PPARβ对成纤维细胞热损伤变性的保护作用及其机制研究

发布时间：2018-07-03 05:29

本文选题：过氧化物酶体增殖物激活受体-β + 成纤维细胞　；参考：《中南大学》2012年博士论文

【摘要】：目的：探讨PPARβ对成纤维细胞热损伤变性的保护作用及其作用机制。方法： 1、采用体外组织细胞培养技术培养离体乳鼠真皮成纤维细胞和NIH3T3成纤维细胞。将培养好的成纤维细胞分为热损伤组(heat injury)(52℃热水浴加热30秒)和正常对照组(normal)。于24h,48h,72h,96h不同时相点采用MTT法检测热损伤组和正常对照组细胞的生长曲线和存活率；于24h采用倒置显微镜、电镜和Western blot技术对热损伤组和正常对照组细胞的形态结构和PPARβ的表达进行比较。 2、运用PPARβ shRNA质粒对乳鼠真皮成纤维细胞和NIH3T3成纤维细胞进行RNA干扰,两种细胞各分为PPARβ shRNA组、shRNA control对照组和空白对照组;采用PPARβ特异激动剂GW0742(10-66M)干预乳鼠真皮成纤维细胞和NIH3T3细胞,两种细胞各分为GW0742组、DMSO对照组和空白对照组。经52℃热水浴加热30秒处理,建立成纤维细胞热损伤变性模型。于24h时相点,分别采用电镜,流式细胞技术,Western blot技术检测乳鼠真皮成纤维细胞和NIH3T3细胞各六组细胞热损伤变性后细胞结构,生长周期及PCNA表达,观察PPARβ对于热损伤变性成纤维细胞结构和生长增殖的影响。 3、采用HSF1表达质粒转染NIH3T3成纤维细胞,建立高表达HSF1的细胞株。运用RT-PCR和Western blot技术检测NIH3T3成纤维细胞热损伤变性(52℃,30s)后PPARβ的表达。多个独立样本采用单因素方差分析,两指标间相关分析用T检验的统计学方法分析结果数据,P0.05为差异有统计学意义。结果： 1.采用52℃,30s热水浴损伤条件,成功诱导出乳鼠真皮成纤维细胞和NIH3T3成纤维细胞热损伤变性模型。热损伤变性的乳鼠真皮成纤维细胞和NIH3T3细胞的生长曲线在热损伤后24h时相点达到最低值,后逐渐开始恢复。24h时相点,热损伤变性的两种成纤维细胞生存率分别为50.6%和49.6%,都有统计学意义(P0.05)。倒置显微镜和透射电镜观察热损伤变性的乳鼠真皮成纤维细胞和NIH3T3细胞均显示细胞胞浆出现大量空泡样物质,线粒体和内质网明显受损,两种细胞的损伤情况十分相似。乳鼠真皮成纤维细胞和NIH3T3细胞热损伤变性后,热损伤组PPARP的表达量与正常对照组比较均增高(P0.05)。 2.用RNA干扰技术沉默PPARβ,乳鼠真皮成纤维细胞和NIH3T3细胞热损伤变性后,电镜观察乳鼠真皮成纤维细胞和NIH3T3细胞shRNA组细胞结构破坏情况相似,结构损伤情况与对照组比较更加严重,出现胞膜和核膜的破坏,核边聚固缩的现象。流式细胞周期检测,shRNA组乳鼠真皮成纤维细胞的G0/G1期细胞百分率为(76.5±1.77),NIH3T3细胞的G0/G1期细胞百分率为(77.5±0.77),分别明显高于乳鼠真皮成纤维细胞空白对照组(50.3±2.28)、阴性对照组(50.7±1.78)和NIH3T3细胞空白对照组(50.3±0.2)、阴性对照组(50.4±1.21)(P0.05)。乳鼠真皮成纤维细胞的G2/M、S期细胞百分率为(7.8±1.23和13.6±0.84),NIH3T3细胞的G2/M、S期细胞百分率为(9.2±1.13和12.8±1.84),分别低于乳鼠真皮成纤维细胞空白对照组(12.5±2.07和36.5±2.38)、阴性对照组(13.0±0.67和36.1±1.46)和NIH3T3细胞空白对照组(16.3±0.39和31.8±1.38)、阴性对照组(16.6±0.39和33.8±2.32)(P0.05)。 