ABSTRACT
Objective:To improve the users’ comfort of steady-state visual evoked potential (SSVEP)-based brain-computer interface (BCI) through high-frequency stimulation and overcome the problem of accuracy decline caused by high frequency by combining dual-frequency encoding.Methods:Two dual-frequency high-frequency 60-instruction paradigms based on left and right visual fields and checkerboard stimuli were designed based on the 25.5 - 39.6 Hz frequency. Thirteen subjects participated in the experiment, and spectrum and spatial characteristics analyses were performed on SSVEP signals. The filter bank parameters were optimized based on the spectrum characteristics. Extended canonical correlation analysis (eCCA), ensemble task-related component analysis (eTRCA), and task-discriminant component analysis (TDCA) were used for SSVEP recognition.Results:Stable SSVEP was successfully induced in both the left and right visual fields and the checkerboard grid paradigm. The left and right visual fields had high signal-to-noise ratios for the fundamental frequency and its harmonics and weak signal-to-noise ratios for intermodulation components, whereas the intermodulation components of the 2 stimulus frequencies of the checkerboard grid, f1 + f2, had significantly higher signal-to-noise ratios than the second harmonic components above 30 Hz, and there was also a f2 ? f1 component and a 2 f1 ? f2 component. Combined with brain topography, it can be seen that the f1 and f2 response components of the left and right visual fields are located on opposite sides of the visual field, while the checkerboard grids are both concentrated in the center of the occipital region. Regarding the lateralization of brain topography amplitude and signal-to-noise ratio, the mean values of the PO3 and PO4 signal-to-noise ratios at the stimulation frequency of the left and right visual fields are consistent with the contralateral response characteristics. The 5 fb ? 1 method is the optimal filter set setting method, and the recognition correctness rate of TDCA for the left and right visual fields is the highest. However, the comparison of the recognition correctness rate of tessellated lattice eTRCA and TDCA is not statistically significant ( P > 0.05). The information transmission rates of the three algorithms all increase and then decrease with the increase in data length. Conclusions:The designed dual-frequency, high-frequency SSVEP-BCI paradigm is able to better balance performance and comfort and provides a basis for practical large instruction set BCI design methods.
ABSTRACT
Objective: To establish a reliable and sensitive method for evaluating quality of Yiqi Jiangzhi Granules (YQJZG). Methods: Ultra performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) was employed for simultaneous determination of eight marker components. Separation was performed on an AQUITY UPLC® HSS T3 column, the mobile phase consisted of acetonitrile as the organic phase and 0.1% (volume percentage) formic acid as the aqueous. Eight marker components, ginsenoside Rg1 (GRg1), ginsenoside Re (GRe), ginsenoside Rb1 (Gb1), typhaneoside (TEO), isorhamnetin-3-O-neohespeidoside (IN), hesperidin (HPD), aurantio-obtusin-6-O-β-D-glucoside (AG) and curcumin (CCM), were detected by multiple reaction monitoring (MRM) mode. The Chinese Pharmacopoeia (2020 edition) was regarded as the guidance document for this method validation. Results: The method showed good linearity (R
ABSTRACT
Objective To explore the effect of using cluster nursing measures on expectoration in mechanical ventilation patients after craniocerebral operation. Methods Convenient sampling and controlled trials at not the same period were used. Sixty-seven mechanical ventilation patients after craniocerebral operation were selected as the research objects in Department of Neurosurgery Intensive Care Unit (ICU) of the First Hospital Affiliated to Wenzhou Medical College. Thirty-two patients treated from June 2015 to June 2016 were assigned in the control group, and they were given routine respiratory nursing care; 35 patients admitted and treated from July 2006 to July 2017 were included in the intervention group, and they were given evidence-based cluster nursing intervention measures on the basis of routine care. The differences in expectoration effect, arterial blood gas analysis index, incidence of pulmonary infection and prognosis