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The number of taking the Examination of Doctor Qualification is rising,while the pass rate is dropping each year.This status has undoubtedly made a big challenge for clinical teaching,and how to improve the effects of clinical lessons has become an important topic for the clinical teachers.The Affiliated Xinqiao Hospital of the Third Military Medical University has made some new changes in clinical teaching mode,which has a study clue of organ and system.Those methods give much help for students to pass the Examination of Doctor Qualification.
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<p><b>BACKGROUND</b>In recent years, many progresses have been made in molecular target therapy for lung cancer, in which anti-angiogenic target therapy is a hot spot drawing researchers' attention. The aim of this study is to explore the expression of canstatin gene transfected into human lymphocytes and its inhibitory effect on growth and metastasis of Lewis lung carcinoma.</p><p><b>METHODS</b>The eukaryotic expression vector of pCMV-Script and the recombinant pCMV-Script/Canstatin vector were separately transfected into lymphocytes by electroporation. The expression of canstatin protein in supernatant of lymphocyues was examined by SDS-PAGE assay. Furthermore, Lewis lung carcinoma cells were subcutaneously inoculated to C57BL mice to make animal model of tumor. When the transplanted tumors on the mice developed to 1cm³, the 30 mice were randomized into 3 groups, which were injected with 0.2mL supernatant of lymphocytes transfected with recombinant vector or naked vector, or 0.2mL NS respectively. After the treatment for 14 days, the size and pathological section of subcutaneous tumors were observed, and the number of pulmonary metastatic node was calculated.</p><p><b>RESULTS</b>Canstatin protein was found in supernatant of the lymphocytes in the recombinant vector group by SDS-PAGE assay. After the treatment, the tumor size in the recombinant vector group (1.49cm³±0.18cm³) was significantly smaller than that in the naked vector group (2.44cm³± 0.19cm³) and NS group (2.53cm³±0.18cm³) (P=0.000). The numbers of pulmonary metastatic node were 3.40±1.14, 7.60±2.61 and 7.60±2.41 in the recombinant vector group, naked vector group and NS group respectively (recombinant vector group vs the other two groups, P=0.013).</p><p><b>CONCLUSIONS</b>The pCMV-Script/Canstatin vector can express canstatin protein in human lymphocytes. Canstatin has strongly inhibitory effect on growth and metastasis of mouse Lewis lung carcinoma.</p>
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Canstatin protein, a newly found angiogenesis inhibitor, has powerful anti-angiogenesis effect. Canstatin is the N terminal fragment of collagen IV alpha2 chain NC1 domain. Its distinct anti-cancer effect in mouse model and low toxicity has not only made it a promising new anti-cancer drug candidate, but also drawn much attention of researchers. The detection, denomination, biological characters and mechanism of canstatin protein, and also the prospect of its application were summarized.
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Animals , Humans , Mice , Angiogenesis Inhibitors , Pharmacology , Antineoplastic Agents , Pharmacology , Collagen Type IV , PharmacologyABSTRACT
<p><b>BACKGROUND</b>Angiogenesis is essential for tumor's growth and metastasis. Canstatin, a newly found potent endogenous angiogenesis inhibitor, has drawn researcher's attention to its powerful biological activities on endothelial cells. The aim of this experiment is to explore the expression and effects of canstatin gene in lung cancer A549 cells and HUV-ECC cells.</p><p><b>METHODS</b>Expression vector of pCMV- Script/Canstatin was transfected into A549 and ECC cells by electroporation, and the positive clone was screened by G418. Growth characteristics of the two cell lines were compared before and after transfection. Expression of canstatin protein in supernatant was examined by SDS-PAGE assay, and cell cycles of the two cell lines were analysed by flow cytometry.</p><p><b>RESULTS</b>The expression of canstatin gene was found in supernatant of the transfected A549 cells and ECC cells. The apoptotic rate in the transfected ECC cells (16.04%) was significantly increased compared with that of the naked plasmid control group (0.43%) and parental cell group (2.92%) (P < 0.01). The growth of the transfected ECC cells was significantly inhibited (P < 0.01). The apoptotic rate in the transfected A549 cells (0.19%) showed no marked difference from the naked plasmid control group (0.13%) and parental cell group (0.07%) (P > 0.05). No significant difference in cell growth was found among the transfected A549 cell, naked plasmid control and parental cell groups.</p><p><b>CONCLUSIONS</b>The results indicate that canstatin gene can express in lung cancer A549 cell line and HUV-ECC cell line, and it can specifically inhibit proliferation of endothelial cell and induce its apoptosis.</p>
