乙型肝炎肝硬化患者脂肪间充质干细胞体外诱导分化为肝细胞及其HBV易感性的研究
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摘要
HBV感染呈世界性流行，据世界卫生组织报道，全球约20亿人曾感染过HBV，其中3.5亿人为慢性HBV感染者。慢性HBV感染是引起肝硬化和肝癌的主要原因，每年全世界约有100万人死于HBV感染所致的肝衰竭、肝硬化和原发性肝癌HCC。肝脏疾病进展到肝硬化、肝癌等终末期肝病，常累及广泛的肝实质细胞受损，肝组织再生能力下降，造成肝功能失代偿，最终导致肝衰竭。肝衰竭是各种严重肝病的终末期表现，目前的治疗条件下，其病死率极高，寻找一种确切有效的治疗手段尤为重要。
近年干细胞的研究在医学领域取得令人瞩目的成就，也为广大临床工作者开拓了新的思路，利用干细胞移植治疗终末期肝病成为新的热点。ADSCs因其与BM-MSCs形态相似亦被称为AD-MSCs。AD-MSCs具有多向分化潜能，不仅可以分化为成骨细胞、软骨细胞、脂肪细胞和肌肉细胞等中胚层间质组织细胞，还可以跨越胚层界限分化为外胚层的神经细胞及内胚层的肝细胞，另外AD-MSCs具有来源丰富、反复取材和较少涉及医学伦理学问题而倍受关注。
本研究旨在建立乙型肝炎肝硬化患者皮下脂肪组织MSCs体外培养体系的基础上，探讨其生物学特性，并与乙型肝炎肝硬化患者骨髓来源MSCs的生物学特性进行比较，同时观察其体外增殖能力及向肝细胞分化的潜能，探讨高滴度HBV环境对其生物学特性的影响及AD-MSCs的体外HBV易感性，为AD-MSCs的基础研究和临床应用提供理论基础和技术平台。
本研究采用胶原酶消化结合AD-MSCs的贴壁生长的特性分离培养乙型肝炎肝硬化患者皮下脂肪组织MSCs，建立AD-MSCs的体外培养体系，观察AD-MSCs的生物学特性，并与乙型肝炎肝硬化患者BM-MSCs的生物学特性进行比较；体外诱导AD-MSCs向肝细胞分化，观察细胞形态变化，免疫组织化学染色和Western blotting技术检测肝细胞系特异性标志物AFP、ALB和CK-18表达，过碘酸雪夫试剂染色检测细胞糖原合成功能，ELISA技术检测细胞白蛋白合成与分泌功能；采用高滴度HBV感染血清体外培养AD-MSCs，观察AD-MSCs生长状况和形态变化，免疫组织化学染色和Western blotting技术检测细胞内HBsAg和HBcAg表达，同时观察高滴度HBV环境对AD-MSCs成肝细胞分化的影响及AD-MSCs的体外HBV易感性。
结果如下：
1.15例乙型肝炎肝硬化患者AD-MSCs的体外培养成功率为100%（15/15），11例乙型肝炎肝硬化患者骨髓MSCs的体外培养成功率为63.6%（7/11）（P<0.05）；AD-MSCs的原代培养时间为（8.6±1.5）d，BM-MSCs的原代培养时间为（16.0±1.9）d，（P<0.05）；两种组织来源MSCs具有相似的形态，均呈梭形的成纤维细胞样细胞，AD-MSCs体外连续传代培养15代细胞无衰老征象，生长状态良好。AD-MSCs和BM-MSCs的体外培养生长曲线均呈“S”形，AD-MSCs在培养3~4d进入对数生长期，第7d进入平台期；BM-MSCs在培养4~5d进入对数生长期，第8d进入平台期。
流式细胞仪检测结果表明：5代AD-MSCs的（G2%+S%）期细胞为（16.52±5.48）%，而BM-MSCs的（G2%+S%）期细胞比例为（7.26±2.34）%，差异有统计学意义。AD-MSCs和BM-MSCs均高表达MSCs的特征性表面标记CD44、CD29和CD105，不表达造血干细胞的表面标志CD34。
乙型肝炎肝硬化患者AD-MSCs和BM-MSCs体外分化潜能无差异，体外均可以诱导分化为脂肪细胞、成骨细胞和神经细胞。
2.三步法体外成功诱导乙型肝炎肝硬化患者AD-MSCs分化为功能性的肝细胞，成肝细胞诱导培养后AD-MSCs细胞形态逐渐由梭形成纤维细胞样细胞分化成多角形，类肝细胞形态的细胞；诱导培养11和18d免疫组织化学染色结果显示：AD-MSCs表达肝细胞特异性标志物AFP、ALB和CK-18，AFP阳性细胞百分率分别为（45.6±6.3）%和（17.6±1.5）%，ALB阳性细胞百分率分别为（45.8±5.2）%和（78.9±8.6）%，CK-18阳性细胞百分率分别为（30.5±4.8）%和（70.4±9.3）%，与未诱导组比较差异有统计学意义（P<0.05）；Western blotting结果与免疫组织化学染色结果一致；诱导分化细胞具有成熟肝细胞特有糖原合成功能，诱导培养11和18d，PAS染色阳性细胞百分率分别为（41.2±8.5）%和（86.9±11.3）%，与未诱导组比较差异有统计学意义（P <0.05）；诱导分化细胞体外合成和分泌白蛋白，诱导培养11和18d培养液上清白蛋白浓度分别为（0.18±0.089）ug/ml和（0.23±0.098）ug/ml，与未诱导组比较差异有统计学意义（P <0.05）。
3.高滴度HBV感染血清培养后AD-MSCs的细胞形态、细胞生长曲线、生长速度及体外成脂肪细胞和成骨细胞分化潜能无改变；免疫组织化学染色和Western blotting技术检测原代培养的乙型肝炎肝硬化患者AD-MSCs、传代培养的3代、5代以及成肝诱导培养18d的AD-MSCs未感染HBV；高滴度HBV环境对AD-MSCs体外成肝细胞分化无影响，细胞形态变化与正常诱导方案诱导过程中细胞形态变化一致；诱导培养11和18dAFP表达阳性率分别为（42.4±7.8）%和（15.3±2.8）%，ALB表达阳性率分别为（39.4±6.8）%和（70.2±7.3），CK-18阳性率分别为（31.3±5.8）%和（63.2±8.3）%，与未诱导组比较差异有统计学意义（P<0.05）；细胞具有糖原合成功能，诱导11和18dPAS染色阳性细胞百分率分别为（37.4±7.6）%和（80.2±12.4）%，与未诱导组比较差异有统计学意义（P<0.05）。
综上研究表明，Ⅰ型胶原酶消化结合AD-MSCs贴壁生长的特性可以从皮下脂肪组织分离获得大量、高活性和高纯度的AD-MSCs；乙型肝炎肝硬化患者AD-MSCs在体外分离培养成功率和增殖速度等方面明显优于BM-MSCs，AD-MSCs体外易于分离培养，增殖迅速，在短期内即可获得大量的细胞；采用在培养过程中添加细胞因子和诱导剂的三步诱导法体外诱导乙型肝炎肝硬化患者AD-MSCs分化为有功能的肝细胞，表达肝细胞系特异性蛋白AFP、ALB和CK-18，具有成熟肝细胞特有的糖原和白蛋白合成功能；乙型肝炎肝硬化患者AD-MSCs未感染HBV，传代培养AD-MSCs和成肝细胞诱导分化AD-MSCs体外不感染HBV；在高滴度HBV环境中，AD-MSCs可以诱导分化为肝细胞，维持成熟肝细胞的具体功能，同时抵抗乙肝病毒感染。
本研究的创新点是在建立乙型肝炎肝硬化患者AD-MSCs体外培养体系的基础上，系统评价了其生物学特性，探讨其向肝细胞分化的潜能，同时观察AD-MSCs的HBV易感性及高滴度HBV环境对AD-MSCs成肝细胞分化的影响，为更加容易且安全的获取AD-MSCs，为临床乙型肝炎肝硬化患者应用自体AD-MSCs移植提供实验基础和理论依据。
Hepatitis B is one of the most common infectious diseases worldwide. There areapproximately350million chronic hepatitis B virus (HBV) carriers all of the world. Thetherapy for hepatitis B is very limited. HBV infection may cause liver cirrhosis andhepatocellular carcinoma. Following chronic liver damage, the regenerative ability ofhepatocyte is lost, which leaves the liver unable to maintain its functional mass. This isclinically so-called “liver failure”. Liver failure is an end-stage hepatic disease. In the presenttreatment condition, its mortality is very high, it is particularly important to find an effectivetreatment method.
In recent years, stem cells research has got outstanding achievement in the field ofmedicine and opened up new ideas for our clinical doctors. Stem cells transplantation in thetreatment of end-stage liver disease becomes the new hot spot. Adipose derived stem cells(ADSCs) morphology is similarity with BM-MSCs, which were also known as adiposederived-mesenchymal stem cells (AD-MSCs). AD-MSCs have the multi-directionaldifferentiation potential, which can differentiate into osteoblasts, chondrocytes, adipocytes,and muscle cells of mesodermal mesenchyme cells, hepatocyte in the mesoderm and neuralcells of the ectoderm. AD-MSCs have a rich source, can be repeatedly drawn, and lessinvolved in medical ethics concern.
In the present study, we isolated AD-MSCs from hepatitis B liver cirrhosis patients andcharacterized for morphology, growth potency, surface phenotype and the differentiationpotential, and compared these with those of BM-MSCs which were isolated from hepatitis Bliver cirrhosis patients. The ability of AD-MSCs to differentiate into hepatocyte wasevaluated. In addition, we investigate the effect of high titer HBV environment on itsbiological characteristics and hepatic differentiation, assessed whether HBV can infect bothAD-MSCs and AD-MSC-derived hepatocyte-like cells in vitro.
AD-MSC were isolated from liver cirrhosis patients subcutaneous fat by collagenasedigestion and adhering to the culture plastic, establish the culture system of AD-MSC in vitro, investigate the biological characteristics of AD-MSC, and compare with BM-MSCs. Theculture ratio and primary culture time were observed.After successive subculture andamplification， the growth curve were drawn, the morphology were observed undermicroscope. The cell cycle and surface marker were detected by flowcytometer. Thedifferentiation potential to osteoblasts、adipocyte and neuronal cell were observed.