of patients in two groups were compared. Results Compared with control group, the amount of expectoration in the intervention group was significantly increased (mL/d: 49.69±9.45 vs. 33.72±10.63, P < 0.05), while the daily number of sputum suction (times: 21.57±2.31 vs. 28.76±22.66), the time needed for each sputum suction(s: 6.81±1.74 vs. 9.28±2.52), respiratory frequency (bpm: 26.26±1.83 vs. 28.58±1.36), incidence of pulmonary infection [0 vs. 12.5% (4/32)], time of mechanical ventilation (days: 6.37±2.51 vs. 8.92±3.32), time of ICU stay (days: 7.49±3.87 vs. 10.33±2.12), time of hospital stay (days: 10.31±1.99 vs. 14.56±3.57), fatality rate [8.6% (3/35) vs. 21.9% (7/32)] in the intervention group were significantly decreased (all P < 0.05); after treatment the arterial partial pressure of oxygen (PaO2) and pulse oxygen saturation degree (SpO2) were significantly increased than those before treatment, and the arterial partial pressure of carbon dioxide (PaCO2) was significantly decreased than that before treatment, and the degrees of improvement in the intervention group on 5 days were significantly better than those in the control group [PaO2(mmHg, 1 mmHg = 0.133 kPa): 60.89±3.44 vs. 57.34±2.49, PaCO2(mmHg): 41.06±4.32 vs. 45.22±4.78, SpO2: 0.986±0.030 vs. 0.963±0.023, all P < 0.05]. Conclusion The cluster nursing measures can effectively improve the expectoration effect for mechanical ventilation patients after craniocerebral surgery, reduce the mortality and incidence of pulmonary infection, shorten the stay in ICU and improve the prognosis, suggesting that the measures be worthy to be applied widely in clinics.
ABSTRACT
Objective To investigate the effect of evidence-based nursing on the clinical curative effect and prognosis of patients with hypertensive cerebral hemorrhage.Methods 120 patients with hypertensive cerebral hemorrhage were selected,and they were randomly divided into observation group(60 cases) and control group(60 cases) according to the digital table.The control group was treated with routine nursing,the observation group was treated with routine nursing and evidence-based nursing.Before and after nursing,the SDS,self rating anxiety scale (SAS),neurological deficit score NIHSS,Barthel score,the incidence of sequelae,hospitalization time,nursing quality score and patients’ satisfaction with nursing were compared between the two groups.Results There were no statistically significant differences in SDS,SAS,NIHSS and Barthel scores between the two groups before nursing intervention(all P ＞ 0.05).After nursing intervention,the SDS,SAS,NIHSS and Barthel scores of the observation group were (38.74 ± 6.21) points,(35.83 ± 8.17) points,(11.24 ± 3.08) points,(92.58 ± 6.46) points,respectively,which in the control group were (44.58 ± 7.10) points,(43.66 ± 8.06) points,(15.34 ± 3.29) points,(84.27 ± 5.82) points,there were significant differences between the two groups (t =4.796,5.285,7.047,7.403,all P ＜0.05).,The incidence rate of venous thrombosis,muscle atrophy and joint ankylosis sequelae of the observation group was 8.33％,which was lower than 40.00％ of the control group,there was significant difference between the two groups(x2 =16.415,P ＜ 0.05).The hospitalization time of the observation group was (9.55 ± 2.43)d,which was shorter than (15.97 ± 4.68) d of the control group (t =9.430,P ＜ 0.05).The health education nursing quality score,ward management score,basic nursing score,nursing care of critical patients score,nursing document writing score of the observation group were (97.66 ± 2.45) points,(98.23 ± 3.46) points,(97.54 ± 3.18) points,(96.88 ± 3.49) points,(98.76 ± 1.31)points,respectively,which were higher than those of the control group [(88.79 ± 2.37) points,(90.72 ±3.52) points,(91.05 ±3.16) points,(91.67 ± 5.34) points,(93.04 ± 1.12) points],there were significant differences between the two groups(t =20.156,11.786,11.214,6.326,25.707,all P ＜ 0.05).The patients’ nursing satisfaction of the observation group (96.67％) was higher than that of the control group (85.00％),there was significant difference between the two groups (x2 =4.904,P ＜ 0.05).Conclusion Evidence -based nursing can effectively relieve patients’ anxiety and depression of patients with hypertensive cerebral hemorrhage,improve the quality of life of patients,reduce the incidence of complications,shorten the hospitalization time,improve patients'satisfaction.
