当归多糖影响小鼠胸腺细胞凋亡的实验研究

目的:观察当归多糖(AP)对小鼠胸腺细胞凋亡的影响。方法:以地塞米松造成胸腺细胞凋亡小鼠模型,观察胸腺细胞形态学的改变,并统计细胞凋亡率及各组小鼠胸腺细胞凋亡指数的变化。结果:模型组具有凋亡细胞典型的形态学特点,低剂量组凋亡细胞数目明显降低,胸腺细胞凋亡指数随着当归多糖剂量的加大而逐渐减少。结论:当归多糖具有抑制地塞米松诱导的胸腺细胞凋亡的作用。

健肾益寿口服液对衰老模型小鼠一氧化氮水平及学习记忆的影响作用

目的:探讨健肾益寿口服液对衰老模型小鼠一氧化氮(NO)水平及学习记忆能力的影响作用。方法:以 D- 半乳糖衰老模型小鼠为研究对象,观察健肾益寿口服液对 D- 半乳糖衰老模型小鼠血清 NO水平及学习记忆的影响作用。结果:该口服液可明显改善衰老小鼠学习记忆能力。并且可使衰老模型小鼠心、脑、肾脏 NO 水平均明显回升,接近正常水平。结论:提示本口服液对衰老机体 NO 水平具有良好的调节功能,此功能也可能是其改善衰老小鼠学习记忆能力的机理之一。

Effects of Low Dose Radiation on Tumor Apoptosis, Cell Cycle and Apoptosis-Related Protein Bcl-2 in Tumor-Bearing Mice

To study the effects of low dose radiation (LDR) on tumor apoptosis, cellcycle progression and changes of apoptosis-related protein Bcl-2 in tumor-bearing mice. Methods:Male mice of Kunming strain were implanted subcutaneously with S180 sarcoma cells in the left inguenas an in situ experimental animal model. Seven days later, the mice were subjected to 75 mGywhole-body γ-irradiation. At 24 and 48 h after the irradiation, all mice were sacrificed. The tumorsizes were measured, and tumor cell apoptosis and cell cycle progression were analyzed by flowcytometry. The expression of apoptosis-related protein Bcl-2 and the apoptotic rate of tumor cellswere observed by immunohistochemistry and electron microscopy. Results: Tumors grew significantlyslower after LDR (P 【 0.05). The tumor cells were arrested in G1 phrase and the expression of Bcl-2protein decreased at 24 h. Apoptotic rate of tumor cells was increased significantly at 48 h afterLDR (P 【 0.01). Conclusion: LDR could cause a G1-phase arrest and increase the apoptosis of tumorcells through the low level of apoptosis-related protein bcl-2 in the tumor-bearing mice. Theorganized immune function and anti-tumor ability are markedly increased after LDR. Our studyprovides practical evidence of clinical application to cancer treatment.

Anti-hepatoma effect of arsenic trioxide on experimental liver cancer induced by 2-acetamidofluorene in rats

AIM： To study the anti-hepatoma efficiency of arsenic trioxide （As2O3） in the treatment of experimental rat hepatocellular carcinoma （HCC） induced by 2-acetamidofluorene （2-FAA） and to elucidate the possible mechanisms. METHODS： SD rats （2 mo old） had been fed with 2-FAA for 8 wk to induce HCC, and then they were treated with As2O3 or matrine. On d 29, the rats were killed and the liver was weighed and liver tumors were counted. The histological changes of liver tissue were observed under microscope, and the cellular dynamic parameters were studied by flow cytometry. Immunohistochemistry （two-step method） was used to observe the expression of vascular endothelial growth factor （VEGF） and micro-vessel density （MVD） on consecutive sections. The pathological parameters were also analyzed, the levels of serum aspartate aminotransferase （AST）, alanine aminotransferase （ALT）, total bilirubin （TBi）, and direct bilirubin （DBi）. RESULTS： The number of liver tumors decreased significantly in groups treated with As2O3, especially in medium-dose （1 mg/kg） group （t = 2.80, P〈0.01）. As2O3 caused HCC cell death via apoptosis; necrosis was seen and apoptosis was common when the dose was 1 mg/kg. Proliferation index decreased sharply in medium-dose （1 mg/kg） group （7.87±4.11 vs24.46±6.49, t= 2087, P〈0.01）, but not in 0.2 mg/kg group. However, S-phase fraction decreased dramatically in both groups, it reached the bottom level only when the dose was i mg/kg compared with control （0.40±0.13 vs3.01±0.51, t= 2.97, P〈0.01）, and it was obviously accompanied with accumulation of cells in G0/G1 （G0/G1 restriction）. The expressions of VEGF and MVD in medium-dose （1 mg/kg） group were significantly lower than normal saline group （0.63±0.74 vs2.44±0.88, P〈0.05; 15.75±3.99 vs47.44±13.41, t= 2.80, P〈0.01）. Compared with normal saline group, mediumand low-dose groups As203 and matrine lowered the levels of ALT in serum （61.46±9.46, 63.75±20.40, 61.18±13.00 vs 108.98±29.86, t= 2.14, P〈0.05）, but had no effect onthe level of serum AST, TBi, and DBi. CONCLUSION： As203 had inhibitory effect on growth of experimental HCC in rats induced by 2-FAA, but had no obvious effect on normal hepatic cells. The mechanisms may involve decrease of cell division, accumulation of cells in G0/G1 phase, apoptosis of tumor cells, and inhibitory effect on angiogenesis through blocking VEGF.

