【作者】 陈红锦；

【导师】 曾莉；

【作者基本信息】 南京中医药大学 ， 中医外科学， 2013， 博士

【摘要】 第一部分结肠荧光原位移植瘤模型构建与生物学功能鉴定目的建立稳定、高表达绿色荧光蛋白标记的裸鼠结肠原位移植瘤模型。方法采用绿色荧光转染的人结肠癌HCT-116细胞建立裸鼠的皮下瘤模型,活体荧光影像观察到绿色荧光标志的肿瘤生长至1Omm×10mm,选取15只BALB/C裸鼠进行结肠原位移植。利用荧光影像系统在不同时间位点观察裸鼠原位移植瘤的生长、转移情况。结果15只裸鼠结肠原位荧光移植瘤模型均成功构建,未发生与手术相关的并发症和死亡。在第3周时,所有动物模型在荧光下观察到肿瘤。肿瘤体积随着时间推移逐步增大,术后3、4、5、6、7周不同时间点整体荧光影像测量原位移植肿瘤计算体积大于体表游标卡尺测量计算的体积,但差异无统计学意义(t=-1.280,—1.115,0.718,—0.199,—0.386,P>0.05)：测量方式与时间的交互作用,两种测量方法的结果差异有统计学意义(F=29.546,P<0.05)。实验终点时存活8只动物模型,其中6/8的动物模型发生肿瘤转移。结论结肠原位荧光移植瘤模型技术可行,成活率高,能够进行体内实时和无创地动态观察和分析肿瘤细胞的生长与转移情况,是研究结直肠肿瘤转移模式和分析药物治疗效果较理想的动物模型。第二部分黄芩苷抑制错配修复基因功能缺失的裸鼠结肠原位移植瘤的实验研究目的研究黄芩苷抑制错配修复基因hMLHl功能缺失的人结肠癌HCT-116细胞裸鼠原位移植瘤生长、转移的作用。方法建立人结肠癌HCT-116细胞裸鼠荧光原位移植瘤模型。60只荷瘤裸鼠随机分为阴性对照组(G1),50mg、100mg、200mg/kg黄芩苷组(G2-G4),每组15只。治疗组分别给予相应剂量的黄芩苷灌胃,2/日；阴性对照组采用等量5％NaHCO3灌胃。荧光影像系统下观察原发肿瘤的体积、血管密度以及肿瘤转移情况,并分析实验动物的体重、生存状况和死亡率。结果药物干预后的第14、21、28天,G2-G4组肿瘤生长速度低于G1组,荧光测量的肿瘤体积(mm3)明显小于G1(14d：832±637；21d：2012±1566;28d：2494±1557),差异具有统计学意义(F=4.433,P<0.05),但治疗组间比较差异无统计学意义。实验终点(第28天)时,G2、G3组实验动物的存活率分别为9/15、13/15,生存状态分析显示G3组明显优于G1组和G4组,差异具有统计学意义(x=4.665、3.980,P<0.05);体内开放观察结果显示各组间原发肿瘤的重量、表面血管密度和肿瘤转移情况比较,差异均无统计学意义。结论黄芩苷对错配修复基因hMLHl功能缺失的结肠癌HCT-116细胞裸鼠原位移植瘤生长具有明显的抑制作用；100mg/kg可能是一个比较理想的治疗剂量。第三部分黄芩苷诱导HCT-116-GFP肿瘤细胞凋亡相关蛋白表达差异影响的研究目的探讨黄芩苷抑制错配修复基因功能缺失的裸鼠结肠原位移植瘤的分子机理。方法建立绿色荧光蛋白标记的裸鼠人结肠癌HCT-116中瘤细胞荧光原位移植瘤模型,三种浓度的黄芩苷溶液(50mg/kg、100mg/kg、200mg/kg)进行干预,检测肿瘤细胞细胞周期、微卫星不稳定变化、相关凋亡基因。结果黄芩苷干预后的肿瘤细胞主要在G2/M期阻滞,与对照组比较差异有统计学显著性意义；黄芩苷具有诱导肿瘤细胞凋亡的作用,与对照组相比差异有统计学意义(G1vs.G2P=0.000, Glvs.G3P=0.004, Glvs.G4P=0.028);实验组与对照组的MSI未见明显变化；与对照组比较,黄芩苷干预后肿瘤细胞的PCNA、P53、hMLH1、hMSH2、TGF-β1、 IGF-IIR基因的蛋白表达发生了明显改变,差异有统计学意义(P<0.05),而凋亡基因Bcl-2以及Bax基因蛋白表达差异无统计学意义(P>0.05)。结论黄芩苷抑制HCT-116细胞移植瘤的作用主要是通过调节PCNA、P53基因、hMLH1基因、hMSH2基因、TGF-β1基因、IGF-IIR基因的蛋白表达,诱导HCT-116肿瘤细胞凋亡,促使肿瘤细胞在G2/M期通路阻滞,但是其功能之间的联系需要做进一步的研究。更多还原

