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SIRT1对氧化应激下人小梁网细胞功能的影响

Effect of SIRT1 on cell function of human trabecular meshwork cell under oxidative stress

来源期刊: 眼科学报 | 2021年6月 第36卷 第6期 405-412 发布时间: 收稿时间:2023/5/22 11:53:48 阅读量:5042
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沉默信息调节因子2相关酶1人小梁网细胞迁移细胞活性
silent information regulator 1 human trabecular meshwork cell migration cell viability
DOI:
10.3978/j.issn.1000-4432.2021.06.16
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目的:通过在人小梁网细胞(human trabecular meshwork cell,HTMC)中过表达沉默信息调节因子2相关酶1(silent information regulator 1,SIRT1),探讨SIRT1对氧化应激下HTMC功能的影响。方法:将SIRT1过表达慢病毒和GFP阴性对照慢病毒按照最佳(multiplicity of infection,MOI)分别转染入HTMC,并用实时定量PCR法对SIRT1是否在细胞中过表达进行验证。实验分为以下4组:正常组、H2O2组、H2O2+Lv-SIRT1-OE(过表达)组、H2O2+Lv-GFP组,分别采用Transwell法和CCK8法检测氧化应激下HTMC的迁移能力和活性。两组间比较采用独立样本t检验。结果:在正常组、H2O2组、H2O2+Lv-SIRT1-OE组、H2O2+Lv-GFP组这4组中,Transwel l实验结果分别为436±73、254±25、510±51、327±46,H2O2+Lv-SIRT1-OE组分别与H2O2组和H2O2+Lv-GFP组差异均有统计学意义(P<0.01)。CCK8法结果显示,H2O2+Lv-SIRT1-OE组分别与H2O2组和H2O2+Lv-GFP组相比差异均有统计学意义(P<0.01)。H2O2+Lv-SIRT1-OE组分别与H2O2组和阴性对照组(H2O2+Lv-GFP)相比,Bax表达水平明显下降,Bcl-2表达水平明显提高,差异均有统计学意义(P<0.01)。ROS活性氧测定显示H2O2+Lv-SIRT1-OE组比H2O2组的细胞活性氧水平显著降低(P<0.05)。结论:在HTMC中过表达SIRT1能有效降低氧化应激对HTMC迁移能力和活性的影响,从而对HTMC起到一定的保护作用,为后续研究SIRT1保护氧化应激下HTMC的调控机制打下基础。
Objective: To explore the effect of Silent Information Regulator 1 (SIRT1) on cell function of human trabecular meshwork cell (HTMC) under oxidative stress by overexpressing SIRT1 in HTMC. Methods: This is an experiment research. HTMCs were transfected with SIRT1-ovexpressed lentivirus and GFP-negative control lentivirus (Lv-GFP) at the optimal multiplicity of infection (MOI). Real-time quantitative PCR was used to verify whether SIRT1 was overexpressed in HTMC. The following experiments were divided into four groups: normal control group, H2O2 group,H2O2+Lv-SIRT1-OE group, H2O2+Lv-GFP group. Cell migration was detected by transwell assay. Cell viability was detected by CCK8 assay. Student’s t-test was used for two groups. P<0.05 was set as statistical signifificance. Results: The number of migration per well of normal control group, H2O2 group, H2O2+Lv-SIRT1-OE group, H2O2+Lv-GFP group were 436±73,254±25, 510±51, 327±46, respectively. Compared with H2O2 group and H2O2+Lv-GFP group, transwell assay demonstrated that the number of migrations per well of H2O2+Lv-SIRT1-OE group significantly increased (P<0.01). Likewise, CCK8 assay indicated that cell viability of H2O2+Lv-SIRT1-OE group was higher than both of H2O2 group and H2O2+Lv-GFP group (P<0.01). Compared with H2O2+Lv-SIRT1-OE group and negative control group (H2O2+Lv-GFP), the expression level of Bax decreased significantly,and the expression level of Bcl-2 increased significantly (P<0.01). ROS assay showed that the ROS level in H2O2+Lv-SIRT1-OE group was significantly lower than that in H2O2 group (P<0.05). Conclusion:SIRT1 overexpressed in HTMC can effectively reduce the effect of oxidative stress on migration ability and proliferation activity of HTMC, which lays a foundation for further study on the regulatory mechanism of SIRT1 protecting HTMC under oxidative stress.
青光眼是世界上第2位致盲性眼病。研究[1]表明:在2040年全球青光眼患者将达到1.118亿人。开角型青光眼(open angle glaucoma,OAG)作为一种主要的青光眼类型,有部分学者[2]认为其主要发病机制是:小梁网细胞(trabecular meshwork cell,TMC)的细胞外基质(extracellular matrix,ECM)异常沉积,使得房水流出阻力增加,最终引起眼压升高。而TMC在氧化应激的作用下会发生ECM的异常沉积[3]。沉默信息调节因子2相关酶1(silent information regulator 1,SIRT1)是一种可以使组蛋白中赖氨酸残基去乙酰化的去乙酰化酶,其广泛参与氧化应激耐受、胰岛素分泌和葡萄糖合成等生理活动。此外,SIRT1还能通过抑制ECM的异常沉积影响纤维化进程,在心肌纤维化[4]、肝纤维化[5]等疾病中起到非常重要的作用。笔者前期研究发现,H2O2处理HTMC后,SIRT1的RNA及蛋白表达水平会显著下降。本研究通过在人小梁网细胞(human trabecular meshwork cell line,HTMC)中过表达SIRT1,探讨SIRT1对氧化应激下HTMC功能的影响,为SIRT1用于青光眼的治疗提供理论基础。