ABSTRACT
BACKGROUND:Hypertrophic differentiation of chondrocytes is the sign of starting endochondral ossification, and it is also an essential step in endochondral ossification, which is a cascade reaction and difficult to be blocked once started. The end result is the formation of bone structure. RNA interference is a post-transcriptional gene silencing. Relevant studies have shown that the use of RNA interference to block the expression of core binding factorα1 (Cbfα1) can effectively inhibit the formation of heterotopic ossification. OBJECTIVE:To use RNA intereference technology to suppress Cbfα1 expression so as to achieve the purpose of blocking the hypertrophic diferentiation of chondrocytes. METHODs: We constructed an adenovirus containing siRNA against Cbfα1 (Ad-Cbfα1-siRNA). Retinoic acid and interleukin-1α were used to induce hypertrophic differetiation of chondrocytes, and then Ad-Cbfα1-siRNA was utilized to inhibit the hypertrophic differentiation of chondrocytes. Immunohistochemistry method was used to analyze the expression of Cbfα1. RESULTS AND CONCLUSION:After induction with retinoic acid and interleukin-1α, the chondrocytes in the negative control virus group appeared to have hypertrophy and the expression of Cbfα1 was positive. In the Ad-Cbα1-siRNA group, the expression of Cbfα1 was negative. These findings suggest that the inhibition of Cbfα1 by RNA interference can be a powerful way to prevent the hypertrophic differentiation of chondrocytes .
ABSTRACT
Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. As current clinical treatments are aimed at restoring biomechanical function and providing symptomatic relief, the methods focused on biological repair have aroused interest and several tissue engineering approaches using different cell types have been proposed. Owing to the unsuitable nature of degenerate cells for tissue engineering, attention has been given to the use of mesenchymal stem cells (MSCs). In this connection, we have made a study on the characteristics of MSCs derived from adult bone marrow and on the feasibility of constructing IVD tissue-engineering cell under a Three-Dimensional Pellet Culture System. The human bone marrow MSCs were isolated and purified with density gradient solution and attachment-independent culture system. MSCs isolated using this method are a homogeneous population as indicated by morphology and other criteria. They have the capacity for self-renewal and proliferation, and the multilineage potential to differentiate.
Subject(s)
Adolescent , Adult , Humans , Young Adult , Bone Marrow Cells , Cell Biology , Cell Culture Techniques , Methods , Cells, Cultured , Chondrogenesis , Physiology , Intervertebral Disc , Intervertebral Disc Degeneration , Therapeutics , Mesenchymal Stem Cells , Cell Biology , Tissue Engineering , MethodsABSTRACT
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.
Subject(s)
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 , Genetics , Recombinant Fusion Proteins , GeneticsABSTRACT
<p><b>BACKGROUND</b>Ras 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.</p><p><b>METHODS</b>Site-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.</p><p><b>RESULTS</b>Two 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.</p><p><b>CONCLUSIONS</b>Two mutant KSR genes are successfully constructed. It provides experimental basement for further functional research of KSR.</p>
ABSTRACT
<p><b>BACKGROUND</b>The 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.</p><p><b>METHODS</b>Using 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).</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSIONS</b>(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.</p>
ABSTRACT
<p><b>BACKGROUND</b>It has been proved that lysosome-associated protein transmembrane 4 beta (LAPTM4B) plays an important role in tumorigenesis, the *2/*2 genotype of LAPTM4B was closely related to liver cancer susceptibity. The aim of this study is to investigate the possible association between the allelic variation of LAPTM4B and the genetic susceptibility of lung cancer.</p><p><b>METHODS</b>The genotype of LAPTM4B was detected in 131 patients with lung cancer and 104 unrelated healthy adult individuals as control by polymerase chain reaction based on special primers. The genotypic distribution of LAPTM4B was analyzed by Chi-square test.