Objective To study the effect of Fungi of Huai Er on human umbilicus vein endothelial cell((HUVEC)) and its inhibiting effect on liver cancer formation by liver cancer cells implanted under nude mice(subcutaneous) tissue, and to study the possible mechanism. Methods HUVEC was cultured with different concentrations of fungi of Huai Er and/or mitomycin(MMC) to observe the effect of Fungi of Huai Er on(HUVEC)′s hyperplasia , motility and adherence ability, and on the generation of blood vessels.In addition,the effect the Fungi of Huai Er on liver(cancer) formation by liver cancer cells implanted in nude mice(subcutaneous) tissue was also observed. Results Fungi ≥2mg/ml, can apparently reduce the hyperplasia(ability),inhibit motility and adherence ability of HUVEC,and the formation of blood vessels. The strongest effect on inhibiting the subcutaneous tumor formation was Fungi(3g/kg)plus MMC(500?g/kg),followed by MMC,and Fungi(3g/kg). Conclusions Possible mechanism of the inhibiting effect of Fungi on liver(cancer) is that the Fungi inhibits the(hyperplatic) ability, motility and adherence ability of HUVEC, and this(inhibits) the growth of blood vessels. Therefore, the growth of blood vessels is reduced and the development of liver cancer is suppressed.

In the present study, 89 porcine reproductive and respiratory syndrome virus (PRRSV) isolates in China during 2007 to 2012 were randomly selected from the GenBank genetic sequence database. Evolutionary characteristics of these isolates were analyzed based on the sequences of non-struc-tural protein 2 (Nsp2) and glycoprotein 5 (GP5). The genetic variations of the isolates were also compared with six representative strains. The results showed that a high degree of genetic diversity exists among the PRRSV population in China. Highly pathogenic PRRSV isolates, with a discon-tinuous deletion of a 30 amino acid residue in the Nsp2 region, remained the most dominant virus throughout 2007-2012 in China. Owing to the extensive use of representative vaccine strains, natu-ral recombination events occurred between strains. Three isolates-HH08, DY, and YN-2011-were more closely related to vaccine strains than the other isolates. Both YN-2011 and DY were the evolu-tionary products of recombination events between strains SP and CH-1R. The results of the present study provide useful information for the epidemiology of PRRSV as well as for vaccine development.

Objective: To describe the prevalence of permanent tooth caries and associated factors among junior high school students in Haikou, and to provide reliable evidence for prevention and control of caries. Methods: A total of 3 573 students from 8 junior high school in Haikou City and towns were selected by the methods of clustered sampling survey. Questionnaire survey and oral health examinations were conducted to analyze the caries rate, mean decayed missing filled tooth (DMFT), filling rate, pit and fissure sealant rate. Logistic regression was used to analyze the influencing factors of caries in permanent tooth. Results: Among the surveyed junior high school students, the caries rate was 47.32%( n =1 691), the mean DMFT was 1.51. The caries rate and mean DMFT were higher in female students (49.59%) than in male students (44.95%), and higher in towns (50.77%) than in urban areas (44.04%), the difference were statistically significant ( χ 2=16.25, 7.72, P <0.05). The filling rate and pit and fissure sealant rate among junior high school students in Haikou were 17.13%, 6.27% respectively. The filling rate and pit and fissure sealant rate were higher in urban areas (18.97%, 7.17%) than towns (15.76%, 5.33%), the difference were statistically significant ( χ 2= 9.57, 5.13, P <0.05). The multivariate Logistic analysis showed that female student, town residence, daily consumption of sweets or sugary drinks (≥1 time), bedtime snack were risk factors for junior high school students suffering from permanent dental caries( OR =1.41, 1.45, 2.63, 2.09, 1.53), while using fluoride toothpaste daily, oral education in school were protective factors( OR =0.44, 0.34)( P <0.05). Conclusion The caries rate of permanent teeth among junior high school students in Haikou is at high level, but the filling rate of caries and pit and fissure sealant rate are lower. The prevention and treatment of dental caries should be carried out in high risk students, and oral health education in school is also needed to improve the oral health level of junior high school students.

BACKGROUND AND OBJECTIVEAtelectasis is a common complication after thoracotomy, and it may threaten patients' life if it was not treated correctly and properly. The aim of this article is to explore and discuss the prevention and treatment for atelectasis during the perioperative period, and also to explore new methods for reducing the perioperative mortality due to atelectasis after thoracotomy.

