本文選題：缺氧 + Rho-ROCK信號通路　； 參考：《廣州醫(yī)學院》2011年碩士論文

【摘要】：1目的 1.1探討缺氧(hypoxia)對人肺微血管內(nèi)皮細胞(HPMVECs)肌動蛋白細胞骨架(F-actin)重構(gòu)的影響,以及Rho-ROCK信號通路在缺氧引起肌動蛋白細胞骨架重構(gòu)過程中的作用。 1.2研究缺氧條件下,HPMVECs內(nèi)半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)活性的變化規(guī)律和重組人白介素10(rhIL-10)對缺氧HPMVECs凋亡的影響。 2方法 2.1在缺氧前用ROCK的特異性抑制劑H-1152,最佳濃度(10 nmol/L)對體外培養(yǎng)的HPMVECs進行預處理0.5h,再分為不同缺氧時間組進行缺氧6h、12h及24h處理,并設(shè)常規(guī)培養(yǎng)正常對照組,每組設(shè)8個復孔。用激光共聚焦顯微鏡(LSCM)觀察缺氧或使用H-1152對HPMVECs細胞肌動蛋白細胞骨架重構(gòu)的影響。流式細胞技術(shù)檢測各組HPMVECs F-actin的平均熒光強度的變化,行統(tǒng)計學分析。 2.2常規(guī)培養(yǎng)HPMVECs細胞株,分為5組:正常對照組(常規(guī)培養(yǎng));缺氧(5% CO2,95% N2)對照組;缺氧培養(yǎng)并加入rhIL-10以(10ng/mL,50ng/mL,100ng/mL)的濃度分別干預的rhIL-10低劑量干預組、rhIL-10中劑量干預組、rhIL-10高劑量干預組。培養(yǎng)4h、12h、24h、48h離心收集細胞,熒光顯微鏡觀察HPMVECs 24h核熒光染色,分析熒光強度,化學比色法檢測細胞核內(nèi)Caspase-3活性檢測,行統(tǒng)計學分析。 3結(jié)果 3.1 HPMVECs 6h各組, LSCM觀察顯示F-actin細胞骨架未發(fā)生明顯改變,流式細胞術(shù)行F-actin熒光強度分析與正常對照組比較差別無統(tǒng)計學意義(P0.05)。缺氧12h組,用LSCM觀察到F-actin細胞骨架已發(fā)生少量解聚,運用流式細胞術(shù)行F-actin熒光強度分析,提示F-actin含量增加與正常對照組比較差別有統(tǒng)計學意義(P0.05);而H-1152+缺氧12h組,細胞骨架及細胞周邊的肌動蛋白絲帶無明顯變化,胞漿中僅有極少量模糊的肌動蛋白,流式細胞術(shù)行F-actin熒光強度分析提示F-actin含量與缺氧組比較差別有統(tǒng)計學意義(P0.05)。缺氧24h組,用LSCM觀察到F-actin細胞骨架已發(fā)生大量解聚,細胞骨架明顯改變,流式細胞術(shù)行F-actin熒光強度分析提示F-actin含量與正常對照組比較差別有統(tǒng)計學意義(P0.05); H-1152+缺氧24h,細胞骨架及細胞周邊的肌動蛋白絲帶有少量變化,胞漿中有模糊的肌動蛋白出現(xiàn),流式細胞術(shù)行F-actin熒光強度分析提示F-actin含量與正常對照組比較及缺氧組比較差別均存在統(tǒng)計學意義(P0.05)。H-1152各時間組在用LSCM觀察F-actin細胞骨架與正常對照組比較無明顯差異,流式細胞術(shù)行F-actin熒光強度分析提示F-actin含量與正常對照組比較差別無統(tǒng)計學意義(P0.05)。HPMVECs經(jīng)缺氧處理,肌動蛋白細胞骨架發(fā)生解聚,應(yīng)力纖維的排列和分布改變;事先使用H-1152再進行缺氧處理,可防止缺氧所致HPMVECs內(nèi)F-actin解聚及其含量增加,同時應(yīng)力纖維在細胞內(nèi)排列和分布趨向正常。 3.2取24h各組行凋亡細胞核Hoechst熒光染色,灰度值分析低氧對照組和低、中、高rhIL-10干預組與正常對照組比較升高(P0.05);低、中、高rhIL-10干預組與缺氧對照組比較降低(P0.05)。凋亡蛋白Caspase-3相對活性,4h各組別無明顯差異(P0.05);12h缺氧對照組、低、中rhIL-10干預組與正常組比較升高(P0.05),高劑量rhIL-10干預組與正常對照組無明顯差異(P0.05),rhIL-10干預各組與缺氧對照組比較降低(P0.05);24h缺氧對照組、rhIL-10干預低、中、高劑量組與正常對照組比較升高(P0.05),低、中、高劑量rhIL-10干預組與缺氧對照組比較明顯降低(P0.05);48h缺氧對照組與低劑量rhIL-10干預組與正常對照組比較升高(P0.05),其余各組別無明顯差異(P0.05);缺氧凋亡峰值見于24h。 4結(jié)論 4.1缺氧所致HPMVECs內(nèi)的F-actin重構(gòu)改變。 4.2 Rho-ROCK信號通路參與缺氧所致HPMVECs內(nèi)F-actin細胞骨架的重構(gòu)改變。 4.3缺氧可促進HPMVECs凋亡,缺氧條件下,Caspase-3在HPMVECs的表達明顯升高。 4.4 rhIL-10可一定程度地抑制缺氧HPMVECs的凋亡。
[Abstract]:1 purposes
1.1 to investigate the effect of hypoxia (hypoxia) on the remodeling of actin cytoskeleton (F-actin) in human pulmonary microvascular endothelial cells (HPMVECs) and the role of Rho-ROCK signaling pathway in the remodeling of actin cytoskeleton induced by hypoxia.
1.2 the changes in the activity of cysteine aspartic proteinase -3 (Caspase-3) in HPMVECs and the effect of recombinant human interleukin 10 (rhIL-10) on the apoptosis of anoxic HPMVECs were studied under the condition of hypoxia.
2 method
2.1 before hypoxia, the specific inhibitor H-1152 of ROCK and the best concentration (10 nmol/L) were pretreated with 0.5h for HPMVECs in vitro, and then divided into different anoxic time groups to perform hypoxia 6h, 12h and 24h treatment, and set up a normal control group with 8 complex holes in each group. The hypoxia or H-1152 to HPM was observed by the excitation confocal microscope (LSCM). The influence of VECs cell actin cytoskeleton remodeling was investigated. The mean fluorescence intensity of HPMVECs F-actin in each group was detected by flow cytometry.
2.2 normal culture HPMVECs cell lines were divided into 5 groups: normal control group (conventional culture), hypoxia (5% CO2,95% N2) control group, hypoxia culture and rhIL-10 (10ng/mL, 50ng/mL, 100ng/mL) concentration intervention group, rhIL-10 middle dose intervention group, rhIL-10 high dose intervention group. HPMVECs 24h nuclear fluorescence staining was used to analyze the fluorescence intensity, and the Caspase-3 activity in the nucleus was detected by chemical colorimetry.
3 Results
