摘要
睾丸肿瘤是生殖系统常见肿瘤,临床上恶性睾丸肿瘤化疗效果不佳是影响治疗结果的重要原因。研究发现与肿瘤多药耐药相关的P-糖蛋白在睾丸毛细血管内皮细胞上高表达形成一种生物学屏障,阻止化疗药物的进入,这可能在睾丸肿瘤的耐药中发挥了重要作用。如何突破这道屏障而逆转耐药,就成为睾丸肿瘤治疗中的难点。本课题拟采用基因沉默的方法,用携带MDR1基因特异性小干扰RNA的真核表达质粒pSEB-siMDR1去抑制血管内皮细胞中P-糖蛋白的表达和功能,从而促进化疗药物进入睾丸组织中。但高效定向的体内基因转染是本研究的关键点,超声微泡的应用为实现体内靶向转染siMDR1基因提供了一种可靠的新方法。携带pSEB-siMDR1质粒的微泡,由静脉注入可通过循环到达睾丸,在超声作用下破坏微泡实现siMDR1小干扰RNA体内向睾丸毛细血管内皮细胞的靶向转染,抑制P-糖蛋白表达和功能,为化疗药物进入睾丸内有效杀灭肿瘤提供了条件,从而提高睾丸肿瘤治愈率。基于以上设想,本课题的研究内容包括以下4部分。
第一部分携带siMDR1的真核表达质粒构建及功能鉴定
目的:构建pSEB-siMDR1真核表达质粒,并通过检验其对内源性MDR1基因及P-糖蛋白表达和功能的抑制效果,筛选出有效的siMDR1序列。
方法:设计4对特异性针对大鼠MDR1基因的siRNA序列,退火后分别与pSEB-HUS质粒连接,重组获得pSEB-siMDR1真核表达质粒,PCR、酶切及测序鉴定插入片段。通过脂质体分别将4种pSEB-siMDR1转染到L2RYC细胞,并设置pSEB-HUS空质粒作为对照,以及等量混合共转染组(siMDR1Pool)。转染48h后提取各组的总RNA和总蛋白,用real-time PCR及Western-blot检测MDR1的mRNA及蛋白表达,并用MTT法检测转染后长春新碱对L2RYC细胞增殖的影响。
结果:重组获得的4种pSEB-siMDR1经PCR扩增均可检测到约300bp的条带,pSEB-HUS空质粒对照无此条带;NotⅠ酶切pSEB-HUS对照可获得1321bp和>5000bp条带各一条,重组成功的质粒仅有>6000bp一条带。重组质粒送测序,结果与所设计的siRNA序列完全一致,4种pSEB-siMDR1质粒构建成功。各组转染后均可见绿色荧光表达,siMDR1-1,-2,-3和-4均能不同程度抑制MDR1基因表达,siMDR1Pool组抑制效率最高,其中siMDR1-1,-2,-3和Pool组与对照组相比有统计学差异(P<0.05),而siMDR1-4与对照相比无统计学差异(P>0.05)。P-糖蛋白表达与MDR1基因表达情况一致。抑制后,长春新碱使L2RYC细胞的增殖降低,siMDR1Pool组最明显
结论:重组pSEB-siMDR1真核表达质粒构建成功,能有效抑制MDR1基因和蛋白的表达,siMDR1Pool能最好地抑制MDR1的内源性表达,因此后续实验将选用siMDR1Pool进行转染。
第一节超声联合微泡体外细胞转染最适条件筛选
目的:探讨超声联合微泡体外转染细胞的主要影响因素,筛选pSEB-siMDR1质粒转染L2RYC细胞的最适条件。
方法:机械振荡法制备脂质微泡,然后用PLL吸附法将pSEB-HUS质粒分层包裹在微泡外表面,制成载基因微泡。用不同强度的超声和不同超声暴露时间组合作为转染的条件,台盼蓝染色检验转染后细胞的存活率,流式细胞仪检测质粒的转染效率。
结果:不同细胞转染的条件不同,随着超声强度和暴露时间增加,转染效率不断增高,超过一定限度后细胞死亡也明显增加而不能用于后续实验。当超声强度0.5W/cm2以及暴露时间为30s时,能保证90%以上细胞存活且转染效率26%。
结论:适合的超声强度和暴露时间能够明显促进质粒转染细胞,并不会对细胞存活产生明显的影响。第二节超声联合微泡促pSEB-siMDR1转染降低细胞对化疗药物耐药
目的:研究超声联合载pSEB-siMDR1微泡的体外转染效率以及转染后对MDR1基因及P-糖蛋白表达和功能的抑制。
方法:将pSEB-siMDR1质粒或制备的携带pSEB-siMDR1质粒的脂质微泡加入培养的细胞中,设置实验分组:①单纯质粒组(Ⅰ组)②质粒+超声组(Ⅱ组)③载基因微泡组(Ⅲ组)④载基因微泡+超声组(Ⅳ组)⑤空白对照组(Ⅴ组)⑥脂质体对照组(Lipo组),用超声参数0.5W/cm~2、30s进行照射,转染后用流式细胞计数检测GFP阳性细胞率,实时荧光定量PCR及Western-blot检测转染后MDR1mRNA及P-糖蛋白的表达。最后,用柔红霉素蓄积实验检测细胞膜上P-糖蛋白的功能,MTT法检测长春新碱和更生霉素对细胞增殖的影响。
结果:仅Ⅳ组和Lipo组转染后L2RYC细胞内有绿色荧光表达,流式细胞计数定量转染率Ⅳ组31.9%,明显高于其余各组(P<0.05)。Ⅳ组转染后L2RYC细胞的MDR1mRNA及P-糖蛋白的表达降低,其余各组无明显变化。Ⅳ组柔红霉素在细胞内蓄积高于其余各组(P<0.05)。Ⅳ组长春新碱和更生霉素IC_(50)为1.34μg/ml和0.11μg/ml,明显低于其余各组(P<0.05)。
结论:仅在超声与微泡造影剂同时存在的情况下,能显著提高pSEB-siMDR1质粒转染L2RYC细胞的效率,并显著抑制MDR1基因及P-糖蛋白的表达和功能。抑制后,L2RYC细胞对化疗药物的敏感性提高。
第三部分超声联合微泡促pSEB-siMDR1体内靶向转染
第一节大鼠睾丸肿瘤动物模型的建立
目的:以卵黄囊瘤为例,建立睾丸肿瘤大鼠动物模型的制备方法。
方法:分别选取3周龄和3月龄的雄性SD大鼠,将培养的L2RYC细胞分别制备成的细胞悬液,无菌操作下将10μl细胞悬液注入右侧睾丸网内。各年龄大鼠分别设3组,接种细胞量为1×106、1×10~7和1×10~8,左侧为自身对照,每个处理组10只。术后每周测量睾丸直径,进行B超检查,观察睾丸肿瘤生长情况,并取睾丸行HE染色及免疫组化检查。
结果:注入1×10~8细胞后,无论3周龄或3月龄鼠均在1-2周内睾丸迅速增大破溃,B超见明显内部液化,病理组织检查提示为坏死灶,大鼠短期内衰竭死亡。细胞量为1×107时,3月龄鼠成瘤率仅12.5%,3周龄鼠在接种后肿瘤逐渐发生,3周成瘤率100%,随着肿瘤生长大鼠全身状态恶化,4周开始先后死亡。成瘤鼠的睾丸B超提示为密度均匀稍低的肿块部分有低回声区;HE染色为疏松网状结构,可见嗜酸性小体和S-D小体;免疫组化Cspg4染色阳性。接种细胞量为1×10~6时,至实验终点未见成瘤。
结论:用1×10~7细胞是制备模型较合适的剂量,细胞过多使睾丸短期内坏死,过少则不能成瘤。制备的大鼠睾丸卵黄囊瘤模型的组织学表现与临床相似,可以用于后续实验。
第二节超声联合微泡促pSEB-siMDR1靶向转染睾丸血管内皮
目的:探讨应用超声联合微泡介导pSEB-siMDR1转染大鼠睾丸毛细血管内皮的可行性,以及转染后对毛细血管内皮细胞中MDR1基因,P-糖蛋白表达和功能的抑制效果。
方法:包载pSEB-siMDR1质粒的脂质微泡由大鼠尾静脉注入,5分钟后在睾丸部位开始连续照射10分钟,设置实验分组:①单纯质粒组(Ⅰ组)②质粒+超声组(Ⅱ组)③载基因微泡组(Ⅲ组)④载基因微泡+超声组(Ⅳ组)⑤空白对照组(Ⅴ组),冰冻切片用荧光显微镜检测pSEB-siMDR1质粒的转染定植部位,PCR和western blot检测转染后MDR1基因和P-糖蛋白的表达。用荧光显微镜检测不同处理后,柔红霉素在睾丸组织中的存留情况。
结果:超声作用后,仅在Ⅳ组的毛细血管壁上可见绿色荧光蛋白的表达,转染后MDR1基因和P-糖蛋白表达明显降低(P<0.05),柔红霉素的红色荧光在Ⅳ组睾丸组织内蓄积明显多于其余各组。
结论:超声联合微泡能促进pSEB-siMDR1对大鼠睾丸毛细血管的转染,抑制P-糖蛋白的表达及其药泵功能,使化疗药物更容易在睾丸组织内积聚。第四部分超声联合微泡靶向转染pSEB-siMDR1提高睾丸肿瘤化
疗效果的实验研究
目的:探讨应用超声联合微泡转染pSEB-siMDR1到荷瘤鼠睾丸内,能否提高其化疗效果。
