| Objective:Tumour is one of import diseases impacting on human health in the 21th.Before,operation,radiotherapy chemotherapy and biotherapy became four measure of management.Human is absorbed in improving four management measure and developing targeted new agents recently. Experiment study approve chemotherapy have became the determinative means of most tumour.The experiment is based on theory that chemotherapeutic drug have the more injury on the more active cells.With a series of experiment index,we discuss the mechanism of EGF chemotherapeutic drug sensitivity.At the same time,the experiment is assistant to animals experiment and clinical therapy. Methods:Rat intestines cancer cell(Colo26) and human laryngeal cance(rHep-2) display growth,and subcultured with two or three days in 5%CO2,37℃incubator,and in RPMI-1640 culture medium(PH=7.4)of containing 10% bovine calf serum,penicillin(100U/ml),streptomycin(100U/ml)and 1% glutamine.When growing rightly,the cells digested as simple cell (cell density 2×104/ml)with 0.25% trypsin are transplanted in 96-hole-plank(200μl/hole)and display growth after 24h. We drop EGF 20μl into each hole at the end consistence 0.01ng/ml,0.1ng/ml,1ng/ml,10ng/ml,100ng/ml separately.Each dose group is repeated three times.We observe the best dose of EGF on the proliferation of Colo26,Hep-2 cells using MTT after 24h. Then,the COLO26 cells digested as simple cell(cell density 2×104/ml)with 0.25% trypsin are transplanted in three block of 96-hole-plank(200μl/hole),and display growth after 24h.We drop EGF 20μl into each hole at the end consistence 0.1ng/ml,1ng/ml,10ng/ml separately.Each dose group is repeated three times.Resembly,the Hep-2 cells digested as simple cell(cell density 2×104/ml)with 0.25% trypsin are transplanted in three block of 96-hole-plank(200μl/hole),and display growth after 24h.We drop EGF 20μl into each hole at the end consistence 0.01ng/ml,0.1ng/ml,1ng/ml separately.Each dose group is repeated three times.We observe the best time effect of EGF on the proliferation of Colo26,Hep-2 cells with MTT at 6,12,24,48 and 72h. By the front course,we gain the best dose and time segment of EGF on the proliferation of Colo26,Hep-2 cells.Afterward,the Colo26 and Hep-2 cells digested with 0.25% trypsin as simple cell(cell density 3×104/ml)are transplanted in 24-hole-plank(1ml/hole),and display growth on a small glass plates in each hole.We bring into effect experiment after 24h,repeat three holes with each chemical drug.The control group:After 48h we drop 5-FU(the end consistence is 250μg/ml)and DDP(the end consistence in 24μg/ml)10μlmerely,take out small glass plates disposed to mensurate PCNA index and HE dyeing.the EGF experiment group:After 24h We drop 100ng/ml EGF 10μl(the end consistence is 1ng/ml in each hole)into Colo26 cells and 1ng/ml EGF 10μl(the end consistence 0.01ng/ml is each hole)into Hep-2 cells,We drop 5-FU(the end consistence is 250μg/ml)and DDP(the end consistence is 24μg/ml)10μl.Observing 48 hour,we take out small glass plates disposed to mensurate PCNA index and HE dyeing. The Colo26 and Hep-2 cells digested as simple cell(cell density 1.5×106/ml)with 0.25% trypsin are transplanted in 24-hole-plank(1ml/hole),and display growth after 24h.Then we bring into effect experiment after 24h,and repeat three holes with each chemical drug.The control group:After 48h we drop 5-FU(the end consistence is 1/16 of 250μg/ml)and DDP(the end consistence is 1/16 of 24μg/ml)10μl merely,collect cells to make cell cycle.The EGF experiment group:After 24h We drop 100ng/ml EGF 10μl(the end consistence is 1ng/ml in each hole)into Colo26 cells and 1ng/ml EGF 10μl(the end consistence 0.01ng/ml is each hole)into Hep-2 cells,we drop 5-FU(the end consistence is 1/16 of 250μg/ml)and DDP(the end consistence is 1/16 of 24μg/ml)10μl.Observing 48 hour,we collect cells to make cell cycle. We mensurate EGFR expression of cancer cells before chemotherapy with EGFR antibody to discuss initially whether EGFR expression is related with the effect of EGFchemotherapeutic drug sensitivity. Result:①We observe the dose-effect relation of EGF in the proliferation of Colo26 cancer cells with MTT:the absorbance values have no significant difference(P﹥0.05)between each dose of EGF experiment group and the control group dose;②We observe the dose-effect relation of EGF in the proliferation of Hep-2 cancer cells with MTT:the absorbance values have significant difference(P﹤0.01)between 0.01ng/ml,0.1ng/ml dose of EGF experiment group and the control group dose;the absorbance values have significant differenc(eP﹤0.05)between 1ng/ml,10ng/ml,100ng/ml dose of EGF experiment group and the control group dose;③We observe the time-effect relation of EGF in the proliferation of Colo26 cancer cells with MTT:the absorbance values have significant differenc(eP﹤0.05)between 24,48,72h time of EGF experiment group and the control group time;the absorbance values have no significant difference(P﹥0.05)between 6,12h time of EGF experiment group and the control group time;④We observe the time-effect relation of EGF in the proliferation of Hep-2 cancer cells with MTT:the absorbance values have significant differenc(eP﹤0.05)between 24,48,72h time of EGF experiment group and the control group time;the absorbance values have no significant difference(P﹥0.05)between 6,12h time of EGF experiment group and the control group time;⑤We mensurate the effect of EGF in chemotherapeutic drug sensitivity with immunohistochemical staining:Colo26 cancer cells:the PCNA index have nosignificant difference(P﹥0.05)between PCNA index of EGF experiment group and that of the control group,but PCNA index of EGF experiment group reduces;Hep-2 cancer cells:the PCNA index have significant difference(P﹤0.01)between PCNA index of EGF experiment group and that of the control group,and PI of EGF experiment group reduces evidently;⑥We mensurate the effect of EGF in chemotherapeutic drug sensitivity with FCM:Colo26 cancer cells:the S phase fraction,G2/M phase fraction and cell proliferation index[PI=(S+G2/M)(/S+G2/M+G0/G1)%] have no significant difference(P﹥0.05)between PI of EGF experiment group and that of the control group,but PI of EGF experiment group reduces;Hep-2 cancer cells:the S phase fraction and PI have significant difference(P﹤0.01)between PI of EGF experiment group and that of the control group,and the S phase fraction and PI of EGF experiment group reduces evidently(especially cell toxicity of cisplatin on Hep-2 cancer cells),but does not reduce G2/M phase fraction;⑦We mensurate possible mechanism of the effect of EGF in chemotherapeutic drug sensitivity with immunohistochemical staining:The average percentages of positive cell of Hep-2 cancer cells are (40±4.10)%,that of Colo26 cancer cells are (15±3.85)%. Conclusions:The EGF experiment group can enhance cytotoxicity of EGF on Colo26 and Hep-2 cancer cells,and chemotherapeutic drug sensitivity acts under the condition with the dose-effect and time-effect relation.In the experiment,EGFR...
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