The Experimental Study Of Treating Pancrease Cancer To Combine Adenovirus-mediated HING4 Gene And Sustaining Low Dose-rate γ Irradiation In Vitro

Pancreas cancer is one of the most common cancer of digestive system .Its morbidity has been rising within the whole world, called "the king of cancer ". Polygenes , many steps and many stages may cause pancreas cancer.Therefore only one method can not reach satisfied treatment effectively in clinic. In recent years, some researchers suggest that gene therapy and other traditional anticancer therapies (eg. radiation therapy, chemotherapy , immunotherapy etc.) combine with each other,which wish to strengthen or cooperate the anticancer effect . That combinative therapy is one kind of the schemes,which has bright prospect comparatively among them. As one new-discovered oncogene, ING4 not only can inhibit growth and induce apoptosis of tumor cells, but also reduce tumor vessel formation and angiogenesis。Because ING4 is fairly anti-tumour function, we use adenovirus-mediated ING4 gene to treat human pancreas cancer panc-1 cells, combining the sustained low dosages the gamma ray radiation to carry out studies about its anticancer effect in vitro and molecular mechanism, providing experimental and theoretical bases in clinical practice.Part One The Experimental Study of Human Pancreas Cancer Panc-1 Cells Infected by the Recombinant Ad-hING4 Adenovirus In VitroObjective To investigate the influence of growth of human pancreas carcinoma cells infected by the recombinant Ad-hING4 and research its antitumor effect in vitro. Methods (1) to increase the recombinant Ad-hING4 and Ad-GFP and detect their potency and identify;(2) to observe their fluorescence expression and their inhibit-growth effect of human pancreas cancer panc-1 cells infected by Ad-hING4 and the Ad-GFP using the inverted microscope and fluorescence microscope;(3) to detect the expression of Ad-hING4 in human pancreas cancer panc-1 cells by indirect immune-fluorescence method;(4) to detect the growing inhibition of the panc-1 cells infected by Ad-hING4 and Ad-GFP using MTT method;(5) to detect of apoptosis induced by Ad-hING4 by flow cytometry.Results (1) to increase the recombinant Ad-hING4 and Ad-GFP and detect their potency and identify;After infection and increasing the recombinant virus many times, the virus potency of Ad-GFP and Ad-hING4 both reached 1010pfu/ml by fluorescence counting method. RT-PCR identified the recombinant virus.Its result indicated that adenovirus-mediated Ad-hING4 gene can be transcripted successfully in Panc-1 cells.(2) to observe their fluorescence expression and their toxic effect of human pancreas cancer panc-1 cells infected by Ad-hING4 and the Ad-GFP using the inverted microscope and fluorescence microscope;To observe under the inverted microscope fluorescence microscope, 90% cells expressed GFP;Ad-hING4 and Ad-GFP both presented very high infection efficiency; Panc-1 cells that infected Ad-hING4 observed under the inverted microscope,appeared to roundout,shed,crimp etc. obviously, which indicated that Ad-hING4 had the obvious inhibit-growth effect to pancreas cancer Panc-1 cells.(3) to detect the expression of Ad-hING4 in human pancreas cancer panc-1 cells by indirect immune-fluorescence method;Panc-1 cells that transfected by Ad-hING4 gene had been expressing the external ING4 protein.(4) to detect the growing inhibition of the panc-1 cells infected by Ad-hING4 and Ad-GFP using MTT method;The panc-1 cells transfected by Ad-hING4 gene inhibited growth obviously, which displayed certain dosages dependence and time dependence. When Ad-hING4 was in 50MOI dosage, panc-1 cells presented more obvious cell-inhibited effect. When ING4 transfected 72 hours, the inhibition rate of panc-1 cells reached 28.7%.(5) to detect of apoptosis induced by Ad-hING4 by flow cytometry.Ad-hING4 group can be seen the apoptotic peak obviously. When 100MOI Ad-hING4 transfected about 48 hours, the apoptosis rate of Panc-1 cells was 24.2%. Conclusions Ad-hING4 can express in the pancrease Panc-1 cells triumphantly,inhibit growth in vitro, and induce apoptosis.Part Two The Experimental Study of the Human Pancreas Cancer Panc-1 Cells Irradiated by Continuous Low Does-rateγray In VitroObjective To investigate the mechanisms of tumor cell death irradiated by continuous low does-rateγray using Iodine-125 seeds and its relationship with dosage, we studied apoptosis and multiplication of human pancreas carcinoma panc-1 cells in vitro. Methods Using self-made irradiation cell unit,Iodine-125 seeds irradiated pancreas cancer panc-1 cells by continuous low does-rateγray:(1) Observation of the morphology change using inverted microscope;(2) Clonogenic assay ;(3) Observation of apoptotic cells by flow cytometry;(4) Detection of apoptosis by DAPI dyeing experiment.Results (1) Observation of the morphology change using inverted microscope Observed under inverted microscope of fluorescence, the Panc-1 cells irradiated by 1Gyγray.90% cells were still adherent and had not seen abnormal in appearance; Panc-1 cells that irradiated by 2 , 4 , 6 , 8 , 10Gy obviously appeared to roundout, shed, crimp etc.,and this change followed by the increasing dosages.