【作者】 曾四平；

【导师】 肖亚军；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 肾癌是起源于肾小管上皮细胞的一种常见肾脏恶性肿瘤，占成人全部恶性肿瘤的2-3％，占肾脏原发性恶性肿瘤的85-90％，发病率仅次于膀胱肿瘤。以往的报道显示肾癌患者确诊时，有30％已合并有远处转移，而其中合并肺转移的患者占很大比率。近年来，近10年来随着影像技术的进步，特别是B超的广泛普及和CT的升级换代，极大促进了肾癌诊疗水平的提高，从而导致肾癌发现比率明显增加。多年来的研究已经证实，肾癌对化疗、放疗、激素治疗效果差。免疫治疗目前被认为是最有价值的治疗手段，但其有效率也在35％以下。肾癌的治疗缺乏一个非常有效的治疗方法，而目前已有的研究显示miRNAs有可能会成为一种潜在的治疗肿瘤的新途径。miRNAs是一类分布十分广泛的大约22个核苷酸大小的内源性非编码RNA，通过控制靶miRNAs达到调控靶基因的表达或翻译。miRNAs在动物、植物、病毒中广泛存在，其序列高度保守。miRNAs在细胞的增殖、分化及凋亡等方面有重要的作用，并对真核生物基因的表达有调控作用。miRNAs对肿瘤的调控作用主要体现在两个方面：可以通过调控原癌基因抑制肿瘤从而起到抑癌基因的作用；另外也可以调控抑癌基因起到癌基因作用。目前研究发现，miRNAs在不同的肿瘤组织中表达水平不同，有些miRNAs在肿瘤组织的表达水平明显低于正常组织，而另外一些肿瘤组织中miRNAs则明显的上调，这些表达异常的miRNAs可能与肿瘤的发生有着密切的关系。我们将has-mir-129通过脂质体介导的方法转染肾癌细胞株，观察其在体外对肾癌细胞的影响。建立肾癌肺转移动物模型，并观察has-mir-129对动物模型肿瘤的形成及肺转移的影响，本研究分为三个部分，现将研究结果报告如下：第一部分has-mir-129转染SN12-PM6肾癌细胞株对其生长影响的实验研究本部分主要研究has-mir-129对肾癌SN12-PM6细胞侵袭和增值能力的影响。我们经过质粒的扩增、抽提等步骤得到has-mir-129(GFP)质粒，采用脂质体介导的基因转染技术，将has-mir-129(GFP)质粒转染肾癌SN12-PM6细胞，用流式细胞仪筛选带有绿色荧光的肾癌SN12-PM6细胞；MTT法检测细胞的生长曲线，检测细胞的同质黏附能力，Boyden小室检测细胞的侵袭及迁移能力，Brdu渗入法检测细胞的增殖能力。结果显示：转染has-mir-129(GFP)后肾癌SN12-PM6细胞的生长明显受到抑制，同质黏附能力下降，细胞的侵袭力和增殖能力明显减弱(P＜0.05)，但对其S期无明显的差别(P＞0.05)。第二部分人肾细胞癌原位移植裸鼠肺转移模型建立的实验研究本部分主要通过建立肾癌原位肺转移动物模型，研究动物模型肾癌移植瘤和肺部转移瘤的生长情况，确定肾癌肺转移的初始时间。人肾癌细胞株SN12-PM6种植于裸鼠右肾实质内，连续观察裸鼠肾脏肿瘤及肺部转移瘤的生长情况。所有采用标本10％甲醛溶液固定，常规石蜡包埋，切片HE染色后分析结果。结果显示：肾脏的成瘤率为100％(39／39)，第5周开始观察到肿瘤的肺转移灶，肺部转移率50％(4／8)，在随后3周其肺部转移率分别为75％(3／4)，100％(8／8)及100％(7／7)，肠道、肝脏、脾脏等脏器未发现明显的转移灶。第三部分has-mir-129影响人肾细胞癌原位移植裸鼠肺转移模型的实验研究本部分主要建立人肾细胞癌原位移植裸鼠肺转移动物模型，研究has-mir-129对肾脏移植瘤和肺部转移瘤的影响。人肾癌细胞株SN12-PM6建立的动物模型作为对照组，感染携带has-mir-129(GFP)慢病毒的肾癌细胞建立的动物模型作为实验组，7周后观察裸鼠肾脏肿瘤及肺部转移瘤的生长情况，并分别计算两组的肾脏成瘤率、肺部转移率及肿瘤的重量；所有的标本均采用10％甲醛溶液固定，常规石蜡包埋、切片、HE染色后分析结果。结果显示：对照组肾脏的成瘤率为100％(8／8)，肺部转移率100％(8／8)，肿瘤的重量平均为(0.7128±0.1474)g；实验组肾脏成瘤率为100％(8／8)，肺部转移率为62.5％(5／8)，肿瘤的重量平均为(0.4815±0.1112)g；肾脏成瘤率两者之间无明显差异，统计学显示两组之间肿瘤的重量和肺部转移率有显著性差异，P＜0.05。结论1．体外转染has-mir-129(GFP)后肾癌SN12-PM6细胞的生长明显受到抑制，同质黏附能力下降，细胞的侵袭力和增殖能力明显减弱。2．转染has-mir-129(GFP)转染后能抑制肾癌SN12-PM6细胞的生长，但其S期无明显差别，推测其机制可能与G1受抑制有关。3．成功构建人肾细胞癌原位裸鼠肺转移动物模型，为肾癌特别是肾癌肺部转移提供了一个非常理想的研究模型。4．人肾细胞癌原位裸鼠肺转移动物模型建立后5周出现肺部转移灶，其肺部转移率为50％，肠道、肝脏、脾脏等器官未见明显的转移灶。5．我们成功建立了人肾细胞癌原位移植裸鼠肺转移动物模型，has-mir-129(GFP)能抑制人肾细胞癌原位移植裸鼠肺转移模型移植瘤的生长与肺部转移瘤的形成。综述microRNAs及其在泌尿系肿瘤的研究进展microRNAs(miRNAs)是一类分布十分广泛的内源性非编码RNA，在动物、植物、病毒中广泛存在。miRNAs与肿瘤的发生发展预后有关，并在肿瘤的增殖、分化及调亡等方面有重要的作用。结合最近相关文献，就miRNAs与泌尿系肿瘤方面的进展作简要概述。更多还原

【Abstract】 Renal cancers are a kind of common renal malignant tumors and originfrom renal tubular epithelial cells, which account for 2-3% of all adultmalignant tumors and 85-90% of primary malignant renal tumors. Theincidence is only the second to bladder cancers. Previous reports indicate that30% of renal cancers have combined with distant metastasis when patientswere diagnosed as renal malignant tumors and there are a large proportion ofpatients with lung metastases. In recent years, advances in imaging technologyhave taken place, especially the popularity of B-ultrasonography and theupgrade of CT have improved the diagnosis and treatment of renal cellcarcinomas, which have resulted in the increase of renal cell carcinomas. Foryears, researches confirm that chemotherapy, radiotherapy, hormone treatmenthave bad effects on renal cell carcinomas. Immunotherapy has currently beenconsidered the most valuable treatment, but the efficient is less