ACE2 and TMPRSS2 Expression in Hepatocytes of Chronic HBV Infection Patients.

Background: Pre-existing liver disease is a risk factor for the worse prognosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We aimed to evaluate whether chronic hepatitis B (CHB) and hepatocellular carcinoma (HCC) affect the expression of viral receptor angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) in the liver. Methods: Twelve pairs of matched liver tissues of HCC and para-carcinoma were collected from the First Affiliated Hospital of Zhejiang University School of Medicine. And 20 liver biopsies from CHB patients were collected from Peking University People's Hospital. The expression of ACE2 and TMRPSS2 were detected using immunofluorescence staining, western blot, and RT-qPCR. The effects of hepatitis B virus (HBV) replication or interferon on ACE2 and TMPRSS2 expression were tested in hepatic cell lines. Results: The mRNA expression of TMPRSS2 in HCC tissues was six-fold higher than that of para-carcinoma tissues (P = 0.002), whereas that of ACE2 was not statistically different between HCC and para-carcinoma tissues. Hepatocellular ACE2 expression was detected in 35% (7/20) of CHB patients and mostly distributed in the inflammatory areas. However, there was no difference in TMPRSS2 expression between areas with or without inflammation. IFN-α2b slightly induced ACE2 expression (2.4-fold, P = 0.033) in HepG2 cells but not in Huh-7, QSG-7701, and L-02 cells. IFN-α2b did not affect TMPRSS2 expression in these cell lines. In addition, HBV replication did not alter ACE2 expression in HepAD38 cells. Conclusions: Although HBV replication does not directly affect the expression of ACE2 and TMPRSS2, intrahepatic inflammation and carcinogenesis may increase their expression in some patients, which, in turn, may facilitate SARS-CoV-2 infection in hepatocytes.

DDX5-targeting fully human monoclonal autoantibody inhibits proliferation and promotes differentiation of acute promyelocytic leukemia cells by increasing ROS production.

Acute promyelocytic leukemia (APL) therapy involves the compounds cytotoxic to both malignant tumor and normal cells. Relapsed APL is resistant to subsequent chemotherapy. Novel agents are in need to kill APL cells selectively with minimal toxicity. DDX5 has been recognized to be a novel target to suppress acute myeloid leukemia (AML). However, the role of DDX5 remains elusive in APL. Here a DDX5-targeting fully human monoclonal autoantibody named after 2F5 was prepared. It is demonstrated that 2F5 selectively inhibited APL cell proliferation without toxicity to normal neutrophil and tissues. Moreover, 2F5 was confirmed to induce G0/G1 phase arrest in APL cells, and promote APL cell differentiation combined with decreased DDX5 expression and increased reactive oxygen species (ROS) production. Knockdown of DDX5 by siRNA also inhibited proliferation, promoted cell differentiation and enhanced ROS production in APL cells. However, the ROS inhibitor reversed the effects of 2F5 on DDX5 and ROS in APL cells. Thus, we conclude that DDX5-targeting 2F5 inhibits APL cell proliferation, and promotes cell differentiation via induction of ROS. 2F5 showed the therapeutic value of fully human monoclonal autoantibody in APL, which provides a novel and valid approach for treatment of relapse/refractory APL.

Role of hepatitis B antibody in predicting reactivation of resolved hepatitis B virus infection in leukemia patients.

