| Antimicrobial peptide is an important component in mammalian immune defense system , which have broad-spectrum antibacterial, antifungal, anti-insects and antiviral activity. In recent years, based on its traditional biological activity , its anti-tumor activity and immunomodulatory effect have much importance,but at present there is no report about the relationship between HCAP-18/LL-37 gene transfection and the tumor occurrence and development. In this study,we will explore its anti-tumor effects in vitro and vivo and study the immunological mechanisms by transient transfecting hCAP-18/LL-37 into Lewis lung cancer cells. ObjectiveIn this study, based on the tumor gene therapy,we will study the effect of hCAP-18/LL-37 to Lewis lung cancer cells proliferative activity, the tumour inhibitory effect and immunological function of tumor-bearing mice from the cellular and molecular level and the overall level separately by applying the molecular biology and immunology techniques and transfecting recombinant vector hCAP-18/LL-37 into lewis lung cancer cells,this will be provide experimental basis to study the anti-tumor effect and mechanism on gene transfection hCAP-18/LL-37 into Lewis lung cancer cells.Methods1.The recombinant gene hCAP-18/LL-37 was transiently transfected into Lewis lung cancer cells by using lipofection method, after cultured for 24 hours in 6-well plates, collecting cells to extract total cellular RNA to do RT-PCR for detecting the expression of target gene;2. Detecting the change of proliferative activity of Lewis lung cancer cells when transiently transfected hCAP-18/LL-37 into Lewis lung cancer cells by MTT method and plate clone formation test in vitro;3. Collecting culture supernatant of tumor cells after transiently transfected hCAP-18/LL-37 into Lewis lung cancer cells for 24h, detecting the effect of tumor cell supernatant on the mouse splenocyte proliferation;4. Detecting the expression of TLR4 mRNA of Lewis lung cancer cells by the method of semi-quantitative RT-PCR;5. Foundating mouse lung cancer model of C57BL / 6 and detecting tumour inhibition ratio of tumor-bearing mice;6. Detecting the chang of immune function on tumor-bearing mice:(1) Detection of splenic index(2) Detecting the proliferative activity of lymphocyte from bearing-cancer mouse by MTT method(3) Detecting the percence of killing activity of NK cell and CTL cell by MTT methodResults1. The mRNA of LL-37 was detected from transfected Lewis lung cancer cells ;2. The recombinant gene hCAP-18/LL-37 was transiently transfected into Lewis lung cancer cells, and after cultured for 24 hours,they were inoculated in 96-well plates .Then we detected the activity of cell proliferation by MTT method at the the next three days.The results showed that cell proliferation was inhibited in recombinant vector transfected group compared with empty vector control group and untransfected cells control group, and the difference has statistical significance(P<0.05);3. The recombinant gene hCAP-18/LL-37 was transiently transfected into Lewis lung cancer cells, and after cultured for 24 hours,they were inoculated in 6-well plates and cultured standingly for a week,then detected colone forming ability of the cells. The results showed that Cell clone-forming ability decreased significantly in recombinant vector transfected group compared with empty vector control group and untransfected cells control group, and the difference has statistical significance(P<0.05);4. The recombinant gene hCAP-18/LL-37 was transiently transfected into Lewis lung cancer cells, collecting the cell culture supernatant after cultured for 24 hours, and co-cultured with mouse lymphocyte for 48h.Then we detected the activity of the lymphocyte proliferation by MTT method. The results showed that when the culture supernatant was diluted at 1:2 , the lymphocyte proliferation has no significant difference in recombinant vector transfected group(0.927±0.219) compared with the lymphocyte control group(1.464±0.11); the lymphocyte proliferation decreased in untransfected cells control group (0.775±0.068)compared with the lymphocyte control group(1.464±0.11), and the difference has statistical significance(P<0.05); the lymphocyte proliferation decreased significantly in empty vector control group (0.667±0.027)compared with the lymphocyte control group(1.464±0.11), and the difference has statistical significance(P<0.01),these results sugesst that the tumor cell culture supernatant of untransfected cells control group and empty vector control group can inhibit the lymphocyte proliferation of mouse .when the culture supernatant was diluted at 1:4, the lymphocyte proliferation has no significant difference in every group which stimulated by the culture supernatant compared with the lymphocyte control group, this result sugessted that when the culture supernatant