大鼠减压应激损伤时脑和肝胞液糖皮质激素受体的改变

目的探讨大鼠减压应激损伤时大脑和肝脏胞液糖皮质激素受体结合量的变化。方法大鼠 3 0只 ,随机分为 5组 ,置于加压舱内 ,进行加减压实验。出舱后 ,以3H地塞米松为配体 ,测定了动物脑、肝胞液糖皮质激素受体结合量的改变 ,同时还监测了动物心前区减压气泡的变化。结果减压应激损伤后 ,动物肝、脑胞液糖皮质激素受体结合量均下降 ,尤其以脑胞液中糖皮质激素受体减少为明显 (P <0 .0 1 ,P <0 .0 5)。实验还观测到 ,减压应激损伤的动物 ,若不采取救治措施 ,可随着时间迁移糖皮质激素受体结合量进一步减少。结论脑和肝胞液糖皮质激素受体结合量改变与减压应激损伤密切相关 ,可作为评价急性减压病损伤的指标之一。

大鼠肝胞液糖皮质激素受体的高效液相分析

目的 :应用高效液相疏水作用层析法 (HPHIC)分离并纯化大鼠肝胞液高、低亲和力糖皮质激素受体 (GRH 和 GRL) ,为临床大剂量糖皮质激素 (GC)冲击治疗提供实验依据。方法 :超速离心大鼠肝组织 ,取上清 ,核素 3 H-地塞米松 (Dex)标记后 ,分别行 Pseudoscatchard分析及 HPHIC分析 ,SDS- PAGE及 Western印迹法鉴定 HPHIC分析后所收集的峰值管蛋白的性质。 结果 :在标准大气压 ,0～ 4℃条件下 ,只出现一个洗脱峰 ,峰值位置在 9～ 11min。而在标准大气压 ,2 5℃且 3 H- Dex≥ 5 0nmol/ L时出现两个洗脱峰 ,峰值位置在 3～ 7min及 9～ 11min。 SDS- PAGE及 Western印迹法显示 5～ 7min和 9～ 11min时出现相对分子质量约 6 0 0 0 0、可与 GC单克隆抗体结合的蛋白质分子。结论 :提示有两种不同大小的 GR分子存在 ,但是否存在 GRH和 GRL

大鼠肝胞液糖皮质激素受体的纯化

近20多年来受体的研究发展很快,所用的研究方法,主要是以同位素标记的配体为探针,对受体作定量及定位研究。而要深入细致地研究受体的结构与功能,则必须先获得一定纯度的受体。由于受体的含量极微,受体本身又极不稳定。

长时间失血性休克大鼠肝胞液糖皮质激素受体与磷酯酶A2变化的研究

长时间失血性休克大鼠肝胞液糖皮质激素受体与磷酯酶Ａ２变化的研究许金廉刘志民１曲莉２贾宁阳１（第二军医大学生理学教研室，上海２００４３３；１第二军医大学长征医院，２解放军１４５医院）长时间失血性休克可致多器官功能衰竭。磷酯酶Ａ２（ＰＬＡ２）作为糖皮质激...

Effect of Quercetin on CYP1A2, CYP2E1, CYP3A2 Activities and its Inhibitory Mechanism Studies in Rat Liver Microsomes

