| ObjectivesThe present study aims to explore therapeutic effectiveness of recombinant adenovirus encoding human p53 tumor suppressor gene (rAd-p53) on lymphoma and its radiosensitivity using rAd-p53 radiotherapy alliance to experiment on lymphoma in vivo and in vitro.Methods1. Human lymphoma Raji and Daudi cell lines were respectively divided into the control and experiment groups. Using flow cytometry, average fluorescence intensity of raji and daudi cells were detected and fluorescence index was calculate; membrane surface coxsackie-adenoviral receptor (CAR) of Raji and Daudi cells was examined and expressions of integrin receptorsαⅤβ3 andαⅤβ5 were integrated.2. The methyl thiazolyl tetrazolium (MTT) was utilized to measure the inhibitory rate on Raji and Daudi of rAd-p53 at different concentrations in 24h, 48h, and 72h, by which the optimal effective dosage and action time could be determined. The inhibitory rate on Raji and Daudi with different exposure doses of radiotherapy was examined to select a suitable radiotherapy dose for the combination reaction with rAd-p53. Morphological change of cells and features of cell apoptosis were observed with inverted microscope and laser confocal scanning microscopy to illustrate the inhibitory effect of rAd-p53 on cell. Cell cycle and cell apoptosis rate were examined with flow cytometry. Expression transformations of mRNA of wild p53 gene and downstream gene bcl-2 and bax were measured by RT-PCR. Protein change of P53 was observed by Western-blotting of protein to compare the effect on Raji and Daudi between rAd-p53, radiotherapy with different exposure doses and their combined therapy.3. Models of athymic mouse transplantation tumor in Raji and Daudi of human lymphoma was set up to observe tumor growth, 5 mice bearing Raji athymic and 4 mice bearing Daudi athymic were successfully obtained. rAd-p53 was injected when tumor reached 0.5cm in diameter. The inhibitory effect of rAd-p53 on tumor growth and survival time of bearing cancer mouse were recorded.Results1. FIs of CAR andαⅤβ3 on the cellular membrane surface of Raji and Daudi exceeded 1 while that ofαⅤβ5 was below 1; expressions of CAR andαⅤβ3 were positive while that ofαⅤβ5 negative.2. MTT results showed that the inhibitory rate of Raji and Daudi was dosage and time dependent on rAd-p53, with the optimal rAd-p53 dose of 2000vp/cell, the optimal time of 48h and the maximum inhibitory rates of (27.5±4.1)% and (28.1±1.6)%, respectively. The results also showed that the inhibitory rate of Raji and Daudi was dosage dependent on reaiotherapy, with the optimal exposure dose being 6Gy and that therapy alliance was the combination of the optimal rAd-p53 dose and the optimal exposure dose.3. Observation under inverted microscope and laser confocal scanning microscopy showed that the morphological changes of Raji and Daudi with the injection of rAd-p53 were not obvious compared with the control group, but that of radiotherapy and therapy alliance was obvious, and showing little difference in radiotherapy and therapy alliance.4. Statistical results based on flow cytometry demonstrated that rAd-p53 did neither clearly arrest cell cycle nor increase cell apoptosis. On the contrary, radiotherapy and therapy alliance did except that there was no significantly difference between them two (P>0.05). In detail, Raji cells were arrested at the phase of G2-M while Daudi cells at the phase of G0-G1; cell apoptosis rates were (35. 6±1.7)%, (37.9±1.3)%, (31.1±2.4)% and (31.5±2.0)% respectively.5. The results of RT-PCR indicated that all three therapies could intensify the expressions of mRNA of wild p53 gene and bax gene compared with the control group, whereas there appeared no obvious change for Raji in mRNA expression of bcl-2 gene and for Daudi the expression of bcl-2 gene was negative. Western-blotting result showed that with the three kinds of therapies increased P53 protein expression of Raji and Daudi. 6. Tumor size of the mice bearing human lymphoma athymic mouse model became larger gradually until its death due to dyscrasia. The longest survival time of bearing Raji athymic mouse model was 37 days; the shortest survival time of bearing Daudi athymic mouse model was about 22 days.Conclusions1. The positive expressions of CAR andαⅤβ3 on the membrane surface of Raji and Daudi cells show that the adenovirus carrier should be, in mostly specialist opinion , be transfected into Raji and Daudi cells.2. rAd-p53 had some inhibitory effect on Raji and Daudi of human lymphoma, intensified the expression of wild p53 gene and downstream bax gene and increased the expression of exogenetic P53 protein. However, the function of exogenetic p53 gene transfected into cells was not exerted effectively. As a result, it did not cause change in cell cycle and apoptosis or obvious change in cell morphology and morphological features of apoptosis. All this indicates rAd-p53 has certain transfection on Radi and Daudi and leads to change at the level of molecular biology, but its action is not effective enough to treat this kind of tumor. At the same time, this study provides evidence that rAd-p53 has no obvious radiosensitivity when treating Raji and Daudi of human lymphoma.3. rAd-p53 has no obvious therapeutic action on Raji cells bearing cancer. Individualdifference exists in the experiments on Daudi cells bearing cancer and thus further research is needed with increased samples.
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