Rho-ROCK信号通路介导缺氧所致人肺微血管内皮细胞肌动蛋白细胞骨架重构和重组人白介素10对缺氧条件下人肺微血管内皮细

发布时间：2018-04-23 10:25

本文选题：缺氧 + Rho-ROCK信号通路　；参考：《广州医学院》2011年硕士论文

【摘要】：1目的 1.1探讨缺氧(hypoxia)对人肺微血管内皮细胞(HPMVECs)肌动蛋白细胞骨架(F-actin)重构的影响,以及Rho-ROCK信号通路在缺氧引起肌动蛋白细胞骨架重构过程中的作用。 1.2研究缺氧条件下,HPMVECs内半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)活性的变化规律和重组人白介素10(rhIL-10)对缺氧HPMVECs凋亡的影响。 2方法 2.1在缺氧前用ROCK的特异性抑制剂H-1152,最佳浓度(10 nmol/L)对体外培养的HPMVECs进行预处理0.5h,再分为不同缺氧时间组进行缺氧6h、12h及24h处理,并设常规培养正常对照组,每组设8个复孔。用激光共聚焦显微镜(LSCM)观察缺氧或使用H-1152对HPMVECs细胞肌动蛋白细胞骨架重构的影响。流式细胞技术检测各组HPMVECs F-actin的平均荧光强度的变化,行统计学分析。 2.2常规培养HPMVECs细胞株,分为5组:正常对照组(常规培养);缺氧(5% CO2,95% N2)对照组;缺氧培养并加入rhIL-10以(10ng/mL,50ng/mL,100ng/mL)的浓度分别干预的rhIL-10低剂量干预组、rhIL-10中剂量干预组、rhIL-10高剂量干预组。培养4h、12h、24h、48h离心收集细胞,荧光显微镜观察HPMVECs 24h核荧光染色,分析荧光强度,化学比色法检测细胞核内Caspase-3活性检测,行统计学分析。 3结果 3.1 HPMVECs 6h各组, LSCM观察显示F-actin细胞骨架未发生明显改变,流式细胞术行F-actin荧光强度分析与正常对照组比较差别无统计学意义(P0.05)。缺氧12h组,用LSCM观察到F-actin细胞骨架已发生少量解聚,运用流式细胞术行F-actin荧光强度分析,提示F-actin含量增加与正常对照组比较差别有统计学意义(P0.05);而H-1152+缺氧12h组,细胞骨架及细胞周边的肌动蛋白丝带无明显变化,胞浆中仅有极少量模糊的肌动蛋白,流式细胞术行F-actin荧光强度分析提示F-actin含量与缺氧组比较差别有统计学意义(P0.05)。缺氧24h组,用LSCM观察到F-actin细胞骨架已发生大量解聚,细胞骨架明显改变,流式细胞术行F-actin荧光强度分析提示F-actin含量与正常对照组比较差别有统计学意义(P0.05); H-1152+缺氧24h,细胞骨架及细胞周边的肌动蛋白丝带有少量变化,胞浆中有模糊的肌动蛋白出现,流式细胞术行F-actin荧光强度分析提示F-actin含量与正常对照组比较及缺氧组比较差别均存在统计学意义(P0.05)。H-1152各时间组在用LSCM观察F-actin细胞骨架与正常对照组比较无明显差异,流式细胞术行F-actin荧光强度分析提示F-actin含量与正常对照组比较差别无统计学意义(P0.05)。HPMVECs经缺氧处理,肌动蛋白细胞骨架发生解聚,应力纤维的排列和分布改变;事先使用H-1152再进行缺氧处理,可防止缺氧所致HPMVECs内F-actin解聚及其含量增加,同时应力纤维在细胞内排列和分布趋向正常。 3.2取24h各组行凋亡细胞核Hoechst荧光染色,灰度值分析低氧对照组和低、中、高rhIL-10干预组与正常对照组比较升高(P0.05);低、中、高rhIL-10干预组与缺氧对照组比较降低(P0.05)。凋亡蛋白Caspase-3相对活性,4h各组别无明显差异(P0.05);12h缺氧对照组、低、中rhIL-10干预组与正常组比较升高(P0.05),高剂量rhIL-10干预组与正常对照组无明显差异(P0.05),rhIL-10干预各组与缺氧对照组比较降低(P0.05);24h缺氧对照组、rhIL-10干预低、中、高剂量组与正常对照组比较升高(P0.05),低、中、高剂量rhIL-10干预组与缺氧对照组比较明显降低(P0.05);48h缺氧对照组与低剂量rhIL-10干预组与正常对照组比较升高(P0.05),其余各组别无明显差异(P0.05);缺氧凋亡峰值见于24h。 4结论 4.1缺氧所致HPMVECs内的F-actin重构改变。 4.2 Rho-ROCK信号通路参与缺氧所致HPMVECs内F-actin细胞骨架的重构改变。 4.3缺氧可促进HPMVECs凋亡,缺氧条件下,Caspase-3在HPMVECs的表达明显升高。 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.

【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

【参考文献】