【作者】 王莹；

【导师】 王峰；

【作者基本信息】 吉林大学 ， 内科学， 2014， 博士

【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。更多还原

【Abstract】 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.更多还原