Objective:To develop a modified University of Wisconsin preservation solution (UW solution) containing α 2-adrenergic receptor agonists (dexmedetomidine) and noble gases (argon) and investigate its protective effect on the isolated amputated skeletal muscle of rats. Methods:Sixty male SD rats were selected to establish a hindlimb cold preservation/perfusion model and were divided into blank control group, hypothermic storage group, UW solution perfusion group, and modified UW solution perfusion group using a random number table, with 15 rats in each group. Simultaneously, a cold preservation model of rat skeletal muscle myoblasts (L6 cells) was established and the rats were also divided into four groups in the same way. Animal models were prepared in different ways: In the blank control group, the hindlimbs received no special treatment; In the hypothermic storage group, the amputated hindlimbs were stored in a dry centrifuge tube at 4℃ for 18 hours; In the UW solution perfusion group, the amputated hindlimbs were perfused with UW solution and then stored in a centrifuge tube containing UW solution at 4℃ for 18 hours; In the modified UW solution perfusion group, the amputated hindlimbs were perfused with modified UW solution (containing 0.1 nmol/L dexmedetomidine and 50% volume fraction of argon) and then stored in a centrifuge tube containing the modified UW solution at 4℃ for 18 hours. Cell models were treated as follows: In the blank control group, L6 cells were cultured under standard conditions; In the hypothermic storage group and UW solution group, L6 cells were treated with conventional culture medium or UW solution, stored in argon-filled sealed bags at 4℃ for 8 hours, and then rewarmed and cultured for 6 hours; In the modified UW solution group, L6 cells were treated with the modified solution, stored in argon-filled sealed bags at 4℃ for 8 hours, and then rewarmed and cultured for 6 hours. After sample collection, skeletal muscle morphology, tissue edema and ultrastructure features were assessed by HE staining, wet-to-dry weight ratio, and transmission electron microscopy, respectively. Additionally, L6 cell morphology was examined by light microscopy. L6 cell viability was determined by cell counting kit-8 (CCK-8) assay (expressed as absorbance A value). Expression levels of glutathione peroxidase 4 (GPX4) protein in both skeletal muscle tissue and L6 cells were evaluated by immunofluorescence staining and Western blot, respectively.Results:After 18 hours of in vitro preservation of rat isolated amputated limbs, the following results were obtained: (1) HE staining results showed that the muscle fiber morphology of the modified UW solution perfusion group was close to that of the blank control group. Moreover, the area ratio of skeletal muscle cells in the modified UW solution perfusion group was significantly higher than that in the hypothermic storage group and UW solution perfusion group ( P<0.05). (2) The wet-dry weight ratio results showed that there was no statistically significant difference among the modified UW solution perfusion group, the blank control group and UW solution group ( P>0.05), with significantly lower ratios in all three groups than that in the hypothermic storage group ( P<0.05). (3) Transmission electron microscopy results revealed that the modified UW solution perfusion group showed no statistically significant differences in ultrastructural metrics, including myofiber diameter, sarcomere length, mitochondrial short-axis/long-axis ratio, and mitochondrial cristae count, compared with those in the blank control group ( P>0.05), and performed significantly better than both the hypothermic storage group and UW solution perfusion group ( P<0.05). (4) Morphological observation of L6 cells showed that the cellular morphology was regular in the modified UW solution perfusion group, close to that in the blank control group, while it was severely damaged in the hypothermic storage group. Moreover, the cells were reduced in number and partially damaged in the UW solution group. The sequence of cell viability expressed as absorbance A value was blank control group >modified UW solution perfusion group > UW solution perfusion group > hypothermic storage group, with statistically significant differences among the four groups ( P<0.05). (5) Immunofluorescence staining showed that there was no statistically significant difference in fluorescence intensity of GPX4 protein expression between the modified UW solution perfusion group and blank control group ( P>0.05), while the fluorescence intensity was higher in the modified UW solution perfusion group than that in the hypothermic storage group and UW solution perfusion group ( P<0.05). Western blot analysis showed that the relative expression level of GPX4 in the modified UW solution group was significantly lower than that in the blank control group ( P<0.05), but higher than that in the hypothermic storage group and UW solution perfusion group ( P<0.05). Conclusion:The modified UW solution can stabilize the expression level of GPX4 protein, thereby inhibiting ferroptosis and alleviating cold preservation injury in both rat amputated isolated limb skeletal muscle tissue and L6 cells.

