骨髓间质细胞致瘤过程中特征基因筛查及其生物学意义的研究

目的:研究骨髓间质细胞(MSCs)诱导致瘤的发生机制。方法:采用荧光差异显示(FDD)技术寻找MSCs诱导致瘤(F6)的关键基因。将回收的差异关键基因片段,进行PCR再扩增,TA克隆cDNA片段,测序后经BLAST软件检索以进行同源性分析。结果:FDD成功分离了MSCs和F6肿瘤细胞之间的不同基因片段,TA克隆得到15个EST片段,GenBank序列比对分析表明,这些片断均与相关基因片断同源,其中C356、G392和G383分别与nu-cleostemin,CyclinⅠ和Siva基因同源,同源百分比分别为95%、97%和100%。结论:F6肿瘤细胞的发生机制是一个涉及到众多基因参与的复杂过程,nucleostemin、CyclinⅠ和Siva等基因在人类骨髓间质干细胞诱导突变转化为F6细胞的过程中起着重要作用。

Preliminary Study on Biological Properties of Adult Human Bone Marrow-derived Mesenchymal Stem Cells

Objective： To establish a method of culture and expansion of adult human bone marrow-derived MSCs in vitro and to explore their biological properties. Methods： Mononuclear cells were obtained from 5 mL adult human bone marrow by density gradient centrifugation with Percoll solution. Adult human MSCs were cultured in Dulbecco＇s Modified Eagle＇s Medium with low glucose （LG-DMEM） containing 10% fetal calf serum at a density of 2×10^5 cell/cm^2. The morphocytology was observed under phase-contrast microscope. The cell growth was measured by MTT method. The flow cytometer was performed to examine the expression of cell surface molecules and cell cycle. The ultrastructure of MSCs was observed under transmission electron microscope. The immunomodulatory functions of MSCs were measured by MTT method. The effects of MSCs on the growth of K562 cells and the dynamic change of HA, IV-C, LN concentration in the culture supernatant of MSCs was also observed. Results： The MSCs harvested in this study were homogenous population and exhibited a spindle-shaped fibroblastic morphology. The cell growth curve showed that MSCs had a strong ability of proliferation. The cells were positive for CD44, while negative for hematopoietic cell surface marker such as CD3, CD4, CD7, CD13, CD14, CD15, CD19, CD22, CD33, CD34, CD45 and HLA-DR, which was closely related to graft versus host disease. Above 90% cells of MSCs were found at G0/G1 phase. The ultrastructure of MSCs indicated that there were plenty of cytoplasmic organelles. Allogeneic peripheral blood lymphocytes proliferation was suppressed by MSCs and the inhibition ratio was 60.68% （P〈0.01）. The suppressive effect was also existed in the culture supernatant of MSCs and the inhibition ratio was 9.00% （P〈0.05）. When lymphocytes were stimulated by PHA, the suppression effects of the culture supernatant were even stronger and the inhibition ratio was 20.91% （P〈0.01）. Compared with the cell growth curve of the K562 ceils alone, the K562 ceils cocultured with MSCs grew slowly and the exponential phase of growth wasn＇t significant. Seeing from the concentration curve, as time passed, the concentration of HA increased quickly, while those of IV-C and LN didn＇t change much. Conclusion： The method for culture and expansion of adult human bone marrow-derived MSCs in vitro has been successfully established in this study. MSCs were a homogenous population that had unique growth phenotype and multilineage potential. Preliminary study proved that it had the abilities of immunomodulatory function, antitumor, hematopoietic supporting and could act as seed cell of tissue engineering.

Feasibility and safety of autologous bone marrow mononuclear cell transplantation in patients with advanced chronic liver disease

