【作者】 魏丽萍；

【导师】 王海昌； 张荣庆；

【作者基本信息】 第四军医大学 ， 内科学， 2009， 博士

【摘要】 研究背景糖尿病与心血管疾病的密切关系得到了研究者的广泛关注。当糖尿病患者发生心血管事件而给予血管再通治疗时再灌注事件发生率增高,证明了缺血再灌注(ischemic reperfusion I/R)在糖尿病病人的易损性,也即糖尿病病人在发生缺血性心脏病后心肌细胞死亡数量、心功能损伤程度及再灌注后无复流现象的发生均远远高于非糖尿病缺血性心脏病患者。糖尿病患者发病后最常出现的病理损伤即是糖尿病微血管并发症,可以累及多个器官,出现复杂多样的临床表现,但多数研究显示其病理基础殊途同归,都与微血管内皮细胞屏障功能损伤有关。这些现象给予研究者提示:糖尿病患者缺血再灌注损伤加重可能与发生缺血事件前即存在心脏微血管功能损害有关。糖尿病微血管并发症早期的病理损害主要是内皮细胞连接破坏,细胞屏障功能降低,使损伤性因子在血管内外的交换加速,损害血管功能并加重血管内皮细胞凋亡,此种病理现象在视网膜、肾脏、心脏组织中均广泛存在,其病理机制认为在高血糖或糖尿病状态下PKC激活,特别是其亚基PKC–βII的激活在血管损伤中起到至关重要的作用。PKC通路的激活导致细胞内信号通路的改变及血管功能的障碍而促使糖尿病微血管病变的发生和发展。高度选择性PKC–β抑制剂Ruboxistaurin(Rx)的应用使PKC–βII激活在糖尿病微血管病变发生中作用的研究更为深入。但PKC–βII激活与心肌微血管屏障功能变化的病理关系研究尚少,具体机制还没有得到明确肯定的结果。而PKC–βII激活是否在糖尿病缺血再灌注后微血管损伤中也具有重要作用,尚未见报道。本课题拟分别从动物和细胞水平观察PKC-βII激活在糖尿病大鼠心肌微血管屏障功能变化中的作用及PKC-βII激活对糖尿病大鼠心肌微血管I/R损伤的影响,选择性PKC–β抑制剂在此病理过程中的重要保护作用。研究目的1.建立糖尿病大鼠模型,明确糖尿病大鼠心脏组织是否存在PKC-βII激活, PKC-βII激活与糖尿病大鼠心肌微血管内皮通透性变化及细胞连接病理性破坏的联系;2.建立糖尿病大鼠心脏I/R模型,观察糖尿病大鼠I/R后心肌微血管损伤特点,并验证PKC-β抑制剂是否具有保护作用,从而证明PKC-βII激活参与糖尿病大鼠I/R后心肌微血管损伤的病理机制;3.在高糖培养心肌微血管内皮细胞实验中观察高糖作用下单层心肌微血管内皮细胞通透性变化及PKC-βII激活影响屏障功能的机制;4.在体外模拟高糖培养心肌微血管内皮细胞缺血再灌注实验中观察单层心肌微血管内皮细胞模拟I/R后,细胞屏障功能的变化及PKC-β抑制剂预处理后的作用。在细胞实验中证实PKC-βII激活参与高糖环境心肌微血管内皮细胞I/R后损伤的病理机制。研究方法1.选取体重120-150g雄性SD大鼠,高脂饲料喂养8w ,腹腔注射50mg/kg STZ,以2次随机血糖≥16.7mmol/L认为糖尿病模型成功。2.戊巴比妥钠(30 mg/kg )腹腔麻醉大鼠,行左侧开胸术,制备心肌缺血/再灌注大鼠模型。大鼠心脏缺血30 min,再灌注72h。3.以硝酸镧为示踪剂透射电镜下检测糖尿病大鼠心肌微血管内皮细胞通透性,以树脂(Mercox,SPI)灌注心脏,扫描电镜下观察微血管铸型。4.免疫荧光方法检测糖尿病大鼠心脏组织PKC-βII及VE-cadherin的表达。免疫组化检测缺血再灌注后心脏Phospho -LIMK2表达。5.八道生理记录仪记录缺血再灌注前后血流动力学指标±LVdp/dtmax差值(缺血后±LVdp/dtmax -缺血前±LVdp/dtmax)变化。6.硫黄素S染色检测缺血再灌注后糖尿病大鼠心脏无复流面积。利用硫黄素S使血管损伤无复流区血管不着色,计算心肌无复流面积。7.无菌分离成年大鼠左心室组织,去除内外膜及冠脉组织,心肌剪为1mm3组织块,经酶消化,条件培养及细胞纯化后获得心肌微血管内皮细胞。8.用In Vitro Vascular Permeability Assay Kit检测单层心肌微血管内皮细胞通透性。9.Western blot方法检测糖尿病大鼠心脏组织及培养细胞中PKC-βII, VE-cadherin,phospho-β-catenin及缺血再灌注后心脏组织及培养细胞中phospho-LIMK2的表达。10.TUNEL染色检测组织和培养心肌微血管内皮细胞的凋亡。以CD31,TUNUL和DAPI三染的方式来计算缺血再灌注72h后糖尿病大鼠CMECs凋亡指数。11.FITC标记的鬼笔环肽染色CMECs细胞骨架F-actin,荧光显微镜下观察。12.以G-actin / F-actin assay kit定量测定F-actin的变化。研究结果1.糖尿病大鼠心脏组织PKC-βII激活改变心肌微血管屏障功能。(1)检测空腹血糖水平升高验证成功制备了糖尿病大鼠模型。(2)免疫荧光显示糖尿病大鼠心脏组织PKC-βII激活及VE-cadherin排列紊乱,给予Rx治疗后,PKC-βII激活减少、VE-cadherin的表达维持连续。(3)Western blot结果证实糖尿病大鼠心脏PKC-βII (1.06±0.09 vs. 0.76±0.06,P<0.01)及phospho-β-catenin (0.74±0.13 vs.0.47±0.11, P<0.01)表达较非糖尿病大鼠增加。Rx治疗可下调PKC-βII(0.79±0.10 vs. 0.97±0.11,P<0.05)及Phospho-β-catenin(0.59±0.07 vs. 0.80±0.11, P<0.05)表达。(4)硝酸镧颗粒示踪电镜观察发现糖尿病大鼠心肌微血管屏障功能破坏,扫描电镜观察Mercox灌注发现心肌微血管内皮细胞连接不完整,而Rx治疗可有明显改善。2.PKC-βII激活参与糖尿病大鼠I/R后心肌微血管损伤加重的发生机制。(1)糖尿病大鼠缺血再灌注前后±LVdp/dtmax差值增加, I/R对心功能损伤加重,给予Rx治疗可保护糖尿病大鼠心功能。(2)糖尿病大鼠I/R后心脏无复流面积明显大于假手术组( 20.0±1.7% vs. 0%)及非糖尿病组(20.0±1.7% vs.14.3±1.8%, P<0.01),Rx治疗可降低糖尿病大鼠无复流面积(16.4±1.9% vs. 19.4±1.6%, P<0.05)。(3)糖尿病大鼠I/R心肌微血管内皮细胞病理损伤加重,肿胀和空泡结构出现。Rx治疗具有保护作用。(4)糖尿病大鼠I/R后CMECs凋亡指数明显高于假手术组( 12.6±2.2% vs.4.8±1.0% , P<0.01 )及非糖尿病对照组( 12.6±2.2% vs.7.8±0.8%, P<0.01),Rx治疗可降低CMECs凋亡指数(9.5±1.5%vs. 12.2±2.1%, P<0.05)。(5)Phospho-LIMK2表达在糖尿病组明显高于假手术(1.01±0.10 vs. 0.66±0.11, P<0.01)及非糖尿病对照组(1.01±0.10 vs. 0.60±0.09, P<0.01), Rx治疗降低Phospho -LIMK2表达(0.76±0.08 vs. 0.97±0.10, P<0.01)。3.高糖培养单层心肌微血管内皮细胞PKC-βII激活,屏障功能降低。