体外构建血管化组织工程心肌

目的:探讨体外构建血管化组织工程心肌的可行性。方法:大鼠骨髓间充质干细胞以浓度为10μmol/L的5-氮胞苷诱导为心肌细胞并用DAPI标记、骨髓源内皮祖细胞以内皮细胞培养基EGM2-MV定向诱导为内皮细胞并用CM-dil标记。标记后的心肌细胞与内皮祖细胞按2:1比例,以4×10~6个/ml的密度种植于Matrigel基质胶支架上。同种密度心肌细胞种植为对照组。对复合体形态、细胞分布等进行观察。结果:H-E染色及荧光显微镜观察可见两组复合体的细胞分布均匀,生长状态良好;单纯心肌细胞种植组可见细胞黏附聚集生长,并未出现网状结构,而2:1混合培养组24 h后可见内皮细胞相互连接成网状结构,出现典型的"成血管现象",且形成的血管样结构明显多于对照组;心肌细胞凋亡数较对照组少。结论:骨髓源性心肌细胞与骨髓源性内皮细胞联合种植于Matrigel基质胶共培养,可于体外成功构建血管化组织工程心肌。     

改进构建后的全生物化组织工程血管的组织结构与生物力学特性

目的改进体外构建的生物化组织工程血管(TEBV),研究其生物力学特性,获得能承载生理血流力学作用的血管替代物。方法用酶消化法制备猪颈总动脉脱细胞支架,加压灌注结合散点注射种植犬胸主动脉平滑肌细胞(VSMC),用自制的血管自动旋转流体培养系统培养3周,随后再用加压灌注法种植犬胸主动脉内皮细胞(EC),继续培养至4周,取材做HE染色。光镜和电镜观察VSMC及EC在支架内的生长情况。用力学测试仪器检测TEBV的应力-应变关系、拉伸弹性回复率以及最大断裂强度和长度。结果在血管自动旋转流体培养系统,VSMC种植3周在中膜层已经大量均匀分布,EC种植7天后已形成连续完整的EC单层;而应用单独的加压灌注法VSMC种植3周时仍不能均匀分布,EC种植1周后仍分布不均匀,未能形成连续的EC单层。TEBV培养至第4周,中膜层有大量VSMC生长,内膜层则形成连续的EC单层;透射电镜下可见VSMC、EC亚微结构与生理状态的细胞结构相似,可见缝隙连接等细胞连接方式,VSMC可产生新的胶原蛋白。扫描电镜可见EC在支架上生长良好,细胞轮廓清晰,形成连续的单层。力学特性检测结果显示:TEBV的粘弹性、拉伸弹性回复率以及最大断裂强度均接近生理血管。结论血管自动旋转流体培养系统在脱细胞血管支架上联合种植VSMC和EC,有利于种子细胞在支架腔面和管壁内均匀生长,改进了TEBV的组织结构。改进构建的TEBV生物力学特性接近生理血管,将有利于TEBV更好地承载血流动力学的作用。     

组织工程化血管移植实验研究

目的 通过体外构建、体内培养的方法 构建组织工程化血管,并将其植入动物的股动脉,探讨利用小口径组织工程化血管进行血管移植的可能性.方法 首先用Brdu标记所有种子细胞,将血管平滑肌样细胞和血管内皮样细胞分层种植于胶原包埋PGA的复合支架表面;然后将细胞和支架的复合体种植于动物皮下构建组织工程化血管,并移植到股动脉;最后行HE染色及免疫荧光等相关检查,了解移植后血管的特点及细胞来源.同时以移植物为单纯支架在皮下培养所形成的管状结构作为对照组.结果 移植后组织工程化血管通畅无血栓形成.HE染色见血管壁和正常生理性血管壁的结构相似.可分内膜、中膜和外膜三层.种植2周后Brdu标记细胞的免疫荧光观察证实血管壁细胞来源于所种植的种子细胞.结论 利用骨髓间充质干细胞分化而来的种子细胞和胶原包埋的PGA支架可以构建组织工程化小口径血管,而且可以移植入动物体内.     