AIM： To evaluate the safety and feasibility of bone marrow cell （BMC） transplantation in patients with chronic liver disease on the waiting list for liver transplantation. METHODS： Ten patients （eight males） with chronic liver disease were enrolled to receive infusion of autologous bone marrow-derived cells. Seven patients were classified as Child-Pugh B and three as Child-Pugh C. Baseline assessment included complete clinical and laboratory evaluation and abdominal MRI. Approximately 50 mL of bone marrow aspirate was prepared by centrifugation in a ficoll-hypaque gradient. At least of 100 millions of mononuclear-enriched BMCs were infused into the hepatic artery using the routine technique for arterial chemoembolization for liver tumors. Patients were followed up for adverse events up to 4 mo. RESULTS： The median age of the patients was 52 years （range 24-70 years）. All patients were discharged 48 h after BMC infusion. Two patients complained ofmild pain at the bone marrow needle puncture site. No other complications or specific side effects related to the procedure were observed. Bilirubin levels were lower at 1 （2.19 ± 0.9） and 4 mo （2.10 ± 1.0） after cell transplantation that baseline levels （238 ± 1.2）. Albumin levels 4 mo after BMC infusion （3.73 ± 0.5） were higher than baseline levels （3.47 ± 0.5）. International normalized ratio （INR） decreased from 1.48 （SD = 0.23） to 1.43 （SD = 0.23） one month after cell transplantation. CONCLUSION： BMC infusion into hepatic artery of patients with advanced chronic liver disease is safe and feasible. In addition, a decrease in mean serum bilirubin and INR levels and an increase in albumin levels are observed. Our data warrant further studies in order to evaluate the effect of BMC transplantation in patients with advanced chronic liver disease.

Direct ex vivo analysis of dendritic cells in patients with hepatocellular carcinoma

AIM： To analyze the phenotype and function of dendritic cells （DC） from patients with hepatocellular carcinoma （HCC） in order to understand their role in this disease. METHODS： Myeloid dendritic cells were enumerated in peripheral blood of HCC patients. CD80, CD83, CD86 and HLA-DR expression on naive and stimulated myeloid dendritic cells from peripheral blood were analyzed. Myeloid dendritic cells were isolated from peripheral blood and their function was tested. Phagocytosis was analyzed using FITC-dextran beads, peptide specific stimulation, the capacity to stimulate allogeneic T cells and secretion of cytokines upon poly dZ：dC was tested. RESULTS： Myeloid dendritic cells were reduced in patients with HCC. No differences in CD80, CD83, CD86 and HLA-DR expression were found on naive and stimulated myeloid dendritic cells from HCC patients and healthy controls. Normal phagocytosis or stimulation of peptide specific T cells was observed in contrast to an impaired allo-stimulatory capacity and a reduced IL-12 secretion. CONCLUSION： Impaired IL-12 production of mDCs in patients could lead to an impaired stimulatory capacity of naive T cells suggesting that IL-12 directed therapies may enhance tumor specific immune responses in HCC patients.

Passage of bone-marrow-derived liver stem cells in a proliferating culture system

AIM： To explore the feasibility of passage of bone- marrow-derived liver stem cells （BDLSCs） in culture systems that contain cholestatic serum.METHODS： Whole bone marrow cells of rats were purified with conditioning selection media that contained 50 mL/L cholestatic serum. The selected BDLSCs were grown in a proliferating culture system and a differentiating culture system. The culture systems contained factors that stimulated the proliferation and differentiation of BDLSCs. Each passage of the proliferated stem cells was subjected to flow cytometry to detect stem cell markers. The morphology and phenotypic markers of BDLSCs were characterized using immunohistochemistry, reverse transcription polymerase chain reaction （RT-PCR） and electron microscopy. The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay.RESULTS： The conditioning selection medium isolated BDLSCs directly from cultured bone marrow cells. The selected BDLSCs could be proliferated for six passages and maintained stable markers in our proliferating system. When the culture system was changed to a differentiating system, hepatocyte-like colony-forming units （H-CFUs） were formed. H-CFUs expressed markers of embryonic hepatocytes （alpha-fetoprotein, albumin and cytokeratin 8/18）, biliary cells （cytokeratin 19）, hepatocyte functional proteins （transthyretin and cytochrome P450-261）, and hepatocyte nuclear factors 1α and -3β）. They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes.CONCLUSION： BDLSCs can be selected directly from bone marrow cells, and pure BDLSCs can be proliferated for six passages. The differentiated cells have hepatocyte-like phenotypes and functions. BDLSCs represent a new method to provide a readily available alternate source of cells for clinical hepatocyte therapy.

