Objective To investigate the reversal effect of antisense phosphorothioate oligonucleotide （ASOND) on human hepatoma resistant cells. Methods Human hepatoma resistant cells SMMC-7721 was transfected with synthetic antisense phosphorothioate oligonucleotide complementary to the 5′ region flanking the AUG initiation codon mediated by lipofectamine. In vitro drug sensitivity was measured by MTT assay. The expression of P-170 was determined by flow cytometry and mRNA was assessed by RT-PCR. Results ASOND inhibited the expression of mRNA and p-170 in SMMC-7721, enhanced the sensitivity of SMMC-7721 to chemotherapeutic drug. The best inhibitory effect was achived by the dose of 0.5μmol/L. Conclusion ASOND enhanced the sensitivity of SMMC-7721 to chemotherapeutic drug and reversed the multidrug resistance of SMMC-7721 partially.
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 establish multidrugresistance cell substrain of human hepatocellular carcinoma and to investigate its characteristics.MethodsSMMC7721 cell strain was cultured in Adriamycin(ADM). The multidrugresistance cell substrain SMMC7721/ADM was harvested after a long period of culture by gradually increasing the concentration of ADM and its characteristics were investigated. Results①The drug resistance of SMMC7721/ADM to ADM increased by 33.3 times, to Vincristine 16.8 times, to Diamminedichloroplatinum 2.8 times. ②The drug resistance cell substrain had almost the same growth velocity as its parental generation. The doubling time was 32.0 hours and 30.5 hours respectively. They had the analogous growth curves. ③The obvious difference between the drug resistance cell substrain and its parental generation was that the former’s microvilli became thick, short and scattered by scanning and transmitting electron microscopy. ④The multidrug resistance cell substrain kept the characteristics of hepatocellular carcinoma, it could be transplanted into the subcutaneous tissue of nude mice. ⑤The drug resistance of the cell substrain reduced to 28.0% and 9.2%after removal of the drug for 1 month and 2 months respectively, its drug resistance could remain stable (35.4 times) after 2 months of culture in ADM (0.04 μg/ml).ConclusionThe SMMC7721/ADM cell substrain has the stable fundamental characteristics of a drug resistance cell strain.
Objective To detect the characteristic of multidrug resistance gene products expressions in gastric cancer tissues, including glutathione-s-transferase π (GST-π), P-glycoprotein (P-gp), topoisomerase Ⅱ (Topo-Ⅱ), thymidylate synthase (TS), and multidrug resistance related protein (MRP), and analyze their clinical significance for the therapy of gastric cancer. Methods SP immunohistochemical stain was used to detect GST-π, P-gp, Topo-Ⅱ, TS and MRP expressions in sample of 48 gastric cancer tissues and 10 normal gastric mucosa. And their corresponding clinical data were comprehensive analyzed. Results The expressions of GST-π, P-gp, Topo-Ⅱ, TS and MRP had notable differences between the gastric cancer tissues and normal gastric mucosa (GST-π： P＜0.01； P-gp： P＜0.01； Topo-Ⅱ： P＜0.01； TS： P＜0.05； MRP： P＜0.05). Positive expression rates of GST-π, P-gp, Topo-Ⅱ, TS and MRP in gastric cancer tissues were 72.9% (35/48), 56.3% (27/48), 83.3% (40/48), 41.7% (20/48) and 39.6% (19/48), and positive expression rates of them in normal gastric mucosa were 10.0% (1/10), 0 (0/10), 0 (0/10), 0 (0/10) and 0 (0/10) corresponding. Their positive expression rates were closely relevant to the degree of differentiation (P＜0.01), but not to the patients’ sex, age, tumour site, size of tumour, invasive depth and lymph node metastasis (Pgt;0.05). Conclusions The expressions of GST-π, P-gp, Topo-Ⅱ, TS and MRP in gastric cancer tissues exist obvious heterogeneity. Their overexpression underlie the multidrug resistance of gastric cancer. The joint detection of GST-π, P-gp, Topo-Ⅱ, TS and MRP can be looked as an important symbol for guiding its chemotherapy.
