Objective To study hepatocyte growth factor (HGF) and its receptor (c-met) expressions in human colorectal cancer and non-neoplasm colorectal mucosa, and the relationship with tumor angiogenesis. Methods Tissue microarrays (TMAs) were made up of 80 cases of colorectal cancer and 80 cases of nonneoplasm colorectal mucosa. The expressions of HGF and c-met were detected by immunohistochemistry (SP). CD105 was used as a marker to account microvessel density (MVD) in tumor tissue. Results HGF was over expressed in 48 cases and c-met was over expressed in 63 cases of colorectal cancer tissue, and the correlation between HGF and c-met positive expression was significant (r=0.231, Plt;0.05). The high expression rate of HGF and cmet in colorectal cancer were significantly higher than that in non-neoplasm colorectal mucosa (χ2=35.387, Plt;0.05; χ2=59.854, Plt;0.05) of colorectal cancer. The overexpression of HGF was correlated with lymph node metastasis (χ2=4.743, Plt;0.05) and TNM staging (χ2=5.576, Plt;0.05). The overexpression of c-met was correlated with differentiation (χ2=15.767, Plt;0.05) and lymph node metastasis (χ2=5.765, Plt;0.05) of colorectal cancer. MVD was different between overexpression and lowexpression colorectal cancer tissues of HGF and cmet (t=2.150, Plt;0.05; t=2.052, Plt;0.05). There was statistical correlation between HGF and cmet overexpression (r=0.259, Plt;0.05). The overexpressions of HGF and cmet were correlated with lymph metastasis in moderate differentiation cancer (χ2=13.154, Plt;0.05; χ2=5.371, Plt;0.05). Conclusions The overexpressions of HGF and c-met in colorectal cancer may be related with tumor angiogenesis. Detecting the expressions of HGF and c-met is valuable to estimate the biological character of colorectal cancer.
ObjectiveTo introduce the relationship between the apoptosis hepatocyte and its genic mediation and the ischemia of portal vein. MethodsThe combination of related literatures and our research findings were made.ResultsPortal vein ischemia may induced hepatocyte apoptosis, p53 and bcl2 gene alternatively adjust hepatocyte apoptosis. Expression of p53 gene is enhanced in hepatic tissue when hepatocyte apoptosis is not obvious, but after 24-72 h of portal vein ischemia, when hepatocyte apoptosis is obvious, enhanced expression of p53 gene or reduced expression of bcl2 gene occur. There exists close relationship between portal vein ischemia and hepatocyte apoptosis. Conclusion Apoptosis hepatocyte is involved in organic atrophy after ischemia of portal vein, and p53 and bcl2 gene alternatively adjust hepatocyte apoptosis. At present, the mechanism of apoptosis of hepatocyte induced by ischemia of portal vein is not clear, which needs further study.
Objective To introduce the studies on gene therapy for peripheral arterial disease(PAD) using plasmid DNA encoding human hepatocyte growth factor(HGF) gene. Methods Recent articles including preclinical and clinical studies were reviewed. Results Intramuscular injection of human HGF plasmid DNA into rat, rabbit, dog and diabetic hindlimb ischemic models, resulted in a significant increase in capillary density, blood flow and blood pressure. but no influence on tumor growth in mice. A clinical trial wasperformed in ischemic limbs of 6 critical limb ischemic patients, the result showed that no side effect caused by gene transfer was detected in all 6 patients.The pain scale and long diameter of ischemic ulcers were reduced. Conclusion Intramuscular injection of naked HGF plasmid DNA could be a safe and potential treatment for PAD.
