【作者】 沈弋桢；

【导师】 闵志廉；

【作者基本信息】 第二军医大学 ， 外科学， 2008， 博士

【摘要】 研究背景:在现代医学技术条件下,肾癌的发病率逐年提高。在美国,肾癌的年发病率从1990年的27200例增长到2005年的36160例,上升了24.8%。欧洲最近的一项大规模临床调查(1987～2004年)显示:肾癌的发病率约占全身恶性肿瘤的3%,占肾脏肿瘤的85%,年均发病率约为7.21/10万,并且以每年平均2.06%的速度递增。对于肾癌的治疗,标准的手术方式为根治性肾切除术。但随着CT等影像技术的普及推广,偶发性肾癌的检出率大大提高,而偶发性肾癌多数为早期肾癌。对于早期肾癌是否采用根治性肾切除术仍存在很大争议。Leibovich等对4～7cm肾细胞癌(RCC)患者行肾部分切除术(NSS)和根治性肾切除术(RN)后,5年生存率的随访显示,两者的癌症特异生存率为98%和86%,无远处转移生存率为94%和83%。考虑到RCC的分期、分级、组织学分型等重要的病理学特性,两者的癌症特异生存率和无远处转移生存率无明显差异。有鉴于此,越来越多的学者倾向于选择肾部分切除术来治疗早期肾癌。目的:行肾部分切除术时,安全切除边距的选择尤为重要,综合国内外的专著和文献报道,传统的观点认为安全的切除范围为1～2cm,进来也有许多学者提出只切除距肿瘤包膜0.5cm以上是安全和可行的。为什么会产生这样的争议呢?一方面:因为肾脏的体积较小,正常成年男性平均长10cm,宽5cm,厚4cm,质量为134～150g;女性肾脏的体积和质量均略小于同龄的男性。如果切除过多,不仅引起有效肾单位的减少,同时还增加了手术难度和并发症出现的几率,如果切除范围过小,手术医生和患者又担心肿瘤的残余,可能会引发肿瘤的破裂和肿瘤的复发。另一方面:随着腹腔镜为代表的微创外科的兴起,传统开放肾部分切除术已经逐渐被腹腔镜肾部分切除术所替代,而以“达芬奇”为代表的机器人外科手术系统的出现更是将外科手术向微创化、精确化方向推进了一大步,因而对于安全切除边距的选择要求更高。基因芯片和组织芯片技术具有高通量、大规模、平行性的特点,可以平行检测多种癌基因和抑癌基因及其相关蛋白产物的组织间表达差异。因此,通过比较癌组织和癌旁不同距离正常组织间肿瘤相关基因和蛋白的差异可以为明确肾部分切除术的安全切除边距提供依据。方法:1)采集第二军医大学附属长征医院和上海市第十人民医院2006年1月～2007年10月间行早期肾癌腹腔镜根治性切除术并经术后病理证实为透明细胞癌的病人共计44例,分别在显微外科放大镜下辨别、测量并采集肿瘤组织(A),距肿瘤边缘0.5cm(B)、1.0cm(C)、2.0cm,(D)厚度为0.2cm的正常肾皮质共计44组。2)全部44组标本常规福尔马林固定、石蜡包埋备用,另选取1组标本液氮冻存备用。3)1组液氮冻存标本送检基因芯片,芯片采用的是Affymetrix Human Genome U133A2.0Array。采用实时荧光定量PCR验证芯片结果,分别检测:T-cell differentiationprotein(MAL);decorin;galectin 1(LGALSl);transforming growth factotbeta-1(TGF-β1);caveolin 1;bcl-2;secreted apoptosis related protein 2(SARP2);secreted frizzled related protein 1(SFRPl);S100 calcium-bindingprotein A2(S100A2)。4)44组石蜡标本构建组织芯片。分别行免疫组化检测组织芯片中CD10,RCC-Ma和EMA的表达差异。结果:一、基因芯片结果:(B)vs(A)上调(对数比≥2)的有1442条基因,下调(对数比≤2)的有593条基因;同样,(C)vs(A)上调的有1336条基因、下调的有631条基因;(D)vs(A)上调的有1259条基因、下调的有737条基因。其中,第一大类基因(凋亡、原癌和抑癌基因)(B)vs(A)上调(对数比≥2)的有125条基因、下调(对数比≤2)的有67条基因;同样,(C)vs(A)上调的有111条基因、下调的有74条基因;(D)vs(A)上调的有103条基因、下调的有87条基因。(A)＼(B),(A)＼(C),(A)＼(D)两两之间无论上调基因还是下调基因均存在总体差异(P＜0.05),经聚类分析后这种差异则更为明显。而(B)＼(C)＼(D)三者之间则无明显差异(P＞0.05)。基因芯片试验结果支持:早期肾癌的安全切除边距为0.5cm。二、荧光定量PCR验证的结果:(A)＼(B)＼(C)＼(D)四份样本的基因芯片结果ABCD和荧光定量PCR验证结果ApBpCpDp高度吻合,基因芯片结果真实可信。三、组织芯片结果:CD10,RCC-Ma和EMA免疫组化染色强阳性率分别为:CD10:A(22.72%),B(72.73%),C(75.00%),D(70.45%)。EMA:A(15.91%),B(84.09%),C(86.36%),D(79.55%)。RCC-Ma:A(18.18%),B(79.55%),C(77.27%),D(75.00%)。(A)＼(B),(A)＼(C),(A)＼(D)两两比较,CD10、EMA和RCC-Ma染色强阳性率均有明显差异(P＜0.05),而(B)＼(C)＼(D)三者间强阳性率无明显差异(P＞0.05)。组织芯片试验结果支持:早期肾癌的安全切除边距为0.5cm。结论:肾部分切除术是早期肾癌治疗的重要术式,行肾部分切除术时切除边距达到0.5cm即为安全可行。更多还原

【Abstract】 BackgroundWith the development of modern medical technology, the detected rate of renal cell carcinoma is increasing year by year. In the United States, the annual incidence of renal cell carcinoma increased by 24.8% from 27,200 cases in 1990 to 36,160 cases in 2005. A recent large-scale European clinical investigation from 1987 to 2004 showed that the incidence of renal cell carcinoma accounted for about 3% of all systemic cancers and 85% of kidney tumors, where the mean annual incidence was about 7.21/100,000, and averagely increased