自体骨髓单核细胞移植对兔肝内缺血型胆道病变的影响

目的 探讨自体骨髓单核细胞移植对血管新生及胆道缺血病变的影响.方法 将30只日本大耳白兔随机分为3组:假手术组(A组)、实验模型组(B组)和骨髓单核细胞移植组(C组),每组10只.3组均游离胆总管、肝总动脉及其间的疏松结缔组织,B组和C组阻断胆总管远端及肝总动脉2 h,C组经肝总动脉注射PKH26标记的自体骨髓单核细胞.术后监测生化指标的变化情况.术后4周开腹行胆道造影.同时,取第一肝门处汇管区肝组织制作石蜡切片,应用免疫组织化学方法观察自体骨髓单核细胞在肝内缺血环境中的分布及分化情况,并检测微血管密度.结果 术后C组生化指标恢复明显优于B组,骨髓单核细胞可分化成血管内皮细胞.术后4周,C组胆道破坏较B组轻,且胆道周围新生毛细血管多于B组.结论 自体骨髓单核细胞移植可以促进局部缺血组织毛细血管的增生,改善局部缺血胆道组织的微循环,从而减轻缺血型胆道病变的程度,甚至预防缺血型胆道病变的发生.     

自体外周血单个核细胞移植治疗实验性大鼠股动脉闭塞症

目的观察自体外周血单个核细胞移植对实验性股动脉闭塞大鼠的疗效.方法选取12只雄性Wistar大鼠,结扎断离双侧股动脉,心脏抽血,经密度梯度离心获得单个核细胞(0.5～2×107/ml),右侧后肢行单个核细胞移植(A组),左侧后肢为注射生理盐水对照(B组).分别在移植当天、移植后14d和28d行双下肢激光多普勒扫描,然后处死动物取双下肢肌肉组织,行HE和免疫组织化学(FⅧ)检查.结果①A组移植后28天与14天相比,血流量明显增加,分别是2.6±0.16和2.34±0.26,P＜0.05;移植后无论14d还是28d,A组较B组下肢血流量均有显著增加移植后28d A组(2.6士0.16),B组(2.12±0.15),P＜0.05;移植后14d A组(2.34±0.26),B组(2.06±0.19),P＜0.05;②A组下肢毛细血管数目显著多于B组,28d A组和B组分别为8.86±1.59与5.46-±-1.06(P＜0.01).结论自体外周血单个核细胞的移植可以显著增加缺血部位血流量和毛细血管的密度,提示单个核细胞在活体缺血环境中有分化为血管内皮细胞的潜能.     

内皮祖细胞移植治疗慢性深静脉血栓形成的实验研究

目的 研究内皮祖细胞(endothelial progenitor cells,EPCs)移植对慢性深静脉血栓的治疗作用. 方法 Ficoll法分离大鼠骨髓单个核细胞(bone marrow-derived mononuclear cells,BMMNCs),采用内皮祖细胞培养基(EGM-2MV)诱导分化为骨髓源性EPCs.建立大鼠慢性深静脉血栓模型,饲养至10 d,并分三组:A组(25只):单纯EPCs组,移植1 ml含有10~6 个EPCs细胞悬液;B组(25只):EGM-2MV培养基对照组,移植1 ml EGM-2MV培养基;C组(25只):空白对照组.移植后第14天取出血栓段下腔静脉及血栓,应用HE染色及vWF免疫组化染色观察血栓机化再通情况,高倍镜下计数血栓内毛细血管数目;Western blot检测血管内皮生长凶子(VEGF)、碱性成纤维细胞生长因子(bFGF)的蛋白表达变化;实验数据采用SPSS13.0软件进行分析.结果 体外成功培养了骨髓源性EPCs和构建了大鼠慢性深静脉血栓模型,EPCs移植后A组VEGF、bFGF的表达有明显的上调(P＜0.05).B组和C组之间差异无统计学意义(P＞0.05).移植后A组血栓再通毛细血管数目明显高于B、C组.免疫组化染色vWF确定再通管道为新牛血管,管道为内皮细胞组成.结论 EPCs为内皮细胞的前体细胞,移植到深静脉血栓中能够促进血管新生,加快血栓的机化和再通.     

