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。     

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.     

门静脉和尾静脉注入小鼠骨髓干细胞向肝脏迁移的比较

目的:探索小鼠骨髓干细胞,在同种异体不同途径移植后向受体肝脏迁移的情况．方法:以红色荧光染料PKH26标记从小鼠骨髓中分离出的干细胞,分别从小鼠的尾静脉和门静脉注入同种异体的CCL4-AAF造成肝损伤的小鼠体内,移植2 wk后分别取肝组织, 通过荧光显微镜观察两种移植途径对小鼠骨髓干细胞向肝脏迁移的影响．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标记阳性的细胞在门静脉移植组20倍镜下每张切片平均为175．4 个,在尾静脉移植组平均172．9个(P>0．05)．结论:骨髓干细胞向肝脏迁移的细胞数与移植途径无关．     

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。     

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。     

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。     

骨髓间充质干细胞诱导分化后脾内移植治疗急性肝损伤的研究

背景：近年干细胞的研究为肝脏疾病的治疗提供了新契机。研究证实骨髓间充质干细胞（MSCs）不仅可分化为与其组织来源一致的细胞．且具有跨系或跨胚层分化为不同组织来源细胞的潜能。目的：探讨骨髓MSCs诱导分化后脾内移植治疗急性肝损伤的可行性。方法：密度梯度离心法联合贴壁培养分离、扩增BALB／c小鼠骨髓MSCs。体外诱导向肝系细胞分化，逆转录聚合酶链反应（RT-PCR）、免疫细胞化学法检测肝系细胞的标志。制备BALB／c小鼠ccL急性肝损伤模型．脾内移植DAPI标记的诱导分化21d的MSCs。荧光显微镜下观察细胞的迁徙、定位．行血清肝功能指标、肝脏组织病理学检测移植效果。结果：诱导后7、14d，均检测到甲胎蛋白（AFP）mRNA和蛋白的表达，14、21d检测到白蛋白（ALB）mRNA和蛋白的表达。移植后实验组肝脏和脾脏均发现DAPI标记细胞。与对照组相比，实验组肝功能显著改善（P〈0．05），肝脏病变程度减轻。结论：脾内移植诱导分化的骨髓MSCs对急性肝损伤具有修复作用。     

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。     

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。     

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.     

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。     

Granulocyte Colony-Stimulating Factor Enhances Bone Marrow Mononuclear Cell Homing to the Liver in a Mouse Model of Acute Hepatic Injury

Background

Experiments have reported that granulocyte colony stimulating factor (G-CSF) can mobilize stem cells. However, few studies have examined the effect of G-CSF on bone marrow mononuclear cell (BMMC) mobilization, in particular regarding their capability to home to acutely injured liver.

Aims

The aim of this study was to evaluate the effort of G-CSF on BMMC homing to the liver following chemically-induced hepatic failure.

Methods

BMMC were isolated from mice, pre-labeled with PKH26 and infused into the mice in which hepatic injury had been induced followed by administration of G-CSF or vehicle. Livers were studied by fluorescent microscopy after transplantation of pre-labeled BMMC.

Results

PKH26 labeled cells were found in liver tissue at 102 ± 10 cells/high power field in the BMMC+G-CSF group and 30 ± 5 cells/high power field in the BMMC group, but none in the G-CSF group and the control group (P < 0.05). In the former two groups the majority of PKH26 labeled cells colocalized with proliferative cell nuclear antigen (PCNA). The number of PCNA positive cells in the BMMC+G-CSF group was 20 ± 4 cells/high power field, while in the BMMC group it was 14 ± 2 cells/high power field, in the G-CSF group 12 ± 2 cells/high power field, and 8 ± 1 cells/high power field in the control group. Moreover, albumin expression was increased in the BMMC+G-CSF treated group (149 ± 7/high power field) relative to the BMMC group (48 ± 6/high power field), the G-CSF group (44 ± 5/high power field) and the vehicle group (30 ± 6/high power field), with the former three groups showing elevated levels as compared to vehicle control (30 ± 6) (P < 0.05).

Conclusion

Transplanted BMMC may home to injured liver, which appears to be enhanced by G-CSF administration.     

骨髓间充质干细胞经心外膜移植到心肌梗死大鼠体内后在肝脾的分布及对肝脾功能影响的实验研究

目的心肌梗死后经心外膜注射骨髓间充质干细胞(bone marrow mesenchymal stem cells,MSCs)后移植细胞在心外脏器(肝脾)的分布及对肝脾功能的影响。方法体外培养扩增雄性Lewis大鼠的MSCs,并用5-溴脱氧尿苷(Bromodeoxyuridine,Brdu)标记,建立心肌梗死后3周的雌性大鼠模型,将MSCs(3×106,50μL)经心外膜注射到大鼠模型(n=22)的心肌梗死边缘区,对照组(n=21)注射同等量磷酸盐缓冲液(Phosphate Buffered Saline,PBS)。4周后,检查大鼠心功能改变情况,并分别取肝脾和外周血液,行免疫组化,HE染色,肝功能检测,血常规,IgM检测等。结果 MSCs移植后4周,MSCs移植组心功能较对照组改善,免疫双染示MSCs向肝样细胞分化,HE染色示肝脾淤血减轻,丙氨酸氨基转移酶(ALT)、直接胆红素(DBIL)显著低于对照组,白蛋白(ALB)高于对照组,脾指数低于对照组。结论经心外膜注射MSCs可以改善心脏功能,移植后少量细胞滞留于肝脾,但对肝脾的功能均无明显影响。     

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.     

骨髓间充质干细胞对大鼠减体积肝移植术后肝细胞再生与修复的作用

目的探讨骨髓间充质干细胞（BMSCs）对大鼠减体积肝移植术后肝脏再生与修复的影响。方法采用生化、免疫组化等方法检测术后不同时间的血清和肝脏标本。（1）取同周龄Wistar大鼠骨髓体外培养BMSCs,标记CSFE荧光后制备悬液;（2）建立大鼠50%减体积肝移植模型（供体鼠为SD大鼠,受体鼠为Wistar大鼠）,分为实验组（取BMSCs悬液经门静脉植入）和对照组（取等量生理盐水经门静脉注射）,观察大鼠生存状态,测量肝再生的相关指标。结果 （1）成功分离培养BMSCs;（2）术后2h,大鼠均自由活动饮水;（3）实验组肝组织中存在大量BMSCs,术后第2、3 d实验组肝体比显著高于对照组。病理显示：实验组肝细胞损伤较对照组轻微。结论植入BMSCs后,可以促进大鼠减体积肝移植术后肝细胞增殖和修复。     

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.