Aim To assess the potential effect of quercetin （QU）, an natural plant estrogen, on CYP1A2, CYP2E1, and CYP3A2 activities in rat liver microsomes; and to identify the magnitude of inhibitory effect and the probable inhibitory mechanism of QU. Methods QU and specific substrate were concurrently incubated, with HPLC detection of the substrate metabolites for data analysis. The magnitude of inhibitory effect of QU on CYP3A2 was compared with those of ketoconazole （Ket） and erythromycin （Ery）. The mechanism of its inhibitory effect on CYP3A2 and CYP2E1 was derived from Lineweaver-Burk plots. Results HPLC methods were in good linear relationship with r〉0.999 1. Relative standard deviations for intra-day and inter-day were〈8.4%. Recovery of each analyte in the concentrations studied was between 91.1% and 107.6 %. QU （up to 8 μmol·L^-1） showed potent induction to CYP1A2 （338.1% of the negative control）while inhibited CYP2E1 （49.2% of the negative control） and CYP3A2 （60.3% of the negative control） activity. The magnitude of inhibitory effect for QU on CYP3A2 was between those for Ket and Ery （Ket〉QU〉Ery）. QU exhibited competitive inhibition of CYP3A2 dextromethorphan N-demethylation reaction and expressed noncompetitive inhibition of CYP2E1 chlorzoxazone-6-hydroxylation reaction. Conclusion HPLC assay has been validated with precision and accuracy. QU is an effective inhibitor of several CYP isoforms. It may cause relevant drug-drug interactions with CYP3A substrates. As a plant flavonoid, QU has potential not only in molecular advantage but also in CYP450 module capability for further application in cancer chemotherapy.

Local recurrence is an important prognostic factor of hepatocellular carcinoma

AIM： To clarify the importance of complete treatment by PELT. METHODS： A total of 140 previously untreated cases of HCC were enrolled in this study from 1988 to 2002. The inclusion criteria were： a solitary tumor less than 4 cm in diameter or multiple tumors, fewer than four in number and less than 3 cm in diameter, without extrahepatic metastasis or vessel invasion. As general principles for the treatment of HCC, the patients underwent transcatheter arterial chemoembolization （TACE） prior to PEIT. After the initial treatment of the patients, ultrasonography and computed tomography were performed, and measurement of serum levels of α- fetoprotein （AFP） was determined. When tumor recurrences were detected, PEIT and/or TACE were repeated whenever the hepatic functional reserve of the patient permitted. We then analyzed the variables that could influence prognosis, including tumor size and number, the serum levels of AFP, the parameters of hepatic function （albumin, bilirubin, ALT, hepaplastin test, platelet number, and indocyanine green retention at 15 rain [ICG-R15]）, combined therapy with TACE, distant recurrence, and local recurrence. RESULTS： Univariate analysis identified the ICG test, serum levels of AFP and albumin, tumor size and number, and local recurrence, but not distant recurrence, as significant prognostic variables. In multivariate analysis using those five parameters, the ICG test, tumor size, tumor number, and local recurrence were identified as significant prognostic factors. In both univariate and multivariate analyses, the relative risk for the ICG test was the highest, followed by local recurrence. CONCLUSION： We found that local recurrence is an independent prognostic factor of HCC, indicating that achieving complete treatment for HCC on first treatment is important for improving the prognosis of patients with HCC. 2005 The WJG Press and Elsevier Inc. All rights reserved.

Correlation and prognostic significance of beta-galactoside alpha-2,6-sialyltransferase and serum monosialylated alpha-fetoprotein in hepatocellular carcinoma

AIM： To investigate the correlation between tissue ST6Gal I and serum msAFP in HCC patients, and to investigate their prognostic significance. METHODS： Preoperative sera, paired tumorous and non-tumorous tissues were collected from 19 consecutive patients who had undergone surgical resection of HCC. ST6Gal I activities in the tissues were measured by an in vitro microsomal enzyme activity assay. The percentages of tumor-specific msAFP in the sera were also estimated by an isoelectric focusing-immunoblotting assay. RESULTS： The tumor ST6Gal I activity was negatively correlated with serum msAFP percentage （r = -0.53, P = 0.019）. Both decreased tumor ST6Gal I activity and increased serum msAFP percentage were associated with poor tumor cell differentiation. Univariate analyses showed that both decreased tumor ST6Gal I activity （P = 0.028）, increased serum msAFP percentage （P = 0.034） and poor tumor cell differentiation （P = 0.031）were associated with shorter overall survival. Multivariate analysis using the Cox regression model showed that the preoperative serum msAFP percentage （P = 0.022） and tumor cell differentiation status （P = 0.048） were independent prognostic indicators for patient overall survival. CONCLUSION： Our results indicate that the presence of msAFP in blood circulation is associated with a decreased activity of ST6Gal I activity in HCC. Both tissue ST6Gal I and serum msAFP are potential prognostic markers for patients with operable HCC.