骨髓单个核细胞自体移植促进心脏血管再生

目的探讨骨髓单个核细胞自体移植于梗死心肌后实现血管再生的能力。方法结扎45只5月龄日本大耳白兔的冠状动脉左前降支建立心肌梗死模型，随机分为骨髓单个核细胞自体移植组(5-溴-2’-脱氧尿嘧啶核苷标记的自体骨髓单个核细胞直接心肌注射至梗死周边区域)，心肌梗死未治疗组及假手术组，分别在细胞移植后2、4、8周时处死，应用免疫组织化学方法检测骨髓单个核细胞在梗死心肌组织中的分化、毛细血管密度以及细胞因子的表达。结果骨髓单个核细胞在梗死心肌组织中分化为血管内皮细胞和血管平滑肌细胞。在骨髓单个核细胞移植早期，心肌白细胞介素-1β(IL-1β)、碱性纤维母细胞生长因子(bFGF)、血管内皮生长因子(VEGF)表达水平显著增高。细胞移植后4、8周骨髓单个核细胞移植组毛细血管密度明显高于心肌梗死未治疗组及假手术组。结论骨髓单个核细胞自体移植于梗死心肌区能促进血管再生，改善侧支循环。     

自体干细胞移植治疗兔后肢缺血中超顺磁氧化铁纳米颗粒标记磁共振体内示踪的研究

目的探讨自体骨髓间充质干细胞（BMSC）移植治疗兔后肢缺血中超顺磁氧化铁纳米颗粒（SPION）标记磁共振成像（MRI）体内示踪的可行性和有效性。方法新西兰大白兔14只，分成A、B两组，各7只。制作后肢缺血模型。A组移植SPION与5-溴脱氧尿嘧啶（BrdU）共标记的自体BMSC；B组作为对照。术后MRI示踪，组织学检查，免疫组织化学染色。结果鉴定显示BMSCCD34和CIM5阴性，CD44阳性。B组中1只死亡。A组毛细血管密度198．9±2．3，B组80．5±5．1．A组毛细血管／肌纤维比值0．84±0．03，B组0．35±0．04，两组差异均有统计学意义（P〈0．01）。A组：电镜发现血管内皮细胞胞质内含SPION；对应部位组织切片血管壁细胞普鲁士兰铁染色、BrdU和CD34染色均阳性；MRI显示术后即时A组注射部位圆形低信号影，术后1—3周向周围扩散，部位与组织学检查结果一致。B组仅能见到普鲁士兰阳性颗粒游离于组织间隙，并非位于血管内皮细胞；MRI低信号影局限于注射局部，无扩散。结论自体BMSC移植治疗兔后肢缺血中SPION标记MRJ体内示踪可行、有效。     

组织工程支架负载骨髓单核细胞促进大鼠下肢缺血组织血管新生

目的 通过对干细胞移植技术的改进,提高干细胞移植效果.方法 采用8周龄雌性SD大鼠48只,结扎左侧股动脉制成下肢缺血模型,随机分为4组,在左下肢内收肌群内,A组行雄鼠骨髓单个核细胞和组织工程支架联合移植;B组行雄鼠骨髓单个核细胞移植;C组行组织工程支架移植;D组行生理盐水注射;2周后测定大鼠下肢活动能力、下肢血管造影并用网格纸计数小血管密度、病理检查计数小血管数目、聚合酶链反应(PCR)检测Y染色体SRY基因评估移植效果.结果 各组活动定向距离分别为(131.92±6.07)、(117.58±3.48)、(63.50±4.52)、(63.17±4.32)cm;血管造影单位面积内血管计数分别为(21.17±2.82)、(18.50±2.80)、(13.58±2.54)、(12.50±4.33)/cm2;病理切片小血管计数分别为(18.50±2.24)、(15.33±2.35)、(5.83±2.17)、(6.54±2.06)/HP,免疫组织化学切片上小血管计数为:(18.67±2.54)、(15.67±2.35)、(6.17±1.64)、(6.67±2.02)/HP.A组的活动距离、血管造影小格计数、病理切片小血管计数、SRY基因量均较其余各组差异有统计学意义(P＜0.05).结论 组织工程支架负载骨髓单个核细胞构建新生血管优于单纯干细胞移植.     