</p><p><b>RESULTS</b>The allelic frequencies of the *1 and *2 were 74.8% and 25.2% in the lung cancer group and 72.1% and 27.9% in the healthy control group respectively, but no significant difference was observed between the two groups (Chi-square=0.433, P=0.510). The genotypic distribution of the *1/*1,*1/*2 and *2/*2 were 53.4%, 42.7% and 3.8% in the lung cancer group, 54.8%, 34.6% and 10.6% in the healthy control group respectively, there was no significant difference between the two groups (Chi-square=4.89, P=0.087). No remarkable association was observed between the genotypic distribution of LAPTM4B and the clinical information in the patients with lung cancer, including pathological type and TNM staging.</p><p><b>CONCLUSIONS</b>The results suggest that the allele of LAPTM4B is not closely associated with genetic susceptibility of lung cancer.</p>
ABSTRACT
<p><b>BACKGROUND</b>Previous 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.</p><p><b>METHODS</b>Site-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.</p><p><b>RESULTS</b>Five 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.</p><p><b>CONCLUSIONS</b>Five 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.</p>
ABSTRACT
<p><b>BACKGROUND</b>The 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.</p><p><b>METHODS</b>N-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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSIONS</b>Eukaryotic 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.</p>
ABSTRACT
<p><b>BACKGROUND</b>Lung cancer is one of the most malignant cancers which is hazarding the people's health and life in the world. At present, it is a highlight to exploit antitumor drug from plant at home and abroad. The aim of this study is to observe the effects of polysaccharid (PS-T) on expression of angiogenic-related gene mRNA in human high-metastatic large cell lung cancer cell line L9981, and to explore its possible molecular mechanism.</p><p><b>METHODS</b>L9981 in vitro was cultured, and the growth data were obtained by trypan blue staining. The mRNA transcript expression of β-catenin, E-cadherin, TIMP-1, CD44V6, MMP-2, endostatin, VEGF was detected in L9981 by RT-PCR before and after treating with PS-T. The ability of invasion of L9981 was determined by Boyden chamber method.</p><p><b>RESULTS</b>(1)PS-T had remarkably inhibitive effects on the growth of L9981 in vitro. The inhibitive rate of PS-T on L9981 was concentration-dependent. No significant difference of inhibitive rate was found among the PS-T (1g/L), cisplatin (3mg/L) and PS-T (0.05g/L) + cisplatin (1.5mg/L)(P > 0.05). (2)The mRNA expression level of β-catenin, E-cadherin, TIMP-1, endostatin and MMP-2 was upregulated, while that of VEGF and CD44V6 was downregulated. Out of them the mRNA expression level of TIMP-1 and endostatin was remarkably upregulated, the expression level of CD44V6 was significanyly downregulated. (3)The in vitro invasive abilities of L9981 was significantly decreased in the PS-T, DDP and PS-T+DDP groups compared with that in blank control group.</p><p><b>CONCLUSIONS</b>(1)PT-S could inhibit the growth of human high-metastatic large cell lung cancer cell line L9981 in vitro, the effect is dose-dependent. (2)PS-T can down- or up-regulate the mRNA transcript expression of some angiogenic-related gene mRNA. (3)PS-T has remarkably coordinating effects with cisplatin in the L9981 lung cancer cell line.</p>
ABSTRACT
<p><b>BACKGROUND</b>nm23-H1 gene is a well-known tumor metastasis suppression gene. Our previous study has found that transfection of wild type nm23-H1 gene can significantly downregulate the ERK1/2 activity of human high-metastatic large cell lung cancer cell line L9981. The aim of this study is to investigate the influence of nm23-H1 and exogenous ERK1/2 pathway inhibitor U0126 on the extracellular signal-regulated kinase (ERK1/2) of human high-metastatic large cell lung cancer cell line L9981 and its malignant biological behaviors.</p><p><b>METHODS</b>The expressive levels of total-ERK1/2, dually phosphorylated ERK1/2 and ERK1/2 relative activity of the human high-metastatic large cell lung cancer cell lines, L9981 (parent cell line with nm23-H1 gene hetero-deletion), L9981-nm23-H1 (transfected with nm23-H1 gene ) and L9981-PLXSN (transfected with vector) were detected by Western blot and immunoprecipitation technique after treating with U0126 (40μmol/L for 20 minutes). The in vitro proliferative and invasive abilities among the above three lung cancer cell lines were determined by MTT and improved Boyden chamber methods.