| The main treatment of advanced liver cancer is Transcatheter arterial chemoembolization(TACE).TACE results in the deficiency of blood and oxygen by blocking the supportive artery hence induces the tumor necrosis.But the shortage of oxygen in the tumor micro environment caused by TACE could lead to adaptive response from the remaining cancer cells,therefore improving the tumor infiltrative capability.How to lower the hypoxia adaptive response of the liver tumor cell is the key to enhancing the long-term follow-up result after TACE.Sorafenib is a multi-kinase inhibitor,having multiple therapeutic targets,and is proved to be able to dramatically lengthen the survival time of liver cancer patients.Whether the combination of Sorafenib and TACE would further advance the conditions of prognosis is the hot spot of the current clinical research.Some studies show the combination of the two has a synergistic effect,but the exact mechanism is unclear.Whether sorafenib had improved the efficiency of TACE by suppressing the hypoxia adaptive response of liver cancer cell is still indistinct.In addition,although sorafenib could lengthen the survival time of liver cancer patients,it did not stick out on the therapeutic reaction rate,part of the benefited patients' tumor even kept increasing.We need new means and methods to evaluate the efficiency of sorafeniba in a more precise way.Sorafenib as a targeted therapeutic drug is very expensive,if it can be decided in an early stage whether it has efficiency or efficiency with what dose amount to a specific patient,we will be able to stop giving the same medicine or change to another in terms of the refractory patients,hence reduce the patients' huge financial burden.We can also monitor and issue reasonable dosage according to the situation of the patients who had side effects and needed the reduction.Based on what have been discussed above,our project proceeded through following three aspects: 1.The research on sorafenib's disturbing effect on hypoxia adaptive response of liver cancer cell.Materials and methods: human liver cancer cell SMMC-7721 conventionally cultured to 70% blended,then divided into following three groups: normally cultured SMMC-7721 cell;under hypoxic condition 24 h cultured SMMC-7721 cell;pre-sorafenib(4?mol/L)-processed SMMC-7721 cell under hypoxic condition 24 h.Used CCK-8 to test the proliferative activity of the different groups;used flow cytometer(FCM)to test cell-cycle distribution;used cell adhesion assay to test adhesive change;used Boyden small room experiment to test cell invasive changes.Result: 1)Cell proliferation activity result: Lack oxygen cultivated group(150?M Co Cl2,24h)compared with control group,proliferative ability had slightly increased,but the difference was not significant;sorafenib(4 ? mol/L 1h)preprocessed lack oxygen cultivated group compared with control group and lack oxygen cultivated group,proliferative ability had dramatically decreased(P<0.01).2)Cell cycle analysis: Lack oxygen cultivated group(150?M Co Cl2,24h)compared with control group,S-phase cell fraction had dropped,G0/G1 phrase and G2/M phrase cell fractions had risen;sorafenibproccessed group compared with control group,S-phrase cells somewhat decreased but not significantly,the increase of G0/G1 phrase cells was obvious(P<0.05),G2/M phrase cells remained unchanged;sorafenibproccessed group compared with lack oxygen cultivated group,S-phrase cell fraction somewhat rose and was close to control group level,G2/M phrase cell fraction dropped obviously and was close to control group level.3)Cell adhesion ability result: Lack oxygen cultivated group cell adhesion ability slightly increased compared to control group,after sorafenibproccess,cell adhesion ability decreased abruptly(P<0.001).4)Boyden small room experiment showed lack oxygen cultivated group(150?M Co Cl2,24h)compared with control group,invasive ability had decreased(P<0.05);sorafenibproccessed group compared with control group and lack oxygen cultivated group,invasive ability had decreased obviously(P<0.01)in both comparisons.Conclusion: 1)Hypoxia could induce SMMC-7721 liver tumor cell initiate hypoxia adaptive response which appeared as tumor proliferation and invasive ability increasing,cell cycle G0/G1 phrase and G2 phrase were also hold up.2)When SMMC-7721 liver tumor cell was lack oxygen cultivated after sorafenib pre-processing,its hypoxia adaptive response was dramatically restrained,which indicted the potential that sorafenib has in restraining the hypoxia adaptive response from liver tumor cell.2.The research on sorafenib's effect on lack oxygen liver tumor cell's tumorigenic ability.Materials and methods: used external GFP(green fluorescent protein)to transect SMMC-7721 liver tumor cell,used puromycin sterilization to select Stable cell line and got SMMC-7721 with