Objective To investigate the effect of HGF on proliferation and migration in cultured human RPE cells. Methods Human RPE cells cultured in serum-free medium were treated with HGF(1，2，10，50，100 mu;g/L), and MT T assay was used to detect the growth of the cells; an in vitro wound healing model was used to count the number of cells that had entered the denudate area in RPE mig ration treated with HGF (1，2，10，50，100 mu;g/L) after 20 h. Results HGF(10,50,100 mu;g/L) increased proliferation rates of cultur ed human RPE (18.2 % to 34.8 %), and at a concentration of 50 mu;g/L on day 3 HGF induced the maximal increase of proliferation (Plt;0.01）; HGF showed effects on migration of 9.3 %, 113.0 %, 91.7 % and 50.3 % at the concentrations of 2,10,50, 100 mu;g/L, respectively. HGF stimulated t he best migratory response in RPE cells at a concentration of 10 mu;g/L (113 %). Conclusion HGF can induce the proliferation and obvious migration of RPE cells, consequently HGF was a mitogen and potent migratory factor for human cultured RPE cells. (Chin J Ocul Fundus Dis, 2001,17:307-310)
【Abstract】Objective To search for the drug that promots the hepatocyte regeneration after partial hepatectomy. Methods Quantitative morphometry technique, 3H-TdR in vivo test and arterial ketone body ratio (AKBR) were employed to evaluate the energy metabolism, DNA synthesis, and liver cell nuclear mitosis and hepatocyte regeneration of the residual liver tissue in rats after partial lobectomy when treated with human growth hormone (hGH) and placebo. Results After partial hepatectomy in the experimental group, liver cell nuclear mitosis，nuclear density, new liver cell numbers, AKBR and 3H-TdR in vivo test were much higher than those in the control group (P＜0.01 or P＜0.05). Conclusion hGH promotes the hepatocyte regeneration of the residual liver.
Objective To investigate the effects of human recombinant hepatocyte growth factor(rh-HGF) on the expression of c-Met in intima of allograft vessels after cardiac transplantation in rats. Methods Heterotopic heart transplantation were established in abdominal cavity with eighty Wistar rats and forty SD rats. Donors’ cardiac grafts from Wistar rats were transplanted to SD rats(allograft) or Wistar rats(isograft).Sixty recipient rats were divided into three groups, control group：20 Wistar rats were injected with normal saline 1ml/kg·d intraperitoneally after transplantation; cyclosporine A (CsA) group:20 SD rats were injected with CsA 5mg/kg·d intraperitoneally on operation day; rhHGF group:20 SD rats were injected with rh-HGF 500μg/kg·d and CsA 5mg/kg·d intraperitoneally on operation day. The cardiac grafts were harvested at the 15th day and 60th day after transplantation. The crosssection of vascular tissues were used for immunohistochemistrical staining of c-Met, and investigated the expression of c-Met messenger ribonucleic acid (mRNA ) in intima of allograft vessels by reverse transcriptionpolymerase chain reaction(RT-PCR). The pathologic changes of allograft coronary vessels were observed with histopathological method. Results The allograft coronary arteries showed minimal intimal thickening, the endothelium and internal elastic lamina remained almost intact in rh-HGF group after transplantation.The expression of c-Met and c-Met mRNA in intima of allograft vessels after transplantation in rhHGF group were significantly higher than those in CsA group and control group（expression of c-Met at 60d: 1.85±0.26 vs. 0.96±0.10, t=8.491,P=0.000;1.85±0.26 vs. 0.58±0.03, t=13.725,P=0.000; expression of c-Met mRNA at 60d: 192±0.22 vs. 0.88±0.07, t=11.940,P=0.000;1.92±0.22 vs. 0.42±0.02,t=19.206,P=0.000）. Conclusion rh-HGF may prevent the progression of cardiac allograft vasculopathy through upregulating the expression of c-Met to stimulate endothelial cell repair and growth.
