Objective To investigate the possible association between serum level of hepatocyte growth factor( HGF) and obstructive sleep apnea hypopnea syndrome( OSAHS) with hypertension.Methods 58 cases of OSAHS without hypertension, 61 cases of OSAHS with hypertension, and 50 normal controls were enrolled. Serum level of HGF was measured by enzyme-linked immunosorbent assay( ELISA) , and the relationships between the serum HGF level and blood pressure( BP) , apnea hypopnea index( AHI) , lowest SaO2 ( LSaO2 ) were analyzed by linear correlation analysis. Results The serum HGF level ( pg/mL) was 761. 46 ±60. 18, 970. 87 ±60. 94, and 487. 34 ±45. 52 in the OSAHS patients without hypertention, OSAHS patients with hypertention, and normal subjects, respectively. Which was significantly higher in the OSAHSpatients than the normal subjects, and highest in the OSAHS patients with hypertension( P lt; 0. 05) . The serum HGF level was positively related to AHI( r = 0. 452, P lt;0. 05) and negatively related to LSaO2 ( r =- 0. 328, P lt;0. 05) in the OSAHS patients without hypertention, positively related to AHI, SBP, DBP( r =0. 670, P lt;0. 01; r =0. 535, P lt;0. 05; r =0. 424, P lt;0. 05) and negatively related to LSaO2 ( r = - 0. 572,P lt;0. 01) in the OSAHS patients with hypertension. Conclusions SerumHGF level increases significantly in patients with OSAHS especialy in OSAHS patients with hypertension, and positively correlates with the severity of OSAHS and hypertension.
ObjectiveTo discuss the possibility of constructing injectable tissue engineered adipose tissue, and to provide a new approach for repairing soft tissue defects.MethodsHuman adipose-derived stem cells (hADSCs) were extracted from the lipid part of human liposuction aspirate by enzymatic digestion and identified by morphological observation, flow cytometry, and adipogenic induction. The hADSCs underwent transfection by lentivirus vector expressing hepatocyte growth factor and green fluorescent protein (HGF-GFP-LVs) of different multiplicity of infection (MOI, 10, 30, 50, and 100), the transfection efficiency was calculated to determine the optimum MOI. The hADSCs transfected by HGF-GFP-LVs of optimal MOI and being adipogenic inducted were combined with injectable fibrin glue scaffold, and were injected subcutaneously into the right side of the low back of 10 T-cell deficiency BALB/c female nude mice (transfected group); non-HGF-GFP-LVs transfected hADSCs (being adipogenic inducted) combined with injectable fibrin glue scaffold were injected subcutaneously into the left side of the low back (untransfected group); and injectable fibrin glue scaffold were injected subcutaneously into the middle part of the neck (blank control group); 0.4 mL at each point. Twelve weeks later the mice were killed and the implants were taken out. Gross observation, wet weight measurement, HE staining, GFP fluorescence labeling, and immunofluorescence staining were performed to assess the in vivo adipogenic ability of the seed cells and the neovascularization of the grafts.ResultsThe cultured cells were identified as hADSCs. Poor transfection efficiency was observed in MOI of 10 and 30, the transfection efficiency of MOI of 50 and 100 was more than 80%, so the optimum MOI was 50. Adipose tissue-like new-born tissues were found in the injection sites of the transfected and untransfected groups after 12 weeks of injection, and no new-born tissues was found in the blank control group. The wet-weight of new-born tissue in the transfected group [(32.30±4.06) mg] was significantly heavier than that of the untransfected group [(25.27±3.94) mg] (t=3.929, P=0.001). The mature adipose cells in the transfected group [(126.93±5.36) cells/field] were significantly more than that in the untransfected group [(71.36±4.52) cells/field] (t=30.700, P=0.000). Under fluorescence microscopy, some of the single cell adipocytes showed a network of green fluorescence, indicating the presence of GFP labeled exogenous hADSCs in the tissue. The vascular density of new-born tissue of the transfected group [(16.37±2.76)/field] was significantly higher than that of the untransfected group [(9.13±1.68)/field] (t=8.678, P=0.000).ConclusionThe hADSCs extracted from the lipid part after liposuction can be used as seed cells. After HGF-GFP-LVs transfection and adipose induction, the hADSCs combined with injectable fibrin glue scaffold can construct mature adipose tissue in vivo, which may stimulate angiogenesis, and improve retention rate of new-born tissue.