ADSCs were induced into hepatocyte in vitro, the morphological changes were observed,The expression of AFP, ALB and CK-18were detected by immunohistochemical staining andwestern blotting technology, periodic acid Schiff (PAS) reagent staining for glycogensynthesis function, ELISA technology for albumin synthesis and secretion; AD-MSC culturein high titer HBV infection serum in vitro, observe the morphological changes and growthcurve, detect the expression of HBsAg and HBcAg by immunohistochemistry and Westernblotting technology, and observe the effect of hepatic differentiation of AD-MSC culturedwith high titer HBV environment. The result is following:
1. The success ration of AD-MSCs and BM-MSCs were100%(15/15) and63.6%(7/11),respectively. The primary passage times of AD-MSCs and BM-MSCs were (8.6±1.5) and(16.0±1.9) days, respectively (p <0.05). AD-MSCs and BM-MSCs have similar morphology,showed a fibroblast like cells of spindle shaped. AD-MSCs continuously culture for15passages in vitro and the cells grew well. The growth curves of MSCs from the two sourceswere “S”shape AD-MSCs came into a logarithmic phase at days3–4, reached the peak at day6, and then came into platform at day7. BM-MSCs came into logarithmic phase at days4–5,reached the peak at days8, and then came into platform at day9. Furthermore, DNA contentof AD-MSCs and BM-MSCs were measured by flow cytometry. The percentage of S-phasenuclei in AD-MSCs was (9.25±1.38)%, which compared with (5.26±1.24)%, the S-phase ofBM-MSCs nuclei, the proportion of S-phase AD-MSCs was significantly higher than that ofBM-MSCs (p<0.05). Both AD-MSCs and BM-MSCs from hepatitis B liver cirrhosis patientshave the potential of adipogenic, osteogenic and neurons differentiation, which are consistentwith previous reports. The surface marker of the5rd generation AD-MSCs and BM-MSCswere analyzed and the results are consistent with previous report. Nearly all the cellsexpressed CD44, CD29and CD105, which are the surface marker characteristics of MSCs. The absence of contaminating hematopoietic cells in the MSCs population was verified by thelack of surface antigen defining hematopoietic progenitor cells (CD34).
2. AD-MSCs can differentiate into hepatocytes by three-step protocol. The fibroblast-like AD-MSCs were changed into hepatocyte-like morphology. The immunocytochemicalstaining results showed that undifferentiated cells were negative for AFP, ALB and CK-18.AD-MSCs were cultured in accordance with the three-step protocol at day11and day18, thepositive rates of AFP were (45.6±6.3)%and (17.6±1.5)%, respectively; the positive rate ofALB were (45.8±5.2)%and (78.9±8.6)%, respectively; the positive rate of CK-18were(30.5±4.8)%and (70.4±9.3)%, respectively. Compared with the un-induced group, thedifferences are statistically significant (p<0.05). The Western blotting results showed thesame results as that of immunocytochemical staining. We assayed glycogen storage of thedifferentiated AD-MSCs by periodic acid-Schiff (PAS) staining. AD-MSCs showed noactivity of glycogen production in undifferentiated group. Glycogen staining was positive inAD-MSCs after they were exposed to hepatic differentiation medium at days11and18d, thepositive rate were (41.2±8.5)%and (86.9±11.3)%, respectively. Compared with theun-induced group, the differences are statistically significant (p<0.05). In addition, ELISAanalyses showed that differentiated AD-MSCs had significantly higher secreted albumin atday11(0.18±0.089μg/mL) and day18(0.23±0.098μg/mL) than undifferentiated AD-MSCs(0.0±0.012μg/mL). Compared with the un-induced group, the differences are statisticallysignificant (p <0.05).
3. There were no influence on the morphology, growth curve and the potential ofadipogenic, osteogenic differentiation of AD-MSCs which were incubated in the high titerHBV infectious environment.The immunocytochemical staining and western blotting resultsshowed that AD-MSCs and the hepatocyte-like cells from AD-MSCs were not susceptible toinfection by HBV.There were no influence on hepatic differentiation of AD-MSCs whichwere were incubated in the high titer HBV infectious environment.. The immunocytochemicalstaining results showed that undifferentiated cells were negative for AFP, ALB and CK-18.AD-MSCs were cultured in accordance with the three-step protocol at day11and day18, thepositive rates of AFP were (42.4±7.8)%and (15.3±2.8)%, respectively; the positive rate ofALB were (39.4±6.8)%and (70.2±7.3)%, respectively; the positive rate of CK-18were (31.3±5.8)%and (63.2±8.3)%, respectively. Compared with the un-induced group, thedifferences are statistically significant (p<0.05). Glycogen staining was positive in AD-MSCsafter they were exposed to hepatic differentiation medium at days11and18, the positive ratewere (37.4±7.6)%and (80.2±12.4)%, respectively. Compared with the un-induced group,the differences are statistically significant (p <0.05).
Comprehensive studies have shown that a number of highly active purified AD-MSCswere isolated by collagenase digestion and adhering to the culture plastic.AD-MSCs fromhepatitis B liver cirrhosis patients have a similar differentiation potential towards the hepaticlineage as BM-MSCs, but their abundance, accessibility and higher proliferation capacitydiffer from BM-MSCs. Under certain defined inducing conditions, they can differentiatetoward a hepatic phenotype in vitro and have hepatic biochemical functions. In addition,AD-MSCs and hepatic differentiated AD-MSCs were not susceptible to infection by HBV.There were no influence on hepatic differentiation of AD-MSCs which were were incubatedin the high titer HBV infectious environment.Therefore, adipose tissue seems to be an idealsource of high large amounts of autologous multilineage MSCs for cell therapy of liverdysfunction for hepatitis B liver cirrhosis patients.
In summary, the innovative point of this study was systematic evaluation of thebiological characteristics of AD-MSCs from liver cirrhosis patients and investigated thedirectional differentiation potential to hepatocytes. At the same time, we observe the HBVsusceptibility of AD-MSCs and the influence of high titer HBV environment ondifferentiation of AD-MSCs into hepatocytes. The results may provide the experimental basisand theoretical basis for the therapy of liver cirrhosis patients.
近年干细胞的研究在医学领域取得令人瞩目的成就，也为广大临床工作者开拓了新的思路，利用干细胞移植治疗终末期肝病成为新的热点。ADSCs因其与BM-MSCs形态相似亦被称为AD-MSCs。AD-MSCs具有多向分化潜能，不仅可以分化为成骨细胞、软骨细胞、脂肪细胞和肌肉细胞等中胚层间质组织细胞，还可以跨越胚层界限分化为外胚层的神经细胞及内胚层的肝细胞，另外AD-MSCs具有来源丰富、反复取材和较少涉及医学伦理学问题而倍受关注。
本研究旨在建立乙型肝炎肝硬化患者皮下脂肪组织MSCs体外培养体系的基础上，探讨其生物学特性，并与乙型肝炎肝硬化患者骨髓来源MSCs的生物学特性进行比较，同时观察其体外增殖能力及向肝细胞分化的潜能，探讨高滴度HBV环境对其生物学特性的影响及AD-MSCs的体外HBV易感性，为AD-MSCs的基础研究和临床应用提供理论基础和技术平台。
本研究采用胶原酶消化结合AD-MSCs的贴壁生长的特性分离培养乙型肝炎肝硬化患者皮下脂肪组织MSCs，建立AD-MSCs的体外培养体系，观察AD-MSCs的生物学特性，并与乙型肝炎肝硬化患者BM-MSCs的生物学特性进行比较；体外诱导AD-MSCs向肝细胞分化，观察细胞形态变化，免疫组织化学染色和Western blotting技术检测肝细胞系特异性标志物AFP、ALB和CK-18表达，过碘酸雪夫试剂染色检测细胞糖原合成功能，ELISA技术检测细胞白蛋白合成与分泌功能；采用高滴度HBV感染血清体外培养AD-MSCs，观察AD-MSCs生长状况和形态变化，免疫组织化学染色和Western blotting技术检测细胞内HBsAg和HBcAg表达，同时观察高滴度HBV环境对AD-MSCs成肝细胞分化的影响及AD-MSCs的体外HBV易感性。
结果如下：
1.15例乙型肝炎肝硬化患者AD-MSCs的体外培养成功率为100%（15/15），11例乙型肝炎肝硬化患者骨髓MSCs的体外培养成功率为63.6%（7/11）（P<0.05）；AD-MSCs的原代培养时间为（8.6±1.5）d，BM-MSCs的原代培养时间为（16.0±1.9）d，（P<0.05）；两种组织来源MSCs具有相似的形态，均呈梭形的成纤维细胞样细胞，AD-MSCs体外连续传代培养15代细胞无衰老征象，生长状态良好。AD-MSCs和BM-MSCs的体外培养生长曲线均呈“S”形，AD-MSCs在培养3~4d进入对数生长期，第7d进入平台期；BM-MSCs在培养4~5d进入对数生长期，第8d进入平台期。
流式细胞仪检测结果表明：5代AD-MSCs的（G2%+S%）期细胞为（16.52±5.48）%，而BM-MSCs的（G2%+S%）期细胞比例为（7.26±2.34）%，差异有统计学意义。AD-MSCs和BM-MSCs均高表达MSCs的特征性表面标记CD44、CD29和CD105，不表达造血干细胞的表面标志CD34。
乙型肝炎肝硬化患者AD-MSCs和BM-MSCs体外分化潜能无差异，体外均可以诱导分化为脂肪细胞、成骨细胞和神经细胞。
2.三步法体外成功诱导乙型肝炎肝硬化患者AD-MSCs分化为功能性的肝细胞，成肝细胞诱导培养后AD-MSCs细胞形态逐渐由梭形成纤维细胞样细胞分化成多角形，类肝细胞形态的细胞；诱导培养11和18d免疫组织化学染色结果显示：AD-MSCs表达肝细胞特异性标志物AFP、ALB和CK-18，AFP阳性细胞百分率分别为（45.6±6.3）%和（17.6±1.5）%，ALB阳性细胞百分率分别为（45.8±5.2）%和（78.9±8.6）%，CK-18阳性细胞百分率分别为（30.5±4.8）%和（70.4±9.3）%，与未诱导组比较差异有统计学意义（P<0.05）；Western blotting结果与免疫组织化学染色结果一致；诱导分化细胞具有成熟肝细胞特有糖原合成功能，诱导培养11和18d，PAS染色阳性细胞百分率分别为（41.2±8.5）%和（86.9±11.3）%，与未诱导组比较差异有统计学意义（P <0.05）；诱导分化细胞体外合成和分泌白蛋白，诱导培养11和18d培养液上清白蛋白浓度分别为（0.18±0.089）ug/ml和（0.23±0.098）ug/ml，与未诱导组比较差异有统计学意义（P <0.05）。
