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

[摘 要] 目的：制备并表征包载CPI-444并偶联CD8抗体的纳米微粒（CNP/αCD8），探讨其对CD8+ T细胞活化、增殖和抗肿瘤作用的影响。方法: 采用复乳溶剂蒸发法和EDC/NHS法制备包载腺苷受体A2A（A2AR）特异性拮抗剂CPI-444（C）或香豆素6（C6）荧光素的纳米微粒并分别在其表面偶联CD8抗体，制得CNP/αCD8和C6NP/αCD8。扫描电镜和NanoPlus粒度测定仪表征纳米微粒形态和粒径，液相色谱与串联质谱联用（LC-MS/MS）法和离心法测定纳米微粒的载药量和药物释放情况，荧光显微镜和流式细胞仪检测CD8+ T细胞内化C6NP/αCD8的情况，流式细胞仪、ELISA和LDH法检测CNP/αCD8对CD8+ T细胞增殖、活化、细胞毒活性和杀瘤能力的影响。结果: CNP/αCD8纳米微粒为圆形、粒径约150 nm，能有效包载CPI-444和偶联CD8抗体，药物包封率和CD8抗体偶联效率分别约为60%和53.4%；CNP/αCD8纳米微粒具有良好稳定性，能被CD8+ T细胞内化，抑制A2AR分子表达。生物学功能实验显示，CNP/αCD8增强CD8+ T细胞的增殖能力、促进T细胞活化、分泌细胞因子及产生颗粒酶B和穿孔素，并增强CD8+ T细胞杀伤肿瘤细胞的能力。结论: CNP/αCD8纳米微粒能显著增强CD8+ T细胞免疫效应功能，其增强CD8+ T细胞功能可能是通过抑制A2AR分子的表达起作用。

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.