ABSTRACT
Objective:To prepare cell membrane-coated nanovesicles with targeted delivery of toll-like receptor 4 (TLR4) agonist, and to explore the effect and mechanism of inducing the polarization of tumor-associated macrophages (TAMs) and treating osteosarcoma.Methods:TLR4 agonist loaded nanovesicles were prepared by polycarbonate membrane extruders. The morphology and size of nanovesicles were detected by transmission electron microscopy (TEM) and particle size analyzer, and the drug loading performance of the nanovesicles to TLR4 agonist was investigated. TLR4 agonist loaded nanovesicles were co-incubated with macrophages in vitro, and the targeting ability of nanovesicles to macrophages and its role in regulating the function of macrophages were detected by confocal fluorescence microscopy. In vitro experiments, a cell co-culture system was established. After the upper layer macrophages were treated by the control group, the TLR4 agonist group and the TLR4 agonist loaded nanovesicle group, the lower layer osteosarcoma cells were collected for CCK-8 and cloning formation experiments to evaluate their effects on the proliferation and migration of osteosarcoma cells. In vivo experiments, an osteosarcoma subcutaneous graft tumor model was established, and mice were randomly divided into the control group, the TLR4 agonist group, and the TLR4 agonist loaded nanovesicle group. After the treatment by caudal vein, the tumor targeting ability of nanovesicles in vivo was explored through the in vivo imaging system, and the volume of tumor tissue was continuously detected. The subcutaneous tumors were stained to detect macrophage-related markers, and their effect on the polarization of macrophages was evaluated. The TUNEL fluorescence of tumor tissues was further detected.Results:TEM showed the round shape of TLR4 agonist loaded nanovesicle and the size was about 200 nm. The co-incubation of 0.05 mg TLR4 agonist with 0.1 mg nanovesicles was the best condition for the preparation of drug-loaded nanovesicles. The drug loading efficiency was about 35% and the drug loading content was about 0.11 mg/mg. The membrane-coated nanovesicles could efficiently load and deliver TLR4 agonist. TLR4 agonist loaded nanovesicles were labeled with DiD red fluorescent dye, and then the labeled nanovesicles were co-incubated with macrophages. It was found by confocal fluorescence microscopy that DiD labeled TLR4 agonist loaded nanovesicles significantly accumulated in macrophages, and the fluorescence of M1-type macrophage marker (iNOS) was significantly enhanced, which could induce M1 polarization of macrophages. In vitro experiments, it was found that the number of osteosarcoma cells in the TLR4 agonist loaded nanovesicle group was significantly reduced under the light microscope, and the cell morphology was wrinkled and rounded. CCK-8 and cloning formation experiments showed that the proliferation and migration ability of osteosarcoma cells in the TLR4 agonist loaded nanovesicle group was significantly reduced compared with the control group and the TLR4 agonist group. A subcutaneous graft tumor model was established. In vivo imaging experiments showed that TLR4 agonist loaded nanovesicles locally accumulated in tumor tissues in vivo, but were not distributed in other organs. The growth of tumor tissue was significantly inhibited in the TLR4 agonist loaded nanovesicle group. Moreover, the fluorescence of M1-type macrophage marker (iNOS) was significantly enhanced (relative fluorescence intensity: 3.27±0.19), while the fluorescence of M2-type macrophage marker (CD163) was significantly decreased (relative fluorescence intensity: 0.14±0.04). TUNEL fluorescence staining showed that the apoptosis level of osteosarcoma cells was significantly increased (relative fluorescence intensity: 9.53±0.21).Conclusion:Membrane-coated nanovesicles could targeted deliver TLR4 agonist to osteosarcoma, induce TAMspolarization, remodel tumor immunosuppressive microenvironment, promote cell apoptosis, and effectively kill osteosarcoma.
