[Objective] To elucidate the effects and mechanism of Naoqingling Decoction(ND)on Gilles de la Tourette syndrome(TS).[Method] Central nervous system of mice was stimulated by amphetamine(AMP)to develop the model of experimental tourette syndrome(ETS).Then we observed the stereotypia and autonomic activities of mice,and determined the levels of monoamine neurotransmitters such as dopamine(DA),norepinephrine(NE),5-hydroxytryptamine(5-HT)and 5-hydroxyindoleacetic acid(5-HIAA)in the brains.[Result] To compare with model group,three-dose ND groups can resist the determinate activities stimulated by AMP;can obviously decrease the autonomic activities of mice and degrade the levels of DA,NE,5-HT and 5-HIAA in the corpora striates.[Conclusion] ND has therapeutical effect on ETS model and it's mechanism may be its effects on the secretion or metabolism of neurotransmitter in the brains.

A 65-year-old man with right central type of lung squamous carcinoma was admitted to our department. Bronchoscopy displayed complete obstruction of right upper lobe bronchus and inifltration of the bronchus intermedius with tumor. Chest contrast computed tomography revealed the tumor invaded right pulmonary artery, superior vena cava, and the persistant letf superior vena cava lfowed into the coronary sinus. hTe tumor was successfully removed by means of bronchial and pulmonary artery sleeve resection of the right upper and middle lobes combined with resection and reconstruction of superior vena cava (SVC) utilizing ringed polytetralfuoroethylene gratf. To the best of our knowledge, this was the ifrst report of complete resection of locally advanced lung cancer involving superior vena cava, right pulmonary artery trunk and main bronchus with persistant letf superior vena cava.

Objective To observe the change of the serum ABCG2 level of patients with non‐small cell lung cancer(NSCLC)be‐fore and after chemotherapy ,and explore its clinical significance .Methods Venous blood specimens of 15 healthy adults and 50 pa‐tients with NSCLC were collected before and chemotherapy ,the serum ABCG2 level of these specimens were detected by ELISA . the relation between the ABCG2 level and the chemosensitivity was investigated .Results The serum ABCG2 level of patients with NSCLC before chemotherapy was significantly higher than that in healthy adults(P<0 .05);the serum ABCG2 level was not related with TNM stage in NSCLC(P<0 .05);The serum ABCG2 level of patients with NSCLC after chemotherapy was significantly high‐er than that of before(P<0 .05);in chemosensitive patients ,the different of serum ABCG2 level was not significantly before and af‐ter chemotherapy(P>0 .05);among chemoresistant patients ,the serum ABCG2 level of patients with NSCLC after chemotherapy was significantly higher than that of before(P<0 .05);the serum ABCG2 level was higher in patients with pleural effusion than the patients without pleural effusion ,but the different was not significantly(P>0 .05) .Conclusion The serum ABCG2 level of patients with NSCLC is higher than that of healthy adults ;serum ABCG2 level may become a useful indicator in predicting the effect of NSCLC chemotherapy .

Objective: To evaluate the value of different sequences magnetic resonance imaging (MRI) in rectal cancer re-staging after neoadjuvant chemoradiation therapy (NCRT). Methods: The clinical data of 117 patients with rectal cancer who underwent NCRT before surgery operation in Peking University cancer hospital from January 2016 to December 2018 were retrospectively analyzed. Among 117 patients, 101 patients underwent MRI scanning before and after NCRT, and 16 patient underwent MRI scanning after NCRT; T₂ weighted imaging (T₂WI) and diffusion weighted imaging (DWI) scanning were performed in all patients, and dynamic contrast enhancement (DCE) scanning was performed in 96 patients. T₂WI, T₂WI combined with DWI, T₂WI combined with DCE were used for T re-staging of rectal cancer after NCRT respectively, and the results of which were compared with those of pathology after operation. Results: The sensitivity of diagnosis of ypT_0-2 rectal cancer after NCRT using T₂WI combined with DWI, T₂WI combined with DCE respectively was significantly higher than that using T₂WI: 52.7% (29/55) and 30.4% (14/46) vs. 10.9% (6/55), and there was statistical difference (P<0.05). The accuracy rate and specificity of diagnosis of ypT₃ and ypT₄ rectal cancer after NCRT using T₂WI combined with DWI were significantly higher than that using T₂WI, with an accuracy rate of 60.7% (71/117) vs. 47.0%(55/117) and 92.3% (108/117) vs. 80.3% (94/117), and a specificity of 55.9% (33/59) vs. 23.7% (14/59) and 92.9% (105/113) vs. 80.5% (91/113), and there were statistical differences (P<0.05). The accuracy rate of down-staging after NCRT using T₂WI combined with DWI was significantly higher than that using T₂WI: 72.3% (73/101) vs. 58.4% (59/101), and there was statistical difference (P<0.05); there was no significant difference in accuracy rate between using T₂WI and using T₂WI combined with DWI and between using T₂WI combined with DWI and using T₂WI combined with DCE (P > 0.05). Conclusions T₂WI combined with DWI is superior to T₂WI in re-staging of rectal cancer after NCRT.

