大鼠脂肪间充质干细胞在部分肝切除模型中向肝细胞分化的研究

目的探讨脂肪间充质干细胞（ADMSCs）在部分肝切除模型中向肝细胞的分化。方法从大鼠脂肪组织中分离出干细胞，并进行体外扩增、传代，取第2代ADMSCs用PKH26标记，制作部分肝切除模型，将标记细胞经门静脉自体植入体内。2周后切下肝脏制成冰冻切片，荧光显微镜下观察标记细胞在肝脏的定位，进行免疫荧光染色检测标记细胞白蛋白的表达。结果从脂肪组织中分离出的ADMSCs能在体外大量扩增，PKH26标记后细胞在荧光显微镜下发红色荧光，细胞标记率约95％；荧光显微镜下可见肝脏冰冻切片中散在分布红色标记细胞，免疫荧光染色显示大多数标记细胞白蛋白染色阳性。结论 大鼠脂肪间充质干细胞在肝再生环境中能向肝细胞分化，有可能在肝部分切除后参与肝再生。     

骨髓干细胞移植治疗急性肝损伤大鼠的实验研究

目的评价骨髓干细胞移植治疗急性肝损伤大鼠的治疗作用。方法采用2-乙酰氨基芴造成急性化学性肝损伤及2/3肝脏切除造成急性物理性肝损伤两种大鼠肝损伤模型,观察门静脉和肝实质内两种移植方式移植骨髓干细胞对以上两种急性肝损伤大鼠的治疗作用。结果在化学性肝损伤大鼠,细胞移植后3周,肝内注射组血清白蛋白明显高于门静脉移植组(P=0.03);在物理性肝损伤大鼠,移植后48h,门静脉移植组和肝内注射组血清谷丙转氨酶均明显升高,且肝内注射组明显高于门静脉移植组(P=0.0009),而谷草转氨酶和血清白蛋白均无明显异常(P0.05);移植后3周,两组各项肝功指标均无明显异常。结论骨髓干细胞移植可以促进化学性肝损伤大鼠血清白蛋白恢复,肝内注射方式较门静脉移植疗效更好,肝实质内移植骨髓干细胞易引起谷丙转氨酶升高。     

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。     

诱导分化的骨髓间充质干细胞不同途径移植治疗慢性肝损伤的研究

目的 探讨诱导分化的骨髓间充质干细胞(MSC)不同途径移植治疗慢性肝损伤的可行性.方法 密度梯度离心法联合贴壁培养分离、扩增骨髓MSC,体外诱导向肝系细胞分化,反转录-聚合酶链反应(RT-PCR)、免疫细胞化学法检测肝系细胞标志,电镜观察超微结构的变化.清洁级Wistar大鼠120只,其中114只建立慢性肝损伤模型(实验组),另6只作为正常对照组.实验组8周内自然死亡12只,将剩余96只根据三种移植途径及体质量分层随机均分为门静脉移植实验组、门静脉移植对照组、脾脏移植实验组、脾脏移植对照组、尾静脉移植实验组和尾静脉移植对照组,各移植实验组移植4',6-二脒基-2'-苯基吲哚(DAPI)标记诱导分化14 d的MSC,观察移植后细胞的迁徙、定位,血清肝功能指标,肝脏组织病理学及移植效果.结果 诱导后第7、14天均检测到甲胎蛋白(AFP)mRNA表达,第14、21天检测到白蛋白(ALB)mRNA、兔抗鼠细胞角蛋白18(CK18)和肝细胞抗原表达.诱导后MSC胞体增大,胞质中含有较多线粒体、粗面内质网、高尔基体、溶酶体和糖原颗粒等.各移植实验组大鼠均发现DAPI标记细胞且丙氨酸转氨酶等肝功能指标及肝脏病变程度均较其移植对照组改善,以脾内移植途径更为显著[(98.13±8.22)U/L比(145.85±15.88)U/L,P＜0.05].结论 诱导分化的骨髓MSC通过三种移植途径均对慢性肝损伤具有修复作用,且脾内移植优于门静脉及尾静脉移植.     