1 材料与方法

1.1 材料

人原代小梁网细胞系购自美国ScienCell公司;细胞培养基购自美国GIBCO公司;SIRT1过表达慢病毒购自上海吉凯基因化学技术有限公司;TRIzol RNA提取剂购自美国Invitrogen公司;第一链cDNA合成试剂盒购自瑞士Roche公司;FastStart Universal SYBR Green Master(ROX)购自美国Thermo公司;30%过氧化氢购自天津市风船化学试剂科技有限公司;Cell Counting Kit-8试剂盒购自日本同仁化工;Trans wel l细胞侵袭迁移培养皿购自美国Corning公司;ROS活性氧检测试剂盒(DCFHDA法)购自上海贝博生物公司;常温超速离心机购自德国Eppendorf公司;CO2培养箱为瑞士HEAL FORCE公司HF 160W型;移液枪、PCR扩增仪购自德国Eppendorf公司;生物安全柜购自北京HDL公司;纯水制备仪购自法国Millipore公司;7500 Fast real-time PCR仪购自美国Applied Biosystems公司。

1.2 方法

1.2.1 HTMC培养
HTMC在含有10%胎牛血清、丙酮酸钠(终浓度为1 mmol/L)、L -谷氨酰胺(终浓度为20 mmol/L)、非必需氨基酸(终浓度为100 μmol/L)、青霉素(终浓度为100 units/mL)及链霉素溶液(终浓度为100 μg/mL)的DMEM培养基中培养。HTMC在恒温37 ℃的5%CO2培养箱中常规培养,当细胞汇合度达70%~80%时进行传代。
1.2.2 SIRT1过表达慢病毒转染HTMC模型的构建
细胞按照5×104/孔种于6孔板中,24 h后弃去培养基;每孔对应取一个新的1.5 mL EP管,往里加入完全培养基1 mL以及适量的polybrene(使polybrene的终浓度为5 μg/mL),再分别加入2 μL、20 μL、200 μL的1×108 TU/mL的SIRT1过表达慢病毒,吹打混匀后将混合液加入到每孔中;12 h后弃去培养基,加入新的完全培养基2 mL,以供后续实验使用。
1.2.3 细胞迁移实验
HTMC以1×105/孔预种于6孔板,24 h后做相应处理;而后用杜尔贝科磷酸盐缓冲液润洗细胞2遍,弃液,0.25%胰蛋白酶消化,离心,弃上清并用1 mL完全培养基重悬细胞,吸50 μL悬液加10%台盼蓝染色计数;提前将Trans wel l小室置于24孔板内,往上室加入100 μL细胞培养基,37 ℃、5% CO2孵箱孵育1 h;吸除小室中的培养基,在下室中加入600 μL完全培养基(血清:DMEM=1:1),用含有0.2%小牛血清的细胞培养基以5×104/mL HTMC种于上室中,上样200 μL,37 ℃、5% CO2孵箱继续培养10~12 h;吸出小室和24孔板内液体,用棉签将小室上表面的细胞轻轻擦掉;配置结晶紫工作液(0.03 g结晶紫粉剂加到6 mL无水乙醇中,再用24 mL生理盐水稀释,全程避光),往小室和24孔板内加入800 μL结晶紫工作液,避光室温孵育20 min;用磷酸盐缓冲液洗3遍,每遍10 min;吸除磷酸盐缓冲液,再将小室倒置晾干,用无菌尖刀片将小室膜切下(注意正反面不要弄错),并置于载玻片上;使用中性树胶封片,滴1滴树胶于小室表面,再盖上盖玻片,注意避免气泡产生;用倒置显微镜观察,按照象限随机选择6~8个视野,并拍照计数。