Objective:To develop a modified University of Wisconsin preservation solution (UW solution) containing α 2-adrenergic receptor agonists (dexmedetomidine) and noble gases (argon) and investigate its protective effect on the isolated amputated skeletal muscle of rats. Methods:Sixty male SD rats were selected to establish a hindlimb cold preservation/perfusion model and were divided into blank control group, hypothermic storage group, UW solution perfusion group, and modified UW solution perfusion group using a random number table, with 15 rats in each group. Simultaneously, a cold preservation model of rat skeletal muscle myoblasts (L6 cells) was established and the rats were also divided into four groups in the same way. Animal models were prepared in different ways: In the blank control group, the hindlimbs received no special treatment; In the hypothermic storage group, the amputated hindlimbs were stored in a dry centrifuge tube at 4℃ for 18 hours; In the UW solution perfusion group, the amputated hindlimbs were perfused with UW solution and then stored in a centrifuge tube containing UW solution at 4℃ for 18 hours; In the modified UW solution perfusion group, the amputated hindlimbs were perfused with modified UW solution (containing 0.1 nmol/L dexmedetomidine and 50% volume fraction of argon) and then stored in a centrifuge tube containing the modified UW solution at 4℃ for 18 hours. Cell models were treated as follows: In the blank control group, L6 cells were cultured under standard conditions; In the hypothermic storage group and UW solution group, L6 cells were treated with conventional culture medium or UW solution, stored in argon-filled sealed bags at 4℃ for 8 hours, and then rewarmed and cultured for 6 hours; In the modified UW solution group, L6 cells were treated with the modified solution, stored in argon-filled sealed bags at 4℃ for 8 hours, and then rewarmed and cultured for 6 hours. After sample collection, skeletal muscle morphology, tissue edema and ultrastructure features were assessed by HE staining, wet-to-dry weight ratio, and transmission electron microscopy, respectively. Additionally, L6 cell morphology was examined by light microscopy. L6 cell viability was determined by cell counting kit-8 (CCK-8) assay (expressed as absorbance A value). Expression levels of glutathione peroxidase 4 (GPX4) protein in both skeletal muscle tissue and L6 cells were evaluated by immunofluorescence staining and Western blot, respectively.Results:After 18 hours of in vitro preservation of rat isolated amputated limbs, the following results were obtained: (1) HE staining results showed that the muscle fiber morphology of the modified UW solution perfusion group was close to that of the blank control group. Moreover, the area ratio of skeletal muscle cells in the modified UW solution perfusion group was significantly higher than that in the hypothermic storage group and UW solution perfusion group ( P<0.05). (2) The wet-dry weight ratio results showed that there was no statistically significant difference among the modified UW solution perfusion group, the blank control group and UW solution group ( P>0.05), with significantly lower ratios in all three groups than that in the hypothermic storage group ( P<0.05). (3) Transmission electron microscopy results revealed that the modified UW solution perfusion group showed no statistically significant differences in ultrastructural metrics, including myofiber diameter, sarcomere length, mitochondrial short-axis/long-axis ratio, and mitochondrial cristae count, compared with those in the blank control group ( P>0.05), and performed significantly better than both the hypothermic storage group and UW solution perfusion group ( P<0.05). (4) Morphological observation of L6 cells showed that the cellular morphology was regular in the modified UW solution perfusion group, close to that in the blank control group, while it was severely damaged in the hypothermic storage group. Moreover, the cells were reduced in number and partially damaged in the UW solution group. The sequence of cell viability expressed as absorbance A value was blank control group >modified UW solution perfusion group > UW solution perfusion group > hypothermic storage group, with statistically significant differences among the four groups ( P<0.05). (5) Immunofluorescence staining showed that there was no statistically significant difference in fluorescence intensity of GPX4 protein expression between the modified UW solution perfusion group and blank control group ( P>0.05), while the fluorescence intensity was higher in the modified UW solution perfusion group than that in the hypothermic storage group and UW solution perfusion group ( P<0.05). Western blot analysis showed that the relative expression level of GPX4 in the modified UW solution group was significantly lower than that in the blank control group ( P<0.05), but higher than that in the hypothermic storage group and UW solution perfusion group ( P<0.05). Conclusion:The modified UW solution can stabilize the expression level of GPX4 protein, thereby inhibiting ferroptosis and alleviating cold preservation injury in both rat amputated isolated limb skeletal muscle tissue and L6 cells.