磁标记大鼠间充质干细胞肝脏移植的磁共振成像活体示踪

目的探讨1.5 T MRI活体示踪磁标记干细胞经门静脉肝脏移植的可行性。方法大鼠间充质干细胞以菲立磁和DAPI标记后,经门静脉注入大鼠肝脏,分别于移植前及移植后1 h、3 d、7 d和14 d行MRI扫描,并与组织荧光显像和铁染色对照。结果移植后1 h、3 d、7 d及14 d肝脏信噪比分别为1.10±0.26、8.18±1.55、11.08±1.30、14.15±1.02,7 d以内肝脏信噪比与移植前比较均有明显降低(P<0.05)。各时相肝组织铁染色与荧光显像的空间分布一致。结论1.5 T MRI活体示踪经门静脉移植的磁标记干细胞是可行的,MRI示踪时间可持续7 d。     

菲立磁标记猪骨髓间充质干细胞自体肝内移植的MR成像

目的:探索标记细胞肝内移植后磁共振威像技术.方法:获取猪自体骨髓间充质干细胞,分离、培养.应用菲立磁(Feridex)标记细胞,普鲁士蓝染色鉴定,标记细胞组n=6)和未标记细胞组(n=4)行经门静脉行肝内移植,分别于移植前,移植后6h、3 d、7d行磁共振T1WI,T2WI,T2*WI序列成像,同时行组织切片普鲁士蓝染色结果:普鲁士蓝染色表明MSCs的标记率达接近100%,磁标记MSCs肝内移植后行磁共振T2*WI序列呈明显低信号改变,并持续至细胞移植后7 d,组织切片普鲁士蓝染色显示7 d后肝内仍有移植的磁标记细胞存在于肝实质及肝血窦中.结论:利用Feridex可以在体外成功标记猪骨髓间充质干细胞,肝脏移植后行磁共振可以对标记细胞进行活体成像.     

Bcl-2基因修饰的骨髓间充质干细胞治疗急性肝衰竭的初步研究

目的 构建Bcl-2基因修饰慢病毒质粒,建立稳定表达Bcl-2基因的骨髓间充质干细胞系,探讨Bcl-2基因修饰的骨髓闸充质干细胞治疗ALF的可行性。方法 PCR合成Bcl-2基因片段并插入GV287空载体后扩增进行慢病毒包装,使用Bcl-2慢病毒液转染大鼠骨髓间充质干细胞,使用单纯未处理骨髓间充质干细胞,移植急性肝损伤模型大鼠,观察移植后大鼠存活情况及其肝脏病理改变。结果 得到纯化的骨髓间充质干细胞细胞株;成功分离Bcl-2基因DNA片段并构建Bcl-2基因重组慢病毒质粒,并成功转染到骨髓间充质干细胞中,经荧光显微镜检测GFP荧光及免疫印迹蛋白检测,转染后细胞可高表达Bcl-2基因,转染后细胞状态较好,可进行移植实验;模型鼠经过尾静脉移植骨髓间充质于细胞后,共聚焦显微镜下观察肝脏冰冻切片病理,骨髓间充质干细胞,可以定植到损伤肝脏组织。结论 初步证实,骨髓间充质干细胞能够在急性肝损伤大鼠肝脏归巢,并且能发挥一定程度治疗作用。     

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.     

间充质干细胞在肝脏的归巢及分化机制

间充质干细胞可通过旁分泌营养机制和分化为肝实质细胞等修复肝组织,移植间充质干细胞将成为临床治疗肝病的一种重要方法。本文介绍了间充质干细胞在肝损伤条件下,归巢到损伤肝脏并分化为肝实质细胞的可能机制。     

