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。     

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.     

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。     

DAPI联合PKH26标记骨髓干细胞在急性肝脏损伤模型小鼠肝内迁移的示踪作用

目的探讨PKH26和DAPI联合标记骨髓干细胞在肝脏组织内迁移过程中的示踪作用。方法用PKH26和DAPI联合标记从Balb/c小鼠骨髓中分离出骨髓干细胞,用CCl4腹腔注射的方法制备小鼠急性肝脏损伤模型后,将标记的BMMC经尾静脉移植入急性肝脏损伤模型小鼠体内,对照组经尾静脉注入磷酸盐缓冲液(PBS)。移植2周后取肝脏组织,通过激光共聚焦荧光显微镜观察骨髓干细胞向肝脏内迁移的情况。结果接受骨髓干细胞移植组小鼠肝脏组织内可发现DAPI和PKH26双重标记的骨髓干细胞。结论 PKH26和DAPI可以联合标记向急性肝损伤模型肝组织迁移的骨髓干细胞,为体内示踪骨髓干细胞脑内迁移提供更加准确的实验方法。     

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。     

门脉移植骨髓间充质干细胞后肝内定居的实验研究

目的探讨同种异体大鼠骨髓间充质干细胞(MSCs)门脉移植后在受体大鼠模型肝内定居分布情况。方法利用密度梯度法和贴壁法纯化大鼠骨髓间充质干细胞。取第3代MSCs经CFSE标记后从门静脉植入同种异体正常组和肝损伤组大鼠体内,10d后取出受体大鼠肝脏,通过冰冻切片行荧光计数法,比较MSCs在大鼠肝脏内定居分布情况。结果门静脉移植后的MSCs均能在正常组和肝损伤组的肝脏内定居；在细胞数量上,肝损伤组明显多于正常组。结论经门静脉移植的MSCs可以定居于肝脏内,定于肝脏的细胞数量与肝脏是否损伤密切相关。     

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标记示踪人脐带间充质干细胞在肝硬化大鼠体内的迁移

目的 用红色荧光染料PKH26标记人脐带间充质干细胞(hUCMSCs),观察PKH26标记的hU CMSCs在肝硬化大鼠体内的迁移情况. 方法 hU CMSCs经复苏培养后,用PKH26对细胞进行标记,测定标记率和单次标记后荧光可检测时长;显微镜下观察标记后细胞形态,四甲基偶氮唑盐法测定细胞生长曲线.将PKH26标记的hU CMSCs通过大鼠尾静脉分别移植给正常大鼠和肝硬化大鼠;48 h后取肝组织做冰冻切片,荧光显微镜下观察hUCMSCs在大鼠体内的迁移情况.数据采用完全随机设计两组均数t检验进行分析. 结果 PKH26对hUCMSCs的标记率为100％,标记后细胞生长状态良好,细胞形态与未标记细胞无明显差别,均为长梭形成纤维样细胞;两组细胞各时间点的吸光度值的差异亦无统计学意义(P＞0.05),细胞增殖未见明显影响;第3～5天细胞生长速度最快,为对数生长期,在对数生长期中,对照组的细胞倍增时间为(2.22±0.04)d,标记组的细胞倍增时间为(2.20±0.04)d,两组的差异无统计学意义(P=0.53).体外单次标记后荧光至少可维持20 d;移植给大鼠后,PKH26标记的hUCMSCs主要分布于硬化肝脏的汇管区、血管及假小叶周围,脾脏、肺亦有少量分布.结论 PKH26是标记hUCMSCs的理想荧光染料,PKH26标记技术可用于hU CMSCs移植治疗肝硬化时的细胞示踪研究.     

SCF和G-CSF对骨髓单个核细胞移植治疗急性肝损伤的研究

目的探索联合应用干细胞因子(SCF)和粒细胞集落刺激因子(G-CSF)动员骨髓单个核细胞向急性肝损伤小鼠肝脏的迁移情况及对肝损伤的修复作用。方法应用CCl4/2-AAF制备急性肝损伤模型,然后行骨髓单个核细胞移植(体外先经PKH26标记)和皮下注射细胞因子SCF、G-CSF(实验按注射细胞因子的种类随机分为4组,A组:SCF+G-CSF;B组:SCF;C组:G-CSF;D组:移植对照组),分别于移植后2周、4周处死小鼠,取其血清检测肝功(ALT、AST、ALB值),取肝组织观察小鼠骨髓单个核细胞向肝脏迁移的情况及损伤肝脏的修复情况。结果 (1)PKH26标记阳性细胞数:A组>C组>B组、D组。A组与C组、B组、D组比较差异均有统计学意义(P<0.05);(2)病理组织学检测:A组小鼠肝脏修复较其他组好;(3)肝功指标:与B组、C组、D组比较,A组ALT,AST值差异有统计学意义(P<0.05),ALB值差异无统计学意义(P>0.05);移植后4周与2周比较差异有统计学意义(P<0.05)。结论 (1)SCF和G-CSF可以通过动员骨髓干细胞促进急性肝损伤小鼠肝脏的修复。(2)联合应用SCF和G-CSF比单独应用更能有效保护受损肝脏,并加快受损肝脏的再生。     

