【作者】 郑旭光；

【导师】 陈晓光；

【作者基本信息】 中国协和医科大学 ， 药理学， 2005， 博士

【摘要】 目前抗肿瘤药物筛选已经转向针对特异靶点，提高选择性、高度自动化的高通量筛选。野生型p53是一种重要的抑癌基因，其生物学功能是监视细胞基因组DNA的完整性。如果DNA受损伤，P53蛋白就使细胞停留在G1期，修复后再进入M期；如损伤不能修复，则诱导其凋亡。目前临床上常用的抗肿瘤药如阿霉素、鬼臼乙叉甙、紫杉醇、阿糖胞苷等以及放疗所采用的UV射线、γ射线等均被认为可以通过诱导p53基因依赖性的细胞凋亡而发挥抗肿瘤作用。野生型P53蛋白可以作为p53野生型表达肿瘤的抗癌药物筛选的靶点。与此同时，p53基因的缺失或突变已被证实存在于不同类型的肿瘤中。而野生型p53基因与肿瘤化疗和放疗所引起的副作用之间的关系已越来越引起研究者的关注。目前临床上常用的抗肿瘤药如阿霉素、鬼臼乙叉甙、紫杉醇等以及放疗所采用的UV射线、γ射线等均被认为是部分通过诱导p53基因依赖性的细胞凋亡而发挥抗肿瘤作用。但对于p53基因的缺失或突变的肿瘤，p53基因依赖性的细胞凋亡在肿瘤组织中不能发挥作用，但却可以诱导一些野生型p53基因高表达的正常组织细胞凋亡。针对p53缺损（突变或缺失）的肿瘤，放化疗的同时如果应用p53抑制剂，可以保护正常组织细胞免遭损害。既达到治疗肿瘤的目的，又可减轻副作用，使放、化疗顺利进行。本文构建了P53蛋白反应性的萤火虫荧光素酶表达质粒，将该质粒稳定转染于p53野生型表达的细胞株MCF-7中，建成一个以野生型P53蛋白为靶点的药物高通量细胞筛选模型，用于p53诱导剂或抑制剂的筛选。我们以该模型研究了9种化合物对P53蛋白表达的影响，发现阿霉素（ADM）、紫杉醇（Tax）和5-氟尿嘧啶（5-FU）具有较强的野生型P53蛋白诱导作用。肾功能不全，尤其慢性肾脏疾病（包括各类慢性肾炎、糖尿病肾病及高血压性肾损害等）是我国最为常见的难治性疾病之一。不论何种病因，也不论是免疫机制或非免疫机制参与，肾组织病变从早期病变均可逐渐进展为晚期的肾小球硬化和（或）肾间质纤维化。近年来国外研究发现，在众多的与肾脏纤维化病变相关的调控因子中，转化生长因子-β（TGF-β）和血管紧张素Ⅱ（AngⅡ）是最为关键的两类因子。其中，AngⅡ可以造成高血压、肾小球内高滤过压，并可促进一系列促细胞增生或促硬化因子的分泌，尤其是可促进TGF-β的分泌。而TGF-β可刺激其它促纤维蛋白因子合成、促进成肌纤维细胞的形成和表型转化、促进多种细胞外基质合成、粘附及沉积并减少其降解，因此被认为是直接影响肾脏纤维化过程的细胞因子。我所谢平副研究员课题组在香豆素结构基础上，进一步修饰和优化结构，以TGF-β₁为靶点，有目的的设计、合成了大量香豆素衍生物，经筛选、结构优化、反复体内药效学验证和初步毒理学研究，筛选出来的具有明确的治疗肾功能不全作用且毒性低、生物活性较高的一个小分子新结构化合物42-5。本文对42-5治疗肾功能不全作用、作用机制和毒理学进行了初步探讨，结果如下：1．对cisplatin所引起肾脏细胞损伤的保护作用体外研究结果表明，42-5对于较低浓度cisplatin造成的大鼠肾系膜细胞（rMC）和人肾小管上皮细胞（HKC）的损伤具有一定的保护作用。0.12—1μmol／Lcisplatin合用33μmol／L或100μmol／L 42-5较之单独应用cisplatin可以降低其对rMC的抑制率。0.12—2.5μmol／L cisplatin合用2μmol／L、10μmol／L以及50μmol／L 42-5较之单独应用cisplatin可以降低其对HKC的抑制率。2．对cisplatin急性肾损伤、5／6肾切除、链脲霉素（STZ）致糖尿病大鼠肾衰模型的保护作用在体内考察了42-5对不同肾功能不全模型的保护作用，无论是cisplatin致急性肾损伤、5／6肾切除还是STZ致糖尿病大鼠肾衰模型，其血尿素氮（BUN）、血肌酐（Scr）、尿蛋白水平均较正常动物显著升高，体重下降。大鼠腹腔注射cisplatin 4天后，BUN、Scr、尿蛋白分别由正常组的27.98±4.01mg／dL、1.14±0.56mg／dL和1.24±0.72mg／24hr上升到237.10±24.34mg／dL、4.41±0.73mg／dL和5.65±3.43mg／24hr。30mg／kg 42-5给药组在造模后4天（给药7天），BUN、Scr和尿蛋白分别下降到125.84±67.06mg／dL、2.83±1.16mg／dL和2.06±1.41mg／24h。5／6肾切除大鼠在造模后16周，BUN、Scr、尿蛋白分别由正常组的25.34±4.52mg／dL、0.98±0.36mg／dL和11.49±5.12mg／24hr上升到46.66±9.38mg／dL、1.65±0.27mg／dL和105.38±38.61mg／24hr。30mg／kg 42-5给药组在造模后16周（给药12周），BUN、Scr、尿蛋白分别下降到37.38±6.67mg／dL、1.07±0.40mg／dL和29.21±13.70mg／24h。STZ致糖尿病大鼠在造模后16周，血糖（Glu）、BUN、Scr、尿蛋白分别由正常组的62.68±8.62mg／dL、22.31±1.46mg／dL、1.41±0.21mg／dL和8.62±1.71mg／24hr上升到494.35±45.89mg／dL、45.64±6.74mg／dL、2.15±0.42mg／dL和14.13±5.10mg／24hr。20mg／kg 42-5给药组在造模后16周（给药14周），Glu、BUN、Scr、尿蛋白分别下降到332.44±84.06mg／dL、32.84±4.67mg／dL、1.72±0.34mg／dL和7.41±2.04mg／24hr。Cisplatin急性肾损伤的肾脏病理病变特点为肾小管上皮细胞肿胀、空泡变性和坏死。42-5给药组多数动物肾小管上皮细胞变性和坏死轻于模型组，其病变程度为中度和轻度。5／6肾切除大鼠肾脏病理检查显示病变主要表现为肾小球的硬化。造模后16周（给药12周），42-5给药组肾小球的硬化程度，小球基底膜增厚及基质系膜区扩大明显轻于模型组。STZ所致糖尿病大鼠肾脏病变主要表现为肾小球血管袢节段性硬化、弥漫性硬化、近端肾小管上皮空泡变性、胞核固缩，染色，严重者满视野均可见这种病变。造模后16周（给药14周）后，42-5给药组。肾小球节段性硬化、弥漫性硬化明显少于模型组；未见球囊渗出病变；肾小管空泡变性发生率和严重程度明显轻于模型组。3．治疗肾功能不全的作用机制体外初步机制研究显示：42-5改善肾功能，抑制纤维化主要与其抑制HKC细胞的凋亡和转分化、抑制脂质过氧化、抑制TGF-β₁受体结合并增加uPA mRNA和MMP-2 mRNA的表达、增加基质金属蛋白酶活性有关。体内研究发现其药理作用与抑制血浆肾素活性、降低血浆TGF-β₁和AngⅡ水平、抑制肾脏TGF-β₁ mRNA表达、减少肾脏组织TGF-β₁、fibronectin和CollagenⅣ的合成和积聚相关。4．初步毒理学研究MTT实验显示42-5对不同组织来源的细胞均未见明显的细胞毒作用；口服一次性给药在小鼠的急性毒性试验MTD＞5g；鼠伤寒沙门氏菌营养缺陷型回复突变（Ames）试验结果阴性；啮齿类动物骨髓嗜多染红细胞微核实验结果阴性。初步毒理学研究表明42-5毒性较低且不具有致突变作用。综上所述，42-5是具有明确肾功能不全治疗作用，且毒性低、生物活性较高、作用机理新，结构全新的小分子化合物。可以作为研制新一代治疗肾功能不全药物的后选化合物。更多还原

