Construction of bait plasmid containing SIGIRR cytoplasmic tail and detection of self-activation in yeast two hybrid system / 重庆医学

Objective To construct the bait plasmid of pSos-single immunoglobin IL-1 receptor related protein (SIGIRR) in Cy-toTrap yeast two hybrid system ,and to test its self-activation .Methods The cDNA fragments of SIGIRR(480 -1 230 bp) were amplified from pReceiver-LV19-SIGIRR and ligated into the bait plasmid pSos to generate the plasmid pSos-SIGIRR .The pSos-SI-GIRR was identified by DNA sequencing and dual-site endonuclease digestion .Then the recombinant plasmid and control plasmid were introduced into the yeast cell cdc25H .The transformants were inoculated on plates of 25 ℃ /SD/Glucose(-UL) ,25 ℃/SD/Ga-lactose(-UL) ,37 ℃ /SD/Glucose(-UL) and 37 ℃ /SD/Galactose ,respectively and the proliferation ability of transformant was ob-served for 6 d .The Western blot was adopted to detect the expression of target protein .Results The pSos-SIGIRR vector was cor-rectly constructed and proved of no self-activation and toxic action .The Western blot showed that the target protein was expressed in a form of fusion protein of 170KD .Conclusion The bait plasmid containing SIGIRR cytoplasmic tail can be applied to the yeast two-hybrid system and lays the important foundation for seeking the interacting protein with SIGRR from the human lung cDNA li-brary in .

Alteration levels of IL-17 and IL-19 inflammatory factors among deadaptation personnel from the plateau / 军事医学

Objective To analyze the change in the concentration of IL-17 and IL-10 inflammatory factors among the deadaptation personnel who returned from the plateau.Methods A total 21 healthy males were investigated who averaged 25 years in age, lived permanently in the plains (200 m), and once stayed to the plateau (Lasha) for 6 months.Their venous blood was collected at three time points:the day before ascending to the plateau(control), the second day after return to the plains(d2) and the 30th day(d30), respectively.Their serum was seperated from the whole blood and the level of IL-17A and IL-10 was detected by ELISA method.Results The concentration of IL-17A and the IL-17A/IL-10 ratio were significant increased at d2 and d30, respectively, compared with control (P<0.05).Compared with d2, IL-17A and the IL-17A/IL-10 ratio were decreased obviously at d30(P <0.05).The level of IL-10 at d2 and d30 was significantly reduced compared with control ( P <0.05), but increased at d30 compared with d2.Correlative analysis showed that there was a negative correlation in the levels of IL-10 and IL-17A between control, d2 and d30, respectively (r1=0.948, P<0.05;r2=0.969, P<0.05;r3=0.972, P<0.05).A significant negative correlation was observed in the alteration levels of IL-10 and IL-17A between the three groups(r4=-0.793, P<0.05; r5=-0.756, P<0.05). Conclusion The concentration of inflammatory factors among the plateau deadaptation patients is imbalanced, but it is gradually reduced with time.The mechanism is still not clear.

Adenovirus-mediated protein-kinase-GIα suppresses the hypoxia-induced proliferation and phenotype-switching of pulmonary arterial smooth muscle cell / 中华内科杂志

Objective To observe the proliferation and phenotype-switching of pulmonary arterial smooth muscle cell (PASMC) induced by hypoxia and interfered by Ad-PKGIα. And to investigate the potential regulative role of PKGIα gene in the molecule mechanism of hypoxia pulmonary vessel remodeling (HPVR). Methods To establish the pure PASMC cultured by tissue-sticking methods. Semi-quantitative reverse transcription and polymerase chain reaction (RT-PCR) and Western blot were used to examine the PKGIα mRNA and protein expression after PASMC were transfected by Ad-PKG. The mRNA and protein expressive change of smooth muscle α actin(SM-α-actin) determined the degree of cell phenotype-switching. The changes of PASMC proliferation were determined by flow cytometry and ~3H-TdR incorporated way. Results Ad-PKGIα could transfect into PASMC and highly express. Hypoxia down-regulated the expression of SM-α-actin protein (44. 25±5.34 in normoxia, 32. 18±4. 19 in 12 h hypoxia condition, 21.90 ±2. 44 in 24 h hypoxia condition, P < 0. 05), that could be blocked by the transfeetion of Ad-PKGIα. Hypoxia could push PASMC mitosis and proliferating(~3H-TdR incorporated way: 7570 ± 371 in normoxia,12 020± 831 in 12 h hypoxia condition,14 924 ± 1491 in 24 h hypoxia condition, P <0. 05), that could be blocked by the transfection of Ad-PKGIα, too. Conclusions The results suggested that PKGIα signaling pathway might play an important role in the molecule mechanism of HPVR. And PKGIα gene might be a target point of gene therapy.