骨髓间充质干细胞移植在肝纤维化治疗中的应用

目的 探讨大鼠骨髓间充质干细胞(BMSC)对肝纤维化大鼠模型的治疗效果,为临床自体BMSC移植治疗肝脏疾病提供实验基础和理论依据.方法 四氯化碳(CCl4)腹腔注射构建SD大鼠肝纤维化模型.以绿色荧光蛋白(GFP)标记的正常SD大鼠BMSC为细胞来源,于造模后第10周经尾静脉植入模型鼠体内,对照组同法注入生理盐水.4周后取大鼠肝脏进行病理学检测,同时采集大鼠血液,检测血清肝纤维化指标和肝功能指标.结果 (1)成功构建SD大鼠肝纤维化模型.(2)体外分离获得了高纯度贴壁生长的大鼠BMSC,并用携带GFP报告基因的慢病毒成功对其进行感染,感染率达80％以上.(3)经BMSC移植的大鼠肝脏中可检测到GFP阳性的细胞,主要沿着中央静脉及汇管区周围纤维间隔分布.免疫组织化学结果显示GFP阳性细胞呈肝样细胞形态,且与肝组织相容.(4)BMSC移植组与模型对照组和生理盐水组相比,病理检测肝纤维化程度减轻,血清学指标好转.结论 BMSC移植能抑制大鼠肝纤维化发展,部分逆转肝纤维化进程,有效改善肝脏功能,为临床自体BMSC移植治疗肝脏疾病提供实验基础.     

大鼠急性肝损伤后经门静脉自体骨髓单个核细胞移植对宿主凝血机制的影响

目的观察经门静脉大鼠自体骨髓单个核细胞移植后是否形成肝内及肝外血栓。方法应用CCl4/2-AAF制备大鼠急性肝损伤模型,从大鼠骨髓分离骨髓単个核细胞。实验A组:经门静脉注射骨髓单个核细胞0.3 mL/只;B组:经门静脉注射生理盐水0.3 mL/只;分别于移植后两周处死大鼠,取心、肝、肺、脑、肾、脾等组织观察大鼠这些器官有无血栓形成。结果各器官血栓形成情况:HE染色观察移植后2周心、肝、肺、脑、肾均无血栓形成。结论通过门静脉移植的骨髓单个核细胞在急性肝损伤的大鼠各个器官均无血栓形成。     

内质网及氧化应激在大鼠慢性肝损伤中的变化

目的研究内质网应激相关基因、氧化应激指标在四氯化碳诱导的大鼠慢性肝损伤中的变化。方法四氯化碳制备大鼠慢性肝损伤模型,通过测定大鼠血清ALT、AST水平,采用HE染色和TUNEL法观察肝组织病理形态和肝细胞凋亡改变,评价成模效果。检测大鼠肝脏葡萄糖调节蛋白78（GRP78）、GRP94、血红素加氧酶（HO）-1 mRNA表达及超氧化物歧化酶（SOD）、丙二醛（MDA）、还原型谷胱甘肽（GSH）变化。结果四氯化碳成功诱导了大鼠慢性肝损伤,与对照组相比,大鼠肝脏GRP78、GRP94、HO—1的表达量、MDA含量均明显增加,SOD活性、还原型GSH显著降低。结论四氯化碳诱导大鼠慢性肝损伤时,内质网应激相关基因的表达增加,氧化应激指标亦发生改变,表明内质网应激、氧化应激共同参与了大鼠慢性肝损伤过程。     

Contribution of mononuclear bone marrow cells to carbon tetrachloride-induced liver fibrosis in rats

AIM： To study the inhibitory effect of mononuclear bone marrow cell （BNC） transplantation on carbon tetrachloride （CCl4） -induced liver fibrosis in rats.
METHODS： Rat liver fibrosis models were induced by CCl4 and alcohol administration. After 8 wk, twenty rats were randomly allocated into treatment group （n = 10） and control group （n = 10）. BMC were infused into the rats in treatment group via the portal vein, while heparinized saline was infused in control group. CCl4 was hypodermically injected into the rats twice a week for 4 wk. At the end of wk 12, all rats were humanely sacrificed. Uver samples were taken and stained with HE or Masson trichrome. The general conditions, liver fibrosis （hydroxyproline and collagen fibre） and liver pathological grades in rats were evaluated.
RESULTS： The general conditions of the rats in treatment group improved markedly, but not in control group. Hydroxyproline was 504.6± 128.8 μg/g in treatment group, and 596.0 ± 341.8 μg/g in control group. The percentage of collagen fibre was 3.75% ± 0.98% in treatment group and 5.02% ± 0.44% in control group. There was a significant difference between the two groups （P 〈 0.05）. Liver pathological grade decreased from grade N to grade 11 partially in treatment group （P 〈 0.05） with no obvious improvement in control group （P 〉 0.05）. There was a significant difference between treatment group and control group （P 〈 0.05）.
CONCLUSION： Transplantation of BMC can improve liver fibrosis due to chronic liver injury in rats.     