3.高滴度HBV感染血清培养后AD-MSCs的细胞形态、细胞生长曲线、生长速度及体外成脂肪细胞和成骨细胞分化潜能无改变；免疫组织化学染色和Western blotting技术检测原代培养的乙型肝炎肝硬化患者AD-MSCs、传代培养的3代、5代以及成肝诱导培养18d的AD-MSCs未感染HBV；高滴度HBV环境对AD-MSCs体外成肝细胞分化无影响，细胞形态变化与正常诱导方案诱导过程中细胞形态变化一致；诱导培养11和18dAFP表达阳性率分别为（42.4±7.8）%和（15.3±2.8）%，ALB表达阳性率分别为（39.4±6.8）%和（70.2±7.3），CK-18阳性率分别为（31.3±5.8）%和（63.2±8.3）%，与未诱导组比较差异有统计学意义（P<0.05）；细胞具有糖原合成功能，诱导11和18dPAS染色阳性细胞百分率分别为（37.4±7.6）%和（80.2±12.4）%，与未诱导组比较差异有统计学意义（P<0.05）。
综上研究表明，Ⅰ型胶原酶消化结合AD-MSCs贴壁生长的特性可以从皮下脂肪组织分离获得大量、高活性和高纯度的AD-MSCs；乙型肝炎肝硬化患者AD-MSCs在体外分离培养成功率和增殖速度等方面明显优于BM-MSCs，AD-MSCs体外易于分离培养，增殖迅速，在短期内即可获得大量的细胞；采用在培养过程中添加细胞因子和诱导剂的三步诱导法体外诱导乙型肝炎肝硬化患者AD-MSCs分化为有功能的肝细胞，表达肝细胞系特异性蛋白AFP、ALB和CK-18，具有成熟肝细胞特有的糖原和白蛋白合成功能；乙型肝炎肝硬化患者AD-MSCs未感染HBV，传代培养AD-MSCs和成肝细胞诱导分化AD-MSCs体外不感染HBV；在高滴度HBV环境中，AD-MSCs可以诱导分化为肝细胞，维持成熟肝细胞的具体功能，同时抵抗乙肝病毒感染。
本研究的创新点是在建立乙型肝炎肝硬化患者AD-MSCs体外培养体系的基础上，系统评价了其生物学特性，探讨其向肝细胞分化的潜能，同时观察AD-MSCs的HBV易感性及高滴度HBV环境对AD-MSCs成肝细胞分化的影响，为更加容易且安全的获取AD-MSCs，为临床乙型肝炎肝硬化患者应用自体AD-MSCs移植提供实验基础和理论依据。
Hepatitis B is one of the most common infectious diseases worldwide. There areapproximately350million chronic hepatitis B virus (HBV) carriers all of the world. Thetherapy for hepatitis B is very limited. HBV infection may cause liver cirrhosis andhepatocellular carcinoma. Following chronic liver damage, the regenerative ability ofhepatocyte is lost, which leaves the liver unable to maintain its functional mass. This isclinically so-called “liver failure”. Liver failure is an end-stage hepatic disease. In the presenttreatment condition, its mortality is very high, it is particularly important to find an effectivetreatment method.
In recent years, stem cells research has got outstanding achievement in the field ofmedicine and opened up new ideas for our clinical doctors. Stem cells transplantation in thetreatment of end-stage liver disease becomes the new hot spot. Adipose derived stem cells(ADSCs) morphology is similarity with BM-MSCs, which were also known as adiposederived-mesenchymal stem cells (AD-MSCs). AD-MSCs have the multi-directionaldifferentiation potential, which can differentiate into osteoblasts, chondrocytes, adipocytes,and muscle cells of mesodermal mesenchyme cells, hepatocyte in the mesoderm and neuralcells of the ectoderm. AD-MSCs have a rich source, can be repeatedly drawn, and lessinvolved in medical ethics concern.
In the present study, we isolated AD-MSCs from hepatitis B liver cirrhosis patients andcharacterized for morphology, growth potency, surface phenotype and the differentiationpotential, and compared these with those of BM-MSCs which were isolated from hepatitis Bliver cirrhosis patients. The ability of AD-MSCs to differentiate into hepatocyte wasevaluated. In addition, we investigate the effect of high titer HBV environment on itsbiological characteristics and hepatic differentiation, assessed whether HBV can infect bothAD-MSCs and AD-MSC-derived hepatocyte-like cells in vitro.
AD-MSC were isolated from liver cirrhosis patients subcutaneous fat by collagenasedigestion and adhering to the culture plastic, establish the culture system of AD-MSC in vitro, investigate the biological characteristics of AD-MSC, and compare with BM-MSCs. Theculture ratio and primary culture time were observed.After successive subculture andamplification， the growth curve were drawn, the morphology were observed undermicroscope. The cell cycle and surface marker were detected by flowcytometer. Thedifferentiation potential to osteoblasts、adipocyte and neuronal cell were observed.
ADSCs were induced into hepatocyte in vitro, the morphological changes were observed,The expression of AFP, ALB and CK-18were detected by immunohistochemical staining andwestern blotting technology, periodic acid Schiff (PAS) reagent staining for glycogensynthesis function, ELISA technology for albumin synthesis and secretion; AD-MSC culturein high titer HBV infection serum in vitro, observe the morphological changes and growthcurve, detect the expression of HBsAg and HBcAg by immunohistochemistry and Westernblotting technology, and observe the effect of hepatic differentiation of AD-MSC culturedwith high titer HBV environment. The result is following:
1. The success ration of AD-MSCs and BM-MSCs were100%(15/15) and63.6%(7/11),respectively. The primary passage times of AD-MSCs and BM-MSCs were (8.6±1.5) and(16.0±1.9) days, respectively (p <0.05). AD-MSCs and BM-MSCs have similar morphology,showed a fibroblast like cells of spindle shaped. AD-MSCs continuously culture for15passages in vitro and the cells grew well. The growth curves of MSCs from the two sourceswere “S”shape AD-MSCs came into a logarithmic phase at days3–4, reached the peak at day6, and then came into platform at day7. BM-MSCs came into logarithmic phase at days4–5,reached the peak at days8, and then came into platform at day9. Furthermore, DNA contentof AD-MSCs and BM-MSCs were measured by flow cytometry. The percentage of S-phasenuclei in AD-MSCs was (9.25±1.38)%, which compared with (5.26±1.24)%, the S-phase ofBM-MSCs nuclei, the proportion of S-phase AD-MSCs was significantly higher than that ofBM-MSCs (p<0.05). Both AD-MSCs and BM-MSCs from hepatitis B liver cirrhosis patientshave the potential of adipogenic, osteogenic and neurons differentiation, which are consistentwith previous reports. The surface marker of the5rd generation AD-MSCs and BM-MSCswere analyzed and the results are consistent with previous report. Nearly all the cellsexpressed CD44, CD29and CD105, which are the surface marker characteristics of MSCs. The absence of contaminating hematopoietic cells in the MSCs population was verified by thelack of surface antigen defining hematopoietic progenitor cells (CD34).
2. AD-MSCs can differentiate into hepatocytes by three-step protocol. The fibroblast-like AD-MSCs were changed into hepatocyte-like morphology. The immunocytochemicalstaining results showed that undifferentiated cells were negative for AFP, ALB and CK-18.AD-MSCs were cultured in accordance with the three-step protocol at day11and day18, thepositive rates of AFP were (45.6±6.3)%and (17.6±1.5)%, respectively; the positive rate ofALB were (45.8±5.2)%and (78.9±8.6)%, respectively; the positive rate of CK-18were(30.5±4.8)%and (70.4±9.3)%, respectively. Compared with the un-induced group, thedifferences are statistically significant (p<0.05). The Western blotting results showed thesame results as that of immunocytochemical staining. We assayed glycogen storage of thedifferentiated AD-MSCs by periodic acid-Schiff (PAS) staining. AD-MSCs showed noactivity of glycogen production in undifferentiated group. Glycogen staining was positive inAD-MSCs after they were exposed to hepatic differentiation medium at days11and18d, thepositive rate were (41.2±8.5)%and (86.9±11.3)%, respectively. Compared with theun-induced group, the differences are statistically significant (p<0.05). In addition, ELISAanalyses showed that differentiated AD-MSCs had significantly higher secreted albumin atday11(0.18±0.089μg/mL) and day18(0.23±0.098μg/mL) than undifferentiated AD-MSCs(0.0±0.012μg/mL). Compared with the un-induced group, the differences are statisticallysignificant (p <0.05).
3. There were no influence on the morphology, growth curve and the potential ofadipogenic, osteogenic differentiation of AD-MSCs which were incubated in the high titerHBV infectious environment.The immunocytochemical staining and western blotting resultsshowed that AD-MSCs and the hepatocyte-like cells from AD-MSCs were not susceptible toinfection by HBV.There were no influence on hepatic differentiation of AD-MSCs whichwere were incubated in the high titer HBV infectious environment.. The immunocytochemicalstaining results showed that undifferentiated cells were negative for AFP, ALB and CK-18.AD-MSCs were cultured in accordance with the three-step protocol at day11and day18, thepositive rates of AFP were (42.4±7.8)%and (15.3±2.8)%, respectively; the positive rate ofALB were (39.4±6.8)%and (70.2±7.3)%, respectively; the positive rate of CK-18were (31.3±5.8)%and (63.2±8.3)%, respectively. Compared with the un-induced group, thedifferences are statistically significant (p<0.05). Glycogen staining was positive in AD-MSCsafter they were exposed to hepatic differentiation medium at days11and18, the positive ratewere (37.4±7.6)%and (80.2±12.4)%, respectively. Compared with the un-induced group,the differences are statistically significant (p <0.05).