1.2.4 CCK8细胞活性检测
将HTMC悬液按照3×104/mL种于96孔板中,每孔上样100 μL,37 ℃、5% CO2孵箱预培养;24 h后根据实验需要处理HTMC;刺激结束后将培养液吸除,并用PBS洗2遍,吸除;按照细胞培养基:CCK8溶液=9:1向每孔加入混合液100 μL(注意避免产生气泡),孵箱继续孵育1~2 h;最后用酶标仪450 nm测定吸光度(OD值)。细胞活力(%)=[OD(被处理过的细胞)–OD(仅有培养基)]/[OD(仅有细胞和培养基)?OD(仅有培养基)]×100。
1.2.5 实时定量PCR法检测基因的表达水平
根据TRIzol试剂的说明书提取HTMC的总RNA ,根据第一链cDNA合成试剂盒将其逆转录成cDNA ,利用随机引物进行扩增。再根据FastStart Universal SYBR Green Master试剂盒说明书加样以及目的基因的相应引物(表1 ),扩增条件为:95 ℃预变性10 min;95 ℃变性15 s,60 ℃退火并延伸60 s,共4 0个循环。其中β-actin作为内参基因,利用2???Ct法计算各组细胞中相关RNA的相对表达量。其中??Ct=(目的基因C T值?内参基因C值)实验组?(目的基因C T值?内参基因C T值)对照组

表1 相关基因的引物序列
Table 1 Primer sequences of related genes

20230522113236_3537.png
1.2.6 ROS测定
按照1 : 1 000用无血清培养基稀释DCFH-DA,将各组处理好的细胞收集后重悬于稀释好的DCFH-DA中,37 ℃细胞培养箱内孵育20 min(避光),每隔4 min颠倒混匀一下。随后用无血清细胞培养基洗涤细胞3次。用96孔板检测,每组样品添加4份,使用多功能酶标仪检测,激发波长488 nm,发射波长525 nm,读取A值,然后以空白对照组为100%,其他各组与之对比,得出的结果为检测值。

1.3 统计学处理

结果均由至少3次的独立实验统计而得。采用SPSS 20.0统计学软件进行数据处理。本研究中各检测指标的计量资料经Kolmogorov-Smimov检验证实呈正态分布,以均数±标准差(x±s)表示。采用完全随机分组单因素干预多水平实验设计,SIRT1过表达组和GFP病毒空载体组组间RNA相对表达量的总体差异比较均采用独立样本t检验分析,多样本组间两两比较先进行方差齐性检验,方差齐采用LSD-t法,方差不齐采用Dunnett T3法。P<0.05为差异有统计学意义。