【Abstract】 The growth suppressor protein p53 is a key regulator of the cell cycle and cell proliferation. Usually, level of p53 in the cell is low due to the short half life of the protein. However, in cases of DNA damage, including those induced by genotoxic anticancer drugs and environmental exposures, p53 is stabilized and induces a G1- or G2-phase arrest of the cell cycle or even causes cell death. Wild type p53 is one of main targets for many anticancer drugs to take action, including ADM, VP-16 and Taxol. Designing efficient ways to induce the expression of p53 in tumor expressing wild type p53 is therefore key issue in anticancer drug research.However, the role of wild type p53 in cancer treatment is not limited to its involvement in killing tumor cells. The wild type p53 gene is highly expressed in several normal tissues, including lymphoid and hematopoietic organs, intestinal epithelia, and the testis, and it is these tissues that are damaged by anticancer therapy. P53-dependent apoptosis occurs in sensitive tissues shortly after gamma irradiation. More than 50% of human primary tumors have lost the wild type p53 suppressor gene and instead express high level of mutant p53 protein. P53-dependented apoptosis is invalid when cure these turners with the above antitumor drugs, and the side effects associated with the activation of p53 become relative serious in normal tissues. Thus it may be an appropriate target for therapeutic suppression to reduce the damage to normal tissue. This approach would be applicable only for tumors that lack functional p53.In order to obtain an experimental tool for the analysing expression of wild type p53 with different compounds, we created a plasmid named p53-luc-pcDNA3.0 carrying a reporter luciferase gene under the control of a p53-responsive promoter with p53-binding sequences of human p21 promotor. The choice of the construct was based on its efficient and specific p53-dependent transcriptional activation. The plasmid was transfected into a human breast cancer cell line （MCF-7） which expressing wild type p53 to constructe wild type p53-specific reporter system. The reconstructed human breast cancer cells, termed p53R-MCF-7 cells can be used to screening p53 inducer or inhibitor through bioluminescent. We tested 9 conventional chemotherapeutic agents in vitro. ADM, Taxol and 5-FU activated p53 activity by about 50％or above 50％under a concentration of 10^-7mol/L. The kidney disease, especially chronic kidney disease （including all kinds of chronic nephritis, diabetic nephropathy and hypertensive nephropathy） is one of the common chronic diseases that are hard to cure. No matter what pathogeny, pathological changes of renal tissue is always advanced to the end-stage glomerular sclerosis and/or interstitial fibrosis from the early-stage changes. Recent studies