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Objective To explore the effects of antisense vector of annexinⅡ gene on the growth of SPC A 1, a human lung cancer cell line. Methods The total RNA was isolated from human lung cancer cell line SPC A 1 and the target DNA fragments were amplified by RT PCR. The antisense expression vector was constructed by double restriction endonuclease cleavage directional clone method. Annexin Ⅱ antisense expression vector was introduced into SPC A 1 cells by liposome transfection reagent. The expression of annexin Ⅱ mRNA was analyzed by semi quantitative RT PCR. The effects of antisense vector of annexinⅡ gene on the growth of SPC A 1 were observed. Results The antisense vector of annexinⅡ gene was constructed and introduced into SPC A 1 cells successfully. Semi quantitative RT PCR showed that the annexin Ⅱ mRNA expression reduced by about two thirds in the transfected cells as compared with that in the untransfected cells. Compared with the untransfected cells, transfected cells decreased significantly in cell growth, clone formation efficiency in plating and DNA synthesis. Cell cycle was blocked in G 0 G 1 phase. Conclusion Annexin Ⅱ could promote the growth of lung cancer cells and may be helpful for the development of lung cancer.
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Objective To explore the relationship between the microsatellite markers on chromosome 6 and the phenotypes of atopy. Methods Sixteen microsatellite markers on chromosome 6 were evaluated in 20 affected sib pair and trios families. Linkage disequilibrium analysis was conducted according to asthmatic phenotypes (total IgE, skin prick test, bronchial hyper responsiveness and eosinophil) by ETDT software system. Results Significant P value( P
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Objective To study the drug sensitivity of chemotherapeutic drugs by MTT method. Methods Twenty five lung cancer specimens obtained by operation were used for primary culture and the sensitivity of the cancer cells to four drugs, adriamycin(ADM), cis dichlorodiamine platinum(DDP), vincristine(VCR) and etoposide(VP16), was tested by MTT assay. TOPOⅡ, p53, bcl 2 and GST ? proteins in 10 cases of lung cancer tissue were determined by immunohistochemistry. Results The successfully obtained results in 21 cases showed that the effective rate of ADM, DDP, VCR and VP16 was 19.0%, 38.1%, 23.8% and 23.8% respectively. Positive expressions of TOPOⅡ, p53, bcl 2 and GST ? proteins were found in lung cancer tissue in the 10 cases which had different multi drug resistance(MDR) to the four drugs. Conclusion It is possible to test the drug sensitivity of the primary cultured lung cancer cells obtained from operation by MTT method and the results are useful for the selection of anticancer drugs. The expressions of TOPOⅡ, p53, bcl 2 and GST ? are factors to determine MDR for lung cancer cells.
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Objective To establish lung adenocarcinoma drug resistance cell strain induced by cis-diaminodichloroplatin and investigate the biologic characteristics and chromosome karyotype of cell strain.Methods Large dosage impact and step by step inducing method were used to establish A549 and SPC-A-1 drug resistance cells: A549/CDDP and SPC-A-1/CDDP.MTT was used to detect the cell drug resistance index. Bioluminescence was used to detect the energy metabolism.Cell cycle was analysed by flow cytometer.The differential staining technique and SKY were used to analyze the chromatosome of A549/CDDP and SPC-A-1/CDDP.Results The drug resistance index of A549/CDDP and SPC-A-1/CDDP were 14.0 and 12.0. The content of ATP,ADP and AMP decreased significantly.The cell cycle of A549/CDDP and SPC-A-1/CDDP were of no notable changes.The results of the differential staining technique and SKY showed that the chromatosome of A549/CDDP and SPC-A-1/CDDP had several derivative chromosomes.Both cells had the same derivative chromosome: der(21)t(21;22).Conclusion CDDP can induce lung adenocarcinoma cell strain drug resistance.The derivative chromosome,including der(21)t(21;22) may have relationship with the drug resistance of cell strains.