Antitumor effect and mechanism of Gecko on human esophageal carcinoma cell lines in vitro and xenografted sarcoma 180 in Kunming mice

AIM: To investigate the anti-tumor effect of Chinese medicine Gecko on human esophageal carcinoma cell lines and xenografted sarcoma 180 in Kunming mice and its mechanism. METHODS: The serum pharmacological method was used in vitro . The growth rates of the human esophageal carcinoma cells (EC9706 or EC1) were measured by a modifi ed 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The transplanted tumor model of the mouse S180 sarcoma was established. Fifty mice were randomly divided into fi ve groups (n = 10). Three Gecko groups were treated respectively with oral administration of Gecko powder at a daily dose of 13.5 g/kg, 9 g/kg, and 4.5 g/kg. The negative group (NS group) was treated with oral administration of an equal volume of saline and the positive group (CTX group) was treated with 100 mg/kg Cytoxan by intraperitoneal injection at the fi rst day. After 2 wk of treatment, the anti-tumor activity was evaluated by tumor tissue weighing. The impact on immune organ was detected based on the thymus index, spleen index, phagocytic rate and phagocytic index. The protein expression of vascular endothelingrowth factor (VEGF) and basic fibroblast growth factor (bFGF) were detected by immunohistochemistry. The cell apoptotic rate was detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay. RESULTS: The A value in each group treated with Gecko after 72 h was reduced signif icantly in EC9706 and in EC1. The tumor weight in each group of Gecko was decreased signifi cantly (1.087 ± 0.249 vs 2.167 ± 0.592; 1.021 ± 0.288 vs 2.167 ± 0.592; 1.234 ± 0.331 vs 2.167 ± 0.592; P < 0.01, respectively). However, the thymus index and Spleen index of mice in Gecko groups had no significant difference compared with the NS group. The immunoreactive score of VEGF and bFGF protein expression of each Gecko group by immunohistochemical staining were lowered signifi cantly. The apoptosis index (AI) of each group was increased progressively with increase of dose of Gecko by TUNEL. CONCLUSION: Gecko has anti-tumor effects in vitro and in vivo; induction of tumor cell apoptosis and the down-regulation of protein expression of VEGF and bFGF may be contributed to anti-tumor effects of Gecko.

Comparative study of histopathology changes between the PS1/APP double transgenic mouse model and Aβ_(1-40)-injected rat model of Alzheimer disease

Objective To identify the genetype of the PS1/APP double transgenie mouse model, then to analyse the histopathological changes in the brain and compare the differences between the transgenie mice models and Aβ1-40-injeeted rats models of Alzheimer disease. Methods The modified congo red staining, Nissl＇s staining and immunohistology staining was used to observe the Aβ deposits, activation of astrocyte respectively. Results ①The PS1/APP transgenic mouse extensively displayed Aβ deposits in the cortex and hippocampal structures, and GFAP positive cells were aggregated in mass and surrounded the congo red-positive plaque. ②The Aβ1-40-intrahippocmnpal-injeeted rat model showed the Aβ plaque deposits in the dentate gyrus of the hippocampus, with the astrocyte surrounded. The neurons loss was significant in the injection point and pin hole of injection with Nissl＇s staining methods. GFAP-positive cells increased significantly compared with the uninjected lateral of the hippocampus. Conclusion Although Aβ1-40-injected rat models could simulate some characteristic pathological features of human Alzheimer diseases, Aβ deposits and neurons loss in partial hippocampal, it would not simulate the progressive degenenration in the brain of AD. The double transgenie PS1/APP mice could simulate the specific pathogenesis and progressive changes of AD, mainly is Aβ deposits and the spongiocyte response , while no neurons loss were observed in this model.

