氯沙坦对肺成纤维细胞转分化和胶原合成的抑制作用

Losartan inhibited angiotensin induced human lung fibroblast transformation and collagen synthesis

目的观察血管紧张素Ⅱ(AngⅡ)对人胚肺成纤维细胞(HLF)的转分化作用和对其结缔组织生长因子(CTGF)表达、胶原合成的影响,并检测 AngⅡ受体拮抗剂氯沙坦对 AngⅡ作用的干预。方法常规培养 HLF 细胞并随机分为对照组、AngⅡ组、AngⅡ+氯沙坦组和氯沙坦组,用免疫荧光法和 Western 免疫印迹法检测各组 HLF 转分化肌成纤维细胞中标志蛋白α-平滑肌动蛋白(α-SMA)的表达,逆转录-多聚酶链反应和免疫组织化学法检测各组 CTGF mRNA 及蛋白表达的改变;分别用 AngⅡ、AngⅡ+氯沙坦孵育 CTGF 反义、正义、错义寡核苷酸转染 HLF 细胞,观察各组细胞Ⅰ型胶原蛋白 mRNA 和α-SMA 的 mRNA 和蛋白水平变化,比色法测定各组细胞培养液中羟脯氨酸含量。结果 AngⅡ组 CTGF mRNA 的吸光度值(0.82±0.07)较对照组(0.29±0.05)、AngⅡ+氯沙坦组(0.51±0.04)、氯沙坦组(0.26±0.04)明显增高;AngⅡ组 CTGF 蛋白的吸光度值(0.24±0.05)明显高于其他组;AngⅡ组胶原蛋白 mRNA 的吸光度值为1.03±0.12,羟脯氨酸含量为(0.62±0.01)ng/ml,明显高于其他3组;AngⅡ孵育 CTGF 反义寡核苷酸转染细胞组α-SMA 的吸光度值(1.14±0.15)和胶原蛋白 mRNA 的吸光度值(0.30±0.04)明显低于正义组(4.25±0.21和0.55±0.08)和错义组(4.34±0.31和0.58±0.06);AngⅡ+氯沙坦孵育 CTGF 反义寡核苷酸转染细胞组α-SMA 和Ⅰ型胶原蛋白 mRNA 的吸光度值(0.85±0.09和0.20±0.02)明显低于 AngⅡ单独孵育时(4.39±0.29和1.03±0.12)。结论 AngⅡ可通过上调 HLF 细胞 CTGF 表达水平诱导其转分化为肌成纤维细胞并促进胶原合成,阻断 CTGF 表达可使 AngⅡ对 HLF 细胞的转分化及胶原诱导合成作用减低,氯沙坦可抑制 AngⅡ对 HLF 细胞的诱导作用。

Objective To observe the induction of cell transdiiferentiation,connective tissue growth factor （CTGF） expression and collagen production of human lung fibroblast （HLF） by angiotensin Ⅱ （ Ang Ⅱ ）, and to investigate the inhibitory effect of Ang Ⅱ receptor antagonist- losartan on this process. Methods HLF cells were cultured and divided into： a control group, an Ang Ⅱ treated group, an Ang Ⅱ ＋ losartan coincubated group and a losartan group. The marker of myofibroblast-α-smooth muscle actin （α-SMA） was detected by immunofluorescence and Western blot respectively, and the expression of CTGF mRNA and protein level were measured by reverse transcription-pelymerase chain reaction （RT-PCR）and immunohistochemisty respectively. Groups of Ang Ⅱ and Ang Ⅱ ＋ losartan incubated with CTGF phosphomthioate antisense, sense and random oligonucleotides transfected HLFs respectively, and collagen Ⅰ （Col）Ⅰ mRNA and α-SMA expression level were compared. The amount of hydroxyproline in the cell culture was measured by colormetric assay. Results In the Ang Ⅱ group, CTGF mRNA level （0. 82 ± 0. 07） was significantly higher than the control group （0. 29 ±0. 05）, the Angll ＋ Losartan group （0. 51 ± 0. 04） and the Losartan group （0. 26 ± 0. 04）. The CTGF protein level in the Ang Ⅱ group （0. 24 ± 0. 05 ） was increased as compared to the other groups. In the Aug Ⅱ group, Col Ⅰ mRNA （1.03 ± 0. 12） and amount of hydroxyproline （0. 62 ±0. 01 ng/ml） were significantly different as compared with the other three groups. The α-SMA expression level （ 1.14 ± 0. 15 ） and Col Ⅰ mRNA （0. 30 ± 0. 04 ） of HLF cells transfected with antisense oligonucleotide incubated with Ang Ⅱ decreased significantly as compared to those of sense and random oligonucleotide transfected cells, α-SMA expression level （0. 85 ± 0. 09） and Col Ⅰ mRNA （0. 20 ± 0. 02） of Losartan ＋ Ang Ⅱ co-incubated antisense oligonucleotide transfected HLF ceils decreased significantly as compared to Ang Ⅱ treated alone. Conclusion Ang Ⅱ promoted HLF ceil transdifferentiation into myofibroblasts and increased collagen production through induction of CTGF expression. Blocking CTGF expression decreased Ang Ⅱ induced transdifferentiaion. Losartan blocked Ang Ⅱ induced HLF ceil transdifferentiation and collagen production.