| The bone marrow contains hematopoietic stem cell (hematopoietic stemcells, HSCs), mesenchymal stem cells (mesenchymal stem cells, MSCs) andendothelial precursor cells. Recent studies show that the bone marrow containsother stem cell populations, such as the myogenic multipotent adult progenitorcells (MACP). MSCs with similar biological properties have also been isolatedfrom other tissue including adipose tissue, skeletal muscles, fetal liver andpancreas, cord blood and amniotic fluid. MSCs have self-renewal andmulti-directional differentiation potential characters when cultured inappropriate conditions, such as: osteoblasts, cardiomyocytes, adipocytes,myocytes, oligodendrocytes and neurons. BM-MSCs posses anti-proliferativeand anti-inflammatory activity, which, together with their privileged immu-nological properties(cause low immune reaction), make them safe forallogeneic and atologous use. They are also easy to transfect and toleratecryopreservation well. MSCs play an important role in cellular and tissueengineering. Bone marrow-derived MSCs have attracted great interest in bothbasic research and clinical practice in recent years.Hepatitis B is one of the most common infectious diseases worldwide.There are approximately350million chronic hepatitis B virus (HBV)carriersall of the world. The therapy for hepatitis B is very limited. HBV infection maycause liver cirrhosis and hepatocellular carcinoma. Following chronic liverdamage, the regenerative ability of hepatocyte is lost, which leaves the liverunable to maintain its functional mass. This is clinically so-called "liverfailure". Currently, orthotopic liver transplantation(OLT) is considered to be the most suitable therapeutic method for patients with hepatic failure. Cell-basedtherapy has been proposed as a potential alternative to OLT. Stem celltransplantation may promote liver regeneration and self-repair,it provides atheoretical approach for liver regeneration. MSCs are easily obtained andextensively expanded in vitro without ethical complication and immunologicrejection and MSCs-based therapy for liver disease is more suitable than otherkind of stem cells. Some researchers conclude that MSCs represent a promisingcandidate for liver stem cell therapy. Several studies have demonstrated thatMSCs can differentiated along hepatogenic lineage in vitro. Studies on animalmodels have shown that MSCs, transplanted by either intrasplenic orintravenous route, can be engrafted into the recipient liver and differentiate intofunctional hepatocytes. To date, some clinical trials of autologous bonemarrow-derived MSCs transplantation have been conducted, and thepreliminary result seems feasible and safe for treating patients with liverdiseases with MSCs.However, there are many different views on the efficacy ofautologous MSCs transplantation in patients with cirrhosis induced by chronicHBV infection and whether HBV infection can affect the function of MSCs fortransplantation remains unknown. Up to now, it is not completely clear aboutthe biological characteristics and differentiation potential of MSCs in patientswith cirrhosis induced by HBV.The purpose of the sudy is to establish the culture system of bone marrowMSCs from liver cirrhosis patients in vitro,observe the biologicalcharacteristics and compare with MSCs from normal donors; MSCs werecultured in Heptozyme-SFM and L-DMEM with20%autologous serum,anddetect the expression of specific markers of liver cell,such as AFP,ALB andCK-18in the level of gene and protein, investigate the hepatic differentiationpotential of bone marrow MSCs from liver cirrhosis patients.We alsoinvestigate the susceptibility of bone marrow MSCs from liver cirrhosis patients to HBV in vitro. The results may provide the experimental basis andtheoretical basis for the therapy of liver cirrhosis patients.MSCs were isolated and purified from liver cirrhosis patients and normaldonors bone marrow by density gradient centrifugation and adhering to theculture plastic. The culture ratio and primary culture time were observed.Aftersuccessive subculture and amplification,the growth curve were drawn,themorphology were observed under microscope. The cell cycle and surfacemarker were detected by flowcytometer. The differentiation potential toosteoblasts,adipocyte and neuronal cell were observed. The HBV marker ofbone marrow MSCs were detected by immunohistochemical method.HBVDNA in MSCs were detected by fluorescence quantitative PCR. Bonemarrow MSCs were cultured in differentiation media of Heptozyme-SFM andL-DMEM with20%autolgous serum, the morphology change anddifferentiated character were observed. Specific markers hepatocyte such asAFP,ALB and CK-18were detected by immunohistochemical method. Theglycogen production were detected by periodic acideschiff (PAS) staining.Theexpression of ALBmRNA were detected by PCR. The result is as following:1. MSCs from liver cirrhosis patients with the density gradientcentrifugation combined adherent method and cultured adhered to the wall withthe colony growth. The culture ratio was73.33%(11/15) and100%(11/11),respectively. The