[E1A gene transfection of human undifferentiated thyroid cancer cell line HTC/3 by nanoparticles].
OBJECTIVE
To prepare nanoparticles containing E1A gene and observe the efficiency and feasibility of transfecting E1A gene into human undifferentiated thyroid cancer cell line HTC/3. To examine the sensitivity of transgene cells to X-ray and X-ray-induced apoptosis in those cells.


METHODS
Nanoparticle-DNA complex was prepared with PLGA coating adenoviral early expression gene E1A, and the package efficiency, release progress in vitro, and size of the complex were determined. The nanoparticle-DNA was transfected into the HTC/3 cells. Lipofectamine was used to transfect E1A gene as a control. RT-PCR was used to examine E1A gene mRNA expression in the transfected cells. The survival ratio of HTC/3-E1A and control cells, and the growth inhibition ratio induced by different doses of X-ray in HTC/3-E1A cells were examined by MTT assay. The apoptosis in HTC/3-E1A cells induced by 2 Gy X-ray iradiation was examined by flow cytometry and DNA electrophoresis.


RESULTS
The package efficiency, release progress in vitro, and size of the nanoparticle-DNA complex were 0.78%, 18 days, and 150-280 nm, respectively when transfected the plasmid at the same level, the nanoparticle group got more positive transgene cell clones than that in lipofectamine group, with a statistically significant difference (P < 0.05). RT-PCR showed that transgenic cells from both nanoparticle-DNA and lipofectamine groups had E1A gene mRNA expression. The HTC/3-E1A cells grew slowly, and their doubling time was prolongated (1.44 times in comparison with that in parental cells). According to IC50, the sensitivity of HTC/3-E1A cells to X-ray was improved 2.9 and 2.8 times, respectively, in comparison with that in HTC/3-Vect and HTC/3 cells. The ratio of subG0/G1 phase of HTC/3-E1A cells was significantly higher than that in HTC/3-Vect and HTC/3 cells (P < 0.01). The ratio of S phase of HTC/3-E1A cells was significantly lower than that in HTC/3-Vect and HTC/3 cells (P < 0.01). A typical DNA ladder pattern of apoptosis in HTC/3-E1A cells was observed by electrophoresis, but not found in HTC/3-Vect and HTC/3 cells.


CONCLUSIONS
A nanoparticle-DNA complex has been successfully prepared, and it may carry a foreign gene into cells. The sensitivity of HTC/3-E1A cells to X-ray is significantly improved. Moreover, apoptosis is induced by x-ray in the E1A gene-transfected cells.
He
Xiang-Liang
XL
Department of Emergency, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China. HXliang1963@Tom.com
He
Dong-Hua
DH
Liao
Xiao-Xing
XX
Zhan
Hong
H
Ma
Zhong-Fu
ZF
Wang
Xi-Fu
XF
Li
Qing
Q
Li
Xin
X
Li
Yu-Jie
YJ
D017500:Adenovirus E1A Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D017209:Apoptosis / Q000528:radiation effects; D002453:Cell Cycle / Q000528:radiation effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004247:DNA / Q000235:genetics; D006801:Humans; D019344:Lactic Acid / Q000737:chemistry; D053758:Nanoparticles; D010316:Particle Size; D010957:Plasmids; D011100:Polyglycolic Acid / Q000737:chemistry; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D012333:RNA, Messenger / Q000378:metabolism; D013964:Thyroid Neoplasms / Q000378:metabolism / Q000473:pathology*; D014162:Transfection; D014965:X-Rays
D017500:Adenovirus E1A Proteins; D012333:RNA, Messenger; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid; D004247:DNA