Relationship between T-lymphocyte cytokine levels and sero-response to hepatitis B vaccines

AIM: To investigate the cellular defects by analyzing the (Th1/Th2) cytokine levels in vaccine responders and non-responders. METHODS: Peripheral blood mononuclear cell (PBMC) from responders and non-responders were stimulated with or with out recombinant HBsAg or PHA. Broad spectrum of cytokines viz (Th1) IFN-γ, IL-2, TNF-α, IL-12 and (Th2) IL-10, IL-4 were measured after in vitro stimulation with recombinant HBsAg and were compared with respective antibody titers. RESULTS: A significant decrease (P = 0.001) in Th1 and Th2 cytokines namely, IL-2, INF-γ, TNF-α and IL-10in non-responders was observed. The level of IL-4 was not significant between the three groups. Furthermore, despite a strong Th1 and Th2 cytokine response, the level of IL-12 was elevated in high-responders compared to other groups (P = 0.001) and demonstrated a positive correlation with anti-HBs titers and Th1 cytokine response. CONCLUSION: Our findings suggest that unrespon-siveness to recombinant hepatitis B vaccines (rHB) is multifactorial, including specific failure of antigen presentation or the lack of both T helper Th1 and Th2 response.

Circulating DNA level is negatively associated with the long-term survival of hepatocellular carcinoma patients

AIM： To quantify the circulating DNA in plasma from patients with hepatocellular carcinoma （HCC） and to evaluate its prognostic value. METHODS： Blood samples were collected from 79 patients with HCC before operation, 20 patients with liver cirrhosis, and 20 healthy volunteers. Circulating DNA was extracted from plasma and quantified. The association between circulating DNA level and prognosis of HCC patients was evaluated. RESULTS： Compared with the healthy volunteers （17.6 ± 9.5 ng/mL）, a significant higher circulating DNA level was found in the patients with HCC （47.1 ± 43.7 ng/ mL, P = 0.000） or with liver cirrhosis （30.0 ± 13.3 ng/ mL, P = 0.002）. The circulating DNA level was closely associated with tumor size （P = 0.008） and TNM stage （P = 0.040）, negatively associated with the 3-year diseasefree survival （DFS） （P = 0.017） and overall survival （OS） （P = 0.001）. CONCLUSION： Large or invasive tumor may release more circulating DNA, and higher level of circulating DNA may be associated with poor prognosis of HCC patients.

CIK cells from patients with HCC possess strong cytotoxicity to multidrug-resistant cell line Bel-7402/R

ABM: To investigate the cytotoxicity of the cytokine-induced killer (CIK) cells from the post-operation patients with primary hepatocellular carcinoma (HCC) to multidrug-resistant (MDR) cell of HCC both in vitro and in vivo. METHODS: A drug-resistant cell line was established by culturing human HCC cell line Bel-7402 in complete RPMI 1640 medium with increasing concentrations of adriamycin from 10 to 2 000 nmol/L. CIK cells were obtained by inducing the peripheral blood mononuclear cells with rhlFN-γ, monoclonal anti-CD3 antibody, rhIL-1α as well as rhIL-2, which were added into the culture. To detect the cytotoxicity of the CIK cells from HCC patients, the Bel-7402/R was taken as target (T) cells and CIK cells as effect (E) cells. Cytotoxic test was performed and measured by MTT. As to in vivo test, CIK cells were transfused into patients with HCC. The tumor specimens of the patients were obtained and immunohistochemistry was carried out to detect CD3, CD45, CD45RO as well as CD68. RESULTS: A MDR 1 HCC cell line Bel-7402/R was established. Its MDR1 mRNA overexpressed which was shown by RT-PCR; the P-glycoprotein expression increased from 1.32% of parent cells to 54%. CIK cells expanded vigorously by more than 70-fold and the CD3+CD56+ increased by more than 600-fold after 3-wk incubation on average. The cytotoxicity of CIK from HCC patients to Bel-7402/R was about 50% and to L-02 below 10% (t = 8.87, P<0.01), the same as that of CIK from normal individuals. Each of the 17 patients received 1-5×1010of CIK cell transfusion. No side effects were observed. After CIK treatment, the tumor tissue nodules formed and a large amount of lymphocytes infiltrated in the liver cancer tissue and CD3, CD45, CD45RO, and CD68 increased greatly which was shown by immunohistochemistry. CONCLUSION: A stable MDR1 HCC cell line has been established which could recover from liquid nitrogen and CIK from HCC patients has strong cytotoxicity to MDR HCC cell. CIK adoptive immunotherapy is safe and has no side effects. Receivers improved their immunity to tumor evidently. CIK treatment may be a better choice for HCC patients after operation to prevent the recurrence, especially when tumors have developed drug resistance.