方法:将建模成功的大鼠分组:①单给化疗药(A组)②微泡+超声+化疗药(B组)③载基因微泡+超声+化疗药(C组)④对照组(D组);监测肿瘤的生长及荷瘤鼠存活率,给药完后1周处死大鼠,取睾丸组织检测指标:比较各组肿瘤的大小、重量;HE染色后观察肿瘤病理变化;PCR检测凋亡相关基因Fas、P53的表达。
结果:加用化疗药物后,C组大鼠肿瘤生长抑制,相对睾丸体积均明显小于其余各组,同一时间点存活率也高于其余各组,肿瘤细胞减少,睾丸部分形态保留;Fas和P53基因表达增加。
结论:超声联合微泡靶向转染pSEB-siMDR1能够提高睾丸肿瘤的化疗效果,增加肿瘤细胞的死亡,使荷瘤鼠存活率提高。
Testicular cancer is the common cancer diagnosis in genitourinarysystem of children. However, the poor effect of chemotherapy of malignanttesticular tumors is a big problem. Tumor existed in testis tissues with theprotection of blood-testis barrier, so the chemotherapy drug is difficult toreach and play role. Our previous study have found that P-glycoprotein(P-gp) is associated with the multidrug resistance of tumor, P-gp exists incapillary endothelial cells of the blood-testis barrier to form biologicalbarrier, which play important roles in drug resistance. Therefore, how tobreak through the barrier and reversal of drug resistance, has become veryimportant in testicular tumor treatment. In this study, we try to construct aeukaryotic expression plasmid pSEB-siMDR1carrying small interferenceRNA fragment for MDR1. By using gene silencing method, P-gpexpression in endothelial cell was inhibited, so chemotherapy drugs wentthrough the blood-testis barrier and entered into the testicular tissue.However, the efficacy of in vivo targeted gene transfection is still low.Herein the application of ultrasound microbubbles provides a new method for in vivo targeting siMDR1gene transfection and chemotherapyorientation. With intravenous injection, microbubbles carryingpSEB-siMDR1plasmid arrived at the testis through circulation, under theeffect of ultrasound targeted microbubble destruction, siMDR1waseffectively transfected esticular capillary endothelial cells so that inhibitedthe expression of P-gp, providing a good condition for tumor chemotherapy.This method can improve the cure rate of testicular tumor. Based on theabove hypothesis, we set up this research at four parts as follows.PART ONE Construction and functional verification of eukaryoticexpression plasmid carrying simdr1
Objective: To construct the eukaryotic expression plasmid carryingsiRNA for MDR1gene and detect inhibition efficiency to screen out theeffective siRNA sequence for later research.
Methods: Four pairs of siRNA sequence for rat MDR1gene weredesigned and annealed in vitro, then cloned in pSEB-HUS vector to obtaineukaryotic expression plasmid pSEB-siMDR1, which was verified by usingPCR, endonuclease cutting and gene sequencing.Then pSEB-siMDR1weretransfected into L2ryc dells by Lipfectamine2000, we set up individualfour pSEB-siMDR1transfection, equimolar amounts of three effectivepSEB-siMDR1transfection as pool group and pSEB-HUS vectortransfection as negative control.
Results: Specific300bp fragment was amplified by PCR frompSEB-siMDR1, compared with no PCR product from pSEB-HUS vector.By using NotⅠendonuclease cutting, pSEB-HUS wasdigested into1321bpand5000bp of DNA fragment,while only7000bp of DNA fragment wasdigested from recombinant pSEB-siMDR1.Four siRNA specific for MDR1were clarified to be correctly cloned in adenovirus vector by genesequencing. GFP was positive in L2ryc cells with different pSEB-siMDR1transfection groups. The mRNA and protein expression of MDR1inL2RYC were significantly inhibited by three of four pairs of siMDR1srespectively(p<0.05), especially by pool siMDR1s.