Objective To investigate the effect of hepatocyte growth factor (HGF) on the barrier function of retinal peigment epithelium (RPE) and to detect the pathological mechanism of retinal detachment (RD) induced by over expression of HGF in RPE. Methods Sub-retina injection of E1/E3deleted adenoviral vectors encoding HGF (Ad CMV.HGF) and green fluorescent protein (Ad CMV.GFP) in adult pigmented rabbits [5times;104 plaque-forming units (pfu)/eye] to set up the model of retinal detachment. The ocular fundus and pathological changes were observed 3, 7, 14, and 28 days after injection. The expression level of HGF in retina and vitreous body was detected by immunohistochemistry and enzyme linked immunosorbent assay (ELISA). Results In the control eyes injected with AdCMV.GFP, expression of GFP only detected in RPE monolayer. The eyes injected with AdCMV.HGF had b HGF immune positive action in RPE cells at the injection site. The expression level of HGF in vitreous body reached the peak 7 days after injection and decreased to the basic level 28 days after injection. Chronic RD and chronic choroidal inflammation were found in the eyes injected with AdCMV.HGF within the time frame of HGF expression. Proliferative RPE cells were found in subretinal space in the region of RD, and multilayered cellular membranes developed in some eyes. Conclusion Over expression of HGF in RPE may induce chronic serous RD with subretinal proliferation of RPE, which suggests that HGF should be further studied as a target for therapeutic intervention in RD. (Chin J Ocul Fundus Dis, 2007, 23: 193-197)
【Abstract】 Objective To observe the effect of cytokines and combinations in the inducement of human umbil icalcord blood-derived CD34+ cells into hepatocyte-l ike cells. Methods The mononuclear cells (MNCs) were derived by density gradient centrifugation and the CD34+ cells were sperated from MNCs. The human umbil ical cord blood-derived CD34+ cells were cultivated through 49 different combinations of cytokines including leukemia inhibitor factor (LIF), oncostatin M, bFGF, aFGF, hepatocyte growth factor, EGF and stem cell factor for 28 days, and the concentrations of the cytokines were 10, 10, 10, 10, 20, 20 and 50 ng/mL, respectively. The mRNAs of cytokeratin 19 (CK-19), CK-18, glutamine synthetase (GS), human albumin (ALB) and α-fetoprotein (AFP) were detected every seven days. The ALB secretion abil ity, detoxification abil ity and hepatin synthesis abil ity of the induced cells were detected by immunofluorescence assay, indocyanine green (ICG) and periodic acid-schiff assay, respectively. The fresh umbil ical cord blood-derived CD34+ cells were detected at the same time as a control. Results The mRNAs of CK-19, CK-18 and GS could be transcribed in all the induced cells, but the transcription of the mRNAs of ALB and AFP which was the special mark of mature hepatocyte and l iver stem cell, respectively, was not found. All the mRNAs could not be found in freshly isolated umbil ical cord blood-derived CD34+ cells. All the cells induced in vitro could not release ALB, and not help the detoxification of ICG which was the fundamental function of mature l iver cells. These results were the same in the control group. The hepatin synthesis abil ity of all the induced cells increased by comparison to the fresh ones. Conclusion Though some mRNAs of proteins which are transcribed in hepatocytes can be found in the induced cells, umbil ical cord blood-derived CD34+ cells could not be transdifferentiated into hepatocyte-l ike cells through cytokines in vitro.
Objective To prepare chitosan microcarriers and to use it to cultivate rat primary hepatocytes. Methods The crosslinked chitosan microcarrier was prepared by the reaction of glutaraldehyde with chitosan. Various factors that influence the preparation were studied and the reaction conditions were optimized. Rat primary hepatocytes cultured on chitosan microcarrier were observed by using phase contrast microscope and scanning electron microscope. Results Chitosan microcarriers with good properties could be prepared by adjusting the concentration of chitosan solution and the quantity of glutaraldehyde. Rat hepatocytes cultured on chitosan microcarriers retained the spherical shape as they have in vivo. And albumin secretion can last over one week. The highest albumin secretion rate reached 26.7μg/24h/ml. Conclusion Chitosan microcarriers is a promising scaffold for hepatocyte attachment, which can be used in bioartificial liver support system.