人血管平滑肌细胞种植构建工程生物血管的实验研究

了解猪血管去细胞后平滑肌细胞种植情况,为猪血管用于血管组织工程提供资料。取猪颈动脉,生物酶预处理猪血管,在自行设计制作的新型动力性生物反应器中,用原代培养的人平滑肌细胞种植在去细胞血管基质材料内,HE染色及银染检测平滑肌细胞种植效果。结果表明生物酶预处理血管后,HE染色及银染检测可见血管腔平滑肌细胞形态正常,沿血管长轴分布,提示经生物酶预处理的猪血管人平滑肌细胞能成功种植,可望构建实用的组织工程血管。     

骨髓间充质干细胞-聚乙醇酸支架复合体种植动物皮下构建组织工程化小口径血管

背景：课题组的前期工作已证实骨髓间充质干细胞可以诱导分化为血管内皮细胞和血管平滑肌细胞,并证实所诱导的细胞和胶原包埋的聚乙醇酸支架具有良好的组织相容性。 目的：探讨利用动物皮下作为生物反应器构建小口径组织工程化血管的可行性。 方法：骨髓间充质干细胞诱导分化为血管平滑肌样细胞和血管内皮样细胞,分层种植于胶原包埋聚乙醇酸支架表面,然后将细胞-支架复合体种植于动物皮下,构建小口径组织工程化血管。 结果与结论：人工血管组织学观察见管壁结构清晰,其大体结构和天然血管相似,可承受26.6 kPa的血管腔内压力不破裂。皮下培养8周免疫荧光观察Brdu标记的部分细胞核呈现明亮的黄绿色荧光。结果说明利用动物的皮下作为生物反应器,采用静态培养的方式构建小口径组织工程化血管是可行的。     

初级组织工程小口径血管的制备

目的 探讨利用组织工程技术构建小口径组织工程化血管的可行性.方法 将犬骨髓细胞诱导为平滑肌细胞和内皮细胞后,用Brdu标记平滑肌细胞,DAPI标记内皮样细胞,观察细胞标记的成功率;血管平滑肌样细胞和血管内皮样细胞分层种植于胶原包埋PGA的复合支架表面:将细胞和支架的复合体种植于犬皮下,并设立对照组,分别于植入后的第4、6、8周取出植入皮下的组织工程化血管,进行相关检查分析.结果 免疫荧光提示Brdu、DAPI标记种子细胞效果良好;核呈蓝色荧光的内皮样细胞均匀排列在支架表面:组织工程化血管材料植入动物皮下取出后观察,实验组管壁结构比较清晰,细胞数量也已减少、胶原成分增多,管壁分层明显,可见内膜、中膜和外膜结构,对照组无此特点;免疫荧光观察,8周实验组组织工程血管壁内有Brdu标记的种子细胞存在.结论 以动物的皮下为生物反应器,采用静态培养的方式可强化组织工程血管壁,该组织的结构与天然血管相似,种子细胞同时参与初级组织工程小口径血管的构建.     

犬平滑肌样细胞和胶原包埋PGA支架组织的相容性

目的 研究骨髓间充质干细胞所诱导的犬平滑肌样细胞和胶原包埋聚羟基乙酸（PGA）支架的组织相容性。方法 胶原包埋PGA构建复合支架,犬骨髓间充质干细胞诱导血管平滑肌样细胞,评价组织相容性。结果 HE染色见胶原包埋PGA组有平滑肌样细胞生长;甲苯胺蓝染色见平滑肌样细胞被染成浅蓝色,胶原包埋PGA组较单纯PGA组明显增多;电镜观察,胶原包埋PGA组可见到细胞在支架上贴附和生长良好。结论 细胞和胶原包埋PGA的支架组织相容性良好。     

