| Objective:Several previous studies have suggested that telomere length of in the peripheral white blood cells of coronary artery disease patients is shorter than that of healthy controls. Telomere complex is located at the end of the chromosomes of eukaryotic cells, which includes DNA, telomere binding proteins, telomerase, and they can play a protective role in the telomere. TRF1, TRF2, POT1 can be directly combined with the telomere DNA, which may play a more critical role. The purpose of this study includes:（1） Compare the clinical features of patients with premature coronary artery diseases （PCAD） and subjects with normal coronary artery angiography （CAG）, as well as TRF1, TRF2, POT1 expression level and telomere length difference in peripheral white blood cells. （2） Compare the difference of the length of telomere DNA and telomere associated protein expression level between PCAD patients and subjects with normal CAG in two kinds of different cells, peripheral blood mononuclear cells （PBMCs） and granulocytes, and explore the white blood cell composition characteristics in PCAD patients. （3） Culture the mononuclear macrophage cells in PCAD patients to explore the dynamic changes of telomere length and telomere associated proteins before and after the action of risk factors.Methods:（1） in the first part,52 PCAD patients and 42 subjects with no cornary artery stenosis were included. All patients were admitted to Peking Union Medical College Hospital for CAG, and arterial blood was collected before CAG for DNA and RNA extraction, to determine the telomere DNA length and three telomere binding protein expression level in peripheral leukocyte cells. （2） in the second part,36 PCAD patients and 10 subjects with no cornary artery stenosis were included. PBMCs were isolated by density gradient centrifugation, and granulocyte were collected by red blood cell lysis buffer dealing with remaining cells. Extract DNA and RNA and determine the differences in expression level of TRF1, TRF2, POT1 and telomere length. （3） in the third part, six PCAD patients and three subjects with no cornary artery stenosis were included. PBMCs were isolated by density gradient centrifugation. After 6 hours adherent culture, monocytes were isolated. Monocytes were stimulated by PMA for 48 hours to induced macrophages. Six different treatments were used, including medium control,50 ug/ml ox LDL+10-3 mol/L nicotine,10 ug/ml oxLDL,50 ug/ml ox LDL,10-6 mol/L nicotine and 10-3 mol/L nicotine. Cells were harvested at 24 hours and 72 hours cell to extract DNA and RNA, to determine the telomere length and TRF1, TRF2, POT1 expression levels.Results:（1） in the first part, PCAD patients have shown shorter telomere length （5.19 ±0.53 vs KB 6.24±2.54 KB, P=0.011） than CAG negative group. It also suggests that in PCAD group, TRF1 expression is lower than the CAG negative group （0.03± 0.88 vs 0.44±0.78, P = 0.024）,while POT1 expression is higher（-0.64±0.84 vs-1.27±0.91,P=0.001）,the differences were statistically significant. Two groups of TRF2 expression was not significantly different. POT 1 expression was negatively correlated with age,POT1 expression =-0.024 age +0.280 r2=0.047, P=0.044). The expression of TRFl,TRF2, POT1 has significant correlation. In addition,TRFl expression is significantnegative correlated with hyperlipidemia（P = 0.022）,POT1 is significantly positively correlated with leukocyte neutrophil proportion（P = 0.003）（2）in the second part, in all selected candidates, the telomeric DNA length of granulocytes is relatively longer than PBMCs（the difference is（-0.67±2.14） KB, P=0.041）. The expression of TRFI,TRF2,POT1 is higher in granulocytes than PBMCs（the difference is 0.55 dbl.05, P=0.002;0.74±0.90, P=0.000; 0.98±1.09, P=0.000, respectively）. The expression level of TRFl,TRF2, POT1 in granulocytes and PBMCs is significantly positively correlated（P =0.011,0.002, 0, respectively）. The telomere length of granulocytes was significantly shorterthan CAG negative population, and the thfference was（-1.43± 1.28） KB,P=0.010; while1。PCAD population,there was no significant difference in telomere length between two kinds of cells.（3） the third part showed after 72 hours culture in vitro, the telomere length of human mononuclear macrophages had no significant change（P=0.226）;withdifferent stimulation,the expression of TRF2, POT1 in PCAD group was higher than CAG negative group（P = 0.002, 0,respectively）,there was no significant difference in TRFl（P=0.n3）; with different culture time,the expression of TRFl,TRF2 and POT1 had no significant difference（P = 0.126, 0.730, 0.389） in two groups. In CAG negative group,10 ug/ml oxLDL and 10-6mol/L nicotine the had the trend th inhthit the expression of TRFl, TRF2, POT1; while, 50 ug/ml oxLDL and 10-3 mol/L nicotine had the trend to promoththe expression of TRFl,TRF2, POT1. Similar situation occured in PC AD group,while, 10 ug/ml oxLDL had the trend th promote the expression of TRF2,10-6mol/L nicotine had the trend to promote the expression of POT1. The difference between the expression level of these thlomere binding proteins with 50 ug/ml oxLDL,10-3mol/L nicotine and the mixture of both was not significant.Conclusion:（1） the expression level of TRF1 is lower in PCAD patients than controls, while POT1 expression level is higher; the expression level of different telomere binding proteins have a certain correlation; POT1 decreases with the process of aging, which indicates POT1 may be potential biomarkers for aging; hyperlipidemia may induced atherosclerosis though reducing TRF1 expression level, and neutrophils though elevating POT1 in the disease process of premature coronary artery disease. （2）In PCAD patients rather than common CAD patients, granulocytes may play a more important role in the pathogenesis. （3） After 72 hours culture in vitro, the telomere length of human mononuclear macrophages had no significant change; stimulating by different risk factors, TRF2, POT1 expression is higher in PCAD group than CAG negative group; relative lower level of risk factors can induce the increase of TRF2 and POT1 in PCAD group, showing their susceptibility. Larger dosage of risk factors may induce cell apoptosis, so the difference of telomere binding protein expression is not significant. |
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