| Objective: To establish adriamycin-resistant and rituximab-resistant human diffuse large B-cell lymphoma(DLBCL)cell lines and to detect the morphology,proliferation,drug sensitivity and the expression of MDR1 respectively.To investigate whether there is cross-resistance between chemotherapeutic drugs and rituximab.We provide a cell model for research of drug resistance of DLBCL,and provide theoretical basis for the clinical medication of DLBCL after secondary resistance.Methods: 1.Human DLBCL cell line(Pfeiffer)were exposed in stepwise escalating concentration of adriamycin and rituximab respectively,untill the resistant cell lines(Pfeiffer/ADM,Pfeiffer/R)were developed.2.The morphology of Pfeiffer,Pfeiffer/ADM and Pfeiffer/R cells were compared under the microscope.3.The cell growth curve of the parents and drug resistant cell lines were drawn by using the cell counting method.4.The proliferation and half inhibitory concentration(IC50)of Pfeiffer,Pfeiffer/ADM and Pfeiffer/R which exposed respectively in ADM,VP-16 and VCR after 72 h were detected by CCK-8 assay.5.The ability of rituximab to mediate CDC in Pfeiffer/ADM andPfeiffer/R compared with the Pfeiffer was assessed by analyzing the percentage of inhibition rate that were treated with human serum as source of complement by CCK-8 assay.6.The expression of MDR1 mRNA in Pfeiffer,Pfeiffer/ADM and Pfeiffer/R were detected by quantitative Real-time PCR.Results: 1.Pfeiffer/ADM cell line was established after about 7 months and Pfeiffer cell line was established after about 1 months.2.Under microscope,there was no significant difference among Pfeiffer,Pfeiffer/ADM and Pfeiffer/R.3.Respectively,The doubling time of Pfeiffer,Pfeiffer/ADM and Pfeiffer/R were(32.28±1.18)h,(31.41±2.03)h and(32.03±1.77)h.The difference was not statistically significant(P>0.05).4.The half inhibitory concentration of ADM,VP16 and VCR detected by CCK-8 assay of Pfeiffer cells were(0.038±0.0090)μg/mL,(0.26±0.045)μg/mL and(0.0093±0.0023)μg/mL.The half inhibitory concentration of Pfeiffer/ADM cells were(0.48±0.049)μg/mL,(3.42±0.45)μg/mL and(0.16±0.0032)μg/mL.Pfeiffer/ADM was resistant to adriamycin,etoposide and vincristine which resistance index was 12.87,13.18 and 16.85 indicating its multidrug-resistant ability.The half inhibitory concentration of Pfeiffer/R cells were(0.038±0.046)μg/mL,(0.25±0.13)μg/mL and(0.0098±0.00014)μg/mL.There was no significant difference between Pfeiffer and Pfeiffer/R(P> 0.05),indicating that Pfeiffer/R is sensitive to ADM VP-16 and VCR.5.Using human serum as source of complement,different concentrations of RTX(1μg /mL,3μg /mL,10μg /mL,30μg/mL)were used for 6hours in vitro.The inhibitory rates of Pfeiffer cells were 28.58%,47.06%,69.00%,77.20%,Pfeiffer/ADM were 30.33%,47.21%,64.63% and 72.53%,respectively.There was no significant difference between Pfeiffer and Pfeiffer/ADM(P> 0.05),indicating that Pfeiffer/ADM is sensitive to RTX.The inhibitory rate of Pfeiffer/R cells were 11.04%,24.12%,40.56% and 53.58%,respectively.The difference was statistically significant between Pfeiffer and Pfeiffer/R(P <0.05),indicating that Pfeiffer/R is resistant to RTX.6.The relative expression of MDR1 mRNA in Pfeiffer and Pfeiffer/ADM cell line were respectively found to be(1.00±0.10)and(82033.27 ± 4187.24)by qRT-PCR.The difference was statistically significant(P<0.001).However,The relative expression of MDR1 mRNA in Pfeiffer and Pfeiffer/R cell line were(1.03±0.33)and(3.00 ± 1.32),there was no significant difference between Pfeiffer and Pfeiffer/R.(P=0.066).Conclusion: By using the method of stepwise escalating concentration,we successfully establish the human DLBCL adriamycin-resistant cell line(Pfeiffer/ADM)and RTX-resistant cell line(Pfeiffer/R).Pfeiffer/ADM cell lines were resistant to ADM,VP-16 and VCR,which showing multidrug resistance,but sensitive to RTX-mediated CDC effect.Pfeiffer/ADM was significantly higher than Pfeiffer in MDR1 Expression.Furthermore,Pfeiffer/R cell lines were resistant to RTX-mediated CDC effect,but chemotherapeutic drugs VP-16,VCR and ADM were still sensitive.Compare with Pfeiffer,there was no significant difference in the expression of MDR1.Suggesting that there is no cross-resistance between chemotherapeutic drugs VP-16,VCR,ADM and RTX.The resistant cell lines were established in this study are the ideal model for the next step in drug resistance or reversal of drug resistance. |
PDF Full Text Request