Improving P-gp expression in human mononuclear cells in vitro transfected by multidrug resistance-1 mRNA.
OBJECTIVE
To evaluate the expression and function activity of P-glycoprotein (P-gp) in human mononuclear cells (MNCs) in vitro transfected by multidrug resistance-1 (MDR1) mRNA.


METHODS
Two MDR1 cDNA vectors, pT7TS_MDR1 and pGEM5Zf(+)_MDR1, were constructed and transcripted in vitro. Vector pGEM5Zf(+)_MDR1 only contained the coding region of mdr1 cDNA, and pT7TS_MDR1 also included Xeponus beta-globin 5' and 3' untranslated region. MNCs were prepared from peripheral blood of parvicellular lung cancer patient. The two human mdrl mRNAs were then transferred into human MNCs in vitro by DOTAP. And the expression efficiency and pump function of P-gp were measured with flow cytometry.


RESULTS
Expression of P-gp significantly elevated in both transferred cells compared with untransferred cells (P < 0.01). And pT7TS_MDR1 showed higher capability in elevating the expression of P-gp than pGEM5Zf(+)_MDR1 (P < 0.01). The P-gp function was elevated in both pT7TS_MDR1 and pGEM5Zf(+) MDR1 groups. The survival ratio of MNCs in erythrocyte-lysis-solution (ELS, 86.07%) and lymphocyte-isolation-solution (LIS, 83.67%) had no significant difference. The CD34+ cells content of the MNCs used for transfection was 2.65% and 1.01% in ELS and LIS group, respectively (P < 0.01).


CONCLUSIONS
It is a feasible approach to improve P-gp expression in human MNCs by transfection of MDR-1 mRNA. And the ELS may be more suitable for purifing MNCs for mRNA transfection than LIS.
Xiang
Yang
Y
Department of Obstetrics & Gynecology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730. xiangy@pumch.ac.cn
Li
Lei
L
Tian
Fang
F
Yang
Xiu-yu
XY
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis* / Q000235:genetics; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D015972:Gene Expression Regulation, Neoplastic; D019450:Genes, MDR; D005822:Genetic Vectors; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D008175:Lung Neoplasms / Q000473:pathology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D012333:RNA, Messenger