OBJECTIVE: To study the expression of type I collagen and its receptor system-integrin alpha 2 beta 1 in different passages of osteoblasts. METHODS: The expression of type I collagen and integrin alpha 2 beta 1 in the primary, sixth and fifteenth passage of osteoblasts were detected by S-P immunohistological staining technique, and their mRNA expression by quantity RT-PCR technique. RESULTS: Type I collagen and integrin alpha 2 beta 1 were expressed in different passages of osteoblasts and there was no significant difference among three passages by immunohistological technique. Their mRNA expression was gradually decreased with subculture. CONCLUSION: Type I collagen promotes the adhesion and phenotype expression of osteoblasts through its receptor-integrin alpha 2 beta 1. The reductive expression of type I collagen-receptor system will decline the phenotype of osteoblasts.
【Abstract】Objective To observe the changeable expressions of vascular endothelial growth factor (VEGF) and integrin β3 during the angiogenetic process of granulation tissue. Methods mRNA and protein of VEGF and integrin β3 in human normal subcutaneous tissue, proliferative granulation tissue and mature granulation tissue were observed by RT-PCR and immunohistochemistry staining. Results The expressions VEGF and integrin β3 were low in normal subcutaneous tissue and were much higher in proliferative granulation tissue. When the granulation tissue was mature, the expression was decreased again. Conclusion VEGF and integrin β3 are important regulating factors in ngiogenesis.
Objective To study relationship between integrins and carcinogenesis, development, treatment or prognosis of gastric cancer. Methods The literatures about integrins and gastric cancer in recent years were reviewed and analyzed. Results The current study found that the β1 subunit integrins and αν subline integrins are closely associated with the gastric cancer. The β1 subunit integrins are associated with the invasion and metastasis of the gastric cancer, the αν subline integrins are associated with the typing, grading, and staging of the gastric cancer, and the ανβ3, ανβ5 and ανβ6 are associated with the prognosis of the gastric cancer, further more, the ανβ6 could be used as an independent effective prognostic factor. Conclusions Integrins are associated with occurrence, development, treatment, and prognosis of gastric cancer. It′s mechanism such as signal transduction pathway is not completely clarified. With further in-depth research, it′s molecular mechanism would be gradually elucidated and provide new ideas and methods for diagnosis, treatment, and prognosis of gastric cancer.
OBJECTIVE: To investigate the selection and identification of human keratinocyte stem cells(KSC) in vitro. METHODS: According to the characteristics of KSC which can adhere to extracellular matrix very fast, we selected 3 groups of different time(5 minutes, 20 minutes and 60 minutes) and unselected as control group. And the cells were identified by monoclone antibody of beta 1-integrin and cytokeratin 19 (Ck19), then the image analysis was done. Furthermore we analyzed the cultured cells with flow cytometer(FCM) and observed the ultrastructure of the cell by transmission electron microscope(TEM). RESULTS: The cell clones formed in all groups after 10 to 14 days, while the cells of 5 minute group grew more slowly than those of the other groups, however, the clones of this group were bigger. The expression of beta 1-integrin and Ck19 were found in all groups. The positive rate of beta 1-integrin was significant difference between 5 minute group and the other groups (P lt; 0.05). And the expression of Ck19 was no significant difference between 5 minute group and 20 minute group(P gt; 0.05), and between 60 minute group and control group. But significant difference was observed between the former and the later groups(P lt; 0.05). The result of FCM showed that most cells of the 5 minute group lied in G1 period of cell cycle, which was different from those of the other groups. At the same time, the cells of 5 minute group were smaller and contained fewer organelles than those of the other groups. CONCLUSION: The above results demonstrate that the cells of 5 minute group have a slow cell cycle, characteristics of immaturity, and behaving like clonogenic cells in vitro. The cells have the general anticipated properties for KSC. So the KSC can be selected by rapid attachment to extracellular matrix and identified by monoclone antibody of beta 1-integrin and Ck19.