Conclusion: Successfully construct the recombinant eukaryoticexpression plasmid pSEB-siMDR1, which can effectively inhibit the geneand protein expression of MDR1. The highest silencing efficiency wasobserved in the pooled plasmids group. Therefore, for the followingexperiment, we chose to use the pooled plasmids to transfect cells.PART TWO Ultrasound-microbubble mediated pseb-simdr1transfection reverse drug resistance in yolk sac carcinoma L2cellin vitroSECTION ONE Optimized condition of Ultrasound-microbubbledestruction for gene delivery in vitroObjective: To investigate the main factors in Ultrasound-microbubble destruction for gene delivery and screen out the best condition ofpSEB-siMDR1transfection in L2RYC cells.
Methods: lipid microbubbles were prepared with mechanical oscillationtion,pSEB-siMDR1plasmid DNA was packaged on the surface of microbubbleby using polylysine adsorption to made siMDR1-loaded lipid microbubble.Cells were exposed to ultrasound with different radiation frequency andsound intensity at different time period. Trypan blue staining was used todetect cell survival, GFP expression was observed under fluorescentmicroscoped, Transfection efficiency was checked by using flowcytometry.
Result: Transfection conditions were different in different cells.Transfection efficiency was improved with increase ultrasound intensityand exposure time, however, exposed to ultrasound intensity of more than0.75W/cm2, cell suvival rates was too low to be used in the study. We foundout ultrasound with intensity of0.5W/cm2for30s could effectivelytransfect plasmids into cells with26%efficiency and90%cell survival.Conclusion: Suitable ultrasonic intensity and exposure time cansignificantly promote plasmid transfection, meanwhile not do obviouseffect on cell survival.
SECTION TWO Ultrasound-microbubble destruction promotespSEB-siMDR1transfection for reversing Drug Resistance in Yolk SacCarcinoma L2Cell
Objective: To study transfection efficiency of siMDR1mediated byultrasound-microbubble in vitro and the inhibition of MDR1geneexpression, P-gp expression and its function.
Methods: pSEB-siMDR1or siMDR1-loaded lipid microbubble were addedon cultured cells. Experimental groups were set up as follows:①Plasmid(group Ⅰ),②P lasmid and microbubble (group Ⅱ),③Plasmid andultrasound (group Ⅲ),④P lasmid, microbubble and ultrasound (groupⅣ),⑤negtive control (groupⅤ),⑥Liposome contro(lgroup Lipo), cells wereexposed on ultrasound intensity of0.5W/cm2for30s. transfectionefficiency was detected with flow cytometry. Real-time PCR andWestern-blot were carried out to evaluate mRNA expression of MDR1andprotein expression of P-gp, respectively. Daunorubicin accumulation assaywas used to detect function of P-gp. MTT assay was also performed todetermine cell viability of L2-RYC cells response to Vincristine andDactinomycin.
Results: GFP fluorescence was observed only in the fourth group and Lipogroup. Compared with other experimental groups, transfection efficiencyof pSEB-MDR1was30%in the fourth group, the mRNA expression ofMDR1and protein expression of P-gp were inhibited significantly.Intracellular accumulation of Daunorubicin in L2-RYC cells of fourthgroup was much more than other groups. The IC50of Vincristine and
Dactinomycin were1.34μg/ml and0.11μg/ml in group Ⅳ which werestatistically different from other groups (P<0.05).Conclusion: Ultrasound-microbubble contrast agent can promote the invitro siMDR1gene transfection in L2RYC cells, and siMDR1caneffectively inhibit the endogenous MDR1mRNA expression, P-gpexpression and its function, while reverse chemical drug resistance ofL2-RYC cells.
PART THREE Ultrasound-microbubble destruction promotespSEB-siMDR1targeting transfection in vivoSECTION ONE An animal model of rat testis tumors
Objective: Take yolk sac tumor as an example to establish thepreparation methods for an animal model of rat testicular tumors.Method: Three-week and3-months old male SD rats were selected,cultured rat L2RYC cells were prepared into a cell suspension of1×10~6,1×10~7and1×10~8/ml. In an aseptic condition,10μl cell suspension wasinjected into the right rete testis, normal left rete testis was self-control.Each group contains10rats. Per week measurement of Testicular diameterwas measured per postoperative week, B-ultrasound was performed toobserve the growth of testicular cancer, and testis was extracted for HEstaining and immunohistochemical examination.Results: With injection of cell suspension at the concentration of1×10~8/ml, the testis of both3-week and3-month old rats rapidly growth within1-2weeks. The testis was partly ruptured, internal liquefaction and pathologicalnecrosis. When the injected cell suspension at the concentration of1×107/ml, no testis tumor formed in3-month old rats whereas inoculatedtumors occurred gradually in3-week old rats, after three weeks, the tumorrate was100%. With tumor growth, the body function of rats deterioratedprogressively and died at fourth week.
Conclusions: The cell suspension at concentration of1×10~7/ml wasappropriate to construct animal model of rat testicular tumors. Too manytumor cells injection resulted in short-term necrosis in testis, while notumor formation with few cells injection. Prepared rat model of testicularyolk sac tumor has similar histological phenotype compared with clinicaltesticular tumor, which can be used for subsequent experiments.
SECTION TWO Ultrasound microbubble-mediated targetedtransfection of pSEB-siMDR1in vivo
Objective: To investigate the application of Ultrasound-microbubbledestruction in in vivo transfection of pSEB-siMDR1into rat testis, anddetect the effect of siMDR1on inhibition of MDR1expression in testicularcapillary endothelial cells.
Methods: The pSEB-siMDR1-loaded lipid microbubble was injected intorat tail vein, after5min, the testis area was continuously exposed to ultrasound for10mins. Experimental groups were set up as follows:①Plasmid alone (group Ⅰ),②Plasmid and microbubble (group Ⅱ),③Plasmid and ultrasound (group Ⅲ),④Plasmid, microbubble andultrasound (group Ⅳ),⑤negtive control (group Ⅴ). Testis was preparedinto frozen section, immunofluoresence was carried out to detectcolonization site of pSEB-siMDR1plasmid transfection, Western blot andimmunohistochemical were used to detect P-gp localization and semi-quantitative expression. Daunorubicin accumulation test was performed toinvestigate the intracellular accumulation of Daunorubicin in testiculartissue of different treatd groups.
Results: After exposed to ultrasound, green fluorescent protein expressionwas visible only in the walls of the capillaries of groupⅣ. MDR1gene andP-gp expression was significantly decreased (P <0.05), the red fluorescenceof daunorubicin in testicular tissue of group Ⅳ accumulated significantlymore than that of other experimental groups.
Conclusion: Ultrasound-microbubble destruction could enhancetransfection of pSEB-siMDR1in rat testis capillaries, inhibit P-gpexpression and its drug pump function, providing a more accessible way tocarry chemotherapy drugs into testicular tissue.
PART FOUR Ultrasound microbubbles-mediated targetedtransfection of pSEB-siMDR1promote testicular tumorchemotherapy
Objective: To investigate whether it can enhance the chemotherapy effectof testis tumor that ultrasonic microbubble-mediated transfection ofpSEB-siMDR1to the testis of tumor-bearing rats.