2 结果

2.1 成功建立SIRT1过表达慢病毒感染HTMC模型

笔者将SIRT1过表达慢病毒和GFP阴性对照慢病毒按照感染复数(multiplicity of infection,MOI)为10转染入HTMC,转染48 h后,荧光显微镜下可观察到HTMC内有明显的报告基因GFP表达,表明SIRT1过表达慢病毒成功转染至HTMC中(图1)。
20230522113335_8069.png

图1 显微镜下观察慢病毒转染HTMC 48 h后的转染情况(×100)
Figure 1 HTMC was observed under the microscope after lentivirus transfection for 48 hours (×100)
(A)光学显微镜下观察转染GFP阴性对照慢病毒48 h后的转染情况(100 μm);(B)荧光显微镜下观察转染GFP阴性对照慢病毒48 h后的转染情况(100 μm);(C)光学显微镜下观察转染SIRT1过表达慢病毒48 h后的转染情况(100 μm);(D)荧光显微镜下观察转染SIRT1过表达慢病毒48 h后的转染情况(100 μm)。
(A) HTMC was observed under the optical microscope after GFP-negative control lentivirus transfection for 48 hours (100 μm);(B) HTMC was observed under the fluorescence microscope after GFP-negative control lentivirus transfection for 48 hours (100 μm);(C) HTMC was observed under the optical microscope after SIRT1-ovexpressed lentivirus transfection for 48 hours (100 μm); (D) HTMCwas observed under the fluorescence microscope after SIRT1-ovexpressed lentivirus transfection for 48 hours (100 μm).

2.2 SIRT1在转染后HTMC的表达水平明显提高

分别提取感染了SIRT1过表达慢病毒或GFP阴性对照慢病毒的HTMC的总RNA,反转录后进行Real-time PCR实验,检测两组中SIRT1的表达水平差异,使用2???Ct法计算基因的相对表达倍数变化,结果显示:与GFP阴性对照组相比,SIRT1过表达组中SIRT1的表达水平明显高于对照组(1.000±0.056 vs 0.095±0.005,P<0.0001);同样地,SIRT1过表达组中SIRT1的蛋白表达水平也明显高于对照组(图2)。
20230522113542_3081.png

图2 SIRT1过表达组与GFP阴性对照组中SIRT1在mRNA水平和蛋白水平的比较
Figure 2 Comparison of SIRT1 mRNA and protein levels between SIRT1 overexpression group and GFP negative control group
与对照组相比,****P<0.0001。
Compared with the control group, ****P<0.0001.

2.3 SIRT1过表达能有效降低氧化应激对HTMC迁移能力的影响

Transwell迁移实验结果显示:H2O2刺激组每孔细胞迁移数量为254±25,正常对照组为436±73,两者在细胞迁移能力上,差异有统计学意义(P<0.001)。另外,H2O2+Lv-SIRT1-OE组每孔细胞迁移数量为510±5 1,阴性对照组(H2O2+Lv-GFP)为327±46,H2O2+Lv-SIRT1-OE组分别与H2O2组和阴性对照组差异均有统计学意义(P <0.01),说明SIRT1过表达能有效降低氧化应激对HTMC迁移能力的影响(图3)。
20230522113742_1679.png

图3 SIRT1过表达对氧化应激下HTMC迁移能力的影响(×100)
Figure 3 Effffect of SIRT1 overexpression on HTMC migration under oxidative stress (×100)
(A)正常组HTMC视野;(B)H2O2刺激2 h下的HTMC;(C)H2O2刺激2 h下的转染了过表达SIRT1慢病毒的HTMC视野;(D)H2O2刺激2 h下的转染了空载体慢病毒的HTMC视野;(E)Transwell迁移实验检测HTMC在不同处理后的迁移数量(F=19.600,P<0.001)。*P<0.05,**P<0.01,***P<0.001,****P<0.0001。
(A) View of normal HTMC; (B) View of HTMC treated by H2O2 for 2 hours; (C) View of HTMC transfected by SIRT1-ovexpressed lentivirus under 2-hour-H2O2 treatment; (D) View of HTMC transfected by GFP-negative control lentivirus under 2-hour-H2O2 treatment;(E) View of the number of HTMC migration after different treatments detected by transwell assay (F=19.600, P<0.001). *P<0.05, **P<0.01,***P<0.001, ****P<0.0001.

