摘要:目的: 探讨选择性内皮素A受体拮抗剂BQ123对人喉癌Hep2细胞裸鼠种植瘤的生长及血管形成的影响。 方法 :将实验动物裸鼠随机分为3组:BQ123［n =8,2mg/(kg·day)］、氟尿嘧啶组［n =8,2mg/(kg·day)］、生理盐水组(n =8),比较各组裸鼠成瘤体积、微血管密度(MVD)。 结果 :BQ123组肿瘤体积为(162±053)cm3,明显小于生理盐水组及氟尿嘧啶组,差异具有统计学意义;BQ123组的肿瘤组织中MVD高倍镜下为232,明显低于生理盐水组(586)及氟尿嘧啶组(395),差异具有统计学意义。 结论 :BQ123对人喉癌Hep2细胞在裸鼠体内有明显抑瘤作用,肿瘤的体积、肿瘤组织MVD显著低于对照组,表明BQ123可通过抑制肿瘤血管生成而显著抑制肿瘤生长。Abstract: Objective: To study the effects of endothelin A receptor blockade BQ123 on the implanted human laryngeal carcinoma angiogenesis of nude mouse. Methods : From March 2008 to July 2009, 24 Balb/c nude mice were randomly divided into three groups: BQ123 group ［〖WTBX〗n =8, BQ123 at 2mg/(kg·day)］, 5Fu group ［〖WTBX〗n =8, fluorouracil at 2mg/(kg·day)］ and the control group (〖WTBX〗n =8, normal saline). The carcinoma volume and microvascular density of each group were compared. Results : The tumor size of BQ123 group, which was (162±053)cm3 in average, was significant smaller than the tumor sizes of the other two group s. The average microvascular density score of the tumors in BQ123 group was 232 per hyper power len (HP), which was also significantly less than the average scores of control groups (586 and 395 respectively). Conclusion : Nude mouse experiments show that the carcinoma volume and microvascular density of BQ123 group are significantly lower than those of the control groups. BQ123 inhibits the growth of carcinoma by its inhibition of carcinoma angiogenesis.
【Abstract】Objective To introduce the possible effect of endogenous angiogenesis inhibitive factors in the therapy of hepatocarcinoma. Methods Recent relevant literatures were reviewed. ResultsEndogenous angiogenesis inhibitive factors can suppress the growth of tumor blood vessels, which might head off the development and metastasis of hepatocarcinoma effectively. This might provide a new approach to the therapy of hepatocarcinoma. ConclusionRecent studies on endogenous angiogenesis inhibitive factors will be helpful in the prevention and treatment of hepatocarcinoma.
Objective To explore the effects of adipose-derived stem cell released exosomes (ADSC-Exos) on the proliferation, migration, and tube-like differentiation of human umbilical vein endothelial cells (HUVECs). Methods Adipose tissue voluntarily donated by liposuction patients was obtained. The ADSCs were harvested by enzyme digestion and identified by flow cytometry and adipogenic induction. The ADSC-Exos were extracted from the supernatant of the 3rd generation ADSCs and the morphology was observed by transmission electron microscopy. The surface proteins (Alix and CD63) were detected by Western blot. The nanoparticle tracking analyzer NanoSight was used to analyze the size distribution of ADSC-Exos. After co-culture of PKH26 fluorescently labeled ADSC-Exos with HUVECs, confocal microscopy had been used to observe whether ADSC-Exos could absorbed by HUVECs. ADSC-Exos and HUVECs were co-cultured for 1, 2, 3, 4, and 5 days. The effect of ADSC-Exos on the proliferation of HUVECs was detected by cell counting kit 8 (CCK-8) assay. The expression of VEGF protein in the supernatant of HUVECs with or without ADSC-Exos had been detected by ELISA after 12 hours. Transwell migration assay was used to detect the effect of ADSC-Exos on the migration ability of HUVECs. The effect of ADSC-Exos on the tubular structure formation of HUVECs was observed by Matrigel experiments in vitro. The formation of subcutaneous tubular structure in vivo was observed in BALB/c male nude mice via the injection of HUVECs and Matrigel with or without ADSC-Exos. After 2 weeks, the neovascularization in Matrigel was measured and mean blood vessel density (MVD) was calculated. The above experiments were all controlled by the same amount of PBS. Results After identification, the cultured cells were consistent with the characteristics of ADSCs. ADSC-Exos were circular or elliptical membranous vesicle with uniform morphology under transmission electron microscopy, and expresses the signature proteins Alix and