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Objective To explore the influence of alpha-fetoprotein (AFP) on the expression of drug-resistance gene MDR1 and chemotherapeutic sensitivity in hepatocellular carcinoma (HCC) cells.Methods A HCC cell line SMMC-7721/AFP, which was stably transfected with AFP gene, was established.mRNA and protein expressions of AFP and MDR1 were detected by real-time PCR and Western Blot,respectively.The sensitivity of SMMC-7721/AFP and SMMC-7721/EGFP cells with or without MDR1 silencing by siRNA to doxorubicin was tested by MTT assay.Immunohistochemistry was used to detect the expression of MDR1 genes-coded protein Pgp in 60 cases of HCC tissues, and the relationship between Pgp expression and serum AFP levels was analyzed.Results AFP mRNA and protein could be detected in SMMC-7721/AFP cells, but not in control cells, indicating that the AFP stably transfected cell line was successfully established.MDR1 mRNA and protein levels were higher in SMMC-7721/AFP cells than those in SMMC-7721/EGFP cells.MDR1 mRNA level in SMMC-7721/AFP cells was (52.7 ± 1.5) times as high as that in SMMC-7721/EGFP cells (P ＜ 0.05).The resistance to doxorubicin was increased by (12.8 ± 1.1) times after AFP transfection (P ＜ 0.05).The chemosensitivity to doxorubicin was increased after the expression of MDR1 was knocked down by siRNA.The expression of Pgp in HCC tissues was positively correlated with the serum AFP levels.Conclusion AFP could induce drug-resistance to doxorubicin in HCC cells by increasing the expression of MDR1.
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Objective To study the relationship between expression of multidrug resistant 1 ( MDR1) gene encoded P-glycoprotein ( P-gp) and multidrug resistance relation protein ( MRP) in ulcerative colitis and the disease behavior. Methods Sections of endoscopic biopsy samples from patients with ulcerative colitis were studied by immunohistochemistry with monoclonal antibody of MDR1 gene encoded P-gp and MRP. Results Expressions of MDR1 gene encoded P-gp and MRP in the 12th month and 18th month of ulcerative colitis were 40. 5% , 45.9% , 48. 6% and 51.4% , respectively, which were all significantly higher than those of early phase ( P < 0.05). P-gp and MRP expressions in active stage and remission stage of ulcerative colitis were 36.4% , 18. 6% , 46. 1% and 25.4% , respectively, which was significantly different between the two stages (P < 0.05 ). At active stage of ulcerative colitis there was no significant difference in expressions of P-gp and MRP among mild, moderate and severe patients (P > 0. 05 ). Conclusion Examination of P-gp and MRP expressions is feasible and dependable in presenting evidence of drug insistence. However, it is not suitable to be an indicator of severity because of its poor differentiation ability in active stage.
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Objective To investigate the clinical application of mutations drug-resistant tuberculosis (MDR-TB) by polymerase chain reaction(PCR)-oligonucleotide probes. Methods 80 cases of MDR-TB were divided into observed group 40 cases and conventional group 40 cases, the resistant gene and the type of mutation were determined by polymerase chain reaction (PCR)-oligonucleotide probes. Results 78 isolates of 4 kinds of resistance genes (KatG,inhA,rpoB,embB) ,the mutation rates were 90.9% ,84.8% ,51.98% and 58.1% ;the gene of rpoB sensitivi-ty of 98.0% and specificity of 100% ,positive predictive value of 82.61% ,the rate of according with traditional drug susceptibility testing were 62.50% by polymerase chain reaction (PCR)-oligonucleotide probes;the serisitivity of the KatG gene mutation is of 50.56% ,specificity of 75% ,positive predictive value of 68.75% ,the rate of according with traditional drug susceptibility testing were 46.43% by polymerase chain reaction (PCR)-oligonucleotide probes;the observed group in focus significant absorption, absorption, invariability, deterioration were 32.5% ,62.5% ,5.0%, 0% ,higher than the conventional group(25.0% ,50.0% ,20.0% ,5.0% ) (χ2=3.98,χ2=3.92, P<0.05;χ2=6.78,χ2=6.80,P<0.01) ;observed group in 3 months after treatment,6 months,9 months sputum the conversion negative rate in observed group (65.0%, 77.5%, 85.0% ) higher than the conventional group (37.5%, 50.0%, 60.0%) (χ2=3.86,χ2=3.85,χ2=3.89,P both<0.05). Conclusion An analysis available 3 kind of MDR-TB, 4 kind gene by PCR-oligonucleotide;the observed group in curative effect is better than the conventional group in pa-tients with MDR-TB.
