肝细胞生长因子及其受体c-met在肝细胞癌中的表达与预后价值

目的研究肝癌切除前、后血清肝细胞生长因子(HGF)的动态变化及其受体c met在癌组织中的表达,探讨HGF/c met对手术切除后肝癌患者的预后价值。方法收集手术切除的25例肝细胞癌患者血清及组织标本,用酶联免疫法(ELISA)测定术前1d、术后第3、7、10天血清HGF浓度,分别用免疫组织化学及逆转录聚合酶链反应(RT PCR)方法检测肝癌及癌旁组织中c met蛋白及mRNA的表达。分析术前血清HGF水平及癌组织中c met表达程度与不同临床病理因素之间的关系及影响手术后血清HGF水平变化的因素。以20例健康献血员为正常对照组,22例慢性乙型肝炎患者及22例乙型肝炎肝硬化肝功能失代偿(ChildB或C级)患者作为慢性乙肝组和肝硬化失代偿组。结果肝癌患者血清HGF浓度高于正常对照组及慢性乙肝组[(1.03±0.09)ng/ml比(0.69±0.02)、(0.74±0.09)ng/ml,P<0.05],但与肝硬化失代偿组[(1.04±0.11)ng/ml]比较差异无统计学意义。术后HGF浓度上升高峰出现在术后第3天,术后第7、10天逐渐下降,但仍高于正常水平。术前、术后各组的血清HGF组间比较,仅术后第3天组差异有统计学意义。影响术前血清HGF水平的主要因素有:肿瘤直径>5cm、伴有结节肝硬化、门静脉癌栓、术前血清甲胎蛋白(AFP)≥400μg/L。从术前到术后第3天,大区段切除比小区段切除者的血清HGF浓度上升更明显(P=0.047)。中度和强阳性c met蛋白表达在肝癌组织中(21/25)明显高于癌旁组织(5/25)。高表达的c metmRNA见于所有肝癌组织中,在癌旁组织中仅6例有高表达。癌组织中c met蛋白表达的强度与有无合并门静脉癌栓有关,与肿瘤大小、血清AFP水平、血清HGF水平、有无肝硬化及肝癌的分化程度无相关性。但在癌旁组织,合并肝硬化组c met蛋白表达高于无肝硬化组。术后有肿瘤复发或转移的病例,其术前血清HGF水平及肿瘤组织中c met蛋白表达显著高于无复发或转移的病例。HGF水平与肿瘤组织中c met表达无相关性。结论肝癌患者有血清HGF及癌组织中c met的高表达,术后血清HGF的持续升高可能与肝癌的早期复发和转移有关。     

Activation of human Kupffer cells by thymostimulin (TP-1) to produce cytotoxicity against human hepatocellular cancer

Background: In a small pilot study, thymostimulin (TP-1) produced tumor regression in almost 50% of patients with hepatocellular cancer (HCC) who were treated with TP-1 alone. However, the mechanism of the TP-1-mediated antitumor effect against HCC is unknown. Methods: Human hepatocytes and Kupffer cells were isolated from liver biopsy specimens by collagenase infusion and counterflow elutriation. Hepatocytes and Kupffer cells were incubated in vitro with clinically relevant doses of TP-1. Cell-free supernatants were collected at various time points after incubation. Hepatocyte and Kupffer cell supernatant levels for a panel of growth factors and monokines were determined by enzyme-linked immunosorbent assay. The cytotoxic activity of TP-1 alone and of TP-1-stimulated hepatocyte and Kupffer cell supernatants against Hep G2 and Hep 3B human HCC cells in vitro was measured by MTT assay. Results: Doses of TP-1 up to 100 µg/ml produced no cytotoxicity against Hep G2 or Hep 3B cells. Furthermore, supernatants from TP-1-treated hepatocytes produced no cytotoxicity against Hep G2 or Hep 3B cells, and TP-1 did not stimulate the release of transforming growth factor (TGF)-, TGF-, or hepatocyte growth factor. TP-1-treated Kupffer cell supernatants produced significant cytotoxicity against Hep G2 cells but produced no cytotoxicity against Hep 3B cells. Kupffer cells stimulated by TP-1 released significant amounts of tumor necrosis factor-alpha (TNF-), interleukin (IL)-1, and IL-6 compared with control Kupffer cells (p<0.01). The activity of TP-1-treated Kupffer cell supernatants against Hep G2 cells was blocked by anti-TNF- antibodies, whereas neither anti-IL-1 nor anti-IL-6 antibodies blocked cytotoxicity. Conclusions: These results demonstrate that TP-1 cytotoxicity against human HCC cells is not mediated directly or through hepatocytes, but occurs through activation of Kupffer cells and release of TNF-. Understanding the mechanism of TP-1 cytotoxicity against human HCC has been used to plan a phase I trial of TP-1 combined with regional infusion of doxorubicin to treat unresectable HCC.Presented at the 49th Annual Cancer Symposium of The Society of Surgical Oncology, Atlanta, Georgia, March 21–24, 1996.     