Assessment of hemodynamics in precancerous lesion of hepatocellular carcinoma:Evaluation with MR perfusion

AIM: To investigate the hemodynamic changes in a precancerous lesion model of hepatocellular carcinoma (HCC). METHODS: Hemodynamic changes in 18 Wistar rats were studied with non-invasive magnetic resonance (MR) perfusion. The changes induced by diethylnitrosamine (DEN) developed into liver nodular lesions due to hepatic cirrhosis during the progression of carcinogenesis. The MR perfusion data [positive enhancement integral (PEI)] were compared between the nodular lesions corresponding well with MR images and pathology and their surrounding hepatic parenchyma. RESULTS: A total of 46 nodules were located by MR imaging and autopsy, including 22 dysplastic nodules (DN), 9 regenerative nodules (RN), 10 early HCCs and 5 overt HCCs. Among the 22 DNs, 6 were low-grade DN (lGDN) and 16 were high-grade DN (HGDN). The average PEI of RN, DN, early and overt HCC was 205.67 ± 31.17, 161.94 ± 20.74, 226.09 ± 34.83, 491.86 ± 44.61 respectively, and their liver parenchyma nearby was 204.84 ± 70.19. Comparison of the blood perfusion index between each RN and its surrounding hepatic parenchyma showed no statistically significant difference (P = 0.06). There were significant differences in DN (P = 0.02). During the late hepatic arterial phase, the perfusion curve in DN declined. DN had an iso-signal intensity at the early hepatic arterial phase and a low signal intensity at the portal venous phase. Of the 10early HCCs, 4 demonstrated less blood perfusion and 6 displayed minimally increased blood flow compared to the surrounding parenchyma. Five HCCs showed significantly increased blood supply compared to the surrounding parenchyma (P = 0.02). CONCLUSION: Non-invasive MR perfusion can detect changes in blood supply of precancerous lesions.

Impact of Cinobufacini injection on proliferation and cell cycle of human hepatoma HepG-2 cells

Objective： The aim of our study was to investigate the effect of Cinobufacini injection on the proliferation and cell cycle of human hepatoma HepG-2 cells. Methods： Cell proliferation was assessed by MTT assay, cell cycle distribution was detected by the flow cytometry （FCM）. The expression of Cyclin A, CDK2 mRNA levels were examined by RT-PCR. Quantitative colorimetric assay was used to analyze Cyclin NCDK2 activity in HepG-2 cells. Results： Cinobufacini injection significantly inhibited HepG-2 cells proliferation in dose- and time-dependent ways; FCM analysis showed Cinobufacini injection induced cell cycle arrest at S phase; RT-PCR assay showed Cinobufacini injection down-regulated Cyclin A, CDK2 expression at mRNA levels; Quantitative colorimetric assay showed Cinobufacini injection deceased Cyclin NCDK2 activity in HepG-2 cells. Conclusion： Cinobufacini injection can inhibit human hepatoma HepG-2 cells growth, induce cell apoptosis and induce cell cycle arrest at S phase, the mechanism of which might be partly related to the down-regulation of Cyclin A, CDK2 mRNA expression and inhibition of Cyclin A/CDK2 activity.