3.1 HPMVECs 6h groups, LSCM observation showed that the F-actin cytoskeleton did not change obviously. There was no significant difference in F-actin fluorescence intensity analysis between the flow cytometry and the normal control group (P0.05). In the hypoxia 12h group, a small amount of depolymerization of the F-actin cytoskeleton was observed with LSCM, and the fluorescence intensity analysis of F-actin was performed by flow cytometry. There was a significant difference between the increase of F-actin content and the normal control group (P0.05), but there was no significant change in the actin ribbons in the cytoskeleton and peripheral cells in the H-1152+ hypoxia group 12h group, and only a very small amount of actin was found in the cytoplasm. The F-actin fluorescence intensity analysis of flow cytometry showed that the F-actin content was worse than the anoxia group. There was no statistical significance (P0.05). In the hypoxia 24h group, a large number of depolymerization of the cytoskeleton in the F-actin cell was observed and the cytoskeleton was obviously changed. The F-actin fluorescence intensity analysis of flow cytometry showed that there was a significant difference between the F-actin content and the normal control group (P0.05); H-1152+ was anoxic 24h, cytoskeleton and the myocutaneous egg surrounding the cells. There was a little change in the white ribbon and the appearance of the fuzzy actin in the cytoplasm. The F-actin fluorescence intensity analysis of flow cytometry showed that there was a significant difference in F-actin content compared with the normal control group and the hypoxia group (P0.05).H-1152 in each time group, there was no significant difference between the F-actin cytoskeleton and the normal control group in LSCM. F-actin fluorescence intensity analysis of flow cytometry showed that there was no significant difference between F-actin content and normal control group (P0.05).HPMVECs through anoxic treatment, actin cytoskeleton depolymerization, the arrangement and distribution of stress fibers, and H-1152 before anoxia treatment, which prevented the F-actin solution in HPMVECs induced by hypoxia. Aggregation and its content increased, and stress fibers arranged and distributed normally in cells.
3.2 Hoechst fluorescent staining of apoptotic nuclei in each group of 24h, gray value analysis of hypoxia control group and low, middle, high rhIL-10 intervention group was higher than normal control group (P0.05); low, middle, high rhIL-10 intervention group and hypoxia control group decreased (P0.05). Apoptosis protein Caspase-3 relative activity, 4H groups have no significant difference (P0.05); 12h hypoxia control Group, low, middle rhIL-10 intervention group was higher than normal group (P0.05), high dose rhIL-10 intervention group and normal control group had no significant difference (P0.05), rhIL-10 intervention group and hypoxic control group decreased (P0.05); 24h hypoxia control group, rhIL-10 intervention low, middle, high dose group and normal control group increased (P0.05), low, middle, high dose rhIL-10 dry. The pregroup and the hypoxic control group were significantly lower (P0.05), while the 48h hypoxia control group and the low dose rhIL-10 intervention group were higher than the normal control group (P0.05), and the other groups had no significant difference (P0.05); the peak of hypoxia apoptosis was found in 24h..
4 Conclusion
Change of F-actin remodeling in HPMVECs induced by 4.1 anoxia.
4.2 the Rho-ROCK signaling pathway is involved in the remodeling of F-actin cytoskeleton in HPMVECs induced by hypoxia.
4.3 hypoxia could promote the apoptosis of HPMVECs, and the expression of Caspase-3 in HPMVECs increased significantly under anoxia.
4.4 rhIL-10 could inhibit the apoptosis of hypoxia HPMVECs to some extent.

【學位授予單位】：廣州醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R363

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