The Animal Experimental Studies Of The Interventional Chemotherapy With Gemcitabine And Cisplation On The Human Lung Cancer A549 Cells

Part I Exploration and application of effective method to establish nude rat xenograft model of human lung cancer cells A549Objective:To optimize the effective model of xenograft tumor in nude rats of human lung cancer cells A549 for providing the experimental basis for cancer interventional studies in vivo.Methods:A549 cell lines were transplanted subcutaneously right arm in nude rats,then prepared as single-cell suspension and tumor tissue block to transplant into postotic skin respectively once again.The tumor production,growth situation and cell cycle of primary xenograft tumor group(A group),the secondary single-cell suspension group(B group)and the secondary tumor block group(C group)were investigated.There were 6 rats in every group.observe general situation、tumor production、growth situation and pathology of xenograft tumor.The change of the cell cycle was assayed by flow cytometry.Results:Nodule were found in place injected or transplanted after four to five days.Diameter of the lump were four to five milimeters,ten to twelve millimeters after 14 days,lobulated、compact、defined margin、good activity、none-liquefaction.The lump magnify with central liquefaction.The tumor production rates were 52.3% 、 63.4% and100% in three groups.There was a significant difference between A group and C group(P<0.01).The tumor production rate of C group were significantly higher than that of other two groups.C group also had a fast proliferation and little variation.Under the microscope,xenograft tumor were found arranged in disorder、obvious atypia of nucleus、magnified nuclear-cytoplasmic ratio、dark and thick chromatin、marked nucleus and easily seen abnormal mitoses.Cell cycle analysis provide that cell proliferation index were(32.31±8.72)%、(47.68±4.72)% and(48.61±5.06)% in A、B、C group.There was a significant difference between A group and B、C group(P<0.05),there was no difference between B group and C group. There was a high S,G2/M rato and cell proliferation index in C group.Conclusion:The secondary A549 tumor block for transplantation(C group) could significantly enhance the tumor formation rate,was an effective model of xenograft tumor in nude rats.Part II Research of inhibiting effect for A549 cell lines treated with gemcitabine and cisplatin by different administration routeObjective:To observe the inhibiting effect for human lung adenocarcinoma treated with gemcitabine and cisplatin by arterial or intravenous chemotherapy,which would be evaluated the optimum way of administration route.Methods:The human lung cancer cells line(A549) was transplanted into nude rats after subculture with the secondary A549 tumor block way and established the model of xenograft tumor in nude rats.The models were divided into 4 groups,normal control group、sham-operation group、arterial or intravenous chemotherapy group,with 10 rats in each group.Then dynamically observed common situation of nude rats、the change of tumor volume、the anti-tumor rate、Bcl-2 and Caspase-3 protein expression、HE stain、detecting of Ki67 protein by immunohistochemical staining and compared the inhibiting effect of these two different administration routes with gemcitabine and cisplatin.Results:Arterial and intravenous chemotherapy groups both had suppression of tumor growth,and arterial chemotherapy group showed a more obvious suppression compared to the other groups(P<0.05).The quality(g) of 4 groups were in arterial chemotherapy group(1.91±0.19)、in intravenous chemotherapy groups(2.61±0.21),which were lower than in bearing cancer group(4.58±0.46)、sham-operation group(4.71±0.53).The difference was statistically significant(P<0.05).Furthermore,arterial chemotherapy group had obvious 57.6% anti-tumor rate compared intravenous one 42.4%(P<0.05).Transplantation tumor HE stain showed the tumor apoptosis both in arterial and intravenous chemotherapy groups,but significant in arterial chemotherapy group.Bcl-2 and Caspase-3 protein expression displayed that both arterial and intravenous chemotherapy could promote the tumor apoptosis effectively.And arterial route showed much more obvious tumor apoptosis.Conclusion:Arterial route of gemcitabine and cisplatin for lung adenocarcinoma treatment is a more effective way to restrain the tumor growth in clinical application.Intraarterial chemotherapy has advantages to intravenous chemotherapy in gemcitabine and cisplatin.Part III Concentratin change of chemotherapeutic agents in plasma and tissue after intraarterial and intravenous injection Gemcitabine and CisplatinObjective:To study the drug concentration change of chemotherapeutic agents in plasma and tissue after arterial and intravenous injection.Methods:Gemcitabine and Cisplatin were injected into the 40 mature nude rats with xenografted tumor.The rats were divided into vein injection group and artery injection group randomly.The rats were given Gemcitabine and Cisplatin respectively.Blood samples were collected at 5,10,20,40,80,120,360,720 min after injection and the tumor tissue specimens were collected at 10,40,120,720 min after injection.Gemcitabine concentration in plasma and tumor tissues were determined by high performance liquid chromatography(HPLC) method,while Cisplatin was determined by inductively coupled plasma-mass spectrometry(ICP-MS) method.The data were analyzed by the pharmacokinetic program.Results:Regular concentration change of the three drugs in plasma and tissues were observed after the intravenous and arterial injection,which met the two-compartment model.The pharmacokinetic parameters of the two drugs after intravenous and arterial injection were different.The peak concentration in plasma of artery injection group [Gemcitabine:(20.84±10.11)μg/m L;Cisplatin:(15.13±7.12)μg/m L] were lower than those of the vein injection group [(Gemcitabine:(28.96±7.02)μg/m L;Cisplatin:(21.64±9.72) μg/m L],the peak concentration in tissues of the arterial injections group [Gemcitabine:(20.18±9.43)μg/m L;Cisplatin:(6.98±0.31)μg/m L] were higher than those of the intravenous injection group [Gemictabine:(18.19±10.30)μg/m L;Cisplation:(3.04±0.11)μg/m L],and area under curve(AUC) value [(Gemcitabine:(2641±411)μg/(min m L);Cisplatin:(6025±870)μg/(min m L)],in tissues of the arterial injections group were higher than those of the intravenous injection group[Gemcitabine:(1663±568)μg/(min m L);Cisplatin:(1780±883)μg/(min m L)].The difference was statistically significant(P<0.05 or P<0.01).Conclusion:Intraarterial chemotherapy has advantages to intravenous chemotherapy in gemcitabine and cisplatin.These advantages depend on the drug pharmacological properties.