Objective To investigate the application and advancement of hepatocyte t ransplantation ( HCT) .Methods Literatures about the advancement of HCT were reviewed and analyzed. Results There have been manynovel technologies and advancement s in the application of HCT. For example , gene modified cell can be used as seedcell , subcutaneous t ransplantation can be taken when combined with giant molecule material and the encap sulationpreconditioning technique can also carried before operation to improve the rate of survival. Conclusion With moreand deeper understanding of hepatocyte t ransplantation and the development of advanced techniques such as the application of giant molecule , HCT will be extensively used in the clinical t reatment of acute and chronic hepatic diseases.
Objective To study the effect of recombinant lentiviral vector mediated human hepatocyte growth factor (hHGF) gene-modified bone marrow mesenchymal stem cells (BMSCs) on the immunological rejection after allograft l iver transplantation in rats, and to reveal the mechanism of immune tolerance. Methods Eight male Sprague Dawley (SD)rats of clean grade (aged 3 to 4 weeks, weighing 75-85 g) were selected for the isolation and culture of BMSCs; 64 adult male SD rats of clean grade (weighing 200-250 g) were used as donors; and 64 adult male Wistar rats of clean grade (weighing 230-280 g) were used as receptors. After establ ishing a stable model of rat allogeneic l iver transplantation, 1 mL sal ine, 2 ×106/mL of BMSCs 1 mL, 2 × 106/mL of BMSCs/green fluorescent protein 1 mL, and 2 × 106/mL of BMSCs/hHGF 1 mL were injected via the portal vein in groups A, B, C, and D respectively. Then the survival time of the rats was observed. The hepatic function was determined and the histological observation of the l iver was performed. The hHGF mRNA expression was detected by RT-PCR, the level of cytokine including hHGF, interleukin 2 (IL-2), IL-4, IL-10, and interferon γ (IFN-γ) by ELISA assay, the level of apoptosis by TUNEL method, and the expression level of prol iferating cell nuclear antigen (PCNA) by immunohistochemical method. Results The survival time of group D was significantly higher than that of groups A, B, and C (P lt; 0.01); the survival time of groups B and C was significantly higher than that of group A (P lt; 0.01), but there was no significant difference between group B and group C (P gt; 0.05). RT-PCR demonstrated the transcription of hHGF mRNA in the grafts of group D; the serum cytokine hHGF reached to (6.2 ± 1.0) ng/mL. Compared with groups B and C, group D exhibited significant inhibitory effect, significantly improved l iver function, and showed mild acute rejection. In addition, the levels of cytokine IL-2 and IFN-γ decreased; the levels of cytokine IL-4 and IL-10 increased; the level of apoptosis reduced; and the expression level of PCNA increased. Except for the expression of IL-4 (P gt; 0.05), there were significant differences in the other indexes between group D and groups B, C (P lt; 0.05). Conclusion BMSCs/hHGF implanting to rat l iver allograft via portal vein can induce immune tolerance. Compared with injection of BMSCs alone, BMSCs/hHGF treatment can alleviate acute rejection and prolong the survival time significantly. The immunosuppressive effect of BMSCs/hHGF is correlated with Th2 shifts up of Th1/Th2 shift, reduced apoptosis, promoted l iver regeneration.
ObjectiveTo provide theoretical and technological support for further study of liver metabolism and disease by comparing the advantages and disadvantages of various artificial liver models (biological). MethodsLiteratures were searched and compared to summarize the requirement for liver donor, isolation, and culture of hepatocyte. ResultsIn the separation method of hepatocyte, mechanical separation method had no requirement for liver donor, and was easy to acquire hepatocyte, while the acquired hepatocyte would be destructed severely, and the survival rate was low. On the other hand, the restriction of the digestion of the hepatocytes to the liver cell samples was unlimited, while the key of the enzyme digestion method was to regulate the balance between enzyme concentration and digestion time, which was limited to function researches of hepatocyte, and research about the responds of hepatocyte against outside, and other few researches. Perfusion digestion method had been widely applied for animal test. The Ca2+, collagenase and perfusion rate, pH value, buffer, and intubation method all play vital roles. During the cultivation, we needed to choose different methods according to several experiments, and add different additives in the appropriate medium. Different biological reactors had different advantages, disadvantages, and applicable conditions. ConclusionsThe donor selection is based on various experimental purposes to harvest hepatocytes from different sources. Whether on the separation process or on the cultivation process, according to the specific circumstances, such as the concentration, perfusion time, and the choice of different kinds of culture medium, we can choose different kinds of bioreactors, but all kinds of methods are still remained with multiple insufficiencies, which require more researchers to improve.
