| 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. |
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