Post-Surgical Recurrence of Hepatocellular Carcinoma

Objective To analyze post-surgical recurrence of hepatocellular carcinoma (HHC) according to pathologic findings, primary tumor and angiographic features of the recurrent tumor.Methods In this series, 100 cases of recurrent HCC were analyzed in following aspects: ( 1) size, tumor nodular numbers, gross and histologic findings of the primary tumor; (2) post-surgical recurrent time; (3) size, tumor nodular numbers, blood supply, staining property, and accumulation of lipiodol oil in the recurrent tumor. Following angiography, arterial chemoembolization was performed. Results In the primary tumor, single nodules were seen in 80 cases, multiple nodules in 16 cases and multiple fused nodules in 4 cases. All tumors were classified as: trabecular type, 65 cases; compact type, 12; sclerotic type, 2 cases; mixed type, 15 cases and cholangiocarcinoma type, 6 cases. 38 cases had incomplete or no capsule. Satellite tumor nodules were grossly identified during operation in 33 cases, but were proven microscopically in 66 cases. Tumor thrombi of portal vein was noted in 18 cases during oeration, but 85 cases in histopathological sections. The recurrent tumors were diagnosed post surgically within 6 months in 67 cases, 6-12 months in 15 cases and after 12 months in 18 cases respectively. On angiography, 67 % recurrent tumors was rich in blood supply and with abundant accumulation of lipiodol after embolization.Conclusion The post-surgical recurrence rate of the HCC patients with massive size, incomplete or no capsule, satellite tumor nodules and portal vein tumor thrombus was high. The patients shoud receive angiography in 1 ?2 months after surgery in order to detect early recurrence and, if confirmed, the patients may be treated by transcatheter arterial chemoembolization

Ultracytochemical localization of H+-adenosine triphosphatase activity in autophagic vacuoles induced by vinblastine in rat liver

H+-adenosine triphosphatase (H+-ATPase) activity was demonstrated oytoohemioally in autophagio vaouoles (AVs) of rat hepatooytes using a modification of the method for the demonstration of neutral p-nitrophenyl phosphatase (p-NPPase) activity [1]. When an inhibitor of H+-ATPase, N-ethylmaleimide (NEM) or 4,4'-diisothiooyanostilbene-2,2'disalfonio aold, di-sodium salt (BIDS) was included in the incubation medium the enyzme activity was abolished indicating that p-NPPase demonstrated in this study represents H+-ATPase. Autophagy was induced by a single intraperitoneal injection of vinblastine sulfate (VBL). The number of AVs increased remarkably in hepatooytes from 40 min after VBL treatment. H+-ATPase activity was observed mainly on the membranes of lysosomes and AVs. However, early forms of AVs containing only incompletely digested material showed no H+-ATPase activity. Most AVs revealing a positive reaction seemed to be in advanced stages of development. Acid phosphatase aotioity was demonstrable in mature but not in early forms of AVs. The present investigation showed that membranes of advanced stage A Vs possess an H+-ATPase which may be derived from lysosomal membranes.

Administration of granulocyte colony stimulating factor after liver transplantation leads to an increased incidence and severity of ischemic biliary lesions in the rat model