</p><p><b>RESULTS</b>The phosphorylated ERK1/2 expression level and relative activity in L9981-nm23-H1 lung cancer cell line were remarkably lower than those in L9981 and L9981-PLXSN lung cancer cell lines after being treated with U0126 (P < 0.01), but there was no significant difference between L9981 and L9981-PLXSN lung cancer cell lines. No significant difference of total ERK1/2 expression level was observed among the three lung cancer cell lines (P > 0.05) after being treated with U0126. The in vitro proliferation and invasion of L9981-nm-23H1 lung cancer cell line were remarkably lower than those of L9981 and L9981-PLXSN lung cancer cell lines (P < 0.01 ), but no significant difference was found between L9981 and L9981-PLXSN lung cancer cell lines (P > 0.05 ); U0126 could significantly down-regulate the in vitro proliferation and invasion of L9981 lung cancer cell line (P < 0.01).</p><p><b>CONCLUSIONS</b>Blocking the activity of ERK1/2 in L9981 lung cancer cell line and transfecting the nm23-H1 gene into the L9981 lung cancer cell line may produce similar cell biological behavior changes, namely the significant reduction of in vitro proliferation and invasion of L9981 lung cancer cell line. These results indicate that the molecular mechanism which nm23-H1 gene reverses invasion and proliferation of the human high-metastatic large cell lung cancer cell line may be related to its effects of down-regulating the activity of the key kinase ERK1/2 of Ras-to-MAPK signal transduction pathway.</p>
ABSTRACT
<p><b>BACKGROUND</b>It has been known that oncogenesis and development of lung can- cer is a complex process regulated many genes and involved in multistages. Recent studies have demonstrated that signal transduction abnormality may play a very important role in these procedures. The aim of this study is to investigate the influence of exogenous MEK1/2 pathway inhibitor U0126 on the expression and activity of extracellular signal-regulated kinase (ERK1/2) of human high-metastatic large cell lung cancer cell line L9981 and its malignant biological behaviors.</p><p><b>METHODS</b>The expressive levels of total-ERK1/2, dually phosphorylated ERK1/2 and ERK1/2 relative activity of the human high-metastatic large cell lung cancer cell line L9981 (parent cell line with nm23-H1 gene hetero-deletion ) were detected by Western blot and immuno-precipitation technique after treating with different doses of U0126. The in vitro proliferative and invasive abilities of the lung cancer cell line were determined by MTT and modified Boyden chamber methods.</p><p><b>RESULTS</b>The ERK1/2 relative activity of L9981 gradually decreased accompanied with increase of U0126 doses, and a highly significant difference of phosphorylated ERK1/2 expression level existed among the different concentration groups of U0126 (P < 0.01), but no significant difference of total ERK1/2 expression was found among the different concentration groups of U0126 (P=0.387). After treatment with same concentration of U0126 for different time, the ERK1/2 relative activity of L9981 gradually decreased as the treatment time of U0126 prolonging, and a highly significant difference of phosphorylated ERK1/2 expression level was observed among different treatment time groups (P < 0.01). But no significant difference of total ERK1/2 expression level was found among different time groups (P=0.689). The inhibition of ERK1/2 pathway by MEK1/2 specific inhibitor U0126 targeting the ERK1/2 pathway of L9981 was dose- and time-dependent. After treatment with different concentration of U0126, the proliferation of L9981 gradually decreased accompanied with increase of U0126 concentration and a highly significant difference existed among different groups of U0126 concentration (P < 0.001). The in vitro invasion of L9981 gradually decreased accompanied with increase of U0126 concentration. No significant difference of in vitro invasion of L9981 was found among 0, 10 and 20μmol/l of U0126 (P > 0.05). A highly significant difference was observed when U0126 concentration increased to 40 and 60μmol/l compared with 0, 10 and 20μmol/l of U0126 (P < 0.001).</p><p><b>CONCLUSIONS</b>The inhibition of Ras-to-MAPK pathway by Ras-to-MAPK specific inhibitor U0126 targeting the Ras-to-MAPK pathway of the human high-metastatic large cell lung cancer cell line L9981 is dose-dependent and time-dependent. Suppressing or blocking of Ras-to-MAPK signal transduction pathway can reverse the invasive and metastatic phenotype of the human high-metastatic large cell lung cancer cell line L9981. These results suggest that the key kinase MEK1/2 of the ERK1/2 pathway may be a potent therapeutic target for human lung cancer.</p>