Changes in serum levels of growth factors in healthy individuals after living related liver donation

INTRODUCTION: To obtain better insight into the kinetics of hepatic growth factors following partial hepatectomy for living related liver donation, we investigated the postoperative changes in serum levels of hepatocyte growth factor (HGF), epidermal growth factor (EGF), vascular epidermal growth factor (VEGF), and transforming growth factor-alpha (TGF-alpha). PATIENTS AND METHODS: Eighteen healthy donors undergoing right hepatectomy for living related donation were enrolled in this study. Serum levels of HGF, EGF, VEGF, and TGF-alpha were measured using enzyme-linked immunosorbent assay kits before surgery, at 2 hours after resection, and daily during 5 days postoperatively. RESULTS: Mean preoperative HGF serum levels in healthy adults were 778 +/- 64 pg/mL. Within 2 hours after operation, they significantly increased to 9608 +/- 3111 pg/mL afterward decreasing to 2726 +/- 241 at day 1 and 2283 +/- 250 pg/mL at day 2. Hereafter HGF serum levels stabilized at increased levels until day 5 (2109 +/- 138, 2047 +/- 219, 2283 +/- 336 pg/mL, respectively). At all time points, the differences between pre- and postoperative HGF levels were significant (P < .01). In contrast, VEGF and EGF serum levels showed no significant differences between pre- and postoperative levels at all time points. TGF-alpha was not detected using a commercially available test with a detection limit of 10 ng/mL, suggesting only low TGF-alpha serum levels following liver resection. CONCLUSION: Significantly increased HGF serum levels after hepatectomy demonstrate its crucial role among the other investigated growth factors in regeneration of the remnant liver tissue during the early period after the operation.     

肝细胞癌合并肝硬化患者肝脾联合切除术后免疫功能变化的研究

目的　探讨肝细胞癌合并肝硬化患者肝癌切除时联合脾切除术后免疫功能的变化。方法　将 16例肝癌合并肝硬化患者分成 2组 ,即肝癌切除联合脾切除组 ( 7例 )和单纯肝癌切除组 ( 9例 ) ,于术前、术后 2个月取外周血 7ml,采用流式细胞仪检测CD4、CD8、CD4 /CD8,ELISA法检测IL 2、IFN γ、IL 10。　结果　 2组患者术前CD4、CD8、CD4 /CD8、IL 2、IFN γ、IL 10水平差异无显著性 ;术后 2个月 ,切脾组CD4 ( 3 8 2 %± 3 7% )、CD4 /CD8( 1 7%± 0 3 % )高于保脾组CD4 ( 3 2 5 %± 4 0 % )、CD4 /CD8( 1 1%± 0 1% ) ,而CD8( 2 3 7%± 3 7% )低于保脾组CD8( 2 9 4 %± 4 0 % ) (P <0 0 5 ) ;切脾组IFN γ[( 10 4 4± 14 9)pg/ml]、IL 2 [( 98 6± 18 6)pg/ml]高于保脾组 [IFN γ( 70 5± 12 6)pg/ml、IL 2 ( 80 9± 13 5 )pg/ml],而IL 10 [( 5 5 5± 11 2 )pg/ml]低于保脾组 [IL 10 ( 89 4± 10 )pg/ml](P <0 0 5 )。　结论肝癌切除时联合脾切除不但没有降低机体T细胞亚群和Th细胞的平衡 ,反而促进其恢复平衡 ,并改善机体抗肿瘤免疫功能     

