【作者】 何葵；

【导师】 陈利生；

【作者基本信息】 广西医科大学 ， 肿瘤学， 2008， 博士

【摘要】 第一部分进展期结直肠癌合并低白蛋白血症形成机制临床研究目的:阐明进展期结直肠癌低白蛋白血症与IL-6、TNF-α诱导的急性时像反应的关联性;探讨进展期结直肠癌组织中IL-6mRNA和PIFmRNA表达及其与进展期结直肠癌低白蛋白血症的关系。方法:收集进展期结直肠癌患者63例病例资料,根据入院24小时内用溴甲酚绿法测定的血浆白蛋白浓度将病例分为两组:低白蛋白血症组(Alb＜35g/L)38例、血浆白蛋白正常组(Alb≥35g/L)25例。采用ELISA和免疫组织化学技术分别检测患者血清中CRP、IL-6、TNF-a浓度和癌组织中IL-6、TNF-a蛋白表达水平;RT-PCR方法检测癌组织和正常结肠粘膜中IL-6mRNA和PIFmRNA表达水平。结果:进展期结直肠癌合并低白蛋白血症组患者中血清C-反应蛋白、纤维蛋白原明显高于血浆白蛋白正常组;其血清总蛋白和血清前白蛋白浓度明显低于血浆白蛋白正常组。低白蛋白血症组血清TNF-a和IL-6浓度均明显高于白蛋白正常组(P=0.000,P=0.003),进展期结直肠癌患者血清TNF-a、IL-6浓度与Alb呈明显负相关,与CRP呈正相关。免疫组化结果显示正常直肠粘膜组织中未见TNF-α和IL-6蛋白表达。两组间TNF-α蛋白的表达无显著性差异(P=0.567),IL-6蛋白的表达有显著差异(P=0.005),低白蛋白血症组IL-6表达明显高于白蛋白正常组(χ~2=2.82 P=0.005)。进展期结直肠癌低白蛋白血症组患者癌组织中IL-6mRNA相对表达量明显高于血浆白蛋白正常组(p＜0.01),癌组织中IL-6mRNA相对表达量与血浆白蛋白浓度呈负相关,与血清IL-6浓度呈正相关。PIFmRNA进展期结直肠癌组织和正常粘膜组织中无表达。结论:进展期结直肠癌低白蛋白血症患者存在一个由IL-6、TNF-a诱导的急性时相反应,其血浆蛋白代谢紊乱表现以血清CRP浓度增高和总蛋白、前白蛋白、白蛋白浓度下降为特征;进展期结直肠癌组织中IL-6mRNA和蛋白高表达,无PIFmRNA表达;进展期结直肠癌组织自身分泌IL-6是引起血清IL-6、TNF-a升高的始动因素,与进展期结直肠癌低白蛋白血症形成密切相关。第二部分重组生长激素对荷瘤小鼠肿瘤生长增殖及血浆蛋白代谢的影响目的:观察外源性重组生长激素对荷瘤小鼠肿瘤生长、细胞增殖和血浆蛋白代谢的影响。探讨外源性重组生长激素对荷瘤机体血浆蛋白合成影响及调节的分子机制。方法:选用近交系BALB/c雄性小鼠30只,接种鼠结肠腺癌细胞株CT26建立移植瘤模型,随机分成三组:对照组(A组)、单纯移植瘤组(B组)、生长激素治疗组(C组、D组、E组)。B组和E组分别在当天到第28天每天给予生理盐水和生长激素皮下注射。C组和D组从第14天起到第28天每天分别给予不同剂量生长激素皮下注射。每天测定移植瘤大小和荷瘤鼠体重,第28天测定荷瘤小鼠血清生化指标(血糖、总蛋白、白蛋白、甘油三脂)和血浆IL-6浓度,Real-Time PCR法测定小鼠肝脏组织中SOCS-3mRNA的表达,免疫组织化学方法测定移植瘤增殖细胞核抗原(PCNA)表达。结果:治疗组(C、D、E组)荷瘤小鼠实验结束时体重、血浆白蛋白浓度明显高于单纯荷瘤组(B组),移植瘤终末体积和重量比较没有差异(P＞0.05);血糖、总蛋白、白蛋白比较有显著性差异(P＜0.05)。治疗组小鼠血浆IL-6浓度低于单纯荷瘤组(B组),比正常对照组(A组)高;免疫组化测定移植瘤中PCNA表达发现,PCNA在移植瘤中均有表达,但移植瘤组织中PCNA阳性细胞百分率不同,有显著性差异(F=3.14v=3 P=0.048),E组最高,B组最低。采用实时荧光定量PCR检测各组小鼠肝脏组织中SOCS-3mRNA表达,小鼠肝脏组织中SOCS-3mRNA相对表达量比较有显著性差异(F=7.88 P=0.00),E组与B组比较有显著性差异(P=0.003)。CDE三组SOCS-3mRNA表达增高。结论:外源性生长激素可以改善荷瘤机体的营养状况,促进血浆蛋白合成。短期、小剂量使用生长激素治疗恶性肿瘤所致低白蛋白血症状况是安全、有益的;长期、大剂量使用外源性生长激素可能促进肿瘤细胞增殖;外源性GH对荷瘤机体血浆蛋白代谢影响的机制可能与GH上调肝脏SOCS-3mRNA表达和调节IL-6诱导的肝脏急性时相蛋白反应相关。更多还原