show that transforming growth factor-β（TGF-β） and AngiotensinⅡ（AngⅡ） are two key factors which relate to renal fibrosis. AngⅡincreases the accumulation of extracellular matrix proteins through the induction of fibrogenic cytokines like transforming growth factor-β1（TGF-β1）. TGF-β1, part of a multimember protein super-family with overlapping functions, stimulates the production of extracellular matrix proteins like collagens and fibronectin as part of a wound repair response to injury, reduces collagenase production, facilitates matrix assembly, inhibits matrix degradation, finally leads to renal scarring.To define TGF-β（one of the important factors that relate to renal fibrosis） as a target in vitro, by purposeful design, synthesis, screening, structure modification, optimization and repeated pharmacodynamics confirmation in vivo, we regard 42-5 as a new structure compound which has the specific effects on renal insufficiency. It is a coumarin derivative synthesized by professor Shiping Xu and Ping Xie. This study aimed to evaluate its protective effects against renal insufficiency and to clarify the mechanism of this protective action. Preliminary study was also made in its toxicological characteristics. Main results cound be summarized as follows:（1） Protection of 42-5 on the cisplatin-caused toxicity in renal cells In vitro, 42-5 was found to protect from damage of rat renal mesangial cells （rMC） and human kidney tubule epithelial cell （HKC） induced by low concentration cisplatin. MTT assay showed the inhibition ratios of rMC induced by cisplatin were lower in incubation with 33μmol/L or 100μml/L 42-5 compared with incubation with cisplatin （0.12—1μmol/L） alone. The inhibition ratios of HKC induced by cisplatin were lower in incubation with 2μmol/L, 10μmol/L or 50μmol/L 42-5 compared with incubation with cisplatin （0.12—2.5μmol/L） alone too.（2） Effects of 42-5 on renal insufficiency of ARF induced by cisplatin, 5/6 nephrectomy and diabetic induced by STZ in rats.In vivo, different renal insufficiency models including ARF induced by cisplatin, 5/6 nephrectomy and diabetic induced by STZ in rats were characterized with renal dysfunction （BUN and Scr increase）, decrease of body weight and the development of proteinuria. Morphological examination showed renal tubule epithelial cell necrosis in acute failure model （ARF induced by cisplatin in rats） and glomerulosclerosis and interstitial fibrosis in chronic renal insufficiency model （5/6 nephrectomy and diabetic induced by STZ in rats）. 42-5 administration exerted functional and morphological protection against ARF induced by cisplatin, 5/6 nephrectomy and diabetic induced by STZ in rats. BUN, Scr and urine protein were 237.10±24.34 mg/dl, 4.41±0.73mg/dl and 5.65±3.43 mg/24hr in rats 4 days following cisplatin injection compared with control values of 27.98±4.01 mg/dl, 1.14±0.56mg/dl and 1.24±0.72 mg/24hr in ARF induced by cisplatin in rats. Treatment with 42-5（30mg/kg） resulted in a significant reduction in BUN（125.84±67.06 