尾加压素对人单核巨噬细胞DNA、RNA及蛋白质合成的影响

Effects of urotensin on synthesis of DNA, RNA and protein of human monocyte macrophages

目的:观察新近发现最强的缩血管活性肽之一的尾加压素对体外培养的人单核巨噬细胞DNA,RNA及蛋白质的合成过程的影响。方法:实验于2004-10/12在南方医科大学心内科实验室及广州军区总医院中心实验室完成。取健康自愿者50mL新鲜静脉血,用聚乙酰胺吡咯烷酮包被的硅胶颗粒液(d=1.130)分离单核细胞。采用24孔塑料培养板,每孔加入有巨噬细胞特性、浓度为2×105个/mL细胞0.5mL,随机分成4组。分别加入1×10-8,1×10-9,1×10-10,0mmol/L尾加压素,每组再随机分成3小组,分别加入3H-Leu,3H-TdR,3H-UR3.7×1010Bq/mL。培养7d,分别于第1、4、7天测定样品3H-Leu,3H-TdR,3H-UR的放射性,并分别代表蛋白质、RNA、DNA的合成状态。细胞蛋白含量测定:将细胞培养于8孔塑料培养板中,随机分成4组,培养至第3天分别加入1×10-8,1×10-9,1×10-10,0mmol/L尾加压素,继续培养4d,收集细胞。Lowery法常规测细胞蛋白含量。结果:①在RPMI1640+300mL/L自体血清中,细胞生长、成熟、分裂状态良好,细胞合成DNA、RNA、蛋白质迅速,尤以3H-UR掺入作用较强。②成熟的人单核巨噬细胞与1×10-8,1×10-9,1×10-10,0mmol/L浓度范围的尾加压素孵育24h,未见细胞有形态学改变。1×10-10mmol/L浓度尾加压素在24h时即对3H-UR,3H-TdR掺入有抑制作用,随浓度升高和时间延长,作用均加强;且在1×10-9,1×10-8mmol/L浓度下对RNA作用较DNA强(0.608±0.005,0.730±0.007,t=18.8,P<0.01;0.500±0.007,0.571±0.004,t=10.4,P<0.01)。③加入1×10-8,1×10-9mmol/L浓度尾加压素与未加入尾加压素组比较对蛋白质的合成有抑制作用[(22.4±0.707)×10-3,(32.5±0.634)×10-3dpm,t=23.8,P<0.01;(27.35±0.636)×10-3,(32.5±0.634)×10-3dpm,t=12.8,P<0.01],但对蛋白质合成的抑制作用明显低于对DNA,RNA合成的抑制作用(0.688±0.003,0.571±0.004,0.500±0.007,t=12.3,17.5,P<0.001;0.840±0.007,0.730±0.007,0.608±0.005,t=9.6,31.2,P<0.001)。④1×10-8mmol/L尾加压素与成熟人单核巨噬细胞共同作用4d,与未加入尾加压素组比较,细胞的蛋白质含量显著降低(0.225±0.012,0.319±0.027,t=5.76,P<0.001),并呈浓度依赖性。结论:尾加压素在1×(10-10～10-8)mmol/L浓度范围内对单核巨噬细胞DNA,RNA合成有明显抑制作用,对RNA较强,对蛋白质作用较弱;其与成熟过程中人单核巨噬细胞较长时间孵育时,对蛋白质合成抑制作用变明显。尾加压素抑制单核巨噬细胞DNA,RNA,蛋白质合成代谢,从而影响细胞生长、分裂增殖及免疫吞噬等功能。

AIM： To study the effects of urotensin, one of the optimal mini-vessel bioactive peptide that found recently on DNA, RNA and protein synthesis of human monocyte macrophages that cultured in vitro. METHODS： The experiment was conducted at the laboratory of Department of Cardiology, Southern Medical University and central laboratory of General Hospital of Guangzhou Military Area Command from October to December 2004. The 50 mL fresh venous blood was gained from healthy volunteers. The monocytes were separated with silica gel granule liquor （d= 1.130） coated with poly-acetamide pyrrolidine. Using the 12 holes plastic culture plate, the 0.5 mL cells at the concentration of 2×10^5 each per mL that had macrophage character in every hole, and divided randomly into 4 groups,1×10^-8 mmol/L, 1×10^-9 mmol/L, 1×10^-10 mmol/L,0 mmol/L urotensin were added, respectively, and the groups were assigned randomly into 3 subgroups, adding with 3H-Leu,3H-TdR,3H-UR 3.7×10^10 Bq/mL, respectively, cultured for 7 days. The radioactivities of 3H-Leu, 3H-TdR, 3H-UR Were detected at the 1st, 4th and 7th days, representing the synthesis status of protein, RNA and DNA, respectively. The detection of protein content： The cells were cultured in the 8-hole plastic cultured board, and assigned randomly into 4 groups. After culturing at the 3^rd day, the 1×10^-8 mmol/I, 1×10^-9 retool/L, 1×10^-10 retool/L, 0 mmol/L urotensin were added, respectively, cultured for 4 days continuously, then the cells were collected. The content of cell protein was detected routinely with the Lowery method. RESULTS： ① In the RPMI 1640＋300 mL/L autobody serum, the growth, mature and splitting status of cells were good. The synthesis of DNA, RNA and protein was rapid; especially the filter effect of 3H-UR was strong.② There was no change of cell morphology of mature human monocyte macrophage and urotensin at the concentration of 1×10^-8 mmol/L, 1×10^-9 mmol/L, 1×10^-10 mmol/L and 0 mmol/L incubated for 24 hours. The urotensin at the concentration of 1×10^-10 mmol/L at the 24^th hour had in- hibitive effects on infiltration of 3H-UR,3H-TdR. With the increase of concentration and prolongation of time, the effect became strong; Besides, the effect of RNA was stronger than that of the DNA at the concentration of 1×10^-9 mmol/L, 1×10^-8 mmol/L （0.608±0.005, 0.730±0.007 ,t=18.8 ,P 〈 0.01;0.500±0.007,0.571±0.004,t=10.4 ,P〈 0.01 ）. ③ Adding with urotensin at the concentration of 1×10^-8 mmol/L and 1×10^-9 mmol/L had inhibitive effects as compared with that without urotensin group [（22.4 ± 0.707）×10^-3, （32.5±0.634）× 10^-3 dpm, t=23.8, P 〈 0.01 ; （27.35±0.636） × 10^-3, （32.5±0.634）×10^-3 dpm, t=12.8,P 〈 0.01], but the inhibitive effects on synthesis of protein was lower significantly than that on the synthesis of DNA and RNA （0.688±0.003,0.571±0.004,0.500±0,007, t=12.3,17.5,P 〈 0.001 ;0.840±0.007,0.730±0.007,0.608±0.005, t=9.6, 31.2,P 〈 0.001）. ④ The 1×10^-8 mmol/L urotensin with mature human monocyte macrophages had corporate effects for 4 days. The content of protein decreased significantly （0.225±0.012,0.319±0.027,t=5.76,P 〈 0.001）, and showed dose-dependent effect, as compared with the unjoined urotensin group. CONCLUSION： The urotensin at the concentration from 1×10-10 to 1×10-8 mmol/L has significantly inhibitive effects on synthesis of DNA and RNA of monocyte macrophages, and the effect is stronger on RNA, weaker on protein. The inhibitive effects on synthesis of protein become signifi- cant, when the time of incubation of mature human monocyte macrophages is long. The urotensin inhibits the synthesis of DNA, RNA and protein of monocyte macrophages in order to affects the function of cell growth, splitting,proliferation and immune phagocytosis etc.