【Abstract】 Chapter 1 Clinical studies on the mechanism of hypoalbumineamia in advanced colorectal cancerObjective To explore the relationship between acute phase protein response induced proinflammatory cytokines（TNF-a and IL-6）and hypoalbuminemia in advanced colorectal cancer patients.Moreover,this study was alsoconducted to clarify the expression levels of IL-6 mRNA and PIF mRNA in tissue of advanced colorectal cancer and to further elucidate the relationship between the expression levels of IL-6 mRNA,PIF mRNA and hypoalbumineamia in patients with advanced colorectal cancer.Methods According to the serum albumin concentration（detected by BCG method at the second day in hospital）,63 patients with advanced colorectal cancer were divided into 2 groups:hypoalbumineamia group with concentration of serum albumin＜35g/L（38 patients）and normal group with concentration of serum albumin≥35g/L（25 patients）.Serum IL-6 and TNF-a concentrations were determined by enzyme-linked immunosorbent assay（Elisa testing）.Serum CRP concertration was measured by an immunoturbinometric method. Immunohistochemistry testing(Supervision^TM)was used to detect the expression of TNF-a and IL-6 in cancer tissues.RT-PCR semi-quantity method was used to detect the expression of IL-6 mRNA and PIF mRNA in the two groups.Results Compared with normal group,hypoalbumineamia group showed significantly higher serum concentration of CRP and serum concentration of FIB, while this group showed significantly lower serum concentration of total protein and prealbumin.The level of serum TNF-a and IL-6 in hypoalbumineamia group was significantly higher than that in normal group（P=0.000,P=0.003）.Significantly,the concentration of serum IL-6 and TNF-a was negatively correlateed with serum albumin concentration and was positively correlated with serum CRP concentration.Furthermore,high IL-6 and high CRP levels were associated with reduced serum albumin concentration.In the normal tissues,expression of IL-6 and TNF-a were not showed by immunohistochemistry in two groups.There was no significant difference between normal group and hypoalbumineamia group（P=0.567）of TNF-a immunohistochemistry,while IL-6 immunohistochemistry were significantly different（P=0.005）,the expression of IL-6 in hypoalbumineamia group was higher than that in normal group（x²=2.82 P=0.005）.Compared with normal group,hypoalbumineamia group showed higher expression levels of IL-6 mRNA（1.39±0.49VS0.62±0.40）in colorectal cancer tissue（t=6.5,P=0.000）.Expression levels of IL-6 mRNA in colorectal cancer tissue were negatively correlated with serum albumin concentration,and positively correlated with serum IL-6 concentration.PIFmRNA in tissue of advanced colorectal cancer were not expressed.Conclusion There is a acute-phase protein response in patiens with advanced colorectal cancer complicating hypoalbumineamia,which is characterized by elevated serum CRP concentration and reduced serum total protein,prealbumin,albumin concentration.There is high expression of IL-6 mRNA and no expression of PIF mRNA in advanced colorectal cancer tissue. IL-6 autocrined of colorectal cancer tissue prossibly play an important role in inducing acute-phase protein response in patiens with advanced colorectal cancer complicating hypoalbumineamia.Hypoalbumineamia in advanced colorectal cancer is associated with acute-phase protein response with serum IL-6 participated.Chapter 2Effect of exogenous recombinant growth hormone on the growth and proliferation of thansplanted colon adenocarcinoma carcinoma and serum protein metabolism in tumor-bearing BLBA/c miceObjective To investigate the effect of exogenous growth hormone on growth of transplanted tumor,proliferation of cancer cell and serum protein metabolism of tumor-bearing mice.In order to explore the molecular mechanisms of exogenous growth hormone enhancing serum protein synthesis on tumor-inducing mice.Methods Thirty male BALB/c mice were randomized into three groups:normal control group（A group）and tumor group（B group）and tumor group+GH treatment group（C,D,E group）.In tumor group and tumor group+GH treatment,a suspension of 10⁶ murine colon 26 adnocarcinoma cells was inoculated s.c into each mouse.The model of thansplanted murine colon 26 adnocarcinoma cells in BALB/c was set up.Saline or GH were respectively administered S.C.daily from day 0 for 28 days for B or E group,Whereas GH were respectively administered s.c.daily from day 14 for 28 days for C and D group.Body weight and tumour volume were measured daily after inoculation.Serum glucose,total protein,albumin,triglyceride and IL-6 concentrations were determined at the day 28 following the inoculation.SOCS-3 mRNA expression levels of tumor-bearing mice’s live tissue were observed by Real-time PCR.PCNA expression levels of transplanted tumor were observed by immunohistochemistry also.Results Compared with group B,the weight and serum albumin concentration of tumor-bearing mice in group C,D and E were increased significantly at the end of experiment.however there was no difference in tumor weight and volmue among B,C,D,E groups（p＞0.05）.Whereas,the concentrations of glucose,total protein,albumin and IL-6 level showed significant difference between the three groups（p＜0.05）.The serum IL-6 concentration in treatment group was lower than that in the tumour group（B group）,but higher than the normal control group（A group）.PCNA expression was always positive in transplantation tumor （F=3.14 v=3 P=0.048）,The percentage of positive cells in each group was different,of which group E the highest,and group B the lowest.There was significant differences of the relative expression of SOCS-3 mRNA in mice liver tissue in each groups（F=7.88 P=0.00）.Group E has the significance difference with group B（P=0.003）.The express of SOCS-3 mRNA in group C,D and E were increased.Conclusion Exogenous growth hormone can amelioration hypoalbumineamia of tumor-bearing mice.The use of short-term,low dose exogenous growth hormone is safe and useful for the cancer-caused hypoalbumineamia,but using growth hormone in long-term and large dose will increase a possibility of cancer cell proliferation.The molecular mechanism of exogenous growth hormone regulating acute-phase protein response induced by IL-6 is possibly associated with the up-regulation of SOCS-3 mRNA expression in liver tissue of tumor-bearing mice.更多还原