Therapeutic effect of transplanting HGF-treated bone marrow mesenchymal cells into CCl4-injured rats

BACKGROUND/AIMS: The autologous transplantation of bone marrow cells is a promising treatment for liver disease. Pluripotent bone marrow stem cells can differentiate into hepatocytes, but few reports address the therapeutic effect of transplanting these stem cells into damaged liver in vivo. Here, we transplanted bone marrow-derived mesenchymal cells (BMMCs) to test their effect in liver-injured rats. METHODS: Rat bone marrow cells were cultivated for 2 weeks in the presence or absence of hepatocyte growth factor (HGF), labeled with a fluorescent marker, and transplanted by injection into CCl(4)-injured rats. Blood samples collected 4 weeks later were analyzed for albumin production and transaminase levels. The amount of fibrosis was determined by histology. RESULTS: RT-PCR analysis detected alpha-fetoprotein and albumin mRNAs in BMMCs cultured with HGF for 2 weeks. Albumin protein was also produced in the BMMC cultures by a subpopulation of cells. Transplantation of the BMMCs into liver-injured rats restored their serum albumin level and significantly suppressed transaminase activity and liver fibrosis. These effects were not seen when the BMMCs were cultured without HGF. CONCLUSIONS: The transplantation of BMMCs cultured with HGF effectively treats liver injury in rats. This is a promising technique for autologous transplantation in humans with liver injury.     

Therapeutic potential and related signal pathway of adipose-derived stem cell transplantation for rat liver injury

Aim:  Liver transplantation is the only currently effective therapy for end-stage chronic liver disease and severe acute liver failure, but its use is limited by high cost and a shortage of allografts. Here we explored the effectiveness of transplanting adipose-derived stem cells (ADSCs) into rats with experimentally induced liver injury.
Methods:  ADSCs obtained from rats were hepatogenic induced in vitro with MAPK pathways inhibitors preconditioning. In vivo , ADSCs were transplanted into rats via different routes and serum liver function markers from post-operative rats were tested.
Results:  When grown in adipogenic induction medium, ADSCs were able to differentiate into adipocytes. In hepatogenic induction medium, ADSCs were able to differentiate into hepatocyte-like cells, with appropriate changes in morphology and appropriately elevated expression of hepatocyte-specific markers. ERK1/2 phosphorylation activity was also significantly upregulated during the hepatogenic differentiation process, and was blocked by the ERK/MAPK pathway-specific inhibitor PD98059. In a rat liver injury model, intravenously injected ADSCs successfully engrafted into recipient livers. We found that injection via the hepatic portal vein was more efficient than via the dorsal vein of the penis. ADSC transplantation into damaged livers significantly decreased the level of serum liver enzymes such as alanine aminotransferase and aspartate aminotransferase, and improved serum albumin level. Both the number of engrafted cells and the improvement of liver function reached a peak two weeks after transplantation.
Conclusion:  Transplanted ADSCs appear to be therapeutically effective in the rat liver injury model, which may ultimately provide a therapeutic alternative to liver transplantation in human patients.     

The protective effects of SA3443, a novel cyclic disulfide, on chronic liver injuries in rats.

