Cav-1-AKF-PD抗肺纤维化治疗新靶点

发布时间：2018-03-17 12:52

本文选题：氟非尼酮　切入点：原代正常肺成纤维细胞　出处：《中南大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的观察氟非尼酮(Fluorofenidone, AKF-PD)抑制转化生长因子-p1(transforming growth factor-β1, TGF-β1)诱导原代正常肺成纤维细胞(Normal Human Lung fibroblasts, NHLFs)活化和细胞外基质(Extracellular matrix,ECM)合成的作用,探讨调控小窝蛋白-1(caveolin-1,cav-1)表达在AKF-PD抗肺纤维化机制中的作用。方法 1. NHLFs的原代培养、鉴定及纯化传代：于肺叶切除术中取病变远端的正常肺组织,采用组织块培养法进行NHLFs的原代培养,将培养的细胞纯化传代至第3代细胞冻存备用。 2.取NHLFs第3-7代使用,首先将实验分为5组(n=5),分别为空白对照组(N组)、TGF-β1(10ng/ml)处理组(M组)、TGF-β1(10ng/ml)+高、中、低浓度AKF-PD(200μg/ml、400μg/ml、800μg/ml)治疗组(A200、A400、A800组),各组于细胞接种后相应处理12h、24h、48h、64h、72h,收集细胞提取总蛋白,采用Western Blot检测cav-1、α-平滑肌动蛋白(Alpha smooth muscle actin,α-SMA)、纤维连接蛋白(fibronectin,FN)的蛋白表达； 3.取NHLFs第3-7代使用,由上海吉玛制药技术有限公司设计、制备得到3个不同序列的正常人cavl-siRNA,再将实验分为7组,分别为空白对照组(N组)、TGF-β1(10ng/ml)处理组(M组)、Co-siRNA转染组、Co-siRNA+TGF-β1(10ng/ml)处理组、cavl-siRNA转染组、cav1-siRNA+TGF-β1(10ng/ml)处理组、cavl-siRNA+TGF-β1(10ng/ml)+AKF-PD(400μg/ml)处理组,各组于细胞接种后相应处理48h,收集细胞提取总蛋白,采用Western Blot检测cav-1、α-SMA、FN的蛋白表达。结果 1.随着TGF-β1(10ng/ml)作用时间的延长,cav-1的表达逐渐减少(p0.05),并且具有时间依赖性;细胞α-SMA、FN的表达先增加后减少(p0.05)。TGF-β1能显著下调细胞cav-1的表达(p0.05),能明显上调细胞α-SMA、FN的表达(p0.05),细胞活化与ECM合成的最佳时间为48h时。 3.随着AKF-PD(200μg/ml、400μg/ml、800μg/ml)浓度的增加,与TGF-β1(10ng/ml)共同接种细胞48h后,细胞cav-1的表达逐渐增加(p0.05),细胞α-SMA、FN的表达逐渐减少(p0.05),并且具有剂量依赖性。AKF-PD作用的最佳浓度为400μg/ml,能显著抑制TGF-β1的作用,能部分恢复细胞cav-1的表达(p0.05)能明显下调细胞α-SMA、FN的表达(p0.05)。 4.Co-siRNA转染对cav-1表达无影响：Co-siRNA转染后,与N组比较,细胞cav-1、α-SMA及FN的表达差异无统计学意义(P0.05)。 5.cavl-siRNA成功干扰cav1的表达：cavl-siRNA转染后,与N组比较,细胞cav-1的表达几乎消失(p0.05),细胞α-SMA、FN的表达差异无统计学意义(P0.05)。而最佳转染序列为cavl-siRNA-2,细胞cav-1沉默最明显(p0.05),细胞α-SMA、FN的表达几乎与N组相同。 6.TGF-β1处理后的Co-siRNA转染组：与单纯Co-siRNA转染组比较,细胞cav-1的表达明显减少(p0.05),细胞α-SMA、FN的表达明显增多(p0.05);与M组比较,细胞cav-1、α-SMA、FN的表达差异无统计学意义(P0.05)。 7.TGF-β1处理后的cav1-siRNA转染组：与单纯cav1-siRNA转染组比较,细胞cav-1的表达有所减少,但差异无统计学意义(P0.05),细胞α-SMA、FN的表达明显增多(p0.05)；与M组比较,细胞cav-1的表达明显减少(p0.05),细胞α-SMA、FN的表达有所减少(p0.05)。 8.TGF-β1+AKF-PD共同处理后的cav1-siRNA转染组：与TGF-β1处理后的cav1-siRNA转染组比较,细胞cav-1、α-SMA、FN的表达差异无统计学意义(P0.05)。结论 1. AKF-PD能有效地抑制TGF-β1诱导的NHLFs活化和ECM合成。 2. AKF-PD能有效地上调TGF-β1刺激的NHLFs上cav-1的表达。 3.调控cav-1的表达是AKF-PD抑制TGF-β1诱导的NHLFs活化和ECM合成的关键机制。
[Abstract]:objective
Observation of fluorofenidone (Fluorofenidone, AKF-PD) inhibit transforming growth factor -p1 (transforming growth factor- TGF- beta 1, beta 1) primary normal lung fibroblasts induced by (Normal Human Lung fibroblasts, NHLFs) activation and extracellular matrix (Extracellular matrix, ECM) synthesis, regulation of caveolin-1 (caveolin-1, -1 Cav-1) expression mechanism in AKF-PD against pulmonary fibrosis in rats.
Method
1. NHLFs of primary culture, subculture and purification of normal lung lobectomy on from distal to the lesion, cultured by tissue culture method of NHLFs, the cultured cells at the third passage cells cryopreserved.
2. NHLFs 3-7 generation, the first experiment were divided into 5 groups (n=5), which were the control group (N group), TGF- beta 1 (10ng/ml) treatment group (M group), TGF- beta 1 (10ng/ml) + high, low concentration of AKF-PD (200 g/ml, 400 g/ml 800, g/ml) treatment group (A200, A400, A800 group), each group in the cell after inoculation with corresponding treatment 12h, 24h, 48h, 64H, 72h, total protein extraction were collected by Western Blot detection of Cav-1, alpha smooth muscle actin (Alpha smooth muscle actin, alpha -SMA, fibronectin (fibronectin), FN the expression of egg white);