BACKGROUND & AIMS: Quantification of anti-HBs and anti-HBc predicts the risk of HBV reactivation (HBVr) in lymphoma patients receiving rituximab treatment. However, it remains unclear whether the quantification is predictive of HBVr in leukemia patients undergoing immunosuppression. METHODS: and patients: Clinical and laboratory data of the leukemia patients with resolved HBV infection diagnosed between January 2013 and March 2018 were retrospectively collected. Data series of HBV seromarkers and HBV DNA levels before the patients receiving chemotherapy and/or hematopoietic stem cell transplantation (HSCT) and during follow-up duration were analyzed. RESULTS: In total, 533 leukemia patients with resolved HBV infection were included. The incidences of HBVr were 5.7% (25/441) and 2.2% (2/92) in patients receiving HSCT and chemotherapy, respectively. In patients receiving HSCT, acute lymphoid leukemia had a significantly higher incidence of HBVr than acute myeloid leukemia (8.9% vs 3.9%, P < 0.05). The incidence varied almost zero to 40% due to the differences in the profiles of HBV antibodies. High anti-HBs (cut-off of 79.2 IU/L) or low anti-HBc levels (cut-off of 4.475, S/CO) at baseline were associated with a low risk of HBVr. Anti-HBe status did not affect the incidence of HBVr. However, the cut-offs were only predictive of HBVr in the patients who had negative anti-HBe. CONCLUSION: The baseline profiles of HBV antibodies are predictive of the risk of HBVr in leukemia patients undergoing immunosuppression. However, seronegative anti-HBe is a prerequisite for using baseline anti-HBs and anti-HBc quantification to predict HBVr risk.

HBsAg stimulates NKG2D receptor expression on natural killer cells and inhibits hepatitis C virus replication.

BACKGROUND: Higher hepatitis B surface antigen (HBsAg) facilitates hepatitis C virus (HCV) clearance in patients with hepatitis B virus (HBV)/HCV co-infection. We investigated the effect of exogenous HBsAg on the inhibition of HCV replication mediated by natural killer (NK) cells. METHODS: After isolated from peripheral blood of 42 chronic hepatitis B (CHB) patients and 16 healthy individuals, NK cells were co-cultured with HCV-infected Huh7 cells, respectively, with or without HBsAg. Three days later, the co-cultured supernatants were collected and HCV RNA levels were measured by real-time quantitative PCR. NKG2D, NKp46 and NKG2A expression levels were measured by flow cytometry. NKG2D on NK cells from CHB responsive subgroup was blocked and HCV RNA levels were examined again. RESULTS: HCV RNA levels in the co-cultured system were significantly reduced by NK cells isolated from healthy donors (P < 0.01) but not from CHB patients. However, HCV RNA levels in CHB cultures were significantly decreased following HBsAg addition (P < 0.05), whereas no such effect was seen in control cultures. No significant difference was observed in basic NKG2D expression between the CHB patients and healthy donors. On NK cells from CHB patients, the expression of NKG2D was increased significantly by HBsAg stimulation (P < 0.01), and higher than that from healthy controls (P < 0.05). HCV RNA levels were increased significantly after the blockage of NKG2D on NK cells from responsive CHB patients in the co-cultured system (P < 0.05). CONCLUSION: Exogenous HBsAg stimulated NKG2D expression on NK cells from CHB patients which inhibit HCV replication, suggesting that HBsAg may facilitate the clearance of HCV in patients with HBV/HCV co-infection.

Serum viral duplex-linear DNA proportion increases with the progression of liver disease in patients infected with HBV.

OBJECTIVE: HBV has two forms of genomic DNA, relaxed-circular DNA (rcDNA) and duplex-linear DNA (dlDNA). Compared to rcDNA, dlDNA has been demonstrated to integrate more frequently into host cellular chromosomes, which may have oncogenic consequences. However, the dlDNA proportion relative to total HBV DNA and its clinical significance in patients remain to be investigated. DESIGN: Based on the structural difference between rcDNA and dlDNA, we developed a peptide nucleic acid (PNA)-mediated quantitative real-time PCR (qPCR) clamping assay to measure the proportions of dlDNA in total HBV DNA in sera obtained from patients with chronic hepatitis B (CHB), liver cirrhosis (LC) or LC-developed hepatocellular carcinoma (HCC). The factors that influence the proportion of dlDNA were also investigated. RESULTS: The average dlDNA proportion was approximately 7% in the sera of chronic HBV-infected patients and was elevated in CHB patients with abnormal levels of alanine aminotransferase. The sera dlDNA proportions increased to approximately 14% and 20% in the patients with LC and HCC, respectively. Interferon-α treatment slightly increased the dlDNA proportion in the responders; and nucleotide analogue therapy spuriously elevated the proportion. Moreover, treatment of human hepatoma cells supporting HBV replication with inflammatory cytokines significantly altered the dlDNA proportion in vitro. CONCLUSIONS: Using a novel PNA-mediated qPCR clamping assay, we first showed that serum dlDNA proportions progressively increased during the development of HBV-related liver diseases. The dlDNA proportion can be regulated by inflammatory cytokines, suggesting an association among inflammation, increased production of HBV dlDNA and development of HCC.