mg/dl）, Scr（2.83±1.16 mg/dl）and urine protein （2.06±1.41 mg/24hr）that after 7 day of treatment. BUN, Scr and urine protein were 46.66±9.38 mg/dl, 1.65±0.27mg/dl and 105.38±38.61 mg/24hr in rats 16 wk following surgery compared with control values of 25.34±4.52 mg/dl, 0.98±0.36mg/dl and 11.49±5.12 mg/24h in 5/6 nephrectomy rats. Treatment with 42-5（30mg/kg） resulted in a significant reduction in BUN（37.38±6.67 mg/dl）, Scr（1.07±0.40 mg/dl）and urine protein （29.21±13.70 mg/24h）that after 12 wk of treatment. Glu, BUN, Scr and urine protein were 494.35±45.89 mg/dl, 45.64±6.74 mg/dl, 2.15±0.42mg/dl and 14.13±5.10mg/24hr in rats 16 wk following STZ injection compared with control values of 62.68±8.62mg/dl, 22.31±1.46 mg/dl, 1.41±0.21mg/dl and 8.62±1.71mg/24hr in diabetic induced by STZ in rats. Treatment with 42-5（20mg/kg） resulted in a significant reduction in Glu （332.44±84.06mg/di）, BUN （32.84±4.67mg/dl）, Scr （1.72±0.34 mg/dl） and urine protein （7.41±2.04mg/24hr）that after 14wk of treatment. Morphological examination showed that 42-5 can attenuate the renal tubule epithelial cell necrosis in ARF induced by cisplatin in rats and glomerular sclerosis and interstitial fibrosis in rats with chronic renal disease.（3） Main mechanisms of the protective actionIn vitro mechanism research demonstrated that the activity of 42-5 correlated with inhibiting apoptosis of HKC, transdifferentiation of HKC, lipid peroxidation, and TGF-β₁ receptor binding, increasing matrix metalloproteinase activity, uPA mRNA and matrix metalloproteinase-2（MMP-2） mRNA expression. In vivo mechanism research demonstrated that the activity of 42-5 correlated with inhibiting plasma renin activity, TGF-β₁ mRNA expression, decreasing serum TGF-β₁ and AngⅡlevel, inhibiting synthesis of TGF-β₁, Fibronectin, and CollagenⅣin kidney tissue.（4） Preliminary study on toxicological characteristics of 42-5Primary safety assay showed that 42-5 had not cytotoxicity on tested different cell lines. Acute toxicity test showed MTD＞5g in mouse orally once. Using His^- type Salmonella Typhimurium TA97, TA98, TA100 and TA102, mutagenesis of 42-5 was determined at concentration of 0.05, 0.5, 5.0, 50.0, 500.0μg/plate with S9 or without S9, according to Ames （1983） revised method. The results showed that the number of colony formation of Salmonella Typhimurium TA97, TA98, TA100 and TA102 induced by 42-5 did not increase by 2 fold. The frequency of micronucleated polychromatic erythrocytes （PCE） in bone marrow cells in mice, induced by 42-5 was not increase either. It suggests that 42-5 has no mutagenesis.In summary, It was found in this study that 42-5, a coumarin derivative, could protect the kidney from several renal insufficiency including models induced by cisplatin, 5/6 nephrectomy and diabetic induced by STZ in rats. Mechanism research indicated TGF-β₁ and AngⅡwere two main targets which 42-5 take actions and it had no definite mutagenesis. It suggests that 42-5 may be a candidate to develop new drug used to cure renal insufficiency.更多还原