(1)FITC-Dextran检测高糖培养基中CMECs单层细胞通透性较普通培养基增加(394.50±36.92 vs. 282.10±14.93, P<0.01)。加入不同浓度Rx (1, 10, 100nmol/L)预处理后,单层细胞通透性明显降低(P<0.01)。( 2 )高糖培养CMECs PKC-βII (1.17±0.13 vs.0.71±0.07, P<0.01) ,phospho-β-catenin(0.92±0.11 vs. 0.56±0.07, P<0.01)表达增高,Rx(10nmol/L)预处理细胞可降低PKC-βII及phospho-β-catenin表达(P<0.01)。VE-cadherin(c-19)蛋白总量无明显变化。4.PKC-βII激活参与高糖环境心肌微血管内皮细胞SI/R后损伤加重的病理机制。(1)高糖培养单层细胞经历SI/R后通透性较Sham组(590.40±34.38 vs. 359.20±22.80, P<0.01)及普通低糖培养基组(590.40±34.38 vs. 383.70±37.58, P<0.01 )显著增加, Rx ( 10nmol/L )预处理后,通透性可明显降低(432.00±30.83 vs. 571.10±32.60 , P<0.01)。(2)高糖培养CMECs给予SI/R后Phospho -LIMK2表达明显高于Sham组(1.11±0.09vs. 0.31±0.09, P<0.01)及普通低糖培养基组(1.11±0.09 vs. 0.43±0.15, P<0.01),Rx预处理可降低Phospho -LIMK2表达(0.60±0.08 vs.1.28±0.11,P<0.01)。(3)高糖培养基CMECs AI明显高于Sham组(13.3±1.3% vs. 5.7±1.4%, P<0.01)及普通低糖培养基(13.3±1.3% vs. 7.8±0.8%, P<0.01)。Rx预处理后AI明显降低(9.1±1.1% vs.12.8±1.8%, P<0.01)。(4)高糖培养基中F-actin / G-actin较Sham组(10.73±1.32 vs. 6.58±1.34, P<0.01)及普通培养基组(10.73±1.32 vs. 7.73±0.97, P<0.01)增高。给予Rx预处理后F-actin / G-actin可降低(7.32±1.02 vs.10.24±1.38, P<0.05)。研究结论1.应用STZ诱导的糖尿病大鼠证实糖尿病状态可激活心脏PKC-βII表达,出现以微血管屏障功能降低为主要表现的病理改变。同时发现phospho-β-catenin表达增加,VE-cadherin排列紊乱可能参与病理机制。2.糖尿病大鼠心脏缺血再灌注后心功能损害加重,心肌无复流面积增加,微血管内皮细胞的通透性恶化、凋亡增加。这一系列的病理变化与糖尿病大鼠PKC-βII激活诱导I/R时Rho激酶异常激活,LIMK2磷酸化增加有关;3.高糖可致培养心肌微血管内皮细胞中PKC-βII激活,从而使黏附连接蛋白β-catenin发生磷酸化改变, VE-cadherin-catenin连接复合体破坏是CMECs通透性增加,屏障功能损害的结构基础;4.培养于高糖环境的心肌微血管内皮细胞PKC-βII激活,SI/R后诱导Rho-Kinase异常激活,使LIMK2磷酸化表达增加,受其调控的下游分子F-actin组分增加。细胞屏障功能维持因素VE-cadherin和F-actin的失平衡是高糖培养心肌微血管内皮细胞SI/R损伤加重的主要机制。研究提示控制糖尿病大鼠PKC-βII激活所诱导的慢性微血管并发症对预防糖尿病大鼠发生严重的心肌微血管缺血再灌注损伤有非常重要的意义。更多还原

【Abstract】 BackgroundAmount of researchers showed interest in close relationships between diabetes and cardiovascular disease. If diabetes patients received revascularization, accidence related with reperfusion often happened. These indicated diabetic heart is more sensitive to ischemic reperfusion injury than the nondiabetic heart. This phenomenon was confirmed by cardiacmyocyte death increase, severe impaired heart function and incidence of no-reflow rose. Cardiac microvascular barrier dysfunction, commonly occurring before microvascular sclerosis, is considered to be one of the initiating mechanisms that underlie the pathogenesis of diabetes microvascular complications. These phenomenons indicate endothelial barrier dysfunction probably significantly contributes to subsequent functional and cellular injury during I/R through a variety of pathological pathways and I/R in diabetes extending into the microvasculature probably is more severe.Early impairment of microvascular complication of diabetes was deterioration of cell junction and cell barrier function. This pathologic change may increase paracellular flux of injury factor, which could worse inflammatory reaction, aggravate apoptosis of endothelium and deteriorate microvascular function. Above pathology phenomena wildly existed in microangiopathy-prone tissues, such as the retina, renal glomeruli and heart. PKC was activated, which is strongly implicated in the pathogenesis of diabetic