【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 study the effects of hepatocyte growth factor (HGF) and receptor c-met on the development of primary breast carcinoma, and the relationship between it and prognosis. Methods The study of HGF and c-met related to breast carcinoma was reviewed by history document and experimental study in recent years. Results HGF is a growth factor which has mitogenic, migrating, invasive and angiogenic activities in breast carcinoma cells. The carcinogenic mechanism of breast carcinoma was more clear with the discovery of the relationship between HGF and its receptor c-met. Conclusion The HGF/c-met plays an important role in the generation and progress of breast carcinoma. Studying the effects of HGF/cmet on breast carcinoma is significant in guiding clinical treatment.
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.
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.
Objective To construct lentiviral vector carrying the human hepatocyte growth factor (hHGF) gene, and then to get hHGF gene/modified bone marrow mesenchymal stem cells (BMSCs) by infecting the BMSCs. Methods The hHGF gene was obtained with PCR from pcDNA-hHGF plasmid. The recombination lentiviral vector plasmid hHGF was constructed with Age I digestion and gene recombinant, then was identified with PCR and sequencing. Mediated by Lipofectamine2000, the three plasmids system of lentiviral vector including pGC-E1-hHGF, pHelper 1.0, and pHelper 2.0 was co-transfected to 293T cells to produce hHGF gene. The supernatant was collected and concentrated by ultracentrifugation and the titer of lentivirus was measured by real-time quantitative PCR. The BMSCs were infected by the constructed lentivirus and the multipl icities of infection (MOI) was identified with fluorescent microscope, the efficiency of infection with flow cytometry (FCM) analysis, the hHGF level with ELISA analysis, and the expression of hHGF gene with RT-PCR. Results Lentiviral vector carrying hHGF gene was constructed successfully. The titer of lentivirus was 1 × 108 TU/mL. The infection efficiency of BMSCs by hHGF lentiviral was high and reached 98% by FCM, and the best MOI was 10. A great mount of green fluorescence was observed with the fluorescent microscope at 28 days after infection. Peak concentration of hHGF secreted by BMSCs/hHGF reached 40.5 ng/mL at 5 days. The concentration could maintain a high level until 28 days after infection. RT-PCR showed that BMSCs/hHGF could express hHGF gene. Conclusion By lentiviral vector, hHGF gene was integrated into BMSCs genome, and it can express stably.
Abstract： Objective To investigate the effects of hepatocyte growth factor（HGF）gene transfected bone marrow mesenchymal stem cells （MSCs）transplantation in pigs with chronic ischemic heart disease. Methods MSCs were isolated from pig bone marrow by density gradient centrifugation and adherent cell culture, purified, and determined by cellsurface antigens（CD34, CD44, CD71, Ⅷ factor and desmin）. MSCs were transfected by adenovirus expressing hepatocyte growth factor（AdHGF）, and the influence of HGF on the biological characteristics of MSCs was tested. The pig model of chronic myocardial ischemia was established by placing Ameroid ring inside the left circumflex coronary artery via leftthoracotomy. A total of 40 pigs were randomly divided into 5 groups （n=8） and were injected 5×106/ml MSCs＋ 4×109 pfu 200 μl AdHGF （MSCs＋ AdHGF group）, 4×109 pfu 200 μl AdHGF （AdHGF group）, 5×106/ml MSCs 200 μl（MSCs group），4×109 pfu 200 μl AdNull （AdNull group）and 1 ml saline（control group） into the ischemic myocardiumrespectively. Echocardiogram, digital subtraction angiography （DSA） of coronary artery, single photon emission computed tomography（SPECT） myocardial perfusion imaging and cardiomyocyte apoptosis were examined after 4 weeks. Results Positive CD44 and CD71 and negative CD34, Ⅷ factorand desmin were detected in MSCs by flow cytometer. HGF had a b influence on stimulating the proliferation and differentiation of MSCs. Echocardiogram examination showed that left ventricular end-diastolic volume（LVEDV），left ventricular ejection fraction（LVEF），fractional shortening（FS）of MSCs＋ AdHGF group were significantly increased after treatment （P＜ 0.05）. DSA detection showed that ischemic neovascularization of MSCs＋ AdHGF group was significantly higher than those of AdHGF group and MSCs group （P＜ 0.05）. SPECT showed that the left ventricular myocardium of MSCs＋ AdHGF group appeared thickened,myocardial perfusion was significantly improved and the myocardial motion was significantly increased （P＜ 0.05）. Vascular density of MSCs＋ AdHGF group was significantly higher than those of AdHGF group and MSCs group by HE stain of myocardium ［（39.4±1.2）/ HPF vs. （36.5±1.4）/ HPF and（34.5±1.7）/ HPF，P＜ 0.05］. Cardiomyocyte apoptosis rate of MSCs＋ AdHGF group was significantly lower than those of AdHGF group and MSCs group by TUNEL stain （P＜ 0.05）. Conclusion Combination transplantation can promote the angiogenesis of chronic ischemic myocardium, inhibit cardiomyocyte apoptosis and improve heart function in pigs with chronic ischemic heart disease. The effect of HGF gene transfected MSCs transplantation is better than that of MSCs or HGF transplantation alone.