Delivery of cell-penetrating peptide-peptide nucleic acid conjugates by assembly on an oligonucleotide scaffold.

Delivery to intracellular target sites is still one of the main obstacles in the development of peptide nucleic acids (PNAs) as antisense-antigene therapeutics. Here, we designed a self-assembled oligonucleotide scaffold that included a central complementary region for self-assembly and lateral regions complementing the PNAs. Assembly of cell-penetrating peptide (CPP)-PNAs on the scaffold significantly promoted endocytosis of PNAs by at least 10-fold in cell cultures, particularly for scaffolds in which the central complementary region was assembled by poly(guanine) and poly(cytosine). The antisense activity of CPP-PNAs increased by assembly on the scaffold and was further enhanced after co-assembly with endosomolytic peptide (EP)-PNA. This synergistic effect was also observed following the assembly of antigene CPP-PNAs\EP-PNAs on the scaffold. However, antigene activity was only observed by targeting episomal viral DNA or transfected plasmids, but not the chromosome in the cell cultures. In conclusion, assembly on oligonucleotide scaffolds significantly enhanced the antisense-antigene activity of PNAs by promoting endocytosis and endosomal escape. This oligonucleotide scaffold provided a simple strategy for assembly of multiple functional peptide-PNA conjugates, expanding the applications of PNAs and demonstrating the potential of PNAs as antiviral therapeutics.

2',3'-cyclic nucleotide 3'-phosphodiesterases inhibit hepatitis B virus replication.

2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) is a member of the interferon-stimulated genes, which includes isoforms CNP1 and CNP2. CNP1 is locally expressed in the myelin sheath but CNP2 is additionally expressed at low levels outside the nervous system. CNPs regulate multiple cellular functions and suppress protein production by association with polyadenylation of mRNA. Polyadenylation of Hepatitis B virus (HBV) RNAs is crucial for HBV replication. Whether CNPs interact with polyadenylation signal of HBV RNAs and interfere HBV replication is unknown. In this study, we evaluated expressions of CNP isoforms in hepatoma cell lines and their effects on HBV replication. We found that CNP2 is moderately expressed and gently responded to interferon treatment in HepG2, but not in Huh7 cells. The CNP1 and CNP2 potently inhibited HBV production by blocking viral proteins synthesis and reducing viral RNAs, respectively. In chronic hepatitis B patients, CNP was expressed in most of HBV-infected hepatocytes of liver specimens. Knockdown of CNP expression moderately improved viral production in the HepG2.2.15 cells treated with IFN-α. In conclusion, CNP might be a mediator of interferon-induced response against HBV.

BST2/Tetherin inhibits hepatitis C virus production in human hepatoma cells.

Hepatitis C virus (HCV) infection is a common cause of chronic hepatitis and is currently treated with alpha interferon (IFN-α)-based therapies. IFN-induced cell membrane protein BST2 (also known as CD317, HM1.24 or tetherin) has been reported to tether a broad range of lipid-enveloped viruses on cell surfaces. However, whether HCV is sensitive to BST2 remains controversial. Here we established a Huh7.5-BST2-TO cell line, in which BST2 expression is regulated by tetracycline. Our results showed that the effect of BST2 on inhibiting HCV production was dependent on its expression level. Highly expressed BST2 reduced the yield of cell-free HCV virions but did not affect the efficiency of HCV infection and genome replication. Co-localization of HCV core protein and BST2 was detected by immunofluorescence in certain cells with high expression, but not in cells with low BST2 expression. Furthermore, inhibition of IFN-α induced BST2 expression in Huh7.5 cells by siRNA technology slightly reduced the antiviral response of the cytokine against HCV, but only at low IFN-α concentration. While overexpression of BST2 inhibited HCV replication in this system, BST2 is therefore not likely to be a major contributor to the antiviral effect of IFN-α.