CD63 with particle size ranging from 30 to 200 nm. Confocal microscopy results showed that ADSC-Exos could be absorbed by HUVECs. The CCK-8 analysis showed that the cell proliferation of the experimental group was better than that of the control group at each time point (P<0.05). The result of Transwell showed that the trans-membrane migration cells in the experimental group were significantly more than that in the control group (t=9.534, P=0.000). In vitro, Matrigel tube-forming experiment showed that the number of tube-like structures in the experimental group was significantly higher than that of the control group (t=15.910, P=0.000). In vivo, the MVD of the experimental group was significantly higher than that of the control group (t=16.710, P=0.000). The ELISA assay showed that the expression of VEGF protein in the supernatant of the experimental group was significantly higher than that of the control group (t=21.470, P=0.000). Conclusion ADSC-Exos can promote proliferation, migration, and tube-like structure formation of HUVECs, suggesting that ADSC-Exos can promote angiogenesisin vitro and in vivo.
Objective To investigate the effects of the MKN-45 gastric cancer cell exosomes carrying microRNA-552 (miR-552) on the proliferation, migration, and angiogenesis of human umbilical vein endothelial cells (HUVEC). Methods ① The MKN-45 cells were divided into MKN-45 blank control group (no transfection), MKN-45 miR-552 inhibitor group [transfection of plasmid inhibiting mir-552 expression (mir-552 inhibitor plasmid)], and MKN-45 negative control group [transfection of negative control plasmid (empty plasmid)], the exosomes were extracted, purified, and identified. Western blotting was used to detect the protein expression of exosomal markers [CD63, CD9, and tumor susceptibility gene 101 (TSG101)]. ② The HUVEC cells were divided into HUVEC control group (added PBS), HUVEC-exosome group (co-cultured with exosomes of MKN-45 cell), HUVEC-negative control exosome group (co-cultured with exosomes of MKN-45 cell transfected with negative control plasmid), and HUVEC-miR-552 inhibitor exosome group (co-cultured with exosomes of MKN-45 cell transfected with miR-552 inhibitor plasmid), exosomes tracing experiment was used to detect whether exosomes entered HUVEC cells. Real-time fluorescent quantitative PCR method was used to detect the expression of miR-552, the MTT method was used to detect the proliferation of HUVEC cells, the Transwell chamber method was used to detect the migration of HUVEC cells, the angiogenesis test was used to detect the angiogenesis ability. Results This study successfully extracted exosomes from MKN-45 gastric cancer cells. Observed by transmission electron microscope, the exosomes were all round or elliptical, with a diameter of 100–150 nm, and the exosomal vesicle structure could be seen. Western blotting detection showed that the surface markers of exosomes (CD63, CD9, and TSG101 protein) were expressed in exosomes. The results of the tracing experiment showed that exosomes derived from MKN-45 cells were successfully internalized by HUVEC cells. After MKN-45 cells were transfected with miR-552 inhibitor plasmid, compared with the MKN-45 blank control group and MKN-45 negative control group, the relative expression level of miR-552 in the exosomes decreased (P<0.05). Compared with the HUVEC control group, the cell proliferation rate at 24, 48 and 74 h increased, as well as number of migration, tubule formation nodes, and relative expression level of miR-552 in the HUVEC-exosomes group increased (P<0.05). Compared with the HUVEC-negative control exosome group, the cell proliferation rate at 24, 48 and 74 h decreased, as well as the number of migration, tubule formation nodes, and relative expression level of miR-552 in the HUVEC-miR-552 inhibitor exosome group decreased (P<0.05). Conclusion The exosomes of gastric cancer cells carrying miR-552 can significantly promote the proliferation, migration, and angiogenesis of HUVEC cells.