METHODSWe retrospectively reviewed the medical records of 374 lung cancer patients who underwent thoracotomy in our department between Jan 2007 and Nov 2009.

RESULTSAtelectasis occurred in 14 patients among all the 374 lung cancer patients who underwent thoracotomy. All the atelectasis returned to reexpansion after treatment.

CONCLUSIONThe incidence of atelectasis in these series is relatively low compared with the reports in literatures. Good perioperative preparation and perioperative treatment can remarkably decrease the incidence and mortality of atelectasis after thoracotomy in the treatment of lung cancer.

Female ; Humans ; Lung Neoplasms ; surgery ; Male ; Middle Aged ; Pulmonary Atelectasis ; prevention & control ; Retrospective Studies ; Thoracotomy ; adverse effects ; methods

A single-stranded oligonucleotides containing a 6 histidine sequence, a hydroxylamine cleavage site, a thrombin cleavage site, and stop codon TAA were inserted into the polylinker's downstream of prokaryotic expression vector pBV220 between BamHI and PstI. The resultant vector is named pBV223. Proteins expressed in this vector will have a 6 histidine tail as affinity handy fused to their C terminus and can be quickly purified by one step immobilized metal affinity chromatography (IMAC). This plasmid is verified by restriction map and DNA sequencing. Subsequently, the metastasis suppressor gene nm23-H1 cDNA (without the stop codon) was cloned into vector PBV223 in frame with the 6-histidine sequence, hydroxylamine and thrombin cleavage sites. The soluble nm23-H1 fusion protein was successfully induced in the bacterial DH5a and easily purified with Ni chromatograph. These results indicated that the strategy to clone the single-stranded oligonucleotides directly into the restriction sties between BamH I and Pst I in the pBV220 vector is the simplest and cost-effective method.
Base Sequence ; Chromatography, Affinity ; methods ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Mutation ; NM23 Nucleoside Diphosphate Kinases ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

BACKGROUNDTo clone DNA sequence of human telomerase reverse transcriptase (hTERT) promoter, and study its transcriptional activities in various human lung cancer cells and normal lung cell.

METHODShTERT promoter of 1.1 kb was amplified with polymerase chain reaction (PCR) method, utilizing DNA of 293 cell as template. After DNA sequencing with correct result, the hTERT promoter was inserted into luci-ferase reporter vectors (pGL3-Basic) to reconstruct a recombinant plasmid named pGL3-hTERTp. Then the reconstructed plasmid was transiently transfected into lung cancer cell lines A549, SPC-A-1, LTEPa-2, NCI-H446, YTMLC, GLC-82, A2 and human embryonic pulmonary fibroblast cell line MRC-5. The transcriptional activities of hTERT promoter in various cells were determined by measuring the luciferase activities after 48 hours of transfection.

RESULTSElectrophoresis demonstrated that cloned hTERT promoter was about 1.1 kb, and DNA sequencing showed a same sequence as registered in GenBank. The cloned hTERT promoter was located between 1 126 bp and 43 bp in upstream of the transcription start site ATG and the length was 1 086 bp. The recombinant plasmid pGL3-hTERTp was confirmed by double digestion and PCR method with correct results. Luciferase assay showed there were different transcriptional activities of hTERTp in various lung cancer cell lines, but not in the MRC-5 cell line.

CONCLUSIONSThe hTERT promoter cloned in this study has transcriptional activities in various lung cancer cell lines but not in normal cell. It may act as control element in tumor-targeting gene therapy with hTERT.

BACKGROUNDScreening metastatic-related genes of lung cancer is helpful to understand the molecular mechanisms of lung cancer invasion and metastasis. In order to screen the differential expression genes related to metastasis of lung cancer, we constructed and preliminarily screened the subtracted cDNA libraries of human large cell lung cancer cell lines with different metastatic potentials in this study.

METHODSSubtracted cDNA library was constructed in the different metastastic potential cell lines NL9980 and L9981 by suppression subtractive hybridization (SSH) method. The positive clones were preliminarily screened by blue-white colony based on the α-complementary principal, and precisely identified by PCR. The forward and reverse subtracted libraries were screened and identified by dot blot to obtain the clones corresponding to differential expression segments.

RESULTSThe subtracted cDNA libraries were successfully constructed in the different metastastic potential cell lines NL9980 and L9981. Three hundred and seven positive clones in the forward subtracted library and 78 positive clones in the reverse subtracted library were obtained by the dot blot method.