Human bone marrow-derived mesenchymal stem cells transplanted into damaged rabbit heart to improve heart function

Objective: The present study was designed to test whether transplantation of human bone marrow-derived mesen- chymal stem cells (hMSCs) in New Zealand rabbits with myocardial infarction can improve heart function; and whether engrafted donor cells can survive and transdifferentiated into cardiomyocytes. Methods: Twenty milliliters bone marrow was obtained from healthy men by bone biopsy. A gradient centrifugation method was used to separate bone marrow cells (BMCs) and red blood cells. BMCs were incubated for 48 h and then washed with phosphate-buffered saline (PBS). The culture medium was changed twice a week for 28 d. Finally, hematopoietic cells were washed away to leave only MSCs. Human MSCs (hMSCs) were premarked by BrdU 72 h before the transplantation. Thirty-four New Zealand rabbits were randomly divided into myocardial infarction (MI) control group and cell treated group, which received hMSCs (MI+MSCs) through intramyocardial injection, while the control group received the same volume of PBS. Myocardial infarction was induced by ligation of the left coronary artery. Cell treated rabbits were treated with 5×106 MSCs transplanted into the infarcted region after ligation of the coronary artery for 1 h, and the control group received the same volume of PBS. Cyclosporin A (oral solution; 10 mg/kg) was provided alone, 24 h before surgery and once a day after MI for 4 weeks. Echocardiography was measured in each group before the surgery and 4 weeks after the surgery to test heart function change. The hearts were harvested for HE staining and immunohistochemical studies after MI and cell transplantation for 4 weeks. Results: Our data showed that cardiac function was significantly improved by hMSC transplan- tation in rabbit infarcted hearts 4 weeks after MI (ejection fraction: 0.695±0.038 in the cell treated group (n=12) versus 0.554±0.065 in the control group (n=13) (P<0.05). Surviving hMSCs were identified by BrdU positive spots in infarcted region and transdifferentiated into cardiomyocytes characterized with a positive cardiac phenotype: troponin I. Conclusion: Transplan- tation of hMSCs could transdifferentiate into cardiomyocytes and regenerate vascular structures, contributing to functional im- provement.

Role of soluble triggering receptor expressed on myeloid cells in inflammatory bowel disease

AIM： To investigate the probable role of soluble triggering receptor expressed on myeloid cells-1 （sTREM-1） in the pathogenesis of inflammatory bowel disease （IBD）.METHODS： Fifty-eight patients were enrolled; nineteen healthy volunteers served as controls; 8 patients were diagnosed with Crohn＇s disease, and 31 with ulcerative colitis, Clinical and endoscopic activity indexes of patients with Crohn＇s disease and ulcerative colitis respectively were estimated, Upon admission blood was sampled; sTREM-1 and TNFα were measured by an immunoassay and malondialdehyde （IDA） by the thiobarbitourate assay, after passage through an HPLC system,RESULTS： Median ± SE of TNFα of controls, patients with Crohn＇s disease and patients with ulcerative colitis were 6.02 ± 3.94, 7.98 ± 5.08 （P = NS vs controls）, and 8.45 ± 4.15 ng/L （P = 0.018 vs controls） respectively. Respective values of sTREM-1 were 53.31 ± 32.93, 735.10 ± 197.17 （P = 0.008 vs controls） and 435.82 ± 279.71 ng/L （P = 0.049 vs controls）, sTREM-1 was positively correlated with Crohn＇s disease activity index and clinical and endoscopic activity indexes of ulcerative colitis （P = 0.002, 0.001 and 0.009, respectively）, sTREM-1 of patients with ulcerative colitis was positively correlated with TNFα （P = 0.001）.CONCLUSION： sTREM-1 seems to behave as a novel mediator in IBD in correlation with the degree of the intlammatory reaction of the intestinal mucosa.

Stem cells and cancer:Evidence for bone marrow stem cells in epithelial cancers

Cancer commonly arises at the sites of chronic inflammation and infection. Although this association has long been recognized, the reason has remained unclear. Within the gastrointestinal tract, there are many examples of inflammatory conditions associated with cancer, and these include reflux disease and Barrett＇s adenocarcinoma of the esophagus, Helicobacter infection and gastric cancer, inflammatory bowel disease and colorectal cancer and viral hepatitis leading to hepatocellular carcinoma. There are several mechanisms by which chronic inflammation has been postulated to lead to cancer which includes enhanced proliferation in an endless attempt to heal damage, the presence of a persistent inflammatory environment creating a pro-carcinogenic environment and more recently a role for engraftment of circulating marrow-derived stem cells which may contribute to the stromal components of the tumor as well as the tumor mass itself. Here we review the recent advances in our understanding of the contributions of circulating bone marrow-derived stem cells to the formation of tumors in animal models as well as in human beings.

