【作者】 何昀；

【导师】 魏光辉；

【作者基本信息】 重庆医科大学 ， 儿科学， 2012， 博士

【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/cm²、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IC₅₀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、1×10⁷和1×10⁸，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1×10⁸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1×10⁶时，至实验终点未见成瘤。结论：用1×10⁷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。更多还原

【Abstract】 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 simdr1Objective: 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 L2CellObjective: 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 tumorsObjective: 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 vivoObjective: 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 tumorchemotherapyObjective: 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.更多还原