【作者】 刘玉荣；

【导师】 陈东；

【作者基本信息】 吉林大学 ， 人体解剖与组织胚胎学， 2007， 博士

【摘要】 本实验研究是建立在既往对三氯化镧（LaCl₃）体外抗肝癌作用初步研究的基础上，观察三氯化镧对小鼠肝癌原位移植模型体内的抗肿瘤作用，并初步研究其对细胞周期相关蛋白PCNA、p16、CyclinD₁及CDK4表达的影响。在体内实验证实三氯化镧确切的抗肿瘤作用之后，体外对其抑制人肝癌细胞SMMC-7721生长和细胞周期阻滞作用加以证实。继而从细胞周期调控相关蛋白及相关基因CyclinD1、p16 mRNA表达及pRb信号传导通路等方面对三氯化镧促进肝癌细胞周期阻滞机理作进一步的探讨，得出以下结论：1．0.5mg/kg LaCl₃组及0.1mg/kg LaCl₃与10mg/kg 5-Fu联合用药组对小鼠肝癌原位移植模型有显著抗肿瘤作用。并使细胞周期相关蛋白CDK4，CyclinD₁及PCNA表达下降，p16表达上调。2．LaCl₃体外能显著抑制人肝癌细胞SMMC-7721的增殖并可通过作用于p16/CDK4/CyclinD₁/pRb通路将人肝癌细胞SMMC-7721细胞阻滞在G₀/G₁周期。本研究首次利用体内、体外实验，从蛋白到基因水平完整的研究了三氯化镧的抗肝癌作用及其机理，为其研发及临床应用提供理论依据和实验基础，并为肝细胞癌的临床治疗开辟新径。更多还原