Objective To investigate the expression of ADAM9 in breast cancer and its clinical significance. Methods The expressions of ADAM9 in normal breast tissues and breast cancer tissues were detected by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, and whose relationship with clinicopathologic features was analyzed. Results The expression of ADAM9 mRNA increased in the breast cancer tissues, but which was not detected in the normal breast tissues. The expression of ADAM9 protein in the breast cancer tissues was significantly higher than that in the normal breast tissues (Plt;0.05), and which in the metastatic lymph nodes was significantly higher than that in the negative lymph nodes or corresponding primary lesions (Plt;0.05). The expression of ADAM9 in the breast cancer tissues was correlated with the lymph node metastasis and histological grade (Plt;0.05). Conclusion ADAM9 is overexpressed in the breast cancer tissues, which might involve in the pathological progression of breast cancer.
Neutrophil extracellular traps (NETs) play an important role in the formation of immunothrombosis. However, how vascular endothelial cells mediate the formation of NETs has not been fully understood. We stimulated neutrophils firmly attached on the endothelial cell surface intercellular adhesion molecule-1 (ICAM-1) with lipopolysaccharide (LPS) or phorbol-12-myristate-13-acetate (PMA) for 4 h, then labeled NETs-DNA with Sytox green dye and the formation of NETs was observed by fluorescent microscopy. The area and fluorescence intensity of NETs-DNA were analyzed to quantify the formation of NETs. The results showed that both PMA and LPS were able to induce firmly adhered neutrophils on ICAM-1 to produce NETs. NETs induced by PMA were independent of neither β2 integrin lymphocyte function-associated antigen-1 (LFA-1) nor macrophage antigen complex-1 (Mac-1). In contrast, LPS-stimulated NETs were mediated by Mac-1 integrin, but not by LFA-1. After inhibition of actin filaments or Talin-1, the formation of NETs irrespective of the stimulus was significantly reduced. This study reveals the mechanism of the direct interaction between neutrophils and endothelial cells to produce NETs under inflammatory conditions, providing a new theoretical basis for the treatment of related diseases and the development of new drugs.
Objective To observe the expression of integrin αVβ3 in vascular endothelium cultured in vitro at different time points under different level of shear stress. Methods（1）We established a vascular culture system in vitro which could provide steady flow with different level of shear stress, and tested the flow stability when loading different level of shear stress. （2） A total of 50 rabbits were randomly divided into low shear stress group （5 dyn/cm2, n=25）and normal shear stress group（20 dyn/cm2, n=25）. Rabbits in each group were further randomly divided into five different time points as 2 h, 4 h, 8 h, 16 h and 24 h（n=5 at each time point）. The descending aorta of rabbits were harvested and cultured in the vascular culture system in vitro under different level of shear stress. The expression sites and intensity of αVβ3-Integrin in vascular endothelium were examined at 5 different time points in both groups by immunohistochemical staining. Results The vascular culture system in vitro was stable in providing laminar flow with different level of shear stress required for the experiment. Vascular endothelium expressions of αVβ3-Integrin in the low shear stress group were in high level at all the 5 time points and reached its summit at 16 h, when the mean optical density（MOD）value was （1.995±0.194）×10-2. In the normal shear stress group, the MOD value decreased time-dependently at the 5 time points. The MOD values at 2 h （0.059±0.005）×10-2 and 4 h（0. 049±0.002）×10-2 were significantly higher than those at other time points （P＜ 0.05）. The αVβ3-Integrin MOD values of the low shear stress group were significantly higher than those of the normal shear stress group at all the 5 respective time points （P=0.000）. Conclusion Low shear stress can significantly promote the expression of αVβ3-Integrin while normal shear stress decreases the expression of αVβ3-Integrin in vascular endothelium cultured in vitro.