stable cell line GFP.Divided 16 nude mice into 4 groups,each had 4 mice,and used 1ml injector to inject 2×106SMMC-7721-GFP cells that were processed under different conditions into the back skin of each mouse.Injected normally cultivated SMMC-7721-GFP cell to group A nude mice;lack oxygen cultivated SMMC-7721-GFPcell to group B;sorafenibproccessed plus lack oxygen cultivated SMMC-7721-GFP cell to group C;group D was treated the same way as group C,except being fed sorafenib everyday after the injection.Put the injected nude mice under fluorescent living imager for living imaging on day 2,9,16,23,30,40 to tell the situation of tumor and tumorigenic rate.Used vernier caliper to measure the length and width of the tumor then calculated the volume of it,drew the tumor growth curve;40 days after the injection,put them to death,immumohistochemical stained the tumor with VEGF,CD34.Result: Puromycin kill curve confirmed that the minimum concentration needed to kill all the not infected cells was 4ug/ml.Used the puromycin with this concentration to get four generations of stable cell line SMMC-7721-GFP,then after different proccession injected them to mice to develop tumors.On day 23 after the tumor formed,size of tumor in group D mice was smaller than the control group(P<0.05);day 30,group C's size was obviously different compared with control group(P<0.05);day 40,group D and group C's tumor size were all significiantlly different from control group(P<0.001).On day 40,the weight of tumor in group D were obviously lighter than the rest three groups(P<0.01),but there were no clear variation among all groups on each time node.Absorbance detection result of all groups showed the absorbancy IOD value could quite accurately reflect the tumor growth change status 4 weeks after the tumor formed through under skin injection,and it matched the actual measured value.Immunohistochemical result showed that compared with the control group the lack oxygen preprocessed group's VEGF and CD34 expression were slightly higher,but the sorafenib preprocessed group had a decreased expression;sorafenib preprocessed plus constant gavage administration group had a dramatically decreased VEGF and CD34 expression compared with control group(P<0.05,P<0.01),which meant sorafenib preprocess and constant gavage administration were able to refrain the expression of VEGF and CD34.Conclusion: 1)Built GFP fluorescent lentivirus plasmid transfection cell SMMC-7721,used puromycin selection to get stable cell line SMMC-7721-GFP;2)Alive nude mice tumor experiment showed lack oxygen processed tumor cell had a more obvious tumor inhibitory effect along with the extension of sorafenib active time;3)GFP fluorescent marked SMMC-7721 cell was able to form the tumor inside the nude mouse,and the growth and metastases of the tumor could be observed through GFP fluorescence imaging,its size could also be predicted and compared;4)The continuous use of sorafenib would restrain the angiogenesis,adhesion and invasion ability of tumor,which was further proved through the VEGF,CD34 immunohistochemical research.3.Use optical molecular imaging to study sorafenib's early therapeutic effect on liver cancer.Materials and methods: human liver cancer cell SMMC-7721 conventionally cultured to 70% blended,divided into three groups according to the method used in research 1.After being adaptive bred for 1 week,12 nude mice were divided into 4 groups,each had 3 mice,then they were formed tumor inside through back skin injection.Injected normally cultivated SMMC-7721 cell to group A nude mice;lack oxygen cultivated SMMC-7721 cell to group B;sorafenib processed plus lack oxygen cultivated SMMC-7721 cell to group C;group D was treated the same way as group C,except being fed sorafenib everyday after the injection.6 weeks after the tumor formed through under skin injection,injected Annexin V marked by fluorochrome Cy5.5 into nude mice to apoptosis image the tumor.Result: The formation of tumor through under skin injection went smoothly.Used Annexin marked by Cy5.5 for intratumoral injection,each mouse was injected 2ug,chemiluminescence assay showed the apoptosis situation of tumor 24 h later: compared with control group,oxygen lacked cobalt dichloride processed group and sorafenib preprocessed group's degree of tumor apoptosis slightly increased,but not obvious;sorafenib processed plus stomach medicine given group could significantly induce the tumor apoptosis,absorbance value had a critical statistic difference(P<0.05).Conclusion: 1)Annexin marked by Cy5.5 could target bind to the tumor apoptosis cells and develop image through molecule optics;2)The effect of so rafenib in treating liver cancer could be observed within 6 weeks by apoptosis optical molecular imaging using Annextin marked by Cy5.5. |
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