Simultaneous segmental obstruction of bile duct and portal vein markedly changes a population of biliary and hepatic cells in human liver

BACKGROUND AND AIMS: No studies have investigated histologic changes caused by simultaneous segmental obstruction of the bile duct and portal vein in human liver. PATIENTS/METHODS: Liver tissues with simultaneous obstruction of the segmental bile duct and portal vein (O(+/+) liver), with segmental bile duct obstruction alone (O(+/-) liver), and without obstruction (O(-/-) liver) were obtained from patients who underwent hepatectomy, and studied morphologically and immunohistochemically. RESULTS: In O(+/+) liver, the proportional area consisting of hepatocytes was significantly less (31.0+/-25.8%) than in O(+/-) liver (78.4+/-18.9%) or O(-/-) liver (86.5+/-9.2%). In contrast, the proportional area consisting of biliary epithelial cells was significantly higher in O(+/+) liver (9.1+/-6.1%) than in O(+/-) liver (1.6+/-1.5%) or O(-/-) liver (0.7+/-0.6%). The proportional area consisting of fibrous tissue also was significantly higher in O(+/+) liver than in the other two groups. In O(+/+) liver, some cells located at the periphery of hepatocyte areas were immunoreactive for both hepatocyte and biliary epithelial cell markers. CONCLUSION: Simultaneous segmental obstruction of the bile duct and portal vein induces a marked ductular increase, periportal fibrosis, and a reduction in the number of hepatocytes in human liver tissue.     

The effects of immunosuppressive agents on the function of human hepatocytes in vitro

Calcineurin inhibitors (tacrolimus) and steroids continue to be an important component of hepatocyte transplantation protocols, despite reports of hepatotoxicity and inhibitory effects of steroids on cell proliferation. The aim of the study was to investigate whether isolated human hepatocytes were more vulnerable to the toxicity of these agents and also to investigate their effects on hepatocyte VEGF secretion, a vascular permeability factor suggested to be involved in the cell engraftment process. Human hepatocytes were isolated from donor livers/segments rejected or unused for orthotopic liver transplantation using a collagenase perfusion technique. Hepatocytes were plated for cell function tests and to determine VEGF production. Tacrolimus (0-50 ng/ml) and methylprednisolone (0-500 ng/ml) were added to the culture media and cells incubated for 24 h. Cell metabolic activity was assessed using the MTT assay, cell number using the SRB assay, and cell attachment from hepatocyte total protein content and protein synthesis using [14C]leucine incorporation. VEGF in culture supernatants was measured by ELISA. Tacrolimus and methylprednisolone had no statistically significant inhibitory effects on metabolic activity or protein synthesis compared to controls at all concentrations of the agents tested when added after plating. There were also no significant effects on cell attachment when tacrolimus or methylprednisolone was added at the time of cell plating. There were no differences in the responses obtained when either fresh or cryopreserved hepatocytes were used. The amount of VEGF secreted by untreated hepatocytes was highly variable (0-1400 pg/10(6) cells/24 h). VEGF levels in the culture supernatant from hepatocytes isolated from < or = 20-year-old donors (687 +/- 59 pg/10(6) cells/24 h) was significantly greater than from older donors (61 +/- 7 pg/10(6) cells/24 h; p = 0.003). Tacrolimus and methylprednisolone did not significantly affect VEGF secretion by hepatocytes. Tacrolimus and methylprednisolone did not have detrimental effects on the metabolic function of human hepatocytes, cell attachment, or VEGF secretion after cell isolation.     

miR-542-3p对肝癌细胞恶性生物学行为的影响及机制探讨

目的 探讨microRNA-542-3p(miR-542-3p)在人肝癌细胞系及组织中的表达水平及其对肝癌细胞增殖、侵袭转移能力的影响.方法 采用RT-PCR检测miR-542-3p在肝癌细胞系(HCCLM3、Hep3B、Huh7、SMMC-7721、MHCC-97H、MHCC-97L)以及人正常肝细胞LO2中的表达;...     