Differentiation of hepatocytoid cell induced from whole-bone-marrow method isolated rat myeloid mesenchymal stem cells

AIM： To explore the expansion and differentiation of hepatocytoid cell induced from myeloid mesenchymal stem cell （MSC） in vib＇o, in order to find suitable resource of hepatocytes for bioartiflcial liver or liver transplantation. METHODS： The rat myeloid MSC was isolated and divided into three groups which were cultured by Friedensteion method, and then were induced by culture fluid, culture fluid plus cholestatic serum and culture fluid plus hepatocyte growth factor （HGF）, respectively. Hepatocytoid cell as well as expression of CK18 and AFP was observed by immunohistochemistry. RESULTS： After the induction for 21 d, hepatocytoid cell was observed, and its expression of CK18 and AFP was detected by immunohistochemistry in MSC cultured with cholestatic serum. Furthermore, on the 35^th d, albumin mRNA was expressed in the cell, suggesting the inducing effect was similar to that by HGF.CONCLUSION： Rat myeloid MSC can differentiate into hepatocyte lineage under appropriate condition. This method is easy to operate.

Morphological Properties of Mesenchymal Stem Cells Derived from Bone Marrow of Rhesus Monkeys

To establish an in vitro system for isolating and culturing the mesenchymal stem cells （MSC） of Rhesus monkeys, and to provide research data for its further application, the bone marrow of Rhesus monkeys was collected and separated by gradient centrifugation to discard most of the blood cells. The MSC contained in the monocyte centrifuging layer was obtained and cultured in Dulbecco＇s modified media （low glucose, L-DMEM） supplemented with 10% Fetal bovine serum （FBS） and 1 ng/ml basic fibroblast growth factor （bFGF）. The non-MSC was screened out by continuously renewing the medium. A passage culture was undertaken while the MSC monolayer formed. The spindle-shaped MSC formed a monolayer after 18 days of primary culturing, and the cells appeared in an oriented array with a swirling and irradiating growth trend. In the anaphase of passage culture, the cell proliferation rate was decreased and the morphology changed into triangular, polygon and flat appearance. These results suggested that mesenchymal stem cells （MSC） of the Rhesus monkey can be passaged in vitro with the established optimized culture system.

Ex vivo expansions and transplantations of mouse bone marrow－derived hematopoietic stem／progenitor cells

To examine the effects of co-culture with bone marrow mesenchymal stem cells on expansion of hematopoietic stem/progenitor cells and the capacities of rapid neutrophil engraftment and hematopoietic reconstitution of the expanded cells, we expanded mononuclear cells (MNCs) and CD34+/c-kit+ cells from mouse bone marrow and transplanted the ex-panded cells into the irradiated mice. MNCs were isolated from mouse bone marrow and CD34+/c-kit+ cells were selected from MNCs by using MoFlo Cell Sorter. MNCs and CD34+/c-kit+ cells were co-cultured with mouse bone marrow-derived mesenchymal stem cells (MSCs) under a two-step expansion. The expanded cells were then transplanted into sublethally irradiated BDF1 mice. Results showed that the co-culture with MSCs resulted in expansions of median total nucleated cells, CD34+ cells, GM-CFC and HPP-CFC respectively by 10.8-, 4.8-, 65.9- and 38.8-fold for the mononuclear cell culture, and respectively by 76.1-, 2.9-, 71.7- and 51.8-fold for the CD34+/c-kit+ cell culture. The expanded cells could rapidly engraft in the sublethally irradiated mice and reconstitute their hematopoiesis. Co-cultures with MSCs in conjunction with two-step expansion increased expansions of total nucleated cells, GM-CFC and HPP-CFC, which led us to conclude MSCs may create favorable environment for expansions of hematopoietic stem/progenitor cells. The availability of increased numbers of ex-panded cells by the co-culture with MSCs may result in more rapid engraftment of neutrophils following infusion to transplant recipients.