Methods: the modeling rats were separated to four groups:①a singlechemotherapy drug (group A),②microbubbles+ultrasound+chemotherapy drug (group B),③gene contained microbubbles+ultrasound+chemotherapy drugs (Group C),④control group (group D).Tumor growth and survival of tumor-bearing rats were monitoredcontinuously. The rats were sacrificed one week post administration. Testistumor size and weight were measured. Tumor pathological changes wereassessed with H.E. staining. Apoptotic genes expression of Fas and p53were detected by RT-PCR.
Results: With the addition of chemotherapy drugs, in group C, tumorgrowth was inhibited and relative testicular volume was significantlysmaller than the other groups. At the same time point, survival was alsohigher than other groups and the tumor cells decreased in group C. Part oftesticular tissues were retained. Apoptotic genes expression of Fas and p53were increased.
Conclusion: Ultrasound microbubble-mediated targeted transfection ofpSEB-siMDR1can enhance the chemotherapy effect of testicular tumor,increase death of tumor cells and improved survival of tumor-bearing rats.
引文
[1]李英存,金先庆,王珊等.多药耐药基因在睾丸恶性肿瘤血睾屏障中的作用研究[J].重庆医学,2004,33(6):845-846.
[2] Fromm, MF. Importance of P-glycoprotein at blood-tissue barriers [J]. TrendsPharmacol Sci,2004,25(8):423-429.
[3] Choo EF, Leake B, Wandel C, et al. Pharmacological inhibition of P-glycoproteintransport enhances the distribution of HIV-1protease inhibitors into brain and testes[J]. Drug Metab Dispos.2000Jun;28(6):655-60.
[4]李奇林,全学模.超声微泡造影剂的临床应用及研究进展[J].国外医学(临床放射学分册),2006,29(6):419-421.
[5] Deng CX, Sieling F, Pan H, et al. Ultrasound-induced cell membrance porosity [J].Ultrasound Med Biol.2004,30(4):519-526.
[6]冉海涛,任红,王志刚等.超声波空化效应对体外培养细胞细胞膜作用的实验研究[J].中华超声影像学杂志,2003,12(8):499-501.
[7] Sheikov N, McDannold N, Sharma S, et al. Effect of focused ultrasound appliedwith an ultrasound contrast agent on the tight junctional integrity of the brainmicrovascular endothelium. Ultrasound Med Biol [J].2008,3(4):1093-1104.
[8] Meairs S, Alonso A. Ultrasound, microbubbles and the blood-brain barrier [J]. ProgBiophys Mol Biol.2007,93(1-3):354-62.
[9] Miller DL, Pislaru SV, Greenleaf JE. Sonoporation: mechanical DNA delivery byultrasonic cavitation [J]. Somat Cell Mol Genet,2002,27(1-6):115-134.
[10]Chen L, Zhan WW, Shen ZJ, et al. Blood perfusion of the contralateral testisevaluated with contrast-enhanced ultrasound in rabbits with unilateral testiculartorsion [J]. Asian J Androl,2009,11(2):253-260.
[11]Bekeredjian R, Kuecherer HF, Kroll RD, et al. Ultrasound-targeted microbubbledestruction augments protein delivery into testes [J]. Urology,2007,69(2):386-9.
[12]Li XH, Li C, Xiao ZQ. Proteomics for identifying mechanisms and biomarkersof drug resistance in cancer [J]. J Proteomics.2011,74(12):2642-9.
[13]O'Connor R, Clynes M, Dowling P, et al. Drug resistance in cancer-searching formechanisms, markers and therapeutic agents [J]. Expert Opin Drug Metab Toxicol.2007,3(6):805-17.
[14]Turner NC, Reis-Filho JS. Genetic heterogeneity and cancer drug resistance [J].Lancet Oncol.2012,13(4):e178-85.
[15]Hall MD, Handley MD, Gottesman MM. Is resistance useless? Multidrug resistanceand collateral sensitivity [J]. Trends Pharmacol Sci,2009,30(10):546-556.
[16]Chen ZS, Tiwari AK. Multidrug resistance proteins (MRPs/ABCCs) in cancerchemotherapy and genetic diseases [J]. FEBS J.2011,278(18):3226-45.
[17]Shekhar MP. Drug resistance: challenges to effective therapy [J]. Curr Cancer DrugTargets.2011,11(5):613-23.
[18]Forster S, Thumser AE, Hood SR, et al. Characterization of Rhodamine-123as aTracer Dye for Use In In vitro Drug Transport Assays [J]. PLoS One.2012,7(3):e33253.
[19]Wittgen HG, van den Heuvel JJ, van den Broek PH, et al. ransport of the CoumarinMetabolite7-hydroxycoumarin Glucuronide is Mediated Via MultidrugResistance-Associated Proteins3and4[J]. Drug Metab Dispos.2012Mar12.
[20]Tamada M, Nagano O, Tateyama S, et al. Modulation of Glucose Metabolism byCD44Contributes to Antioxidant Status and Drug Resistance in Cancer Cells [J].Cancer Res.2012Mar15,72(6):1438-1448.
[21]Backos DS, Franklin CC, Reigan P. The role of glutathione in brain tumor drugresistance [J]. Biochem Pharmacol.2012,83(8):1005-12.
[22]Marioni G, Agostini M, Bedin C, et al. Survivin and laryngeal carcinoma prognosis:nuclear localization and expression of splice variants [J]. Histopathology.2012Mar14.
[23]Aller SG, Yu J, Ward A, et al. Structure of P-glycoprotein reveals a molecular basisfor poly-specific drug binding [J]. Science,2009,323(5922):1718-1722.
[24]Sharom FJ. The P-glycoprotein multidrug transporter [J]. Essays Biochem.2011,50(1):161-78.
[25]Tournier N, Declèves X, Saubaméa B, et al. Opioid transport by ATP-bindingcassette transporters at the blood-brain barrier: implications forneuropsychopharmacology [J]. Curr Pharm Des.2011,17(26):2829-42.
[26]Geley S, Müller C. RNAi: ancient mechanism with a promising future [J]. ExpGerontol.2004,39(7):985-98.
[27]Tomari Y, Zamore PD. Perspective: machines for RNAi [J]. Genes Dev.2005,19(5):517-29.
[28]Cole KA, Huggins J, Laquaglia M, et al. RNAi screen of the protein kinomeidentifies checkpoint kinase1(CHK1) as a therapeutic target in neuroblastoma [J].Proc Natl Acad Sci U S A.2011,108(8):3336-41.
[29]Pushparaj PN, Aarthi JJ, Manikandan J, et al. siRNA, miRNA, and shRNA: in vivoapplications [J]. J Dent Res.2008,87(11):992-1003.
[30]Luo Q, Kang Q, Song WX, et al. Selection and validation of optimal siRNA targetsites for RNAi-mediated gene silencing [J]. Gene,2007,395(1-2):160-169.