Objective To establ ish a new induction method from human embryonic stem cells (hESCs) differentiating into hepatocyte-l ike cells using an adherent culture system with single-step induction. Methods Undifferentiated hESCs were cultured on Matrigel-coated culture plates for 4 days, hepatic differentiation was initiated at 60%-70% confluence by adding Activin A for 5 days. Then the induction medium was replaced by hepatocyte induction medium (HIM) supplemented with fibroblast growth factor 1 (FGF-1) and bone morphogenetic protein 4 (BMP-4) for another 6 days. Finally, the cells were treated with HIM adding hepatocyte growth factor (HGF) and Oncostatin M (OSM) for 5-7 days. The characteristics of differentiated cells were determined by morphology, immunofluorescence staining, RT-PCR, and Periodicacid- Schiff (PAS) test. Results Differentiated cells treated with Activin A, FGF-1, BMP-4, HGF, and OSM sequentially were morphologically larger and became spherical, oval or polygon. Some cells had 2 or 3 nuclei, suggesting that the cells have a hepatocyte-l ike morphology. Differentiated cells at first induction stage could be stained positive by SOX17 and Forkhead (FOX)A2 after induction by Activin A. Then they turned to be α fetoprotein (AFP) and α1 antitripsin positive cells at second induction stage after induction by FGF-1 and BMP-4. Finally, the differentiated cells treated with HGF and OSM showed PAS possitive for glycogen detection. The differentiated cells at various stages also expressed at early (SOX17, FOXA2, and GATA-4),middle (AFP, albumin, and cytokeratin 18), and mature (alcohol dehydrogenase 1C and Cytochrome P4501B1) stage hepatic genes, respectively. Conclusion Using a simple-step induction method and by suppl ied with cytokines consequently, hESCs can be induced to differentiate into hepatocyte-l ike cells. The differentiation method can provide seed cells for hepatic tissue engineering or cell-therapy.
【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.
To investigate the effect of hepatocyte growth factor (HGF) on prol iferation of cultured human eccrine sweat gland epithel ial cells (hESGc) and the involvement of phosphorylation of ERK1/2. Methods hESGc were cultured in keratinocyte serum free medium (KSFM) and the first generation of hESGc was harvested. The expression of C-met was detected by immunocytochemistry. MTT assay was used to detect the effect of HGF on the prol iferation of hESGc. The cells were divided into blank group, control group and experimental group. The culture density was 2 × 103 cells/hole in control group and experimental group. Two hundred μL KSFM with HGF in different levels was added to every hole. hESGcwere cultured in KSFM with HGF at different levels (2, 20, 40 and 80 ng/mL) in experimental group, in KSFM without HGF incontrol group, and in KSFM without HGF and no hESGc in blank group. The cell prol iferation was observed in xperimental group 2 and 4 days later. Western blot was used to detect the expression of phosphorylated ERK1/2 at 40 ng/mL HGF after 0, 5, 30, 90 and 120 minutes. Results The results were positive for anti-C-met staining in the cytoplasm. HGF (40 ng/mL and 80 ng/mL) significantly improved the prol iferation of hESGc (P lt; 0.05). When cultured in the KSFM with 40 ng/mL HGF, the cell prol iferation rate and the absorbance were 74.2%, 0.239 3 ± 0.070 9 at 2 days and 74.8%, 0.287 8 ± 0.074 3 at 4 days; showing significant differences when compared with control group (P lt; 0.05). When cultured in KSFM with 80 ng/mL HGF, the cell prol iferation rate and the absorbance were 54.5%, 0.212 3 ± 0.059 2 at 2 days and 40.3%, 0.231 0 ± 0.056 7 at 4 days; showing significant differences when compared with control group (P lt; 0.05). The expression of p-ERK1/2 reached to the maximum after stimulation of 40 ng/mL HGF for 5 minutes, and relative integral absorbance (RIA) was 0.593 2 ± 0.192 2, increased 8.1 times compared with instant stimulation (P lt; 0.01). Conclusion HGF could induce the prol iferation of hESGc and activate the phosphorylation of ERK1/2 protein.
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.