(2) Clonogenic assayThe clone formation-rate of underexposed group was 32.2%.The panc-1 cells irradiated by 1 , 2 , 4 , 6 , 8 , 10Gyγray, and the clone suppression rate ascended with rised dosages.(3) Observation of apoptotic cells by flow cytometryIn the normal control group and the 2Gy dose irradiation group, the apoptosis rate of Panc-1 cells were 1.3% and 22.5% respectively.In every group, the proportion of necrosis cells was small (<1%).(4) Detection of apoptosis by DAPI dyeing experimentThe cell nucleus of pancreas cancer panc-1 cells broke into cluster of unequal size fragment irradiated by 125I seeds, formed apoptosis body containing nucleus fragment having a film to wrap up.Conclusions The continuous low does-rateγray irradiation by Iodine-125 seeds can inhibit growth of pancreas cancer panc-1 cells, induce apoptosis and have certain toxic effect.The suppression rate of panc-1 cells increased with the uptrendγray dosages.Part Three The Experimental Study of Treating Pancrease Cancer to Combine Adenovirus-mediated HING4 Gene and Sustaining Low Dose-rateγRay In VitroObjective To investigate its effect of treating pancrease cancer in vitro and mechanisms of tumor cell death,we combined adenovirus–mediated hING4 gene and sustaining Low Dose rateγray using Iodine-125 seeds to provide the experimental basis in clinic. Methods To combine 2Gy dosage with Ad-hING4 gene therapy,the experiment had 5 groups:①normal control group;②Ad negative-control group;③Ad-hING4 group;④single 125I seeds group;⑤Ad-hING4 and 125I seeds combination group. Observation and detection:(1) Observation of the morphology change using the inverted microscope and the inverted microscope of fluorescence;(2) Detection of cell proliferation by clonogenic assay;(3) Observation of apoptotic cells by flow cytometry;(4) Detection of the expression of mRNA of the apoptosis-inhibited protein survivin gene by RT-PCR;(5)Detection of the expression of the apoptosis-relevance protein activated caspase-3 by Western-blotting(6)Detection of the expression of the tumour markers CA199,CA242 and CA153 in supernatant fluid by Protein chip detectionResults (1) Observation of the morphology change using the inverted microscope and the inverted microscope of fluorescenceTo observe under the inverted microscope fluorescence microscope, 90% cells expressed GFP,and Ad-hING4 present very high infection efficiency; Panc-1 cells of Ad-hING4 group,single 125I seeds group and combination group appeared to roundout, shed, crimp(CPE) etc. obviously,while normal control group and Ad negative-control group can not appear these changes.(2) Detection of cell proliferation by clonogenic assayThe clone formation-rate of these groups were respectively (32.93±0.61)%,(32.13±0.41)%,(21.87±0.81)%,(12.93±0.12)%,(7.73±0.76)%。Compared with normal control group and Ad group,the combination group had very obvious difference equally(P<0.001).The clone formation inhibition rate of the single ING4 group and 2Gy group was respectively (33.56±3.67)% and(60.83±0.88)%,while that of combination group amounted to(76.51±2.37)%,the latter surpassed the formers obviously(P<0.001).(3) Observation of apoptotic cells by flow cytometryAd-GFP had certain antitumour effect, but there was no obvious difference compared with normal control group (P>0.05). Compared with normal control group and the Ad-GFP group, Ad-hING4 group,single 125I seeds group and combination group all can increase apoptosis rate of Panc-1 cells, respectively were(17.80±3.03)%,(23.43±0.86)%,(51.23±5.05)%.But the apoptosis rate of combination group was higher than which of single 125I seeds group obviously, reached (51.23±5.05)%。(4) Detection of the expression of mRNA of the apoptosis-inhibited protein survivin gene by RT-PCRAd-hING4 group and single 125I seeds group both can down-regulate the expression of mRNA of the apoptosis-inhibited protein survivin gene, but the down-regulation effect of combination group was more obvious.(5) Detection of the expression of the apoptosis-relevance protein activated caspase-3 by Western-blottingWestern-blotting detection indicated that combination group could more obviously up-regulate the expression of the apoptosis-relevance protein activated caspase-3 than other groups.(6) Detection of the expression of the tumour markers CA199,CA242 and CA153 in supernatant fluid by Protein chip detectionNormal control group and Ad negative-control group had no obvious difference(P>0.05);comparing with normal control group and Ad negative-control group,the tumour markers CA199,CA242 and CA153 in supernatant fluid of Ad-hING4 group,single 125I seeds group and combination group were all reduced,and which of combination group came down most obviously.Conclusions The combination group can inhibit growth of the pancreas cancer cells in vitro.Ad-hING4 maybe can close the apoptosis-inhibited protein survivin, and up-regulate expression of the relevance proliferating protein activated caspase-3 to inhibit proliferation and accelerate apoptosis of tumour cells to cure pancrease cancer cooperatively .