不同途径移植骨髓干细胞治疗急性肝损伤小鼠模型的效果比较

目的探讨4种途径移植骨髓干细胞治疗的急性肝损伤小鼠的肝脏迁移情况及肝损伤的修复情况。方法雄性BALB/c小鼠分为A、B、C、D、E、F 6组,每组10只,A、B、C、D为移植组,E组为骨髓干细胞供体组,F组为急性肝损伤模型组。用CCL4/2-乙酰氨基芴制备小鼠急性肝损伤模型,分离小鼠骨髓干细胞,用红色荧光染料PKH26标记后经门静脉(A组,n=10)、尾静脉(B组,n=10)、腹腔(C组,n=10)及脾内(D组,n=10)输入到急性肝损伤小鼠体内,2周后处死小鼠,血清检测肝功能(ALT、AST、Alb),肝组织病理观察骨髓干细胞向肝脏迁移的情况及肝损伤小鼠的肝脏修复情况。F组小鼠于第8天处死检测ALT、AST及Alb值。计量资料2组间比较采用t检验,多组间比较采用单因素方差分析。结果显微镜下4个移植组移植的细胞均迁移到肝脏且通过病理图片均可见新生的肝细胞; ALT、AST、Alb值A、B、C、D 4组分别与F组比较差异均有统计学意义(ALT:t值分别为2. 372、2. 473、2. 354、2. 383,P值均0. 05; AST:t值分别为2. 534、2. 423、2. 437、2. 643,P值均0. 05; Alb:t值分别为2. 336、2. 243、2. 373、2. 352,P值均0. 05)。结论骨髓干细胞促进急性肝损伤小鼠肝脏的修复,其修复程度与移植途径无关。     

PKH26荧光示踪剂在小鼠骨髓单个核细胞肝内迁移过程中标记作用的研究

目的 探讨PKH26荧光示踪剂在小鼠骨髓单个核细胞肝内迁移过程中的标记作用。方法以红色荧光染料PKH26标记从小鼠骨髓中分离出的骨髓单个核细胞，从小鼠的尾静脉注入CCIA—AAF造成肝损伤的同种异体的小鼠体内，移植2周后取肝组织，通过荧光显微镜观察实验组小鼠骨髓干细胞向肝脏迁移的情况。结果受体组小鼠的肝小叶中央静脉及汇管区均可见新生的PKH26标记阳性的肝细胞。结论PKH26可用于标记向急性肝损伤小鼠肝脏迁移的骨髓单个核细胞。     

自体骨髓单个核细胞移植对急性肝损伤小鼠组织成瘤性的实验研究

目的观察急性肝损伤小鼠接受自体骨髓单个核细胞(BMMNC)移植后能否导致肿瘤的发生,从而对自体骨髓干细胞(BMSC)移植的安全性进行初步探讨。方法采用CCl4/2-AAF制备小鼠急性肝损伤模型,随机分为6组,A组:BMMNC移植对照组;B组:腹部皮下注射BMMNC0.3ml;C组:经尾静脉注射BMMNC0.3ml;D组:经尾静脉注射BMMNC0.9ml;E组:模型检测组;F组:供体组。BMMNC采用PKH26标记。病理观察小鼠接受BMMNC移植后皮下及肝脏内有无肿瘤形成。结果 C组及D组肝脏内未见肿瘤形成;B组皮下形成一肿物,病理证实为脂肪及淋巴细胞的炎性坏死所形成的包裹。结论理论上自体BMSC移植虽有形成肿瘤的可能性,但在本实验中未见肿瘤形成,自体BMSC移植能否诱发肿瘤还有待进一步探讨。     

