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  本文選題：氟非尼酮　切入點：原代正常肺成纖維細胞　出處：《中南大學》2013年碩士論文　論文類型：學位論文

【摘要】：目的 觀察氟非尼酮(Fluorofenidone, AKF-PD)抑制轉(zhuǎn)化生長因子-p1(transforming growth factor-β1, TGF-β1)誘導原代正常肺成纖維細胞(Normal Human Lung fibroblasts, NHLFs)活化和細胞外基質(zhì)(Extracellular matrix,ECM)合成的作用,探討調(diào)控小窩蛋白-1(caveolin-1,cav-1)表達在AKF-PD抗肺纖維化機制中的作用。 方法 1. NHLFs的原代培養(yǎng)、鑒定及純化傳代：于肺葉切除術(shù)中取病變遠端的正常肺組織,采用組織塊培養(yǎng)法進行NHLFs的原代培養(yǎng),將培養(yǎng)的細胞純化傳代至第3代細胞凍存?zhèn)溆谩?2.取NHLFs第3-7代使用,首先將實驗分為5組(n=5),分別為空白對照組(N組)、TGF-β1(10ng/ml)處理組(M組)、TGF-β1(10ng/ml)+高、中、低濃度AKF-PD(200μg/ml、400μg/ml、800μg/ml)治療組(A200、A400、A800組),各組于細胞接種后相應處理12h、24h、48h、64h、72h,收集細胞提取總蛋白,采用Western Blot檢測cav-1、α-平滑肌動蛋白(Alpha smooth muscle actin,α-SMA)、纖維連接蛋白(fibronectin,FN)的蛋白表達； 3.取NHLFs第3-7代使用,由上海吉瑪制藥技術(shù)有限公司設計、制備得到3個不同序列的正常人cavl-siRNA,再將實驗分為7組,分別為空白對照組(N組)、TGF-β1(10ng/ml)處理組(M組)、Co-siRNA轉(zhuǎn)染組、Co-siRNA+TGF-β1(10ng/ml)處理組、cavl-siRNA轉(zhuǎn)染組、cav1-siRNA+TGF-β1(10ng/ml)處理組、cavl-siRNA+TGF-β1(10ng/ml)+AKF-PD(400μg/ml)處理組,各組于細胞接種后相應處理48h,收集細胞提取總蛋白,采用Western Blot檢測cav-1、α-SMA、FN的蛋白表達。 結(jié)果 1.隨著TGF-β1(10ng/ml)作用時間的延長,cav-1的表達逐漸減少(p0.05),并且具有時間依賴性;細胞α-SMA、FN的表達先增加后減少(p0.05)。TGF-β1能顯著下調(diào)細胞cav-1的表達(p0.05),能明顯上調(diào)細胞α-SMA、FN的表達(p0.05),細胞活化與ECM合成的最佳時間為48h時。 3.隨著AKF-PD(200μg/ml、400μg/ml、800μg/ml)濃度的增加,與TGF-β1(10ng/ml)共同接種細胞48h后,細胞cav-1的表達逐漸增加(p0.05),細胞α-SMA、FN的表達逐漸減少(p0.05),并且具有劑量依賴性。AKF-PD作用的最佳濃度為400μg/ml,能顯著抑制TGF-β1的作用,能部分恢復細胞cav-1的表達(p0.05)能明顯下調(diào)細胞α-SMA、FN的表達(p0.05)。 4.Co-siRNA轉(zhuǎn)染對cav-1表達無影響：Co-siRNA轉(zhuǎn)染后,與N組比較,細胞cav-1、α-SMA及FN的表達差異無統(tǒng)計學意義(P0.05)。 5.cavl-siRNA成功干擾cav1的表達：cavl-siRNA轉(zhuǎn)染后,與N組比較,細胞cav-1的表達幾乎消失(p0.05),細胞α-SMA、FN的表達差異無統(tǒng)計學意義(P0.05)。而最佳轉(zhuǎn)染序列為cavl-siRNA-2,細胞cav-1沉默最明顯(p0.05),細胞α-SMA、FN的表達幾乎與N組相同。 6.TGF-β1處理后的Co-siRNA轉(zhuǎn)染組：與單純Co-siRNA轉(zhuǎn)染組比較,細胞cav-1的表達明顯減少(p0.05),細胞α-SMA、FN的表達明顯增多(p0.05);與M組比較,細胞cav-1、α-SMA、FN的表達差異無統(tǒng)計學意義(P0.05)。 7.TGF-β1處理后的cav1-siRNA轉(zhuǎn)染組：與單純cav1-siRNA轉(zhuǎn)染組比較,細胞cav-1的表達有所減少,但差異無統(tǒng)計學意義(P0.05),細胞α-SMA、FN的表達明顯增多(p0.05)；與M組比較,細胞cav-1的表達明顯減少(p0.05),細胞α-SMA、FN的表達有所減少(p0.05)。 8.TGF-β1+AKF-PD共同處理后的cav1-siRNA轉(zhuǎn)染組：與TGF-β1處理后的cav1-siRNA轉(zhuǎn)染組比較,細胞cav-1、α-SMA、FN的表達差異無統(tǒng)計學意義(P0.05)。 結(jié)論 1. AKF-PD能有效地抑制TGF-β1誘導的NHLFs活化和ECM合成。 2. AKF-PD能有效地上調(diào)TGF-β1刺激的NHLFs上cav-1的表達。 3.調(diào)控cav-1的表達是AKF-PD抑制TGF-β1誘導的NHLFs活化和ECM合成的關(guān)鍵機制。
[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.

【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R563

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