Negative regulation of cells proliferation by Gax gene and its mechanisms / 生物医学工程学杂志

Gax gene is a newly found negative transcriptional regulator of cells proliferation. This paper introduces the detection and structural features of Gax, details the inhibitory effect of Gax on the proliferation of vascular smooth muscle cells, vascular endothelial cells and cancer cells, and explains the putative mechanisms therein involved. The potential for providing therapeutic insights into human diseases by modulating Gax activity is prospected.

Construction of eukaryotic expression vector of Der p2 gene and its expression in mouse dendritic cells / 中国组织工程研究

BACKGROUND: Dendritic cells, the most potent antigen presenting cells known at present, have been extensively used in the immunotherapy as adjuvant. OBJECTIVE: The present study was to construct Der p 2 eukaryotic expression vector and validate its expression in the mouse bone marrow-derived dendritic cells. DESIGN, TIME AND SETTING: A single sample observation was performed at the Institute of Respiratory Disease,Xinqiao Hospital, Third Military Medical University of Chinese PLA between May and December 2005. MATERIALS: C57BL/6 mice were included. Plambd-Der p 2 was the product of Heska Company, USA.pCI-neo plasmid was provided by the Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University of Chinese PLA.METHODS: Mouse bone marrow-derived dendritic cells were in vitro isolated and cultured.Complete Der p 2 cDNA was spliced from prokaryotic expression vector plambd-Der p 2, and then cloned into eukaryotic expression vector pCI-neo (pCI-neo-Der p 2).The positive recombinants pCl-neo-Der p 2 transfected into dendritic cells.Non-transfected and blank vector pCI-neo-transfected dendritic cells were used as controls. MAIN OUTCOME MEASURES: ①Identification of pCI-neo-Der p 2 recombinant plasmid.②Detection of Der p 2 mRNA and protein expression by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot techniques. RESULTS: Sequencing results showed Der p 2 cDNA in pCI-neo-Der p 2 was in coincidence with the sequence registrated in Gene Bank.RT-PCR and Western Blot results showed that expression of Der p 2 mRNA and protein could be detectable in the pCI-neo-Der p 2-transfected dendritic cells. CONCLUSION: The Der p 2 cDNA was successfully constructed into the eukaryotic expression vector, and Der p 2 gene and protein could be expressed efficiently in dendritic cells.

Effect of Ad-Gax transfection on apoptosis of human lung adenocarcinoma A549 cells and its mechanism / 中国肺癌杂志

<p><b>BACKGROUND</b>Apoptosis is closely related to development of lung cancer. It is a strategy of lung cancer therapy to induce apoptosis. The aim of this study is to explore the effects of growth arrest-specific homeobox (Gax) transfection on apoptosis and expression of Bcl-2 and Bax proteins of human lung adenocarcinoma A549 cells.</p><p><b>METHODS</b>A549 cells were transfected with Gax gene by a replication-deficient adenovirus expressing the hemagglutinin-tagged Gax cDNA (Ad-Gax). Apoptosis of A549 cells was observed by transmission electronic microscope and terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) positive staining. Apoptotic rate of A549 cells was evaluated by flow cytometry. Expressions of Bcl-2 and Bax proteins in A549 cells were detected by immunocytochemistry.</p><p><b>RESULTS</b>Before Ad-Gax transfection, none or few of TUNEL-positive A549 cells were detected. After Ad-Gax transfection, a marked increase in TUNEL-positive staining occurred, especially at 24 h later. The ratio of apoptosis of A549 cellsin non-transfection group and transfection groups at 12 h, 24 h, 48 h were 0.25%, 12.57%, 17.29%, 15.03%, respectively. Compared with non-transfection group, the apoptotic rates of transfection groups increased significantly (Chi-square value was 7.357, 11.126 and 9.943 respectively, P < 0.01). The average optical density (AOD) of Bcl-2 protein in A549 cells in non-transfection group and transfection groups at 12 h, 24 h, 48 h were 2.02±0.07, 1.79±0.02, 1.25±0.51 and 1.21±0.24 respectively. Compared with non-transfection group, AOD of Bcl-2 protein in A549 cells in transfection groups decreased significantly (t value was 6.651, 7.089 and 7.438 respectively, P < 0.01). On the other hand, Bax protein expression in transfection groups increased, the AODs of Bax were 4.49±0.61, 4.24±0.37 and 3.95±0.43, respectively. Compared with non-transfection group (3.12±0.42), AOD of Bax protein in A549 celle in transfection groups increased significantly (t value was 7.469, 7.287 and 6.473 respectively, P < 0.01). In the Ad-Gax transfection groups the lower Bcl-2/Bax ratio was, the higher the apoptotic rate of A549 cells was (r=-0.49, P < 0.01).</p><p><b>CONCLUSIONS</b>Ad-Gax transfection can induce A549 cells apoptosis. Possible mechanism is that Gax can downregulate Bcl-2 protein expression and upregulate Bax protein expression, and A549 cells apoptosis is related to the Bcl-2/Bax ratio.</p>