microangiopathy. PKC-βII isoforms exhibit a greater increase in the membrane fractions of many vascular tissues than the other isoforms in diabetes. Ruboxistaurin(Rx), a high selective PKC-βinhibitor, make the study about the role of PKC activation in microvascular complication more deeply. However, research about the role of PKC-βII activation in cardiac microvascular barrier function was seldom and the mechanism didn’t confirm. It has not been previously established whether PKC-βII activation play important role in I/R injury of cardiac microvessels in diabetes. Under this condition, this study paid attention to the role of PKC-βII activation in cardiac microvascular barrier function and ischemia reperfusion injury in cardiac micvasculture on diabetic rats.AIM1. Diabetic rat model was established and to determine whether PKC-βII activation existed in heart tissue of diabetic rats. Pathologic relationship between cardiac microvascular barrier dysfunction and PKC-βII activation was explored;2. The model of diabetic rat subjected to I/R was established and to observe characters of cardiac microvascular impairment in diabetic rat. In addition, to demonstrate whether this effect could be prevented by a PKC-βinhibitor and to prove PKC-βII activation involved in the mechanism by which CMECs were impaired during I/R.3. To observe permeability of CMECs monolayer in high glucose environment and the mechanism of PKC-βII activation influence barrier function.4. To elucidate whether barrier function aggravated in CMECs monolayer cultured in high glucose medium subjected to SI/R. To proved PKC-βII activation involved in the mechanism by which CMECs impairment worse during I/R.Methods1. Male Sprague-Dawley rat, 120-150 g, dieted with high glucose and high cholesterol for 8 weeks, diabetes was induced with a single intraperitoneal injection of streptozotocin (50 mg/kg, Sigma). Random serum glucose greater than 16.7mmol/L were considered success.2. Diabetic rats were anesthetized with sodium pentobarbital (30 mg/kg, i.p.), and experienced left thoracotomy. And established I/R experimental models. Rats were subjected to 30 min myocardial ischemia and 72 h reperfusion.3. Cardiac microvascular permeability and ultrastructure were compared with Lanthanum as a tracer. Casting of cardiac microvessels perfused by Mercox observed under SEM.4. PKC-βII location and VE-cadherin rearrangement in diabetic heart tissue were examined by immunofluorescence. Phosphorylated LIM kinase 2 in heart tissue after I/R was determined by immunohistological method.5.±LVdp/dtmax of after I/R and±LVdp/dtmax of before I/R were recorded by 8–orbits physiological grapher.6. Thioflavin S was injected into the heart to define the region of no-reflow. Thioflavin S is used as a standard marker for identifying zones of no-reflow and to calculated no-reflow area.7. After removal of the endocardial endothelium and the epicardial coronaries, the left ventricles were cut into small pieces and incubated in 2 ml 0.2% collagens, isolated and purified .Cardiac microvascular endothelium