【Abstract】 Objective To detect the expression of lung resistance protein (LRP) and investigate its significance in pancreatic carcinoma cell lines (SW1990, PCT-2, PCT-3, PCT-4, Aspc-1, Capan-1, Mia-PaCa-2 and Panc-1). Methods Reverse transcription PCR (RT-PCR) and immunocytochemistry (ICC) were carried out to investigate the expression of LRP. Results LRP mRNA was absent in PCT-2 cell line by RT-PCR. Mild to moderate expression level was found in other pancreatic carcinoma cell lines. PCT-4, Aspc-1 and Panc-1 presented the highest LRP mRNA expression level, in contrast, SW1990, PCT-3, Capan-1 and Mia-PaCa-2 showed moderate LRP mRNA expression. The median value was 0.56±0.33. LRP was further validated by ICC. Absent to weak protein expression of LRP was found in PCT-2 and PCT-3. Overexpressed LRP was present in SW1990, Capan-1 and Aspc-1, furthermore, the highest expression of LRP was found in Panc-1, Mia-PaCa-2 and PCT-4 cell lines. Conclusion All these data showed that LRP might play an important role in multidrug resistance of pancreatic carcinoma.
【Abstract】ObjectiveTo construct a mrp1 expression vector and investigate its biological characteristics in HepG2 cells in vitro. MethodsThe 6.5 kb multidrug resistanceassociated protein （MRP) cDNA obtained from plasmid pGEM-mrp1 was cloned into the pCI-neo mammalian expression vector, which was later transferred into human hepatocarcinoma cell line HepG2 by liposome. Then the HepG2 cells resisting G418 were clustered and proliferated, and the mrp1 mRNA and MRP in these HepG2 cells were detected by means of RT-PCR and FCM respectively. ResultsThe mrp1 expression vector was established successfully, and the stable MDR hepatocarcinoma cell line (HepG2/mrp1) was developed as well. The content of the specific fragment of mrp1 mRNA was (56.8±6.37)% and MRP was 7.89 in the HepG2/mrp1 cells, the corresponding value in HepG2 cells was (9.67±3.26)% and 0.79 respectively. The difference was statistically significant (P＜0.05). ConclusionIt is practicable to establish MDR hepatocarcinoma cell line by transferring mrp1 cDNA into HepG2 cells, which is useful in the research of MDR mechanism.
ObjectiveTo explore the prognostic risk factors of bloodstream infections caused by Acinetobacter baumannii in the hospital, to provide a basis for clinical diagnosis and treatment.MethodsA retrospective analysis was performed on the medical records of patients diagnosed with Acinetobacter baumannii bloodstream infection in Guangxi Zhuang Autonomous Region People’s Hospital between January 2013 and December 2018. The patients were divided into survival group and non-survival group according to the outcome within 30 days after blood culture was collected. Univariate and multivariate logistic analyses were used to identify the risk factors of Acinetobacter baumannii bloodstream infections.ResultsA total of 123 patients were included, including 48 in the survival group and 75 in the non-survival group. Third generation cephalosporins [odds ratio (OR)=2.492, 95% confidence interval (CI) (2.125, 2.924), P<0.001], carbapenems [OR=1.721, 95%CI (1.505, 1.969), P<0.001], multidrug resistant-Acinetobacter baumannii infection [OR=1.240, 95%CI (1.063, 1.446), P=0.006], post-operation [OR=0.515, 95%CI (0.449, 0.590), P<0.001], mechanical ventilation [OR=1.182, 95%CI (1.005, 1.388), P=0.043], indwelling central venous catheter [OR=0.116, 95%CI (0.080, 0.169), P<0.001], mixed infection or septic shock [OR=3.935, 95%CI (2.740, 5.650), P<0.001], APACHE Ⅱ score (≥15) [OR=5.939, 95%CI (5.029, 7.013), P<0.001], chronic kidney disease [OR=1.440, 95%CI (1.247, 1.662), P<0.001], immune system disease [OR=28.620, 95%CI (17.087, 47.937), P<0.001], use of corticosteroids [OR=0.520, 95%CI (0.427, 0.635), P<0.001], and combined antifungal agents [OR=0.814, 95%CI (0.668, 0.992), P=0.041] were independent factors for predicting the prognosis of patients with bloodstream infections caused by Acinetobacter baumannii.ConclusionsThe third generation cephalosporins, carbapenem, MDR-Acinetobacter baumannii infection, post-operation, mechanical ventilation, indwelling central venous catheter, mixed infection or septic shock, APACHE Ⅱ score (≥15), chronic kidney disease, immune system disease, use of corticosteroids, and combined antifungal agents were independent factors for predicting the prognosis of patients with bloodstream infections caused by Acinetobacter baumannii. In the clinical work, it is needed to carry out timely detection of microbial etiology, timely report, and reasonable treatment.