【Abstract】 IntroductionHepatocellular carcinoma（HCC） is one of the most malignancy cancer and with a worst prognosis. which attract rate is higher in our country and 90% of liver caner is hepatocellular carcinoma.Though the therapeutic way is numerous ,the effect is very limited for this reason,searching more effective therapeutic schedule is increasingly important..Plenty of reach results have certificated that cell cycle out control is the centre accident in ontogenesis ,if we can find some ways to prevent the anomaly regulation then can prevent tumour development, infiltration, and the metastasis of carcinoma.It has been proved the rare earth compounds, which obviously restrain the growth of the tumor in vitro and in vivo, have various biological activity and functions. On the other hand, the repressing tumors’ occurrence of rare earth was found by the epidemic investigation. It has been indicated more meaningful that the rare earth compounds posses the characteristic of selectively repressing or killing tumor cells with less affect on the normal cell, and the light rare earth has been accumulated primarily in the liver, such as lanthanum and cerium, etc. The stydy about the chlorinated lanthanum on cell cycle of hepatocellular carcinoma and its mechanism have not been reported in domestic or foreign literatures.Objective:（1） To study the anti-carcinoma effects on hepatocellular carcinoma in orthotropic transplantation tumor model of mice by LaCl₃ and to examine the cell cycle hold back effect in situ in vivo.（2） To conform cell cycle hold effect in human hematoma cells SMMC-7721 induced by LaCl₃ and to study the influences on expressions of associated cell cycle protein （PCNA,CDK4,CyclinD1 and p16,）and gene（cyclinD1,p16）and cell proliferation signal transduction pathway （p16/cyclinD1/CDK4/pRb） in SMMC-7721 cells in the course of inducing cell cycle blockage by LaCl₃（3） To provide scientific experimental basis for befallen to HCC therapy. Materials and methods1 .Study on the anti-carcinoma effects of orthotopic transplantation tumor model of hepatoma in mice by bufalin in vivo（1）To fix on the dose of LaCl₃in vivo: 18 healthy male ICR mice were divided into high dose （2mg/kg）, moderate dose （1.0 mg/kg） and low dose of LaCl₃ group（0.5mg/kg） randomly. LaCl₃was injected intraperitoneally at the three doses above for day1-10, respectively. Body weight, appetite and behavior of mice were observed, and the anti-carcinoma doses of LaCl₃ for fixed on 1.0mg/kg, 0.5mg/kg and 0.1 mg/kg.（2）The success rate value: we established the orthotopic transplantation tumor model of mice by H₂₂ cell line injection directly .In order to evaluate the circumstance of tumour genesis, 10 mice were injection with H₂₂ into liver ,after 8 days, the tumour were found in all of the mice liver（3） 90 orthotopic transplantation tumor model of hepatoma in mice were divided into LaCl₃ groups, NS group, 5-Fu group and joint group randomly. LaCl₃ was injected imtraperitoneally at dose of 1.0 mg/kg, 0.5mg/kg and 0.1 mg/kg for dayl-10,respectively. NS group were injected equal volume saline as above ;5-FU group were injected 20mg/kg 5-Fu;jiont group were injected with 0.1mg/kg LaCl₃and 10mg/kg 5-Fu.continue injection imtraperitoneally for 10 days.All of the mice in each group were killed at day2 after withdrawal and detected on morphological and ultrastructural changes in spleen,liver, kidneys and tumor tissues by phase contrast microscope.. The cell cycle associated protein were examined by immunohistochemistry with SPsystem and the cell cycle associated gene expression were examined by RT-PCR.（4） The another 90 orthotopic transplantation tumor model of hepatoma in mice were used to observed the survival time .2.Growth suppression and cell cycle blokage induction in hepatoma cells SMMC-7721 acted on LaCl₃The growth curve, clone inhibition ratio eneral histological variety and the AFP value of SMMC-7721 cells were observed at different time after treated by different doses of LaCl₃ in vitro. At the same time, the related indexes about changing of cell cycle were detected by flow cytometry.3. Influences on signal transduction pathway p16/CDK4/cyclinD1/pRb of human hepatoma cells SMMC-7721The changes of cell cycle associated protein CDK4,CyclinD1, PCNA,p16 by treated with different doses of LaCl₃ were determined by immunohistochemystry with SP system, and the changes of gene mRNA,cyclinD1 and p16 were detected by RT-PCR. The changes of protein CaM,pRb,p-pRb, Cdc25A, were determined by Western blotting.Result1 .Study on the anti-carcinoma effects of orthotopic transplantation tumor model of hepatoma in mice by bufalin in vivoWe established the orthotropic transplantation tumor model of hematoma in mice successfully The tumors genesis rate is 100% . we fixed on the anti-carcinoma doses of LaCl₃ for 1.0mg/kg, 0.5mg/kg and 0.1 mg/kg.and those doses are safety to mice.The inhibitory rate of 1,0.5,0.1mg/kg LaCl₃ groups, joint group were32.2%、47.8%、24.3% and 69.5%, single 5-Fu group is 57.3%, among of them ,0.5mg/kgLaCl₃ joint group and positive group >40%, the inhibitory rate of tumors is effective . The survival time in 0.5mg/kg LaCl₃ group and joint group were prolonged significantly （compared with NS group, P<0.05 and P<0.01）.There are no significant morphological changes detected in the myocardium, spleen, liver and kidney tissues and no significant influence on weight by LaCl₃. Tumor tissues were mainly necrosis in severe or moderate degree in 0.5, 1mg/kg LaCl₃LF groups, and mild degree or moderate degree is in 0.1 mg/kg LaCl₃ and NS group.The liver cancer tissue were remained for immunohistochemistry detect, the results show that positive expression rate of PCNA,CDK4,CyclinD1 were low and the p16 were significantly higher ,mostly of which expression in moderate degree. The RT-PCR results indicated that the expression level of cyclinD1 mRNA degrade and pl6 upgrade by deal with LaCl₃. 0.50, 0.1 mg/kg LaCl₃ and LF can significantly inhibited the secretary of AFP of orthotropic transplantation tumor model of hematoma in mice2.Growth suppression and cell cycle blokage induction in hepatoma cells SMMC-7721 acted on LaCl₃The SMMC-7721 cells were observed to be shrunken and decreased in size by LaCl₃ under invert microscope. As time prolonged and concentration increased by LaCl₃, more and more SMMC-7721 cells suspended in culture medium. 0.05 mmol/L LaCl₃ group cells have no different compare with without drugs group, 0.1 mmol/L LaCl₃ group and DDP group cell begein changes from the third day after treated with LaCl₃.then the 0.5 and 1 mmol/L LaCl₃ begin changed from the first day ,only several cells are alive in fivth day ,and none live cell in seventh day. without drugs were in active growth with pathological mitoses and various cell nuclear.MTT results manifest thatLaCl₃ could inhibit the growth of SMMC-7721 cells significantly and the inhibition was concentration-dependence manner in a range of concentration. The inhibition rates of 0.05 mmol/L LaCl₃ were not increased obvious.（P>0.05）0.1、0.5、1 mmol/L LaCl₃ and DDP groups can significantly reduce the cells number compare with the control group （P<0.05）.then,LaCl₃ have no influence on human normal cells （P>0.05）.The SMMC-7721 cells clone forming were significantly inhibited by 0.1、0.5、1mmol/L LaCl₃ After two weeks . the inhibitory rate were 51%、67%、97%..Flow cytometry result show that cell percent rate of G₁/G₀ gradually increase and the number of cells in S phase was decreased while the number in G₁/G₀ phase was increased by flow cytometry. These results indicate that LaCl₃ restrain the cell proliferation by blocking the cell cycle at G₁/G₀ phase in SMMC-7721 cells.The result of AFP determine in supper serum of the culture cell show that 0.1、0.5 and 1mmol/L LaCl₃ can significantly inhibited the AFP secrete value compare with negative control group（ P<0.01 ）.3.Influences on signal transduction pathway p16/CDK4/cyclinD1/pRb of human hepatoma cells SMMC-7721Immunohistochemistry result show that Low expression of PCNA、CDK4 and CyclinD1 by deal with 0.10、0.50 and 1.00 mmol/LLaCl₃ compare with control（P<0.01）, and the concentration is higher the positive color is weaker ,then the positive stain color of p16 is obvious enhance compare with negative control group, （P<0.01） which have a dose reelection.The results of semi quantitative reverse transcription polymerase chain reaction show that Cyclin D1 mRNA gene was not to be expressed and p16 mRNA gene was to be more expressed in SMMC-7721 cells with raised concentrations by LaCl₃.Western-blotting result show that Rb protein value was obvious increased by LaCl₃ in SMMC-7721,but Rb （ p-pRb ） protein was decreased on the contrary, CaM protein is decrease too, then the Cdc25A protein be in no varsity.Conclusions（1） 0.5mg/kg LaCl₃ and joint group have significant anti-carcinoma effects on orthotropic transplantation tumor model of hematoma (H₂₂) in mice and decreasing the cell cycle associate protein CDK4, CyclinD1 and PCNA, increasing the expression of p16.（2） LaCl₃ has inhibitive effects and cell cycle blockage effect induction to SMMC-7721 cells, To induce cell cycle blockage of tumor cells may be the one of anti-tumor mechanism of LaCl₃（3） LaCl₃ blockage the cell cycle in G₀/G₁ by p16 / CDK.4 / CyclinD1 / pRb cell proliferation pathway.更多还原