Mesenchymal stem cells： a new strategy for immunosuppression and tissue repair

Mesenchymal stem cells （MSCs） have great potential for treating various diseases, especially those related to tissue damage involving immune reactions. Various studies have demonstrated that MSCs are strongly immunosuppressive in vitro and in vivo. Our recent studies have shown that un-stimulated MSCs are indeed incapable of immunosuppression; they become potently immunosuppressive upon stimulation with the supernatant of activated lymphocytes, or with combinations of IFN-γ, with TNF-α, IL-1α or IL-1β. This observation revealed that under certain circumstances, inflammatory cytokines can actually become immunosuppressive. We showed that there is a species variation in the mechanisms of MSC-mediated immunosuppression： immunosuppression by cytokine-primed mouse MSCs is mediated by nitric oxide （NO）, whereas immunosuppression by cytokine-primed human MSCs is executed through indoleamine 2, 3-dioxygenase （IDO）. Additionally, upon stimulation with the inflammatory cytokines, both mouse and human MSCs secrete several leukocyte chemokines that apparently serve to attract immune cells into the proximity with MSCs, where NO or IDO is predicted to be most active. Therefore, immunosuppression by inflammatory cytokine-stimulated MSCs occurs via the concerted action of chemokines and immune-inhibitory NO or IDO produced by MSCs. Thus, our results provide novel information about the mechanisms of MSC-mediated immunosuppression and for better application of MSCs in treating tissue injuries induced by immune responses.

Immunosuppressive properties of cloned bone marrow mesenchymal stem cells

Mesenchymal stem cells （MSCs）, derived from adult tissues, are multipotent progenitor cells, which hold great promise for regenerative medicine. Recent studies have shown that MSCs are immunosuppressive in vivo and in vitro in both animals and humans. However, the mechanisms that govern these immune modulatory functions of MSCs remain largely elusive. Some studies with bulk populations of MSCs indicated that soluble factors such as PGE2 and TGFβ are important, while others support a role for cell-cell contact. In this study, we intended to clarify these issues by examining immunosuppressive effects of cloned MSCs. We derived MSC clones from mouse bone marrow and showed that the majority of these clones were able to differentiate into adipocytes and osteoblast-like cells. Importantly, cells from these clones exhibited strong inhibitory effects on TCR activation-induced T cell proliferation in vitro, and injection of a small number of these cells promoted the survival of allogeneic skin grafts in mice. Conditioned medium from MSC cultures showed some inhibitory effect on anti-CD3 induced lymphocyte proliferation independent of PGE2 and TGFβ. In comparison, direct co-culture of MSCs with stimulated lymphocytes resulted in much stronger immunosuppressive effect. Interestingly, the suppression was bi-directional, as MSC proliferation was also reduced in the presence of lymphocytes. Taking together, our findings with cloned MSCs demonstrate that these cells exert their immunosuppressive effects through both soluble factor（s） and cell-cell contact, and that lymphocytes and MSCs are mutually inhibitory on their respective proliferation.

Rat bone marrow mesenchymal stem cells differentiate into hepatocytes in vitro

AIM: To investigate the mechanism and regulation of differentiation from bone marrow mesenchymal stem cells (MSCs) into hepatocytes and to find a new source of celltypes for therapies of hepatic diseases. METHODS: MSCs were isolated by combining gradient density centrifugation with plastic adherence. The cells were cultured in osteogenic or adipogenic differentiation medium and determined by histochemical staining. MSCs were plated in plastic culture flasks that were not coated with components of extracellular matrix (ECM). When MSCs reached 70% confluence, they were cultured in low glucose Dulbecco's modified Eagle's medium supplemented with 10 mL/L fetal bovine serum, 20 ng/mL hepatocyte growth factor (HGF) and 10 ng/mL fibroblast growth factor-4 (FGF-4). The medium was changed every 3 d and stored for albumin, alpha-fetoprotein (AFP) and urea assay. Glycogen store of hepatocytes was determined by periodic acid-Schiff staining.RESULTS: By combining gradient density centrifugation with plastic adherence, we isolated a homogeneous population of cells from rat bone marrow and differentiated them into osteocytes and adipocytes. When MSCs were cultured withFGF-4 and HGF, approximately 56.6% of cells became smallround and epithelioid on d 24 by morphology. Compared with the control, levels of AFP increased significantly from d 12 to 15.5±1.4 μg/L (t = 2.31, P＜0.05) in MSCs cultured with FGF-4and HGF, and were higher (46.2±1.5 μg/L)ond 21 (t = 41.926, P＜0.01), then decreased to 24.8±2.2 μg/L on d 24 (t = 10.345, P＜0.01). Albumin increased significantly on d 21 (t= 3.325, P＜0.01) to 1.4±0.2 μg/mL,and to 2.1±0.7 μg/mL on d 24 (t= 3.646, P＜0.01). Urea(2.3±0.4 mmol/L) was first detected on d 21 (t = 6.739, P＜0.01), and continued to increase to 2.6±0.9 mmol/Lon d 24 (t= 4.753, P＜0.01). Glycogen storage was first seen on d 21.CONCLUSION: The method combining gradient density centrifugation with plastic adherence can isolate MSCs. Rat MSCs may be differentiated into hepatocytes by FGF-4 and HGF. Cytokines may play a more important role in differentiation from rat MSCs into hepatocytes.

