沉默信息调节因子1对缺氧心肌细胞凋亡和自噬的影响

目的:分析沉默信息调节因子1(Sirt1)对缺氧条件下心肌细胞凋亡和自噬的影响。方法:将心肌H9c2细胞分为Control组、Hypoxia组(缺氧处理24h)、NC+Hypoxia组(转染阴性对照并缺氧处理24h)和Sirt1+Hypoxia组(转染Sirt1过表达载体并缺氧处理24h)。以qRT-PCR和Western blot测定心肌细胞中Sirt1mRNA和蛋白表达水平,MTT方法评估细胞存活率,流式细胞术测定细胞总凋亡水平,Western blot检测凋亡蛋白Bax、Cleaved Caspase-3水平和自噬蛋白LC3-Ⅱ/Ⅰ、Beclin-1水平。结果:与Control组比较,Hypoxia组细胞中Sirt1mRNA和蛋白表达水平降低,细胞存活率降低,细胞凋亡率和Bax、Cleaved Caspase-3蛋白水平升高,LC3-Ⅱ/Ⅰ、Beclin-1蛋白水平降低(P0.05)。与NC+Hypoxia组比较,Sirt1+Hypoxia组细胞中Sirt1mRNA和蛋白表达水平升高,细胞存活率升高,细胞凋亡率和Bax、Cleaved Caspase-3蛋白水平降低,LC3-Ⅱ/Ⅰ、Beclin-1蛋白水平升高(P0.05)。结论:Sirt1可抑制缺氧心肌细胞凋亡并诱导细胞自噬。     

The Search for New Hypoxic Cell Radiosensitizers

A number of newly synthesized compounds whose chemical structure suggested possible or remotely possible ability to radiosensitize hypoxic mammalian cells were studied in an in-vitro system. Those compounds that were not excluded because of insolubility or extreme cytotoxicity were tested for radiosensitizing ability. The correlation between chemical structure and radiosensitizing ability will be used for the rational design of additional compounds with a high probability of being effective hypoxic cell radiosensitizers. It is hoped that this will contribute to attempts to improve the cure rate of patients with malignant tumors through the use of radiation therapy and hypoxic cell radiosensitizers.     

Lipopolysaccharide-Induced Proliferation of the Vasa Vasorum in a Rabbit Model of Atherosclerosis as Evaluated by Contrast-Enhanced Ultrasound Imaging and Histology

Whether lipopolysaccharide (LPS) can promote vasa vasorum (VV) proliferation for atherosclerosis in vivo is unclear. Eighteen rabbits with atherosclerosis were randomly assigned into one of three groups of six. Group A received biweekly injections of 10 mL saline after 2 weeks of balloon injury. Groups B and C received biweekly intravenous injections of 3.0 μg LPS in 10 mL saline at weeks 10 and 4, respectively, until study termination. LPS significantly increased the levels of triglycerides and C-reactive protein and decreased the level of high-density lipoprotein cholesterol. Group C had significant larger plaques and more macrophages than group A (p = 0.01 and p < 0.001, respectively). Contrast enhancement ultrasound imaging and histological detection demonstrated that plaques in group C had a significantly higher VV density than that in group A (p = 0.009 and p = 0.002, respectively). In summary, VV proliferation for plaque destabilization can be accelerated by LPS-induced systemic inflammation and changes in lipid profiles.     

兔骨髓间充质干细胞来源的心肌(样)细胞的诱导分化研究

目的体外诱导骨髓间充质干细胞(Mesenchymal stem cells,MSCs)向肌源性细胞分化,探索诱导后的MSCs移植于心肌梗死区的存活和分化情况。方法提取、分离、培养兔的MSCs。经5-氮胞苷诱导后,进行免疫组化,电镜观察。4',6二乙酞基-2-苯基吲哚(DAPI)标记MSCs,建立兔心肌梗死模型。实验动物随机分两组:实验组(n=10)在心梗区域注入经诱导后的MSCs;对照组(n=10)在心梗区域注入不含MSCs的培养液。移植4周后,进行病理标本观察和免疫组化检测。结果5-氮胞苷诱导MSCs4周,部分细胞表达肌钙蛋白T(troponin T),电镜观察到肌丝形成。MSCs在体外用DAPI标记,用荧光显微镜观察细胞发蓝色荧光。移植4周后,在实验组中用荧光显微镜观察可见梗死区组织标本中可见DAPI标记带蓝色荧光的供体细胞核,移植细胞表达troponin T。结论MSCs经5-氮胞苷诱导后可向心肌细胞转化。移植细胞可在心肌存活,并向心肌细胞(样)转化。     