BST2/tetherin inhibits dengue virus release from human hepatoma cells.

UNLABELLED: Type I interferons (IFN) have been shown to play an important role for inhibiting Dengue virus (DENV) infection. Identifying IFN-induced cellular proteins are essential for understanding its mechanisms against DENV. Here we established stable Huh7-derived cell lines expressing the IFN-induced cell membrane protein BST2 (Huh7-BST2) or its variant bearing a V5 tag at the C-terminal (Huh7-BST5CV5). These cell lines were infected with DENV to determine proteins modulating their anti-DENV response. We found that expression of BST2 did not affect the efficiency of DENV infection and intracellular replication. Rather, it significantly reduced the virion yield of the infected cells, particularly at low MOI infection. In addition, BST2 also decreased the foci formation and the size of infectious foci in cultured Huh7 monolayers with media containing methocellulose. The addition of the V5 tag at C-terminal inhibited the GPI modification of BST2 and blocked its shift from endoplasm to cytoplastic membrane. BST2CV5 did not affect DENV infection and foci formation in Huh7 cells but reduced virion yield by 1 log at low MOI infection. Interestingly, intracellular BST2CV5 expression was reduced by high level of DENV production. CONCLUSION: Our results imply that BST2 is a functional mediator of the IFN response against DENV infection. BST2 inhibits the release of DENV virions from Huh7 cells and limits viral cell-to-cell transmission. BST2CV5 variant is unable to inhibit DENV release but impairs viral infection in cells.

Characterization of microRNA expression profiles associated with hepatitis B virus replication and clearance in vivo and in vitro.

BACKGROUND AND AIM: Alpha interferon (IFN-α) is an approved treatment for chronic hepatitis B (CHB). MicroRNA (miRNA) are currently known as a part of IFN-mediated antiviral defense. We aimed at characterizing the miRNA expression associated with hepatitis B virus (HBV) replication and IFN-mediated HBV clearance. METHODS: We investigated the expression patterns of cellular miRNA induced by HBV replication and/or IFN-α treatment in HepG2 cells, and also analyzed the miRNA response in peripheral blood mononuclear cells in CHB patients on IFN-α treatment. The differentially expressed miRNA were verified using quantitative real-time polymerase chain reaction and an miRNA expression pattern was classified based on the final virological response. RESULTS: A total of 223 miRNA were differentially expressed (> 1.5 folds) between the HepG2.2.15 and HepG2 cells, including 24 highly differentially expressed miRNA (> 5 folds). With 12 h of IFN-α treatment, 23 totally differentially expressed miRNA were identified in HepG2 cells; whereas only five miRNA were identified in HepG2.2.15 cells. Similar amounts of the miRNA were regulated in patients with HBeAg or non-HBeAg seroconversion; whereas levels of eight miRNA were significantly differentially expressed between the two groups. CONCLUSIONS: HBV replication alters miRNA expression profiles and impairs IFN-inducible miRNA response in HepG2 cells. The miRNA expression pattern of peripheral blood mononuclear cells in CHB patients with IFN therapy can be associated with their therapeutic outcome.

Interferons accelerate decay of replication-competent nucleocapsids of hepatitis B virus.

Alpha interferon (IFN-alpha) is an approved medication for chronic hepatitis B. Gamma interferon (IFN-gamma) is a key mediator of host antiviral immunity against hepatitis B virus (HBV) infection in vivo. However, the molecular mechanism by which these antiviral cytokines suppress HBV replication remains elusive. Using an immortalized murine hepatocyte (AML12)-derived cell line supporting tetracycline-inducible HBV replication, we show in this report that both IFN-alpha and IFN-gamma efficiently reduce the amount of intracellular HBV nucleocapsids. Furthermore, we provide evidence suggesting that the IFN-induced cellular antiviral response is able to distinguish and selectively accelerate the decay of HBV replication-competent nucleocapsids but not empty capsids in a proteasome-dependent manner. Our findings thus reveal a novel antiviral mechanism of IFNs and provide a basis for a better understanding of HBV pathobiology.