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。     

Bone marrow mononuclear cell transplant therapy in mice with CCl4-induced acute liver failure

Background/aims: Stem cell transplantation has theoretical potential for the treatment of certain liver diseases. However, the use of bone marrow mononuclear cells as a therapy for liver disease has received little attention. The present study was to examine whether bone marrow mononuclear cells might be useful in the management of acute liver failure in an animal model. Materials and Methots: Bone marrow mononuclear cells were harvested from BALB/c mice and then labeled with the fluorescent dye PKH26. The labeled cells were subsequently infused into the tail veins of mice in which hepatic injury had been induced by CCl4 toxicity. After transplantation, the labeled cells in the liver were studied by fluorescent microscopy, and the levels of proliferating cell nuclear antigen and albumin were quantified in bone marrow mononuclear cell-treated and untreated groups. Serum aminotransferase activity was also monitored at various time points post-liver injury. Results: Transplanted bone marrow mononuclear cells labeled with PKH26 were found to populate the damaged liver around the portal and centrolobular regions, and they appeared to differentiate into albumin-producing hepatocyte-like cells. Animals that received bone marrow mononuclear cells also showed a trend toward improved liver enzymes as well enhanced survival rates, relative to controls. Conclusions: These findings suggest that systemically delivered bone marrow mononuclear cells may relocate to and be retained by the injured liver; transplantation of bone marrow mononuclear cells showed an overall beneficial effect in a murine model of acute liver failure.     

Differentiation of rat bone marrow stem cells in liver after partial hepatectomy

AIM: To investigate the differentiation of rat bone marrow stem cells in liver after partial hepatectomy. METHODS: Bone marrow cells were collected from the tibia of rat with partial hepatectomy, the medial and left hepatic lobes were excised. The bone marrow stem cells (Thy CD3-CD45RA- cells) were enriched from the bone marrow cells by depleting red cells and fluorescence-activated cell sorting. The sorted bone marrow stem cells were labeled by PKH26-GL in vitro and autotransplanted by portal vein injection. After 2 wk, the transplanted bone marrow stem cells in liver were examined by the immunohistochemistry of albumin (hepatocyte-specific marker). RESULTS: The bone marrow stem cells (Thy CD3-CD45RA- cells) accounted for 2.8% of bone marrow cells without red cells. The labeling rate of 10μM PKH26-GL on sorted bone marrow stem cells was about 95%. There were sporadic PKH26-GL-labeled cells among he-patocytes in liver tissue section, and some of the cells expressed albumin. CONCLUSION: Rat bone marrow stem cells can differentiate into hepatocytes in regenerative environment and may participate in liver regeneration after partial hepatectomy.     

骨髓干细胞在大鼠肝再生环境中的分化

研究大鼠骨髓干细胞在部分肝切除后肝再生环境中的分化。从部分肝切除模型大鼠的胫骨中提取骨髓细胞,应用流式细胞仪富集骨髓干细胞,以PKH26-GL体外标记后通过门静脉进行自体移植,2周后行白蛋白和角蛋白8免疫组化检查。结果肝板肝细胞间PKH26-GL标记骨髓干细胞表达白蛋白、角蛋白8。提示骨髓干细胞在部分肝切除后肝再生环境中能分化为肝细胞,骨髓干细胞可能参与部分肝切除后的肝再生过程。     

骨髓干细胞在大鼠肝纤维化形成环境中的分化

目的 研究骨髓干细胞在肝纤维化形成环境中向肝细胞定向分化。 方法 采用四氯化碳皮下注射法诱导大鼠肝纤维化,应用流式细胞仪分选富集Thy+CD3-CD45RA-的骨髓干细胞,采用红色荧光染料PKH26-GL对其标记后进行自体移植,6周后通过免疫组织化学方法检测大鼠肝组织白蛋白、ck 8、α-平滑肌肌动蛋白表达。 结果 PKH 26-GL标记的细胞在纤维化肝脏中表达白蛋白和ck8,占肝细胞总数的(0.17±0.02)％;未见表达α-平滑肌肌动蛋白。 结论 骨髓干细胞在肝纤维化形成环境中可以向肝细胞定向分化,不向肌成纤维样细胞分化。     