cells(CMECs).8. Quantitatively assess permeability of CMECs monolayer using In Vitro Vascular Permeability Assay kit.9. PKC-βII, VE-cadherin, phospho-β-catenin and Phosphorylated LIMK2 was determined by western blot analysis.10. TUNEL staining detected apoptosis of CMECs in diabetic rat and in high glucose medium subjected to I/R. Three staining of CD31, TUNUL and DAPI were used to obtain AI of CMECs of diabetic rats after I/R.11. F-actin was stained with FITC-phalloidin and cells were visualized with fluorescence microscopy.12.G-actin / F-actin assay kit was used to quantitatively detected F-actin.Results1. PKC-βII activation play a key role in microvascular barrier dysfunction on diabetic rat’s heart.(1) Successfully established diabetic rat model with FPG increase.(2) IF examined PKC-βII activation and VE-cadherin rearrangement in diabetic rats’heart. PKC-βII was downregulated and VE-cadherin expression was regular after Rx treatment(3) Relative expression of PKC-βII (1.06±0.09 vs. 0.76±0.06,P<0.01)and P-β-catenin(0.74±0.13 vs.0.47±0.11, P<0.01)in diabetic rats increased compared with that in non-diabetic control. PKC-βII expression (( 0.79±0.10 vs. 0.97±0.11,P<0.05) and P-β-catenin (0.59±0.07 vs. 0.80±0.11, P<0.05) were significantly downregulated by pretreatment with Rx.(4) Lanthanum as a tracer to found cariac microvascular dysfunction in diabetic rat and the surfaces of cardiac microvascular casting weren’t integrity inder electron microscope. Rx administration could attenuate this disturbance.2. PKC-βII activation had important role in cardiac microvascular function impairment aggravated in diabetic rat during I/R. (1)±LVdp/dt decreased in DM group compared with non-diabetic control and sham, and Rx administration could improve±LVdp/dt in DM group.(2) No-reflow size in diabetic rats significantly larger than sham (20.0±1.7% vs. 0%) and non-diabetic control (20.0±1.7% vs.14.3±1.8%, P<0.01), Treatment with Rx significantly reduced the no-reflow size (16.4±1.9% vs. 19.4±1.6%, P<0.05).(3)CMECs were severely impaired after I/R in diabetic rats. The edema and vacuolus were represented in diabetic rats. Rx pretreatment could attenuate this severe pathology.(4)AI of CMECs in diabetic heart increased compared with that in sham (12.6±2.2% vs.8.1±1.7%, P<0.01)and non-diabetic control(12.6±2.2% vs.7.8±0.8%, P<0.01). AI of CMECs in Rx treated group reduced(9.5±1.5% vs. 12.2±2.1%, P<0.05).(5)Relative expression of Phospho-LIMK2 increased compared with that in Sham (1.01±0.10 vs. 0.66±0.11, P<0.01)and non-diabetic control(1.01±0.10 vs. 0.60±0.09, P<0.01 ) . Phospho -LIMK2 expression was significantly downregulated by Rx administration(0.76±0.08 vs. 0.97±0.10, P<0.01).3. PKC-βII activation decrease the barrier function of CMECs monolayer in High glucose medium.(1) Permeability of monolayer cell in high glucose medium was higher than in normal medium (394.50±36.92 vs. 282.10±14.93, P<0.01).However, the addition of PKC-βII i?nhibitor, Rx (1, 10, 100nmol/L), ?resulted in permeability decreased (P<0.01).