2.4 SIRT1过表达能有效降低氧化应激对HTMC细胞活性的影响

CCK8增殖实验结果显示: H2O2刺激组与正常对照组在细胞活性上,差异有统计学意义( P < 0.01)。同时,H2O2+Lv-SIRT1-OE组分别与H2O2组和阴性对照组(H2O2+Lv-GFP)相比差异均有统计学意义(P <0.01),且与正常组的差异没有统计学意义(P>0.05),表明SIRT1过表达能有效降低氧化应激对HTMC细胞活性的影响(图4 )。同时检测凋亡标志蛋白Bax和Bcl-2,H2O2+Lv-SIRT1-OE组分别与H2O2组和阴性对照组( H2O2+Lv-GFP)相比,Bax表达水平明显下降,Bcl-2表达水平明显提高,差异均有统计学意义(P<0.01,图5)。此外,通过ROS活性氧测定,笔者发现H2O2+Lv-SIRT1-OE组比H2O2组的细胞活性氧水平显著地降低(P <0.05),表明SIRT1过表达能有效降低氧化应激状态下HTMC的活性氧水平(表2)。
20230522114058_2011.png

图4 SIRT1过表达对氧化应激下HTMC细胞活性的影响
Figure 4 Effect of SIRT1 overexpression on cell viability of HTMC under oxidative stress
CCK8 法检测 HTMC 在不同处理下的细胞增殖活性(F=9.248,P<0.001)。*P<0.05,**P<0.01。
CCK8 assay was used to detect the cell viability of HTMC under different treatments (F=9.248, P<0.001). *P<0.05, **P<0.01.

20230522114223_7482.png

图5 SIRT1过表达对凋亡标志蛋白表达的影响
Figure 5 Effect of SIRT1 overexpression on expression of apoptotic marker proteins
(A)各组中Bax的mRNA表达水平比较;(B)各组中Bcl-2的mRNA表达水平比较;(C)各组中Bax和Bcl-2蛋白水平的比较。与normal组比较,**P<0.01;与H2O2组比较,##P<0.01。
(A) Comparison of mRNA expression levels of Bax between each group; (B) Comparison of mRNA expression levels of Bcl-2 between each group; (C) Comparison of protein levels of Bax and Bcl-2 between each group. Compared with the Normal group, **P<0.01;compared with the H2O2 group, ##P<0.01.

表2 SIRT1过表达对氧化应激下HTMC活性氧水平的影响
Table 2 Effffects of SIRT1 overexpression on reactive oxygen species of HTMC under oxidative stress