Stromal cell derived factor-1 enhances bone marrow mononuclear cell migration in mice with acute liver failure

AIM： To evaluate the number of bone marrow mononuclear cells （BMMC） that are migrated to the liver following transplantation of murine BMMC into mice with acute liver injury.
METHODS： BMMC were isolated from the bone marrow of mice in a lymphocyte separation medium and then labeled with PKH26. The labeled cells were subsequently infused into the caudal veins of BALB/c mice with hepatic injury induced by carbon tetrachloride and 2-acetylaminofluorene. Mice in experimental group were treated with stromal cell-derived factor-1 （SDF-1） which was injected intraperitoneally after trans- plantation of BMMC. Mice in control group were injected intraperitoneally with 0.1 mL of saline （0.9% NaCl） after transplantation of BMMC. After 2 wk, migration of the cells in experimental group was studied by fluorescence microscopy. The expression of proliferating cell nuclear antigen and albumin was quantified with manual methods in both groups. The serum transaminase levels at different time points were compared between the two groups.
RESULTS： The labeled ＂cells＂ were found in the portal region and central veins of hepatic Iobules. The PKH26labeled cells appeared at an average frequency of 108 ± 8/high power field in the experiment group and 65 ± 8/high power field in the control group （P 〈 0.05）. The total number of positive cells was 29 ± 7/high power field in the experimental group and 13 ± 2/high power field in the control group. The albumin expression level was also higher in the experimental group than in the control group （29 ± 7 vs 13 ± 2, P 〈 0.05）. The total number of crossing points was 156 ± 5/high power field in the experimental group and 53 ± 5/high power field in the control group （P 〈 0.05）. The serum alanine aminotransferase levels in experimental and control groups were measured at different time points （120 ± 40 vs 118.50 ± 1.75, P 〉 0.05; 80.60 ± 6.50 vs 101.08 ± 5.67, P 〈 0.05; 50.74 ± 5.38 vs 80.47 ± 4.62, P 〈 0.05; 30.54 ± 2.70 vs 60.72 ± 4.37, P 〈 0.05; 30.77 ± 5.36 vs 40.47 ± 6.50, P 〈 0.05）. At the same time, the serum aspartate aminotransferase levels were measured in experimental and control groups at different time points （122.55 ± 1.46 vs 120.70 ± 4.22, P 〉 0.05; 54.26 ± 6.50 vs 98.70 ± 8.20, P 〈 0.05; 39.47 ± 5.39 vs 78.34 ± 4.50, P 〈 0.05; 28.94 ±2.70 vs 56.44 ± 4.28, P 〈 0.05; 30.77 ± 5.45 vs 42.50 ± 6.28, P 〈 0.05）.
CONCLUSION： SDF-1 can promote the migration of BMMC to the liver of mice with acute liver failure.     

经门静脉骨髓间充质干细胞移植治疗小鼠急性肝损伤的疗效

目的 评价经门静脉途径行骨髓间充质干细胞(hMSCs)移植对对乙酰氨基酚所致小鼠急性肝损伤的疗效. 方法 建立对乙酰氨基酚导致的急性药物性肝损伤动物模型,经门静脉途径行hSMCs移植,采用肝功能检查、免疫荧光、荧光显微镜,网状纤维染色等方法 观察hMSC移植前后严重联合免疫缺陷病小鼠肝腺泡结构的恢复与肝功能的改善情况. 结果免疫缺陷病小鼠肝功能在经门静脉移植组与对照组比较明显改善(P＜0.05).免疫荧光显示经门静脉移植的hMSCs在肝脏有大量定植、分化与增殖.免疫荧光观察到门静脉移植后肝腺泡结构改善明显. 结论 经门静脉途径的hMSCs移植能显著改善急性肝损伤小鼠肝功能,经门静脉移植的hMSCs在小鼠肝内生长良好,是hMSCs移植治疗的良好途径.     

骨髓干细胞移植治疗急性肝衰竭的实验研究

目的探讨骨髓干细胞移植治疗实验性肝衰竭大鼠的效率及可行性。方法以D-氨基半乳糖及内毒素制备雌性大鼠肝衰竭模型,然后动员和富集雄性大鼠骨髓干细胞,并经门静脉移植至雌性大鼠肝脏;移植后观察受体雌鼠临床表现、生化改变、肝脏病变及肝组织Y染色体阳性率。结果受体肝衰竭雌鼠在细胞移植后,精神、食欲逐渐好转,生化异常及肝脏病变得以恢复,肝脏可检出Y染色体阳性的供体源性肝细胞;未接受骨髓干细胞移植的对照组雌鼠则全部死亡。结论骨髓干细胞移植对实验性肝衰竭大鼠具有明确治疗作用。