ObjectiveTo find the role of oncogene cmet and suppressor gene p53 in the process of tumor angiogenesis and their clinical significance. MethodsBy immunohistochemical method and computer image analysis technique, microvessel count and cmet, p53 protein expression were quantitatively determined in 80 cases of breast carcinoma and 20 cases of breast fibroadenoma. ResultsThe high microvessel count and the positive expression of cmet, p53 were significantly correlated with histologic grade, lymph node metastasis and the stage of the tumor (P＜0.01). The high microvessel count was significantly correlated with the positive expression of cmet and p53 (P＜0.01).ConclusionBoth oncogene cmet and suppressor gene p53 modulate tumor angiogenesis of breast carcinoma.
ObjectiveTo investigate the effect of transverse tibial bone transport on the expression of angiogenesis-related growth factors in the serum of diabetic foot patients.MethodsBetween January 2018 and December 2018, 10 patients who suffered from diabetes mellitus accompanied with Wagner stage 4 diabetic foot underwent transverse tibial bone transport. There were 5 males and 5 females with an average age of 59.2 years (range, 51-70 years). The duration of diabetes was 2-60 months, with an average of 24.2 months. The duration of diabetic foot was 30-120 days, with an average of 54.1 days. Peripheral venous blood was taken at 1 day before operation and at 1, 4, 11, 18, 28, and 35 days after operation. The serum was centrifuged and subjected to ELISA test to detect the expression levels of serum vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF).ResultsThe levels of serum VEGF, bFGF, and EGF increased rapidly at 11 days after operation, and the expression levels of the factors at 11, 18, 28, and 35 days were significantly higher than those before operation (P<0.05). The expression level of PDGF increased suddenly at 18 days after operation, and the expression level of PDGF at 18, 28, and 35 days was significantly higher than that before operation (P<0.05).ConclusionTransverse tibial bone transport for the treatment of diabetic foot can significantly increase the expression of serum angiogenesis-related growth factors in early stage, which may be the mechanism of promoting the healing of diabetic foot wounds.
Tenascin-C (TNC) is an extracellular matrix glycoprotein, which is usually highly expressed in embryonic tissues and tumor tissues, but is not expressed or just lowly expressed in mature tissues. TNC is involved in various complex signaling pathways during tumor metastasis, especially through modulating FAK, RhoA, Wnt and Notch pathways by interacting with syndecan-4, integrinα5β1, matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF). As a result, TNC affects epithelial mesenchymal transition, tumor cell adhesion, proliferation and angiogenesis, which eventually enhances the invasion and metastasis ability of many tumors. Further studies have demonstrated that TNC could be used as prognosis or metastasis marker of patients with malignant tumor.
Objective To study the expression of inducible nitric oxide synthase (iNOS)，endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) in human gastric cancer and their relationship with tumor angiogenesis and to investigate the interaction of NOS and VEGF in gastric cancer. Methods The expression and distribution of VEGF, iNOS and eNOS in 34 gastric cancer specimens were detected with immunohistochemistry. Microvessel density (MVD) was counted with FⅧRAg immune specific staining. Results The expression rates of iNOS, eNOS and VEGF in 34 gastric cancers were 73.5%, 82.4% and 91.2% respectively. The expression of VEGF had a significant positive relation with iNOS, but not with eNOS. The MVDs of VEGF or iNOS positive gastric cancers were obviously higher than those of VEGF or iNOS negative gastric cancers. There was no significant difference between the MVDs of eNOS positive gastric cancers and eNOS negative ones. Conclusion MVD increases with increase of expression of VEGF and iNOS in gastric cancer. It is indicated that VEGF and iNOS can promote gastric cancer angiogenesis. VEGF and iNOS have a significant positive correlation, which suggests that in human gastric cancer, iNOS plays an important role in the production and action of VEGF.