Effects of Gax gene transfection on proliferation and expression of proto-oncogenes in A549 cells / 中国肺癌杂志

<p><b>BACKGROUND</b>Lung cancer is the leading cancer of malignant tumor in China.It is the direction that poeple make efforts to seek gene therapy of lung cancer. The aim of this study is to explore the effects of transfected growth arrest-specific homeobox gene (Gax gene) on the proliferation and expressions of c-fos and c-jun mRNA in A549 cells.</p><p><b>METHODS</b>A549 cells were transfected with Gax gene by adenovirus. Expressions of Gax mRNA and protein were detected by RT-PCR and immunocytochemistry. The expressions of c-fos and c-jun mRNA were evaluated by RT-PCR. The proliferation inhibition effect of Gax transfection on A549 cells was evaluated by MTT assay.</p><p><b>RESULTS</b>Only in the A549 cells transfected with Gax gene the Gax expression was confirmed by RT-PCR and immunocytochemistry. Compared with that in the control group, c-fos and c-jun mRNA level decreased significantly in Gax-transfected A549 cells (t=7.755, P < 0.01; t= 5.938 , P < 0.01). MTT assay showed that the proliferation inhibition rates of A549 cells transfected by Ad-Gax for 24h, 48h and 72h were (47.35±5.36)%, (54.96±1.78)%, and (65.39±5.11)% respectively. And these proliferation inhibition rates were significantly higher than those in the control group (Chi-Square=7.152, 9.431 and 12.847, P < 0.01).</p><p><b>CONCLUSIONS</b>Gax gene can inhibit the proliferation of A549 cells. Its molecular mechanism may be through down-regulating the expressions of c-fos and c-jun.</p>

New Clinical Teaching Mode To Cultivate Practitioner / 中华医学教育探索杂志

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.

Expression of canstatin gene in human lymphocytes and its inhibitory effect on growth and metastasis of Lewis lung carcinoma / 中国肺癌杂志

<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>

Summarization in the research of canstatin / 生物医学工程学杂志

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.

Dynamic changes of apoptotic rate of peripheral blood polymorphonuclear neutrophils in the patients with chronic obstructive pulmonary disease / 中国组织工程研究

BACKGROUND: Chronic obstructive pulmonary disease(COPD) is characterized of chronic inflammation in airway, pulmonary parenchyma and pulmonary vessels. The mechanism of the increment and activity changes of these inflammatory cells is unclear at present.OBJECTIVE: To study the apoptotic character of peripheral blood polymorphonuclear neutrophils(PMNs) and its relationship with COPD to provide a reference for early intervention and function surveillance for COPD patients.DESIGN: An observatory comparative study based on COPD patients and healthy population as controls.SETTING: Department of pulmonary medicine in a military medical university of Chinese PLA affiliated hospital.PARTICIPANTS: Totally 98 COPD patients were admitted by the Department of Pulmonary Medicine, Research Institute of Surgery, Daping Hospital, Third Military Medical University of Chinese PLA between February 2003 and December 2003 due to COPD acute attack. Eighteen patients including 12 males and 6 females aged between 48 and 70 years old [mean of(56 ± 7) years old]were randomly selected into COPD group according to random number table.Totally 14 healthy adults including 10 males and 4 females aged between 50 and 70 years old [mean of (59 ± 8) years old] who were individuals came for physical check up in our hospital were selected in control group.METHODS: PMNs were separated from peripheral blood by density gradient centrifugation. The dynamic changes of PMNs apoptosis in peripheral blood was observed by flow cytometer and TUNEL method.MAIN OUTCOME MEASURES: Comparison of PMNs apoptotic rate in peripheral blood among groupsRESULTS: As indicated by flow cytometric analysis, PMNs apoptotic rate at early apoptotic phase in COPD patients at paracmasis was(8.5 ± 1.3)%,which was significantly lower than(12.5 ± 1.8)% of normal control group( t=6.25, P ＜ 0. 01); PMNs apoptotic rate was(5.1 ±0.6)% at acute aggravation stage, which was significantly lower than that of paracmasis group ( t =5.66, P ＜001). As indicated by TUNEL analysis, PMNs apoptotic rate at paracmasis was(12.42 ±2.7)% , which was significantly lower than (21.5±4.8)% of normal control group(t=5.76, P ＜ 0.01); PMNs apoptotic rate was(4. 9 ±0.4)% at acute aggravation stage, which was significantly lower than that of paracmasis group( t = 6. 12, P ＜ 0. 01 ) . PMNs changes at the late phase of apoptosis/necrosis had a contrary tendency, i. e.,PMNs rate at late apoptotic phase/necrosis was(2. 8 ± 0.5)% in COPD patients at paracmasis, which was significantly higher than(1. 3 ±0.4)% of normal control group ( t= 6. 37, P ＜ 0. 01 ); PMNs rate was (3.7 ± 0. 3) % at acute aggravation stage, which was significantly higher than that of paracmasis group(t=5.81, P ＜0.01).CONCLUSION: PMNs abnormal apoptosis might be an important reason that induces PMNs aggregation in airway and lung tissues in COPD. This process might have an important significance in the generation and development of chronic airway inflammation, which provides an etiologic basis for the primary rehabilitative intervention of COPD.