[31]Jackson AL, Linsley PS. Recognizing and avoiding siRNA off-target effects fortarget identification and therapeutic application [J]. Nat Rev Drug Discov,2010,9(1):57-67.
[32]Caffrey DR, Zhao J, Song Z, et al. siRNA Off-Target effects can be reduced atconcentrations that match their individual potency [J]. PLoS One2011,6: e21503.
[33]Parsons BD, Schindler A, Evans DH, et al. A direct phenotypic comparison ofsiRNA pools and multiple individual duplexes in a functional assay [J]. PLoS One2009,4:e8471.
[34]Shi Z, Liang YJ, Chen ZS, et al. Reversal of MDR1/P-glycoprotein-mediatedmultidrug resistance by vector-based RNA interference in vitro and in vivo [J].Cancer Biol Ther,2006,5(1):39-47.
[1] Karshafian R, Bevan PD, Williams R, et al. Sonoporation by ultrasound-activatedmicrobubble contrast agents: effect of acoustic exposure parameters on cellmembrane permeability and cell viability [J]. Ultrasound Med Biol,2009,35(5):847-860.
[2] Wang F, Cheng Y, Mei J, et al. Focused ultrasound microbubbledestruction-mediated changes in blood-brain barrier permeability assessed bycontrast-enhanced magnetic resonance imaging [J]. J Ultrasound Med,2009,28(11):1501-1509.
[3] Dang SP, Wang RX, Qin MD, et al. A novel transfection method for eukaryotic cellsusing polyethylenimine coated albumin microbubbles [J]. Plasmid,2011,66:19-25.
[4] Wang X, Ito A, Li X, et al. Signal molecules-calcium phosphate coprecipitation andits biomedical application as a functional coating [J]. Biofabrication,2011,3:022001.
[5] Sun B, Tran KK, Shen H. Enabling customization of non-viral gene deliverysystems for individual cell types by surface-induced mineralization [J].Biomaterials,2009,30:6386-6393
[6] Posadas I, Guerra FJ, Ce a V. Nonviral vectors for the delivery of small interferingRNAs to the CNS [J]. Nanomedicine (Lond),2010,5:1219-1236.
[7] Juffermans LJ, van Dijk A, Jongenelen CA, et al. Ultrasound andmicrobubble-induced intra-and intercellular bioeffects in primary endothelial cells[J]. Ultrasound Med Biol,2009,35(11):1917-1927.
[8] Lavorini-Doyle C, Gebremedhin S, Konopka K, et al. Gene delivery to oral cancercells by nonviral vectors: why some cells are resistant to transfection [J]. J CalifDent Assoc,2009,37(12):855-858.
[9] Kedar RP, Cosgrove D, McCready VR, et al. Microbubble contrast agent for colorDoppler US: effect on breast masses. Work in progress [J]. Radiology,1996,198(3):679-686.
[10]Ferrara K, Pollard R, Borden M. Ultrasound microbubble contrast agents:fundamentals and application to gene and drug delivery [J]. Annu Rev Biomed Eng,2007,9:415-47.
[11]Yip GW, Chandrasekaran K, Miller TD, et al. Feasibility of continuous venousinfusion of SonoVue for qualitative assessment of reversible coronary perfusiondefects in stress myocardial contrast echocardiography [J]. Int J CardiovascImaging,2003,19(6):473-481.
[12]Clark LN, Dittrich HC. Cardiac imaging using Optison [J]. Am J Cardiol,2000,86(4A):14G-18G.
[13]汪朝霞,王志刚,许川山等.一种载基因及多聚赖氨酸的脂质超声微泡造影剂制备的实验研究[J].中国超声医学杂志,2009,25(2):101-104.
[14]Lawrie A, Brisken AF, Francis SE, et al. Microbubble-enhanced ultrasound forvascular gene delivery [J]. Gene Ther,2000,7(23):2023-2027.
[15]Guo Z, Hong S, Jin X, et al. Study on the multidrug resistance1gene transfectionefficiency using adenovirus vector enhanced by ultrasonic microbubbles in vitro [J].Mol Biotechnol,2011,48:138-146.
[16]Belova V, Gorin DA, Shchukin DG, et al. Selective ultrasonic cavitation onpatterned hydrophobic surfaces [J]. Angew Chem Int Ed Engl,2010,49(39):7129-7133.
[1] Kohn, DB. Gene therapy for childhood immunological diseases [J]. Bone MarrowTransplant,2008,41(2):199-205.
[2] Montana CL, Corbo JC. Inherited diseases of photoreceptors and prospects for genetherapy [J]. Pharmacogenomics,2008,9(3):335-347.
[3] Santilli G, Thornhill SI, Kinnon C, et al. Gene therapy of inheritedimmunodeficiencies [J]. Expert Opin Biol Ther,2008,8(4):397-407.
[4] Paul A, Jardin BA, Kulamarva A, et al. Recombinant baculovirus as a highly potentvector for gene therapy of human colorectal carcinoma: molecular cloning,expression, and in vitro characterization [J]. Mol Biotechnol,2010,45(2):129-139.
[5] Liu T, Tang A, Zhang G, et al. Calcium phosphate nanoparticles as a novel nonviralvector for efficient transfection of DNA in cancer gene therapy [J]. Cancer BiotherRadiopharm,2005,20(2):141-149.
[6] Choi EW, Shin IS, Youn HY, et al. Gene therapy using non-viral peptide vector in acanine systemic lupus erythematosus model [J]. Vet Immunol Immunopathol,2005,103(3-4):223-233.
[7] Miller AD. The problem with cationic liposome/micelle-based non-viral vectorsystems for gene therapy [J]. Curr Med Chem,2003,10(14):1195-1211.
[8] Stone D. Novel viral vector systems for gene therapy [J]. Viruses,2010,2(4):1002-1007.
[9] Pozzi D, Fattibene P, Viscomi D, et al. Use of EPR and FTIR to detect biologicaleffects of ultrasound and microbubbles on a fibroblast cell line [J]. Eur Biophys J,2011,40(10):1115-1120.
[10]Guo DP, Li XY, Sun P, et al. Ultrasound/microbubble enhances foreign geneexpression in ECV304cells and murine myocardium [J]. Acta Biochim BiophysSin (Shanghai),2004,36(12):824-831.
[11]Guo DP, Li XY, Sun P, et al. Ultrasound-targeted microbubble destructionimproves the low density lipoprotein receptor gene expression in HepG2cells [J].Biochem Biophys Res Commun,2006,343(2):470-474.
[12]冉海涛,任红,王志刚等.超声波空化效应对体外培养细胞细胞膜作用的实验研究[J].中华超声影像学杂志,2003,12(8):499-501.
[13]Zhigang W, Zhiyu L, Haitao R, et al. Ultrasound-mediated microbubble destructionenhances VEGF gene delivery to the infarcted myocardium in rats [J]. Clin Imaging,2004,28(6):395-398.