than 35%. The treatment of renal cell carcinomas is a lack of effective methods. Studieshave indicated that miRNAs are likely to be a new potential way for tumors.MicroRNAs (miRNAs) are a widely distributed class of endogenous smallnon-coding RNAs with 22 nucleotides in length. MicroRNAs control targetgene expression or translation by target mRNAs. MicroRNAs with highlyconserved sequences exist in a wide range including animals, plants andviruses. They have an important role in cell proliferation, differentiation andapoptosis, and regulate eukaryotic gene expression. There are two aspects thatmiRNAs regulate tumors. At first, miRNAs can control proto-oncogene andthus inhibit tumors. Secondly, they can also regulate tumor suppressor genesand play a role of ongenes. The current studies find that miRNAs havedifferent levels in different tumor tissues and many miRNAs in tumor tissuesare significantly lower than normal tissues, but the expression of miRNAsincreases in some tumor tissues, which indicates that the abnormal expressionof miRNAs may be closely related to the genesis of tumors.We have transfected renal cancer cell lines (SN12-PM6) with has-mir-129by the liposome-mediated method and observed the impact on renal cancercell lines in vitro. The model of renal cancer with lung metastasis has beenestablished. And we have observed that has-mir-129 impacted orthotopicmouse model of human renal cell carcinoma with pulmonary metastasis. The study has been divided into three parts and the results are as follows.Part One. The study that has-mir-129 has impacted on the growth of renalcancer cell lines (SN12-PM6) after SN12-PM6 was transfected withhas-mir-129The part is the major role that has-mir-129 impacts the growth,the invasion ability, reproductive activity and the cell cycle of renal carcinomacell lines (SN12-PM6). After plasmid amplification and extraction, theplasmid of has-mir-129(GFP) can be obtained. SN12-PM6 cells weretransfected with has-mir-129(GFP) and screened by flow cytometry.SN12-PM6 cells with green fluorescent were cultured. The cell growth curvewas detected by MTT method. The homogeneous adhesion capacity, theinvasion and migration capacity (Boyden chamber), the proliferative capacity(Brdu method) has been detected. The results indicate that the growth ofSN12-PM6 cells was inhibited, the homogeneous adhesion, invasion andmigration capacity decreased, (P＜0.05), but DNA replication capacity was nosignificant difference in S phase after SN12-PM6 cells were transfected withmiR-129.Part Two. Establishment of orthotopic mouse model of human renal cell carcinoma with pulmonary metastasisThe part is the role to construct orthotopic mouse model ofhuman renal cell carcinoma with pulmonary metastasis and to study the firsttime of pulmonary metastasis in nude mice. SN12-PM6 was transplanted intothe right kidey of nude mice. Tumorigenicity and metastasis were evaluatedsubsequently. All tumor tissues were fixed in 10% formalin and embedded inparaffin routinely. Paraffin-embedded tissues were stained with hematoxylinand eosin (HE) for routine histological examination. The results indicate thatthe incidences of tumorigenicity were 100% (39/39). In the fifth weekpulmonary metastasis were observed and the incidences of metastasis were50% (4/8). In the