Effects of canstatin gene transfection on growth and apoptosis of lung cancer A549 cells and HUV-ECC cells / 中国肺癌杂志

<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>

A study on acid-base disorders and causes of death in patients with cor pulmonale / 第三军医大学学报

The results of analysing 179 patients with pulmonary heart disease (PHD) show an acid-base disorder just before death.Acid-base disorder,chronic respiratory acidosis is the most commoa Triple acid-base disorder,chronic respiratory acidosis plus metabolic acidosis and chronic respiratory acidosis plus metabolic alkalosis are in succession.Chronic respiratory alkalosis ( CRA),CRA plus metabolic alkalosis and metabolic acidosis are seldom.The major cause of death in patients with PHD is supper gastrointestinal hemorrhage,the next is acid-base disorder,especially triple acid-base disorders.

THE STATUS QUO AND PROSPECT OF ACUTE LUNG INJURY AND ACUTE RESPIRATORY DISTRESS SYNDROME / 解放军医学杂志

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) refers to acute progressive respiratory failure with stubborn hypoxemia induced by different predisposing factors except cardiogenic causes. The definition of ALI/ARDS have been renewed and standardized in recent years, thus there is a new understanding in the pathogenesis of ALI/ARDS. The new diagnostic criteria and the combining therapeutic measures of ALI/ARDS have been proposed. With the progress in research pertaining to ALI/ARDS and the new insight into the pathogenesis of intractable systemic inflammation, new therapeutic measures directing at controlling and regulating inflammatory responses may be expected to achieve a breakthrough in the treatment of ALI/ARDS.

Recent progress in research on the diagnosis and treatment of acute lung injury and acute respiratory distress syndrome / 解放军医学杂志

Acute lung injury(ALI) and acute respiratory distress syndrome(ARDS) are commonly encountered serious diseases.The pathogenesis of ALI and ARDS remains unclear at present.The mortality rate of ALI and ARDS remains high due to the Absence of sensitive method for early diagnosis and specific and effective therapy.The present paper will focus on the latest developments of diagnosis and treatment for ALI and ARDS.Diagnosis:Althongh theoretically it is possible to look for the diagnostic markers and target of injury by painstaking study of its pathogenesis,but as yet an ideal pathognomonic marker with due sensitivity and specificity remains unavailable.Diffuse alveolar injury is a major pathological feature and may act as a reliable parameter for the diagnosis of ALI and ARDS.However,it is difficult to obtain the lung tissues from living patients with ALI/ARDS,and dynamic monitoring of arterial blood gas,combined with clinical symptoms,is still the main criterion for early diagnosis of API and ARDS in clinical setting.Therapy:The use of corticosteroids to subdue rampant inflammatory reaction had not given a promising result in clinical study,so it is not recommended for routine use,however short-term corticosteroids may be considered for allergy-or aspiration-induced ALI in early stage.With the treatment of primary diseases as the main strategy,the main therapeutic measures for ALI/ARDS should be integrated treatment of multiple targets and correction of hypoxia.Mechanical ventilation is the main substitutive therapy for correction of hypoxia.Protective ventilation with adequate tidal volume and positive end-expiratory pressure(PEEP) are recommended.Briefly,management of primary disease,correct use of mechanical ventilation,nutritional support and specific treatment for pathological changes and clinical manifestation are important for ALI and ARDS patients.Hereafter,emphasis should be placed on seeking the methods for early diagnosis and effective therapies for diffuse alveolar injury and massive inflammation.