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.
【摘要】目的评价骨髓间充质干细胞(BMSCs)向肝样细胞诱导的可行性。方法2008年1月2009年1月,以肝细胞生长因子(HGF) 20 ng/mL,成纤维细胞生长因子4(FGF4) 10 ng/mL为诱导剂,从细胞形态变化,并通过RTPCR、免疫组化方法分别对诱导第7、14、21及28天的细胞进行白蛋白(ALB)、甲胎蛋白(AFP)、细胞角蛋白18(CK18)等检测。人L02肝细胞及未诱导的BMSCs分别为阳性和阴性对照结果BMSCs诱导7 d出现类圆形或多角形细胞,并出现铺路石样结构;诱导14 d细胞呈现典型的铺路石状;诱导21 d,同前;诱导28 d,细胞排列紊乱,局部细胞的形态不规则、细胞边界不清。BMSCs诱导第7、14、21天ALB、CK18、AFP等mRNA表达阳性;未诱导BMSCs均为阴性;肝细胞ALB、CK18、AFP等mRNA表达均阳性。免疫细胞化学检测结果同RTPCR。结论以HGF及FGF4为主的诱导体系可有效诱导BMSCs向肝样细胞转化,BMSCs可以作为一种新的肝细胞来源。
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 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 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.
【Abstract】ObjectiveTo explore the effect of hepatocyte growth factor/scatter factor (HGF/SF) on apoptosis of colorectal cancer cells induced with curcumin. MethodsMTT assay was used to evaluate the cytotoxicity of curcumin to colorectal cancer cells. Flow cytometry was used to detect the antiapoptosis effect of HGF. ResultsFlow cytometry showed only 64 μg/ml curcumin could play the proliferationinhibiting role in Caco-2 cells leading to their apoptosis; at the same time, different concentrations of HGF could antagonize this inhibitory effect resulting in the decrease of apoptosis, but HGF worked without a concentration-dependent manner. The study on MAPK pathway showed that the protective effect of HGF on the apoptosis of Caco-2 cells was not influenced by inhibiting p42/p44 MAPK and p38 MAPK pathway. ConclusionHGF/SF antagonizes the apoptosis of Caco-2 cells induced with curcumin, but MAPK signaling pathway might not participate in this process.
ObjectiveTo study the expression of c-Met in colorectal carcinoma cells and the effect of hepatocyte growth factor (HGF) on proliferation and invasion of colon carcinoma cells SW480. MethodsReal-time PCR and Western blot methods were respectively used to detect the expressions of c-Met mRNA and protein in the different colorectal carcinoma cells in order to screen the high c-Met expression cells. The SW480 cells were incubated with different concentrations (0, 20, 40, and 70 ng/mL) HGF. MTT assay and Transwell test were used to evaluate the effects of proliferation and invasion in the SW480 cells. Results①The c-Met was expressed in each colorectal carcinomar cells, especially highly expressed in the colon carcinoma cells SW480 in vitro.②MTT assay showed that the HGF could promote the proliferation of SW480 cells in a dose-dependent manner with some extent.③Transwell test showed that the HGF could increase the invasion of SW480 cells. ConclusionThe c-Met is highly expressed in colorectal carcinoma cells and HGF could promote proliferation and increase invasion of colorectal carcinoma cells in vitro.
This research aims to construct a lentiviral expression vector carrying the extracelluar domain (ED) of human hepatocyte growth factor receptor (C-Met), and to express it in transfected 293T cells. The extracellular domain of C-Met was amplified by RT-PCR, ligated with lentiviral expression vector p RRL-CMV-ED, and then expressed in 293T cell line. The expressed protein was purified and identified by RT-PCR and Western blot. The enzyme digestion and sequence analysis showed that the lentiviral expression vector p RRL-CMV-ED was constructed correctly. The size of amplified genes was about 2 700 bp. The purified protein with Ni-affinity column was about 105 kD analyzed by SDS-PAGE. The Western blot and ELISA results showed that the expressed protein which could bind to HGF specifically was the extracelluar domain of human hepatocyte growth factor receptor. This research may lay a foundation for further study of anti-C-MET monoclonal antibody and neutralizing antibody.