by 2.06% yearly.Radical nephrectomy is the standard surgical treatment of renal cell carcinoma. With the popularization and spread of imaging technology such as CT, more cases of sporadic renal cell carcinoma have been detected, and most of them are primary renal cell carcinoma. In their five-year-survival follow up study of patients with 4 to 7cm renal cell carcinoma (RCC) who underwent partial nephrectomy (NSS) and radical nephrectomy (RN), Leibovich et al found that the cancer-specific survival rate was 98% and 86%, with a non-distant metastasis survival rate of 94% and 83% respectively. The stage, grade, histological type and other important pathological characteristics of RCC considered, there was no significant difference in both cancer-specific survival rate and survival rate without distant metastasis between the two groups. In the light of these findings, more and more researchers tend to use partial nephrectomy to treat primary renal cell carcinoma.ObjectiveSafe selection of the resection margin is particularly important in partial nephrectomy. An overview of the literature shows that a 1-2cm resection range is traditionally accepted as the safe margin, but many researchers come to agree that a 0.5cm resection range from the tumor capsule is safe and feasible. Why is there such a controversy? On the one hand, the mean size and volume of the kidney is relatively small in male adults measuring about 10cm long, 5cm wide and 4cm thick and weighing 134-150g, and those in female adults of the same age are even a bit smaller. Excessive resection would not only decrease effective renal units but also increase surgical difficulty and risks of complications, while inadequate resection may worry both surgeons and patients for possible tumor residuals, tumor rupture and recurrence. On the other hand, with the advent of laparoscopy as the representative of minimally invasive surgery, traditional open partial nephrectomy has been gradually replaced by laparoscopic partial nephrectomy, and the emergence of the robot surgical system with representative "Da Vinci" promotes the rapid development of surgery in terms of minimal invasion and precise direction, which calls for a higher requirement on the selection of a safe resection margin.DNA microarray and Tissue microarray have three features: high-flux, large scale, and parallelism. DNA microarray and Tissue microarray can be used for the researching of many oncogenes, anti-oncogenes, and gene-associated protein. Accordingly, to compare the differences of tumor-related genes and proteins of tumor and para-tumor normal renal tissue of different margins, can be helpful for the selecting of safe margin about partial nephrectomy of primary renal cell carcinoma.Methods1) Included in the present study were 44 patients with primary clear cell carcinoma of kidney confirmed by postoperative pathology who underwent laparoscopic radical nephrectomy in