Functional analysis of two Sp1/Sp3 binding sites in murine Nanog gene promoter

Nanog gene plays a key role in maintaining pluripotency of ES cells and early embryonic cells. A 5＇ flank sequence of the Nanog gene has been reported to be regulated differentially, and two regulatory elements within the Nanog promoter, namely Oct-4 and Sox-2 binding sites, have been identified to regulate the transcriptional activity ofNanog gene. In this report, we identified the role of two putative Spl binding sites located in the Nanog gene 5＇-flanking region in regulation ofmurine Nanog gene transcription. Mutation studies showed that the two sites were essential for the Nanog promoter activity. Gel shift and supershift analysis showed that both sites specifically bind Spl and Sp3. Furthermore, overexpression of dominant-negative Spl or Sp3 mutants significantly inhibits Nanog promoter activity. These results suggest that the transcription factor Spl and Sp3 are important for Murine Nanog gene expression.

Delayed ethyl pyruvate therapy attenuates experimental severe acute pancreatitis via reduced serum high mobility group box 1 levels in rats

AIM： To investigate the effect of delayed ethyl pyruvate （EP） delivery on distant organ injury, survival time and serum high mobility group box 1 （HMGB1） levels in rats with experimental severe acute pancreatitis （SAP）. METHODS： A SAP model was induced by retrograde injection of artificial bile into the pancreatic ducts of rats. Animals were divided randomly into three groups （n = 32 in each group）： sham group, SAP group and delayed EP treatment group. The rats in the delayed EP treatment group received EP （30 mg/kg） at 12 h, 18 h and 30 h after induction of SAP. Animals were sacrificed, and samples were obtained at 24 h and 48 h after induction of SAP. Serum HMGB1, aspartate arninotransferase （AST）, alanine arninotransferase （ALT）, blood urea nitrogen （BUN）, and creatinine （Cr） levels were measured. Lung wet-to-dry-weight （W/D） ratios and histological scores were calculated to evaluate lung injury. Additional experiments were performed between SAP and delayed EP treatment groups to study the influence of EP on survival times of SAP rats. RESULTS： Delayed EP treatment significantly reduced serum HMGB1 levels, and protected against liver, renal and lung injury with reduced lung W/D ratios （8.22 ±0.42 vs 9.76 ± 0.45, P 〈 0.01）, pulmonary histological scores （7.1 ± 0.7 vs 8.4 ± 1.1, P 〈 0.01）, serum AST （667 ± 103 vs 1 368 ± 271, P 〈 0.01）, ALT （446 ± 91 vs 653 ± 98, P 〈 0.01） and Cr （1.2 ± 0.3 vs 1.8 ± 0.3, P 〈 0.01） levels. SAP rats had a median survival time of 44 h. Delayed EP treatment significantly prolonged median survival time to 72 h （P 〈 0.01）. CONCLUSION： Delayed EP therapy protects against distant organ injury and prolongs survival time via reduced serum HMGBllevels in rats with experimental SAP. EP may potentially serve as an effective new therapeutic option against the inflammatory response and multiple organ dysfunction syndrome （MODS） in SAP patients.