was diluted at 1:4, it had no inhibition on the lymphocyte proliferation of mouse.5. The recombinant gene hCAP-18/LL-37 was transiently transfected into Lewis lung cancer cells ,after cultured for 24 hours in 6-well plates, collecting cells to extract total cellular RNA to do RT-PCR for detecting the expression of TLR4 mRNA of Lewis lung cancer cells in recombinant vector transfected group, empty vector control group and untransfected cells control group,the results showed that the expression of TLR4 mRNA decreased in recombinant vector transfected group compared with the empty vector control group and untransfected cells control group.6. The recombinant gene hCAP-18/LL-37 was transiently transfected into Lewis lung cancer cells ,after cultured for 24 hours in 6-well plates, collecting cells and injected 0.2ml into every mouse by the left flank at the concentration of 1×10 7/ml,and set up the untransfected cells control group and empty vector transfection group, the results showed that all mouse have tumour growth and the tumor size in each group has no difference, this result sugessted that LL-37 gene transfected group had no significant inhibitory effect.7. Effection of hCAP-18/LL-37 gene transfection to the immune function of tumor-bearing mice :(1) Detection of splenic index: Spleen index was significantly increased in recombinant vector transfected group(0.8%) compared with untransfected cells control group(0.5%)(P<0.05),but has no difference in compared with empty vector control group(0.8%).(2)Detecting the proliferative activity of lymphocyte from bearing-cancer mouse by MTT method,the result showed that there were no difference among the three groups for SI, so the proliferative activity of lymphocyte from bearing-cancer mouses in three groups have no difference.(3) Detecting the percence of killing activity of NK cell and CTL cell by MTT method, the results showed that when the ratio on effector cells to target cells at 50:1,the percence of killing activity of NK cell was significantly increased in recombinant vector transfected group(48.4±0.091) compared with untransfected cells control group(33.3±0.071),the difference was significant (P<0.05),but has no difference in compared with empty vector control group(48.9±0.078);when the ratio on effector cells to target cells at 25:1,the percence of killing activity of NK cell was increased in recombinant vector transfected group(39.0±0.088) compared with untransfected cells control group(30.9±0.044)and empty vector control group(36.39±0.116) ,the difference was no significant; when the ratio on effector cells to target cells at 10:1,the percence of killing activity of NK cell was increased in recombinant vector transfected group(39.7±0.063) compared with untransfected cells control group(35.3±0.097) ,the difference was no significant;when the ratio on effector cells to target cells at 10:1 and 25:1,the percence of killing activity of CTL cell was significantly increased in recombinant vector transfected group(6.63±0.040,18.69±0.0991) compared with untransfected cells control group(1.27±0.0063,2.03±0.0204),the difference was significant(P<0.05),the percence of killing activity of CTL cell was increased in recombinant vector transfected group compared with empty vector control group(5.37±0.0408,15.69±0.0691),the difference was no significant; when the ratio on effector cells to target cells at 50:1,the percence of killing activity of CTL cell was significantly increased in recombinant vector transfected group(49.75±0.1861) compared with untransfected cells control group(14.53±0.05),the difference was significant(P<0.01), the percence of killing activity of CTL cell was increased in recombinant vector transfected group(49.75±0.1861) compared with empty vector control group(39.25±0.1112),the difference was no significant .Conclusions1. The mRNA of LL-37 was detected from transfected Lewis lung cancer cells;2.hCAP-18/LL-37 gene transfection decreased the ability of proliferation and the clone-forming of Lewis lung cancer cells;3. hCAP-18/LL-37 gene transfection can reduce the inhibition of cell culture supernatant to the mouse lymphocyte proliferation ;4. When the recombinant gene hCAP-18/LL-37 was transfected into Lewis lung cancer cells,the expression of TLR4 mRNA of the cells was decreased, the result suggested that the proliferation of Lewis lung cancer cells may be associated with the expression of TLRs,and hCAP-18/LL-37 have an impact on the proliferation of Lewis lung cancer cells by regulating the expression of TLRs through some mechanism;5. When the recombinant gene hCAP-18/LL-37 was transfected into Lewis lung cancer cells,the effect of anti-tumor was not obvious in tumor-bearing mice ,but Gene transfection have a catalytic role on NK, CTL activity in tumor-bearing mice.
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