Effect of Alloxan-diabetes on the Metabolism of Rabbit Colon Smooth Muscle

The metabolism of colon smooth muscle from normal and alloxan-diabetic rabbits was studied in vitro. Glucose uptake, incorporation of glucose-¹⁴C into glycogen and incorporation of leucine-¹⁴C into protein were determined. These three parameters were all depressed in diabetic smooth muscle incubated in a medium containing 5.6 mM glucose. Raising the glucose concentration in the medium to 22.2 mM almost doubled the glucose uptake both in diabetic and normal smooth muscle and at this glucose concentration no significant difference in this parameter was found. Insulin (0.1 U/ml) added in vim) stimulated glucose uptake, incorporation of glucose-¹⁴C into glycogen and incorporation of leucine-¹⁴C into protein in diabetic colon smooth muscle. Insulin stimulated glucose uptake to the same degree in normal and diabetic colon smooth muscle, while its effect on glucose-¹⁴C and Ieucine-¹⁴C incorporation tended to be somewhat more pronounced in diabetic colon muscle.     

甲鱼油对胰岛素抵抗细胞模型胰岛素敏感性和葡萄糖代谢的影响

采用高浓度胰岛素诱导建立胰岛素抵抗(IR)HepG2细胞模型,评价甲鱼油对IR细胞模型胰岛素敏感性和葡萄糖代谢的影响.利用甲鱼油处理IR细胞模型,通过葡萄糖氧化酶及3H-D-葡萄糖参入实验分别检测其葡萄糖消耗和3H-D-葡萄糖参入率.结果表明,HepG2细胞模型的葡萄糖参入率在各种浓度的胰岛素刺激下均低于对照细胞(P＜0.05),并且HepG2细胞模型与对照细胞的3H-D-葡萄糖参入率随着胰岛素浓度的升高而升高,但前者升高的幅度低于后者.MTT(甲基噻唑基四唑)结果显示甲鱼油均具有促进对照细胞和IR细胞模型增殖作用.与对照组细胞相比,采用不同浓度胰岛素诱导的模型细胞经甲鱼油处理后其葡萄糖摄取和消耗显著增加(P＜0.05).甲鱼油明显增加该IR细胞模型的葡萄糖消耗和3H-D-葡萄糖参入率,提高IR细胞模型的胰岛素敏感性,改善其糖代谢.     

Simultaneous Measurement of Contraction and Calcium Transients in Stem Cell Derived Cardiomyocytes

Induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) provide a powerful platform for disease modeling and drug development in vitro. Traditionally, electrophysiological methods or fluorescent dyes (e.g. calcium) have been used in their functional characterization. Recently, video microscopy has enabled non-invasive analysis of CM contractile motion. Simultaneous assessments of motion and calcium transients have not been generally conducted, as motion detection methods are affected by changing pixel intensities in calcium imaging. Here, we present for the first time a protocol for simultaneous video-based measurement of contraction and calcium with fluorescent dye Fluo-4 videos without corrections, providing data on both ionic and mechanic activity. The method and its accuracy are assessed by measuring the effect of fluorescence and background light on transient widths and contraction velocity amplitudes. We demonstrate the method by showing the contraction-calcium relation and measuring the transient time intervals in catecholaminergic polymorphic ventricular tachycardia patient specific iPSC-CMs and healthy controls. Our validation shows that the simultaneous method provides comparable data to combined individual measurements, providing a new tool for measuring CM biomechanics and calcium simultaneously. Our results with calcium sensitive dyes suggest the method could be expanded to use with other fluorescent reporters as well.     

后肢接振对家兔组织中血管内皮活性物质的影响

目的探讨局部振动对组织中血管内皮活性物质的影响及意义.方法将家兔随机分为不同接振强度组进行振动负荷试验,于试验结束后测定各组大脑及肾组织中内皮素、血管紧张素Ⅱ、一氧化氮的浓度.结果随接振强度的增大,ET、AnⅡ呈显著增高、NO呈显著降低的趋势(P＜0.05,P<0.01).结论局部振动所致的组织中血管内皮活性物质的变化,可能与局部组织缺血、功能紊乱有关.     