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Objective To study the efficiency of reversing MDR by suppressing MDR1 gene with RNAi in K562/ADM cells. Methods For reversing MDR by RNAi technology, two different short hairpin RNAs (shRNAs) were designed and constructed into pGenSil-1 plasmid, respectively. They were then transfected into the highly adriamycin-resistant K562/ADM cell line. The RNAi effect on MDR was evaluated by real-time PCR, and Rhodamine123 (Rh123) efflux assy. Results The stable transfected clones showed varied degrees of reversal of MDR phenotype. Surprisingly, the MDR phenotype was completely reversed in two transfected clones. Conclusion This study demonstrates that MDR can be reversed by the shRNA-mediated RNAi in K562/ADM cells, which provides a valuable clue as to sensitizing multidrug-resistant hepatoma cells to anti-cancer drugs.
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Objective To investigate the modulation for multidrug resistance cell line K562/A02 us- ing a specific siRNA against mdrl,GST?.Methods siRNA were synthesized targeting the coding region se- quences of mdrl(79～99 nt)and GST?(308～327nt)respectively,and cloned to plasmid pSilence2.1-U6.The cloned products pSilenee-mdr1 and pSilence-GST? were transfected into K562/A02 cells.Expression of mdr1 and GST? mRNA were assayed by SYBR Green Ⅰ real-time PCR.The apoptosis of cell line K562/A02 was examined by Flow cytometry,50% inhibition concentration(IC_(50))of doxorubicin on K562/A02 cell was deter- mined by MTT method.Results The siRNA expression vector against mdr1,GST? mRNA was constructed successfully.After transfected with pSilenee-mdr1,the expression of mdr1 mRNA in K562/A02 in was re- duced 71.5 % compared to the mock transfeetion,from(2.8?1.65)?10~8 copy/?g RNA to(3.9?2.37)?10~7 copy/?g RNA(P
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Objective To study P-glycoprotein(P-gp),the mdr-1 gene product,expression and response to pirarubicin containing regimen in recurrent and metastatic breast carcinoma.Methods P-gp expression in 40 postoperative patients with recurrent and metastatic breast carcinoma was detected by SABC immunohistochemical method.The patients were treated with combination regimen of cyclophosphamide,pirarubicin and 5-fluorouracil.The correlation between P-gp expression and chemotherapeutic response was analyzed.Results The positive rate of P-gp expression in patients with lung or liver metastasis was higher than that in skin or lymphnode metastasis(P=0.049);The overall response rate was 52.5% in 40 patients;The responserate 84.2% of the P-gp negative group was higher than that 33.3% of the P-gp positive group(P
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Objective To determine the frequency of polymorphism at exon 26 (C3435T) of muhidrug resistance 1 gene (MDR1) in epileptic patients in the southern Chinese and to study the association of this polymorphism with pharmacoresistance.Methods DNA samples were obtained from 134 patients,of whom 72 were resistant to antiepileptic drug treatment and 62 were responsive to the treatment. Genotypes of the C3435T polymorphism were determined by polymerase chain reaction (PCR) followed by restriction digestion and gel electrophoresis.Genotype and allele frequencies in the drug resistant group were compared to those in the response group by Chi-square analysis.Results Of all 134 patients,33 (24.6%) had CC genotype,72 (53.7%) had CT genotype,and 29 (21.6%) had TT genotype.The frequency of CC genotype was significantly higher in the pharmaeoresistance group (33.3%) than that in the responsive group (14.5%,P=0.012).The frequency of the C allele was also significantly higher in the pharmacoresistance group (57.6%) than that in the responsive group (44.4%,P=0.03).When patients were divided by types of seizure into three groups:generalized seizure group,partial seizure group,and undefined seizure group,the CC genotype and C allele were associated with pharmacoresistance in the partial seizure group.Conclusions In the southern Chinese,the CC genotype and C allele are associated with resistance to the antiepileptic treatment.This finding needs to be verified in studies with larger sample size.
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Objective To explore the relationship between mdr1 gene expression of hepatocellular carcinoma (HCC) and pathological characteristics,chemotherapy and prognosis. Methods The 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. Results The 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 (? 2=4.39, 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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Objective:To study the reversal effect of tamoxifen(TAM)on the expression of mdr1 in human pancreatic cancer cell lines.Methods:The inhibtion effect of TAM on pancreatic cancer cell line SW1999、Capan-Ⅱ and mammary cancer cell line T47D were studied;The influence of TAM on mdr1 protein/mRNA expression in SW1999、Capan-Ⅱ and T47D were studied using the method of flow cytometer and RT-PCR.Results:The high dose TAM could inhibit Capan-Ⅱ cell line.TAM could reverse the expression of mdr1 protein and mRNA in Capan-Ⅱ cell line and T47D cell line. Conclusion:The high dose TAM could inhibit Capan-Ⅱ cell line,maybe related with reduction of the expression of mdr1 protein and mRNA.