ABSTRACT
Secondary ion mass spectrometry ( SIMS) as a powerful surface analysis technique has been widely applied in semiconductor industry and geology research. Recently, with the development of instrumental technology, SIMS is attracting more and more attention in life sciences. SIMS can provide surface MS spectra, 2D/3D chemical images and depth profiling of substances simultaneously. The minimal lateral resolution of 2D SIMS imaging is 80 to 100 nm, and the longitudinal resolution of 3D SIMS imaging is about 1-5 nm. However, due to lack of specific ions to render the structures of organelles, SIMS imaging for single cells still has great challenges. Optical microscopy, in particular laser scanning confocal microscopy ( LSCM) , has been emerged to be an indispensable technique for single cell imaging and can obtain high spatial 2D/3D imaging to visualize the structures of organelles. Thus, the combinational use of SIMS and LSCM, which takes advantages of SIMS for molecular imaging and LSCM for morphological imaging, has greatly extended the application of SIMS imaging and ensured its accuracy at single cells level, providing novel insights into better understanding of the biological events inside cells. In this review, we focus on the development and application of SIMS imaging and the correlated SIMS and LSCM imaging in the research of cell biology and drug discovery. We anticipate that the combinational use of SIMS and LSCM imaging has promising future in biomedicine and life sciences.
ABSTRACT
Objective To investigate the p53,Bax,bcl-2 gene in NaAsO2-induced human embryonic lung fibroblasts(HELF)apoptosis.Methods HELF was divided into HELF cells transfected with p53 plasmid(p53 group),HELF cells transfected with PC plasmid(PC group)and normal cultured HELF cells(normal group).The mRNA expression of p53,Bax and bcl-2 gene was detected by real-time PCR,the protein expression of p53,Bax and bcl-2 was assessed by immunohistochemical SABC and the cell apoptosis of HELF was detected by flow cytometry(FCM),in a 6-well plate and cultured for 48 hours,which was exposed to different doses(0,3,9,15mmol/L)NaAsO2 for 24 hours.Results The p53 gene mRNA expression level of p53 group(0.51±0.29)was lower than that of the normal group and PC group [ (1.00 ± 0.20), (1.32 ± 0.26), all P < 0.05 ]. The p53 protein expression level of p53 group(4.10 ± 1.20) was lower than the PC group and normal group[ (8.00 ± 1.63), (7.90 ± 1.79), allP < 0.05]. In p53 group, PC group, normal group exposed to 0,3,9,15 mmol/L NaAsO2 doses, the apoptotic rate [(0.57 ± 0.28)%, (22.91 ± 4.86)%, (40.05 ± 3.93)%, (44.87 ± 3.58)%; (0.65 ± 0.24)%, (14.09 ± 3.49)%,(20.31 ± 3.66)%, (32.42 ± 3.63)%; (0.56 ± 0.25)%, (12.14 ± 3.70)%, (19.61 ± 3.63)%, (30.43 ± 2.83)%], Bax mRNA expression level[(12.73 ± 3.96), (25.12 ± 6.42), (104.96 ± 26.77), (154.04 ± 30.52); (14.63 ± 3.57),(36.75 ± 3.67), (272.26 ± 66.11), (846.12 ± 243.36); (14.75 ± 5.65), (37.22 ± 11.27), (278.51 ± 37.42),(861.67 ± 369.29) ], Bax protein expression level [ ( 15.07 ± 0.83 ) %, ( 23.79 ± 3.99 ) %, (38.51 ± 1.58 ) %, (53.86 ±1.74)%;(15.43 ± 1.45)%,(36.11 ± 1.37)%, (56.86 ± 1.97)%, (76.09 ± 2.01)%; (15.20 ± 1.03)%,(35.25 ±1.09)%, (55.56 ± 2.17)%, (74.48 ± 2.85)% ] was respectively increased in a dose-dependent manner with the increased concentration of NaAsO2(all P < 0.05). The bel-2 mRNA expression level [ (443.00 ± 244.47), (156.79 ±53.18), (62.13 ± 13.66), (23.10 ± 6.44); (420.55 ± 110.77), (48.15 ± 10.02), (14.91 ± 6.53), (7.54 ± 2.62);(577.75 ± 123.22), (49.68 ± 10.11), (12.41 ± 1.28), (7.22 ± 1.89)], bcl-2 protein expression level[(47.20 ±3.77)%, (41.80 ± 2.94)%, (36.00 ± 2.36)%, (29.00 ± 2.91)%; (45.90 ± 4.15)%, (35.70 ± 2.77)%, (29.80 ±2.78)%, (24.80 ± 2.66)% ; (46.70 ± 3.47)%, (36.20 ± 2.90)%, (30.10 ± 3.21)%, (25.10 ± 2.28)% ] wasdecreased in a dose-dependent manner with the increased concentration of NaAsO2(all P < 0.05 ). In 3,9,15 mmol/L NaAsO2, apoptotic rate of p53 group, mRNA expression of bcl-2, protein expression of bcl-2 was higher than that ofnormal group and PC group, respectively (all P < 0.05), but mRNA expression of Bax, protein expression of Bax was respeetivelylower than that normal group and the PC group(P < 0.05 ). Conclusion p53 gene reduced the apoptosis induced by NaAsO2 in HELF, possibly by changing the apoptosis pathway.