目的 建立以及验证人肝细胞L-02缺血再灌注损伤(IRI)模型.方法 分别利用液体石蜡、氯化钴(CoCl2)以及缺氧培养箱构建人肝细胞株L-02 IRI模型,采用细胞计数试剂盒(CCK-8)法检测细胞活力,利用相关试剂盒检测细胞超氧化物歧化酶(SOD)、丙二醛(MDA)水平,利用异硫氰酸荧光素标记的膜联素Ⅴ/碘化丙锭(Annexin V-FITC/PI)细胞凋亡检测试剂盒检测细胞凋亡水平,蛋白质印迹(Western blot)法测定凋亡相关蛋白天冬氨酸特异性半胱氨酸蛋白酶-3(Caspase-3)以及B细胞淋巴瘤-2相关X蛋白(bax)表达水平,通过以上方法对3种方式构建肝细胞IRI效果进行验证以及统计分析.两组间的比较采用t检验,多组间比较采用单因素方差分析.结果 利用液体石蜡、CoCl2 以及缺氧培养箱构建的肝细胞IRI模型的细胞活力均低于对照组,各组细胞450 nm吸光度分别为(0.698±0.021、0.622±0.029、0.725±0.025 比 1.086±0.123),差异有统计学意义(F=30.02,P＜0.01),其中液体石蜡组、缺氧培养箱组的细胞活性要高于CoCl2组(t=3.660、4.605,P均＜0.05);各组 SOD 水平均低于对照组[(117.678±9.797)、(100.095±5.588)、(105.614±4.880)U/mg 蛋白 比(172.378±6.208)U/mg 蛋白,F=92.56,P＜0.01],其中液体石蜡组 SOD 水平高于 CoCl2 组(t=3.118,P＜0.05);各组 MDA 水平高于对照组[(62.684±4.584)、(70.931±1.849)、(63.472±2.316)nmol/mg 蛋白 比(16.163±2.106)nmol//mg 蛋白,F=219.90,P＜0.01],CoCl2组MDA高于液体石蜡组和缺氧培养箱组(t=2.889、4.347,P均＜0.05);IRI各组细胞凋亡率高于对照组[(26.087±4.107)％、(36.797±3.453)％、(31.945±2.859)％比(1.840±0.489)％,F=103.50,P＜0.01],其中 CoCl2 组凋亡率高于液体石蜡组(t=3.992,P＜0.01).各组P-Caspase-3相对表达水平较对照组升高(2.460±0.102、2.629±0.059、2.574±0.107 比 1.000,F=291.23,P＜0.01],bax相对表达水平同样高于对照组(1.506±0.012、1.618±0.055、1.631±0.080 比 1.000,F=90.80,P＜0.01),液体石蜡、CoCl2和缺氧培养箱三组之间的凋亡蛋白相对表达量无明显差异(F=4.33、0.92,P均＞0.05).结论 采用液体石蜡、CoCl2以及缺氧培养箱均可成功诱导肝细胞的IRI,在无缺氧培养箱的条件下,与CoCl2比较,可优先选用液体石蜡的方法来构建肝细胞的IRI模型.

颈动脉粥样硬化斑块破裂是导致脑卒中的重要原因之一,大量研究证实颈动脉斑决内新生血管是导致斑块内出血、斑块破裂的重要因素.炎症因子及各类细胞通过斑块内新生血管进入斑块,导致斑块稳定性破坏,但影响斑块内新生血管形成的重要相关因子和主要机制目前尚未完全明确,因此识别斑块内新生血管、探索斑块内新生血管形成的相关因子及机制是研究斑块内新生血管致斑块不稳定性的关键.抑制斑块内新生血管生成,可能成为防治颈动脉斑块破裂、降低脑栓塞事件发生的新策略.本综述旨在探讨颈动脉斑块内新生血管形成的相关因子、机制以及检测成像的最新研究进展,为动脉粥样硬化的诊疗提供支持.

Objective:To investigate the effect of competitive inhibition of β-catenin/T-cell factor and β-catenin/forkhead box protein O1 (FoxO1) signal downstream of transforming growth factor (TGF)-β 1 on angiogenesis and plaque stability in carotid plaque.Methods:Plaque tissues of patients with carotid stenosis after CEA were collected ( n=21). Vulnerable and stable plaques were identified by hematoxylin-eosin (HE) staining, and the expression sites and levels of CD31 and VE-Cadherin in each tissue were analyzed by immunohistochemistry (IHC). Model of carotid stenosis was established in ApoE -/-mice. Follwoing groups were set up ( n=12/group). Group T was given TGF-β 1 (50 μg/kg) + normal saline (5 mg/kg). Group TI was given TGF-β 1 (50 μg/kg) + ICG-001 (5 mg/kg). Group I was given normal saline (50 μg/kg) + ICG-001 (5 mg/kg). Group N was given normal saline (5.05 mg/kg). All groups were treated by their own treatment for 2 weeks, then the carotid tissues were taken.After HE staining, vulnerable and stable plaques were divided according to the pathological characteristics. The expression sites and levels of CD31 and VE-Cadherin in each tissue were detected by IHC. All data were analyzed by Imageplus 6.0 and SPSS 25.0 statistical software. Results:The vulnerable rate in groups T and TI was 60.00% and 33.33% respectively, which was lower than in group N (72.73%). The expression of VE-Cadherin in groups T and N was 0.156±0.007 and 0.191±0.005 respectively, which was statistically significant ( F=1.615, P<0.05). The expression of VE-Cadherin in group T was significantly higher than that in group TI (0.133±0.010, F=0.068, P<0.05). The expression of CD31 in groups TI and T was 0.119±0.004 and 0.136±0.010 respectively, which was statistically significant ( F=3.451, P<0.05). The expression of CD31 and VE-Cadherin in stable plaque was low, and that in group TI was lower than in the rest groups. Conclusion:Specific blocking β-catenin/TCF downstream of TGF-β can inversely enhance the inhibitory effect of β-catenin/FOXO1 on carotid atherosclerosis intraplaque angiogenesis, thereby having a positive impact on stabilizing plaque.