Primary hepatocytes outperform Hep G2 cells as the source of biotransformation functions in a bioartificial liver.

OBJECTIVE: Metabolic activity of transformed human liver (Hep G2) cells and primary rat hepatocytes were compared during in vitro application of a gel entrapment bioartificial liver. BACKGROUND: Clinical trials of bioartificial liver devices containing either transformed liver cells or primary hepatocytes have been initiated. A study comparing transformed liver cells and primary hepatocytes in a bioartificial liver under similar conditions has not been reported previously. METHODS: Gel entrapment bioartificial liver devices were inoculated with 100 million cells, Hep G2 cell line (n = 4), or rat hepatocytes (n = 16), and studied for up to 60 days of in vitro cultivation. RESULTS: Hep G2 cells grew to confluence within the gel entrapment configuration with a doubling time of 20 +/- 3 hours. Rat hepatocytes significantly outperformed Hep G2 cells at confluence in all categories of biotransformation, including ureagenesis (3.5 +/- 0.7 vs. 0.3 +/- 0.1 mumol/hr, p < 0.05), glucuronidation (630 +/- 75 vs. 21 +/- 2 nmol/hr, p < 0.005), sulfation (59 +/- 13 vs. 5 +/- 2 nmol/hr, p < 0.05), and oxidation (233 +/- 38 vs. < 1 nmol/hr, p < 0.005). At the conclusion of one experiment, Hep G2 cells were found in the extracapillary compartment of the bioartificial liver, analogous to the patient's compartment during clinical application. CONCLUSIONS: Primary rat hepatocytes were superior to the Hep G2 cell line as the source of hepatic function in a bioartificial liver and avoided the potential risk of tumor transmigration from the bioartificial liver into the patient's circulation.     

转肝细胞生长因子基因肝细胞模型的建立

目的：利用脂质体介导法在体外建立转肝细胞生长因子（HGF）基因的人肝细胞模型。方法：建立HGF真核细胞表达载体，利用脂质体介导法在体外将HGF基因转染入人肝细胞，利用荧光显微镜观察、免疫组织化学、原位杂交方法检测HGF真核细胞表达载体的转录和表达情况。结果：以阳离子脂质体LipofectAMINE为载体将HGF基因转染人肝细胞后，经400mg/L的G418筛选后可形成抗性克隆；Neo基因原位杂交结果显示转染基因的细胞有阳性表达；荧光显微镜下观察到有绿色荧光；免疫组织化学证实转染HGF基因的肝细胞有HGF蛋白的表达。结论：HGF基因可被成功转染入人肝细胞并能有效表达，这可能为肝病的基因治疗提供一种新途径。     

Transplantation of highly differentiated immortalized human hepatocytes to treat acute liver failure

BACKGROUND: Temporary support of a damaged liver by a bioartificial liver (BAL) devise is a promising approach for the treatment of acute liver failure. Although human primary hepatocytes are an ideal source of hepatic function in BAL, shortage of human livers available for hepatocyte isolation is the limiting factor for the use of this modality. A clonal human hepatocyte cell line that can grow economically in culture and exhibit liver-specific functions should be an attractive solution to this problem. METHODS: To test this alternative, primary human fetal hepatocytes were immortalized using Simian virus 40 large T antigen. To investigate the potential of the immortalized cells for BAL, we transplanted the cells into the spleen of adult rats and performed a 90% hepatectomy 12 hr later. RESULTS: One of the cloned human liver cell lines, OUMS-29, showed highly differentiated liver functions. Intrasplenic transplanting of 20x10(6) OUMS-29 cells protected the animals from hyperammonemia and the associated hepatic encephalopathy. Survival was significantly prolonged in 90% of hepatectomized rats receiving OUMS-29 cells. CONCLUSIONS: A highly differentiated immortalized human hepatocyte cell line, OUMS-29, was able to provide metabolic support during acute liver failure induced by 90% hepatectomy in rats. Essentially unlimited availability of OUMS-29 cells may be clinically useful for BAL treatment.     