Preventive effects of chitosan on peritoneal adhesion in rats

AIM： To study the effects of chitosan gel and blending chiston/gelatin film on preventing peritoneal adhesion in rats. METHODS： SD rats were randomly divided into 2 groups, group A treated with chitosan gel and group B with blending chiston/gelatin film. In group A, rats were randomly subdivided into 3 subgroups as groups A1, A2 and A3, and different methods were used to induce peritoneal adhesions at the dead end of vermiform process in each group as follows： Group A1 with trauma, A2 with talc powder and A3 with ligation of blood vessel. In each subgroup, rats were redivided into control group and experimental group whose treated vermiform processes were respectively coated with chitosan gel and normal saline immediately after the adhesioninduced treatments. In group B, all the rats received traumatic adhesion-induced treatments and then were randomly divided into 4 groups （groups B1, B2, B3, B4）. Group B1 served as control group and were coated with normal saline in the vermiform processes immediately after the treatments, and groups B2, B3 and B4 with 100% chitosan film, chitosan film containing 10% gelatin and chiston film containing 50% gelatin, respectively. At 2 and 4 wk after the above treatments, half of the rats in each terminal group were belly opened, and the peritoneal adhesive situation was graded and histopathological changes were examined. RESULTS： （1） In group A, regarding peritoneal adhesion situation： At both 2 and 4 wk after the treatments, for groups A1 and A3, the adhesive grades of experimental groups were significantly lower than those of the control group （2 wk： H = 4.305, P 〈 0.05 for A1, H = 6.743, P 〈 0.01 for A3; 4 wk： H = 4.459, P 〈 0.05 for A1, H =4.493, P 〈 0.05 for A3）. However, of group A2, there was no significant difference between the experimental and control groups （2 wk： H = 0.147, P 〉 0.05; 4 wk： H = 1.240, P 〉 0.05）. Regarding pathological changes： In groups A1 and A3, the main pathological change was fibroplasia. In group A2, the main changes were massive foreign-body giant cell reaction and granuloma formation with fibroplasia of different degrees. （2） In group B, regarding degradation of film： With increase of the blended gelatin concentration, degrading speed of the film accelerated significantly. Regarding peritoneal adhesion situation： At both 2 and 4 wk after the treatments, the adhesive grades of B1 were the lowest among the four subgroups of B （2 wk： H = 29.679, P 〈 0.05; 4 wk： H = 18.791, P 〈 0.05）. At 2 wk after the treatments, the grades of group B2 were significantly lower than that of groups B3 and B4 （H = 4.025, P 〈 0.05 for B2 vs B3; H = 4.361, P 〈 0.05 for B2 vs B4）. At 4 wk, there were no significant differences of the grades between groups B2, B3 and B4. Regarding pathological changes： Inflammatory cell infiltration and fibroplastic proliferation were observed in the local treated serous membranes, which was the mildest in group B1. Slight foreign-body giant cell reactions were also found in groups B2, B3, and B4. CONCLUSION： （1） Chitosan gel has preventive effect on traumatic or ischemic peritoneal adhesion, but no obvious effect on foreign body-induced peritoneal adhesion. （2） Chitosan film may exacerbate the peritoneal adhesion. Blending with gelatin to chitosan film can accelerate the degradation of the film, but can simultaneously facilitate the formation of peritoneal adhesion.

Salt-responsive genes in rice revealed by cDNA microarray analysis

We used cDNA microarrays containing ～9,000 unigenes to identify 486 salt responsive expressed sequence tags (ESTs) (representing ～450 unigenes) in shoots of the highly salt-tolerant rice variety, Nona Bokra (Oryza sativa L. Ssp.Indica pv. Nona). Some of the genes identified in this study had previously been associated with salt stress. Howeverthe majority were novel, indicating that there is a great number of genes that are induced by salt exposure. Analysis of the salt stress expression profile data of Nona provided clues regarding some putative cellular and molecular processes that are undertaken by this tolerant rice variety in response to salt stress. Namely, we found that multiple transcription factors were induced during the initial salt response of shoots. Many genes whose encoded proteins are implicated in detoxification, protectant and transport were rapidly induced. Genes supporting photosynthesis were repressed and those supporting carbohydrate metabolism were altered. Commonality among the genes induced by salt exposure with those induced during senescence and biotic stress responses suggests that there are shared signaling pathways among these processes. We further compared the transcriptome changes of the salt-sensitive cultivar, IR28, with that of Nona rice. Many genes that are salt responsive in Nona were found to be differentially regulated in IR28. This study identified a large number of candidate functional genes that appear to be involved in salt tolerance and further examination of these genes may enable the molecular basis of salt tolerance to be elucidated.