[High replicated hepatitis B virus induces apoptosis of hepatocytes].

OBJECTIVE: To investigate the effects of high level hepatitis B virus (HBV) replication on the hepatocytes. QSG-7701 cells. METHODS: Human hepatocytes of the line QSG-7701 were cultured and transfected with the plasmid pUC18-HBV1.2 or pUC18 containing 1.2 full length HBV DNA by the standard calcium phosphate precipitation method. Other QSG-7701 cells were transfected with the plasmid pUC18 as controls. Cell growth curves were drawn for 7 days after transfection. Four 4 days after transfection, HBV DNA in the culture medium was detected by using fluorescence quantitative real-time PCR. Cell apoptosis was detected by using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and electronic microscopy. Differential expressed genes were analyzed by using Oliga signal pathway micro-array. RESULTS: The curves of cell growth showed that the amount of control QSG-7701 cells increased by (8.3 +/- 1.2) times, significantly faster than the pUC18-HBV1. 2 transfected QSG-7701 cells that increased only by (1.1 +/- 0.2) times (P < 0.01). Four days after transfection, the HBsAg positive rate of the pUC18-HBV1.2 transfected cells was 35.4% +/- 6.7%, and the apoptotic rate was 15.2% +/- 4.3%. The HBV DNA level in the culture supernatant peaked 4 days adder transfection with the maximum value of (5.8 +/- 2.6) x 10(6) copies/ml. Genes related to cell growth and apoptosis, such as CASP3 (2.7981) ,CASP7 (2.2643), 3-Apr (3.5013), CDC2 (0.4380), MAPK6 (0.4447), and MAP3K2 (0.2785), were differentially expressed. CONCLUSION: High replicated HBV markedly inhibits the growth of hepatocytes and induces cell apoptosis.

[Subcellular distribution and translocation of hepatitis B virus core protein in HepG2.2.15 cells].

OBJECTIVES: The hepatitis B virus core protein has been found in nuclei, cytoplasm, or both of hepatocytes transfected with HBV DNA. It is still unclear whether intact core particles could pass through nuclear pores and what could be the mechanism regulating the subcellular localization of the core protein. This study on the distribution of core protein in hepatocytes and its translocation has a potential advantage to learn more about the HBV life cycle. METHODS: Dimethyl sulphoxide (DMSO, 2%), which effects hepatic differentiation, and/or 1 micro mol/L heteroaryldihydropyrimidine Bay41-4109, which interferes with the assembly of core particles, were added into HepG2.2.15 cell culture system for 4 days. The hepatitis B virus core antigen (HBcAg) and hepatitis B virus surface antigen (HBsAg) were stained with fluorescent immunocytochemistry and then observed under a confocal microscope. HBcAg in cytoplasm and nuclei were respectively extracted and analyzed using Western blot. HBV covalently closed circular DNA (cccDNA) was detected by using selective PCR method. RESULTS: The HBcAg was mostly expressed in the cytoplasm and weak signals of cccDNA were detected in the control HepG2.2.15 cells. After DMSO treatment, the expression of HBcAg in cytoplasm was increased about 2.5-fold; the expression of HBcAg and cccDNA in nuclei also increased. With the use of Bay41-4109, the signal of HBcAg in cytoplasm decreased 2/3, but it increased in the nuclei, and cccDNA decreased in the nuclei. When the HepG2.2.15 cells were treated both with DMSO and Bay41-4109, cord-liked distribution of HBsAg was observed in the cytoplasm. HBcAg in cytoplasm was decreased 1/2 but the HBcAg in the nuclei increased about 5-fold, whereas the cccDNA was almost negative. CONCLUSION: In HepG2.2.15 cells, the core protein is mainly assembled as a formation of core particles in the cytoplasm and they are blocked by the nuclear membrane. Bay41-4109 interferes with the assembly of core particles and the dissociated core proteins are able to enter the nuclei. DMSO promotes the nuclear entry of core protein/core particles and facilitates the formation of cccDNA.