The effects of (4R)-hexahydro-7,7-dimethyl-6-oxo-1,2,5-dithiazocine-4- carboxylic acid (SA3443), a novel cyclic disulfide compound, on the development of chronic liver injury were studied in rats, using two types of models, carbon tetrachloride (CCl4)-induced chronic liver injury and heterologous serum (swine serum)-induced liver fibrosis. SA3443 (30-100 mg/kg, p.o.) significantly suppressed increases in serum transaminase and alkaline phosphatase activity induced by CCl4-treatment for 10 weeks. This compound also inhibited increases in hepatic lipids and hydroxyproline content in CCl4-treated rats. In the histopathological studies, treatment with SA3443 resulted in a decrease in the degree of hepatic necrosis, fibrosis and steatosis. On the other hand, 8-weeks treatment with swine serum revealed hepatic fibrosis without appearance of necrosis or fatty accumulation. In this model, SA3443 (30 mg/kg, p.o.) reduced the hepatic hydroxyproline level, and diminished the formation of connective tissue in the liver. These findings indicate that SA3443 protects the liver against chronic liver injuries induced by CCl4 and heterogeneous serum.     

Effects of extract from Ginkgo biloba on carbon tetrachloride-induced liver injury in rats

AIM: To study the effects of extract from Ginkgo biloba (EGb) containing 22% flavonoid and 5% terpenoid on chronic liver injury and liver fibrosis of rats induced by carbon tetrachloride (CCl4). METHODS: All rats were randomly divided into control group, CCl4-treated group, colchicine-treated group and EGb-protected group. Chronic liver injury was induced in experimental groups by subcutaneous injection of CCl4 and fed with chows premixed with 79.5% corn powder, 20% lard and 0.5% cholesterol (v/v). EGb-protected group was treated with EGb (0.5 g/kg body weight per day) for 7 wk. At the end of wk 8, all the rats were killed. Liver function, liver fibrosis, oxidative stress and expression of transforming growth factorβ1 (TGF-β1), a-smooth muscle actin (α-SMA) and typeⅠcollagens in liver were determined. In addition, pathology changes of liver tissue were observed under light microscope. RESULTS: The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and albumin (Alb) in EGb-protected group were notably improved as compared with the CCL4-treated group (P < 0.01). The contents of serum hyaluronic acid (HA), typeⅢprocollagen (PCⅢ), typeⅣcollagen (CIV) and the expression of hepatic tissue TGF-β1,α-SMA and typeⅠcollagen in EGb-protected group were significantly lower than those in CCL4-treated groups (P < 0.05, P < 0.01). The degrees of liver fibrosis in EGb-protected groups were lower than those in CCL4-treated groups (6.58±1.25 vs 9.52±2.06, P < 0.05). Compared to the CCL4-treated group, the levels of plasma glutathoine peroxidase (Se-GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA) were strikingly improved also in EGb-protected group (P < 0.05, P < 0.01). CONCLUSION: EGb resists oxidative stress and thereby reduces chronic liver injury and liver fibrosis in rats with liver injury induced by CCl4     

银杏叶提取物对肝硬化大鼠门静脉压及肝窦病理变化的影响

目的探讨银杏叶提取物（EGb）对肝硬化大鼠肝窦微循环及门静脉高压的影响。方法25只雄性Wistar大鼠分为3组：正常对照组（5只）,CCl_4组（10只）,EGb治疗组（10只）。肠系膜上静脉插管测定各组大鼠灌注EGb前后门静脉压力;肝组织匀浆中检测丙二醛（MDA）,血管内皮素（ET-1）,血小板活化因子（PAF）,一氧化氮（NO）,构生型一氧化氮合成酶（cNOS）和诱生型一氧化氮合成酶（iNOS）水平;电镜观察各组肝窦微循环特征。结果EGb灌注后CCl4组门静脉压由（8．7±0．8）mm Hg降至（7．4±0．6）mmHg,降低幅度15%,t 4．11,P＜0．01; EGb治疗组与CCl_4组比较,门静脉压、肝组织MDA、ET-1、PAF、NO和iNOS水平显著降低（P＜0．05或P＜0．01）,肝窦内胶原沉积、微血栓形成、肝窦毛细血管化程度减轻。结论银杏叶提取物能改善肝窦微循环障碍,降低门静脉压力,其可能机制是降低MDA、ET 1、PAF含量,下调iNOS的表达调节NO含量,对慢性肝病治疗具有重要意义。