by suppression subtractive hybridization technique / 中华微生物学和免疫学杂志

Objective To screen differential Mycobacterium tuberculosis genes between Xinjiang clinical strains and H37Rv by suppression subtractive hybridization( SSH), and to analyze the function of these specifically pathopoiesis genes. Methods Both M. tuberculosis Xinjiang clinical strains and H37Rv as tester and driver each other, most identical genome was drived whereas some distinctive genes was re-mained and enriched by utilization SSH technique. Meanwhile through inserting differential genes to E. coli all of sequences that we have cloned were determined by BLAST in GenBank. The function of differential genes between M. tuberculosis H37Rv and Xinjiang clinical strains were analyzed. Results We cloned and analyzed six different DNA fragments that only existed in Xinjiang clinical strains. One is the fragment of a gene ceding monooxygenase, flavin-binding family identified by Glimmer2. One fragment belongs to acyl-transferase family protein. One for aminotransferase, class Ⅱ, acyl carrier protein. One fragment belongs to chromosomal replication initiator protein DNA and one for M. tuberculosis paralogous family 11-pyridoxamine 5'-phosphate oxidase-related. Meanwhile, we cloned ten DNA fragments only in H37Rv. Conclusion SSH technique can efficiently screen differential genes in M. tuberculosis in Xinjiang clinical strains. They are possible key genes that M. tuberculosis survive and fortify virulence in mal-environment as same as their ho-mogenic genes, such as enhanced adsorbability in wall-held protein, counteracted digestion by nitro-oxygen-ase, elevated composition capability in the acyhransferase, control chromosomal replication initiator protein, synthesized aminotransferase acyl cartier protein and pyridoxamine 5'-phosphate oxidase.

Construction and identification of differential expression genes of peripheral blood cells in radon-exposed mice / 中华放射医学与防护杂志

Objective To screen and identify the differential expression genes on peripheral blood cells of mice based on the experimental animal model of radon exposure. Methods BALB/c mice were exposed in a type HD-3 multifunctional radon-room, with the accumulative doses of radon-exposure group at 105 WLM and control group at 1 WLM. Total RNA was extracted from peripheral blood cells and the methods of SMART for dscDNA synthesis and SSH for gene screening was applied. With the construction of the cDNA library enriched with differentially expressed genes, the pMD 18-T plasmid containing LacZ operator at the multiple cloning site was used to allow a blue-white screening. The TA clones were amplified by nested PCR and the reverse Northern blot was used to identify up and down regulation of the clones. The differently expressed cDNA was then sequenced and analyzed. Results The subtracted cDNA libraries were successfully constructed. A total of 390 recombinant white colonies were randomly selected. Among the 312 cDNA monoelones selected from bath forward- and reverse-subtracted libraries,41 clones were chosen to sequence for their differential expressions based on reverse Northern blot. Among the 41 sequenced clones, 10 clones with known function/annotation and 3 new ESTs with the GenBank accession numbers were obtained. Most of the known function/annotation genes were revealed to be related with cell proliferation, metabolism, cellular apoptosis and carcinogenesis. Conclusions The animal model of radon exposure was established and the cDNA library of peripheral blood cells was suceessfully constructed. Radon exposure could up- and down-regulate a series of genes. Differentially expressed genes could be identified by using SSH technique and the results may help exploring mechanisms of random exposure.

Exploring upregulated genes during osteogenic differentiation of hMSCs

Human bone marrow mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tenden, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells could be isolated from marrow aspirates of human and animals. This study was designed to identify and characterize genes specifically expressed by osteogenic supplements-treated cells by suppression subtractive hybridization(SSH) method. The results were as follows: 1. 2 genes were upregulated genes in osteogenic diffeentiation of hMSCs, which is further proved by Northern blot analysis. 2. IGFBP-2 has been identified playing an important role in bone formation. 3. HF1 was also upregulated during osteogenic differentiation, but its role in bone formation is not clear yet.

Identification of genes related to induced resistance to ceftriaxone in Neisseria gonorrhoeae using suppression subtractive hybridization and DNA microarray / 中华皮肤科杂志

Objective To elucidate the molecular basis for induced resistance of N. gonorrhoeae to ceftriaxone in vitro. Methods The reference strain ATCC49226 and clinical isolate ZSSY00205 of N. gon-orrhoeae were exposed to subinhibitory concentration of ceftriaxone for the induction of resistance. Then,suppression subtractive hybridization was performed with the pre-induction parent strains as drivers and post-induction mutant strains as testers to create a subtractive cDNA library. Following that, a total of 192 clones were randomly selected from the library, and arrayed by spotting onto nylon membranes. Finally, dif-ferentially expressed genes were screened by hybridization with labeled-RsaI restriction fragments from the sensitive and resistant N.gonorrhoeae strains respectively, and analyzed by sequencing and homology research using Blast program. Results A subtractive library for these resistant N.gonorrhoeae strains was generated by SSH technique. Microarray analysis and homology research confirmed 5 genes related to ceftriaxone resistance, i.e. mtrR, mtrC, gyrB, rpsJ and PJD1. Conclusions The induced resistance of N. gonorrhoeae to ceftriaxone may be associated with mtrR, mtrC, gyrB, rpsJ and PJD1 genes which probably mediate the resistance by enhancing the activity of efflux pump system.