AIM： Recently it has been reported that granulocyte colony stimulating factor （G-CSF） can induce hypercoagulability in healthy bone marrow donors. It is conceivable that the induction of a prothrombotic state in a recipient of an organ graft with already impaired perfusion might cause further deterioration in the transplanted organ. This study evaluated whether G-CSF treatment worsens liver perfusion following liver transplantation in the rat model. METHODS： A non-arterialized rat liver transplantation model was employed to evaluate the effect of G-CSF treatment on the liver in a syngeneic and allogeneic strain combination. Study outcomes included survival time and liver damage as investigated by liver enzymes and liver histology. Observation times were 1 d, 1 wk and 12 wk. RESULTS： Rats treated with G-CSF had increased incidence and severity of biliary damage following liver transplantation. In these animals, hepatocellular necrosis was accentuated in the centrilobular region. These lesions are indicative of impaired perfusion in G-CSF treated animals. CONCLUSION： G-CSF should be used with caution in recipients of liver transplantation, as treatment might enhance preexisting, undetected perfusion problems and ultimately lead to ischemia induced biliary complications .

The impact of Cinobufacini injection on proliferation and apoptosis of human hepatoma HepG-2 cells

Objective： The aim of our study was to investigate the effect of Cinobufacini injection on the proliferat（on and apoptosis of human hepatocarcinoma HepG-2 cells. Methods： Cells proliferation was assessed by MTT assay, cells morphologic was observed by the inverted microscopy, Annexin V/PI stain was used to detect the apoptosis and necrosis of the tumor ceils. The expression of TOPOI mRNA and TOPO Ⅱ mRNAwere examined by RT-PCR. Results： Cinobufacini injection significantly inhibited HepG-2 cells proliferation in dose- and time-dependent ways. After Cinobufacini injection intervention, HepG-2 cells showed typical morphological changes： cells changed from polygon into round, chromatin looseness and karyolysis were observed. The percentages of apoptosis were 88.49%, 76.02%, 61.73% corresponding to the 48 h interference of 0.42 μg/mL, 0.21 μg/mL, 0.105 μg/mL Cinobufacini injection, perspectively. RT-PCR assay showed that Cinobufacini injection down-regulated TOPOI and TOPO Ⅱ expression at mRNA level. Conclusion： Cinobufacini can inhibit human hepatocarcinoma HepG-2 cells growth and induce tumor cells apoptosis, the mechanism of which might partly relate to the down-regulation of TOPOI mRNA and TOPO Ⅰ mRNA induced by Cinobufacini injection.

Xuebijing injection alleviates liver injury by inhibiting secretory function of Kupffer cells in heat stroke rats

OBJECTIVE： To evaluate the effects of Xuebijing （XBJ） injection in heat stroke （HS） rats and to inves- tigate the mechanisms underlying these effects. METHODS： Sixty anesthetized rats were random- ized into three groups and intravenously injected twice daily for 3 days with 4 mL XBJ （XBJ group） or phosphate buffered saline （HS and Sham groups） per kg body weight. HS was initiated in the HS and XBJ groups by placing rats in a simulated climate chamber （ambient temperature 40℃：, humidity 60% ）. Rectal temperature, aterial pressure, and heart rate were monitored and recorded. Time to HS onset and survival were determined, and serum concentrations of tumor necrosis factor （TNF）-α, in-terleukin （IL）-1β, IL-6, alanine-aminotransferase （ALT）, and aspartate-aminotransferase （AST） were measured. Hepatic tissue was harvested for patho- logical examination and electron microscopic ex- amination. Kupffer cells （KCs） were separated from liver at HS initiation, and the concentrations of se- creted TNF-α, IL-1β3 and IL-6 were measured. RESULTS： Time to HS onset and survival were signif- icantly longer in the XBJ than in the HS group. Moreover, the concentrations of TNF-α, IL-1β, IL-6, ALT and AST were lower and liver injury was milder in the XBJ than in the HS group. Heat-stress in- duced structural changes in KCs and hepatic cells were more severe in the HS than in the XBJ group and the concentrations of TNF-α, IL-1β and IL-6 se- creted by KCs were lower in the XBJ than in the HS group. CONCLUSION： XBJ can alleviate HS-induced sys- temic inflammatory response syndrome and liver injury in rats, and improve outcomes. These protec- tive effects may be due to the ability of XBJ to inhib- it cytokine secretion by KCs.