Abstract
Many investigators have studied the effect of low density lipoprotein (LDL) on the proliferation of arterial smooth muscle cells (SMC). The methodology is not well studied. The purpose of this study was to investigate the method for analysis of the effect of LDL on DNA synthesis in cultured human arterial SMC.
Human arterial SMC were cultured from splenic artery with MEM containing 5% of LPDS (lipoprotein deficient serum from normal subjects). LDLs were ultracentrifugally prepared from normal subjects (n=8) and diabetics (n=12). 0.2×105 cells/dish were cultured with 5% LPDS-MEM for 48 hours. This medium was replaced with fresh 5% LPDS-MEM containing LDL (25 microgr./ml) and 0.5 micro Ci of 3H-Thymidine. After 6 hours intracellular radioactivity was measured and DNA synthesis calculated as cpm/microgr, cell protein. This experiment was repeated for 6 times during 36 hours. In another experiment DNA synthesis in these SMC was studied during 36 hours after 3H-Thymidine addition.
DNA synthesis in SMC cultured without LDL addition was almostly constant during 36 hours, however, the value of cpm/microgr, protein was variated in each 7-experiment. Therefore, we calculated the value of Index as shown below: Index (%)
This Index was useful in comparison of data among 7-experiments. The time point of the maximum value of Index in the culture with each LDL was variated from 18 to 36 hours after LDL addition in the experiment repeatedly performed for 6 hours during 36 hours. The maximum Index was higher in the culture with diabetic LDL than in that with normal LDL. When DNA synthesis was studied during 36 hours after 3H-Thymidine addition, Index in the culture with diabetic LDL was significantly (p<0.05) higher than that in the culture with normal LDL.
We concluded that (1) Index of DNA synthesis was useful for analysing the effect of LDL on SMC in vitro, (2) DNA synthesis should be studied for 36 hours after addition of LDL and 3H-Thymidine.