Changes of L-selectin expression on peripheral blood polymorphonuclear leukocytes and their significance in rats with acute lung injury / 中国病理生理杂志

AIM: To explore the changes of L-selectin expression on peripheral blood polymorphonuclear leukocytes (PMNs) and their significance in rats with acute lung injury (ALI). METHODS: ALI model in rat was established by intravonous injection of E. coli endotoxin (ET). The expression of L-selectin on peripheral blood PMNs was measured by immunofluorescence and flow cytometry.RESULTS: The contribution of L-selectin fluorescence signal was on the surface of PMNs membrane. The expression of L-selectin on poeripheral blood PMN was significantly lower at 5 min after injection of ET and the lowest during 15 min to 30 min, then gradually increased, but the expression of L-selectin on PMN was lower at 60 min after injection of ET than that of control animal.CONCLUSION: In physiological state, L-selectin were expressed on the surface of PMN membrane. The protein expression of L-selectin on PMNs reduced rapidly after injection of ET and the lowest at 15 min, then gradually increased. L-selectin may play a role in the development of ALI.

Analysis of chromosome variation of lymphocytes and comparative genomic hybridization of lung carcinoma in patients with long survival after operation / 中国癌症杂志

Background and Purpose:To analyse the chromosome of peripheral lymphocytes and tissue samples of lung cancer for the long term survival patients after surgery,which could provide the rationale for the screening of relative genes of carcinogenesis and clinical treatment of lung cancer.Methods:Twenty patients with over 5 year survival after operation were defined as long term survival group.There were 9 patients with squamous cell carcinoma,6 with adenocarcinoma and 5 with small cell carcinoma in the long term survival group.The control group was defined as the samples from the patients with lung cancer who had surgery within one year and had to be randomly chosen,8 patients were histologically proven as squamous cell carcinoma,7 as adenocarcinoma and 5 as small cell carcinoma in the control group.The samples from healthy donors were defined as the normal group.The chromosome variations of peripheral lymphocytes in all these 3 groups were analyzed by routine methods.The comparative genomic hybridization of the embed-paraffin tissue sample of lung carcinoma was done by the DeVries methods.Results:In the long term survival group,the chromosome variations rate of lymphocytes was 3.15%,which was lower than that of the control group(3.15% vs 10.85%,P

Clinical detection and modulation strategy of lung cancer multidrug resistance with membrane transport proteins as the target / 解放军医学杂志

The major problem in lung cancer chemotherapy is the emergence of inherent and acquired drug resistance of the cancer cells. The cancer becomes resistant not only to the drugs used initially, but also to those to which it has not yet been exposed, This condition is known as multidrug resistance (MDR). MDR has been found to be related to three members of the ABC-superfamily of membrane transport proteins (transporters) and one member of the non-ABC-superfamily of transporter. The formers are P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and breast cancer resistance pro-tein (BCRP). The latter is lung resistance-related protein (LRP). These findings provided new molecular targets to predict drug resistance, and an atraumatic detection method by using Tc-99m methoxyisobutyl isonitrile ( 99m Tc-MIBI ) SPECT has been developed for predicting MDR in lung cancer. Based on these transporters, different strategies are tried attempting to reverse drug resistance in lung cancer. Encouraging results have been acquired, but further research is necessary.

Construction of eukaryotic expression system of a cell surface molecule——human CD40 ligand / 第三军医大学学报

Objective To construct human eukaryotic expression system expressing T cell surface molecule CD40L and identify the expression product in SCCL cell line h446. Methods The expression vector pcDNA3.1-CD40L was constructed by recombinant DNA technology, then transfected into h446 cell line of SCCL by lipidosome methods, and the expressed membrane protein was identified by flow cytometry. Results The pcDNA3.1(+)-CD40L was constructed successfully and the coding region was inserted into the vector correctly. The membrane protein was observed by flow cytometry on transfected h446 cell line of SCCL. Conclusion Human CD40L was expressed successfully in eukaryotic system, which is very important in the anti-tumor vaccination study with a recombinant human CD40L in clinic.

Canstatin high level expression system and its biology effects on lung cancer / 中国癌症杂志

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