Biological role of HGF/MET pathway in renal cell carcinoma

PURPOSE: Several lines of evidence show that hepatocyte growth factor (HGF) and its receptor MET play a significant role in the progression of various cancers including renal cell carcinoma (RCC). Our objectives were to evaluate the gene expression of HGF and MET in RCC, and to examine the effect of HGF on the biological activities of cultured RCC cells. MATERIALS AND METHODS: We examined the gene expression of HGF and MET in 27 primary RCC tumors by quantitative competitive RT-PCR. The effects of HGF on in vitro chemoinvasion assay and the expression of matrix metalloproteinase-9 (MMP-9), and the induction of Fas-induced apoptosis were studied by transfection of HGF cDNA to cultured RCC cells, Caki-1. RESULTS: HGF mRNA and MET mRNA were detected in all surgical specimens. The level of expressed HGF mRNA was proportional with the volume of tumor (r = 0.50, p = 0.015). Caki-1 cells overexpressing HGF cells showed enhanced in vitro invasiveness in the chemoinvasion assay and increased activity of 92 kDa type IV collagenase (MMP-9). The sensitivity to Fas-induced cell death was reduced in HGF transfectants, which was reversed by the presence of anti-HGF antibody. CONCLUSIONS: HGF enhanced the invasive properties of cultured RCC cells and inhibited Fas-induced apoptosis in vitro. Both HGF and MET mRNA were expressed in RCC tissues tested. Our results indicate that HGF/MET pathway may have a significant role in the progression of RCC.     

Mir-145在肝癌细胞及肝癌组织中的表达及其意义

目的 观察微小RNA(miRNA)mir-145在正常胚肝细胞株L02、肝癌细胞株MHCC97和SMMC7721中的表达,同时检测该miRNA在正常肝组织及肝细胞癌组织中的表达,探讨其在肝癌发生发展中的作用和意义.方法 应用实时定量聚合酶链反应(PCR)方法检测mir-145在正常细胞株及肝癌细胞株中的表达,同时收取肝细胞癌手术患者正常组织、癌周组织、癌组织,检测其表达并分析其意义.结果 mir-145在肝癌细胞(MHCC97、SMMC7721)中的表达比正常肝细胞L02显著降低(0.312±0.025、0.396±0.076、0.954±0.037,P＜0.05),在癌组织中表达显著低于正常组织与癌周组织(0.162±0.002、0.972±0.054、0.956±0.018,P＜0.05),正常组织及癌周组织比较差异无统计学意义(P＞0.05).结论 mir-145在肝癌细胞及肝癌组织中的低表达,可能与肝癌发生发展密切相关,并可能作为肝癌的诊断及预后的指标.

Abstract：

Objective To explore the role of miR-145 in human hepatocellular carcinoma ( HCC). We investigate the expression of miR-145 in human embryonic hepatocytes ( L02 cells),hepatocellular carcinoma cell lines (MHCC97 ,SMMC7721) ,HCC tissues and normal liver. Methods We detected the expressions of miR-145 in human embryonic hepatocytes, hepatocellular carcinoma cell lines ( MHCC97,SMMC7721) ,HCC tissues,cancer organizations and normal liver by real-time PCR. Result The expression of miR-145 in hepatocellular carcinoma cell lines ( MHCC97,SMMC7721) is significantly lower than the human embryonic hepatocytes ( 0. 312 ± 0. 025,0. 396 ± 0. 076,0. 954 ± 0. 037, P ＜ 0. 05), and compared with the cancer organizations and normal liver,the expression of miR-145 in HCC tissues is significantly decreased (0. 162 ±0.002,0. 972 ±0.054,0.956 ±0. 018,P＜0. 05). Conclusion These results indicate that the miR-145 probably involved in HCC pathogenesis and was the indicator of diagnosis and prognosis of the HCC.     