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Objective To investigate effects of endotoxin on 11?-HSD2 gene transcription in vascular endothelial cells to observe the role of p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway. Methods The effects of endotoxin in the presence or absence of p38 MAPK specific inhibitor SB203580 on the transcription of 11?-HSD2 in vascular endothelial cells was evaluated by reverse transcription DNA polymerase chain reaction. Results Treatments of endotoxin (1.0, 10, 20, 50, 100 ?g/ L) for 24 h increased the ratios of 11?-HSD2mRNA/?-actin mRNA in vascular endothelial cells. The induction of 11?-HSD2 mRNA by endotoxin could be inhibited partially by 10 mmol/ L SB203580. Conclusion Endotoxin stimulated the transcription of 11?-HSD2 gene in vascular endothelial cells. The activation of p38 MAPK might be an important mechanism of 11?-HSD2 gene induced by endotoxin.
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monocarboxylate transporter (MCT) are vital transmembran e transporters involved in multiple cellular functions including the regulation of intracellul ar pH and lactate transport. At least eight isoforms of MCT have been cloned and characterized to date, they constitute a new gene family of mammal transporters . These isoforms have the differences in substrate and inhibitor specificities a nd tissue distribution. Thus it may provide a new way of diagonosis and treating for dieases such as cancer by investigating on the structure and function and r egulational mechanism of MCT.
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Objective To clone the partial positive regulatory fragment of Na+/H+ exchanger-1 (NHE-1) gene from human lung cancer cells. Methods After BamHⅠ and EcoRⅠ cut sites were added to the 5' ends of the upstream and downstream primers respectively, the partial positive regulatory sequence of NHE-1 gene was cloned with the length of 170 bp from genomic DNA of lung cancer cell line A549 cells with PCR method. The cloned fragment was ligated to plasmid pUC18. Finally, the constructed recombinant was identified with enzyme cut, PCR and DNA sequencing. Results The cloned fragment was about 170 bp in size and successfully ligated to pUC18 with identifiation of double enzyme cut and PCR. DNA sequencing approved that the fragment cloned was objective one with 168 bp in length. Compared with the reported sequence, two t were lost. Conclusion The positive regulatory fragment of NHE-1 gene from human lung cancer cells was successfully cloned.
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Objective To study the effect of transfecting anti-sense expression vector of the first subtype of the monocarboxylate transporter (MCT1) gene into human lung adenocarcinoma cells on intracellular pH (pHi) regulation, lactate transportation and cell growth. Methods MCT1 antisense gene recombinant vector pLXSN-MCT1 was introduced into human lung cancer cells A549 with electroporation. The cell colonies resistant to G418 were selected. Positive clones were examined by PCR to confirm the integration of genomic A549 DNA and antisene MCT1 gene. The changes of pHi and lactate transportation were detected with spectrophotometry. Cell growth was studied with cell growth curve. Results pHi and lactate transport were remarkably decreased in the transfected cells, and the cell growth was inhibited compared with the cells without transfection（P<0.001）. Conclusion MCT1 gene may play an important role in pHi regulation, lactate transport and cell growth in lung tumor cells.
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Objective To establish a multidrug resistance cell line from human lung adenocarcinoma. Methods The human lung adenocarcinoma cell line SPC-A-1 was exposed to cisplatin of a high and then increasing concentration for 192 d to establish multidrug resistance cell line (SPC-A-1/CDDP). The relative resistance was tested with MTT assay. The morphology of the cells was observed with transmission and scanning electron microscopy and the chromosome of them was analyzed with Giemsa stained specimens. Results The resistance index of SPC-A-1/CDDP cells to cisplatin was 11.2 and the cells showed various cross-resistance to 5-Fu, doxorubicin, mitomycin, vincristine and etoposide, but not to hydroxycamptothecine. Electron microscopy showed the cells with irregular and enlarged nuclei and abundant microvilli. Conclusion A multidrug resistance cell line (SPC-A-1/CDDP) from human lung adenocarcinoma is established. It can be used to downstream experiment.