Hepatogenic differentiation of human mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal stem cells

AIM： To investigate and compare the hepatogenic transdifferentiation of adipose tissue-derived stem cells （ADSC） and bone marrow-derived mesenchymal stem cells （BMSC） in vitro. Transdifferentiation of BMSC into hepatic cells in vivo has been described. Adipose tissue represents an accessible source of ADSC, with similar characteristics to BMSC. METHODS： BMSCs were obtained from patients undergoing total hip arthroplasty and ADSC from human adipose tissue obtained from lipectomy. Cells were grown in medium containing 15% human serum. Cultures were serum deprived for 2 d before cultivating under similar pro-hepatogenic conditions to those of liver development using a 2-step protocol with sequential addition of growth factors, cytokines and hormones. Hepatic differentiation was RT-PCR-assessed and liver-marker genes were immunohistochemically analysed.RESULTS： BMSC and ADSC exhibited a fibroblastic morphology that changed to a polygonal shape when cells differentiated. Expression of stem cell marker Thyl decreased in differentiated ADSC and BMSC. However, the expression of the hepatic markers, albumin and CYPs increased to a similar extent in differentiated BMSC and ADSC. Hepatic gene activation could be attributed to increased liver-enriched transcription factors （C/EBPβ and HNF4α）, as demonstrated by adenoviral expression vectors.CONCLUSION： Mesenchymal stem cells can be induced to hepatogenic transdifferentiation in vitro. ADSCs have a similar hepatogenic differentiation potential to BMSC, but a longer culture period and higher proliferation capacity. Therefore, adipose tissue may be an ideal source of large amounts of autologous stem cells, and may become an alternative for hepatocyte regeneration, liver cell transplantation or preclinical drug testing.

Local signals in stem cell-based bone marrow regeneration

The cellular basis of bone marrow （BM） tissue development and regeneration is mediated through hematopoietic stem cells （HSCs） and mesenchymal stem cells （MSCs）. Local interplays between hematopoietic cells and BM stromal cells （BMSCs） determine the reconstitution of hematopoiesis after myelosuppression. Here we review the BM local signals in control of BM regeneration after insults. Hematopoietic growth factors （HGFs） and cytokines produced by BMSCs are primary factors in regulation ofBM hematopoiesis. Morphogens which are critical to early embryo development in multiple species have been added to the family of HSCs regulators, including families of Wnt proteins, Notch ligands, BMPs, and Hedgehogs. Global gene expression analysis of HSCs and BMSCs has begun to reveal signature groups of genes for both cell types. More importantly, analysis of global gene expression coupled with biochemical and biological studies of local signals during BM regeneration have strongly suggested that HGFs and cytokines may not be the primary local regulators for BM recovery, rather chemokines （SDF- 1, FGF-4） and angiogenic growth factors （VEGF-A, Ang- 1） play instructive roles in BM reconstitution after myelosuppression. A new direction of management of BM toxicity is emerging from the identification of BM regenerative regulators.

Protective effects of ACLF sera on metabolic functions and proliferation of hepatocytes co-cultured with bone marrow MSCs in vitro

AIM： To investigate whether the function of hepatocytes co-cultured with bone marrow mesenchymal stem cells （MSCs） could be maintained in serum from acute-on- chronic liver failure （ACLF） patients.METHODS： Hepatocyte supportive functions and cy- totoxicity of sera from 18 patients with viral hepatitis B-induced ACLF and 18 healthy volunteers were evalu- ated for porcine hepatocytes co-cultured with MSCs and hepatocyte mono-layered culture, respectively. Chemo- kine profile was also examined for the normal serum and liver failure serum.RESULTS： Hepatocyte growth factor （HGF） and Tumor necrosis factor; tumor necrosis factor （TNF）-a were re- markably elevated in response to ACLF while epidermal growth factor （EGF） and VEGF levels were significantly decreased. Liver failure serum samples induced a higher detachment rate, lower viability and decreased liver sup- port functions in the homo-hepatocyte culture. Hepato-cytes co-cultured with MSCs could tolerate the cytotoxic- ity of the serum from ACLF patients and had similar liver support functions compared with the hepatocytes cul- tured with healthy human serum in vitro. In addition, co- cultured hepatocytes maintained a proliferative capability despite of the insult from liver failure serum.CONCLUSION： ACLF serum does not impair the cell morphology, viability, proliferation and overall metabolic capacities of hepatocyte co-cultured with MSCs in vitro.