血管基质片段细胞携血管内皮生长因子对移植颗粒脂肪血管化的影响

目的 探讨血管内皮生长因子(vascular endothelial growth factor,VEGF)复合血管基质片段细胞( stromal vascular fraction cells,SVFs)细胞疗法对移植颗粒脂肪组织血管化的影响,寻找一种安全、有效、简单的细胞辅助脂肪移植术式.方法 取患者植皮术后废弃的皮下脂肪,提取SVFs.用DiI染色观察SVFs染色标记情况,锥虫蓝染色观察SVFs活性.将0.3 ml待移植的脂肪颗粒分别与0.2 ml下列细胞混合:①复合有VEGF的SVFs(A组);②SVFs(B组);③DMEM完全培养基(C组).按照随机化原则将3组脂肪组织混合物分别注射移植于裸鼠背部皮下,每只裸鼠背上注射3个点(共12只).观察移植物外形、成活情况、湿重、直径,HE染色及CD31染色镜下观察并计数毛细血管密度.使用SPSS 16.0进行方差分析检验.结果 新鲜提取的SVFs可被DiI标记染色,加用VEGF仍能保持较好的细胞活性,移植术后2个月3组移植脂肪的湿重为:A组(191.90 ±9.81)mg、B组( 177.01±10.50)mg、C组(92.05±8.30)mg,A组＞B组＞C组(P＜0.01).移植物直径为:A组(0.49±0.24)cm、B组(0.40±0.26) cm、C组(0.32±0.28) cm,A组＞B组＞C组(P＜0.01).CD31染色镜下,每高倍镜视野微血管密度:A组(14.58±2.06)个、B组(11.55±2.18)个、C组(7.87±1.55)个,与B组和C组比较,A组较少见纤维结缔组织增生及坏死凋亡细胞.结论 结合VEGF因子的SVFs辅助脂肪移植是一种临床可操作性强、简便可行的较理想的细胞疗法,比单纯SVFs更能有效地促进移植脂肪血管化.     

PDGF-B基因表达在组织工程化皮肤移植后血管重建中的作用

目的构建含有血小板衍化生长因子B(PDGFB)基因的组织工程化皮肤,进行动物移植实验,研究PDGFB基因表达在真皮血管重建中的作用。方法构建PDGFB真核表达质粒,用脂质体LipofectAMINE介导转染成纤维细胞。分别构建3种不同类型的组织工程化皮肤角质形成细胞 脱细胞猪真皮基质(A组),角质形成细胞 脱细胞猪真皮基质 成纤维细胞(B组),角质形成细胞 脱细胞猪真皮基质 PDGFB基因转染的成纤维细胞(C组),分别移植于大鼠背部创面,观察术后2、4、6周真皮血管重建情况。结果术后2周C组真皮浅层内可见较多新生毛细血管长入,B组次之,A组毛细血管长入较少(P<005);术后4周各组真皮浅层内毛细血管数逐渐增加,但C组毛细血管数仍明显高于B、A两组(P<005);术后6周各组织工程化皮肤内毛细血管数差异无显著性意义。结论PDGFB基因在组织工程化皮肤移植后早期真皮血管重建中发挥了重要的作用,为移植后皮片成活提供了保障。     

自体静脉段移植修复胆管缺损的实验研究

目的 探讨胆管缺损的修复材料和方法.方法 本地白山羊40 只随机分成4 组,每组10 只,A 组、B 组、C 组分别整段切除2、3、4 cm 的胆总管,缺损通过移植相应长度内置硅胶管的自体大隐静脉进行修复,移植物周围以带蒂大网膜包裹,同时切除胆囊;D 组为对照组,仅行胆囊切除及胆管周围松动.观察各组动物的活动情况、黄疸及排便情况,分析意外死亡的原因,分别记录术前、术后1、3、6 个月血生化变化,在术后6 个月处死动物观察移植血管的病理改变.结果 A组、B 组、D 组三组动物各组内部术前及术后1、3、6 个月各时间点血生化检查结果,各组之间术前及术后1、3、6 个月各时间点血生化检测无显著性差异;A 组、B 组、C 组所移植血管存活情况有差异.A 组与B 组、C 组比较差异有统计学意义(P&lt;0.001).结论 山羊采用自体大隐静脉段移植加内支架并带蒂大网膜包裹来修复胆管短段(&lt;3 cm)缺损是可行的,进一步的实验研究对指导临床具有重要意义.     

