| This study consists of two parts as described as below.The first part is that the preparation of nanoliposomes of TTF-1 promoter operated miR-7 expression vector.The second part is that the effect of nanoliposomes deliver TTF-1 promoter operated miR-7 expression vector on the growth of human lung cancer in nude mice model.Objective:Part One To construct the eukaryotic vector of TTF-1 promoter operated expression of miR-7,and to prepare the nanoliposomes of TTF-1 promoter operated miR-7 expression vector by thin film dispersion method.Part Two To observe the possible effects of nanoliposomes deliver TTF-1promoter operated miR-7 expression vector on the growth of human lung cancer cells in vivo using nude mice model of human lung cancer through related experimental technique including HE staining,immunofluorescence(Ki-67 and TUNEL),Western blot and Real-time PCR assay.At the same time,the safety of nanoliposomes of TTF-1 promoter operated miR-7 expression vector in vivo also was estimated.Methods:Part One We constructed an eukaryotic vector of TTF-1 promoter operated expression of miR-7,and then prepared the nanoliposomes of TTF-1 promoter operated miR-7 expression vector by thin film dispersion method.The outline of nanoliposomes were tested by transmission electron microscope.Laser particle size and Zeta potential analyzer were used to test its diameter of particle,zeta potential and PDI.The encapsulation efficiency and stability were tested by dialysis method.Part Two Human lung cancer model using nude mice was established.Human lung cancer cell line 95 D cells were injected subcutaneously into left flank of Balb/c nude mice(n=6).11 days later,the nanoliposomes of encapsulated p-T-mi R-7 vector(100?g)and p-T vector(100?g)were respectively injected through tail vein in nudemice,subsequently injection every 3 days,the process continuous 3 times.Then,tumor growth was measured accordingly.After last injection,3 days later,all mice were killed.The tumor tissue and lung tissue were obtained and stained by HE staining.The expression level of miR-7 was detected by Real-time PCR assay.The expression of EGFP was observed by Co-focus technique.Moreover,the tumor growth-associated factors(CDK2,CDK3,CDK4 and CDK6)and tumor metastasis-associated factors(MMP-2,MMP-9,E-cadherin and CXCR4)were detected by Real-time PCR assay respectively.The expression of Ki-67 was analyzed by immunofluorescence assay.And the apoptosis of cells was detected by TUNEL method.Morevoer,the expression of associated signal pathway,including p-Akt,p-Erk,and NDUFA-4 were detected by western blot assay.The change of organs and tissues,as well as biochemistry indicators,in nude mice model of human lung cancer were analyzed.Finally,the expression levels of miR-7 and distribution of plasmid copies in major organs of nude mice model were analyzed by Real-time PCR assay.Results:Part One TTF-1 promoter operated miR-7 eukaryotic expression vector(pEGFP-N1-TTF-1-mi R-7,named p-T-miR-7)and TTF-1 promoter vector(pEGFP-N1-TTF-1,named p-T)were successfully constructed.Then,the nanoliposomes of p-T-miR-7 and the nanoliposomes of p-T were prepared by thin film dispersion method.Results showed that transmission electron microscope observed the outline of nanoliposomes were spherical or nearly spherical,and the average particle diameter of p-T-miR-7 nanoliposomes was 72.15 nm,the zeta potential was-3.15 mV,the PDI was 0.258,the average particle diameter of p-T nanoliposomes was 78.10 nm,the zeta potential was-18.88 mV,and the PDI is 0.255.The encapsulation efficiency and stability were tested by dialysis method,and the results showed that the encapsulation efficiency of p-T-miR-7 nanoliposomes was67%,the encapsulation efficiency of p-T nanoliposomes was 64%,and the encapsulation efficiency was stable.Part Two Human lung cancer model using nude mice was established.And the growth of tumor in p-T-miR-7 nanoliposomes group significantly decreased when compared with that in p-T nanoliposomes group(P<0.05).HE staining of lung tissue in nude mice showed that there were no clearly pulmonary metastatic nodules inp-T-miR-7 nanoliposomes group.HE staining further showed that there were large areas of necrosis in tumor mass in p-T-miR-7 nanoliposomes group.The expression level of miR-7 in tumor tissue in p-T-miR-7 nanoliposomes group was significantly higher than that in the p-T nanoliposomes(P<0.01).Moreover,there was EGFP expression in tumor mass.Next,the expression of tumor growth-associated factors(CDK2,CDK3,CDK4 and CDK6)and tumor metastasis-associated factors(MMP-2,MMP-9,E-cadherin and CXCR4)in tumor tissue was significantly decreased in p-T-miR-7 nanoliposomes group compared with in p-T nanoliposomes group(P<0.05).In addition,mmunofluorescence assay further showed that the Ki67 of tumor cells decreased obviously in p-T-miR-7 nanoliposomes group,conversely,the apoptosis of tumor cells increased significantly.At the same time,the expression level of p-Akt,p-Erk and NDUFA-4 in tumor tissue were decreased significantly in p-T-miR-7 nanoliposoms group(P<0.05).Importantly,there were no significant difference on the morphology and histology of 7 major organs and tissues,including heart,liver,spleen,kidney,brain,intestine and lymph nodes between p-T-miR-7nanoliposomes group and p-T nanoliposomes group(P>0.05).Moreover,we found that there were no significant difference on the weight of 7 major organs and tissues.Besides,we also found that there were no significant change on the biochemistry indicators such as TC,TG,GLU,AST and ALT.Finally,the expression level of miR-7and distribution of plasmid copies in major organs and tissues were analyzed by RT-PCR,and the results demonstrated that the highest expression level of miR-7 and the distribution of plasmid copies in lung of the nude mice model.Conclusion:1.Prepared the nanoliposomes of TTF-1 promoter operated mi R-7 expression vector by thin film dispersion method.2.Nanoliposomes deliver TTF-1 promoter operated miR-7 expression vector significantly inhibited tumor growth of human lung cancer in nude mice model. |
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