[14]Wang ZX, Wang ZG, Ran HT, et al. The treatment of liver fibrosis induced byhepatocyte growth factor-directed, ultrasound-targeted microbubble destruction inrats [J]. Clin Imaging,2009,33(6):454-461.
[15]Wu J and Nyborg WL. Ultrasound, cavitation bubbles and their interaction withcells [J]. Adv Drug Deliv Rev,2008,60(10):1103-1116.
[16]Belova V, Gorin DA, Shchukin DG et al. Selective ultrasonic cavitation onpatterned hydrophobic surfaces [J]. Angew Chem Int Ed Engl,2010,49(39):7129-7133.
[17]Hernot S and Klibanov AL. Microbubbles in ultrasound-triggered drug and genedelivery [J]. Adv Drug Deliv Rev,2008,60(10):1153-1166.
[18]Kimmel E. Cavitation bioeffects [J]. Crit Rev Biomed Eng,2006,34(2):105-161.
[19]ter Haar G. Safety and bio-effects of ultrasound contrast agents [J]. Med Biol EngComput,2009,47(8):893-900.
[20]Danialou G, Comtois AS, Dudley RW, et al. Ultrasound increases plasmid-mediatedgene transfer to dystrophic muscles without collateral damage [J]. Mol Ther,2002,6(5):687-693.
[21]Patiroglu T, Unal E, Ozdemir MA, et al. Accidental intramuscular overdoseadministration of vincristine [J]. Drug Chem Toxicol,2012,35(2):232-234.
[22]Li L, Cui D, Zheng SJ, et al. Regulation of Actinomycin D induced upregulation ofMdm2in H1299cells [J]. DNA Repair (Amst),2012,11(2):112-119.
[1] Stiller CA. International variations in the incidence of childhood carcinomas [J].Cancer Epidemiol Biomarkers Prev,1994,3(4):305-310.
[2] Kato K, Ijiri R, Tanaka Y, et al. Testicular immature teratoma with primitiveneuroectodermal tumor in early childhood [J]. J Urol,2000,164(6):2068-2069.
[3] Secchiero P, Zorzet S, Tripodo C, et al. Human bone marrow mesenchymal stemcells display anti-cancer activity in SCID mice bearing disseminated non-Hodgkin'slymphoma xenografts [J]. PLoS One,2010,5(6): e11140.
[4] Ostrand-Rosenberg, S. Animal models of tumor immunity, immunotherapy andcancer vaccines [J]. Curr Opin Immunol,2004,16(2):143-150.
[5] Gao Y, Cai QQ, Li S, et al. Effects of arsenic trioxide under different administrationways on T-cell lymphoma xenografts in nude mice: in vivo and in vitroexperiments [J]. Ai Zheng,2009,28(2):127-131.
[6] Vesely J, Riman J, Seifert J. A quantitative and qualitative study of postirradiationmyelogenic leukaemia in C57black mice [J]. Neoplasma,1960,7:172-186.
[7] Hummel KP. Mammary tumors in mice of strains C3H and DBA/1and their F1hybrids [J]. Cancer Res,1960,20:467-469.
[8] Minton JP, Abou-Issa H, Foecking MK, et al. Caffeine and unsaturated fat dietsignificantly promotes DMBA-induced breast cancer in rats [J]. Cancer,1983,51(7):1249-1253.
[9] Wogan GN and Newberne PM. Dose-response characteristics of aflatoxin B1carcinogenesis in the rat [J]. Cancer Res,1967,27(12):2370-2376.
[10] Fergusson RJ, Carmichael J, Smyth JF. Human tumour xenografts growing inimmunodeficient mice: a useful model for assessing chemotherapeutic agents inbronchial carcinoma [J]. Thorax,1986,41(5):376-380.
[11]高岿然,史铁梅,邸晶等.宫颈癌移植瘤大鼠动物模型的建立[J].中国组织工程研究与临床康复,2008,12(28):5494-5498.
[12]何文飞,何大维,马超等.C57BL/6j小鼠异种移植瘤模型的建立[J].中华实验外科杂志,2011,28(1):145-147.
[13]Roomi MW, Ivanov V, Kalinovsky T, et al. In vivo antitumor effect of ascorbic acid,lysine, proline and green tea extract on human prostate cancer PC-3xenografts innude mice: evaluation of tumor growth and immunohistochemistry [J]. In Vivo,2005,19(1):179-183.
[1] Suzuki R and Maruyama K. Effective in vitro and in vivo gene delivery by thecombination of liposomal bubbles (bubble liposomes) and ultrasound exposure [J].Methods Mol Biol,2010,605:473-486.
[2]李英存,金先庆,王珊等.多药耐药基因在睾丸恶性肿瘤血睾屏障中的作用研究[J].重庆医学,2004,33(6):845-846.
[3] Taniyama Y, Azuma J, Rakugi H, et al. Plasmid DNA-based gene transfer withultrasound and microbubbles [J]. Curr Gene Ther,2011,11(6):485-490.
[4] Geers B, Lentacker I, Cool S, et al. Ultrasound responsive doxorubicin-loadedmicrobubbles; towards an easy applicable drug delivery platform [J]. J ControlRelease,2010,148(1): e59-60.
[5] Tsivgoulis G, Culp WC, Alexandrov AV. Ultrasound enhanced thrombolysis in acutearterial ischemia [J]. Ultrasonics,2008,48(4):303-311.
[6] Emoto M, Tachibana K, Iwasaki H, et al. Antitumor effect of TNP-470, anangiogenesis inhibitor, combined with ultrasound irradiation for human uterinesarcoma xenografts evaluated using contrast color Doppler ultrasound [J]. CancerSci,2007,98(6):929-935.
[7] Ren ST, Zhang H, Wang YW, et al. The preparation of a new self-mademicrobubble-loading urokinase and its thrombolysis combined with low-frequencyultrasound in vitro [J]. Ultrasound Med Biol,2011,37(11):1828-1837.
[8] Korpanty G, Chen S, Shohet RV, et al. Targeting of VEGF-mediated angiogenesis torat myocardium using ultrasonic destruction of microbubbles [J]. Gene Ther,2005,12(17):1305-1312.
[9] Wang ZX, Wang ZG, Ran HT, et al. The treatment of liver fibrosis induced byhepatocyte growth factor-directed, ultrasound-targeted microbubble destruction inrats [J]. Clin Imaging,2009,33(6):454-461.
[10]Hajri Z, Boukadoum M, Hamam H, et al. An investigation of the physical forcesleading to thrombosis disruption by cavitation [J]. J Thromb Thrombolysis,2005,20(1):27-32.
[11]Zhou S, Li S, Liu Z, et al. Ultrasound-targeted microbubble destruction mediatedherpes simplex virus-thymidine kinase gene treats hepatoma in mice [J]. J Exp ClinCancer Res,2010,29:170.