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3 讨论

SIRT1存在于眼部近乎所有组织中,如角膜、睫状体、葡萄膜、晶状体、视网膜等[6]。在青光中,HTMC对氧化应激的反应在房水流出通路的生理过程中起重要作用[7-8]。研究[9]显示:与正常HTMC相比,SIRT1在青光眼HTMC中的表达量显著较低,且SIRT1可以使HTMC免受氧化应激所致的细胞凋亡和DNA双链损伤。表明SIRT1在一定程度上参与到青光眼的发病机制当中,并发挥重要作用。
白藜芦醇(resveratrol,RSV)作为SIRT1的激动剂,可通过激活SIRT1来保护细胞免受外界刺激的损伤,提高其抗氧化能力[10]。在青光眼方面,RSV可抑制小梁网细胞中活性氧的产生[11]及多种炎症因子的表达[12]。多数学者[2-3]认为:ECM的异常积聚是开角型青光眼的主要发病机制之一[2],而氧化应激是导致ECM异常沉积的重要机制之一[3]。笔者前期研究发现:在300 μmol/L H2O2刺激2 h下,经过RSV处理的HTMC,其ECM的转录水平和翻译水平都比只用H2O2处理的HTMC要低[13];同样,在300 μmol/L H2O2刺激2h下,在HTMC中过表达SIRT1,其ECM的转录水平和翻译水平都比只用H2O2处理的HTMC要低,证明SIRT1也参与到了调控人小梁网细胞ECM生成的过程中。为探究氧化应激对HTMC细胞功能的影响,本研究应用了CCK-8及Transwell实验进行评估,结果显示:氧化应激会使得HTMC活性降低,同时也会对细胞迁移有抑制作用。而在HTMC中,过表达SIRT1能有效降低氧化应激对HTMC活性以及迁移能力的影响。同时,过表达SIRT1能显著减少细胞内Bax表达水平,减少ROS的产生。因此,笔者认为激动氧化应激下HTMC中的SIRT1将极有可能会对HTMC产生保护作用。
SIRT1抵抗氧化应激可能存在多种调控机制。氧化应激状态下,SIRT1可调控多种靶蛋白,例如FOXO、p53等。研究[14]表明:高糖诱导人晶状体上皮细胞处于氧化应激状态下,FOXO3是应激反应早期敏感的指标,氧化应激状态下,SIRT1与FOXO3在细胞中形成复合物,并去除FOXO3的乙酰化水平,从而保护细胞避免凋亡,提高对氧化应激的抵抗力。另一项研究[15]显示:p53蛋白稳定性增加或者被激活都能诱导细胞凋亡的发生,SIRT1能够降低p53乙酰化水平,从而阻止氧化应激与DNA损伤诱导的凋亡。此外, SIRT1能够调高抗氧化酶MnSOD的活性,提高H9C2心肌细胞中氧化物的清除率从而实现抗氧化应激的作用[16]
青光眼滤过手术(glaucoma filtration surgery,GFS)是一种针对降眼压药物和激光治疗不理想时的主要手术方式,而GFS的失败主要是由于瘢痕形成、ECM增生及纤维化,最终导致滤过泡功能减退[17]。因此,通过激动SIRT1,比如使用SIRT1的激动剂——RSV,这将有可能应用于GFS术后,减少术后ECM沉积及瘢痕化,从而更好地维持滤过泡的形成。
笔者前期曾采用Agilent长链非编码RNA芯片对分别转染了SIRT1过表达慢病毒和GFP阴性对照慢病毒的HTMC样本进行长链非编码RNA表达谱分析。结果发现,Notch信号通路[18-19]、FoxO信号通路[20-21]、MAPK信号通路[22]、mTOR信号通路[23]以及PI3K-Akt信号通路[24-25]均可能参与开角型青光眼的发病过程,而这些信号通路与细胞外基质的异常沉淀、氧化应激和细胞凋亡密切相关。笔者认为SIRT1将在其中扮演重要角色。但是本研究还有不足之处,本研究仅在体外完成,还需要更多的体内实验来验证结论的准确性。此外,本研究只进行了RNA分析,后续研究中还需要进行具体的蛋白表达分析。关于SIRT1过表达能够降低氧化应激对HTMC迁移能力的影响,笔者考虑是否可能是通过促进HTMC的上皮间质转化(epithelial-mesenchymal transition,EMT)完成的,还需要补充EMT相关分子的表达水平变化。
综上,在HTMC中过表达SIRT1能有效降低氧化应激对HTMC活性以及迁移能力的影响,对病理状态下的HTMC起到保护作用。通过结合前期高通量基因芯片的结果,笔者将筛选出差异表达的长链非编码RNA,并结合上述所提及的通路,进一步探究SIRT1保护氧化应激状态下HTMC功能的具体调控机制,为青光眼临床治疗提供真实有效的科学依据。
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1、国家自然科学基金 (82070961);深圳市三名工程项目 (SZSM201812091);深圳市科创委国际合作项目 (GJHZ20190929145402153)。
This work was supported by the National Natural Science Foundation (82070961), Sanming Project of Medicine in Shenzhen (SZSM201812091) and the International Science and Technology Cooperation Research Project of Shenzhen Science and Technology Innovation Committee (GJHZ20190929145402153), China.()
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