分泌型与包涵体型G-CSF促进骨髓单个核细胞向肝脏迁移比较

目的 比较分泌型与包涵体型G—CSF促进骨髓单个核细胞向急性肝损伤小鼠肝脏迁移的作用。方法 以红色荧光染料PKH26标记从小鼠骨髓中分离出骨髓单个核细胞，从小鼠的尾静脉注入同种异体的CCl4-AAF造成肝损伤的小鼠体内，移植2周后取肝组织，通过荧光显微镜观察实验组及对照组小鼠骨髓干细胞向肝脏迁移的情况。结果 两组鼠的肝小叶中央静脉及汇管区均可见新生的肝细胞，PKH26标记阳性的细胞在20倍镜下实验组每张切片平均为（75．76±70．00）个，对照组平均（79．84±80．98）个（P〉0．05）。结论 分泌型与包涵体型G-CSF在促进骨髓单个核细胞向急性肝损伤小鼠的肝脏迁移作用无明显差异。     

粒细胞集落刺激因子促进自体骨髓单个核细胞向急性肝损伤小鼠肝脏迁移的实验研究

目的 探索粒细胞集落刺激因子促进自体骨髓单个核细胞向急性肝损伤小鼠肝脏迁移的作用.方法 以红色荧光染料PKH26标记从小鼠骨髓中分离出的骨髓单个核细胞,从小鼠的尾静脉注入同种异体的CCL4-AAF造成肝损伤的小鼠体内,移植2周后取肝组织.通过荧光显微镜观察实验组及对照组小鼠骨髓干细胞向肝脏迁移的情况.结果 两组小鼠的肝小叶中央静脉及汇管区均可见新牛的肝细胞,PKH26标记阳性的细胞在实验组20倍镜下每张切片平均为(102.76±37.304)个,在对照组平均(53.84±29.987)个(P＜0.05).结论 重组粒细胞集落刺激凶子可以促进骨髓单个核细胞向急性肝损伤小鼠的肝脏迁移.     

小鼠急性肝损伤后自体骨髓单个核细胞移植对宿主凝血机制的影响

目的观察小鼠自体骨髓单个核细胞移植后是否形成肝内及肝外血栓,以及宿主凝血机制有无其他异常表现。方法应用CCl4/2-AAF制备小鼠急性肝损伤模型,然后行骨髓单个核细胞移植。实验按注射骨髓单个核细胞的数量随机分为3组：A组为移植对照组;B组为经尾静脉注射骨髓单个核细胞0.3 mL;C组为经尾静脉注射骨髓单个核细胞0.9 mL;分别于移植后1周、2周处死小鼠,取其血清检测D-二聚体值,取肝、肺、心、脑、肾组织观察小鼠骨髓单个核细胞向这些器官迁移情况及有无血栓形成。结果 PKH26标记阳性细胞数：脑部未发现荧光;同一时间点,C组与其他组比较,同一实验组移植后1周与2周相比差异均有统计学意义（P〈0.05）。各器官血栓形成情况：HE染色观察发现,移植后1周及2周肝脏、心脏、肺脏及肾脏均有血栓形成,第2周血栓较第1周明显;脑部未发现血栓。D-二聚体检测：乳胶凝集法检测血清D-二聚体值,各时间点各组间均未发现差异。结论 BALB/C小鼠尾静脉注射BMMNC 0.3 mL及0.9 mL均可致多器官血栓形成。BALB/C小鼠尾静脉注入BMMNC剂量越大形成血栓的机会越大。乳胶凝集法测定D-二聚体值对检测BALB/C小鼠尾静脉移植BMMNC后是否有血栓形成没有帮助。     