[Comparisons of the characteristics and mechanisms of HBV replication in QSG-7701 and HepG2 cell lines].

OBJECTIVE: To gain some insights into the critical events relating to HBV transcriptional regulation by comparing HBV replicative characteristics in different cell lines. METHODS: Hepatic cell lines QSG-7701 and HepG2 were transfected with plasmid PUC18-HBV 1.2 by standard calcium phosphate precipitation method, and 1.0 microg pSEAP2-control vector was included in the transfection procedures to serve as an internal control monitoring the transfection efficiency. Hepatitis B surface antigen (HBsAg) in the medium was detected by ELISA method and HBV DNA was quantitated using fluorescent quantitative PCR. The intracellular HBsAg and HBcAg were detected with immunofluorescent staining. The gene expression profiles of QSG-7701 and HepG2 were compared using oligonucleotide microarray; partial differentially expressed genes were verified with quantitative RT-PCR. RESULTS: In the medium of the cultured HepG2 cells, HBsAg and HBV DNA could be detected 6 days after the transfection, whereas in QSG-7701 cells, the HBsAg and HBV DNA could be detected for 2 weeks. The HBV DNA in the culture medium of QSG-7701 was about 50 times more than that of the HepG2 cells which were kept in 1 x 10(7)copies/ml(-3) x 10(7)copies/ml for 0 to 10 days after the transfection. On the 4th day after the transfection, 20% to 30% of the QSG-7701 cells were positive with HBsAg and HBcAg immunofluorescent staining. The gene microarray analysis showed that most transcription factors involved with HBV life cycle in QSG-7701 and HepG2 cells had similar levels, whereas some factors involved with HBV transcriptional regulation and core particle disassembly, such as interleukin-6 (R=5.1340), retinoid X receptor, alpha (R=5.1268), hepatic leukemia factor (R=3.2538), serine protease PRRS23 (R=2.8356), hepatitis B virus x interacting protein (R=0.4939), serine protease inhibitor Kazal type 1 (R=0.0740) and matrix metalloproteinase 3 (negative in QSG-7701) were all differentially expressed by HepG2 cells. CONCLUSION: Different than HepG2 cells, the QSG-7701 cells could support a high level and relatively stable HBV replication after HBV DNA transient transfection. The HBV core particles were probably recycled in the QSG-7701 cells. The differential gene expressions between QSG-7701 and HepG2 might explain the mechanism of the different HBV replication patterns. Hepatic cell line QSG-7701 might serve as a useful tool for HBV transcriptional regulation research.

[Cyclosporine A improves the nuclear entry of hepatitis B virus core protein in HepG2.2.15 cells].

OBJECTIVE: The present aimed to observe the effect of phosphatase inhibitor cyclosporine A on the subcellular location and on expression of HBcAg in HepG2.2.15 cells. METHODS: Thirty micrograms/ml of cyclosporine A (CSA) was added into HepG2.2.15 cell culture system and on days 2 and 4 HBcAg and HBsAg were respectively stained with fluorescent immunocytochemistry and observed under confocal microscope. Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling method. RESULTS: HBcAg was mostly expressed in cytoplasm in the control HepG2.2.15 cells. After 2 days CSA administration of the expression of HBcAg and HBsAg in cytoplasm significantly decreased and the signals of HBcAg in nucleus increased , whereas the HBcAg was still mainly expressed in nucleus in about 1/4 of the cells. Cell apoptosis was observed in about 30% of the cells. CONCLUSION: CSA improves the nuclear entry of core protein. The increase of HBcAg in nucleus was likely to be related with it's phosphorylation and cell aging or apoptosis.

[A study on stem cell mobilization after partial liver transplantation in rats].