ABSTRACT
<p><b>BACKGROUND</b>Metastasis is not only the malignant characteristics of lung can- cer, but also the chief cause of failure to cure and high mortality of lung cancer. To better explore and understand the mechanism of lung cancer metastasis and to search for potential markers for early diagnosing and reversing lung cancer metastasis, differential proteomic analysis is conducted in two human large cell lung cancer cell lines with high metastasis potentials (L9981) and low metastasis potentials (NL9980) by two-dimension gel electrophoresis (2-DE).</p><p><b>METHODS</b>The total proteins of the two cell lines were separated by immobilized pH gradient (IPG)-based 2-DE. The differentially expressed proteins of the two cell lines were analyzed using image analysis software.</p><p><b>RESULTS</b>A high resolution and reproducible 2-DE image was successfully obtained. Average deviations for protein position in IEF direction were (0.858±0.076)mm and (1.514±0.127)mm in SDS-PAGE direction. The relative standard deviation for protein volume was (12.06±0.580)% in L9981 and (12.22±0.640)% in NL9980. The average total number of protein spots was 902±169 in L9981 cells and 941±173 in NL9980 cells in three repeated experiments. Image analysis of siliver-stained 2-DE image revealed that 4 protein spots had significant differential expressions in L9981 and NL9980 (student's t-test, P < 0.05). Fifteen protein spots were only detected in L9981, and 27 protein spots were only detected in NL9980.</p><p><b>CONCLUSIONS</b>The results in this study suggest that an obviously differential proteomic expression exists between the human high- and low-metastatic large cell lung cancer cell lines. It will be helpful to further understand the molecular mechanisms of lung cancer invasion and metastasis, and provide new experimental evidence for searching metastatic-related molecule of lung cancer.</p>
ABSTRACT
<p><b>BACKGROUND</b>Lung cancer is the leading cause of malignant tumor death among Chinese population. It has been known that the development of lung cancer may be associated with genetic po-lymorphism of some lung cancer related genes. The aim of this study is to evaluate the relationship between genetic polymorphism of metabolizing enzymes and susceptibility of lung cancer in Chinese population.</p><p><b>METHODS</b>Polymorphism of CYP2E1 RsaI/PstI and GSTM1 was detected in 99 patients with lung cancer and 66 patients with benign pulmonary disease by PCR-RFLP and PCR. The association between genetic polymorphism and susceptibility of lung cancer was analyzed.</p><p><b>RESULTS</b>No significant difference in three RsaI/PstI genotype distribution of CYP2E1 was found between lung cancer group and control group (Chi-Square=1.374, P=0.241). (2) The frequency of GSTM1-null genotype in lung cancer group was significantly higher than that in control group (57.6% vs 40.9%, Chi-Square=4.401, P=0.036). (3) The individuals who carried with GSTM1-null genotype had a 1.96 fold increased risk of lung cancer (OR=1.96, 95%CI=1.042-3.689, P=0.037) than those who carried with GSTM1-present genotype. (4) When data were stratified by smoking status, the smokers who carried with c1/c1 genotype had a significantly higher risk of lung cancer (OR=3.525, 95%CI=1.168- 10.638, P=0.025) than those never-smokers who carried with at least one c2 allel. (5) When combination of polymorphism of CYP2E1 RsaI/PstI genotype and GSTM1 genotype was analyzed, compared with individuals who had concurrent present of GSTM1 and at least one c2 allel genotype, the risk of lung cancer for combination of GSTM1 null and c1/c1 genotype was increased significantly (OR=3.449, 95%CI=1.001- 11.886, P=0.050). Considering smoking status, compared with never-smokers who had concurrent present of GSTM1 and at least one c2 allel genotype, the risk of lung cancer for combination of GSTM1 null and c1/c1 genotype was remarkably increased (OR=11.553, 95%CI=1.068-124.944, P=0.044), as well as that for combination of GSTM1 null and at least one c2 allel genotype (OR=13.374, 95%CI=1.258-142.166, P= 0.032).</p><p><b>CONCLUSIONS</b>(1)GSTM1 null genotype is an important factor associated with increased risk of lung cancer. (2) The combination of c1/c1 and GSTM1-null genotype can remarkably increase risk of lung cancer both in smokers and non-smokers.</p>
ABSTRACT