兔骨髓内皮祖细胞在组织工程血管构建中的实验研究

建立体外分离、培养及鉴定兔骨髓血内皮祖细胞（endothelial progenitor cell,EPCs）的方法,并探讨其在血管组织工程构建过程中的功能。采用密度梯度离心法分离单个核细胞,经培养鉴定为EPCs后作为种子细胞接种于人纤维连接蛋白包被（FN）的组织工程血管支架上,加入血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）进行体外诱导培养,同时设置未包被纤维连接蛋白及未添加血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）的培养方法作为对照组,体外培养10 d后,对构建的组织工程血管进行鉴定分析。分离培养的骨髓单个核细胞呈典型的＂铺路石样＂外观。经免疫荧光检测、细胞吞噬功能鉴定为内皮祖细胞;种植细胞10 d后结果显示：加入纤维连接蛋白和血管内皮生长因子的血管支架可见细胞种植密度明显高于对照组,扫描电子显微镜观察到,血管内腔面较为完整的覆盖内皮细胞。HE染色显示：内皮细胞在血管支架上成活并较为均匀;免疫组化结果显示分化为成熟血管内皮细胞并表达VEGFR-2、vWF、CD34。兔骨髓单个核细胞体外培养可以诱导分化为内皮祖细胞,血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）和纤维连接蛋白（FN）的组合更有利于内皮祖细胞在血管支架上增殖和分化,为人工血管制备创造了条件。     

Computational Models of Ventricular- and Atrial-Like Human Induced Pluripotent Stem Cell Derived Cardiomyocytes

The clear importance of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) as an in-vitro model highlights the relevance of studying these cells and their function also in-silico. Moreover, the phenotypical differences between the hiPSC-CM and adult myocyte action potentials (APs) call for understanding of how hiPSC-CMs are maturing towards adult myocytes. Using recently published experimental data, we developed two computational models of the hiPSC-CM AP, distinguishing between the ventricular-like and atrial-like phenotypes, emerging during the differentiation process of hiPSC-CMs. Also, we used the computational approach to quantitatively assess the role of ionic mechanisms which are likely responsible for the not completely mature phenotype of hiPSC-CMs. Our models reproduce the typical hiPSC-CM ventricular-like and atrial-like spontaneous APs and the response to prototypical current blockers, namely tetrodotoxine, nifedipine, E4041 and 3R4S-Chromanol 293B. Moreover, simulations using our ventricular-like model suggest that the interplay of immature I _Na, I _f and I _K1 currents has a fundamental role in the hiPSC-CM spontaneous beating whereas a negative shift in I _CaL activation causes the observed long lasting AP. In conclusion, this work provides two novel tools useful in investigating the electrophysiological features of hiPSC-CMs, whose importance is growing fast as in-vitro models for pharmacological studies.     

Identification of an Invasive, N-Cadherin-Expressing Epithelial Cell Type in Endometriosis Using a New Cell Culture Model

Studies of molecular, cellular, and pathophysiological parameters in endometriosis are primarily hampered by a lack of in vitro model systems, such as endometriotic cell lines. To overcome this we successfully established cell lines from peritoneal endometriotic biopsies and characterized them at the molecular and cellular level. Two types of cells could be transformed: one exhibiting stromal cell features (cytokeratin/E-cadherin-negative), the other epithelial-like (cytokeratin-positive/E-cadherin-negative, invasive in vitro). Using a Matrigel assay the epithelial-like cell lines proved as invasive as metastatic carcinoma cells, possibly through the influence of N-cadherin implicated as a path-finding cadherin allowing cellular invasion and migration in both normal and pathophysiological processes. Our results support the idea that endometriosis, although not neoplastic, shares features with malignant cells and that metastasis in endometriosis may include mechanisms proposed for micrometastasis in cancer. Thus our cell lines will not only be useful tools for analyzing molecular and cellular events relating to endometriosis, but may also represent a paradigm for invasion and metastasis in general.     