Changzheng Hospital and Shanghai 10th people’s hospital between Jan 2006 and Oct 2007. Tumor tissue (A), and 0.5 cm (B), 1.0cm (C) and 2.0cm (D) para-tumor normal renal cortex were identified, measured and collected with the help of the microsurgery magnifying glass. A total of 44 groups of specimen sections with a 0.2 cm thickness were obtained.2) A total of 44 groups of specimen were fixed routinely with formalin, and paraffin-embedded for use. One group of specimen was frozen in liquid nitrogen for use.3) One group of specimen was frozen in liquid nitrogen for DNA microarray, the chip is Affymetrix Human Genome U133A 2.0 Array DNA microarray. Real time quantitive PCR was used for the proving of the results of gene chip, Including: T-cell differentiation protein (MAL) ; decorin; galectin 1 (LGALS1); transforming growth factor beta-1(TGF-β1); caveolin 1; bcl-2; secreted apoptosis related protein 2 (SARP2); secreted frizzled related protein 1 (SFRP1); S100 calcium-binding protein A2 (S100A2).4) Tissue microarrays of A/B/C/D paraffin-embedded specimens of the 44 groups were constructed in a tissue microarray instrument for use. The express difference of CD10, RCC-Ma and EMA were researched through immunohistochemistry.Results 1) DNA microarray results:(B) vs (A) up (log-ratio>2) is 1442, down(log-ratio<2) is 593; as the same, (C) vs (A) up is 1336, down is 631; (D) vs (A) up is 1259, down is 737. In the first gene group (apoptosis; oncogenes and anti-oncogenes), (B) vs (A) up (log-ratio>2) is 125, down(log-ratio<2) is 67; as the same, (C) vs (A) up is 111, down is 74; (D) vs (A) up is 103, down is 87. The difference between (A)\(B); (A)\(C) and (A)\(D) is prominent (p<0.05), and the difference is more noticeable through cluster analysis. (B)\(C)\(D) is no difference (p>0.05). The results of DNA microarray support the conclusion: The safe resection margin about partial nephrectomy of primary renal cell carcinoma is 0.5cm2) Real time quantitive PCR results:For the group of specimen was frozen in liquid nitrogen, the DNA microarray results A\B\C\D were as same as the results of Real time quantitive PCR Ap\Bp\Cp\Dp. The DNA microarray results were reliable.3) Tissue microarray results:The percentage of intensive immunostaining of CD10, RCC-Ma and EMA is as follow:CD10: A(22.72%), B(72.73%), C(75.00%), D(70.45%);EMA: A(15.91%), B(84.09%), C(86.36%), D(79.55%);RCC-Ma: A(18.18%), B(79.55%), C(77.27%), D(75.00%);The difference between (A)\(B); (A)\(C) and (A)\(D) about CD10; EMA; and RCC-Ma is prominent (p<0.05), (B)\(C)\(D) is no difference (p>0.05). The results of Tissue microarray support the conclusion: The safe resection margin about partial nephrectomy of primary renal cell carcinoma is 0.5cm Conclusions:Partial nephrectomy is very important method of the treatment about primary renal cell carcinoma. The safe resection margin about partial nephrectomy of primary renal cell carcinoma is 0.5cm.更多还原