干细胞移植治疗大鼠后肢缺血中HIV-Tat-CLIO标记磁共振体内示踪的研究

目的 探讨人类免疫缺陷病毒tat蛋白转导域交连超顺磁氧化铁纳米颗粒(HIV-Tat-CLIO)标记、磁共振成像(MRI)无创体内示踪移植大鼠骨髓间充质干细胞(BMSC)的可行性和有效性.方法 SD人鼠28只,分为4组,每组7只,建立右后肢缺亦模型.A组移植HIV-Tat-CLIO标记的BMSC;B组移植HIV-Tat-CLIO与BrdU复合标记的BMSC;C组移植未标记的BMSC;D组注射无菌磷酸盐缓冲液(PBS).术后MRI示踪移植细胞,评估缺血改善程度,以及组织学检查.结果 B、C组分别死亡2只和1只.A、B组MRI结果相似:术后第3天,注射部位圆形低信号影,1周低信号灶离心性扩散成颗粒状,第2周低密度影进一步扩散,信号强度减弱,第4周信号强度进一步减弱,低密度颗粒扩散范围局限于原部位7 mm处.C、D组MRI阴性.缺血后肢平均皮肤温度、血管造影评分以及毛细血管密度A、B、C组间差异无统计学意义(P>0.05),均显著高于D组(P<0.05).D组的损伤评分明显高于其他3组,其他3组间差异无统计学意义.组织学检查与MRI结果一致.结论 HIV-Tat-CLIO标记、MRI可以有效地体内示踪植入大鼠缺血后肢的BMSC.对BM-SC改善缺血的效果无明显影响.     

Effects of autologous bone marrow mononuclear cells implantation on cholangiocytes apoptosis in model of intrahepatic ischemic type biliary lesion in rabbits

Aim

To investigate the effects of autologous bone marrow mononuclear cells (BM-MNCs) implantation on regulation of cholangiocyte apoptosis in a model of intrahepatic ischemic type biliary lesion (ITBL) in rabbits.

Materials and Methods

Thirty Japanese white rabbits were divided randomly into 3 groups (10 per group) including controls (group A), ITBL model (group B), and BM-MNCs implantation groups (group C). All rabbits underwent the same surgical procedure to prepare the liver for graft removal during transplantation. Subsequently, no additional vascular intervention was performed in group A. In group B, the hepatic artery and common bile duct were clamped with microvascular clips for 2 hours, where after the clips were removed to recover the blood supply. Group C received, BM-MNCs (10⁸ cells per rabbit) injected through the hepatic artery after removing the clips. The animals were killed 4 weeks after operation. The survival rate, histopathologic examination, cholangiocyte apoptosis with terminal uridine nick-end labeling (TUNEL) staining and expressions of Bcl-2 and Bax proteins were examined using immunohistochemical staining.

Results

Group A animals showed a survival of 100%; the rates in groups B and C were both 90%. Histopathologic examination revealed normal intrahepatic cholangiocytes in group A, obviously damaged ones in group B, and alleviated damage in group C. TUNEL staining indicated apoptosis of cholangiocytes in group B was more serious than that in group A or group C. Immunohistochemical staining demonstrated significantly decreased Bcl-2 expression in group B compared with that in group A; Bcl-2 expression in group C returned to the level of group A. Simultaneously, the Bax expression presented adverse results; the ratios of Bcl-2/Bax were ranked as group A > group C > group B.

Conclusion

Implantation of autologous BM-MNCs significantly reduced apoptosis of intrahepatic cholangiocytes and prevented or abated intrahepatic ITBL.     