ObjectiveTo investigate the in vitro effect of pseudolaric acid B (PAB) on apoptosis of protoscolece cells and its regulatory effects on angiogenesis and cell apoptosis in the the lesion-host microenvironment tissue in vivo, as well as its possible mechanisms, in order to provide a basis for the clinical development of new alternative drugs for Echinococcus multilocularis. MethodsIn vitro experiments: the protoscoleces, vesicles, germinal cells, human foreskin fibroblasts (HFFs) and normal human liver cells were treated with different concentrations of PAB (0, 2.5, 5, 10, 20, 40, 80, 160 and 320 μmol/L) for 7, 5, 5, 5 and 5 days, then evaluated the survival rate of the protoscoleces, the release level of phosphoglucose isomerase (PGI) from the vesicles, the viability of the germinal cells, as well as the viability of HFFs and normal human liver cells. The protoscoleces and vesicles were fixed with 2.5% glutaraldehyde and used for scanning electron microscopy and transmission electron microscopy observation. Animal experiments: the protoscoleces were isolated from the abdominal lesions of the protected gerbils, and then infected 18 C57BL/6J mice by intraperitoneal injection to establish models, dividing into 3 groups with 6 mice in each group. The model group was given 0.3 mL of PBS by gavage daily, the albendazole (ABZ) group was given 0.3 mL ABZ (100 mg/kg) daily by gavage, the PAB group was given 0.3 mL of PAB (40 mg/kg) by gavage daily. After continuous gavage for 6 weeks, the lesion host microenvironment tissue was taken and ELISA was used to detect the expression levels of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS) and cysteinyl aspartate specific proteinase3 (caspase3), the expression levels of nitric oxide (NO) was detected using a biochemical detection kit, Western blot was used to detect the expression levels of BCL2-associated X protein (Bax), B-cell lymphoma-2 (Bcl2), caspase3, cleaved-caspase3, VEGF, vascular endothelial growth factor receptor 2 (VEGFR2), phosphatidylinositol 3 kinase (PI3K), phosphorylated PI3K (p-PI3K), protein kinase B (AKT) and phosphorylated AKI (p-AKT) protein. ResultsIn vitro experiments: the protoscoleces of Echinococcus multilocularis were cultured with different concentrations of PAB for 7 days in vitro, the protoscoleces of 40, 80, 160 and 320 μmol/L group all died after 6, 4, 2 and 1 day, respectively; PAB exhibited a certain time and concentration dependence on the protoscoleces of Echinococcus multilocularis. After PAB treatment, the release of PGI in culture supernatant of Echinococcus multilocularis gradually increased with the increase of PAB concentration [concentration for 50% of maximal effect value was (24.40±1.42) μmol/L], the vitality of germinal cells was significantly inhibited [half maximal inhibitory concentration value was (15.94±2.55) μmol/L]. PAB had no significant toxicity to mammalian cells. When 20 μmol/L PAB intervention in the protoscoleces for 3 days, the expression levels of Bax and caspase3 proteins were upregulated, while the expression level of Bcl2 protein was downregulated. Animal experiments: compared with the model group, the wet weight of lesions in the PAB and ABZ groups decreased (P<0.01), and the inhibition rates of lesion growth in the PAB and ABZ groups were 91.03% and 74.44%, respectively. The expression of proliferation and angiogenesis indicators (Ki67, CD34, VEGF, VEGFR2, eNOS, NO) were downregulated in the lesion host microenvironment tissues of mice in the ABZ and PAB groups (P<0.05), while the expression of apoptosis related proteins (caspase3, cleaved-caspase3 and Bax) were upregulated and the expression of PI3K/AKT signaling pathway related proteins (p-PI3K and p-AKT) were downregulated (P<0.05). ConclusionPAB has a strong in vitro and in vivo effect against Echinococcus multilocularis, and its mechanism may be related to the inhibition of PI3K/AKT signaling pathway, leading to increased apoptosis and decreased angiogenesis.