shRNA组乳鼠真皮成纤维细胞增殖指数(0.22),NIH3T3细胞增殖指数(0.22),分别明显低于乳鼠真皮成纤维细胞空白对照组(0.49)、阴性对照组(0.49)和NIH3T3细胞空白对照组(0.49)、阴性对照组(0.50)(P0.05)。 Western blot显示,shPNA组的乳鼠真皮成纤维细胞和NIH3T3细胞的PNCA表达量与对照组比较分别降低了61.5%和52.7%(P0.05)。 3.采用特异性激动剂GW0742激动PPARβ,乳鼠真皮成纤维细胞和NIH3T3细胞热损伤变性后,电镜观察乳鼠真皮成纤维细胞和NIH3T3细胞GW0742组细胞损伤情况十分相似,细胞结构与对照组相比更为完整,胞浆内空泡结构减少,核仁清晰。流式细胞周期检测,GW0742组乳鼠真皮成纤维细胞的G0/G1期细胞百分率为(38.5±3.06), NIH3T3细胞的G0/G1期细胞百分率为(40.1±3.27),分别明显低于乳鼠真皮成纤维细胞空白对照组(50.4±1.43)、阴性对照组(50.2±2.15)和NIH3T3细胞空白对照组(56.4±1.43)、阴性对照组(56.2士2.15)(P0.05)。乳鼠真皮成纤维细胞的G2/M、S期细胞百分率为(21.3士1.57和42.8±1.38),NIH3T3细胞的G2/M、S期细胞百分率为(16.8±3.17和44.3±1.15),分别高于乳鼠真皮成纤维细胞空白对照组(10.8±2.56和38.7±2.29)、阴性对照组(10.5±1.26和38.4±1.49)和NIH3T3细胞空白对照组(10.7±2.56和31.6±2.29)、阴性对照组(11.5士1.26和33.4±1.49)(P0.05)。GW0742组乳鼠真皮成纤维细胞增殖指数(0.62),NIH3T3细胞增殖指数(0.61),分别明显高于乳鼠真皮成纤维细胞空白对照组(0.50)、阴性对照组(0.49)和NIH3T3细胞空白对照组(0.42)、阴性对照组(0.42)(P0.05)。Western blot显示,GW0742组的乳鼠真皮成纤维细胞和NIH3T3细胞的PNCA表达量与对照组比较分别增加了67.7%和47.3%(P0.05)。 4.上调表达HSF1的NIH3T3细胞遭受热损伤后,采用RT-PCR和Western blot显示PPARβ的表达量均增加(P0.05)。结论： 1.采用52℃,30s热水浴成功诱导离体鼠类成纤维细胞热损伤模型。热损伤变性的乳鼠真皮成纤维细胞和NIH3T3细胞的细胞结构和生长情况相似：胞浆结构损伤严重,胞核未见明显损伤,24h时相点成纤维细胞生长增殖减低最严重,后逐渐恢复正常,生存率明显降低。热损伤乳鼠真皮成纤维细胞和NIH3T3细胞均显示PPARβ的表达量增高。 2. shRNA组乳鼠真皮成纤维细胞和NIH3T3细胞结构损伤更为严重,处于分裂期的细胞百分比降低,处于静止期细胞百分比增高,增殖能力降低。而GW0742组乳鼠真皮成纤维细胞和NIH3T3细胞经热损伤变性后,细胞结构损伤比较对照组细胞减弱,处于分裂期的细胞百分比增高,增殖能力提高。PPARβ对热损伤变性的乳鼠真皮成纤维细胞和NIH3T3细胞的结构和生长增殖具有保护作用。 3.转染HSF1的NIH3T3成纤维细胞热损伤变性后,该细胞PPARβ的表达上调,初步证实HSF1通过上调PPARβ而对热损伤变性后成纤维细胞发挥保护作用。
[Abstract]:Objective:
Objective to investigate the protective effect and mechanism of PPAR beta on heat damage and degeneration of fibroblasts.