骨髓间充质干细胞构建组织工程化小口径血管

目的采用在动物体外构建初级组织工程化血管、体内强化的方法,探讨构建小口径组织工程化血管的可能性.方法体外培养骨髓间充质干细胞(BMSC),用含全反式维甲酸(AT-RA)、双丁酰环磷酸腺苷(db-cAMP)的DMEM-LG培养液和含血管内皮细胞生长因子(VEGF)的培养液诱导BMSC分别向血管平滑肌样细胞和血管内皮样细胞分化.免疫荧光观察平滑肌样细胞β肌动蛋白的表达和内皮样细胞vWF的表达.电镜观察超微结构的改变.诱导的血管平滑肌样细胞和血管内皮样细胞,分层种植于胶原包埋聚乙醇酸(PGA)的复合支架表面,将细胞和支架复合体种植于动物皮下,于植入后第4、8周再次麻醉动物,取出植入皮下的组织工程化血管,行组织学检查、压力实验及免疫荧光检查.结果诱导14 d后,BMSC能够分化为血管平滑肌样细胞和血管内皮样细胞:β肌动蛋白和vWF呈阳性表达,电镜证实细胞出现了相应的形态学改变.人工血管组织学观察见管壁结构清晰.单纯支架组可承受100～150 mm Hg(1mm Hg=0.133 kPa)的血管腔内压力,实验组则均可承受200mm Hg的血管腔内压力不破裂.实验组皮下培养8周Brdu标记细胞的免疫荧光结果显示部分细胞核呈现明亮的黄绿色荧光.结论以动物皮下为生物反应器可构建出组织工程化血管,其大体结构和天然血管相似.     

细胞凝集态在钙结节形成过程中的显微结构观察

目的 观察兔骨髓基质细胞来源的细胞凝集态在体外钙结节形成过程的形态变化.方法 通过全骨髓培养方法分离培养兔骨髓基质细胞,并在培养瓶中进行扩增,使用成骨诱导液作为培养基;再通过四环素标记、碱性磷酸酶染色、茜素红染色、扫描电镜及透射电镜方法,研究细胞凝集态在钙结节形成过程中的形态变化.结果荧光显微镜下观察可见,四环素标记的...     

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。     

自动测量造影图像中血管中心线和宽度的新方法

在血管造影图像中，精确估计血管中心线和宽度是血管疾病量化与可视化诊断的先决条件。本研究在优化处理血管特征并获取细化的血管骨架之后，提出了一种基于截面梯度轮廓（CSGP）的血管参数（中心线和宽度）测量新算法：该算法采用了统计均值和亮度控制的搜索技术。实验中，利用了多种方法进行理想的和实际的造影图像血管参数测量。通过量化误差比较，CSGP新算法明显好于现有较好的引力中心法：解决了经典方法不提供血管宽度，以及无法测量节点（交叉、分支）处血管中心线的缺陷。     

Higher blood vessel density in comparison to the lymphatic vessels in oral squamous cell carcinoma

Introduction: Oral squamous cell carcinoma (OSCC) is characterized by local invasion and the development of cervical metastasis. In the tongue, an association between the invasion of the lymphatic vessels and the development of metastasis in the regional lymph nodes has been demonstrated. Moreover, invasion of the blood vessels is associated with greater recurrence and poorer prognoses. Therefore, the presence and density of lymphatic and blood vessels in intra- and peritumoral tissues should play an important role in the progression, dissemination and metastasis of carcinomas. However, the evidence regarding OSCC is inconclusive. The aim of this study was to determine the comparison and association between the lymphatic (D2-40) and blood vessel (CD34) densities in intratumoral OSCC tissue. Materials and Methods: Thirty-seven cases diagnosed as OSCC between the years 2000 and 2008 were obtained from the Anatomic Pathology Service of the School of Dentistry, University of Chile. The immunohistochemical markers D2-40 and CD34 were used, and the densities (mm²) of lymphatic vessels (LVD) and blood vessels (BVD) in the intratumoral region were determined. The relationship between LVD and BVD values was evaluated. Results: There were significant association between the CD34 and D2-40 expression (rho=0.4, P<0.05) and between the LVD and the location in the tongue (P=0.019). The BVD was greater (128.0 vessels/mm²) than the LVD (42.9 vessels/mm²), and there was a positive correlation between the LVD and BVD. Conclusions: In OSCC, the BVD is greater than the LVD, and there is a moderate correlation between the two quantities.     

