Objective To observe recombinant plasmids were constructed with the macrophage colony-stimulating factor ( GM-SCF) , interleukin -21 (IL-21) and retinoic acid early transcription factor-1 (Rae-1), and observe the inhibitory effects in subcutaneous liver cancer model in mice with the recombinant plasmids.Methods The recombinant plasmids of GM-SCF, IL-21 and Rae-1 were constructed with RT-PCR method, mouse model was constructed, the model mice were randomly divided into six groups including control, IRES/GFP, IRES/IL21, IRES/GM-SCF, IRES/GM-SCF-IL21 and IRES/combination with 10 mice included in each group, each groups (15 mice) were treated with the corresponding gene therapy.The survival rate were observed after 60 days.The blood levels of interferon -γ(IFN-γ) and interleukin -2 (IL-2) were detected in each group.Results The pGM-CSF-GFP-IRES-Rae-1-IL-21 has been successfully constructed.All mice had demised 14 and 16 days after treatment in the control and IRES/GFP groups, respectively.There were 2, 1, 11 mice remaining after 60 days of treatment in the IRES/GM-SCF, IRES/IL21 and IRES/GM-SCF-IL21 groups respectively.The survival rate of mice at 60 days of treatment was 73.33%, 13.33%, and 6.67% for groups IRES/GM-SCF-IL21, IRES/GM-SCF and IRES/IL21, respectively.The survival rate of the mice was significantly higher in IRES/GM-SCF-IL21 than the other groups.The levels of IL-2 and INF-γof mice 1-6 days after treatment gradually increased in the IRES/combination groups, including IRES/GM-SCF-IL21, IRES/GM-SCF and IRES/IL21.They were highest in the IRES/combination group and lowest (P<0.01) in the IRES/GM-SCF and IRES/IL21 groups, with the IRES/GM-SCF-IL21 group showing intermediate levels.By 6-10 days after treatment, IL-2 and INF-γlevels had stably increased in the IRES/combination groups, but had gradually decreased in the IRES/GM-SCF-IL21, IRES/GM-SCF and IRES/IL21 groups.At the end of treatment, IL-2 and INF-γlevels were significantly (P<0.01) higher in the IRES/GM-SCF-IL21 than were found in either the IRES/GM-SCF group or IRES/IL21 group, which were also significantly (P<0.01) higher than either the IRES/GFP or control groups.The levels of IL-2 and INF-γwere highest in the IRES/combination group ( P<0.01) and not significantly different among the IRES/GM-SCF, IRES/IL21, IRES/GFP, and control groups.Conclusion The inhibitory effects in subcutaneous liver cancer model in mice were obvious significantly, and its mechanism maybe be related to the activation of the body's immune.

Objective : To investigate the role of member septin family (SEPT12)in human spermatogenesis and its influence on sperm motility and sperm ultrastructure. Methods : Whole exome sequencing (WES) was performed on peripheral blood DNA extracted from 375 patients with asthenoteratozoospermia , and a patient with idiopathic infertility carrying compound heterozygous mutation of SEPT12 was screened out. Sanger sequencing was performed to verify the mutation , and co⁃segregation analysis was performed in the family. The morphological abnormalities of sperm were analyzed by hematoxylin⁃eosin (HE) staining and scanning electron microscopy (SEM) , and the ultrastructural defects of sperm were analyzed by transmission electron microscopy (TEM) . Then the effects of the mutation on the level and position of the protein and the changes of the location and level of the defect structure markers were analyzed by Western blot and immune⁃fluorescence (IF) . Results : The compound heterozygous mutations c.C332A (p. T111K) and c. 406_416 del TGCTCGTATTG (p. q136 VFS ∗39) in the SEPT12 gene were screened and identified in a patient with asthenoteratozoospermia. The mutations were verified by Sanger sequencing , which was consistent with the co⁃segregation genetic pattern of the family. The mutations resulted in loss of protein expression , decreased sperm motility and sperm morphological deformities , mainly including short tail , curly tail and irregular sperm head. The ultrastructure of sperm showed that the annulus between the mid⁃piece and the principlepiece was missing , the acrosome membrane of sperm head fell off and the nucleus contained vacuoles. In the midpiece of sperm flagella , the arrangement of mitochondrial sheath was disordered , most of flagella axoneme central pair was absent , microtubules doublet was missing or disordered , and some radical spoke was absent. By Western blot and IF , the marker proteins of related structural components were detected , and the results showed that the level of SEPT4 protein decreased , SEPT6 protein unchanged , acrosomal related proteins ACTL7A and ACROSIN protein missing , and the expression levels of mitochondrial and axoneme related proteins TOMM20 , SPAG6 and RSPH3 protein significantly decreased. Conclusion The deletion of SEPT12 protein caused by SEPT12 gene mutation leads to the deletion of the annulus between the mid⁃piece and the principle⁃piece , and the abnormal assembly of sperm acrosome , mitochondrial sheath and flagella.