subsequent 3 weeks the incidences of metastasis were 75%(3/4), 100% (8/8) and 100% (7/7), respectively. The metastasis of Intestinaltracts, livers, spleens was not observed obviously.Part Three. Study that has-mir-129 impacted orthotopic mouse model ofhuman renal cell carcinoma with pulmonary metastasisThe part is the role to construct orthotopic mouse model ofhuman renal cell carcinoma with pulmonary metastasis and to study theimpact of has-mir-129(GFP) on renal transplantation tumor and lungmetastases. Human renal cancer cell line SN12-PM6 set up the animal model as the control group, and Human renal cancer cell lines SN12-PM6 beinginfected the lentivirus with has-mir-129(GFP) were cultured in order to set upthe animal model as the experimental group. After seven weekstumorigenicity and metastasis were evaluated subsequently. In the two groups,the percentage of tumorigenicity and metastasis was calculated; at the sametime, the weight of tumor was also taken into account. All tumor tissues werefixed in 10% formalin and embedded in paraffin routinely. Paraffin-embeddedtissues were stained with hematoxylin and eosin (HE) for routine histologicalexamination. Results: In the control group, the percentage of tumorigenicitywas 100% (8 / 8), the percentage of metastasis was 100% (8 / 8) and theaverage tumor weight was (0.7128±0.1474) g. But in the experimental thepercentage of tumorigenicity was 100% (8 / 8), the percentage of metastasiswas 62.5% (5 / 8) and the average tumor weight was (0.4815±0.1112) g.Statistics show that the percentage of tumorigenicity was no significantdifference between the two groups, but the average tumor weight and thepercentage of metastasis was significantly different between the two groups, P＜0.05.Review articles. MicroRNAs and Advances of Urinary TumorsMicroRNAs(miRNAs) are universally small non-coding RNA that are widely reported in plants, animals, and even viruses. It has beendemonstrated that miRNAs are associated that development and prognosis oftumors, and miRNAs play an important role of proliferation, differentiationand apoptosis of tumor. In this review article, we will discuss the progress onthe study of miRNAs and urinary tumors.Conclusion①The growth of SN12-PM6 cells was inhibited, the homogeneous adhesion,invasion and migration capacity decreased after SN12-PM6 cells weretransfected with has-mir- 129 (P＜0.05).②Has-mir-129 can inhibit the growth of SN12-PM6 cells, but DNAreplication capacity was no significant difference in S phase after SN12-PM6cells were transfected with has-mir-129(GFP), and its mechanism may berelated to inhibition of the G1 phase.③We have successfully constructed orthotopic mouse model of human renalcell carcinoma with pulmonary metastasis and provided an ideal researchmodel of renal cell carcinoma, especially lung metastasis of renal cellcarcinoma.④Five weeks later pulmonary metastasis were observed and the incidencesof metastasis were 50%. The metastasis of Intestinal tracts, livers, spleens was not observed obviously.⑤We have successfully constructed orthotopic mouse model of human renalcell carcinoma with pulmonary metastasis and found that has-mir-129 caninhibit tumor growth and pulmonary metastasis of orthotopic mouse model ofhuman renal cell carcinoma with pulmonary metastasis.更多还原