激光热源止血刀闭合血管的可行性研究

目的研发一种具有克服生物组织对激光选择性吸收的激光热源止血刀,通过血管封合在体实验,探讨其闭合猪动脉血管的可行性。方法设计一个激光热源刀头,采用石墨作为激光吸收介质用于激光止血刀刀头,该吸收介质将1470 nm激光能量转化为热能。用该刀头闭合小型猪不同部位不同直径的离体动脉血管,闭合后测量血管的爆破压力作为血管闭合强度的指标。术后,将未进行爆破压实验的血管制成切片,观察闭合处血管的对合情况。结果激光热源止血刀成功闭合了4 mm以下的猪离体血管,小血管(1~2 mm)、中血管(3~4 mm)、大血管(5~6 mm)的平均爆破压分别为(238.83±26.68)mm Hg、(358.83±29.39)mm Hg、(60.40±0.17)mm Hg。显微镜下观察闭合血管的切片,闭合处血管对合好。结论激光热源止血刀封合小血管和中血管有较高的闭合强度和可靠性。     

Histological characteristics of tumor in vessels and lymph nodes are significant predictors of progression of invasive ductal carcinoma of the breast: a prospective study

Invasive ductal carcinomas (IDCs) of the breast are composed of primary invasive tumors as well as tumor cells in blood vessels and lymph nodes. The purpose of this study was to determine whether the histological characteristics of tumor in the vessels and nodes are significantly associated with outcome. In a series of 393 patients, multivariate analyses showed that in IDCs without nodal metastasis and with fibrotic focus dimension, lymph vessel tumor emboli with >6 apoptotic figures and those invading >3 mm from the tumor margin had significantly higher hazard rates (HRs) for recurrence (P<0.05). In IDCs with 1 to 3 nodal metastases, >2 apoptotic figures in tumor emboli in blood vessels and >5 invaded lymph vessels were associated with significantly higher HRs for tumor recurrence and death (P<0.005). In IDCs with 4 or more nodal metastases, nodal tumors with >5 mitotic figures and >5 nodes with extranodal extension were associated with significantly higher HRs for tumor recurrence or death (P<0.05). We conclude that several histological characteristics of tumors in vessels and nodes have significant implications for the progression of IDCs.     

Lymphatic vessel density in pulmonary adenocarcinoma immunohistochemically evaluated with anti-podoplanin or anti-D2-40 antibody is correlated with lymphatic invasion or lymph node metastases

In lung cancers, lymph node metastasis of cancer cells is one of the most important prognostic factors, and lymphatic vessel invasion (LVI) is very important in the stage preceding lymph node metastases. Recently, it has been reported that lymphatic vessel density (LVD) is associated with lymph node metastasis. The aim of the present study was to evaluate the relationship between LVD and LVI based on the immunohistochemical expression of podoplanin or D2-40, which are new specific markers for lymphatic endothelium. Using 76 cases of pulmonary adenocarcinoma, the relationship between LVD and LVI, lymph node metastases, vascular endothelial growth factor C (VEGF-C), VEGF-D or hepatocyte growth factor (HGF) expression was investigated. LVD was significantly associated with LVI, lymph node metastases and VEGF-D expression. LVI was also associated with lymph node metastases, histological subtype, VEGF-C or VEGF-D expression. High LVD, induced by VEGF-C or VEGF-D expression of cancer cells, is a good indicator of lymphatic metastases and LVI in pulmonary adenocarcinoma.     

