To Detect Gene Therapeutic Effect Of HTRAIL For Lung Cancer A549 Cells Nude Mice Transplantated Tumor In Vivo By Luciferase Bioluminescence Imaging

Objective: To evalue effect of intratumorally injecting adenovirus vector, ad-luc-hTRAIL to treat the A549 cells xenograft tumour of nude mice, ad-hTRAIL,ad-luc and PBS as control groups, and exploring whether the expression of report gene luciferase(luc) could reflect the expression of theoretical gene human tumor necrosis factor-related apoptosis ligand(hTRAIL), and detect the therapeutic informations.Methord:1 Establishment of lung cancer A549 cells xenograft of nuce mice models: The lung cancer A549 cells were cultured in vitro routinly, and the cells were collected. After sterilizing the skin of the back of the mude mice, the A549 cells were injected in hypodermic into the nude mouse to establish the xenograft of nude mice models.2 Observing the antitumour therapeutic effect: The adenovirus vectors(ad-luc-hTRAIL,ad- hTRAIL,ad-luc) were injected respectively at several points intratumorally, the control group was received PBS. Dynamic detected the informations of the tumour growth at different time points(day 4,7,10,14,21,28 after started the gene therapy). the volume of the tumours were measured, when the experiment was completed, draw the tumour time growth curve and calculated the inhibition rate of the tumour.3 Detecting the activity of luciferase: After gene therapy was started, the mice in all groups were taken bioluminescence imaging exmination, to detect the activity of luciferase of tumour's location, by using high-sensitivity cooled-charged coupled device camera at differert time pionts.4 Immunohistochemistry staining to detcet the expression of hTRAIL protein: After gene therapy started, a part of nude mouse were sacrificed at different time points to get the tissues of tumour, observing the expression of hTRAIL protein in group ad-hTRAIl and ad-luc-hTRAIL,PBS by immunohistochenical staining. Immunohistochemical scores (IHS) was done for analysis.5 TUNLE to detect the apoptosis of tumour cells:After gene therapy started, a part of nude mouse were sacrificed at different time points to get the tissues of tumour, observing the apoptosis of tumour cells in group ad-hTRAIl and ad-luc-hTRAIL,PBS by TUNLE methods, and the apoptosis cells rate were calculated.Result: the A549 cells xenograf tumour of nude mice models were established successfully, the rate of establishment was100 %., hTRAIL could inhibit the growth of xenograft tumours. The growing speed of group ad-luc-hTRAIL and group ad-hTRAIL were lower than that of group ad-luc and group PBS. At day 4,7, 10,14,21,28, the tumour inhibition rate of group ad-luc-hTRAIL and group ad-hTRAIL were 30.20%,43.93%,52.02%,65.67%,55.37%,51.20% and 34.20%,52.67%,55.70%,58.53%,56.67%,57.50% respectivly。Comparing the tumour volume of each group ,the results were: there is significant difference between group ad-luc-hTRAILand group ad-luc, between group ad-luc-hTRAILand group PBS, between group ad-hTRAILand group ad-luc, between group ad-hTRAILand group PBS(P<0.05); there is no significant difference between group ad-luc-hTRAILand group ad-hTRAIL(P > 0.10). The result of bioluminescence imaging examine displayed that: the location of xenograft tumour in group ad-luc,ad-luc-hTRAIL could express luciferase after gene therapy strarted, and the others couldn't; group ad-luc-hTRAIL reached its peach at day 7, and after that the activity of luciferase decreased obviously; group ad-luc kept high level of expression from day 4 to 14, and decreased slightly when the time point was day 21,28. The result of immunohistochemistry examine displayed that: both of the group ad-luc-hTRAIL and group ad-hTRAIL could express hTRAIL protein substantially,and reached their peak at day 7(the IHS peak value were 6.25+/-2.06,6.5+/-2.887 separately), after that gradualy declined. TUNLE detecting the apoptosis of tumour cells in the tumour tissues displayed that: there were a great quantity of apoptosis of tumour cells in group ad-luchTRAIL,ad-hTRAIL, the apoptosis rate reached their peak value were 60.75+/-8.06%,61.50+/-8.47% separately), after that their apoptosis rategradualy declined. Conclusion:1 The double expressing adenovirus vector- ad-luc-TRAIL could efficiently make the target gene transducted to lung cancer A549 cells xenograft of nude mouse models, and independently expressed TRAIL and luciferase at a high level.2 The expression of TRAIL induced by adnovirus produced significantly growth inhibition and contributed to cell apoptosis , in lung cancer A549 cells xenograft of nude mouse models.3 The expression of luc detected by high sensitivity,cooled-charged coupled device camera in vivo could monitor the expression of the target gene hTRAIL, and also could efficiently monitor the therapeutical effect of hTRAIL.