Hepatitis E virus chimeric DNA vaccine elicits immunologic response in mice

AIM： To construct the plasmid pcHEV23 containing fragments of HEV ORF2 and ORF3 chimeric gene and to assess its ability to elicit specific immunologic response in mice. METHODS： The gene encoding the structural protein of HEV ORF2 fragment and full-length ORF3 was amplified by PCR. The PCR products were cloned into an eucaryotic expression plasmid pcDNA3. The resulting plasmid pcHEV23 was used as a DNA vaccine to inoculate BALB/c mice intramuscularly thrice at a dose of 100 or 200 ug. Mice injected with empty pcDNA3 DNA or saline served as control and then specific immune responses in the mice were detected. RESULTS： After 2-3 times of inoculation, all mice injected with pcHEV23 had anti-HEV IgG seroconversion and specific T lymphocyte proliferation. The lymphocyte stimulation index in the group immunized with pcHEV23 （3.1＋0.49） was higher than that in the control group （0.787±0.12, P〈0.01）. None in the control group had a detectable level of anti-HEV IgG. CONCLUSION： DNA vaccine containing HEV ORF2 and ORF3 chimeric gene can successfully induce specific humoral and cellular immune response in mice.

Effects of emodin on treating murine nonalcoholic fatty liver induced by high caloric laboratory chaw

AIM: To investigate the effects of emodin on the treatment of non-alcoholic fatty liver in rats induced by high caloric laboratory chaw. METHODS: Non-alcoholic fatty liver model was successfully established by feeding with high caloric laboratory chaw for 12 wk. Then the model rats were randomly divided into 3 groups, namely model control group, emodin group and dietary treatment group. The rats in emodin group were given emodin at dose of 40 mg/(kg·d) while animals in other groups were given distilled water of the same volume. The rats in model control group were fed with high caloric laboratory chaw while animals in other groups were fed with normal diet. Four weeks later, liver index (liver/body weight ratio), serum activities of liver-associated enzymes, blood lipid, fasting blood glucose, fasting plasma insulin, HOMA insulin resistance index (HOMA-IR), hepatic triglyceride content and histology features of all groups were assayed. The expression of hepatic peroxisomal proliferator activated receptor (PPAR) gamma was determined by RT-PCR. RESULTS: The body weight, liver index, serum activities of alanine aminotransferase (ALT), blood lipid, hepatic triglyceride content of model control group were significantly elevated, with moderate to severe hepatocyte steatosis. The expression of hepatic PPAR gamma mRNA was obviously reduced in model control group. Compared with model control group, the body weight, liver index, serum activities of ALT, blood lipids and hepatic triglyceride of emodin group significantly decreased and hepatic histology display was also greatly improved. Meanwhile, the expression of hepatic PPAR gamma mRNA was elevated. However, high serum activities of ALT and hyperlipidemia were persisted in dietary treatment group although liver index was decreased and liver histology was somewhat improved. CONCLUSION: It is suggested that emodin might be effective in the treatment of non-alcoholic fatty liver in rats. Its therapeutic mechanism could be associated with increasing the expression of hepatic PPAR gamma mRNA.

Presence and integration of HBV DNA in mouse oocytes

AIM: Hepatitis B is a worldwide public health problem. To explore the feasibility of hepatitis B virus (HBV) vertical transmission via oocytes, the presence and integration of HBV DNA in mouse oocytes were studied. METHODS: Genomic DNA was isolated and metaphases were prepared, respectively from mouse oocytes cocultured with pBR322-HBV DNA plasmids. PCR, Southern blot, dot hybridization and fluorescence in situ hybridization (FISH) were performed to explore the existence and integration of HBV DNA in oocytes.RESULTS: PCR detected positive bands in the tested samples, and then Southern blot revealed clear hybridization signals in PCR products. Final washing solutions were collected for dot hybridization and no signal for HBV DNA was observed, which excluded the possibility that contamination of washing solutions gave rise to positive results of PCR and Southern blot. FISH demonstrated that 36 of 1 000 metaphases presented positive signals. CONCLUSION: HBV DNA sequences are able to pass through the zona and oolemma to enter into oocytes and tointegrate into their chromosomes. HBV DNA sequences might be brought into embryo via oocytes as vectors when they are fertilized with normal spermatozoa.