脑缺血再灌流家兔血液中循环内皮细胞变化及针刺对其的影响

利用缺血再灌流家兔模型研究变化规律，并探讨了脂质过氧化物与循环内皮细胞之间的关系，以期了解缺血再灌流损伤血管内皮细胞的受损情况及发生机理。实验共用家兔３３只，缺血组９只，其循环内皮细胞比假手术组增多（Ｐ＜０．０５）。再灌流组９只，其循环内皮细胞显著增多（与假手术组、缺血组比较，Ｐ均＜０．００１），结果显示了再灌流血管内皮细胞损伤程度较缺血组明显，同时可看到脂质过氧化物变化与循环内皮细胞数量变化有一致性。实验同时观察到针刺对再灌流出现的上述异常改变大多有明显的改善作用     

模拟失重时兔血液中血管内皮细胞数量的变化

模拟失重时兔血液中血管内皮细胞数量的变化沈羡云１陈建和１孟京瑞１董欣１王玉清１钱冠清２刘会齐２近几年来，国内外进行了循环内皮细胞数（ＣＥＣ）检测技术及其临床应用的研究［１，３］。研究结果表明，血液中ＣＥＣ的变化可以反映许多病理情况下血管内膜的损伤程度...     

兔骨髓内皮祖细胞在组织工程血管构建中的实验研究

建立体外分离、培养及鉴定兔骨髓血内皮祖细胞(endothelial progenitor cell,EPCs)的方法,并探讨其在血管组织工程构建过程中的功能。采用密度梯度离心法分离单个核细胞,经培养鉴定为EPCs后作为种子细胞接种于人纤维连接蛋白包被(FN)的组织工程血管支架上,加入血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)进行体外诱导培养,同时设置未包被纤维连接蛋白及未添加血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)的培养方法作为对照组,体外培养10 d后,对构建的组织工程血管进行鉴定分析。分离培养的骨髓单个核细胞呈典型的"铺路石样"外观。经免疫荧光检测、细胞吞噬功能鉴定为内皮祖细胞;种植细胞10 d后结果显示:加入纤维连接蛋白和血管内皮生长因子的血管支架可见细胞种植密度明显高于对照组,扫描电子显微镜观察到,血管内腔面较为完整的覆盖内皮细胞。HE染色显示:内皮细胞在血管支架上成活并较为均匀;免疫组化结果显示分化为成熟血管内皮细胞并表达VEGFR-2、vWF、CD34。兔骨髓单个核细胞体外培养可以诱导分化为内皮祖细胞,血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)和纤维连接蛋白(FN)的组合更有利于内皮祖细胞在血管支架上增殖和分化,为人工血管制备创造了条件。     

血管内皮细胞损伤在家兔多器官衰竭发病过程中的病理意义

利用家兔多器官衰竭(MOF)模型,从形态和机能两方面结合观察了血管内皮细胞的变化。结果平铺切片和电镜观察均显示MOF家兔的血管内皮细胞受到明显损伤。血浆Fn含量锐减,而ⅧR:Ag含量却明显增加,其72h值与实验前比、与对照组比均有显著差异(P<0.05);血浆TXB_2含量随时间明显增加,而6—酮—PGF_(1a)含量则明显下降,故二者的平衡失调。提示内皮细胞损伤在MOF发病过程中可能是重要环节,由此引起的种种变化可能是机体微血栓形成、微循环障碍和器官功能障碍的病理基础之一。     

Resveratrol Has Anabolic Effects on Disc Degeneration in a Rabbit Model

This study was done to evaluate whether injections of resveratrol, a natural compound found in the skin of grapes, had anabolic effects on degenerated intervertebral discs in a rabbit model. Two non-continuous lumbar discs were punctured in rabbits to induce disc degeneration. Four weeks and 6 weeks after puncture, the rabbits were treated by injections with dimethylsulfoxide (DMSO) or resveratrol. At 4, 8, and 16 weeks after initial injection, rabbits were sacrificed and the spine was extracted for magnetic resonance image (MRI), mRNA expression, and histological staining. Resveratrol treatment resulted in stronger signal intensity in T2-weighted images. MRI grade showed significantly lower in the resveratrol group than the DMSO group (P = 0.039). In the resveratrol group, aggrecan gene expression was significantly increased than that in the DMSO group at 16 weeks after injection (P = 0.027). MMP-13 mRNA levels in the resveratrol group were significantly decreased than those in the DMSO group at 8 and 16 weeks (P = 0.006 and P = 0.048, respectively). In hematoxylin and eosin stain, resveratrol-treated discs showed the features of regeneration. Histologic grade revealed improvement in resveratrol-treated discs, compared with DMSO-treated discs (P = 0.024). These anabolic effects on degenerated discs indicate that resveratrol is a promising candidate for treatment of degenerative disc disease.