Objective To investigate the reversal of the multidrug resistant gene mdr1 in vivo by antisense oligodeoxynucleotide （ASODN） on the basis of study in vitro. Methods The cultured drug-resistant human hepatocellular carcinoma cells were injected under the skin of axilla to establish the tumor model of nude mice. mdr1 ASODN accompanied by Lipofectamine were injected locally and ADM was injected intraperitoneally. Control 1 and control 2 were locally injected by Lipofectamine and normal saline separately, and ADM was also injected intraperitoneally. Results As time went on the tumor size increased and from the 5th day on alterations were marked, tumor size in different time phase showed marked difference to the prior time phase with significant difference (P＜0.05）. Tumor size in group ASODN was marked smaller than that of other 3 groups after the 5th day (P＜0.05)，while tumor size of group control 1,2 and group SODN in different phase showed no significant difference (Pgt;0.05). The results suggested that SODN and Lipofectamine showed no marked effect on tumor growth of nude mice and ASODN had marked inhibition effect on tumor growth. Conclusion mdr1 ASODN can also reverse multidrug resistance of drug-resistant human hepatocellular carcinoma cells in vivo. After the treatment the tumor’s growth in nude mice will slow down in a range of time.
Objective To investigate the effect of phosphorothioate antisense oligonucleotides(AS-ODN) on suppressing multidrug resistance-associated protein gene(MRP) in human drug-resistant hepatocellular carcinoma cell line (SMMC-7721/ADM). Methods Cell line was transfected with a synthetic S-ODN complementary to the coding region of MRP mRNA, Lipofectamine acting as carrier. The drug sensitivity was measured by MTT assay. The expression of MRP mRNA was detected by RT-PCR and the expression of P190 was detected by flow cytometry. Results AS-ODN inhibited expression of MRP mRNA and P190 and promoted sensitivity to daunorubicinum and adriamycinum. Conclusion AS-ODN can reduce the expression of MRP gene. MDR caused by MRP is an important cause of multidrug resistance of SMMC-7721/ADM.
【Abstract】ObjectiveTo construct an mdr1 expression vector and detect its expression in HepG2 cells in vitro. MethodsThe 4.5-kb mdr1 cDNA was obtained from the plasmid pHaMDR1 cloned into the PCIneo mammalian expression vector, which was later transferred into human hepatocarcinoma cell line HepG2 by liposome. Then the HepG2 cells resisting G418 were clustered and proliferated，and the specific fragment of mdr1 cDNA, mRNA and the Pgp in these HepG2 cells were detected by means of PCR, RT-PCR and FCM respectively. ResultsThe mdr1 expression vector was constructed successfully，and the stable multidrug resistance(MDR) hepatocarcinoma cell line （HepG2/mdr1） was developed as well. The outcome of PCR analysis showed that the specific fragment of mdr1 cDNA could be found in HepG2/mdr1 cells, but not in the nontransfection HepG2 cells. Furthermore,the content of the specific fragment of mdr1 mRNA and the expression of P-gp in HepG2/mdr1 cells were (59.7±7.9)% and (12.5±5.45)% respectively, the corresponding value in HepG2 cells were (16.9±3.2)% and (4.63±2.59)% respectively. The difference was statistically significant (P＜0.05). ConclusionIt is praticable to develop MDR hepatocarcinoma cell line by transferring mdr1 cDNA into HepG2 cells, which is useful in the research of MDR mechanism.
ObjectiveTo construct the recombinant adenovirus vector carrying antisense multidrug resistanceassociated protein (MRP) and transfect the human drugresistant hepatocellular carcinoma cell line(SMMC7721/ADM). MethodsThe fragment of MRP gene encoding 5′region was cloned reversely into the shuttle plasmid pAdTrackCMV, with the resultant plasmid and the backbone plasmid pAdEasy1,the homologous recombination took place in the bacteria and the recombinant adenoviral plasmid was generated. The adenoviruses were packaged and amplified in 293 cells. Then the cell line of SMMC7721/ADM was transfected with the resultant adenoviruses.ResultsThe recombinant adenovirus vector carrying antisense MRP was constructed successfully. The viral titer was 2.5×109 efu/ml, and more than 90% SMMC7721/ADM cells could be transfected when the multiplicity of infection（MOI） was 100. ConclusionThe recombinant adenovirus vector constructed by us could introduce the antisense MRP into the human drugresistant hepatocellular cell line effectively, which would provide experimental basis for the mechanisms and reversal methods of the multidrug resistance in human hepatocellular carcinoma.