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.

BACKGROUNDThe results of our previous studies have proven that nm23-H1 gene can suppress metastasis of lung cancer, which may be associated with suppression of the Wnt signal pathway through up-regulating the activity of glycogen synthase kinase 3β (GSK-3β), the key kinase of the Wnt signal pathway. The aim of this study is to investigate the effect of point mutation of nm23-H1 gene on GSK-3β activity in cytoplasm and nucleus in human high-metastatic large cell lung cancer cell line L9981.

METHODSUsing immunoprecipitation and a radioactive isotope scintillation counter, the activity of GSK-3β was detected in cytoplasm and nucleus of human low-metastatic large cell lung cancer cell line NL9980, human high-metastatic large cell lung cancer cell line L9981, L9981-pEGFP (transfected with vector), L9981-nm23-H1-pEGFP (transfected with wild type nm23-H1), L9981-nm23-H1 S44A -pEGFP mutant (transfected with serine 44 to alanineon of nm23-H1 gene), L9981-nm23-H1 P96S -pEGFP mutant (transfected with proline 96 to serine of nm23-H1 gene), L9981-nm23-H1 H118F -pEGFP mutant (transfected with histidine 118 to phenylalanine of nm23-H1 gene) and L9981-nm23-H1 S120G -pEGFP mutant (transfected with serine 120 to glycine of nm23-H1 gene).

RESULTSThe GSK-3β activity in cytoplasm and nucleus was remarkably decreased in the transgene lung cancer cell lines transfected with mutant nm23-H1 cDNA (L9981-nm23-H1 S44A -pEGFP, L9981-nm23-H1 P96S -pEGFP, L9981-nm23-H1 H118F -pEGFP and L9981-nm23-H1 S120G -pEGFP). Significant differences of GSK-3β activity in cytoplasm and nucleus were observed (P < 0.05) when L9981-nm23-H1-pEGFP cell line was compared with L9981-nm23-H1 S44A -pEGFP, L9981-nm23-H1 P96S -pEGFP, L9981-nm23-H1 H118F -pEGFP and L9981-nm23-H1 S120G -pEGFP lung cancer cell lines. There was a highly significant difference in GSK-3β activity in the cytoplasm between L9981-nm23-H1-pEGFP cell line and L9981-nm23-H1 P96S -pEGFP lung cancer cell line (P < 0.01 ). A highly significant difference in GSK-3β activity in the nucleus was observed (P < 0.01) when L9981-nm23-H1-pEGFP lung cancer cell line was compared with L9981-nm23-H1 S44A -pEGFP, L9981-nm23-H1 P96S -pEGFP and L9981-nm23-H1 H118F -pEGFP lung cancer cell lines.

CONCLUSIONS(1) Point mutation of nm23-H1 gene can significantly influence the regulating effects on the GSK-3β activity in the cytoplasm and nucleus in the human high-metastatic large cell lung cancer cell line L9981. (2) The effects of nm23-H1 gene on metastatic phenotype may be related to the upregulation of GSK-3β activity in human high-metastatic large cell lung cancer cell line L9981.

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.