Objective: : To investigate the effects of GBX2 gene on the proliferation, migration and invasion of human cervical carcinoma SiHa cells and to explore the mechanism. Methods: Recombinant plasmid over-expressing GBX2 gene (pCMV6-entry-GBX2, experimental group) and empty vector plasmid (pCMV6-entry, negative control group) were transfected into cervical cancer SiHa cells by plasmid transfection technique. The proliferation, colony formation and cell cycle of transfected cells were detected by WST-1 method, Colony formation assay and flow cytometry, respectively. The cell migration and invasion were detected by wound healing assay and Transwell assay. The expression level of IL-6 in cell culture supernatant was detected by ELISA. WB was used to detect the expression changes of EMT-related proteins and to explore its possible mechanism. Results: Compared with the SiHa/pCMV6 negative control group, after up-regulation of GBX2, (1) the proliferation, colony formation, migration and invasion of SiHa/GBX2 cells in the experimental group were significantly enhanced (all P<0.01); The proportion of cells in G0/G1 phase decreased while the proportion of cells in S phase and G2/M phase increased (all P<0.01); (2) the expression of E-cadherin decreased, and the expressions of N-cadherin, vimentin and snail increased (all P<0.01); (3) the expression of IL-6 in the culture supernatant of SiHa/GBX2 cells was significantly up-regulated (P<0.01); (4) STAT3 phosphorylation in SiHa/GBX2 cells was enhanced, and could be inhibited by STAT3 inhibitor S31-201 (P<0.01). Conclusion: GBX2 may induce EMT of cervical cancer SiHa cells through IL-6/STAT3 pathway, thereby promoting the proliferation, migration and invasion of cervical cancer cells.

Objective:To investigate the cytotoxic effects of CTL cell induced by DCs loaded with exosomes derived from hepatoma Huh-7 cells(T-exo).Methods: Exosomes derived from hepatoma Huh-7 cells were isolated and purified by combination of ultrafiltration centrifugation and sucrose density gradient centrifugation.Morphology of exosomes was observed under transmission electron microscopy and the expression of CD 9,CD63,HSP70 and AFP was detected by Western blot.DCs were induced with peripheral blood monocytes isolated from healthy donors.Flow cytometry was used to analysis surface markers of the DCs loaded with T-exo.WST-1 light absorption measurement was adopted to evaluate the T cell proliferation ability.Annexin-V/PI Flow cytometry were respectively used to examined cytotoxicity against the tumor cells.Results:Exosomes isolated and extracted from culture supernatant of Huh-7 cells presented as circular or elliptical vesicle with bilayer membrane , unequal in size , and with diameter of 50 to 100 nm.Western blot showed that the T-exo expressed CD9,CD63,HSP70 and AFP molecules.DCs loaded with T-exo caused significantly higher T cell pro-liferation and cytotoxic effect against AFP positive Huh-7 cells as compare to gainst AFP negative SMMC 7721 cells and un-loaded control group ( P<0.05 ).Conclusion: T-exosome loaded-DC can promote proliferation and induce significant cytotoxic effect of CTL against Huh-7 cells.

We constructed a recombinant plasmid encoding VP1 gene of O type foot-and-mouth disease virus fused to a molecular adjuvant, goat complement C3d gene. The goat C3d gene was cloned and three copies were tandem-linked with the linker (G4S)2 sequence. VP1 gene of O type foot-and-mouth disease virus was linked to three tandem repeats of C3d through the linker sequence and cloned into pUC19 to obtain the recombinant plasmid pUC19-VP1-C3d3. The VP1-C3d3 fusion gene was then subcloned into the eukaryotic vector pcDNA3.1(+) that had been modified to contain the tissue plasminogen activator (tPA) leader sequence to obtain pcDNA3.1-tPA-VP1-C3d3. HeLa cells were transfected with pcDNA3.1-tPA-VP1-C3d3 by Lipofectamine 2000. Indirect immunofluorescent assay and Western blot assay showed that VP1-C3d3 fusion gene was successfully expressed in HeLa cells. The fusion protein with the expected size 133 kD could be secreted outside the cells. This study laid a good foundation to further research on the novel vaccine against foot-and-mouth disease virus by using goat C3d as a molecular adjuvant to enhance the immunogenicity of VP1.
Animals ; Capsid Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Complement C3d ; biosynthesis ; genetics ; immunology ; Female ; Foot-and-Mouth Disease Virus ; genetics ; Goats ; HeLa Cells ; Humans ; Immunologic Factors ; biosynthesis ; genetics ; immunology ; Plasmids ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Transfection