Establishment Of Ie2 Gene-bearing Glioma Mice And Effect Of Gene On Tumor Self-state

ObjectHuman cytomegalovirus(HCMV)is a double-stranded DNA virus.It has a high infection rate and molecular weight.It belongs to the herpes virus family.In the early stages of a viral infection,an acute infection occurs,after which HCMV establishes a latent infection that eventually persists with the life of the host until the host dies.In the early stage of viral infection,the early ie2 gene of HCMV is expressed first,and the IE86 protein encoded by this gene plays an important regulatory role in the process of viral infection.However,HCMV has strict species specificity,which makes us face great challenges in host selection.Therefore,we constructed an animal model of transgenic mice and carried out tumor-bearing transformation of ie2 gene mice.Based on this,we studied the interaction between gene and tumors.Methods1.Tumor bearing of HCMV ie2 gene and non-transgenic wild mice,and observing and recording the growth state of mice after tumor injection.After the mice were sacrificed,the isolated tumors were measured and detected to determine the ie2 gene.The effect on the growth state of mice before and after tumor-bearing.2.In the parallel group experiment,the transgenic mice and the wild mice were subjected to tumor-bearing experiments in the same way.After successful tumorbearing experiments,the death test was not performed and the death date of the tumorbearing mice was recorded.Also the survival rate was calculated and compared to determine the effect of ie2 gene on the survival state of the tumor-bearing mice.3.The tumor tissues of tumor-bearing mice were stained with HE to further observe the difference of tumor morphology between IE positive tumor-bearing mice and negative tumor-bearing mice.4.Qualitative PCR was used to qualitatively detect the glioma marker transcription factor ATF5,and then real-time PCR was used to quantitatively detect the expression of ATF5 in tumor tissues and brain tissues of tumor-bearing mice.The relationship between the expression of ATF5 and the expression of IE86 protein in tumor tissues of transgenic tumor-bearing mice was detected by western-blotting.Results1.Two kinds of glioma cells GL261 and G422 were selected for cell culture,and GL261 cells with the easiest culture and the highest value-added rate were selected for later stage tumor-bearing experiment.After tumor-bearing,the tumor growth status of ie2 mice was more rapid and the survival time of mice is shortened.2.Paraffin sections were taken from isolated tumor tissues.HE staining showed that the cell morphology of the negative mice was clear and the cells were loose.The cells of the positive mice were irregular,the boundaries were unclear and the cells were dense.3.Tumor-bearing mice and non-transgenic mice were tumor-bearing.Tumor tissues and brain tissues were exfoliated after tumor-bearing,and then ATF5 was quantitatively detected by real-time PCR.It was found that ATF5 was highly expressed in tumors of both HCMV ie2 transgenic mice and non-transgenic mice,and no ATF5 expression or low expression of ATF5 was observed in brain tissues.4.Protein detection: Protein extraction was performed on tumor tissues of isolated positive mice.The quantitative relationship between ATF5 and IE86 protein was detected by Western-blotting method.The results showed that ATF5 was highly expressed in transgenic mice.Conclusions1.A mouse model of glioma bearing transgenic ie2 gene was successfully established to study the mechanism of HCMV IE gene and its encoding protein IE86 in the development of glioma.2.Compared with ie2-negative glioma-bearing mice,ie2-transgenic gliomabearing mice had worse survival status and shorter survival time.The gene and protein expression levels of the transcription factor ATF5 are elevated.The above evidence suggests that the expression of HCMV ie2 gene is related to the malignant degree of glioma.