Comprehensive studies have shown that a number of highly active purified AD-MSCswere isolated by collagenase digestion and adhering to the culture plastic.AD-MSCs fromhepatitis B liver cirrhosis patients have a similar differentiation potential towards the hepaticlineage as BM-MSCs, but their abundance, accessibility and higher proliferation capacitydiffer from BM-MSCs. Under certain defined inducing conditions, they can differentiatetoward a hepatic phenotype in vitro and have hepatic biochemical functions. In addition,AD-MSCs and hepatic differentiated AD-MSCs were not susceptible to infection by HBV.There were no influence on hepatic differentiation of AD-MSCs which were were incubatedin the high titer HBV infectious environment.Therefore, adipose tissue seems to be an idealsource of high large amounts of autologous multilineage MSCs for cell therapy of liverdysfunction for hepatitis B liver cirrhosis patients.
In summary, the innovative point of this study was systematic evaluation of thebiological characteristics of AD-MSCs from liver cirrhosis patients and investigated thedirectional differentiation potential to hepatocytes. At the same time, we observe the HBVsusceptibility of AD-MSCs and the influence of high titer HBV environment ondifferentiation of AD-MSCs into hepatocytes. The results may provide the experimental basisand theoretical basis for the therapy of liver cirrhosis patients.
引文
[1] Liaw, Y.F.; Chu, C.M. Hepatitis B virus infection. Lancet2009,337,582–592.
[2] Hou, J.; Schilling, R.; Janssen, H. L., et al. Genetic characteristics of hepatitis B virusgenotypes as a factor for interferon-induced HBeAg clearance[J]. J Med Virol,2007,79(8):1055-1063.
[3] Jardi, R.; Rodriguez-Frias, F.; Schaper, M., et al. Analysis of hepatitis B genotypechanges in chronic hepatitis B infection: Influence of antiviral therapy[J]. J Hepatol,2008,49(5):695-701.
[4] Liu, C. J.; Kao, J. H. Genetic variability of hepatitis B virus and response to antiviraltherapy[J]. Antivir Ther,2008,13(5):613-624.
[5] Piccini JP，Hranitzky P. Diagnostic monitoring strategies in heart failure management[J]. Am Heart J,2007,153(4Suppl):12-17.
[6]宫铁红,刘玉乔,马艳丽,等.人工肝血浆置换治疗肝衰竭疗效观察[J].现代中西医结合杂志,2011,20(19):2374-2375.
[7]邢汉前,刘俊薇,王开利,等.持续缓慢血浆透析滤过治疗肝功能衰竭的临床疗效分析[J].2013,3,17(2):152-155.
[8] Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS,Jones JM. Embryonic stem cell lines derived from human blastocysts. Science1998;282:1145-1147
[9] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S.Induction of pluripotent stem cells from adult human fbroblasts by defned factors. Cell2007;131:861-872.
[10] Campagnoli C, Roberts IA, Kumar S, et al. Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. Blood,2001,98:2396-2402
[11] Hu Y, Liao L, Wang Q, Ma L, et al. Isolation and identification of mesenchymal stemcells from human fetal pancreas. J. Lab. Clin. Med,2003,141:342-349.
[12] Romanov YA, Svintsitskaya VA and Smirnov VN.Searching for alternative sources ofpostnatal human mesenchymal stem cells: candidate MSC-like cells from umbilicalcord. Stem Cells,2003,21,105-110.
[13] Krause DS, Theise ND, Collector MI, et al.Multi-Organ, Multi-Lineage Engraftmentby a Single Bone Marrow-Derived Stem Cell. Cell,2001,105:369-377.
[14] Pettenger MF, Mackay AM, Beck SC, et al. Multilineage Potential of Adult HumanMesenchymal Stem Cells. Science,1999,284:143-147.
[15] Woodbury D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrowstromal cells differentiate into neurons. J. Neurosci. Res,2000,61::364-370.
[16]殷莉波,赵文秀,尹震宇,等.体内外人脂肪间充质干细胞向肝样细胞分化的实验研究.中华肝胆外科杂志,2011,17(4):322-327
[17] Yang XF, He X, He J, et a1. High efficient isolation and systematic identification ofhuman adipose-derived mesenchymal stem cells[J]. J Biomed Sci,2011,18:59-68．
[18]闵敏,张雪静,马红,等.人脂肪间充质干细胞的原代培养及体外成骨成脂诱导分化,江苏大学学报(医学版),2013,23(3):185-190.
[19] Yang XF, He X, He J, et a1. High efficient isolation and systematic identification ofhuman adipose-derived mesenchymal stem cells[J]. J Biomed Sci,2011,18:59-68.
[20] Gruber HE, Somayaji S, Riley F, et a1. Human adipose-derived mesenchymal stemcells: serial passaging, doubling time and cell senescence[J]. Biotech Histochem,2012,87:303-311.
[21] Wen-Li Zhou, Claire N Medine, Liang Zhu, David C Hay. Stem cell differentiationand human liver disease. World J Gastroenterol.2012,18(17):2018-2025.
[22] Guenahel H. Danet, Jennifer L.Luongo, Gary Butler et al. C1qRpdefines a newhuman stem cell population with hematopoietic and hepatic potential. PNAS,2002;99(16):10441-10445.
[23] Friendenstein A, Chailakhyan R, Gerasimov U.Bone marrow osteogenic stem cells:invitro cultivation and transplantion in diffusion chambers[J]. Cell Tissue Kinet,1987,20(3):263-272.
[24] Thesis ND, Badve S, Saxena R, et al. Derivation of hepatocutes from bone marrowcells in mice after radiation-induced myeloablation[J]. Hepatology,2000,31(1):235-240.
[25] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult humanmesenchymal stem cells[J].Science,1999,284(5411):143-147.
[26] Zuk PA, Zhu M, Ashjian P, et a1. Human adipose tissue is a source of multipotent stemcells.Mol Biol Cell,2002,13l:4279-4295.
[27] Gruber HE, Somayaji S,Riley F, et a1. Human adipose-derived mesenchymal stemcells: serial passaging, doubling time and cell senescence[J]. Biotech Histochem,2012,87:303-311.
[28] Guenahel H. Danet,Jennifer L.Luongo,Gary Butler, et al. C1qRpdefines a new humanstem cell population with hematopoietic and hepatic potential. PNAS,2002;99(16):10441-10445.
[29] Encina NR, Billotte WG, Hofmann MC, et al. Immunomagnetic isolation ofosteoprogenitors from human bone marrow stromal. Lab Invest,1999,79:449-457.
[30] Zohar R, Sodek J, Mc Cullon CA. Characterization of stromal progenitor enriched byflowcytometry[J]. Blood,1998,90(9):3471-3481
[31] Schwartz RE, Reyes M, Koodie L, et al. Multipotent adult progenitor cells from bonemarrow differentiate into functional hepatocyte-like cells. J Clin Invest,2002,109:1291-1302.
[32]范颍,杜珍武,张玉成,等.成人脂肪间充质于细胞的分离、培养与鉴定.中国体视学与图像分析,2009,14(4):406-412.
[33]李传江,王万明.兔脂肪组织来源干细胞体外生长特性及培养的适宜条件.中国组织工程研究与临床康复,2009,13(19):3685-3688．
[34]杨民,徐祝军,董利军,等.大鼠脂肪间充质干细胞体外培养及其分化研究.皖南医学院学报,2009,28(1):4-8.
[35] Gronthos S, Franklin DM,Leddy HA,et a1. Surface protein characterization of humanadipose tissue-derived stromal cells.Cell Physi.2001:189(1)54-63
[36]房林,宋维铭.脂肪间充质干细胞多向分化潜能的特征.中国组织工程研究与临床康复,2008,12(29):5704-5708．
[37] De Ugarte DA, Morizono K, Elbarbary A, et a1. Comparison of mufti-lineage cellsfrom human adipose tissue and bone marrow. Cells Tissues Organs,2003,174(3):101-109.
[38] Winter A, Breit S, Parsch D, et a1. Cartilage-1ike gene expression in differentiatedhuman stem cell spheroids: a comparison of bone marrow-derived and adipose tissue-derived stromal cells. Arthritis Rheum,2003,48(2):418-429.
[39] Brzoska M, Geiger H, Gauer S, et al. Epithelial differentiation of human adiposetissue-derived adult stem cells. Biochem Biophys Res Commun.2005,330(11):142-150.
[40] Ashjian PH, Elbarbary AS, Edmonds B, et a1. In vitro differentiation of humanprocessed lipoaspirate cells into early neural progenitors. Plast ReconstrSurg.2003;111(6):1922-1931.
[41] Nakagami H, Morishita R, Maeda K, et a1. Adipose tissue-derived stromal cells as anovel option for regenerative cell therapy. J Atheroscler Thromb.2006,13(2):77-81.
[42] Fraser JK, Wulur I, AIfonso Z, et al. Fat tissue: an underappreciated source of stemcells for biotechnology.Trends Biotechn01.2006,24(4):150-154.
[43] Phanette Gir, Georgette Oni, Spencer A. Brown, et al. Human Adipose Stem Cells:Current Clinical Applications. Plastic and Reconstructive Surgery.2012,129(6):1277-1290.
[44] Mitchell JB,McIntosh K, Zvonic S,et al. Immunophenotype of human adipose-derivedcells: temporal changes in stromal-associated and stem cell-associated markers. StemCells,2006,24(2):376-385.
[45] Gimble JM, Guilak F. Differentiation potential of adipose derived adult stem (ADSC)cells. Curr. Top. Dev. Biol,2003,58:137-160.
[46] De Ugarte DA, Alfonso Z, Zuk PA, et al. Differential expression of stem cellmobilization-associated molecules on multi-lin eage cells from adipose tissue andbone marrow. Immunol Lett,2003,89(23):267-270.
[47] Varma MJ, Breuls RG, Schouten TE,et al. Phenotypical and functional characterizationof freshly isolated adipose tissue-derived stem cells [J].Stem Cells Dev.2007,16(1):91-104.
