本文選題：過(guò)氧化物酶體增殖物激活受體-β + 成纖維細(xì)胞　； 參考：《中南大學(xué)》2012年博士論文

【摘要】：目的： 探討PPARβ對(duì)成纖維細(xì)胞熱損傷變性的保護(hù)作用及其作用機(jī)制。 方法： 1、采用體外組織細(xì)胞培養(yǎng)技術(shù)培養(yǎng)離體乳鼠真皮成纖維細(xì)胞和NIH3T3成纖維細(xì)胞。將培養(yǎng)好的成纖維細(xì)胞分為熱損傷組(heat injury)(52℃熱水浴加熱30秒)和正常對(duì)照組(normal)。于24h,48h,72h,96h不同時(shí)相點(diǎn)采用MTT法檢測(cè)熱損傷組和正常對(duì)照組細(xì)胞的生長(zhǎng)曲線和存活率；于24h采用倒置顯微鏡、電鏡和Western blot技術(shù)對(duì)熱損傷組和正常對(duì)照組細(xì)胞的形態(tài)結(jié)構(gòu)和PPARβ的表達(dá)進(jìn)行比較。 2、運(yùn)用PPARβ shRNA質(zhì)粒對(duì)乳鼠真皮成纖維細(xì)胞和NIH3T3成纖維細(xì)胞進(jìn)行RNA干擾,兩種細(xì)胞各分為PPARβ shRNA組、shRNA control對(duì)照組和空白對(duì)照組;采用PPARβ特異激動(dòng)劑GW0742(10-66M)干預(yù)乳鼠真皮成纖維細(xì)胞和NIH3T3細(xì)胞,兩種細(xì)胞各分為GW0742組、DMSO對(duì)照組和空白對(duì)照組。經(jīng)52℃熱水浴加熱30秒處理,建立成纖維細(xì)胞熱損傷變性模型。于24h時(shí)相點(diǎn),分別采用電鏡,流式細(xì)胞技術(shù),Western blot技術(shù)檢測(cè)乳鼠真皮成纖維細(xì)胞和NIH3T3細(xì)胞各六組細(xì)胞熱損傷變性后細(xì)胞結(jié)構(gòu),生長(zhǎng)周期及PCNA表達(dá),觀察PPARβ對(duì)于熱損傷變性成纖維細(xì)胞結(jié)構(gòu)和生長(zhǎng)增殖的影響。 3、采用HSF1表達(dá)質(zhì)粒轉(zhuǎn)染NIH3T3成纖維細(xì)胞,建立高表達(dá)HSF1的細(xì)胞株。運(yùn)用RT-PCR和Western blot技術(shù)檢測(cè)NIH3T3成纖維細(xì)胞熱損傷變性(52℃,30s)后PPARβ的表達(dá)。多個(gè)獨(dú)立樣本采用單因素方差分析,兩指標(biāo)間相關(guān)分析用T檢驗(yàn)的統(tǒng)計(jì)學(xué)方法分析結(jié)果數(shù)據(jù),P0.05為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果： 1.采用52℃,30s熱水浴損傷條件,成功誘導(dǎo)出乳鼠真皮成纖維細(xì)胞和NIH3T3成纖維細(xì)胞熱損傷變性模型。熱損傷變性的乳鼠真皮成纖維細(xì)胞和NIH3T3細(xì)胞的生長(zhǎng)曲線在熱損傷后24h時(shí)相點(diǎn)達(dá)到最低值,后逐漸開(kāi)始恢復(fù)。24h時(shí)相點(diǎn),熱損傷變性的兩種成纖維細(xì)胞生存率分別為50.6%和49.6%,都有統(tǒng)計(jì)學(xué)意義(P0.05)。倒置顯微鏡和透射電鏡觀察熱損傷變性的乳鼠真皮成纖維細(xì)胞和NIH3T3細(xì)胞均顯示細(xì)胞胞漿出現(xiàn)大量空泡樣物質(zhì),線粒體和內(nèi)質(zhì)網(wǎng)明顯受損,兩種細(xì)胞的損傷情況十分相似。乳鼠真皮成纖維細(xì)胞和NIH3T3細(xì)胞熱損傷變性后,熱損傷組PPARP的表達(dá)量與正常對(duì)照組比較均增高(P0.05)。 2.用RNA干擾技術(shù)沉默PPARβ,乳鼠真皮成纖維細(xì)胞和NIH3T3細(xì)胞熱損傷變性后,電鏡觀察乳鼠真皮成纖維細(xì)胞和NIH3T3細(xì)胞shRNA組細(xì)胞結(jié)構(gòu)破壞情況相似,結(jié)構(gòu)損傷情況與對(duì)照組比較更加嚴(yán)重,出現(xiàn)胞膜和核膜的破壞,核邊聚固縮的現(xiàn)象。流式細(xì)胞周期檢測(cè),shRNA組乳鼠真皮成纖維細(xì)胞的G0/G1期細(xì)胞百分率為(76.5±1.77),NIH3T3細(xì)胞的G0/G1期細(xì)胞百分率為(77.5±0.77),分別明顯高于乳鼠真皮成纖維細(xì)胞空白對(duì)照組(50.3±2.28)、陰性對(duì)照組(50.7±1.78)和NIH3T3細(xì)胞空白對(duì)照組(50.3±0.2)、陰性對(duì)照組(50.4±1.21)(P0.05)。乳鼠真皮成纖維細(xì)胞的G2/M、S期細(xì)胞百分率為(7.8±1.23和13.6±0.84),NIH3T3細(xì)胞的G2/M、S期細(xì)胞百分率為(9.2±1.13和12.8±1.84),分別低于乳鼠真皮成纖維細(xì)胞空白對(duì)照組(12.5±2.07和36.5±2.38)、陰性對(duì)照組(13.0±0.67和36.1±1.46)和NIH3T3細(xì)胞空白對(duì)照組(16.3±0.39和31.8±1.38)、陰性對(duì)照組(16.6±0.39和33.8±2.32)(P0.05)。 