ObjectiveTo investigate the value of integrin αvβ3 targeted microPET/CT imaging with 68Ga-NODAGA-RGD2 as radiotracer for the detection of osteosarcoma and theranostics of osteosarcoma lung metastasis.MethodsThe 68Ga-NODAGA-RGD2 and 177Lu-NODAGA-RGD2 were prepared via one-step method and their stability and integrin αvβ3 binding specificity were investigated in vitro. Forty-one nude mice were injected with human MG63 osteosarcoma to established the animal model bearing subcutaneous osteosarcoma (n=21), osteosarcoma in tibia (n=5), and osteosarcoma pulmonary metastatic (n=15). The microPET-CT imaging was carried out in 3 animal models at 1 hour after tail vein injection of 68Ga-NODAGA-RGD2. Biodistribution study of 68Ga-NODAGA-RGD2 was performed in animal model bearing subcutaneous osteosarcoma at 10, 60, and 120 minutes. The animal model bearing pulmonary metastatic osteosarcoma was injected with 177Lu-NODAGA-RGD2 at 7 weeks after model establishment to observe the therapeutic effect of pulmonary metastatic osteosarcoma. Histological and immunohistochemistry examinations were also done to confirm the establishment of animal model and integrin β3 expression in animal models bearing subcutaneous osteosarcoma and bearing pulmonary metastatic osteosarcoma.Results68Ga-NODAGA-RGD2 and 177Lu-NODAGA-RGD2 had good stability in vitro with the 50% inhibitory concentration value of (5.0±1.1) and (6.5±0.8) nmol/L, respectively. The radiochemical purity of 68Ga-NODAGA-RGD2 at 1, 4, and 8 hours was 98.5%±0.3%, 98.3%±0.5%, and 97.9%±0.4%; while the radiochemical purity of 177Lu-NODAGA-RGD2 at 1, 7, and 14 days was 99.3%±0.7%, 98.7%±1.2%, and 96.0%±2.8%. 68Ga-NODAGA-RGD2 microPET-CT showed that the accumulation of 68Ga-NODAGA-RGD2 in animal models bearing subcutaneous osteosarcoma and osteosarcoma in tibia and in lung metastasis as small as 1-2 mm in diameter of animal model bearing pulmonary metastatic osteosarcoma. Biodistribution study of 68Ga-NODAGA-RGD2 in animal model bearing subcutaneous osteosarcoma revealed rapid clearance from blood with tumor peak uptake of (3.85±0.84) %ID/g at 120 minutes. The distribution of 177Lu-NODAGA-RGD2 in lung metastasis was similar with 68Ga-NODAGA-RGD2. The number and size of osteosarcoma metastasis decreased at 2 weeks after 177Lu-NODAGA-RGD2 administration and integrin targeting specificity was confirmed by pathology examination.Conclusion68Ga-NODAGA-RGD2 was potential for positive imaging and early detection of osteosarcoma and metastasis. Targeted radiotherapy with 177Lu-NODAGA-RGD2 was one potential alternative for osteosarcoma lung metastasis.
ObjectiveTo summarize the relationship between integrins, tumor metabolism, and tumor cells with pancreatic stellate cells in the tumor microenvironment, in order to provide targets and ideas for the treatment of pancreatic ductal adenocarcinoma.MethodTo review the literatures on pancreatic stellate cells, integrins, and amino acid metabolism as therapeutic targets for pancreatic ductal adenocarcinoma in the domestic and overseas.ResultsThe drug research for pancreatic ductal adenocarcinoma was currently under vigorous development, but remain in the animal and clinical test stage. As a new therapeutic protein, ProAgio could inhibit the expression of integrin αvβ3, activation and secretion of pancreatic stellate cells, and alanine metabolism in the microenvironment of pancreatic ductal adenocarcinoma, so as to achieve the dual effects of anti-fibrosis and anti-tumor.ConclusionsThe roles of activated pancreatic stellate cells, ProAgio, integrin αvβ3, and alanine metabolism in pancreatic ductal adenocarcinoma have been partially elucidated, but the specific mechanism still needs further investigation and may become a completely new therapeutic target someday.