Method:
1, the cultured rat dermal fibroblasts and NIH3T3 fibroblasts were cultured in vitro tissue cell culture. The cultured fibroblasts were divided into heat injury group (heat injury) (52 centigrade hot water bath heating for 30 seconds) and normal control group (normal). In 24h, 48h, 72h, and 96h, the thermal injury group and normal control group were detected by MTT method. The growth curve and survival rate of the cells were compared with the morphological structure and the expression of PPAR beta in the heat injured group and the normal control group by the inverted microscope, the electron microscope and the Western blot technique in 24h.
2, PPAR beta shRNA plasmids were used to interfere with the RNA cells of dermis fibroblasts and NIH3T3 fibroblasts in milk rats. The two cells were divided into PPAR beta shRNA group, shRNA control control group and blank control group. PPAR beta specific agonist GW0742 (10-66M) was used to intervene the dermis fibroblasts and NIH3T3 cells, and the two kinds of cells were divided into groups. The control group and the blank control group were treated by hot water bath at 52 C for 30 seconds, and the heat damage degeneration model of fibroblasts was established. At 24h phase point, the cell structure, growth cycle and PCNA expression were detected by electron microscope, flow cytometry and Western blot technique in six groups of cells of dermis fibroblasts and NIH3T3 cells. To observe the effect of PPAR beta on the structure, growth and proliferation of heat damaged denatured fibroblasts.
3, NIH3T3 fibroblasts were transfected with HSF1 expression plasmid to establish a cell line with high expression of HSF1. RT-PCR and Western blot were used to detect the expression of PPAR beta in the heat damage degeneration (52 degrees, 30s) of NIH3T3 fibroblasts. Multiple independent samples were analyzed by single factor analysis of variance, and the results of statistical analysis by T test of two inter finger correlation analysis were analyzed by T. The data, P0.05, were statistically significant.
Result:
1. with the damage condition of 52 C and 30s hot water bath, the heat damage degeneration model of the dermal fibroblasts and NIH3T3 fibroblasts in the milk rat was successfully induced. The growth curve of the dermis fibroblasts and the NIH3T3 cells of the heat damaged milk rat reached the lowest value at the 24h phase point after the heat damage, and then gradually began to recover the phase point of.24h, and the heat damage degeneration was found. The survival rates of two kinds of fibroblasts were 50.6% and 49.6% respectively (P0.05). The inverted microscope and transmission electron microscope showed that the dermis fibroblasts and NIH3T3 cells of the rats with heat damage degeneration showed that a large number of vacuoles appeared in the cytoplasm, the mitochondria and endoplasmic reticulum were obviously damaged, and the damage of the two cells was very different. The expression level of PPARP in the thermal injury group was higher than that in the normal control group (P0.05) after the thermal injury and degeneration of neonatal rat dermal fibroblasts and NIH3T3 cells.
2. RNA interference technique was used to silence PPAR beta, the dermis fibroblasts and NIH3T3 cells of the milk rat were damaged by heat damage. The damage of the cell structure of the dermis fibroblasts and the shRNA group of the NIH3T3 cells was similar. The damage of the structure was more serious than the control group, and the destruction of the membrane and nuclear membrane and the condensation of the nuclear side. Cell cycle detection, the percentage of G0/G1 phase cells in shRNA group of dermis fibroblasts was (76.5 + 1.77), and the percentage of G0/G1 cells in NIH3T3 cells was (77.5 + 0.77), which was significantly higher than that in blank control group (50.3 + 2.28), negative control group (50.7 + 1.78) and NIH3T3 cell blank control group (50.3 + 0.2), negative The control group (50.4 + 1.21) (P0.05). The percentage of G2/M and S phase cells in the dermis fibroblasts was (7.8 + 1.23 and 13.6 + 0.84), and the percentage of G2/M and S cells in NIH3T3 cells was (9.2 + 1.13 and 12.8 + 1.84), which were lower than that in the blank control group (12.5 + 2.07 and 36.5 + 2.38) in the dermis fibroblasts, and the negative control group. And NIH3T3 cell blank control group (16.3 + 0.39 and 31.8 + 1.38), negative control group (16.6 + 0.39 and 33.8 + 2.32) (P0.05). The proliferation index of dermal fibroblast (0.22) and NIH3T3 cell proliferation index (0.22) in shRNA group were significantly lower than that of blank control group (0.49), negative control group (0.49) and NIH3T3 cell blank group, respectively. The control group (0.49), negative control group (0.50) (P0.05). Western blot showed that the PNCA expression of dermis fibroblasts and NIH3T3 cells in shPNA group was 61.5% and 52.7% (P0.05) compared with the control group respectively.