(2) PKC-βII (1.17±0.13 vs.0.71±0.07, P<0.01)and phospho-β-catenin(0.92±0.11 vs. 0.56±0.07, P<0.01) expression in CMECs incubated with high glucose medium increased compared with that in normal medium. PKC-βII and phospho-β-catenin expression was significantly down regulated given Rx (10nmol/L) pretreatment (P<0.01). VE-cadherin(c-19)didn’t alter in CMECs cultured in high glucose medium.4. PKC-βII activation was involved in the mechanism by which CMECs impairment worse during I/R.(1) Permeability of monolayer cell in high glucose medium was higher than in sham (590.40±34.38 vs. 359.20±22.80, P<0.01) and in normal medium (590.40±34.38 vs. 383.70±37.58 P<0.01).However, the addition of Rx (10nmol/L) could resulte in permeability decreased (432.00±30.83 vs. 571.10±32.60P<0.01).(2) Phospho -LIMK2 of CMECs in high glucose medium increased compared with that in sham (1.11±0.09vs. 0.31±0.09, P<0.01)and in normal medi( 1.11±0.09vs. 0.43±0.15, P<0.01 ) . Phospho -LIMK2 expression was significantly downregulated by pretreatment with Rx ( 0.60±0.08 vs.1.28±0.11,P<0.01).(3) AI of CMECs in high glucose medium subjected to I/R increased compared with that in sham (13.3±1.3% vs. 5.7±1.4%, P<0.01)and in normal medium (13.3±1.3% vs. 7.8±0.8%, P<0.01). AI could be decreased by pretreatment with Rx(9.1±1.1% vs.12.8±1.8%, P<0.01).(4)The ratios of F-actin to G-actin in CMECs incubated with high glucose medium increased compared with that in sham (10.73±1.32 vs. 6.58±1.34, P<0.01)and in normal medium (10.73±1.32 vs. 7.73±0.97, P<0.01).Pretreatment with Rx (10nmol/L) could reduce it.(7.32±1.02 vs.10.24±1.38, P<0.05).Conclusion1. STZ induced diabetic rat model was used to verify that diabetic state induced activation of PKC-βII and microvascular complication displaying in permeability increase or microvascular barrier dysfunction. Phospho-β-catenin expression and VE-cadherin rearrangement involved in this mechanism; 2. Exaggerated no-reflow areas accompanying with severe impairment of cardiac function and aberrant microvascular barrier function presented in diabetic rat model after I/R and this effect could be prevented by a PKC-βinhibitor pretreatment. PKC-βII-dependent Rho kinase activation in diabetes in response to I/R and phosphor- LIMK2 increase contributed to microvascular damage and the severe no-reflow phenomenon;3. High glucose microenvironment could activate PKC-βII in cultured CMECs and lead to phospho-β-catenin increase. It promoted the dissociation of VE-cadherin–catenin complex and deteriorated the CMECs barrier function;4. PKC-βII was activated in CMECs cultured in high glucose, and in this state subjected to I/R, Rho kinase was excessively activated and phospho-LIMK2 increased. Phospho-LIMK2 was involved in structural alterations in cytoskeletal filaments and polymerization of F-actin which formed concentrical force. Loss of equilibrium in VE-cadherin and F-actin could aggravate CMECs impairment after I/R.These results indicated pretreatment for diabetic microvascular complication induced by PKC-βII could be helpful to attenuate the risk of I/R injury.更多还原