Vascular endothelial growth factor and angiopoietins regulate sinusoidal regeneration and remodeling after partial hepatectomy in rats

AIM： To study the regulatory mechanisms of sinusoida regeneration after partial hepatectomy. METHODS： We invesldgated the expression of angiopoietin （Ang）-1, Ang-2, Tie-2, and vascular endothelial growth factor （VEGF） in regenerating liver tissue by quantitative reverse-transcription polymerase chain reaction （RT- PCR） using a LightCycler （Roche Diagnostics） and also immunohistochemical staining after 70% hepatectomy in rats. In the next step, we isolated liver cells （hepatocytes, sinusoidal endothelial cell （SEC）, Kupffer cell, and hepatic stellate cells （HSC）） from regenerating liver tissue by in situ collagenase perfusion and counterflow elutriation, to determine potential cellular sources of these angiogenic factors after hepatectomy. Proliferation and apoptosis of SECs were also evaluated by proliferating cell nuclear antigen （PCNA） staining and the terminal deoxynucleotidyl transferase d-uridine triphosphate nick end labeling （TUNEL） assay, respectively. RESULTS： VEGF mRNA expression increased with a peak at 72 h after hepatectomy, decreasing thereafter. The expression of Ang-1 mRNA was present at detectable levels before hepatectomy and increased slowly with a peak at 96 h. Meanwhile, Ang-2 mRNA was hardly detected before hepatectomy, but was remarkably induced at 120 and 144 h. In isolated cells, VEGF mRNA expression was found mainly in the hepatocyte fraction. Meanwhile, mRNA for Ang-1 and Ang-2 was found in the SEC and HSC fractions, but was more prominent in the latter. The PCNA labeling index of SECs increased slowly, reaching a peak at 72 h, whereas apoptotic SECs were detected between 120 h and 144 h. CONCLUSION： Ang-Tie system, together with VEGF, plays a critical role in regulating balance between SEC proliferation and apoptosis during sinusoidal regeneration after hepatectomy. However, the VEGF system plays a more important role in the early phase of sinusoidal regeneration than angiopoietin/Tie system.

A novel gain of function mutant in C-kit gene and its tumorigenesis in nude mice

AIM： To transfect mutant C-kit cDNA at codon 579 into human embryonic kidney cell line to observe its role in the pathogenesis of gastrointestinal stromal tumor （GIST）. METHODS： Eukaryotic expression vectors of pcDNA3- Kit-NW and pcDNA3-Kit-W were constructed. Then pcDNA3-Kit-NW and pcDNA3-Kit-W plasrnids were transfected into human embryonic kidney cell line by Upofectamine. The resistant clone was screened by G418 filtration and identified by sequencing, Western blotting, and immunocytochemical staining. Human embryonic kidney cells were divided into three groups including pcDNA3-Kit-NW, pcDNA3-Kit-W, and vector control groups. Absorbency value with a wavelength of 574 nm was detected by MTT analysis. Mice were injected with three groups of cells. Volume, mass, and histological examinations of the tumors in different groups were measured and compared. RESULTS： The C-kit gene and mutant C-kit gene were successfully cloned into the eukaryotic expression vector pcDNA3, pcDNA3-Kit-NW and pcDNA3-Kit-W were successfully transfected into human embryonic kidney cell line and showed stable expression in this cell line. Cell proliferating activity had significant differences between pcDNA3-Kit-NW and pcDNA3, pcDNA3-Kit- NW and pcDNA3-Kit-W （P〈0.05）, respectively. Tumors were only observed in nude mice implanted with cells transfected with pcDNA3-Kit-NW. CONCLUSION： Mutation of C-kit gene increases the proliferation activity of human cells and plays an important role in the malignant transformation of GIST.

Expressed genes in regenerating rat liver after partial hepatectomy

AIM: To reveal the liver regeneration (LR) and its controlas well as the occurrence of liver disease and to study the gene expression profiles of 551 genes after partial hepatectomy (PH) in regenerating rat livers.METHODS: Five hundred and fifty-one expressed sequence tags screened by suppression subtractive hybridization were made into an in-house cDNA microarray, and the expressive genes and their expressive profiles in regenerating rat livers were analyzed by microarray and bioinformatics. RESULTS: Three hundred of the analyzed 551 genes were up- or downregulated more than twofolds at one or more time points during LR. Most of the genes were up- or downregulated 2-5 folds, but the highest reached 90 folds of the control. One hundred and thirty-nine of themshowed upregulation, 135 displayed downregulation, and up or down expression of 26 genes revealed a dependence on regenerating livers. The genes expressedin 24-h regenerating livers were much more than those in the others. Cluster analysis and generalization analysis showed that there were at least six distinct temporal patterns of gene expression in the regenerating livers, that is, genes were expressed in the immediate early phase, early phase, intermediate phase, early-late phase, late phase, terminal phase. CONCLUSION: In LR, the number of down-regulated genes was almost similar to that of the upregulated genes; the successively altered genes were more than the rapidly transient genes. The temporal patterns of gene expression were similar 2 and 4 h, 12 and 16 h, 48 and 96 h, 72 and 144 h after PH. Microarray combined with suppressive subtractive hybridization can effectively identify the genes related to LR.