【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 evaluate the effect of neoadjuvant chemotherapy and find the mechanism of multidrug resistance. MethodsTwenty patients with gastric cancer and 31 patients with colorectal cancer underwent neoadjuvant chemotherapy and then operations. The preoperative specimens were stained by immunohistochemical techniques for testing p53,multidrug resistanceassociated protein (MRP), glutathione S transferase（GST）, telomerase. Resection specimens were evaluated for chemotherapy effect by routine histology; at the same time, the postoperative morbidity and mortality were observed. ResultsIn 51 patients, the response rate of neoadjuvant chemotherapy was 27.45%(14/51),so multidrug resistance was a kind of common phenomena in gastrointestinal carcinomas. The postoperative morbidity was 15.69%(8/15), the main operation complication was infection,the mortality was 1.96%(1/51),only one person died from severe infection.The expression rate of p53, MRP, GST, telomerase was 58.0%,51.0%,66.7%,74.0%respectively, the location of p53 was at cell nucleus,location of MRP,GST was at cell memberane and cytoplasm,location of telomerase was at cytoplasm.The response rate had nothing to do with age, sex and metastasis. But it was related with p53 and telomerase expression. ConclusionNeoadjuvant chemotherapy is an effective, safe therapy. But the rate of drug resistance is high in gastrointestinal carcinomas, and the response rate is related to p53, telomerase expression.
Objective To review the application advancements of ATP-binding cassette (ABC) transporter in medical research.Methods Relevant literatures about the applications of ABC families in medical research were reviewed. Results ABC families mainly took roles in transporting substances across cell membrane. Some of them were useful for the prediction of drug resistance and the prognosis of malignant tumors. Others were target s for molecular researches. Their expressions or mutations might be related with the occurrence of diseases. Conclusion ABC families are very important in the diagnosis and therapy for diseases. Thus they are very promising tools for future medical research.
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.
Objective To investigate the detection of multidrug-resistant organisms (MDRO) by targeted monitoring in a tertiary hospital, and to understand the distribution of MDRO. Methods We retrospectively analyzed the detection and distribution of methicillin-resistantStaphylococcus aureus (MRSA), carbon black alkeneAcinetobacter baumannii (CRABA), carbapenem-resistantPseudomonas aeruginosa (CRPAE), vancomycin-resistantEnterococci (VRE) and carbapenem-resistantEnterobacter (CRE) in clinical samples collected from 2013 to 2015. Results A total of 990 multidrug-resistant bacteria strains were isolated from 2013 to 2015, of which 445 were MRSA (44.95%), 328 were CRABA (33.13%), 99 were CRPAE (10.00%), 12 were VRE (1.21%), and 106 were CRE (10.71%). They were mainly distributed in the Department of Burn, Comprehensive ICU, Department of Neurosurgery, Department of Respiratory Medicine and Department of Orthopedic Surgery. The detection rates of multidrug-resistant organisms of 2013-2015 were 10.85% (352/3 244), 9.20% (304/3 303), and 7.11% (334/4 699) respectively, which reduced year by year with significant difference (χ2= 34.42,P< 0.001). The detection rates of CRPAE, CRE and VRE all reduced with significant differences (P< 0.05). Conclusions The detection rate of multidrug-resistant organisms under targeted monitoring shows an obvious downward trend. MRSA and CRABA are still the major MDROs, which show no obvious change. The detection rates of CRPAE, VRE and CRE show obvious downward trend. Department of Burn, Comprehensive ICU, Department of Neurosurgery, Department of Respiratory Medicine and Department of Orthopedic Surgery have the highest risks of MDRO. In the future, we should strengthen the monitoring of high-risk departments, and focus on the reasonable choice of special antimicrobial agents to avoid special MDROs.
【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.