CONCLUSIONSSSH is proved to be an efficient tool for differential expression gene cloning. The forward and reverse subtracted cDNA libraries of different metastastic potential cell lines are constructed by this method. The differential expression genes related to tumor metastasis might exist in the human large cell lung cancer cell lines with different metastasis potential.

BACKGROUNDRas to MAPK pathway plays a critical role in the transmission of many growth and developmental signals. A new component of this pathway which is termed kinase suppressor of Ras (KSR) was found in 1995. KSR is as a scaffolding protein that coordinates the assembly of a multiprotein complex containing mitogen-activated protein kinase (MAPK) and its upstream regulators. It has been proven that KSR has many phosphorylation sites, and phosphorylation state changes response to signaling events. Site-directed mutagenesis can precisely change the base sequence and get mutant proteins. The aim of this study is to construct two mutant proteins of KSR by using site-directed mutagenesis, and to express and purify them, therefore to provide basement for studying the functional and biochemical mechanisms of KSR.

METHODSSite-directed mutagenesis of pCMV-Tag2b-KSR gene was performed by modified QuikChangtm site-directed mutagenesis kit method. Two pairs of mutagenic primers were synthesized in vitro and two mutations desired, the recombinant plasmids were verified by restriction enzyme analysis and DNA sequencing. Then positive clones were transfected into 293T cell line. The purified mutant proteins were analyzed by Western blot.

RESULTSTwo mutants were successfully constructed. The results of DNA sequencing confirmed that the base sequences of the mutant genes were completely concordant with experiment design, which could be used to be transfected into 293T cell line. The purified mutants were identified by Western blot.

CONCLUSIONSTwo mutant KSR genes are successfully constructed. It provides experimental basement for further functional research of KSR.

BACKGROUNDThe present experimental data have showed that the function of kinase suppressor of Ras (KSR) is mainly as a scaffold protein that coordinates the assembly of a multiprotein complex containing MAPK and its upstream regulators. But whether KSR has kinase activity is still the point of argument until now. The aim of this study is to construct eukaryotic expression vectors of carboxyl terminus and amino terminus of KSR and to detect their expression in 293T cell line.

METHODSN-KSR and C-KSR were amplified by PCR. The eukaryotic expression vectors of pCMV-Tag2b-N-KSR and pCMV-Tag2b-C-KSR were constructed by gene recombination technique and the recombinant plasmids were verified by restriction enzyme analysis and sequencing. Then positive clones were transfected into 293T cell line. Expression of target proteins was analyzed by Western blot.

RESULTSThe sequences and open read frames of the two vectors were both completely concordant with experiment design. The target proteins could be observed in transfected 293T cells by Western blot.

CONCLUSIONSEukaryotic expression vectors of pCMV-Tag2b-N-KSR and pCMV-Tag2b-C-KSR are successfully constructed, and they can be expressed in 293T cells. It provides an experimental base for further research work.

BACKGROUNDPrevious researches have proven that nm23-H1 gene is a tumor metastatic suppressor gene, however, it is still unknown about its exact molecular mechanisms. Site-directed mutagenesis can correctly change the base sequence and get mutant proteins. The aim of this study is to construct nm23-H1 mutant and EGFP fusion genes by site-directed mutagenesis, and to provide basement for studying the functional and biochemical mechanisms of nm23-H1 gene.

METHODSSite-directed mutagenesis of nm23-H1 gene was performed by modified QuikChange™ Site-directed Mutagenesis Kit method. Pure plasmid containing fusion gene of nm23-H1 and EGFP (PLXSN-nm23-H1-EGFP) was mini-prepared. Four pairs of mutagenic primers were synthesized in vitro and the desired five mutations, S44A, P96S, H118F, S120G and P96S-S120G were introduced into nm23-H1-EGFP fusion gene by PCR.

RESULTSFive nm23-H1 mutant and EGFP fusion genes, nm23-H1(S44A)-EGFP, nm23-H1(P96S)-EGFP, nm23-H1 (H118F)-EGFP, nm23-H1(S120G)-EGFP and nm23-H1(P96S-S120G)-EGFP, were successfully constructed. The results of DNA sequencing confirmed that the base sequences of the mutant fusion genes were completely concordant with experiment design.

CONCLUSIONSFive nm23-H1 mutant and EGFP fusion genes are successfully constructed, which can be used in further studies. QuikChange™ site-directed mutagenesis is a simple, rapid and efficient method.