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کاربرد نوع شرط:
- جایگاه : پژوهشی
- مجله: Iranian Journal of Biotechnology
- نوع مقاله: Journal Article
- کلمات کلیدی: Hemophilia B,Plasmid,Hepatocyte,Aldolase B intronic enhancer,Beta-globin introns,Human factor IX (hFIX)
- چکیده:
- چکیده انگلیسی: Background: Human FIX (hFIX) gene transfer into hepatocytes has provided a novel approach for treatment of hemophilia B. To obtain an improved expression of hFIX, the functional hFIX-expressing plasmids with appropriate intron-derived fragments which facilitate transcription and promote an efficient 3′-end formation of mRNAs are required.
Objectives: We aim to evaluate the functions of the heterologous intron-derived fragments intra and extra hFIX-cDNA coding region with respect to the hFIX expression in the hepatocytes and kidney cells.
Materials and Methods: HepG2 cells as differentiated hepatocytes and Hek-293T cells as embryonic kidney cells were transfected with the different hFIX-expressing plasmids containing various combinations of the two human beta-globin (hBG) introns within the hFIX-cDNA and Kozak sequence. In the next stage, as a hepatocyte-specific sequence, the rat aldolase B intronic enhancer sequence (rABE), was isolated from the first intron of the rat aldoase B gene and inserted within the upstream CMV promoter (CMVp) and efficacies of the engineered vectors were investigated in the stably-transfected HepG2 cells.
Results: Our data indicate that the intron-less construct and hBG intron-I containing construct are more effective with regard to hFIX expression compared to other constructs in Hek-293 cells. In HepG2 cells, the rABE in combination with CMVp in context of intron-less plasmid induced an increase in total expression of hFIX protein dramatically; ranging from 2.3 to 40 folds increase compared to other constructs. The rABE in combination with CMVp in the hBG intron-I, hBG intron-II, and hBG intron-I,II containing plasmids induced 3.7, 2, and 1.6-fold increase in the total expression of hFIX protein, respectively. The presence of both hBG intronic sequences within the hFIX-cDNA induced a higher secretion level of hFIX than either intron-I or II alone and provided correctly spliced hFIX transcripts in HepG2 and kidney cell lines. The intron-less construct with or without rABE induced the highest hFIX mRNA levels in HepG2 and Hek-293T cells respectively compared to other constructs.
Conclusions: The embryonic kidney cells in addition to the differentiated hepatic cell lines could be successfully targeted by plasmid vectors. The intron-less and hBG intron-I containing plasmids represent a particular interest in producing recombinant hFIX in Hek-293T cells. The synergistic function on the hFIX expression that was achieved by combining the CMVp with the liver-specific rABE would be a useful approach for future designing of the expression cassettes for hepatocyte-mediated gene expression in hemophilia B.- انتشار مقاله: 28-08-1395
- نویسندگان: Mohammad Reza Sam,Alireza Zomorodipour,Aliakbar Haddad-Mashadrizeh,Mahdi Ghorbani,Gholam Ali Kardar,Sohrab Sam
- مشاهده
- جایگاه : پژوهشی
- مجله: Asia Oceania Journal of Nuclear Medicine and Biology
- نوع مقاله: Journal Article
- کلمات کلیدی: Monte Carlo simulation,Beta emitters,absorbed fraction,analytical function,internal radiotherapy
- چکیده:
- چکیده انگلیسی: Objective(s): The purpose of this research is to study the effect of beta spectrum on absorbed fraction ( ) and to find suitable analytical functions for beta spectrum absorbed fractions in spherical and ellipsoidal volumes with a uniform distribution for several radionuclides that are commonly used in nuclear medicine.
Methods: In order to obtain the beta particle absorbed fraction, Monte Carlo simulations were performed by using the MCNPX code. The validation of the simulations was performed by calculating the absorbed fractions in spheres and comparing the results with the data published by other investigators. The absorbed fractions were calculated and compared by using an actual beta energy spectrum with those obtained through the mean beta energy of 14C, 199Au, 177Lu, 131I, 90Sr, 153Sm, 186Re, 32P, 90Y, 38Cl and 88Rb radionuclides.
Results: The maximum difference between the absorbed fractions for beta particles accounting for the whole beta spectrum of all the considered nuclides was 29.62% with respect to the mean beta energy case. Suitable analytical relationships were found between the absorbed fraction and the generalized radius, and the dependence of the fitting parameters from beta spectrum energy was discussed and fitted by appropriate parametric functions.
Conclusion: The results allowed the calculation of the absorbed fractions from the above stated beta sources uniformly distributed in spherical and ellipsoidal volumes of any ellipticity and volume, in a wide range of practical volumes that are not only used for internal dosimetry in nuclear medicine applications, but also in radiological protection estimates of
doses from internal contamination.- انتشار مقاله: 16-02-1397
- نویسندگان: Mahdi Ghorbani,Marjan Hashempour,Ernesto Amato,Courtney Knaup
- مشاهده