[48] Linyi Peng, Zhuqing Jia, Xinhua Yin, et al. Comparative Analysis of MesenchymalStem Cells fromBone Marrow, Cartilage, and Adipose Tissue.Stem cella anddevelopment.2008,17:761-774.
[49] Oh SH, Miyazaki M, Kouchi H, et al. Hepatocyte growth factor induce differentiationof adult rat bone marrow cells into a heoatocyte lineage in vitro[J]. Biochem BiophysRes Commun,2000,279:500-504.
[50] Pournasr B, Mohamadnejad M, Bagheri M, Aghdami N, Shahsavani M, et al. In vitrodifferentiation of human bone marrow mesenchymal stem cells into hepatocyte-likecells.Arch Iran Med2011,14:244-249
[51]张淑芹,叶东霞,刘殊荣.肝细胞生长因子诱导骨髓间充质干细胞表达甲胎蛋白和白蛋白.中国组织工程研究与临床康复杂志.2010,14(27):5041-5045.
[52]赵文静,陈亚杰,赵振国,等.肝病患者血清诱导人骨髓间充质干细胞表达甲胎蛋白和白蛋白的实验研究,2006,4(14):300-301
[53]赵文静,刘淑荣,刘薇,等.酒精性肝硬化患者血清诱导人骨髓间充质干细胞定向分化为肝细胞样细胞.中国组织工程研究与临床康复,2009,13(6):1025-1029
[54]方艳秋,赵文静,齐亚玲,等.损伤肝组织匀浆诱导间充质干细胞甲胎蛋白和白蛋白的表达[J].吉林大学学报(医学版),2008,34(6):971-974.
[55] Lange C, Bassler P, Lioznov, MV, et al. Hepatocytic gene expression in cultured ratmesenchymal stem cell[J].Thansplant Proc,2005,37(1):276-279.
[56] Schwartz RE, Reyes M, Koodie L, et al. Multipotent adult progenitor cells from bonemarrow differentiate into functional hepatocyte-like cells[J]. J Clinic Ivest,2002,109(10):1291-1302.
[57] Pengyu Huang, Ludi Zhang, Yimeng Gao, et al. Direct reprogramming of humanfibroblasts to functional and expandable hepatocyte. Cell stem cell,2014,14:370-384.
[58] Petersen B, Grossbard B, Hatch H, Pi L, Deng J, Scott EW. Mouse A6positive hepaticoval cells also express several hematopoietic stem cell markers. Hepatology2003;37:632–640.
[59] Lagasse E, Connors H, AlDhalimy M,et a1. Purified hematopoietic stem cells candifferentiate into hepatocytes in vivo. NatMed,2000,6:1229-1234.
[60] Ju S, Teng GJ, Lu H, Jin J, Zhang Y, Zhang A, NiY. In vivo differentiation ofmagnetically labeled mesenchymal stem cells into hepatocytes for cell therapy torepair damaged liver. Invest Radiol2010,45:625-633.
[61] Zhao DC, Lei JX, Chen R, et al. Bone marrow derived medenchymal stem cellsprotect against experimental liver fibrisis in rats.World J Gastro-enterol,2005,11:3431-3435
[62]周播江,钟脆平,顾云娣,等.肝再生大鼠血清诱导骨髓干细胞向肝细胞分化的实验研究[J].中华肝脏病杂志,2004,12(12):730-733.
[63] Abdel Aziz MT, Atta HM, Mahfouz S, et a1. Therapeutic potential of bone marrowderived mesenchymal stem cells on experimental liver fibrosis．Clin Biochem,2007,40:893-899.
[64] Mohammadnejad M, Alimoghaddam K, Mohyeddin-B0nab M, et al. Phase Ⅰtrail ofautilogous bone marrow mesenchymal stem cell transplantation in patients withdecompensated liver cirrhsis.Arch Iran Med,2007,10:459-468.
[65] Kharaziha P, Hellstrom, PM, Noorinayer B, et al. Improvement of liver function inliver cirrhosis patients after autologous mesenchymal stem cells injection: a phase Ⅰ-Ⅱclinical trial, Eur J Gastroenterol Hepatol,2009,21(10):1199-1205.
[66] Peng L, Zhu HP, Zheng YB, Xie C, Chong YT. Mesenchymal stem cells differentiateinto hepatocyte-like cells under different induction systems in hepatitis B patients withliver failure. Afr. J. Biotechnol.,2011,10:840-847.
[67] Feras AB, Joery DK, Tamara V, et al. Current status of human adipose-derived stemcells: differentiation into hepatocyte-like cells. The scientific world journal.2011,11:1568-1581.
[68] Seo MJ, Suh SY, Bae YC, et a1. Difierentiation of human adipose stromal cells intohepatic lineage in vitro and in vivo. Biochem Bioph Res Co,2005,328:258-264.
[69] Talens-Visconti, R., Bonora, A., Jover, R., et al. Hepatogenic differentiation of humanmesenchymal stem cells from adipose tissue in comparison with bone marrowmesenchymal stem cells. World J. Gastroenterol.2006,12,5834–5845.
[70] Banas, A.; Teratani, T. Adipose tissue-derived mesenchymal stem cells as a sources ofhuman hepatocytes. Hepatology2007,46,219–228.
[71] Aurich, I.; Mueller, L.P. Functional integration of hepatocytes derived from humanmesenchymal stem cells into mouse livers. Gut2007,56,405–415.
[72] Lee, J.H.; Lee, K.H. Possibility of undifferentiated human thigh adipose stem cellsdifferentiating into functional hepatocyte. Arch. Plast. Surg.2012,39,593–599.
[73] Banas, A., Teratani, T., Yamamoto, Y., et al. Rapid hepatic fate specification ofadipose-derived stem cells and their therapeutic potential for liver failure. J.Gastroenterol. Hepatol.2009,24,70–77.
[74] Snykers, S., Vanhaecke, T., De Becker, A., et al. Chromatin remodeling agenttrichostatin A: a key-factor in the hepatic differentiation of human mesenchymal stemcells derived of adult bone marrow. BMC Dev. Biol.2007,7,24.
[75]向贤宏,李家平,谭国胜,等.同种异体肝细胞与脂肪间质干细胞移植治疗肝硬化的实验对照研究.中华普通外科学文献.2011,5(1):12-16.
[76]柳林,杨海山,等.自体脂肪间充质干细胞治疗晚期肝病的初步临床研究.中国实验诊断学杂志,2007,11(4)473－476.
[77] Puglisi MA, Saulnier N, Piscaglia AC, et a1. Adipose tissue-derived mesenchymalstem cells and hepatic differentiation:old concepts and future perspectives[J]. Eur RevMed Pharmacol Sci,2011,15:355-364.
[78] De la Garza-Rodea AS, Van der Velde-van Dijke I, Boersma H, et al. Myogenicproperties of human mesenchymaI stem cells derived from three different sources [J].Cell Transplant,2012,21:153-173.
[79] Banas A, Teratani T, Yamamoto Y, et al. IFATS collection: in vivo therapeutic potentialof human adipose tissue mesenchymal stem cells after transplantation into mice withliver injury.Stem cells,2008,26:2705-2712.
[80] Salama H, Zekri AR, Bahnassy AA, et a1. Autologous CD34+and CDl33+stem cellstransplantation in patients with end stage liver disease．World J Gastroenterol,2010,16:5297-5305.
[81] Levicar N, Pai M, Habib NA, et al.Long term clinical results of autologous infusionof mobilized adult bone marrow derived CD34+cells in patients with chronic liverdisease.Cell Prolif,2008,41:115-125．
[82] Lyra AC, Soares MB, Silva VF, et al. Feasibility and safety of autologous bonemarrow mononuclear cell transplantation in patients with advanced chronic liverdisease. World J Gastroenterol.2007,13:1067-1073.
[83] Nikeghbalian S, Pournasr B, Aghdami N, et al. Autogolous transplantation of bonemarrow-derived mononuclear and CDl33+cells in patients with decompensatedcirrhosis. Arch Iran Med.2011,14:12-17.
[84] LIn H, Xu R, Zhang Z, et al. Implications of the immunoregulatory functions ofmesenchvmal stem cells in the treatment of human liver diseases. Cel Mol Immunol,2011,8:19-22.
[85] Fan, B.J.; Piao, Y.F. Bone marrow mesenchymal stem cell from chronic hepatitis Bpatients differentiation into hepatocyte-like cells. Afr. J. Microbiol. Res.2012,6,3866–3873.
[86] Liang Peng, Hua Li, Lin Gu, et al.Comparison of biological characteristics of marrowmesenchymal stem cells in hepatitis B patients and normal adults.World JGastroenterol2007,13(11):1743-1746.
[87] Yue-Si Zhong, Nan Lin, Mei-Hai Deng, et al. Deficient Proliferation of Bone Marrow-Derived Mesenchymal Stem Cells in Patients with Chronic Hepatitis B ViralInfections and Cirrhosis of the Liver. Dig Dis Sci,2010,55:438–445.
[88] Galmiche MC, Koteliansky VE, Briere J, et al. Stromal cells from human long termmarrow cultures are mesenchymal cells that differenti-ate following a vascularsmooth muscle differentiation pathway [J]. Blood,1993,82(1):66-67.
[89] Peng L, Zhu HP, Zheng YB, Xie C, Chong YT. Mesenchymal stem cells differentiateinto hepatocyte-like cells under different induction systems in hepatitis B patients withliver failure. African Journal of Biotechnology.,2011,10:840-847.
[90] Kou TK, Hung SP, Chuang CH, Chen CH, Shih YR, Fang SC, Yang VW. Stem celltherapy for liver disease:parameters governing the success of using bone marrowmesenchymal stem cell. Gastroenterology.,2008,134:2111-2121.
[91] Russo FP, Alison MR, Bigger BW, et al. The bone marrow functionally contribute toliver fibrosis[J].Gastroenterology,2006,130:1807-1821.
[92]中华医学会肝病学分会,中华医学会感染病学分会.慢性乙型肝炎防治指南(2010版).胃肠病学.2011,16(6):351-366.
[93] De Ugarte DA, Morizono K, Elbarbary A, Alfonso Z, Zuk PA, Zhu M, Dragoo JL,Ashjian P, Thomas B, Benhaim P, et al. Comparison of multi-lineage cells from humanadipose tissue and bone marrow. Cells Tissues Organs.2003;174:101–109
[94] Hong L, Peptan IA, Colpan A, Daw JL. Adipose tissue engineering by humanadipose-derived stromal cells. Cells Tissues Organs.2006;183:133–140.