自体骨髓单个核细胞移植对肝硬化患者血清白蛋白的影响

目的探讨经肝动脉插管自体骨髓单个核细胞(BM-MNCs)移植对肝硬化患者血清白蛋白(ALB)的影响。方法 201例肝硬化患者中,131例进行自体骨髓单个核细胞移植,70例为对照。骨穿采集自体骨髓,分离纯化单个核细胞将其经肝动脉插管移植入肝脏。分别于移植后4、8、12及24周观察ALB变化情况,比较不同Child-Pugh分级肝硬化患者ALB的改变。结果移植组患者移植前血清ALB平均为(29.33±3.92)g/L,移植后第4、8、12、24周分别为(32.37±4.63)g/L、(32.82±4.84)g/L、(32.95±5.10)g/L和(32.22±5.87)g/L。移植后24周内血清ALB水平较移植前显著升高,而对照组则无明显变化,两组比较差异有统计学意义。移植组中,Child-PughA、B级患者血清ALB改善程度大于C级患者。移植后无严重不良事件发生。结论自体骨髓单个核细胞移植能提高肝硬化患者血清ALB水平,对Child-PughA、B级患者改善明显。     

基质细胞衍生因子1对骨髓单个核细胞向肝脏迁移分化的影响

目的 探索基质细胞衍生因子1对骨髓单个核细胞向肝脏迁移、分化的影响。方法建立小鼠CCl4-AAF肝损伤模型，从小鼠骨髓中分离出骨髓单个核细胞，以荧光染料PKH26标记后经尾静脉输入肝损伤模型的小鼠体内，实验组立即给予肝内注射基质细胞衍生因子1，对照组给予肝内注射盐水，12d后取肝组织，在荧光显微镜下观察两组骨髓单个核细胞向肝脏迁移的差异，并用免疫组化法测定移植细胞的白蛋白表达。结果 PKH26标记阳性的细胞20倍镜下实验组中每张切片平均迁移数为（195．40±9．095）个，对照组平均迁移数为（169．80±7.983）个（P〈0．05）。免疫组化显示移植细胞可以表达白蛋白。结论 基质细胞衍生因子1可以促进骨髓单个核细胞向肝脏迁移，并且迁移至肝脏的骨髓单个核细胞可以向肝细胞分化。     

基质细胞衍生因子-1对小鼠急性肝损伤修复的实验研究

目的探索基质细胞衍生因子对急性肝损伤修复的影响,并与单纯骨髓单个核细胞移植治疗肝损伤疗效相比较。方法建立小鼠CCl4-AAF肝损伤模型,从小鼠骨髓分离骨髓单个核细胞。实验A组经腹腔向模型小鼠体内注入基质细胞衍生因子,实验B组经尾静脉向模型小鼠体内注入单个核细胞,对照C组经尾静脉向模型小鼠体内注入生理盐水,在第2周、3周、4周,从各组取出相同的小鼠处死,通过测肝功能指标（AST、ALT、TBil）和肝组织病理切片,比较各组小鼠肝损伤修复的差异。结果分别在第2周、3周、4周测得的SDF-1组（A组）和单个核细胞组（B组）的数值比较（U/L）：ALT（第2周：19.0±2.0 vs 19.7±4.7,P〉0.05;第3周：19.0±5.0 vs 14.5±2.5,P〉0.05;第4周：40.0±17.0 vs 15.0±3.0,P〉0.05）;AST（第2周：117.1±18.0 vs 116.7±20.0,P〉0.05;第3周：97.5±5.0 vs 104.5±23.5,P〉0.05;第4周：177.3±61.0 vs 105.0±49.1,P〉0.05）;TBil（第2周：1.8±0.1 vs 1.9±0.3,P〉0.05;第3周：1.75±0.55 vs 1.5±0.4,P〉0.05;第4周：1.8±0.6 vs 1.5±0.3,P〉0.05）。在病理方面第2周、4周时A组及B组肝细胞仍有肿胀,但组织结构较急性损伤时有明显改善。结论基质细胞衍生因子和单个核细胞都能够促进肝损伤的修复,在病理方面骨髓单个核细胞对损伤修复效果更明显一些。     