成人骨髓基质干细胞分化来源的肝细胞的冻存与复苏研究

目的在体外诱导人骨髓基质干细胞向肝细胞分化成熟的基础上,将细胞深冻保存,观察冻存复苏后细胞存活率及功能状况。方法在含有多种细胞因子的条件培养基中体外诱导成人骨髓基质干细胞,当细胞显示成熟肝细胞特征后,将细胞分别冻存于90%胎牛血清(FBS)和10%二甲亚砜(DMSO)(A组)、10%FBS、30%甘油和60%培养基(B组)和10%FBS、10%DMSO和80%威斯康星器官保存液(UW液)(C组)中。冻存4周后复苏细胞,测定各组细胞存活率及白蛋白合成情况。结果成人骨髓基质干细胞分化来源的肝细胞经冻存复苏后可恢复功能细胞形态,A、B和C组冻存液中细胞复苏后细胞存活率分别为(60·0±3·3)%、(91·0±2·6)%和(89·0±1·4)%,培养24h后检测上清液白蛋白浓度分别为(0·210±0·005)g/L、(0·340±0·020)g/L和(0·330±0·030)g/L。结论成人骨髓基质干细胞分化来源的肝细胞冻存复苏后仍保持较高的细胞存活率和功能,可作为临床肝细胞移植及生物人工肝的重要肝细胞来源。     

Evaluation of methods of hepatotrophic stimulation in rat heterotopic hepatocyte transplantation using polymers.

BACKGROUND: Hepatocyte transplantation has been studied as an alternative to organ transplantation. Hepatocyte transplant models should provide sufficient cell mass for replacement function and hepatotrophic stimulation of the transplanted cells in heterotopic locations. METHOD: The authors used three-dimensional porous polyvinyl-alcohol matrices as cell carriers, which were implanted between mesenteric leaves of the intestine. In this study, different methods were evaluated for hepatotrophic stimulation. Fifty million transplanted hepatocytes (approximately 10% liver mass) were implanted in Lewis rats. We compared 70% partial hepatectomy, portacaval shunt, cotransplantation of enterocytes, cotransplantation of islets of Langerhans, and methylprednisolone injection to a control group with only hepatocyte transplantation. Portacaval shunt and islet cotransplantation also were used in combination. Specimens were harvested 2 weeks after transplantation, and area per histological cross section compromised by hepatocytes was measured. RESULTS: Seventy percent partial hepatectomy, enterocyte cotransplantation, and methylprednisolone injection resulted in hepatocyte maintenance similar to control group (3,100 +/- 7,592 microm2). Portacaval shunt (96,866 +/- 55,039 microm2) and islet cotransplantation (173,020 +/- 75,977 microm2) yielded a highly significant increase in hepatocyte area. The combination of portacaval shunt and islet cotransplantation resulted in a significant increase compared with using these methods individually (288,930 +/- 86,726 microm2). Additional immunohistochemical stains for active DNA synthesis, insulin, and glucagon demonstrated the proliferative abilities of the hepatocytes and the synthesis of insulin and glucagon in the cotransplanted islets. CONCLUSION: Hepatocyte transplantation can be performed using polymer carriers and that hepatocyte survival and maintenance can be improved with portacaval shunt and islet cotransplantation.     

Transplantation of human hepatocytes cultured with deleted variant of hepatocyte growth factor prolongs the survival of mice with acute liver failure

BACKGROUND: Considering the scarcity of donor livers, it is extremely important to establish a functional culture method for isolated hepatocytes. As a tool for maintaining hepatocyte functions in vitro, dHGF, a variant of HGF (hepatocyte growth factor) with a deletion of five amino acids, attracted our attention because it is less cytotoxic compared with HGF. METHODS: We evaluated growth, albumin production, metabolizing abilities of ammonia, lidocaine, and diazepam of human hepatocytes in the presence of dHGF (10-1000 ng/ml). The gene expression of liver markers was comparatively analyzed. The effect of intrasplenic transplantation of dHGF-treated human hepatocytes into severe combined immunodeficient (SCID) mice was evaluated in an acute liver failure (ALF) model induced by D-galactosamine (D-gal). RESULTS: When 100 ng/ml of dHGF was utilized, metabolism rates of ammonia, lidocaine, and diazepam and albumin production per unit cell significantly increased. The gene expression analysis demonstrated the enhanced expression of albumin, HNF-4alpha, and C/EBPalpha in the hepatocytes treated with 100 ng/ml of dHGF. Transplantation of such hepatocytes prolonged the survival of the SCID mice with ALF induced by D-gal. CONCLUSIONS: The present work clearly demonstrates the usefulness of dHGF (100 ng/ml) for maintaining the differentiated functions of human hepatocytes in tissue culture.     