3. NHLFs 3-7 generation, designed by Shanghai genepharma Co. Ltd., was prepared by 3 different sequence of normal cavl-siRNA, and then were divided into 7 groups, namely control group (group N), TGF- beta 1 (10ng/ml) treatment group (M group), Co-siRNA transfection group beta 1 (10ng/ml), Co-siRNA+TGF- treatment group, cavl-siRNA transfection group, cav1-siRNA+TGF- beta 1 (10ng/ml) treatment group, cavl-siRNA+TGF- beta 1 (10ng/ml) +AKF-PD (400 g/ml) treatment group, each group in the cell after inoculation with corresponding treatment 48h, extraction of total protein were collected by Western Blot detection of Cav-1, -SMA alpha, FN expression the protein.
Result
With the TGF- 1. beta 1 (10ng/ml) the prolongation of time, the expression of Cav-1 decreased gradually (P0.05), and a time dependent cell; alpha -SMA, FN expression increased first and then decreased (P0.05) expression of.TGF- beta 1 can significantly decrease the Cav-1 cells (P0.05), can significantly increase cell alpha -SMA, FN the expression (P0.05), the best time to cell activation and ECM synthesis of 48h.
With 3. AKF-PD (200 g/ml, 400 g/ml, 800 g/ml) concentration increased, and TGF- beta 1 (10ng/ml) Co inoculation of 48h cells, the expression of Cav-1 increased gradually (P0.05), alpha -SMA cells, the expression of FN decreased gradually (P0.05), and has the best dose dependent.AKF-PD the effect of 400 g/ml can significantly inhibit TGF- beta 1 expression, can partially restore Cav-1 cell (P0.05) could significantly reduce cell alpha -SMA expression of FN (P0.05).
The transfection of 4.Co-siRNA had no effect on the expression of Cav-1: after Co-siRNA transfection, there was no significant difference in the expression of Cav-1, alpha -SMA and FN (P0.05) compared with the N group.
The expression of 5.cavl-siRNA cav1: cavl-siRNA interference successfully after transfection, compared with N group, the expression of Cav-1 almost disappeared, alpha -SMA (P0.05) cells, no statistically significant difference between the expression of FN (P0.05). The best transfection sequence is cavl-siRNA-2, the most obvious cell Cav-1 silencing (P0.05), alpha -SMA expression of FN cells. Almost the same with the N group.
The Co-siRNA transfection group after 6.TGF- beta 1 treatment: compared with the simple Co-siRNA transfection group, the expression of Cav-1 was significantly decreased (P0.05), the expression of -SMA and FN increased significantly (P0.05), compared with M group, there was no significant difference in the expression of cell Cav-1, alpha -SMA and M.
Cav1-siRNA transfection group 7.TGF- beta 1 after treatment: compared with cav1-siRNA transfection, the expression of Cav-1 decreased, but the difference was not statistically significant (P0.05), cell alpha -SMA FN expression increased significantly (P0.05); compared with M group, the expression of Cav-1 was significantly reduced (P0.05), alpha cell -SMA, the expression of FN decreased (P0.05).
There was no significant difference in the expression of Cav-1, alpha -SMA and FN between the cav1-siRNA transfection group treated with 8.TGF- and 1+AKF-PD and the TGF- transfection group treated with TGF- 1.
conclusion
1. AKF-PD can effectively inhibit the activation of NHLFs and ECM synthesis induced by TGF- beta 1.
2. AKF-PD can effectively increase the expression of Cav-1 on NHLFs stimulated by TGF- beta 1.
3. regulation of the expression of Cav-1 is the key mechanism of AKF-PD inhibition of NHLFs activation and ECM synthesis induced by TGF- beta 1.

【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R563

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