primary passage time of MSCs from the two groups was (16.0±1.8) days and (12.0±1.5) days, respectively(P＜0.05). All BM-MSCsappeared similar to fibroblasts, with a characteristic spindle shaped fusiformmorphology. After observation on difference of appearance between chronichepatitis B patients and normal donors on days4and8after inoculation at5thand7thgeneration, we found that BM-MSCs grew slower and were easier toexpand, spread out and age in liver cirrhosis patients than in normal donors.The results from flow cytometry show that, most of the cells of the two groups are relatively inactive and in the quiescent period, only a small part inthe active proliferation state. The percentage of S-phase nuclei in MSCs fromliver cirrhosis patients grown was (4.58±0.96)%, which compared with(7.58±1.24)%,the S-phase of the normal donors MSCs nuclei, the proportionof S-phase MSCs from liver cirrhosis patients was significantly lower than thatof normal donors(P＜0.05).Our results indicated that both MSCs from liver cirrhosis patients andnormal donors have the potential of adipogenic, osteogrnic and neuroal diff-erentiation(Fig2). Nearly all the cells expressed CD44,CD29and CD105,CD166, which are the surface marker characteristics of MSCs. The absence ofcontaminating hematopoietic cells in the MSCs population was verified by thelack of surface antigen defining hematopoietic progenitor cells (CD34).2. Bone marrow MSCs from liver cirrhosis patients differentiated tohepatocyte-like cell. The fibroblastic morphology of MSCs developed a broad,flattened shape. The results showed that undifferentiated cells stained negativefor ALB, CK-18and AFP. BM-MSCs were exposed to growth factors at days7,14,21and the positive rates of AFP were (55.6±9.3)%,(20.8±5.2)%and(7.5±1.2)%, respectively(Fig4-C,D,E); the positive rate of ALB were(12.8±4.2)%,(40.5±7.6)%and (70.5±10.9.)%, respectively(Fig4-F,G,H); thepositive rate of CK-18were(10.6±3.8)%,(50.4±9.3)%and (81.2±12.3)%,respectively(Fig4-I,J,K); Compared with un-induced group, the difference areall statistically significant (p <0.05). The liver cell culture medium inducesbone marrow MSCs to express AFP, ALB and CK-18. With the prolongation ofculture time the AFP expression decreased, with the prolongation of culturetime the ALB and CK-18expression gradually increased; The liver cell culturemedium can induce the bone marrow MSCs of patients with liver cirrhosistohave unique glycogen synthesis function, and the ability to express ALBmRNA3. The6liver cirrhosis patients were male, HBVDNA content in serum were6.56E+4～7.52E+8and ALT, CHE, ALB, TBIL level were abnormal.The bone marrow were cultured in DMEM medium containing20%autologousserum, The MSCs developed a broad, flattened shape.Cultured7,1421days,the expression of ALB, CK-18and AFP were positive, the expressionof ALB and CK-18significantly increased in a time-dependant manner, withthe highest expression level at day21. However,AFP was at its highestexpression level at day7. Glycogen staining and ALBmRNAwas present inBM-MSCs after they were exposed to growth factors. Liver cirrhosis patientsbone marrow MSCs cultured with autologous serum in vitro were not infectedwith HBV.Comprehensive studies have shown that a number of highly active purifiedbone marrow MSCs were isolated by density gradient centrifugation combinedwith adherent method. MSCs from liver cirrhosis bone marrow were difficult tocultivate,the success rate was significantly lower than healthy donors and theprimary culture time were longer.Bone marrow MSCs from liver cirrhosispatients were not infected with HBV,which surface markers and differentiationpotential were consistent with MSCs from normal donors.The MSCs from livercirrhosis patients differentiate into hepatocyte-like cells, express ALB, AFPand CK-18, may synthesis glycogen, which were cultured in media ofHeptozyme-SFM.The MSCs from liver cirrhosis patients, which were culturedin DMEM medium containing20%autologous serum, may differentiate intohepatocyte-like cells, express ALB, AFP, CK-18and ALBmRNA andsynthesis glycogen. But different bone marrow MSCs induced by autologousserum of cirrhotic patients have different expression in liver cell lineage-specific markers positive rate. For the2patients whose serum biochemicalindex change is not obvious, the expression level of AFP, ALB and CK-18islow, so is the glycogen synthesizing function. It is presumably related to thelevel of serum cell factors in patients with liver cirrhosis, so we need some deeper research, and to establish suitable microenvironment for thedifferentiation from bone marrow MSCs to hepatocytes.In summary, the innovative point of this study was systematic evaluationof the biological characteristics of MSCs from liver cirrhosis patients andinvestigate the directional differentiation potential to the liver cell lines. Wecultured the MSCs in DMEM with20%autologous serum and investigate itsdifferentiated potential. The results may provide the experimental basis andtheoretical basis for the therapy of liver cirrhosis patients.
|
PDF Full Text Request