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Chemotherapy for lung cancer is expected to prolong the survival of lung cancer patients. multidrug resistance is considered to be a major cause of failure of treatment. Many multidrug resistance mechanisms have contributed to lung cancer drug resistance, such as P glycoprotein (P gp), multidrug resistance associated protein (MRP), lung resistance related protein (LRP). The drug efflux pump mechanisms are the focus of recent researches.
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Objective To study the effect of IFN-γ inhalation via aerosol on cytokines of the immunocompromised rats. Methods Immunocomprised rat model was established with cortisol acetate injection for 14 d and then Candida albicans fluid was injected by tracheal for establishing am immuno comprised with pulmonary infection model. IFN-γ was inhaled with aerosol 1 d before the bacterium injection and then for 1, 3 and 7 d respectively. The activity of TNF-α, and the levels of IL-1β and IL-6 in the supernatant of the cultured alveolar macrophage(AM), the activity of IFN-γ and TNF-α in bronchial alveolar lavage fluid (BALF), the expressions of IFN-γ,TNF-α, IL-1β, and IL-6 of the lung tissues, the level of IFN-γ,IL-1β, and IL-6 in the serum were investigated. Results The activity of TNF-α, and the levels of IL-1β and IL-6 in the culture supernatant of the AM of the rats treated with IFN-γ were significantly higher than those of the control. The activity of IFN-γ and TNF-α in BALF was higher in the IFN-γ inhaled rats than in the control (except the activity of TNF-α on the 7th day). The expressions of IFN-γ and IL-1β in lung tissues was higher in the rats treated with IFN-γ than in the control. The expression of TNF-α in the rats treated with IFN-γ was less than that in the control rats. The expression of IL-6 had no difference between 2 groups. And no difference was found in the activity of IFN-γ, and the levels of IL-1β and IL-6 in the serum between 2 groups(except IL-1β on the 3rd day). Conclusion Administration of IFN-γ via aerosol can obviously increase the activity or levels of some cytokines in the lung of the immunocompromised rats, but has no effect on them in serum of the immunocompromised rats.
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Objective To study the expression of a new drug resistance-related gene cDNA fragment from human lung adenocarcinoma cell line in several types of lung cancer and their adjacent normal tissues. Methods The in situ hybridization histochemistry (ISHH) and Northern blot were employed to study the location and expression of the cDNA fragment in lung cancer cells. Results The results of ISHH and Northern blot showed that the expression of the fragment was discovered in 6 cases of 12 patients with lung adenocarcinoma, but not observed in the lung cancer tissues of other types and normal tissues(P
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Objective To construct an effective expression system for secretory canstatin, and to study its biological effects. Methods Canstatin cDNA was ligated with the cDNA of CEA signal peptide by SOE PCR, and the new fused fragment was cloned into eukaryotic expression vector pCMV-Script. The recombination vector pCMV-CEAS-Cans was transferred into human umbilical vein endothelial cells (HUV-EC) and human lung adeno-carcinoma cell line A549 by cationic liposome. The expression of canstatin mRNA together with CEAS mRNA in the transformed cells were detected by real time PCR method. Results Canstatin mRNA was detected in both transformed cells. The 3 H-TdR intake rate in pCMV-CEAS-Cans transformed HUVEC cells is significant lower than that of the pCMV-Script transformed cells (P
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objective In the former research work, a differential-expressed gene was cloned from multi-drug resistance lung cancer cell line (SPC-A-1/CDDP) with suppression substractive hybridization, and in this study we further analyze the site of this gene on the chromosome, and appraise its function related to multi-drug resistance in lung cancer cells. Methods The cDNA sequence data of the gene was input to computer and analyzed to ascertain its site on human chromosome by screening the gen bank on the www.ncbi.nlm.nih.gov. With DNA recombination technique, the gene was reversedly inserted to the vector pLXSN to get an antisense expression recombinant vector pLXSN-R, which was then transfected into SPC-A-1/CDDP cells with the aid of electroporation technique. And the semi-quantitative RT-PCR technique was used to quantify the mRNA content of gene in the transfected cells. Finally, the chemosensitivity of the transfected cells was tested with MTT method. Results The gene was located on the human chromosome 19q13.3-19q13.4 locus. The antisense gene recombinant vector was successfully constructed and transfected into SPC-A-1/CDDP cells as shown by its inhibitory activity on the mRNA expression of the gene. The drug-resistance indexes of transfected SPC-A-1/CDDP cells for cisplatin, doxorubicin, 5-fluorouracil, vincristin, hydroxycamptothecine and etoposide were obviously decreased. Conclusion The function of this gene is related to multi-drug resistance in human lung cancer cell, and its locus on the human chromosome is at 19q13.3-1913.4.