Establishment of an animal model of biliary ischemic stenosis with clamping in mice

OBJECTIVE: The objective of this study was to explore a method to establish biliary ischemic stenosis in mice. METHODS: After the optimal time of biliary ischemia was determined, 20 Kunming mice were equally divided into 2 groups. In the experimental group a 0.4-cm length of common bile duct was clamped for 90 minutes with 2 micro-vessel clamps (width = 0.1 cm). The common bile duct was not clamped in the control group. Twenty-one days later, biliary tract visualization was performed in all mice. Blood samples were collected from the inferior vena cava to determine the serum levels of total bilirubin (TBIL) and alanine aminotransferase (ALT). Meanwhile, samples of the common bile duct and liver tissue were extracted for microscopic examination to observe morphological changes. RESULTS: In the experimental group, obvious dilatation of the common bile duct appeared over the clamp site. There was no dilatation of the common bile duct in the control group. Twenty-one days later, serum levels of TBIL and ALT were significantly higher among the experimental compared with the control group. Microscopic examination showed that the part of common bile duct at the clamp site was significantly expanded, with a smaller or occluded bile duct lumen necrotic mucosa with determination, and tubular wall with fibrosis and excrustation. A few dead liver cells and many inflammatory cells were observed in liver tissue samples. CONCLUSIONS: A biliary ischemic stenosis model was established using a clamping method in mice, which may provide a reliable technique for basic and clinical research into mechanisms of biliary ischemic stenosis after liver transplantation.     

钳夹法建立小鼠胆管缺血狭窄模型

目的 探讨建立小鼠胆管缺血狭窄模型的方法。方法 先探索昆明小白鼠胆管缺血最适时间点，尔后选取昆明小白鼠随机分为正常对照组和模型组，每组10只，模型组用2枚显微血管夹（宽0．1cm）夹闭一段长约0．4cm的胆总管90min，正常对照组仅暴露胆管未夹闭。术后21d两组小鼠均行胆道造影，下腔静脉取血，分离血清进行总胆红素及谷丙转氨酶含量测定，取胆总管和肝组织石蜡包埋，HE染色，观察病理改变。结果 模型组钳夹部位以上胆管明显扩张，胆管造影示胆管扩张，壁光滑，狭窄部位以下胆管未显影；正常对照组肝内胆管未显影，肝外胆管显影。血清总胆红素模型组高于正常对照组5倍以上，谷丙转氨酶模型组显著高于正常对照组6倍以上。病理示缺血胆管管腔变小或闭塞，胆管黏膜坏死脱落，管壁纤维化增厚；病变部位以上胆管管腔增大，管壁变薄。肝脏可见少量肝细胞坏死，炎性细胞浸润。结论 血管夹钳闭缺血法成功地建立了小鼠胆管缺血狭窄模型，为肝移植术后胆管缺血狭窄并发症发生机制的实验研究和临床研究提供可靠的实验方法。     

脂肪基质血管成分移植促进随意皮瓣成活的实验研究

目的探讨脂肪基质血管成分(Stromal vascular fraction,SVF)移植是否可以促进随意皮瓣成活,及其作用的相关机制。方法分离4周龄Wistar大鼠脂肪中的SVF及骨髓中的单个核细胞(BM-MNCs),RT-PCR检测VEGF和bFGF在两种细胞中的表达。根据移植细胞的不同,将24只Wistar大鼠分成3组,分别为对照组、BM-MNCs组和SVF组。在大鼠背部设计一个10 cm×3 cm大小的矩形皮瓣,分别将含有5×107个SVF及BM-MNCs的混悬液各1 mL均匀注射至皮下组织层,对照组单纯注射1 mL PBS。2 d后,皮瓣掀开原位缝合,术后7 d统计皮瓣的成活率。取各组皮瓣相同部位的组织,实时定量PCR检测组织中VEGF和bFGF基因的表达。结果 SVF和BM-MNCs细胞中VEGF和bFGF的表达无明显差别(P>0.05)。皮瓣原位缝合后7 d,SVF组和BM-MNCs组皮瓣的成活率分别为(72.2±2.0)%和(76.4±3.1)%,均明显高于对照组的(56.8±4.6)%(P<0.05)。实时定量PCR检测发现SVF组和BM-MNCs组皮瓣组织中VEGF和bFGF基因的表达明显升高。结论 SVF移植入皮瓣后可以通过旁分泌生长因子如VEGF和bFGF等增加皮瓣的成活面积。     

