Effects of Leukaemia Inhibitory Factor Receptor on the Early Stage of Osteogenic Differentiation of Human Bone Marrow Mesenchymal Cells.

Leukaemia inhibitory factor (LIF) has a wide variety of biological activities. While recent studies have focused on the role of LIF in osteoblast differentiation, the exact role of LIFR during the early stage of osteogenic differentiation remains unclear. We observed that LIFR expression gradually decreased during the early stage of osteogenic differentiation of hMSCs. To evaluate how LIFR regulates osteogenic differentiation in greater depth, we transfected hMSCs with LIFR overexpression and siRNA lentiviral plasmids. Cells were divided into four groups: a negative overexpression control group, a LIFR overexpression group, a negative siRNA control group, and a LIFR siRNA group. On different days (0, 3, and 6) of the osteogenic differentiation of hMSCs, alkaline phosphatase (ALP) activity was assayed with an ALP staining and activity assay kit. Cells were harvested to assess the mRNA and protein expression of LIF, LIFR, and osteogenesis-related factors (ALP; RUNX2; osteonectin) by qRT-PCR and western blot analyses, respectively. In addition, culture supernatants were tested for the LIF content by ELISA. Our results showed that overexpression of LIFR significantly suppressed the osteoblast differentiation of hMSCs. In contrast, LIFR siRNA markedly improved this osteoblast differentiation as determined by ALP staining and activity measurements. Moreover, RUNX2, ALP, and ONN expression was also significantly changed by altering LIFR expression. We further analysed the expression of LIF and LIFR, revealing consistent LIF and LIFR trends during the osteogenic differentiation of hMSCs. Together, these results suggested that LIFR may be a novel negative regulator during the early stage of hMSC osteogenic differentiation.

The expression of insulin-like growth factor II, hepatitis B virus X antigen and p21 in experimental hepatocarcinogenesis in tree shrews.

The purpose of this paper was to study the mechanism of synergistic effect in hepatocarcinogenesis induced by hepatitis B virus (HBV) infection and aflatoxin B1 (AFB1) intake. Immunohistochemical staining was used in formalin-fixed, paraffin-embedded sections of cancer and liver tissues. The incidence of hepatocellular carcinomas (HCCs) was 52.9% in experimental tree shrews that received both HBV and AFB1. It was significantly higher than that of animals exposed to HBV (11.1%, Group B), or (AFB1) (15.8%, group C) alone. HCC was not found in the control animals (group D). The expressions of insulin-like growth factor II (IGF-II) were 82.4%, 22.2%, 26.3% and 0 in groups A, B, C and D, respectively. The significant differences of IGF-II were observed between groups A and B, C and D (P < 0.05). The expressions of p21 were 29.4%, 11.1%, 15.8% and 0 in group A, B, C and D, respectively. The positive rate of hepatitis B x antigen (HbxAg) was significantly higher in the group A than that in the group B (52.9% vs. 11.1%, P < 0.05). The parallel relations between the incidence of HCC and the overexpressions of these genes protein have been found in each group. On the other hand, the expressions of these genes in tumour-bearing tree shrews were significantly higher than that in nontumour-bearing animals. These findings suggest a synergistic effects of HBV and AFB1 in activation of these genes in tree shrews. Overexpressions of these genes may take an important role in the course of hepatocarcinogenesis in tree shrews.

Synergistic effect of hepatitis B virus and aflatoxin B1 in hepatocarcinogenesis in tree shrews.

An animal experiment with tree shrews was performed to detect the synergistic effects of hepatitis B virus (HBV) infection and dietary aflatoxin B1 (AFB1) in hepatocarcinogenesis. Adult healthy tree shrews (Tupaia belangeri chinensis) were divided into four groups: Group A (HBV + AFB1)--animals were infected with human HBV serum at first, then fed AFB1 diluted with milk, 150 ug/kg.bw/day, 6 days/week for 105 weeks. Group B (HBV)--animals were infected with human HBV as Group A, but no AFB1 treatment. Group C (AFB1)--animals were treated with AFB1 as Group A but no HBV infection. Group D--animals were treated neither with human HBV nor AFB1. During the experiment, blood samples and liver biopsies were taken regularly from all animals in each group. All the animals were sacrificed on the 160th week when the experiment ended. The samples of sera and liver tissues were checked for HBV markers and histological changes. Hepatocellular carcinomas (HCCs) were found only in Group A and Group C, with incidences of 67% and 30% respectively. The average time for HCC occurrence in Group A and Group C was 120.3 +/- 16.6 and 153.3 +/- 5.8 weeks respectively (P < 0.01). Even though no HCC occurred in Group B, 1 animal which died before the end of the experiment showed two large hepatocellular nodules. These results showed that there is synergistic effect between HBV and AFB1 in tree shrews' hepatocarcinogenesis, even though the hepatocarcinogenic effect played by HBV alone is rather weak.

Human hepatitis B virus and hepatocellular carcinoma. II. Experimental induction of hepatocellular carcinoma in tree shrews exposed to hepatitis B virus and aflatoxin B1.