[12]Maeda H, Tominaga K, Iwanaga K, et al. Targeted drug delivery system for oralcancer therapy using sonoporation [J]. J Oral Pathol Med,2009,38(7):572-579.
[13]Warram JM, Sorace AG, Saini R et al. A triple-targeted ultrasound contrast agentprovides improved localization to tumor vasculature [J]. J Ultrasound Med,2011,30(7):921-931
[14]Wu J and Nyborg WL. Ultrasound, cavitation bubbles and their interaction withcells [J]. Adv Drug Deliv Rev,2008,60(10):1103-1116.
[15]Belova V, Gorin DA, Shchukin DG, et al. Selective ultrasonic cavitation onpatterned hydrophobic surfaces [J]. Angew Chem Int Ed Engl,2010,49(39):7129-7133.
[16]Kodama T, Tomita Y, Koshiyama K, et al. Transfection effect of microbubbles oncells in superposed ultrasound waves and behavior of cavitation bubble [J].Ultrasound Med Biol,2006,32(6):905-914.
[17]Miller DL, Reese JA, Frazier ME. Single strand DNA breaks in human leukocytesinduced by ultrasound in vitro [J]. Ultrasound Med Biol,1989,15(8):765-771.
[18]Aggeli C, Giannopoulos G, Lampropoulos K, et al. Adverse bioeffects of ultrasoundcontrast agents used in echocardiography: true safety issue or "much ado aboutnothing"?[J]. Curr Vasc Pharmacol,2009,7(3):338-346.
[19]McDannold N, Vykhodtseva N, Hynynen K. Targeted disruption of the blood-brainbarrier with focused ultrasound: association with cavitation activity [J]. Phys MedBiol,2006,51(4):793-807.
[20]Walker KW, Pantely GA, Sahn DJ. Ultrasound-mediated destruction of contrastagents: Effect of ultrasound intensity, exposure, and frequency [J]. Invest Radiol,1997,32(12):728-734.
[21]Guzmán HR, McNamara AJ, Nguyen DX, et al. Bioeffects caused by changes inacoustic cavitation bubble density and cell concentration: a unified explanationbased on cell-to-bubble ratio and blast radius [J]. Ultrasound Med Biol,2003,29(8):1211-1222.
[22]Zarnitsyn VG and Prausnitz MR. Physical parameters influencing optimization ofultrasound-mediated DNA transfection [J]. Ultrasound Med Biol,2004,30(4):527-538.
[1] Zhang C, Wang QT, Liu H, et al. Advancement and prospects of tumor genetherapy [J]. Chin J Cancer,2011,30(3):182-8.
[2] Wagner E. Advances in cancer gene therapy: tumor-targeted delivery of therapeuticpDNA, siRNA, and dsRNA nucleic acids [J]. J BUON,2007,12Suppl1: S77-82.
[3] Song MS, Jeong JS, Ban G, et al. Validation of tissue-specific promoter-driventumor-targeting trans-splicing ribozyme system as a multifunctional cancer genetherapy device in vivo [J]. Cancer Gene Ther,2009,16(2):113-25.
[4] Maeda H, Tominaga K, Iwanaga K, et al. Targeted drug delivery system for oralcancer therapy using sonoporation [J]. J Oral Pathol Med,2009,38(7):572-579.
[5] Lawrie A, Brisken AF, Francis SE, et al. Ultrasound enhances reporter geneexpression after transfection of vascular cells in vitro [J]. Circulation,1999,99(20):2617-2620.
[6] Stringham SB, Viskovska MA, Richardson ES, et al. Over-pressure suppressesultrasonic-induced drug uptake [J]. Ultrasound Med Biol,2009,35(3):409-415.
[7] Skyba DM, Price RJ, Linka AZ, et al. Direct in vivo visualization of intravasculardestruction of microbubbles by ultrasound and its local effects on tissue [J].Circulation,1998,98(4):290-293.
[8] Carson AR, McTiernan CF, Lavery L et al."Gene therapy of carcinoma usingultrasound-targeted microbubble destruction [J]." Ultrasound Med Biol,2011,37(3):393-402.
[9] Kojima Y, Honda K, Hamada H, et al. Oncolytic gene therapy combined withdouble suicide genes for human bile duct cancer in nude mouse models [J]. J SurgRes,2009,157(1): e63-70.
[10]He X, Tsang TC, Luo P, et al. Enhanced tumor immunogenicity through couplingcytokine expression with antigen presentation [J]. Cancer Gene Ther,2003,10(9):669-677.
[11]Liu Z, Gao S, Zhao Y, et al. Disruption of tumor neovasculature by microbubbleenhanced ultrasound: a potential new physical therapy of anti-angiogenesis [J].Ultrasound Med Biol,2012,38(2):253-261.
[12]王佚,金先庆,王荞等. mdr1基因转染骨髓造血细胞移植联合超剂量化疗治疗兔VX2肝癌的实验研究[J].中华小儿外科杂志,2004,25(3):274-278.
[13]Clarke P, Beckham JD, Leser JS, et al. Fas-mediated apoptotic signaling in themouse brain following reovirus infection [J]. J Virol,2009,83(12):6161-70.
[14]Li YY, Wang R, Zhang GL, et al. An archaeal histone-like protein mediatesefficient p53gene transfer and facilitates its anti-cancer effect in vitro and in vivo[J]. Cancer Gene Ther,2007,14(12):968-975.
[15]Guo Z, Hong S, Jin X, et al. Study on the multidrug resistance1gene transfectionefficiency using adenovirus vector enhanced by ultrasonic microbubbles in vitro [J].Mol Biotechnol2011,48:138-146.
[16]Yan XD, Pan LY, Yuan Y, et al. Identification of platinum-resistance associatedproteins through proteomic analysis of human ovarian cancer cells and theirplatinum-resistant sublines [J]. J Proteome Res,2007,6(2):772-780.
[17]Benedetti V, Perego P, Luca Beretta G, et al. Modulation of survival pathways inovarian carcinoma cell lines resistant to platinum compounds [J]. Mol Cancer Ther,2008,7(3):679-687.
[1]黄澄如.实用小儿泌尿外科学[M].北京:人民卫生出版社,2006
[2]佘亚雄.小儿外科学[M].第三版.北京:人民卫生出版社,2006
[3] Allaway M, Nseyo UO, Kandzari SJ. Primary testicular sarcoma [J]. Journal ofUrology,2000,163(6):187l
[4] Skoog ST. Benign and malignant pediatric scrotal masses [J]. Pediatr Clin NoahAm,1997,44:1229-1250
[5] Frey P, Fliegel C, Herzog B. Testicular tumors in infancy and children: A review ofl0germ cell tumors and10non germ cell tumors [J]. Z Kinder Chir,1990,45:229—231
[6] Mizukami H, Hamamatsu A, Mori S, et al. Autopsy and genetic diagnosis of21-hydroxylase deficiency with bilateral testicular tumors in a case under nomedication for over one year [J]. Forensic Sci Int,2011,206(1-3):e71-5.