ObjectiveTo study the local vascular remodeling, inflammatory response, and their correlations following acute spinal cord injury (SCI) with different grades, and to assess the histological changes in SCI rats.MethodsOne hundred and sixteen adult female Sprague Dawley rats were randomly divided into 4 groups (n=29). The rats in sham group were received laminectomy only. A standard MASCIS spinal cord compactor was applied with drop height of 12.5, 25.0, or 50.0 mm to establish the mild, moderate, or severe SCI model, respectively. Quantitative rat endothelial cell antigen 1 (RECA1) and CD68 positive areas and the correlations were studied by double immunofluorescent (DIF) staining at 12 hours, 24 hours, 3 days, 7 days, and 28 days following SCI. Moreover, qualitative neurofilament-H (NF-H) and glial fibrillary acidic protein (GFAP) positive glial cells were studied by DIF staining at 28 days. ELISA was used to detect the levels of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-6 in spinal cord homogenates at 12 hours, 24 hours, and 3 days, and the correlations between TNF-α, IL-1β, or IL-6 levels and microvascular density (RECA1) were accordingly studied. Moreover, the neural tissue integrity and neuron damage were assessed by HE staining at 12 hours, 24 hours, 3 days, 7 days, and 28 days, and Nissl’s staining at 28 days following SCI, respectively.ResultsDIF staining revealed that the ratio of RECA1 positive area was the highest in moderate group, higher in mild and severe groups, and the lowest in sham group with significant differences between groups (P<0.05). The ratio of CD68 positive area was the highest in severe group, higher in moderate and mild groups, and the lowest in sham group with significant differences between groups (P<0.05), except the comparisons between mild and moderate groups at 24 hours and 28 days after SCI (P>0.05). There was no significant correlation between the RECA1 and CD68 expressions in sham group at different time points (P>0.05). At 12 and 24 hours after SCI, the RECA1 and CD68 expressions in mild and moderate groups showed significant positive correlations (P<0.05), while no significant correlation was found in severe group (P>0.05). No significant correlations between the RECA1 and CD68 expressions was shown in all SCI groups at 3 days and in severe group at 7 days (P>0.05), while the negative correlations were shown in mild and moderate groups at 7 days, and in all SCI groups at 28 days (P<0.05). In mild, moderate, and severe groups, the axons became disrupted, shorter and thicker rods-like, or even merged blocks with increased injury, while the astrocytes decreased in number, unorganized and condensed in appearance. ELISA studies showed that TNF-α, IL-1β, and IL-6 levels in sham group were significantly lower than those in other 3 groups at different time points (P>0.05). The differences in TNF-α, IL-1β, and IL-6 levels between SCI groups at different time points were sinificant (P<0.05), except IL-1β levels between the mild and moderate groups at 12 hours (P>0.05). Three inflammatory factors were all significantly correlated with the microvascular density grades (P<0.05). Histological analysis indicated that the damage to spinal cord tissue structure correlated with the extent of SCI. In severe group, local hemorrhage, edema, and infiltration of inflammatory cells were found the most drastic, the grey/white matter boundary was disappeared concurrently with the formation of cavity and shortage of normal neurons.ConclusionIn the acute stage following mild or moderate SCI, progressively aggravated injury result in higher microvessel density and increased inflammation. However, at the SCI region, the relation between microvessel density and inflammation inverse with time in the different grades of SCI. Accordingly, the destruction of neural structures positively relate to the grades of SCI and severity of inflammation.