脱细胞基质管腔人工仿真胆管的体内重建：动物实验初步报告

目的探讨脱细胞管腔基质人工仿真胆管支架材料（人工仿真胆管）修复胆管缺损的动物体内实验疗效。方法本实验采用前期制备的人工仿真胆管,应用于封闭群内江猪（18只）胆管缺损动物实验模型,探讨其体内修复效果,并根据术中是否安置支架分为支架组、无支架组,另设假手术组为对照每组各6只。术后不同时期检测相关指标,探讨人工胆管修复胆管缺损的实验疗效。结果体内实验发现,支架组、假手术组动物无死亡,无支架组实验动物分别植入术后第2、7、8和40天全部死亡。支架组大体标本未见明显胆漏,吻合口无明显狭窄,植入段胆管未见明显狭窄及管腔塌陷;无支架组大体标本可见植入段胆管狭窄,呈急性梗阻性化脓性胆管炎表现。实验室检查显示：支架组、假手术组胆管植入术后血清总胆红素（TB）无明显升高,无支架组动物在术后不同时间均有不同程度的血清TB升高,最高达到413．2μmol／L。组织学观察显示：植入术后第100天,形成较为完整的类似新生胆管样结构,整个胆管腔基本被类似正常胆管上皮的组织覆盖,无支架组标本可见吻合口狭窄明显,人工胆管挛缩、纤维化、管腔阻塞,局部组织结构混乱,炎性细胞浸润;免疫荧光染色示,在植入术后第100天支架组人工仿真胆管植入段的细胞角蛋白19（CK19）和上皮细胞黏附分子（HEA125）表达与植入段吻合口的上、下端正常胆管黏膜上皮的CK19和HE125表达相接近。结论人工仿真胆管的研发可能会为临床胆管缺损性疾病的治疗提供潜在的治疗途径。     

Intra-arterial bone marrow cell transplantation induces angiogenesis in rat hindlimb ischemia

BACKGROUND: Bone marrow (BM) cells have been shown to augment local angiogenesis by differentiating vessels themselves and/or secreting paracrinally angiogenic growth factors. Herein, the angiogenic effects of intra-arterial BM mononuclear cell (BM-MNC) transplantation were evaluated in a rat ischemic hindlimb model. METHODS: Unilateral hindlimb ischemia was created by excising the femoral artery and its branch in Lewis rats. BM-MNCs were isolated by centrifugation through a Histopaque density gradient. One week after excision of the unilateral femoral artery, BM-MNCs (5 x 10(6) cells, Group A, n = 6) or PBS (Group B, n = 7) were injected into the ischemic thigh skeletal muscles at the six points with a gauge needle. Another injection of BM-MNCs (3 x 10(7) cells, Group C, n = 6) or PBS (Group D, n = 7) was administered via the indwelling catheter in the right common iliac artery. RESULTS: Four weeks after the BM-MNC transplantation, angiographic examination revealed the development of collateral vessels in both BM-MNC-transplanted groups. The difference in skin temperature between right and left hindlimbs was significantly reduced in both BM-MNC-transplanted groups (0.93 +/- 0.15 vs. 2.84 +/- 0.35 vs. 1.20 +/- 0.26 vs. 2.61 +/- 0.37 degrees C, Group A vs. Group B vs. Group C vs. Group D, p < 0.05). Moreover, immunohistochemical analysis demonstrated that capillary endothelial cells were increased in both BM-MNC-transplanted groups. CONCLUSION: BM-MNC implantation was able to induce functional neovascularization in rat ischemic hindlimb. The intra-arterial administration offered similar levels of angiogenic activity as intramuscular injection.     

TGF-β1及CTGF在胆管缺血性损伤修复过程中的表达及意义

目的 观察TGF-β1 及CTGF 在胆管损伤修复过程中的表达,探讨良性胆管狭窄形成机制.方法 建立大鼠胆管缺血损伤模型,观察术后胆总管及肝脏组织病理改变;免疫组化检测两组胆管组织TGF-β1 及CTGF的表达,计算阳性细胞数.结果 实验组夹闭处胆管管腔狭窄伴纤维组织增生,钳夹部位以上胆管扩张;肝脏炎性细胞浸润.对照组...     