ABSTRACT
Objective To provide regional anatomical data for clinical application through observing the type, length and diameter of variation in the medial cubital vein of upper limb. Methods Dissected the medial cubital vein of upper limb in 35 (70 sides) adult cadavers (male 38, female 7), and observed the morphological structure. The length and vessel diameter of the medial cubital vein were measured. Results The variation of the medial cubital vein of upper limb was divided into six type. TypeⅠincluded 29 cases (41. 43%) of male and 8 cases (11. 43%) of female;Type Ⅱ included 14 cases (20. 00%) of male and 4 cases (5. 71%) of female;Type Ⅲ included 7 cases (10. 00%) of male and 2 cases (2. 86%) of female;TypeⅣincluded 3 cases (4. 29%) of male and 0 cases of female;TypeⅤincluded 2 cases (2. 86%) of male and 0 cases of female;TypeⅥincluded 1 cases (1. 43%) male and 0 cases of female. Conclusion This study enriched anthropology data of the medial cubital vein and it has the role of guiding in clinical applications.
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To explore the expression of Beclin1 in osteosarcoma and investigate the effects of down-regulation of autophagy on the chemotherapeutic sensitivity to cisplatin (DDP), the expression of Beclin1 in 28 specimens of osteosarcoma (group A) and 19 specimens of normal bone tissues (group B) were immunohistochemically detected. The expression of Beclin1 mRNA in MG63 cells treated with different concentrations of DDP was examined with RT-PCR. After down-regulation of autophagy in MG63 cells by an autophagy inhibitor, 3-methyladenine (3-MA), the cell proliferation inhibition rate of MG63 cells treated with DDP was evaluated by using the MTT assay. The positive rates of Beclin1 were 67.85% in group A and 94.73% in group B. Its expression was lower in osteosarcoma than in normal bone tissues, with a significant difference found between them (P<0.05). RT-PCR showed that the expression of Beclin1 mRNA in the cells treated with high-dose DDP were higher than that in the non-treated cells, and no significant difference in the expression of Beclin1 mRNA was found between the cells treated with low-dose DDP and the non-treated cells. There was a positive correlation between the level of Beclin1 mRNA expression and the concentration of DDP. MTT assay showed that the proliferation inhibition rates of the cell treated with 3-MA and DDP combined were substantially increased when compared with those treated with DDP alone (P<0.01). This study demonstrated that autophagy may be implicated in the carcinogenesis of osteosarcoma, and DDP may induce autophagy in the MG63 cells. It also suggests that the down-regulated autophagy could increase chemotherapeutic sensitivity of DDP to osteosarcoma.
ABSTRACT
In some conditions,autophagy serves a protective role;but in other conditions it has an anti-cancer role. There is still contradiction on the mechanisms that autophagy induces tumor cells death. During the genesis and development of tumors,autophagy has tumor promoting or inhibiting properties at different times.
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Non-enzymatic glycosylation reaction, which proceeded at an accelerated rate in diabetes, directly caused sharp diminution of total haemoglobin due to glycosylated proteins including haemoglobin digested by macrophages. The diminution contributed to hypoxia of tissue that repressed the enzymatic activities in the respiratory chain as well as in the tri-carboxylic acid cycle [TCA] and Embden-Meyerhof-Parnas [EMP] pathways. It was postulated that non-enzymatic glycosylation reaction accelerated the rise of blood glucose. The theory was further proven by the hypoglycaemic activities of the extract from Tremella aurantialba broth [TBE]. TBE inhibited the formation of advanced glycosylation end-products [AGEs] in vitro [IC[50] =1.7 mg/ml] and in vivo. TBE, when given in vitro, increased the concentration of total haemoglobin and supply of oxygen, enhanced respiration of tissue, decreased the levels of NADH, speeded up catabolism of glucose and finally generated significant anti-hyperglycaemic and hypoglycaemic effect on alloxan-induced diabetic rats. In addition it elevated plasma insulin level. Oral administration of TBE [100 mg/Kg bw] once a day for 4 weeks resulted in significant reduction in the plasma levels of glucose, fructosamine and the ratio of glycosylated haemoglobin to total haemoglobin. Moreover, oral administration of TBE increased the total haemoglobin and plasma insulin levels and enhanced some key enzymatic activities of EMP, TCA pathways and respiratory chain in the blood of diabetic rats compared with control