Induction of apoptosis by shikonin through a ROS/JNK-mediated process in Bcr/Abl-positive chronic myelogenous leukemia （CML） cells

This study examined the signaling events induced by shikonin that lead to the induction of apoptosis in Bcr/ Abl-positive chronic myelogenous leukemia （CML） cells （e.g., K562, LAMA84）. Treatment of K562 cells with shikonin （e.g., 0.5 pM） resulted in profound induction of apoptosis accompanied by rapid generation of reactive oxygen species （ROS）, striking activation of c-Jun-N-terminal kinase （JNK） and p38, marked release of the mitochondrial proteins cytochrome c and Smac/DIABLO, activation of caspase-9 and -3, and cleavage of PARP. Scavenging of ROS completely blocked all of the above-mentioned events （i.e., JNK and p38 phosphorylation, cytochrome c and Smac/DIABLO release, caspase and PARP cleavage, as well as the induction of apoptosis） following shikonin treatment. Inhibition of JNK and knock-down of JNK1 significantly attenuated cytochrome c release, caspase cleavage and apoptosis, but did not affect shikonin-mediated ROS production. Additionally, inhibition of caspase activation completely blocked shikonin-induced apoptosis, but did not appreciably modify shikonin-mediated cytochrome c release or ROS generation. Altogether, these findings demonstrate that shikonin-induced oxidative injury operates at a proximal point in apoptotic signaling cascades, and subsequently activates the stress-related JNK pathway, triggers mitochondrial dysfunction, cytochrome c release, and caspase activation, and leads to apoptosis. Our data also suggest that shikonin may be a promising agent for the treatment of CML, as a generator of ROS.

Differentiation of rat bone marrow stem cells in liver after partial hepatectomy

AIM： To investigate the differentiation of rat bone marrow stem cells in liver after partial hepatectomy. METHODS： Bone marrow cells were collected from the tibia of rat with partial hepatectomy, the medial and left hepatic lobes were excised. The bone marrow stem cells （Thy^＋CD3^-CD45RA^- cells） were enriched from the bone marrow cells by depleting red cells and fluorescence-activated cell sorting. The sorted bone marrow stem cells were labeled by PKH26-GL in vitro and autotransplanted by portal vein injection. After 2 wk, the transplanted bone marrow stem cells in liver were examined by the immunohistochemistry of albumin （hepatocyte-specific marker）. RESULTS： The bone marrow stem cells （Thy^＋CD3^-CD45RA^- cells） accounted for 2.8% of bone marrow cells without red cells. The labeling rate of 10 μM PKH26- GL on sorted bone marrow stem cells was about 95%. There were sporadic PKH26-GL-labeled cells among hepatocytes in liver tissue section, and some of the cells expressed albumin. CONCLUSION： Rat bone marrow stem cells can differentiate into hepatocytes in regenerative environment and may participate in liver regeneration after partial hepatectomy.

Myeloidcell-lineage and premylocytic-stage-specific-expression of the mouse myeloperoxidase gene is controlled at initiation as well as elongation levels of transcription

The myeloperoxidase (MPO) is an important microbicidal protein present at high concentration in the primary granule of mature granulocyte and its expression is regulated in both myeloidcell-lineage and premyelocytic-stagespecific manners. A better understanding of the underlying control mechanisms should provide insights into the temporal and co-ordinate regulation of the gene expression during granulopoiesis. We have identified its promoter by mapping the start(s) of transcription using various molecular approaches together with demonstrating the promoter function of the relevant DNA segment in a transient transfection reporter assay. Besides the major start of transcription mapped at G residue, 11 nucleotide upstream of the 3’ end of exon 0, the usage of that is specific to the MPO expressing cell lines, we have shown that irrespective of the MPO-expression status of the hematopoietic cells, transcription occurs broadly within a two kb region upstream of the 5’ proximity of the gene, and is largely terminated in nitron 2. These data support a model of the pre myelocytic-stage-specific MPO expression, the control of which is operated at initiation as well as elongation levels of transcription.