低分子肝素预防自体骨髓干细胞移植血栓形成的动物实验研究

目的探讨低分子肝素预防小鼠自体骨髓单个核细胞移植后血栓形成的作用。方法应用CC14／2-AAF制备小鼠急性肝损伤模型,然后行骨髓单个核细胞移植。实验A组于经尾静脉注入骨髓干细胞悬液;实验B组于经尾静脉先注入低分子肝素后注入骨髓干细胞悬液。分别于移植2周后处死小鼠,取2组小鼠心、肺、肝、肾、脑等重要脏器于显微镜下观察有无血栓。结果HE染色观察发现：A组肝脏、心脏、肺脏、肾脏均有血栓形成（脑未发现血栓）;B组肝脏、心脏、肺脏、肾脏及脑均无血栓形成。结论低分子肝素可预防BALB／c小鼠尾静脉移植BMMCs后导致的血栓形成。     

Combined deficiencies of factor vlll (ahf) and factor xi (pta)

Combined deficiencies of Factor VIII and Factor XI associated with moderate degree of bleeding symptoms were found in 3 brothers. Examination of Factor VIII activity and Factor VIII-related antigen revealed that the Factor VIII activity/ Factor VIII-related antigen ratio was significantly decreased in their mother and maternal grandmother consistent with the carrier state of hemophilia. Factor XI deficiency was found in 2 siblings, the father, and 2 of his sisters. The paternal grandmother was thought to carry the abnormal Factor XI gene, although her Factor XI level was normal, because of a significant bleeding history. It was concluded that the combined Factor VIII and XI deficiencies in the 3 brothers represent the coincidental inheritance of 2 separate and independent abnormal genes.     

Autoimmune antibody (IgG Kansas) against the fibrin stabilizing factor (factor XIII) system.

Serum from a patient who died from massive hemorrhage within 4 months after onset of an acquired bleeding disorder at age 85 contained a potent inhibitor of fibrin stabilization. Other parameters of coagulation and fibrinolysis and his bleeding time were within normal limits. The inhibitor was shown to be an IgG with kappa light chains (IgG Kansas); its specific target was the factor XIII system itself. Although IgG Kansas combined with the virgin [ab] form of the zymogen, it did not block the thrombin-catalyzed conversion to [a'b]. However, IgG Kansas prevented the subsequent Ca2+-mediated activation of [a'b] to a + b, where a denotes the catalytically competent factor XIIIa species. IgG Kansas, in contrast to a previously studied autoimmune antibody from a similar bleeding disorder (IgG Warsaw), could also inhibit the transamidating activity of the preactivated a enzyme.     

Studies on the inhibition of ellagic acid-activated Hageman factor (factor XII) and Hageman factor fragments

Hageman factor (HF, factor XII) that has been exposed to Sephadex- ellagic acid gels is a single-chain species (HFea) with amidolytic properties for the synthetic substrate H-D-phenylalanyl-L-pipecolyl-L- arginine p-nitroanilide. Earlier we reported that amidolysis was suppressed by incubation of HFea with specific antiserum. The present study provides additional evidence that the amidolytic properties of preparations of HFea are ascribable to this substance through an examination of a number of protease inhibitors. HFea's amidolytic properties were inhibited by alpha 2-plasmin inhibitor, antithrombin III in the presence of heparin, and Cl esterase inhibitor (Cl-INH). Additionally, it was inhibited by popcorn inhibitor, leupeptin, hexadimethrine bromide, protamine sulfate, dansyl-arginine N-(3-ethyl- 1,5-pentanediyl) amide (DAPA), diisopropylphosphofluoridate (DFP), aprotinin, and at excessively high concentrations, soybean and lima bean trypsin inhibitors. The spectrum of action of agents that did or did not inhibit HFea supports the view that amidolysis by preparations of HFea is attributable to this enzyme. In general, the enzymatically active carboxy-terminal fragment of HF (HFf) was inhibited by the same agents that inhibited HFea, but aprotinin, protamine sulfate and hexadimethrine bromide were more effective against HFf than HFea, while the reverse was true of lima bean trypsin inhibitor.