【Abstract】Objective To investigate the injury of calcium on the liver. Methods By using collagenase-containing solution to perfuse the rat livers, the rat liver suspension with different viability was prepared, preserved hypothermically, and the cytosolic calcium comcentration was detected with Fura2/AM. Results ① The concentration of cytosolic calcium after 2-hour storage: Experiment group 1(viability 5%) (1055.0±30.79) nmol/L, experiment group 2(viability 10%) (853.0±20.42) nmol/L, experiment group 3(viability 30%) (616.0±13.20) nmol/L, experiment group 4(viability 50%) (562.0 ±26.06) nmol/L, experiment group 5(viability 70%-80%) (318.0±13.01) nmol/L, experiment group 6(viability 90%) (114.6±6.11) nmol/L. ②The concentration of cytosolic calcium after 24hour storage: Experiment group A(viability 10%) (1704.0±70.11) nmol/L, experiment group B(viability 50%) (1125.0±23.22) nmol/L. The results showed that the lower was the viability, the higher was the cytosolic calcium concentration.With the same viability the cytosolic calcium concentration elevated more than two times higher than the original concentration with the time lengthened. Conclusion Calcium overload is one of the main factors which attribute to the ischemiareperfusion injury of the hepatocytes.
Objective To investigate the effect of combined therapy of granulocyte colony stimulating factor (G-CSF) and bone marrow mesenchymal stem cells (BMSCs) carrying hepatocyte growth factor (HGF) gene on the angiogenesis of myocardial infarction (MI) in rats and the mechanisms of the synergistic effect. Methods BMSCs were aspirated from the femur and tibia of 3-week-old Sprague Dawley (SD) male rats. The third generation of BMSCs were harvested and transfectedwith Ad-HGF. The MI models were establ ished in 44 SD male rats (weighing 200-250 g) by l igating the left coronary artery. At 4 weeks after l igation, the shorting fraction (FS) of the left ventricle being below 30% was used as a criteria of model success. The BMSCs (5 × 107/ mL) transfected with Ad-HGF were transplanted into the infarct zone of 12 SD rats, and the expression of HGF protein was detected by Western blot method at 2, 7, and 14 days after transplantation. At 4 weeks, the other 32 SD rats were randomly divided into 4 groups (n=8). The 0.1 mL normal sal ine was injected into the infarct zone in control group; 0.1 mL normal sal ine was injected combined with intraperitoneal injection G-CSF [100 μg/ (kg•d)] for 5 days in G-CSF group; 0.1 mL BMSCs (5 × 107/ mL) transfected with Ad-HGF was injected into the infarct zone in HGF group; 0.1 mL BMSCs (5 × 107/ mL) transfected with Ad-HGF was injected combined with intraperitoneal injection G-CSF [100 μg/ (kg•d)] for 5 days in combined therapy group. At 2 weeks after transplantation, heart function was detected by cardiac ultrasound and hemodynamic analysis, and then myocardial tissue was harvested to analyse the angiogenesis of the infarct zone, and the expression of VEGF protein by immunofluorescence staining. Results The expression of HGF protein in vivo was detected at 2 days and 7 days of BMSCs transfected with Ad-HGF transplantation. There was no significant difference in left ventricular systol ic pressure (LVSP), left ventricular end-diastol ic pressure (LVEDP), dP/dtmax, and FS between G-CSF group and control group (P gt; 0.05). When compared with the control group, LVEDP decreased significantly; LVSP, FS, and dP/dtmax increased significantly (P lt; 0.05) in HGF group and combined therapy group. When compared with HGF group, FS and dP/dtmax increased significantly in combined therapy group (P lt; 0.05). Immunofluorescence staining showed that the vascular endothel ial cells were observed in myocardial infarction border zone. The vascular density and the expression of VEGF protein were significantly higher in combined therapygroup than in other 3 groups (P lt; 0.05). Conclusion The combined therapy of G-CSF and BMSCs carrying HGF gene has a synergistic effect and can enhance infarct zone angiogenesis through inducing the expression of VEGF protein.