[95] Alonso, M.; Claros, S. The effect of type I collagen on osteochondrogenicdifferentiation in adipose-derived stromal cells in vivo. Cytotherapy2008,10,597–610.
[96] Cardozo AJ, Gomez DE, Argibay PF. Neurogenic differentiation of human adipose-derived stem cells: relevance of different signaling molecules, transcription factors,and key marker genes. Gene.2012;511:427–436
[97] Gwak SJ, Bhang SH, Yang HS, Kim SS, Lee DH, Lee SH, Kim BS. In vitrocardiomyogenic differentiation of adipose-derived stromal cells using transforminggrowth factor-beta1. Cell Biochem Funct.2009;27:148–154
[98] Sterodimas A, de Faria J, Nicaretta B, Pitanguy I. Tissue engineering with adipose-derived stem cells (ADSCs): current and future applications. J Plast Reconstr AesthetSurg.2010;63:1886–1892.
[99] Eun-Hee Kim, Chan Yeong Heo. Current applications of adipose-derived stem cellsand their future perspectives.World J Stem Cells.2014;6(1):65–68.
[100] Zhou Z, Chen Y,Zhang H.et al.Comparison of mesenchymal stromal cells from humanbone marrow and adipose tissue for the treatment of spinal cord injury. Cytotherapy.2013,15(4):434-48.
[101] Locke M, Windsor J, Dunbar PR. Human adipose-derived stem cells: isolation,characterization and applications in surgery. ANZ J Surg.2009;79:235–244
[102]李春明,刘毅.脂肪干细胞及其在脂肪组织工程中的应用[J].中国美容医学,2006,4:466-468.
[103] Brayfield C, Marra K, Rubin JP. Adipose stem cells for soft tissue regeneration.Handchir Mikrochir Plast Chir.2010;42:124–128
[104] Cai L, Johnstone BH, Cook TG, Tan J, Fishbein MC, Chen PS, March KL. IFATScollection: Human adipose tissue-derived stem cells induce angiogenesis and nervesprouting following myocardial infarction, in conjunction with potent preservation ofcardiac function. Stem Cells.2009;27:230–237
[105] Wosnitza M, Hemmrich K, Groger A, Graber S, Pallua N. Plasticity of human adiposestem cells to perform adipogenic and endothelial differentiation. Differentiation2007;75:12–23.
[106] Strioga M, Viswanathan S, Darinskas A, Slaby O, Michalek J. Same or Not the Same?Comparison of Adipose Tissue-Derived Versus Bone Marrow-Derived MesenchymalStem and Stromal Cells. Stem Cells Dev.2012;21:2724–2752.
[107] Maumus M, Peyrafitte JA, D’Angelo R, Fournier-Wirth C, Bouloumie A, et al. Nativehuman adipose stromal cells: localization, morphology and phenotype. Int J Obes.2011;35:1141–1153.
[108] Crop M, Baan CC, Korevaar SS, Ijzermans JN, Weimar W, et al. Human adiposetissue-derived mesenchymal stem cells induce explosive T-cell proliferation. StemCells Dev.2010;19:1843–1853.
[109] Bunnel BA, Flaat, M, Gagliardi, et al. Adipose-derived stem cels: isolation, expansionand differentiation. Methods,2008,45:115-120.
[110] Han Chao, Zhang Liang, Song Lin, et al. Human adipose-derived mesenchymal stemcells: a better cell source for nervous system regeneration Chinese Medical Journal2014;127(2):329-337.
[111]李翰昊,晏雪生,明安萍,等.肝脏细胞条件培养基诱导大鼠骨髓间质细胞分化为肝细胞的作用[J].中西医结合病杂志,2005,15(1)28-30.
[112] Takano H, Qin Y, Hasegawa H, et al. Effects of G-CSF on left ventricular remodelingand heart failure after acute myocardial infraction[J]. J Mol Med2006,84(3):185-193.
[113] Jiang J, Jahagirdar RL, Reinhart RL, et al. Pluripotency of mesenchymal stem cellsderived from adult marrow. Nature2002,418(6893):41-49.
[114]何中杰,方驰华,马俊勋,等.肝细胞生长因子与表皮细胞生长因子联合诱导大鼠骨髓间充质干细胞分化为类肝细胞[J].解放军医学杂志,2006,5(31):446-449.
[115]胡维亭,任军.人乙型肝炎病毒DN阳性血清对人骨髓间充质干细胞向肝细胞分化的影响.北京大学学报,2002,4(5):459-464.
[116] Chan Xie, Yu-Bao Zheng, Hai-Peng Zhu, et al. Human bone marrow mesenhymalstem cells are resistant to HBV infection during differentiation in vivo and in vitro.Cell Biology International,2009,33:493-500.
[117] Ruipin Ma, Quantai Xing, Lihua Shao, et al. Hepatitis B virus and replication inhuman bone marrow mesenchymal stem cells.Virology Journal.2011,8:846-494.
[118] Wang Y, Weil BR, Herrmann JL, et a1. MEK, p38and PI-3K mediate cross talkbetween EGFR and TNFR in enhancing hepatocyte growth factor production fromhuman mesenchymal stem celis[J1．Am J Physiol Cell Physiol,2009,297(5):1284-1293
[119] Colletti E, Airey JA, Liu W, Simmons PJ, Zanjani ED, Porada CD, Almeida-Porada G.Generation of tissue-specific cells by MSC does not require fusion or donor to hostmitochondrial/membrane transfer. Stem Cell Res.2009;2:125–138.
[120] Valerie D. Roobrouck, Kim Vanuytsel, Catherine M. Verfaillie. Concise Review:Culture Mediated Changes in Fate and/or Potency of Stem Cells. Stem Cells;2011,29:583-589.
[2] Hou, J.; Schilling, R.; Janssen, H. L., et al. Genetic characteristics of hepatitis B virusgenotypes as a factor for interferon-induced HBeAg clearance[J]. J Med Virol,2007,79(8):1055-1063.
[3] Jardi, R.; Rodriguez-Frias, F.; Schaper, M., et al. Analysis of hepatitis B genotypechanges in chronic hepatitis B infection: Influence of antiviral therapy[J]. J Hepatol,2008,49(5):695-701.
[4] Liu, C. J.; Kao, J. H. Genetic variability of hepatitis B virus and response to antiviraltherapy[J]. Antivir Ther,2008,13(5):613-624.
[5] Piccini JP，Hranitzky P. Diagnostic monitoring strategies in heart failure management[J]. Am Heart J,2007,153(4Suppl):12-17.
[6]宫铁红,刘玉乔,马艳丽,等.人工肝血浆置换治疗肝衰竭疗效观察[J].现代中西医结合杂志,2011,20(19):2374-2375.
[7]邢汉前,刘俊薇,王开利,等.持续缓慢血浆透析滤过治疗肝功能衰竭的临床疗效分析[J].2013,3,17(2):152-155.
[8] Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS,Jones JM. Embryonic stem cell lines derived from human blastocysts. Science1998;282:1145-1147
[9] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S.Induction of pluripotent stem cells from adult human fbroblasts by defned factors. Cell2007;131:861-872.
[10] Campagnoli C, Roberts IA, Kumar S, et al. Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. Blood,2001,98:2396-2402
[11] Hu Y, Liao L, Wang Q, Ma L, et al. Isolation and identification of mesenchymal stemcells from human fetal pancreas. J. Lab. Clin. Med,2003,141:342-349.
[12] Romanov YA, Svintsitskaya VA and Smirnov VN.Searching for alternative sources ofpostnatal human mesenchymal stem cells: candidate MSC-like cells from umbilicalcord. Stem Cells,2003,21,105-110.
[13] Krause DS, Theise ND, Collector MI, et al.Multi-Organ, Multi-Lineage Engraftmentby a Single Bone Marrow-Derived Stem Cell. Cell,2001,105:369-377.
[14] Pettenger MF, Mackay AM, Beck SC, et al. Multilineage Potential of Adult HumanMesenchymal Stem Cells. Science,1999,284:143-147.
[15] Woodbury D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrowstromal cells differentiate into neurons. J. Neurosci. Res,2000,61::364-370.
[16]殷莉波,赵文秀,尹震宇,等.体内外人脂肪间充质干细胞向肝样细胞分化的实验研究.中华肝胆外科杂志,2011,17(4):322-327
[17] Yang XF, He X, He J, et a1. High efficient isolation and systematic identification ofhuman adipose-derived mesenchymal stem cells[J]. J Biomed Sci,2011,18:59-68．
[18]闵敏,张雪静,马红,等.人脂肪间充质干细胞的原代培养及体外成骨成脂诱导分化,江苏大学学报(医学版),2013,23(3):185-190.
[19] Yang XF, He X, He J, et a1. High efficient isolation and systematic identification ofhuman adipose-derived mesenchymal stem cells[J]. J Biomed Sci,2011,18:59-68.
[20] Gruber HE, Somayaji S, Riley F, et a1. Human adipose-derived mesenchymal stemcells: serial passaging, doubling time and cell senescence[J]. Biotech Histochem,2012,87:303-311.
[21] Wen-Li Zhou, Claire N Medine, Liang Zhu, David C Hay. Stem cell differentiationand human liver disease. World J Gastroenterol.2012,18(17):2018-2025.
[22] Guenahel H. Danet, Jennifer L.Luongo, Gary Butler et al. C1qRpdefines a newhuman stem cell population with hematopoietic and hepatic potential. PNAS,2002;99(16):10441-10445.
[23] Friendenstein A, Chailakhyan R, Gerasimov U.Bone marrow osteogenic stem cells:invitro cultivation and transplantion in diffusion chambers[J]. Cell Tissue Kinet,1987,20(3):263-272.
[24] Thesis ND, Badve S, Saxena R, et al. Derivation of hepatocutes from bone marrowcells in mice after radiation-induced myeloablation[J]. Hepatology,2000,31(1):235-240.
[25] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult humanmesenchymal stem cells[J].Science,1999,284(5411):143-147.