[7] Bosl GJ, Motzer RJ. Testicular germ-cell cancer [J]. N Engl J Med,1997,337(4):242-53
[8] Zeitlin SI, Hakim LS, Crowley AR. Malignant Sertoli cell tumor of the testicle in ablack man [J]. J Urol,1996,156(6):2006
[9] Winter C, Albers P. Testicular germ cell tumors: pathogenesis, diagnosis andtreatment [J]. Nat Rev Endocrinol,2011,7(1):43-53
[10]Zhu YP, Zhang SL, Ye DW, et al. Yolk sac tumor in a patient with transversetesticular ectopia [J]. World J Surg Oncol,2011,9:91
[11]Morales-Barrera R, Valverde C, Rodón J, et al. Bilateral testicular germ celltumours: a single hospital experience [J]. Clin Transl Oncol,2010,12(4):299-302.
[12]Risk MC, Masterson TA. Intratubular germ cell neoplasms of the testis and bilateraltesticular tumors: clinical significance and management options [J]. Indian J Urol,2010,26(1):64-71.
[13]Holzbeierlein JM, Sogani PC, Sheinfeld J. Histology and clinical outcomes inpatients with bilateral testicular germ cell tumors: the Memorial Sloan KetteringCancer Center experience1950to2001[J]. J Urol,2003,169(6):2122-5.
[14]Albino G, Nenna R, Inchingolo CD, et al. Hydrocele with surprise. Case report andrewiew of literature [J]. Arch Ital Urol Androl,2010,82(4):287-90.
[15]Ginsberg JP, Hobbie WL, Ogle SK, et al. Prevalence of and risk factors forhydrocele in survivors of Wilms tumor [J]. Pediatr Blood Cancer,2004,42(4):361-3.
[16]Oliva E, Alvarez T, Young RH. Sertoli cell tumors of the ovary: a clinicopathologicand immunohistochemical study of54cases [J]. Am J Surg Pathol,2005,29(2):143-56.
[17]Mati W, Lam G, Dahl C, et al. Leydig cell tumour--a rare testicular tumour [J]. IntUrol Nephrol,2002,33(1):103-5.
[18]Kanto S, Saito H, Ito A, et al. Clinical features of testicular tumors in children [J].Int J Urol,2004,11(10):890-3.
[19]Kratzik C, Schatzl G, Lackner J, et al. Transcutaneous high-intensity focusedultrasonography can cure testicular cancer in solitary testis [J]. Urology,2006,67(6):1269-73.
[20]Kuo WJ, Chang RF, Chen DR, et al. Data mining with decision trees for diagnosisof breast tumor in medical ultrasonic images [J]. Breast Cancer Res Treat,2001,66(1):51-7.
[21]Cui H, Yang X. In vivo imaging and treatment of solid tumor using integratedphotoacoustic imaging and high intensity focused ultrasound system [J]. Med Phys,2010,37(9):4777-81.
[22]何川蔡迪明刘霆.睾丸淋巴瘤的超声影像学和临床特点及文献复习[J].中国医学影像技术,2007,23(4):592-594.
[23]Coret A, Leibovitch I, Heyman Z, et al. Ultrasonographic evaluation and clinicalcorrelation of intratesticular lesions: a series of39cases [J]. Br J Urol,1995,76(2):216-9.
[24]Stille JR, Nasrallah PF, McMahon DR. Testicular capillary hemangioma: anunusual diagnosis suggested by duplex color flow ultrasound findings [J].J Urol,1997,157(4):1458-9.
[25]Fernández Gómez JM, Guate Ortiz JL, Martín Huescar A, et al.[Clinicalpresentation of testicular germinal cancer][J]. Arch Esp Urol,2002,55(8):915-22.
[26]Sesterhenn IA, Davis CJ Jr. Pathology of germ cell tumors of the testisPathology ofgerm cell tumors of the testis [J]. Cancer Control,2004,11(6):374-87.
[27]Grady RW, Ross JH, Kay R. Epidemiological features of testicular teratoma in aprepubertal population [J]. J Urol,1997,158(3Pt2):1191-2.
[28]Aydin GB, Hayran M, Büyükpamuk u M. Undulant course of AFP: a sign of tumoractivation or not?[J]. ediatr Hematol Oncol,2009,26(1):30-5.
[29]Ulbright TM. The most common, clinically significant misdiagnoses in testiculartumor pathology, and how to avoid them [J]. Adv Anat Pathol,2008,15(1):18-27.
[30]Basunia MA, Hossain MS. Testicular tumour-a diagnostic dilemma [J].Mymensingh Med J,2011,20(2):308-11.
[31]Tasaki Y, Nakagawa M, Hanada T Testis sparing surgery for infantile synchronousbilateral teraomas of the testis [J]. Int J Uro1,1998,5:50l-504.
[32]Rushton HG,Belman AB.Testis-sparing surgery for benign lesions of theprepubertal testis [J]. Urol Clin North Am,1993,20:27—37.
[33]Khouni H, Ghozzi S, Ramzi K, et al.Surgery for testicular cancer: indication,results and technical aspects [J]. Tunis Med.2005,83Suppl12:78-83.
[34]Connolly SS, D'Arcy FT, Bredin HC,et al. Value of frozen section analysis withsuspected testicular malignancy [J]. Urology,2006,67(1):162-5.
[35]Griffin GC,Raney RB,Snyder HD,et al.Yolk sac carcinoma of the testis in children[J]. J Urol,1987,137(11):954.
[36]Kaplan GW,Cromie WC,Kelalis PP,et al.Prepubertal yolk sac testiculartumor-report of the testicular tumor registry [J]. J Urol,1988,140(10):1109.
[37]Oztürk H, Tander B, Aydin A,儿et al. The effects of chemical sympathectomy ontesticular injury in varicocele [J]. BJU Int,2001,87(3):232-4.
[38]Bernal F, Raman JD. Exploration of treatment options for the management of stageI testicular seminoma [J]. Expert Rev Anticancer Ther,2008,8(7):1081-90. Review.
[39]Terenziani M, Massimino M, Casanova M, et al. Childhood malignant ovarian germcell tumors: a monoinstitutional experience [J]. Gynecol Oncol.2001,81(3):436-40.
[40] Gillis AM, Sutton E, Dewitt KD, et al. Long-term outcome and toxicities ofintraoperative radiotherapy for high-risk neuroblastoma [J]. Int J Radiat Oncol BiolPhys,2007,69(3):858-64.
[41]Richardson LC, Neri AJ, Tai E, et al. Testicular cancer: a narrative review of therole of socioeconomic position from risk to survivorship [J]. Urol Oncol,2012,30(1):95-101.
[42] Vlayen J, Vrijens F, Devriese S, et al. Quality indicators for testicular cancer: Apopulation-based study [J]. Eur J Cancer,2011Nov18.[Epub ahead of print]