观察重组人肝细胞生长因子裸质粒表达产物在人体血液中的含量及其抗原性变化

目的 研究血浆中重组人肝细胞生长因子裸质粒(pCK-HGFX7)表达产物肝细胞生长因子(hepatocyte growth factor,HGF)含量在体内的变化,以及应用本品后是否发生免疫反应产生HGF抗体.方法 选择21例严重下肢缺血性疾病患者(Rutherford分级4～6级),随机分为4mg、8mg、12mg、16mg 4个剂量组.将每组剂量等量平分后在试验期第1天及第15天于下肢缺血部位的供血动脉走行和侧支循环可能建立的部位肌内注射给药.采集第1(第1次给药前)、8、15(第2次给药前)、21、59天血浆样本测定HGF含量;采集第1、15、28、59、91天血浆样本测定HGF抗体含量.结果 (1)局部肌内注射给药后受试者血浆中HGF含量范围为216～1189.75 pg/mL;(2)未检测到受试者血浆中存在HGF抗体,提示外周血中未见HGF抗体生成.结论 重组人肝细胞生长因子裸质粒局部肌内注射后,其表达产物HGF在外周血中含量未见明显异常波动,应用本品后人体没有发生免疫反应.     

Immediate early genes and p21 regulation in liver of rats with acute hepatic failure

It has been observed that liver regeneration in acute hepatic failure (AHF) is suppressed [Eguchi et al. Hepatology 1996;24(6):1452-9]. The molecular mechanism regulating this inhibition is not known. We previously reported that in AHF rats, hepatocyte proliferation was significantly impaired with elevation in serum IL-6, TGF-beta1, and HGF [Kamohara et al. Biochem Biophys Res Commun 2000;273(1):129-35]. Following either 70% partial hepatectomy (PH) or liver injury, quiescent mature hepatocytes are "primed" to re-enter the cell cycle. The process of "priming" appears to be triggered by extracellular cytokines (IL-6 and TNF-alpha) and is characterized by expression of immediate early genes. Under the stimulation of growth factors such as HGF, "primed" hepatocytes exit the G1 phase of the cell cycle. G1-associated cyclins and their inhibitors play a pivotal role in G1/S cell cycle transition. Here, we demonstrate that immediate early gene (i.e. c-myc, c-fos) expression and AP-1 activity are preserved in AHF rat livers despite absence of hepatocyte proliferation. In contrast, p21 mRNA and protein are both over-expressed in AHF livers compared to livers from rats undergoing PH; this elevation leads to inhibition in Cdk2 activity, resulting in G1 cell cycle arrest and inhibition of regeneration.     

肝细胞癌门静脉癌栓中肝细胞生长因子及其受体的表达

目的研究肝细胞生长因子（HGF）及其受体C-Met在肝细胞癌门静脉癌栓中的表达，探讨HGF及C-Met的表达与门静脉癌栓形成与发展的关系。方法用半定量逆转录-聚合酶链反应（RT-PCR）技术检测HGFmRNA及C-MetmRNA在20例肝细胞癌门静脉癌栓及其癌旁组织、10例无门静脉癌栓形成的肝细胞癌和癌旁组织及10例正常肝组织中的表达。结果HGF及C-Met在肝癌及癌栓中的阳性表达率均显著高于正常肝组织及癌旁组织，差异有统计学意义（P〈0．05），HGF及C—Met在癌栓中及肝癌中表达的阳性率比较差异无统计学意义（P〉0．05），HGF与C—Met在各组织中的表达阳性率高低差异无统计学意义（P〉0．05）。HGF及C—Met在肝癌及癌栓中的表达量均显著高于正常肝组织及癌旁组织，差异有统计学意义（P〈0．05），HGF及C-Met在癌栓中表达量显著高于其在肝癌中表达量（P〈0．05），HGF与C-Met在各组织中的表达量差异无统计学意义（P〉0．05）。结论HGF及C-Met参与了肝癌的形成，而肝癌患者HGF及C-Met量的升高可能是肝癌发展形成门静脉癌栓的原因之一，HGF及C—Met可作为门静脉癌栓形成的监测指标。