Objective:To observe the effect of macrophage polarization regulated by exogenous transforming growth factor-β1 (TGF-β1) on the stability of carotid plaque in ApoE -/-mice. Methods:Twenty ApoE -/-mice aged 6 to 8 weeks were randomly divided into two groups: saline group ( n = 10) and TGF-β1 intervention group ( n = 10). After 6 weeks of high-fat feeding, the right common carotid artery was separated and ligated in series to construct the model of vulnerable carotid plaque. After 5 weeks of high fat feeding, mice in the two groups were given normal saline and TGF-β1 intraperitoneal injection, 50 μg/kg respectively, once a day for 2 weeks. The body weight of mice in both groups was measured before and after the experiment. Total cholesterol (TC), low density lipoprotein (LDL) and interleukin-10 (IL-10) in the blood were determined by enzyme-linked immunosorbent assay (ELISA) after 2 weeks. The right common carotid artery was taken for hematoxylin-eosin (HE) staining to observe the pathological morphology of plaque. The contents of tumor necrosis factor-α (TNF-α) and mannitol receptor (CD206) in macrophages in carotid plaques were detected by immunohistochemistry. The MI and M2-type macrophages in the plaques were labeled by immunofluorescence staining. The SPSS 23.0 statistical software was used to analyze the measurement data. Results:After the experiment, the body weight of mice in normal saline group [(29.720±1.946) g] and TGF-β1 intervention group [(30.890±1.265) g] showed no significant difference between the two groups ( t=1.671, P>0.05). The content of serum TC in saline group and TGF-β1 intervention group was (27.420±6.704) and (24.260±1.468) respectively, and there was no significant difference between the two groups ( t=1.126, P>0.05). The content of serum LDL in saline group and TGF-β1 intervention group was (2.560±0.227) and (2.660±0.367) respectively, and there was no significant difference between the two groups ( t=0.552, P>0.05). The content of serum IL-10 in saline group and TGF-β1 intervention group was (11.230±2.744) and (27.980±18.330) respectively, and there was significant difference between the two groups ( t=2.564, P<0.05). The percentage of plaque area to lumen area in the normal saline group was (81.280±17.780)%, and that in the TGF-β1 intervention group was (50.160±26.170)%, and there was significant difference between the two groups ( t=2.828, P<0.05). The number of M1-type cells in the normal saline group was (46.390±15.010) cells and that in the TGF-β1 intervention group was (28.330±9.041) cells, and there was significant difference between the two groups ( t=2.460, P<0.05). The number of M2-type macrophages in the normal saline group [(11.060±1.913) cells] and the TGF-β1 intervention group [(27.730±11.860) cells] showed significant difference between the two groups ( t=3.112, P< 0.05). TNF-α absorbance was (39.360±20.470) in the normal saline group, and (18.570±3.227) in the TGF-β1 intervention group and there was significant difference between the two groups ( t=2.456, P<0.05). CD206 absorbance was (5.060±2.621) in the normal saline group, and (10.900±4.594) in the TGF-β1 intervention group and there was significant difference between the two groups ( t=2.506, P<0.05). Conclusion:Exogenous TGF-β1 promotes the polarization to M2, which plays a positive role in stabilizing vulnerable carotid plaques and delaying atherosclerosis.

临床缺血性脑卒中发病因素中，颈动脉粥样斑块占约25%。关于颈动脉粥样斑块引起缺血性脑卒中的机制多集中于斑块破裂方面，目前研究结论尚不充足，本研究就颈动脉粥样斑块破裂主要机制——炎症反应进一步研究讨论。本研究在ApoE -/-小鼠颈动脉粥样斑块模型构建成功的基础上给予重组人转化生长因子-β1干预，其可充分诱导小鼠脾脏组织中的调节性T细胞分化，抑制全身及局部粥样斑块中的炎症反应，促进抗炎因子分泌以及抑制斑块中促进斑块破裂的蛋白分泌，对斑块的稳定性具有积极作用。

Objective The femoral artery of C57BL/6 ApoE-/-mice was ligated to make the model of hind-limb artery stenosis.Angiography was used to evaluate the stenosis rate of hind-limb artery in mice.Methods The ligated femoral arteries were divided into the experimental group and the contralateral femoral arteries were the control group.We exposed the right femoral artery in the operative field using blunt dissection technique,parallel placed the metal wire with femoral artery,used thread to bound the femoral artery and the metal wire,drawn out the metal wire after ligation,and then sutured the skin wound.The contralateral was dealt with the same method but not ligated.Mter 4 weeks of high fat diet,the mice were killed to expose their hearts,and the contrast medium was pushed from the left ventricle,then put into the X-ray imaging system of small animals for angiography.The bilateral femoral arteries were taken from the mice after angiography.Results There were 13 plaques in the experimental group and 4 in the control group.The angiography results showed that the femoral artery stenosis rate of 16-week-old C57BL/6 ApoE-/-mice model was measured to be (48.43 ± 25.13)％,the theoretical stenosis rate was (45.63 ±9.96)％,F =7.956,P ＞0.05.The diameter of femoral artery was (0.97 ±0.11) mm.Conclusion The method of femoral artery ligation is a reliable method to the establishment of the model of hind-limb artery stenosis in C57BL/6 ApoE-/-mice and angiography is one of the methods to evaluate the rate of stenosis.