【Abstract】 Objective To explore the new therapy for pulmonary fibrosis by observing the effects of insulin-like growth factor 1 ( IGF-1) treated mesenchymal stemcells ( MSCs) in rats with bleomycin-induced pulmonary fibrosis. Methods Bone marrowmesenchymal stemcells ( BMSCs) were harvested from6-week old male SD rats and cultured in vitro for the experiment. 48 SD rats were randomly divided into 4 groups, ie.a negative control group ( N) , a positive control group/bleomycin group ( B) , a MSCs grafting group ( M) ,and an IGF-1 treated MSCs grafting group ( I) . The rats in group B, M and I were intratracheally injected with bleomycin ( 1 mL,5 mg/kg) to induce pulmonary fibrosis. Group N were given saline as control. Group M/ I were injected the suspension of the CM-Dil labled-MSCs ( with no treatment/pre-incubated with IGF-1 for 48 hours) ( 0. 5mL,2 ×106 ) via the tail vein 2 days after injected bleomycin, and group B were injected with saline ( 0. 5 mL) simultaneously. The rats were sacrificed at 7,14,28 days after modeling. The histological changes of lung tissue were studied by HE and Masson’s trichrome staining. Hydroxyproline level in lung tissue was measured by digestion method. Frozen sections were made to observe the distribution of BMSCs in lung tissue, and the mRNA expression of hepatocyte growth factor ( HGF) was assayed by RTPCR.Results It was found that the red fluorescence of BMSCs existed in group M and I under the microscope and the integrated of optical density ( IOD) of group I was higher than that of group M at any time point. But the fluorescence was attenuated both in group M and group I until day 28. In the earlier period, the alveolitis in group B was more severe than that in the two cells-grafting groups in which group I was obviously milder. But there was no significant difference among group I, M and group N on day 28.Pulmonary fibrosis in group B, Mand I was significantly more severe than that in group N on day 14, but itwas milder in group M and I than that in group B on day 28. Otherwise, no difference existed between the two cells-grafting groups all the time. The content of hydroxyproline in group B was significantly higher than that in the other three groups all through the experiment, while there was on significant difference betweengroup I and group N fromthe beginning to the end. The value of group M was higher than those of group I and group N in the earlier period but decreased to the level of negative control group on day 28. Content of HGF mRNA in group Nand group I was maintained at a low level during the whole experiment process. The expression of HGF mRNA in group I was comparable to group M on day 7 and exceeded on day 14, the difference of which was more remarkable on day 28. Conclusions IGF-1 can enhance the migratory capacity of MSCs which may be a more effective treatment of lung disease. The mechanismmight be relatedto the increasing expression of HGF in MSCs.
Objective To evaluate the effect of hepatocyte growth factor(HGF) on intestinal permeability and bacterial translocation after small bowel transplantation in rats. Methods Twenty Wistar rats were as recptors and twenty SD rats as donors. After heterotopic intestinal grafting, cyclosporine A was administered at 6mg/kg·day intramuscularly for inhibiting rejection. The SD rats were divided into 2 groups(n=10). HGF was administered at 150 μg/kg·day (HGF group) and normal saline was administered at 150 μg/kg·day (controlgroup). Intestinal permeability and bacterial translocation to the mesenteric lymph nodes and portal vein were assessed at the 8th postoperative day. Results The lactulose and lactulose/ mannitol of control group (0.0931%±0.008 5% and 0.132± 0.021) were higher than those of normal reference value (0.015 0%±0.002 0% and 0.020±0.005)（Plt;0.05）. The lactulose and lactulose/ mannitol of HGF group (0.039 6%±0.009 0% and 0.056±0.013) were also higher than those of normal reference value（Plt;0.05）.The bacterial culture positive proportion of lymphaden in HGF group and control group were 10% and 60%, showing statistically significant difference（Plt;0.05）. The bacterial culture positive proportion of portal vein in HGF group and control group were 10% and 20% respectively（P＞0.05）. Conclusion HGF can decrease intestinal permeability and bacterial translocation from the lumen of the graft to the mesenteric lymph nodes,thus improve gut barrier function, may be of help to reduce the incidence of septic complications after intestinal grafting.