Leptin administration exacerbates thioacetamide-induced liver fibrosis in mice

AIM： To investigate the effects of leptin administration on liver fibrosis induced by thioacetamide （TAA）. METHODS： Twenty-four male C57BI/6 mice were randomly allocated into four groups, which were intra-peritoneally given saline （2 mL/kg）, leptin （1 mg/kg）, TAA （200 mg/kg）, TAA （200 mg/kg） plus leptin （1 mg/kg） respectively, thrice a week. All mice were killed after 4 wk. The changes in biochemical markers, such as the levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in serum and superoxide dismutase （SOD）, malondialdehyde （MDA） in liver were determined. For histological analysis, liver tissues were fixed with 10% buffered formalin, embedded with paraffin. Hematoxylin-eosin （HE） staining and picric acid-Sirius red dyeing were performed. The level of α1（I） procollagen mRNA in liver tissues was analyzed by RT-PCR. RESULTS： Apparent liver fibrosis was found in TAA group and TAA plus leptin group. Compared to saline group, the levels of ALT and AST in serum and MDA in liver increased in TAA group （205.67±27.69 U/L vs 50.67±10.46 U/L, 177.50±23.65 U/L vs76.33±12.27 U/L, 2.60±0.18 nmol/mg pro vs 1.91±0.14 nmol/mg pro, P〈0.01） and in TAA plus leptin group （256.17±22.50 U/L vs 50.67±10.46 U/L, 234.17±27.37 U/L vs76.33±12.27 U/L, 2.97±0.19 nmol/mg pro vs 1.91±0.14 nmol/mg pro, P〈0.01）. The level of SOD in livers decreased （51.80±8.36 U/mg pro vs 81.52±11.40 U/mg pro, 35.78±6.11 U/mg pro vs81.52± 11.40 U/mg pro, P〈0.01） and the level of α1（I） procollagen mRNA in liver tissues also increased （0.28±0.04 vs0.11± 0.02, 0.54±0.07 vs0.11±0.02, P〈0.01）. But no significant changes were found in leptin group and saline group. Compared to TAA group, ALT, AST, MDA, and α1（I） procollagen mRNA and grade of liver fibrosis in TAA plus leptin group increased （256.17±22.50 U/L vs 205.67± 27.69 U/L, P〈0.05; 234.17±27.37 U/L vs 177.50±23.65 U/L, P〈0.05; 2.97±0.19 nmol/mg pro vs 2.60±0.18 nmol/mgpro, P〈0.05, 0.54±0.07 vs0.28±0.04, P〈0.01, 3.17 vs 2.00, P〈0.05）, and the level of SOD in liver decreased （35.78±6.11 U/rag pro vs 51.80±8.36 U/rag pro, P〈0.05）. There were similar changes in the degree of type I collagen deposition confirmed by picric acid-Sirius red dyeing. CONCLUSION： Leptin can exacerbate the degree of TAA - induced liver fibrosis in mice. Leptin may be an important factor in the development of liver fibrosis.