【Abstract】 Part I:Establishment of orthotiopic model of human colon cancer marked by green fluorescent protein and its biological characteristicsObjective To establish a stable orthotiopic model with high green fluorescent protein (GFP) expressing in nude mice.Methods Human HCT-116colon cancer cells transfected with GFP were used to build a subcutaneous tumor model in nude mice. Fifteen BALB/C nude mice were selected to undergo orthotiopic transplantation of colon when the GFP-labeled tumor grew to lOmm×lOmm as observed by in vivo fluorescent microscopy. The growth and metastasis of orthotopically implanted colon cancer cells were observed with fluorescent imaging system at different time points.Results GFP-labeled colon cancer models were successfully established in all the15nude mice, and there was no surgery-related complications or death. Tumors marked by GFP were observed under fluoroscope in week3. The size of the tumors progressively increased with time. The volumes of the orthotopically transplanted tumors obtained from global measurement using fluorescent imaging system was larger than those measured by peripheral vernier calipers at postoperative week3,4,5,6,7, while no statistically significant difference was observed (t=-1.280,-1.115,0.718,-0.199,-0.386, P>0.05); There was a significant difference in the interation of measure method and different time points (F=29.546, P(?)0.05). Eight nude mice survived at the end of the experiment, and tumor metastasis was observed in6mice.Conclusion It is technically feasible to construct GFP-labeled colon cancer orthotiopic transplantation model with high survival rate. The mice model could be used for real-time, in vivo, no-invasive and dynamic observation and analysis of the growth and metastasis of tumor cells. PartⅡ:Effects of Baicalin on an orthotopic transplantation mouse model of mismatch repair gene deficient colorectal cancerObjective To study the anticancer effects of Baicalin on an orthotopic transplantation mouse model of mismatch repair gene deficient colorectal cancer.Methods Sixty orthotopic transplantation mice model of human colon cancer cell line HCT-116expressing GFP were established, which were divided randomly into negative controlled group (5%NaHCO3) and50,100,200mg/kg Baicalin groups. The nude mice were treated with intragastric infusion twice a day. Nude mice growth state, average weigh, inhibition rate of transplanted tumor, tumor metastasis and survival state were observed.Results At14,21and28days after treatment with different dose of Baicalin, tumor growth velocity was significantly slower in the treatment groups, and tumor volume was significantly smaller than the controlled group (there were832±637,2012±1566and2494±1557mm3respectively in14,21and28days)(F=4.433, P<0.05). At the end point of study, survival state of100mg/kg group(13/15)was superior to controlled group (8/15) and200mg/kg group (8/15)(x2=4.665and3.980, P<0.05). However, there were no significant differences in tumor metastasis and tumor surface vessel density.Conclusions Baicalin has statistically significant effects in inhibiting tumor growth in an orthotopic transplantation mouse model of mismatch repair gene deficient colorectal cancer, and100mg/kg may be an ideal treatment dose. PartⅢ:Mechanism of inhibitation of Baicalin on an orthotopic transplantation mouse model of mismatch repair gene deficient colorectal cancerObjective To study the mechanism of Baicalin on an orthotopic transplantation mice model of mismatch repair gene deficient colorectal cancer.Methods Sixty orthotopic transplantation mice model of human colon cancer cell line HCT116expressing GFP were established, which were divided randomly into four equal-sized groups, including a negative control group (G1) and three treatment groups that received50mg/kg (G2),100mg/kg (G3), or200mg/kg (G4) of baicalin. The cell cycle, apoptosis, microsatellite, and relative gene were detected.Results The proportion of tumor cells in G2/M phase in experimental groups was higher than that in control group signigicantly (G2vs G1, p=0.000; G3vs G1, p=0.003; G4vs G1,p=0.004). The apoptotic rates were significantly increase in experimental groups when compared with the control group (G2vs Gl,p=0.000; G3vs G1,p=0.004; G4vs G1,p=0.028). There were higer significant differences in PCNA, P53, hMLH1, hMSH2, TGF-β1, and IGF-IIR genes, but no difference in MSI, Bcl-2, and Bax genes.Conclusion The mechanism of the inhibitory effects of Baicalin is related to inhibit the expression of PCNA. P53、hMLH1、hMSH2、TGF-β1and IGF-ⅡR, induce apoptosis of tumor cells, and cell cycle progression is blocked at G2/M phase.更多还原