[26] Zuk PA, Zhu M, Ashjian P, et a1. Human adipose tissue is a source of multipotent stemcells.Mol Biol Cell,2002,13l:4279-4295.
[27] Gruber HE, Somayaji S,Riley F, et a1. Human adipose-derived mesenchymal stemcells: serial passaging, doubling time and cell senescence[J]. Biotech Histochem,2012,87:303-311.
[28] Guenahel H. Danet,Jennifer L.Luongo,Gary Butler, et al. C1qRpdefines a new humanstem cell population with hematopoietic and hepatic potential. PNAS,2002;99(16):10441-10445.
[29] Encina NR, Billotte WG, Hofmann MC, et al. Immunomagnetic isolation ofosteoprogenitors from human bone marrow stromal. Lab Invest,1999,79:449-457.
[30] Zohar R, Sodek J, Mc Cullon CA. Characterization of stromal progenitor enriched byflowcytometry[J]. Blood,1998,90(9):3471-3481
[31] Schwartz RE, Reyes M, Koodie L, et al. Multipotent adult progenitor cells from bonemarrow differentiate into functional hepatocyte-like cells. J Clin Invest,2002,109:1291-1302.
[32]范颍,杜珍武,张玉成,等.成人脂肪间充质于细胞的分离、培养与鉴定.中国体视学与图像分析,2009,14(4):406-412.
[33]李传江,王万明.兔脂肪组织来源干细胞体外生长特性及培养的适宜条件.中国组织工程研究与临床康复,2009,13(19):3685-3688．
[34]杨民,徐祝军,董利军,等.大鼠脂肪间充质干细胞体外培养及其分化研究.皖南医学院学报,2009,28(1):4-8.
[35] Gronthos S, Franklin DM,Leddy HA,et a1. Surface protein characterization of humanadipose tissue-derived stromal cells.Cell Physi.2001:189(1)54-63
[36]房林,宋维铭.脂肪间充质干细胞多向分化潜能的特征.中国组织工程研究与临床康复,2008,12(29):5704-5708．
[37] De Ugarte DA, Morizono K, Elbarbary A, et a1. Comparison of mufti-lineage cellsfrom human adipose tissue and bone marrow. Cells Tissues Organs,2003,174(3):101-109.
[38] Winter A, Breit S, Parsch D, et a1. Cartilage-1ike gene expression in differentiatedhuman stem cell spheroids: a comparison of bone marrow-derived and adipose tissue-derived stromal cells. Arthritis Rheum,2003,48(2):418-429.
[39] Brzoska M, Geiger H, Gauer S, et al. Epithelial differentiation of human adiposetissue-derived adult stem cells. Biochem Biophys Res Commun.2005,330(11):142-150.
[40] Ashjian PH, Elbarbary AS, Edmonds B, et a1. In vitro differentiation of humanprocessed lipoaspirate cells into early neural progenitors. Plast ReconstrSurg.2003;111(6):1922-1931.
[41] Nakagami H, Morishita R, Maeda K, et a1. Adipose tissue-derived stromal cells as anovel option for regenerative cell therapy. J Atheroscler Thromb.2006,13(2):77-81.
[42] Fraser JK, Wulur I, AIfonso Z, et al. Fat tissue: an underappreciated source of stemcells for biotechnology.Trends Biotechn01.2006,24(4):150-154.
[43] Phanette Gir, Georgette Oni, Spencer A. Brown, et al. Human Adipose Stem Cells:Current Clinical Applications. Plastic and Reconstructive Surgery.2012,129(6):1277-1290.
[44] Mitchell JB,McIntosh K, Zvonic S,et al. Immunophenotype of human adipose-derivedcells: temporal changes in stromal-associated and stem cell-associated markers. StemCells,2006,24(2):376-385.
[45] Gimble JM, Guilak F. Differentiation potential of adipose derived adult stem (ADSC)cells. Curr. Top. Dev. Biol,2003,58:137-160.
[46] De Ugarte DA, Alfonso Z, Zuk PA, et al. Differential expression of stem cellmobilization-associated molecules on multi-lin eage cells from adipose tissue andbone marrow. Immunol Lett,2003,89(23):267-270.
[47] Varma MJ, Breuls RG, Schouten TE,et al. Phenotypical and functional characterizationof freshly isolated adipose tissue-derived stem cells [J].Stem Cells Dev.2007,16(1):91-104.
[48] Linyi Peng, Zhuqing Jia, Xinhua Yin, et al. Comparative Analysis of MesenchymalStem Cells fromBone Marrow, Cartilage, and Adipose Tissue.Stem cella anddevelopment.2008,17:761-774.
[49] Oh SH, Miyazaki M, Kouchi H, et al. Hepatocyte growth factor induce differentiationof adult rat bone marrow cells into a heoatocyte lineage in vitro[J]. Biochem BiophysRes Commun,2000,279:500-504.
[50] Pournasr B, Mohamadnejad M, Bagheri M, Aghdami N, Shahsavani M, et al. In vitrodifferentiation of human bone marrow mesenchymal stem cells into hepatocyte-likecells.Arch Iran Med2011,14:244-249
[51]张淑芹,叶东霞,刘殊荣.肝细胞生长因子诱导骨髓间充质干细胞表达甲胎蛋白和白蛋白.中国组织工程研究与临床康复杂志.2010,14(27):5041-5045.
[52]赵文静,陈亚杰,赵振国,等.肝病患者血清诱导人骨髓间充质干细胞表达甲胎蛋白和白蛋白的实验研究,2006,4(14):300-301
[53]赵文静,刘淑荣,刘薇,等.酒精性肝硬化患者血清诱导人骨髓间充质干细胞定向分化为肝细胞样细胞.中国组织工程研究与临床康复,2009,13(6):1025-1029
[54]方艳秋,赵文静,齐亚玲,等.损伤肝组织匀浆诱导间充质干细胞甲胎蛋白和白蛋白的表达[J].吉林大学学报(医学版),2008,34(6):971-974.
[55] Lange C, Bassler P, Lioznov, MV, et al. Hepatocytic gene expression in cultured ratmesenchymal stem cell[J].Thansplant Proc,2005,37(1):276-279.
[56] Schwartz RE, Reyes M, Koodie L, et al. Multipotent adult progenitor cells from bonemarrow differentiate into functional hepatocyte-like cells[J]. J Clinic Ivest,2002,109(10):1291-1302.
[57] Pengyu Huang, Ludi Zhang, Yimeng Gao, et al. Direct reprogramming of humanfibroblasts to functional and expandable hepatocyte. Cell stem cell,2014,14:370-384.
[58] Petersen B, Grossbard B, Hatch H, Pi L, Deng J, Scott EW. Mouse A6positive hepaticoval cells also express several hematopoietic stem cell markers. Hepatology2003;37:632–640.
[59] Lagasse E, Connors H, AlDhalimy M,et a1. Purified hematopoietic stem cells candifferentiate into hepatocytes in vivo. NatMed,2000,6:1229-1234.
[60] Ju S, Teng GJ, Lu H, Jin J, Zhang Y, Zhang A, NiY. In vivo differentiation ofmagnetically labeled mesenchymal stem cells into hepatocytes for cell therapy torepair damaged liver. Invest Radiol2010,45:625-633.
[61] Zhao DC, Lei JX, Chen R, et al. Bone marrow derived medenchymal stem cellsprotect against experimental liver fibrisis in rats.World J Gastro-enterol,2005,11:3431-3435
[62]周播江,钟脆平,顾云娣,等.肝再生大鼠血清诱导骨髓干细胞向肝细胞分化的实验研究[J].中华肝脏病杂志,2004,12(12):730-733.
[63] Abdel Aziz MT, Atta HM, Mahfouz S, et a1. Therapeutic potential of bone marrowderived mesenchymal stem cells on experimental liver fibrosis．Clin Biochem,2007,40:893-899.
[64] Mohammadnejad M, Alimoghaddam K, Mohyeddin-B0nab M, et al. Phase Ⅰtrail ofautilogous bone marrow mesenchymal stem cell transplantation in patients withdecompensated liver cirrhsis.Arch Iran Med,2007,10:459-468.
[65] Kharaziha P, Hellstrom, PM, Noorinayer B, et al. Improvement of liver function inliver cirrhosis patients after autologous mesenchymal stem cells injection: a phase Ⅰ-Ⅱclinical trial, Eur J Gastroenterol Hepatol,2009,21(10):1199-1205.
[66] Peng L, Zhu HP, Zheng YB, Xie C, Chong YT. Mesenchymal stem cells differentiateinto hepatocyte-like cells under different induction systems in hepatitis B patients withliver failure. Afr. J. Biotechnol.,2011,10:840-847.
[67] Feras AB, Joery DK, Tamara V, et al. Current status of human adipose-derived stemcells: differentiation into hepatocyte-like cells. The scientific world journal.2011,11:1568-1581.
[68] Seo MJ, Suh SY, Bae YC, et a1. Difierentiation of human adipose stromal cells intohepatic lineage in vitro and in vivo. Biochem Bioph Res Co,2005,328:258-264.
[69] Talens-Visconti, R., Bonora, A., Jover, R., et al. Hepatogenic differentiation of humanmesenchymal stem cells from adipose tissue in comparison with bone marrowmesenchymal stem cells. World J. Gastroenterol.2006,12,5834–5845.
[70] Banas, A.; Teratani, T. Adipose tissue-derived mesenchymal stem cells as a sources ofhuman hepatocytes. Hepatology2007,46,219–228.
[71] Aurich, I.; Mueller, L.P. Functional integration of hepatocytes derived from humanmesenchymal stem cells into mouse livers. Gut2007,56,405–415.
[72] Lee, J.H.; Lee, K.H. Possibility of undifferentiated human thigh adipose stem cellsdifferentiating into functional hepatocyte. Arch. Plast. Surg.2012,39,593–599.
[73] Banas, A., Teratani, T., Yamamoto, Y., et al. Rapid hepatic fate specification ofadipose-derived stem cells and their therapeutic potential for liver failure. J.Gastroenterol. Hepatol.2009,24,70–77.
[74] Snykers, S., Vanhaecke, T., De Becker, A., et al. Chromatin remodeling agenttrichostatin A: a key-factor in the hepatic differentiation of human mesenchymal stemcells derived of adult bone marrow. BMC Dev. Biol.2007,7,24.