OBJECTIVE: To explore the role of stem cell mobilization on regeneration of partially grafted livers. METHODS: Rats models with cross-sex 50% PLTx (partial liver transplantation) were established. The rats were divided into three groups: PLTx, WLTx (whole liver transplantation) and sham operation groups. Bone marrow and liver samples were collected on days 1, 3, 5, 7 postoperatively (each n = 6). The quantitative variations of the cells with stem cell markers in the bone marrow, including beta2m-/Thy-1.1+, CD45+/CD34+, Flt2/3+ and c-kit+ markers, were detected using flow cytometry. Sry gene positive cells in donor livers were detected by fluorescent in situ hybridization (FISH), and the expressions of CD34, c-kit and Thy-1.1 were detected by immunohistochemistry technique. RESULTS: Compared with the WLTx and sham operation groups, beta2m-/Thy-1.1+, CD45+/CD34+ cells in bone marrows in the PLTx group increased on the first postoperative day and decreased on the following days. The CD34, c-kit and Thy-1.1 positive cells detected in portal tract areas peaked during the 3-5 postoperative days. CD34+/CD45+ positive cells could be detected. The expressions of CD34, c-kit and Thy-1.1 positive cells were rare in the WLTx and sham operation groups. Sry+ cells could be detected in portal tract areas and few Sry+/CD34+ and Sry+/Thy-1.1+cells were detected. CONCLUSION: In the PLTx group, the stem cells in the bone marrow were mobilized and stem cells in the liver were activated.

[Granulocyte colony-stimulating factor-mobilized autologous bone marrow stem cells promote the liver regeneration of partial liver transplant: an experiment with rats].

OBJECTIVE: To investigate the effects of granulocyte colony-stimulating factor (G-CSF)-mobilized autologous bone marrow stem cells on the liver regeneration of partial liver transplant. METHODS: A 50% partial liver transplantation model as established by transplanting parts of the liver of female rats to the male rats with the equal weights that had had parts of their livers resected. Then the transplanted male rats were randomly divided into 3 equal groups: G-CSF + PLTx group that was injected hypodermically with recombinant human G-CSF (rhG-CSF) daily for 5 days and then underwent liver resection and 50% partial liver transplantation (PLTx); PLTx + G-CSF group that underwent PLTx and 3 hours after the transplantation received injection of rhG-CSF daily for 5 days, and PLTx control group that underwent PLTx and 3 hours later received injection of normal saline daily for 5 days. One, 3, 5, 7, and 14 days after the PLTx blood samples and livers were collected from 6 rats from each group, and one hour before the liver was taken bromodeoxyuridine (BrdU) was injected intraperitoneally to be integrated into the synthesis of DNA in the liver cells. Immunohistochemistry was used to detect the CD34 and BrdU-positive cells. In situ hybridization was used to detect the sry (sex-determining region) gene so as to determine the origin of the proliferating cells in the transplanted liver. RESULTS: The 14-day survival rate of the G-CSF + PLTx group was 90%, significantly higher than those of the G-CSF + PLTx (60%) and PLTx group (50%) (both P < 0.05) with a significant difference between the latter 2 groups too (P < 0.05). The mitosis index of liver cells 3 days after the transplantation of the PLTx + G-CSF group was 30% +/- 5%, significantly higher than those of the G-CSF + PLTx group (24% +/- 7%) and PLTx group (24% +/- 6%) (both P < 0.05) without a significant difference between the latter 2 groups. The rate of BrdU-positive cells of the PLTx + G-CSF group was 42% +/- 6%, significantly higher than those of the G-CSF + PLTx group (38% +/- 4%) and PLTx group (34% +/- 8%) (both P < 0.05). The transplantation the mitosis index and rate of BrdU-positive cells decreased since the 7th days after transplantation in all 3 groups, however, with the same relationship among them. The numbers of CD34(+) cell around the portal area of the 2 G-CSF groups increased since the 3rd day after transplantation in comparison with the PLTx group, was the highest on the 3rd day for the G-CSF + PLTx group, and continued to increase in the PLTx + G-CSF group. Since the 3rd day after transplantation, sry-positive cells were seen in the hepatic sinusoid and portal area in the 2 G-CSF groups, and were rarely seen in the PLTx group. CONCLUSION: G-CSF treatment after 50% PLTx significantly promotes liver regeneration and ameliorates the liver damage, thus raising the survival rate.