3. after PPAR beta was excited by the specific agonist GW0742, the dermis fibroblasts and NIH3T3 cells in the milk rat were damaged by heat damage. The damage of the cells of the dermis fibroblasts and the GW0742 group of the NIH3T3 cells was very similar. The cell structure was more complete than the control group, the cytoplasmic vacuoles were reduced, the nucleolus was clear. The percentage of G0/G1 phase cells in GW0742 group of dermis fibroblasts was (38.5 + 3.06), and the percentage of G0/G1 cells in NIH3T3 cells was (40.1 + 3.27), which was significantly lower than that of blank control group (50.4 + 1.43), negative control group (50.2 + 2.15) and NIH3T3 cell blank control group (56.4 + 1.43), negative control. Group (56.2 and 2.15) (P0.05). The percentage of G2/M and S phase cells in the dermis fibroblasts was (21.3, 1.57 and 42.8 + 1.38), and the percentage of G2/M and S cells in NIH3T3 cells was (16.8 + 3.17 and 44.3 + 1.15), higher than that in the blank control group (10.8 + 2.56 and 38.7 + 1.15), and the negative control group and the negative control group. NIH3T3 cell blank control group (10.7 + 2.56 and 31.6 + 2.29), negative control group (11.5, 1.26 and 33.4 + 1.49) (P0.05).GW0742 group, the proliferation index of dermal fibroblast (0.62) and NIH3T3 cell proliferation index (0.61) were significantly higher than that of blank control group (0.50), negative control group (0.49) and NIH3T3 cell blank control group (0.61). In the control group (0.42), the negative control group (0.42) (P0.05).Western blot showed that the PNCA expression of the dermis fibroblasts and NIH3T3 cells in the GW0742 group increased by 67.7% and 47.3% respectively (P0.05).
4. upregulated the expression of PPAR beta in NIH3T3 cells expressing HSF1 after being damaged by heat. The expression of PPAR beta increased by RT-PCR and Western blot (P0.05).
Conclusion:
1. the heat damage model of isolated rat fibroblasts was successfully induced by 30s hot water bath at 52 C. The cell structure and growth of the dermis fibroblasts and NIH3T3 cells of the heat injured rat were similar: the cytoplasm structure was seriously damaged and the nucleus was not obviously damaged, and the growth and proliferation of the phase point fibroblast in 24h was the most serious, and then gradually restored. The survival rate of the injured rats was significantly reduced. The expression of PPAR beta increased in the dermal fibroblasts and NIH3T3 cells of the heat injured suckling mice.
In 2. shRNA group, the damage of the dermal fibroblasts and NIH3T3 cells was more serious, the percentage of cells in the split stage decreased, the percentage of cells in the resting stage increased and the proliferation ability decreased. The damage of cell structure in the dermis fibroblasts and NIH3T3 cells in the GW0742 group was weaker than that of the control group. The percentage of cells in the split stage increased, and the proliferation ability enhanced.PPAR beta to protect the structure and growth of the dermis fibroblasts and NIH3T3 cells of heat injured rats.
3. NIH3T3 fibroblasts transfected with HSF1 were heat damaged and denatured, and the expression of PPAR beta was up-regulated. It was preliminarily confirmed that HSF1 protects fibroblasts after heat damage by up regulation of PPAR beta.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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