On the basis of the successful establishment of an animal model in tree shrews experimentally infected with human hepatitis B virus (HBV), a study on the hepatocarcinogenic effects of HBV and/or aflatoxin B1 (AFB1) was conducted. The results showed that the incidence of hepatocellular carcinoma (HCC) was significantly higher in the animals both infected with HBV and exposed to AFB1 (52.94%) than in those solely infected with HBV (11.11%) or exposed to AFB1 (12.50%). No HCC of precancerous lesions were found in the controls that were neither HBV-infected nor AFB-1 exposed. Precancerous lesions, including liver cell dysplasia and enzyme-altered hyperplastic hepatocyte foci, were observed before the occurrence of HCC, and the frequency of their appearance correlated well with the incidence of HCC. HBV DNA and the protein it encodes were detected in the cancer cells and/or the surrounding hepatocytes. Integration of HBV DNA into the host liver genome was found during hepatocarcinogenesis among the animals infected by HBV. These results suggest that exposure to HBV and AFB1 may play a synergistic role in the development of HCC, and support the viewpoint of an aetiological relationship between HBV and HCC.

Human hepatitis B virus and hepatocellular carcinoma. I. Experimental infection of tree shrews with hepatitis B virus.

Tree shrews (Tupaia belangeri chinenesis) can be experimentally infected with human hepatitis B virus (HBV) by inoculation with human serum positive for HBV, the experimental infection rate being 55.21%. Successive infections have been passed through five generations among the tree shrews inoculated with HBV-positive sera from the infected animals, the average infection rate being 94.0%. The experimental infection of tree shrews with HBV may be prevented by immunization with hepatitis B vaccine, the protection rate being 88.89%. Standard serum containing HBV at 10(8) CID (chimpanzee infection dose)/ml, was diluted 10(-6), 10(-7), 10(-8), 10(-9), and 10(-10) and produced infection rates of 80.0%, 88.8%, 66.7%, 55.6% and 42.9% respectively. Thus the CID50 in tree shrews may reach a dilution of 10(-9), which shows that tree shrews are sensitive to HBV infection. These results successfully establish tree shrews as a reliable and useful animal model for research on HBV infection and its relation to hepatocarcinogenesis.

Reliability of a short-term test for hepatocarcinogenesis induced by aflatoxin B1.

The reliability of a short-term test for hepatocarcinogenesis induced by aflatoxin B1 (AFB1) was tested by comparing the early appearance of gamma-glutamyl transpeptidase (GGT)-positive foci with the occurrence of primary liver cancer at a later stage. All rats received a basic short-term treatment with AFB1 intraperitoneally, during which three experimental groups received Chinese green tea or 2000 or 5000 ppm butylated hydroxyanisole in the diet and a control group received basic diet. Some of the rats in each group were sacrificed at the end of the short-term procedure, and the remainder were observed up to 92 weeks. The livers of all animals were examined for GGT-positive foci or primary liver tumours. The GGT-positive foci were most numerous and largest and the incidence of liver tumours was highest in the control group. These findings suggest that GGT-positive foci are a valuable preneoplastic marker for AFB1-induced hepatocarcinogenesis, that the short-term model is fairly reliable, and that both Chinese green tea and butylated hydroxyanisole inhibit AFB1-induced hepatocarcinogenesis.

[A study on primary liver cancer in tree shrews induced by human hepatitis B virus and aflatoxin B1].

Among 41 tree shrews exposed to aflatoxin B1(AFB1), 17 were experimental infected by human hepatitis B virus (HHBV) and 24 were uninfected. After 158 weeks, 9 cases (52.94%) of primary liver cancer (PLC) were found out of the 17 tree shrews infected by HHBV and only 3 cases (12.5%) developed PLC in the 24 uninfected animals. Significant difference of PLC incidence was seen between the HHBV-infected and uninfected groups (P less than 0.05). Moreover, 1/9 of the tree shrews that had been infected by HHBV but without exposure to AFB1 developed PLC at the 83rd week. No PLC was found in 6 tree shrews that had neither been infected with HHBV nor been exposed to AFB1. These results demonstrate the possible etiologic relationship between HHBV infection and PLC, and the synergistic effects of HHBV and AFB1 during PLC development.

[Effect of six edible plants on the development of AFB1-induced gamma-glutamyltranspeptidase-positive hepatocyte foci in rats].

Six edible plants, green tea (GT), black tea (BT), Lentinus edodes (berk) Sing (LE), Hericium erinaceus (Bull. ex Fr.) Pers. (HE), Mixture of Ganoderma Lucidum (Ley ss ex Fr.) Karst et Ganoderma Japanium (Fr.) Lloyd (MGLJ) and mung bean (MB), were tested for the effect on the development of AFB1-induced gamma-glutamyltranspeptidase positive hepatocyte foci (gamma-GT foci) using an in vivo short-term test model in rats. The rats received intraperitoneally 12 doses of initiator AFB1, 400 micrograms/kg per dose for 2 successive weeks. Two weeks after the initiation, the rats were submitted to a modified "Solt-Farber promotion program", i.e., a two weeks' feeding of a diet containing 0.015% acetylaminofluorene plus a two-third partial hepatectomy (PH) on day 7. The rats were sacrificed 10 days after PH and the livers were processed to gamma-glutamyltranspeptidase staining. The tested substances were powdered and mixed with the basal diet at the concentration level of 30% for MB and 5% for the others. The rats were fed with the diet-containing tested substances from 10 days before the AFB1 initiation to 3 days after the AFB1 conclusion. Consequently, the liver of the rats which had consumed GT showed significantly less and smaller gamma-GT foci, and those which had consumed BT, HE and LE showed somewhat less and significantly smaller foci than the control groups. It is indicated that the four diets have an inhibiting effect on AFB1-induced gamma-GT foci in different degrees. MB and MGLJ show no significant influence on the foci.