Objective To determine the concentration of hepatocyte growth factor (HGF) in vitreous of diabetic retinopathy (DR) and investigate the result and influence of HGF in neovascularization in proliferative diabetic retinopathy (PDR). Methods The high sensitive sandwich enzyme-linked immunosorbent assay (ELISA) was used to measure the level of HGF in vitreous of normal group for 10 eyes, simple DR group for 7 eyes, PDR without iridal neovascularization group for 24 eyes, PDR with neovascularization of iris group in 9 eyes, other retinopathy group associated with neovascularization for 8 eyes. Results The mean value of HGF level in vitreous of the former groups was: (3.3 ±1.9) μg／L in normal group; (4.8±2.5) μg／L in simple DR group; (13.0 ±5.2) μg／L in PDR without iridal neovascularization group; (18.6±7.2) μg／L in PDR with neovascularization of iris group；(12.1±8.9) μg／L in other retinopathy associated with neovascularization group. Stastistics showed that HGF level in PDR group and other retinal diseases associated with neovascularization were significantly higher than that in normal group (P＜0.01), PDR with neovascularization of iris group showed a higher mean vitreous HGF concentration than those in PDR without iridal neovascularization and simple DR group (P＜0.05 or P＜0.01). Conclusion Our results indicate that vitreous HGF may play an important role in retinal neovascularization in PDR and other retinal diseases. (Chin J Ocul Fundus Dis, 2002, 18: 131-133)
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.
ObjectiveTo study the protective effect of hepatocyte growth factor(HGF) on grafted mucous membrane of transplanted small bowel.MethodsTotal small bowel transplantation was made in inbred Wistar (RT1k) rats heterotopically,either total parenteral nutrition (control group,n=10) or hepatocyte growth factor supplemented TPN (HGF group,n=10) was given to the recipients from the 2nd day to the 10th day postoperatively. Morphological parameters of the transplanted intestinal mucosa, such as mucosal villous height, villous width, crypt depth, mucosal thickness and villous surface area were observed. Variation of ultrastructure of transplanted epithelial cells was observed. Composition of mucosal protein and DNA content were tested. ResultsComparison between HGF group and the control group were as follows. Mucosal villous height (298.79±22.31） μm vs （176.45±14.62） μm, P=0.001, villous width (107.46±12.34） μm vs （74.20±16.85） μm, P=0.004, crypt depth (104.56±11.17） μm vs （74.45±8.34） μm, P=0.000 5, mucosal thickness (409.53±35.83） μm vs （259.38±24.65） μm, P=0.003, and villous surface area (0.101±0.011) mm2 vs (0.041±0.005) mm2, P=0.001 were found significantly increased in HGF group compared with control group, there were no obvious difference decrease as compared to pretransplant parameters.Mucosal protein composition was higher in HGF group than that in control group （89.65±9.28) mg/g wet wt vs (53.73±11.45) mg/g wet wt, P=0.012, baseline (92.64±10.52) mg/g wet wt, nearly equal to baseline; DNA composition was also high in HGF group （0.89±0.09) mg/g wet wt vs (0.51±0.06) mg/g wet wt, P=0.008, baseline (0.91±0.09) mg/g wet wt. Nearly normal ultrastructure of the graft was maintained in HGF group, atrophied microvilli and broken mitochondrial crista were observed in control group.ConclusionHepatocyte growth factor can improve mucosal structure, preserve enterocyte ultrastructure of isograft after small bowel transplantation in rat.
Objective To solve the shortage of hepatocytes for l iver tissue engineering, to explore the possibil ity of prol iferation of rat bone marrow mesenchymal stem cells (BMSCs) and the feasibil ity of differentiation of BMSCs into hepatocyteswith a culture system containing cholestatic rat serum and hepatocyte growth factor (HGF) in vitro. Methods Myeloid cellsof femur and tibia were collected from the female healthy Wistar rats at the age of 6 weeks, the BMSCs were isolated, purified and identified. Normal and cholestatic rat serum were prepared from 40 healthy Wistar rats at the age of 12-14 weeks. The 3rd passage of BMSCs were harvested and added different cultures according to the following grouping: group A, DMEM plus 10%FBS; group B, hepatocyte growth medium (HGM) plus 5%FBS; group C, HGM plus 5% normal rat serum; group D, HGM plus 5% cholestatic rat serum; group E, HGM plus 5% cholestatic rat serum plus 25 μg/L HGF. The changes of cell morphology were observed, MTT assay was used to measure cell growth; the expression of alpha-fetoprotein (AFP) and cytokeratin 18 (CK18) were detected by immunocytochemistry; the glycogen deposit was examined by periodic acid-schiff (PAS) staining; and the urea content in culture supernatant was determined by glutamate dehydrogenase. Results Polygonal cells and binuclear cells were observed in groups D and E, while the shapes of cells in groups A, B, and C did not obviously change. The cell growth curve demonstrated that the speed of cells proliferation in group C was the fastest, the one in group B was the slowest; showing significant differences when compared with groups A, D, and E (P lt; 0.05). On the 7th day in groups D and E, the positive expressions of AFP and CK18 emerged, on the 14th day the positive expression of glycogen emerged. At the same period, the expression ratio was higherin group E than in group D (P lt; 0.05). The urea concentration increased gradually with induction time in groups D and E, the concentration was higher in group E than in group D (P lt; 0.05). No expressions of AFP, CK18, glycogen, and change of the urea concentration were observed in groups A, B, and C. Conclusion Normal rat serum can obviously promote the growth of BMSCs; cholestatic rat serum which promote the growth of BMSCs can induce to differentiate into hepatocyte; and a combination of cholestatic serum and HGF can increase the differentiation ratio.