视网膜血管高血压交叉压迫征的量化研究

研究视网膜血管高血压交叉压迫征的量化方法,采用局部血管图像增强的匹配滤波进行预处理、数学形态学的闭合运算进行图像平滑、线化无级扩大法确定血管边界,实施管径可逆量化自动寻优算法,为获取相关疾病诊断的数据提供一种辅助手段。     

Prognostic significance of the intra-vessel tumor characteristics of invasive ductal carcinoma of the breast: a prospective study

Invasive ductal carcinomas (IDCs) of the breast are composed of stroma-invasive tumors and tumors in vessels. The purpose of this study was to prospectively investigate whether the histological characteristics of the tumors in vessels were more significantly associated with the outcome of 393 IDC patients than well-known histological parameters. Multivariate analyses showed greater than six apoptotic figures in tumor cells in lymph vessels to be significantly associated with increased hazard rates (HRs) of tumor recurrence and death in IDC patients without nodal metastasis (P<0.05). Among IDC patients with nodal metastasis whose tumors were positive for estrogen receptors (ERs) or progesterone receptors (PRs) or both, greater than six apoptotic figures in tumor cells in lymph vessels and greater than four mitotic figures in tumor cells in lymph vessels significantly increased the HR of tumor recurrence and the HR of death, respectively (P<0.05). Among IDC patients with nodal metastases whose tumors were negative for ERs and PRs, multivariate analyses showed that greater than two apoptotic figures in the blood vessel tumor emboli significantly increased the HRs of tumor recurrence and death (P<0.005). We conclude that apoptotic figures and mitotic figures in tumor cells in vessels are very important prognostic indicators for patients with IDC of the breast.     

内皮素在蛛网膜下腔出血诱发脑血管痉挛中的作用

目的：探讨实验动物蛛网膜下腔出血诱发脑血管痉挛时内皮素-1(Endothelin—1，ET—1)基因在大鼠脑底动脉的表达变化及其诱发脑血管痉挛中的作用。方法：56只Wistar大鼠随机分为正常组、蛛网膜下腔出血组(2、3、7、14d)、生理盐水组和假手术组，应用逆转录—聚合酶链反应(RT—PCR)，以B—actin为内参照基因，分别检测各组实验动物脑基底动脉ET—1 mRNA的表达变化。结果：蛛网膜下腔出血组，在出血2d、3d组实验动物脑基底动脉ET—1 mRNA表达较正常组明显增高，出血后7d组仍增高，14d组趋于正常。结论：ET-1参与了蛛网膜下腔出血诱发脑血管痉挛的病理生理过程，提示ET-1在蛛网膜下腔出血诱发脑血管痉挛中起着重要的作用。     

Hyperhomocysteinemia Associates with Small Vessel Disease More Closely Than Large Vessel Disease

Background: Hyperhomocysteinemia was believed to be an independent risk factor for stroke and associate with small vessel disease (SVD) related stroke and large vessel disease (LVD) related stroke differently. However it''s still unclear which type of stroke associated with homocysteine (HCY) more strongly because the conclusions of previous studies were contradictory. In this study we focused on the subclinical angiopathies of stroke, i.e., SVD and LVD instead of stroke subtypes and sought to compare the associations between HCY level and different angiopathies. Methods: 324 non-stroke patients were enrolled. Sex, age, HCY level and other vascular risk factors were collected. MRI and angiographies were used to determine the type of angiopathies and their severity, i.e., the scores of leukoaraiosis (LA), plaques and numbers of silent brain infarctions (SBI). LVD was defined as the presence of atherosclerotic plaques of cerebral arteries. SVD was defined as the presence of either LA or SBI. 230 patients were deemed to have LVD; 180 patients were deemed to have SVD. Spearman''s correlation test and logistic regression were used to analyze the association between HCY level and different angiopathies. Results: The correlation between HCY level and scores of plaques was weaker than that of the scores of LA and numbers of SBI. Hyperhomocysteinemia was an independent risk factor for SVD (OR = 1.315, P <0.001), whereas the association between HCY level and LVD was not that significant (OR = 1.058, P = 0.075). Conclusion: HCY level associated with SVD more strongly than LVD.     

食管壁内淋巴管的研究

应用光镜、电镜观察和研究了新生儿尸体的食管壁肉淋巴管的细微分布及超微结构。结果发现食管粘膜层仅见到毛细淋巴管；在粘膜下层、肌层及外膜层均见到毛细淋巴管及淋巴管。