[75]向贤宏,李家平,谭国胜,等.同种异体肝细胞与脂肪间质干细胞移植治疗肝硬化的实验对照研究.中华普通外科学文献.2011,5(1):12-16.
[76]柳林,杨海山,等.自体脂肪间充质干细胞治疗晚期肝病的初步临床研究.中国实验诊断学杂志,2007,11(4)473－476.
[77] Puglisi MA, Saulnier N, Piscaglia AC, et a1. Adipose tissue-derived mesenchymalstem cells and hepatic differentiation:old concepts and future perspectives[J]. Eur RevMed Pharmacol Sci,2011,15:355-364.
[78] De la Garza-Rodea AS, Van der Velde-van Dijke I, Boersma H, et al. Myogenicproperties of human mesenchymaI stem cells derived from three different sources [J].Cell Transplant,2012,21:153-173.
[79] Banas A, Teratani T, Yamamoto Y, et al. IFATS collection: in vivo therapeutic potentialof human adipose tissue mesenchymal stem cells after transplantation into mice withliver injury.Stem cells,2008,26:2705-2712.
[80] Salama H, Zekri AR, Bahnassy AA, et a1. Autologous CD34+and CDl33+stem cellstransplantation in patients with end stage liver disease．World J Gastroenterol,2010,16:5297-5305.
[81] Levicar N, Pai M, Habib NA, et al.Long term clinical results of autologous infusionof mobilized adult bone marrow derived CD34+cells in patients with chronic liverdisease.Cell Prolif,2008,41:115-125．
[82] Lyra AC, Soares MB, Silva VF, et al. Feasibility and safety of autologous bonemarrow mononuclear cell transplantation in patients with advanced chronic liverdisease. World J Gastroenterol.2007,13:1067-1073.
[83] Nikeghbalian S, Pournasr B, Aghdami N, et al. Autogolous transplantation of bonemarrow-derived mononuclear and CDl33+cells in patients with decompensatedcirrhosis. Arch Iran Med.2011,14:12-17.
[84] LIn H, Xu R, Zhang Z, et al. Implications of the immunoregulatory functions ofmesenchvmal stem cells in the treatment of human liver diseases. Cel Mol Immunol,2011,8:19-22.
[85] Fan, B.J.; Piao, Y.F. Bone marrow mesenchymal stem cell from chronic hepatitis Bpatients differentiation into hepatocyte-like cells. Afr. J. Microbiol. Res.2012,6,3866–3873.
[86] Liang Peng, Hua Li, Lin Gu, et al.Comparison of biological characteristics of marrowmesenchymal stem cells in hepatitis B patients and normal adults.World JGastroenterol2007,13(11):1743-1746.
[87] Yue-Si Zhong, Nan Lin, Mei-Hai Deng, et al. Deficient Proliferation of Bone Marrow-Derived Mesenchymal Stem Cells in Patients with Chronic Hepatitis B ViralInfections and Cirrhosis of the Liver. Dig Dis Sci,2010,55:438–445.
[88] Galmiche MC, Koteliansky VE, Briere J, et al. Stromal cells from human long termmarrow cultures are mesenchymal cells that differenti-ate following a vascularsmooth muscle differentiation pathway [J]. Blood,1993,82(1):66-67.
[89] Peng L, Zhu HP, Zheng YB, Xie C, Chong YT. Mesenchymal stem cells differentiateinto hepatocyte-like cells under different induction systems in hepatitis B patients withliver failure. African Journal of Biotechnology.,2011,10:840-847.
[90] Kou TK, Hung SP, Chuang CH, Chen CH, Shih YR, Fang SC, Yang VW. Stem celltherapy for liver disease:parameters governing the success of using bone marrowmesenchymal stem cell. Gastroenterology.,2008,134:2111-2121.
[91] Russo FP, Alison MR, Bigger BW, et al. The bone marrow functionally contribute toliver fibrosis[J].Gastroenterology,2006,130:1807-1821.
[92]中华医学会肝病学分会,中华医学会感染病学分会.慢性乙型肝炎防治指南(2010版).胃肠病学.2011,16(6):351-366.
[93] De Ugarte DA, Morizono K, Elbarbary A, Alfonso Z, Zuk PA, Zhu M, Dragoo JL,Ashjian P, Thomas B, Benhaim P, et al. Comparison of multi-lineage cells from humanadipose tissue and bone marrow. Cells Tissues Organs.2003;174:101–109
[94] Hong L, Peptan IA, Colpan A, Daw JL. Adipose tissue engineering by humanadipose-derived stromal cells. Cells Tissues Organs.2006;183:133–140.
[95] Alonso, M.; Claros, S. The effect of type I collagen on osteochondrogenicdifferentiation in adipose-derived stromal cells in vivo. Cytotherapy2008,10,597–610.
[96] Cardozo AJ, Gomez DE, Argibay PF. Neurogenic differentiation of human adipose-derived stem cells: relevance of different signaling molecules, transcription factors,and key marker genes. Gene.2012;511:427–436
[97] Gwak SJ, Bhang SH, Yang HS, Kim SS, Lee DH, Lee SH, Kim BS. In vitrocardiomyogenic differentiation of adipose-derived stromal cells using transforminggrowth factor-beta1. Cell Biochem Funct.2009;27:148–154
[98] Sterodimas A, de Faria J, Nicaretta B, Pitanguy I. Tissue engineering with adipose-derived stem cells (ADSCs): current and future applications. J Plast Reconstr AesthetSurg.2010;63:1886–1892.
[99] Eun-Hee Kim, Chan Yeong Heo. Current applications of adipose-derived stem cellsand their future perspectives.World J Stem Cells.2014;6(1):65–68.
[100] Zhou Z, Chen Y,Zhang H.et al.Comparison of mesenchymal stromal cells from humanbone marrow and adipose tissue for the treatment of spinal cord injury. Cytotherapy.2013,15(4):434-48.
[101] Locke M, Windsor J, Dunbar PR. Human adipose-derived stem cells: isolation,characterization and applications in surgery. ANZ J Surg.2009;79:235–244
[102]李春明,刘毅.脂肪干细胞及其在脂肪组织工程中的应用[J].中国美容医学,2006,4:466-468.
[103] Brayfield C, Marra K, Rubin JP. Adipose stem cells for soft tissue regeneration.Handchir Mikrochir Plast Chir.2010;42:124–128
[104] Cai L, Johnstone BH, Cook TG, Tan J, Fishbein MC, Chen PS, March KL. IFATScollection: Human adipose tissue-derived stem cells induce angiogenesis and nervesprouting following myocardial infarction, in conjunction with potent preservation ofcardiac function. Stem Cells.2009;27:230–237
[105] Wosnitza M, Hemmrich K, Groger A, Graber S, Pallua N. Plasticity of human adiposestem cells to perform adipogenic and endothelial differentiation. Differentiation2007;75:12–23.
[106] Strioga M, Viswanathan S, Darinskas A, Slaby O, Michalek J. Same or Not the Same?Comparison of Adipose Tissue-Derived Versus Bone Marrow-Derived MesenchymalStem and Stromal Cells. Stem Cells Dev.2012;21:2724–2752.
[107] Maumus M, Peyrafitte JA, D’Angelo R, Fournier-Wirth C, Bouloumie A, et al. Nativehuman adipose stromal cells: localization, morphology and phenotype. Int J Obes.2011;35:1141–1153.
[108] Crop M, Baan CC, Korevaar SS, Ijzermans JN, Weimar W, et al. Human adiposetissue-derived mesenchymal stem cells induce explosive T-cell proliferation. StemCells Dev.2010;19:1843–1853.
[109] Bunnel BA, Flaat, M, Gagliardi, et al. Adipose-derived stem cels: isolation, expansionand differentiation. Methods,2008,45:115-120.
[110] Han Chao, Zhang Liang, Song Lin, et al. Human adipose-derived mesenchymal stemcells: a better cell source for nervous system regeneration Chinese Medical Journal2014;127(2):329-337.
[111]李翰昊,晏雪生,明安萍,等.肝脏细胞条件培养基诱导大鼠骨髓间质细胞分化为肝细胞的作用[J].中西医结合病杂志,2005,15(1)28-30.
[112] Takano H, Qin Y, Hasegawa H, et al. Effects of G-CSF on left ventricular remodelingand heart failure after acute myocardial infraction[J]. J Mol Med2006,84(3):185-193.
[113] Jiang J, Jahagirdar RL, Reinhart RL, et al. Pluripotency of mesenchymal stem cellsderived from adult marrow. Nature2002,418(6893):41-49.
[114]何中杰,方驰华,马俊勋,等.肝细胞生长因子与表皮细胞生长因子联合诱导大鼠骨髓间充质干细胞分化为类肝细胞[J].解放军医学杂志,2006,5(31):446-449.
[115]胡维亭,任军.人乙型肝炎病毒DN阳性血清对人骨髓间充质干细胞向肝细胞分化的影响.北京大学学报,2002,4(5):459-464.
[116] Chan Xie, Yu-Bao Zheng, Hai-Peng Zhu, et al. Human bone marrow mesenhymalstem cells are resistant to HBV infection during differentiation in vivo and in vitro.Cell Biology International,2009,33:493-500.
[117] Ruipin Ma, Quantai Xing, Lihua Shao, et al. Hepatitis B virus and replication inhuman bone marrow mesenchymal stem cells.Virology Journal.2011,8:846-494.
[118] Wang Y, Weil BR, Herrmann JL, et a1. MEK, p38and PI-3K mediate cross talkbetween EGFR and TNFR in enhancing hepatocyte growth factor production fromhuman mesenchymal stem celis[J1．Am J Physiol Cell Physiol,2009,297(5):1284-1293
[119] Colletti E, Airey JA, Liu W, Simmons PJ, Zanjani ED, Porada CD, Almeida-Porada G.Generation of tissue-specific cells by MSC does not require fusion or donor to hostmitochondrial/membrane transfer. Stem Cell Res.2009;2:125–138.
[120] Valerie D. Roobrouck, Kim Vanuytsel, Catherine M. Verfaillie. Concise Review:Culture Mediated Changes in Fate and/or Potency of Stem Cells. Stem Cells;2011,29:583-589.