PROTECTION OF CARBON MONOXIDE INHALATION ON LIPOPOLY-SACCHARIDE-INDUCED MULTIPLE ORGAN INJURY IN RATS

Objective To observe the protection of carbon monoxide （CO） inhalation on lipopolysaccharide （LPS）-induced rat multiple organ injury. Methods Sprague-Dawley rats with multiple organ injury induced by 5 mg/kg LPS intravenous injection were exposed to room air or 2.5 × 10 ^-4 （V/V） CO for 3 hours. The lung and intestine tissues of rats were harvested to measure the expression of heme oxygenase-1 （ HO-1 ） with reverse transcription-polymerase chain reaction, the levels of pulmonary tumor necrosis factor-or （ TNF-α）, interleukin-6 （ IL-6）, and intestinal platelet activator factor （ PAF）, intercellular adhesion molecule-1 （ICAM-1） with enzyme-linked immunosorbent assay, the content of maleic dialdehyde （MDA） and the activity of myeloperoxidase （MPO） with chemical method, the cell apoptosis rate with flow cytometry, and the pathological changes with light microscope. Results CO inhalation obviously up-regulated the expression of HO-1 in lung （5.43 ± 0. 92） and intestine （6. 29 ± 1.56） in LPS ＋ CO group compared with （ 3.08 ± 0. 82） and （ 3.97 ± 1.16 ） in LPS group （ both P 〈 0. 05 ）. The levels of TNF-ot, IL-6 in lung and PAF, ICAM-1 in intestine ofLPS ＋ CO group were 0. 91 ±0. 25,0. 64 ±0.05, 1. 19 ± 0. 52, and 1.83 ±0. 35 pg/mg, respectively, significantly lower than the corresponding values in LPS group （ 1.48 ± 0. 23, 1.16 ± 0. 26, 1.84 ± 0. 73, and 3.48 ± 0. 36 pg/mg, all P 〈 0. 05 ）. The levels of MDA, MPO, and cell apoptosis rate in lung and intestine of LPS ＋ CO group were 1.02 ± 0. 23 nmol/mg, 1.74 ± 0. 17 nmol/mg, 7.18 ± 1.62 U/mg, 6. 30 ±0. 97 U/mg, 1.60% ±0. 34%, and 30. 56% ±6. 33%, respectively, significantly lower than the corresponding values in LPS group （ 1.27 ± 0. 33 nmol/mg, 2. 75 ± 0. 39 nmol/mg, 8. 16 ± 1.49 U/mg, 7. 72 ± 1.07 U/mg, 3.18% ±0. 51%, and 41.52% -＋3.36%, all P 〈0.05）. In addition, injury of lung and intestine induced by LPS was attenuated at presence of CO inhalation. Conclusion CO inhalation protects rat lung and intestine from LPS-induced injury via anti-oxidantion, anti-inflammation, anti-apoptosis, and up-regulation of HO-1 expression.

Bone marrow-derived mesenchymal stem cells protect against experimental liver fibrosis in rats

AIM: Recent reports have shown the capacity of mesenchymal stem cells (MSCs) to differentiate into hepatocytes in vitro and in vivo. MSCs administration could repair injured liver, lung, or heart through reducing inflammation, collagen deposition, and remodeling. These results provide a clue to treatment of liver fibrosis. The aim of this study was to investigate the effect of infusion of bone marrow (BM)-derived MSCs on the experimental liver fibrosis in rats. METHODS: MSCs isolated from BM in male Fischer 344 rats were infused to female Wistar rats induced with carbon tetrachloride (CCI4) or dimethylnitrosamine (DMN). There were two random groups on the 42nd d of CCI4: CCl4/MSCs, to infuse a dose of MSCs alone; CCI4/saline, to infuse the same volume of saline as control. There were another three random groups after exposure to DMN: DMN10/MSCs, to infuse the same dose of MSCs on d 10; DMN10/saline, to infuse the same volume of saline on d 10; DMN20/MSCs, to infuse the same dose of MSCs on d 20. The morphological and behavioral changes of rats were monitored everyday. After 4-6 wk of MSCs administration, all rats were killed and fibrosis index were assessed by histopathology and radioimmunoassay. Smooth muscle alpha-actin (alpha-SMA) of liver were tested by immunohistochemistry and quantified by IBAS 2.5 software. Male rats sex determination region on the Y chromosome (sry) gene were explored by PCR. RESULTS: Compared to controls, infusion of MSCs reduced the mortality rates of incidence in CCl4-induced model (10% vs 20%) and in DMN-induced model (20-40% vs 90%).The amount of collagen deposition and alpha-SMA staining was about 40-50% lower in liver of rats with MSCs than that of rats without MSCs. The similar results were observed in fibrosis index. And the effect of the inhibition of fibrogenesis was greater in DMN10/MSCs than in DMN20/MSCs. The sry gene was positive in the liver of rats with MSCs treatment by PCR. CONCLUSION: MSCs treatment can protect against experimental liver fibrosis in CCMnduced or DMN-induced rats and the mechanisms of the anti-fibrosis by MSCs will be studied further.