<p><b>BACKGROUND</b>It has been proved that Tanshinone has obvious anticancer effect, but its mechanisms of anticancer are still unknown. Anticancer Ketonon is complex antitumor drug which Tanshinone is combined with other anticancer elements. This study aims to explore the antineoplastic effects of Anticancer Ketonon on Lewis lung cancer and the mechanisms in mice.</p><p><b>METHODS</b>The mice were divided into three groups: Ketonon group, 5-fluorouracil (5-Fu) group and control group. The former two groups were treated with responsive drugs after subcutaneous inoculation of Lewis lung cancer. The last group was only treated with normal saline after inoculation. Apoptosis index and cell cycle were measured by flow cytometry.</p><p><b>RESULTS</b>Two experiments were carried out in male and female mice respectively. The tumor inhibitory rates of Anticancer Ketonon were 38.9% and 32.2% respectively, those of 5-FU were 59.6% and 53.9%. Compared with those of control groups, the tumor weights in Ketonon group and 5-Fu group were statistically decreased (P < 0.05). Metastasis rates of the lung in the three groups were not statistically different (P > 0.05). The apoptosis index of Ketonon group was significantly higher than that of control group (P < 0.05), but the cell cycle was not statistically changed compared with that of control group (P > 0.05).</p><p><b>CONCLUSIONS</b>Anticancer Ketonon has antineoplastic effect on Lewis lung cancer in mice and the mechanism may be associated with inducing apoptosis of tumor cells.</p>
ABSTRACT
<p><b>BACKGROUND</b>The human FHIT gene at chromosome 3p14.2 is a tumor suppressor gene, and its abnormality in structure and function is related to carcinogenesis and progression of lung cancer. It is postulated that FHIT and NIT1 likewise collaborate in biochemical or cellular pathway in mammalian cells, but their molecular mechanisms in lung cancer cell are still unknown. The aim of this study is to construct procaryotic expression vector of human NIT1, providing a foundation to explore the expression of human NIT1 protein and to study the interaction of NIT1 and FHIT genes in lung cancer cell lines.</p><p><b>METHODS</b>The NIT1 cDNA was acquired by RT-PCR. EcoRI/NotI digested PCR product was directly cloned into procaryotic expression vector pET-32a.</p><p><b>RESULTS</b>The sequence of NIT1 cDNA clone exactly corresponded with the sequence of NIT1 cDNA in GenBank. The expectant fragments of DNA were obtained after recombinant procaryotic expression vector was digested by EcoRI and NotI.</p><p><b>CONCLUSIONS</b>The procaryotic expression vector of human NIT1 is successfully constructed by RT-PCR and direct clone. It provides an important basis to detect the expression of NIT1 protein and to further explore the relationship between NIT1 and FHIT genes in oncogenesis and development of human lung cancer.</p>
ABSTRACT
<p><b>BACKGROUND</b>Genetic polymorphism in metabolic enzymes, which are involved in metabolism of environmental carcinogens, have been thought to be related to susceptibility of lung cancer. The aim of this study is to investigate the cytochrome P450 2D6(CYP2D6) genetic polymorphism distribution in Han population in Sichuan, China, and to evaluate the relationship between CYP2D6 genetic polymorphism and lung cancer susceptibility.</p><p><b>METHODS</b>PCR-RFLP was used to identify CYP2D6ch genotypes among 150 patients with primary lung cancer and 152 healthy controls, in Han population in Sichuan, China, and case-control study was used to analyze the relationship between genetic polymorphism and lung cancer susceptibility.</p><p><b>RESULTS</b>(1) The distribution frequency of CYP2D6ch C and T allele were 39.5% and 60.5% in control group and 46.3% and 53.7% in lung cancer group, respectively. There was no significant difference between the two groups (P=0.089). (2)The distribution frequency of C/C, C/T and T/T genotypes were 18.4%, 42.1% and 39.5% in control group, and 22.7%, 47.3% and 30.0% in lung cancer group, respectively. No significant difference was found between the two groups (P=0.215). (3) The individuals who carried with Non-T/T genotypes had a 2.084-fold increased risk with squamous cell carcinoma (95%CI 1.024-4.244, P=0.043) than those who carried with T/T genotype. (4) The lighter smokers ( < 30 pack-years) who carried with Non-T/T genotypes had a 2.92-fold increased risk with lung cancer (95%CI 1.087-7.828, P=0.033) than those who carried with T/T genotype.</p><p><b>CONCLUSIONS</b>CYP2D6ch Non-T/T genotypes are factors associated mail:zhouqh@mail.sc.cninfo.net) with increased risk of squamous cell carcinoma and also increase risk of lung cancer among lighter smokers.</p>