ObjectiveTo explore the relationship between mdr1 gene expression of hepatocellular carcinoma (HCC) and pathological characteristics,chemotherapy and prognosis. MethodsThe mdr1 gene expression of HCC in 56 patients with the methods of immunohistochemistry was studied. The results were analysed with the pathological data by statistic methods. ResultsThe positive expression of mdr1 gene in cancer tissues and pericancerous tissues of HCC were 30/56(53.6%) and 19/56 (33.9%) respectively. The difference was statistically significant (χ2=4.39,P＜0.05). The positive expression of mdr1 gene in cancer tissues of untreated patients and in recurrent patients were 22/48(45.8%) and 8/8(100%) respectively.The expression of mdr1 gene was not associated with tumor size, number, tumor thrombus, differentiation, HBsAg and liver cirrhosis. The patients with positive mdr1 expression had a shorter survival time than that of negative ones. But the difference was not statistically significant. Conclusion The positive expression of mdr1 in HCC is 53.6%. It is not associated with tumor size, number, tumor thrombus, tumor differentiation, HBsAg and liver cirrhosis. There are innate multidrug resistance in HCC.
Objective To dynamically study the formation of multidrug resistance(MDR) of human hepatocellular carcinoma cell SMMC-7721 induced by Adriamycin (ADM) and the role of multidrug resistance-associated protein(MRP) in its mechanisms.Methods Hepatocellular carcinoma cell SMMC-7721 was cultured in RPMI-1640 medium containing ADM with progressively increased concentration or directly cultured in medium containing different concentrations of ADM. Resistant index of drug-resistant variants of SMMC-7721 cell was determined by drawing cell dosage-reaction curves.Levels of MRP mRNA expression were detected by reverse transcription-polymerase chain reaction(RTPCR). Intracellular rubidomycin(DNR) concentration was examined by flow cytometry(FCM).Results With progressive increasing of ADM concentration in medium resistant index and levels of MRP mRNA expression were correspondingly increased but intracellular DNR concentration was markly reduced. When parental cells were directly cultured in medium containing different concentrations of ADM, the higher the ADM concentration, the higher the level of MRP mRNA expression, but intracellular DNR concentration was kept at the similar high level and most cells died. Conclusion ADM may progressively induce SMMC-7721 cell resistant to multiple chemotherapeutic drugs with reduced intracellular DNR accumulation associated with the overexpression of MRP gene.
ObjectiveTo analyze the distribution and drug resistance of Enterobacteriaceae in West China Hospital of Sichuan University, to provide long-term monitoring data references for clinical practice.MethodsThe clinical information of non-repetitive Enterobacteriaceae isolates from 2006 to 2015 was collected and analyzed. All the isolates were identified by VITEK-2 Compact Automatic Microbial Identification Analyzer (Bio Merieux, France). The statistic informations were analyzed by WHONET 5.6 and iLabDataforMDR 1.03.ResultsA total of 38 487 strains of Enterobacteriaceae were isolated from 2006 to 2015, mainly including 14 862 stains of Escherichia (38.6%), 12 894 stains of Klebsiella (33.5%), 6 277 stains of Enterobacter (16.3%), 1 758 stains of Proteus (4.6%), 1 257 stains of Serratia (3.3%), 933 stains of Citrobacter (2.4%), and 506 stains of Morganella (1.3%). The top three sample types were sputum (46.9%), urine (18.7%), and secretions (11.5%). The drug resistance rate of Enterobacteriaceae showed a downward trend to most antibacterials. The average resistance rate of Enterobacteriaceae to ampicillin, ampicillin/sulbactam, and cefazolin was 85.3%, 52.6%, and 72.9%, respectively. The resistance rates to ceftriaxone, cefepime, gentamicin, and tobramycin were significantly reduced. The resistance rates to other antibiotics showed decreasing or slow increasing trends. The isolation rate of extended-spectrum β-lactamases (ESBL)-producing strains in Escherichia did not change, but the rate in Klebsiella decreased significantly. The isolation rate of multidrug-resistant organisms (MDRO) showed a slow decrease.ConclusionsThe overall antimicrobial resistance and the isolation rates of MDRO and ESBL-producing organisms showed a downward trend in investigating period. However, the carbapenem-resistant Enterobacteriaceae was rising continuously. Long-term monitoring of drug resistance is of notable value to antibiotic management policies.