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Purpose:To study the biological effects of high secreted canstatin on human umbilical vein endothelial cells (HUVEC) and tumor model. Methods:Hypoxic responsive elements (HREs) were inserted in the upper stream of canstatin cDNA of a recombinant vector we constructed in a former research. The new recombinant vector named pCMV-3HRE-CEAS-Cans was transferred into A549 cells by cationic liposome; canstatin mRNA expression in the transformed cells under oxic and anoxic condition was detected by Taqman PCR. Then we designed a co-cultured assay: HUVECs were co-cultured with recombinant vector transformed A549 cells with Transwell plates, and the proliferation and apoptosis of co-cultured HUVECs were evaluated with 3H-thymidine incorporation method, TUNEL method respectively. Finally the vector was introduced into tumor tissues of lung cancer-bearing nude mice, and the biological activity in the tumor tissues was tested by micro-vessel count (MVC). A vector of canstatin we constructed before was used as controls in above experiments.Results:The pCMV-3HRE-CEAS-Cans vector was successfully constructed and transferred into A549 cells. canstatin mRNA was detected both in the recombinant vector transformed A549 cells and the pCMV-CEAS-Cans transformed A549 cells, moreover the expression of canstatin in the new vector transformed A549 was significantly higher than that of the controls under hypoxic condition. ~(3)H-TdR intake rate of HUVECs was decreased markedly, and a large amount of them underwent apoptosis when they were co-cultured with recombinant vector transformed A549 cells. Significant differences of ~(3)H-TdR intake rate and apoptotic rate of co-cultured HUVEC were found between pCMV-3HRE-CEAS-Cans group and any of the controls (P
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AIM: To study the effect of IFN-? inhalation on the anti-infection ability of the lungs in the immunocompromised host. METHODS: The immunological factors in the immunocompromised rats and the immunocompromised rats administrated IFN-? via aerosol were investigated after 1, 3, 7 days when they were injected Candida albicans via tracheal The Canidda albicans count of the left lung was also determined after 7 days when injecting pathogen. RESULTS: The Canidda albicans count of the left lung in IFN-? group was significantly less than that of control group. The phagocyting and bactericidal percentages, Ia antigen expression percentages, the levels of TNF-?, IL-1? and IL-6 in the culture supernatant of the AM, the activity of IFN-? and TNF-? in BALF (except the TNF-? on 7 th day) in IFN-? group were markedly higher than those in control group. The expression of IFN-? and IL-1? pulmonary tissues in IFN-? group was higher than that in control group. The expression of TNF-? in IFN-? group was less than that in control group. The expression of IL-6 was no changes between two groups. The levels of IFN-?, IL-1? and IL-6 in the blood (except IL-1? on 3 rd day), and the killing ability of the lymphocytes in blood had no difference between two groups. CONCLUSION: Administration of IFN-? via aerosol obviously enhanced the anti-infection ability of the lungs in the immunocompromised host, but has no influence on the whole body cellular immunity.