shRNA組乳鼠真皮成纖維細(xì)胞增殖指數(shù)(0.22),NIH3T3細(xì)胞增殖指數(shù)(0.22),分別明顯低于乳鼠真皮成纖維細(xì)胞空白對(duì)照組(0.49)、陰性對(duì)照組(0.49)和NIH3T3細(xì)胞空白對(duì)照組(0.49)、陰性對(duì)照組(0.50)(P0.05)。 Western blot顯示,shPNA組的乳鼠真皮成纖維細(xì)胞和NIH3T3細(xì)胞的PNCA表達(dá)量與對(duì)照組比較分別降低了61.5%和52.7%(P0.05)。 3.采用特異性激動(dòng)劑GW0742激動(dòng)PPARβ,乳鼠真皮成纖維細(xì)胞和NIH3T3細(xì)胞熱損傷變性后,電鏡觀察乳鼠真皮成纖維細(xì)胞和NIH3T3細(xì)胞GW0742組細(xì)胞損傷情況十分相似,細(xì)胞結(jié)構(gòu)與對(duì)照組相比更為完整,胞漿內(nèi)空泡結(jié)構(gòu)減少,核仁清晰。流式細(xì)胞周期檢測(cè),GW0742組乳鼠真皮成纖維細(xì)胞的G0/G1期細(xì)胞百分率為(38.5±3.06), NIH3T3細(xì)胞的G0/G1期細(xì)胞百分率為(40.1±3.27),分別明顯低于乳鼠真皮成纖維細(xì)胞空白對(duì)照組(50.4±1.43)、陰性對(duì)照組(50.2±2.15)和NIH3T3細(xì)胞空白對(duì)照組(56.4±1.43)、陰性對(duì)照組(56.2士2.15)(P0.05)。乳鼠真皮成纖維細(xì)胞的G2/M、S期細(xì)胞百分率為(21.3士1.57和42.8±1.38),NIH3T3細(xì)胞的G2/M、S期細(xì)胞百分率為(16.8±3.17和44.3±1.15),分別高于乳鼠真皮成纖維細(xì)胞空白對(duì)照組(10.8±2.56和38.7±2.29)、陰性對(duì)照組(10.5±1.26和38.4±1.49)和NIH3T3細(xì)胞空白對(duì)照組(10.7±2.56和31.6±2.29)、陰性對(duì)照組(11.5士1.26和33.4±1.49)(P0.05)。GW0742組乳鼠真皮成纖維細(xì)胞增殖指數(shù)(0.62),NIH3T3細(xì)胞增殖指數(shù)(0.61),分別明顯高于乳鼠真皮成纖維細(xì)胞空白對(duì)照組(0.50)、陰性對(duì)照組(0.49)和NIH3T3細(xì)胞空白對(duì)照組(0.42)、陰性對(duì)照組(0.42)(P0.05)。Western blot顯示,GW0742組的乳鼠真皮成纖維細(xì)胞和NIH3T3細(xì)胞的PNCA表達(dá)量與對(duì)照組比較分別增加了67.7%和47.3%(P0.05)。 4.上調(diào)表達(dá)HSF1的NIH3T3細(xì)胞遭受熱損傷后,采用RT-PCR和Western blot顯示PPARβ的表達(dá)量均增加(P0.05)。 結(jié)論： 1.采用52℃,30s熱水浴成功誘導(dǎo)離體鼠類成纖維細(xì)胞熱損傷模型。熱損傷變性的乳鼠真皮成纖維細(xì)胞和NIH3T3細(xì)胞的細(xì)胞結(jié)構(gòu)和生長(zhǎng)情況相似：胞漿結(jié)構(gòu)損傷嚴(yán)重,胞核未見(jiàn)明顯損傷,24h時(shí)相點(diǎn)成纖維細(xì)胞生長(zhǎng)增殖減低最嚴(yán)重,后逐漸恢復(fù)正常,生存率明顯降低。熱損傷乳鼠真皮成纖維細(xì)胞和NIH3T3細(xì)胞均顯示PPARβ的表達(dá)量增高。 2. shRNA組乳鼠真皮成纖維細(xì)胞和NIH3T3細(xì)胞結(jié)構(gòu)損傷更為嚴(yán)重,處于分裂期的細(xì)胞百分比降低,處于靜止期細(xì)胞百分比增高,增殖能力降低。而GW0742組乳鼠真皮成纖維細(xì)胞和NIH3T3細(xì)胞經(jīng)熱損傷變性后,細(xì)胞結(jié)構(gòu)損傷比較對(duì)照組細(xì)胞減弱,處于分裂期的細(xì)胞百分比增高,增殖能力提高。PPARβ對(duì)熱損傷變性的乳鼠真皮成纖維細(xì)胞和NIH3T3細(xì)胞的結(jié)構(gòu)和生長(zhǎng)增殖具有保護(hù)作用。 3.轉(zhuǎn)染HSF1的NIH3T3成纖維細(xì)胞熱損傷變性后,該細(xì)胞PPARβ的表達(dá)上調(diào),初步證實(shí)HSF1通過(guò)上調(diào)PPARβ而對(duì)熱損傷變性后成纖維細(xì)胞發(fā)揮保護(hù)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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

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