Therapeutic angiogenesis of bone marrow mononuclear cells (MNCs) and peripheral blood MNCs: transplantation for ischemic hindlimb

We investigated bone marrow mononuclear cells (BM-MNCs) and peripheral blood mononuclear cells (PB-MNCs) for therapeutic angiogenesis in the ischemic hindlimb. BM-MNCs were isolated and injected into ischemic skeletal muscles in mice. Laser Doppler and histological evaluation were performed after the surgical procedure. Fifteen patients suffering from critical lower limb ischemia received subcutaneous injections of recombinant human granulocyte colony-stimulating factor (G-CSF) to mobilize progenitor cells, and PB-MNCs were harvested and transplanted directly into the ischemic limb. Endothelial cells derived from BM-MNCs were plated, then induced to form three-dimensional networks by invading a Matrigel. Four weeks after implantation of BM-MNCs, laser Doppler analysis showed that the blood flow ratio was significantly increased (0.67 +/- 0.02 vs. 0.44 +/- 0.02). Alkaline phosphatase and immunohistochemical analyses showed that capillary density was significantly increased (95.25 +/- 0.07% vs. 39.6 +/- 0.04%). Two months after implantation of PB-MNCs, in both subgroups, ankle-brachial index values, walking distance, pain scale, and transcutaneous oxygen pressure (TcO(2)) were significantly improved (p < 0.005). A total of six of 15 limb ulcers of transplanted patients were healed after cell transplantation. BM-MNC implantation was able to induce functional angiogenesis in mice ischemic hindlimb. This clinical trial shows that G-CSF-based PB-MNC transplantation is a feasible treatment for the ischemic hindlimb.     

普通超声及超声造影检测兔胆道缺血模型胆道异常的可行性研究

目的探讨普通超声及超声造影应用于检测兔胆道缺血模型胆道异常的可行性。方法将20只健康新西兰兔随机分为两组,对照组及肝动脉和胆总管联合阻断2h(HBO2h)组(各10只)。HBO2h组用无损伤动脉夹阻断肝动脉及胆总管下段2h后去除动脉夹制作胆道缺血模型,对照组不进行任何手术处理。术后5d两组实验兔先后采用普通超声、超声造影观察胆总管-左叶胆管情况,然后处死动物取胆总管-左叶胆管标本行苏木素-伊红(HE)染色,对胆管组织缺血损伤程度进行评分,CD34免疫组织化学染色后行微血管密度(microvascular density,MVD)定量分析。结果普通超声表现:对照组门静脉管径/胆管管径比值为3.47±0.23,所有胆管(100%)均表现为管壁连续性好、无增厚,管腔清晰;HBO2h组胆管明显扩张,门静脉管径/胆管管径比值为1.25±0.42,与正常组比较差异有统计学意义(P<0.01);所有胆管均表现为管壁毛糙、增厚。超声造影表现:对照组所有胆管壁(100%)较肝实质提早增强,动脉期胆管壁呈细线状高增强,门静脉期及延迟期呈等增强;HBO2h组中60%(6/10)表现为动脉期胆管壁呈增强水平明显降低,门静脉期及延迟期呈低增强;40%(4/10)表现为增强扫描全过程均呈无增强;与对照组相比,HBO2h组各时相胆管壁增强表现差异均有统计学意义(均为P<0.05)。病理学表现:对照组胆管黏膜上皮细胞和黏膜下腺体上皮细胞呈柱状,排列紧密、完整,无上皮细胞变性、坏死、脱落等,胆管损伤评分为0分,MVD为(6.1±2.5)个。HBO2h组胆管壁增厚,胆管黏膜上皮细胞及黏膜下腺体上皮变性、坏死、脱落,管壁炎症细胞浸润,胆管损伤评分为(2.1±0.6)分,MVD为(2.2±1.6)个,与对照组比较差异有统计学意义(均为P<0.01)。结论普通超声及超声造影能较早发现胆道缺血兔模型的胆道异常,为缺血型胆道病变动物实验研究提供了一种简便、无创、动态监测病程发展的有效手段。