小干扰RNA抑制卵巢癌模型裸鼠移植瘤葡萄糖调节蛋白94的表达及意义

背景：一些实验在模型鼠移植瘤中葡萄糖调节蛋白94被敲除后,细胞黏附中断,刺激肝起源细胞增殖,进而促进肝肿瘤的发生,推测葡萄糖调节蛋白94在肝癌中起到保护作用。 目的：应用小干扰RNA技术抑制裸鼠卵巢癌模型皮下移植瘤内质网分子伴侣葡萄糖调节蛋白94的基因表达,探讨移植瘤中葡萄糖调节蛋白94基因及蛋白表达的变化对移植瘤生长的影响。 方法：从GeneBank中选取人类葡萄糖调节蛋白94基因序列,构建受控于人RNA聚合酶Ⅲ启动子U6的真核表达载体psiSTRIKETM/葡萄糖调节蛋白94。建立人卵巢癌HO-8910细胞株裸鼠皮下接种模型,并将真核表达载体转染入裸鼠移植瘤体内,观察肿瘤生长情况。卵巢癌裸鼠模型经过不同给药方案干预后分为特异性小干扰RNA组,非特异性小干扰RNA组和生理盐水对照组, RT-PCR和免疫组化SP法检测葡萄糖调节蛋白94 mRNA和蛋白在肿瘤内的表达情况,并观察模型裸鼠的移植瘤生长情况。 结果与结论：成功构建RNA干扰质粒载体,所有裸鼠模型均接种成功,5 d后即可见皮下肿瘤形成,14 d左右肿瘤直径达7-10 mm。转染质粒完毕后抑瘤率为65.1%,与非特异性小干扰RNA组和生理盐水对照组比较,差异有显著性意义（P＜0.01）。psiSTRIKETM/GRP94治疗后瘤体内葡萄糖调节蛋白94 mRNA和蛋白显著下调（P ＜0.01）。说明靶向葡萄糖调节蛋白94 mRNA的小干扰RNA可显著抑制人卵巢癌裸鼠移植瘤的生长,其机制可能是诱导葡萄糖调节蛋白94 mRNA和蛋白表达下调所致。     

多西环素对大肠癌裸鼠移植瘤的影响

目的观察金属蛋白酶组织抑制剂（多西环素）对大肠癌裸鼠皮下移植瘤的影响，为大肠癌的治疗寻求一个新的可能治疗途径。方法将10只BALB／C（nu／nu）裸鼠随机分为对照组和实验组，每组5只，将人大肠癌细胞系LoVo细胞种植入裸鼠皮下，建立裸鼠皮下人大肠癌移植瘤模型。对照组应用生理盐水瘤内多点直接注射；实验组应用多西环素瘤内多点直接注射。记录2组裸鼠肿瘤大小，观察肿瘤生长曲线，计算生长抑瘤率。结果第26天肿瘤体积大小和肿瘤生长抑制率：实验组分别为（889．8±80．8）mm^3和49．7％，对照组分别为（1755．8±203．8）mm^3和0，2组比较，差异均有统计学意义（P均〈0．05）。结论多西环素可以抑制大肠癌裸鼠皮下移植瘤的生长，有可能腐用于大肠痛的治疗。     

放疗联合高强度聚焦超声治疗裸鼠胰腺癌的疗效分析

目的:探讨放疗联合高强度聚焦超声(HIFU)治疗裸鼠胰腺癌的疗效。方法:将人胰腺癌细胞系PANC-1接种于24只裸鼠并成功建立移植瘤模型,随机分为4组:空白对照组,HIFU组,放疗组(10Gy×3次),HIFU+放疗组。观察肿瘤体积变化、治疗急性损伤情况,再取肿瘤标本用免疫组化方法检测Ki-67、Caspase-3、STAT-3、HMGB-1的表达情况。结果:空白对照组肿瘤体积明显增大,HIFU组、放疗组、放疗联合HIFU组肿瘤抑制率分别为36.32%、84.49%、70.49%,组间差异有统计学意义(P0.05)。放疗组与对照组无急性治疗损伤,HIFU组与HIFU+放疗组各有2例休克反应及4例皮肤烫伤反应。HMGB-1表达在HIFU组和HIFU+放疗组明显高于其他两组(P0.05),放疗组与空白对照组未见差异。Ki-67与STAT-3在各组均为高表达,Caspase-3在各组均低表达。结论:HIFU联合放射治疗胰腺癌裸鼠皮下瘤未显示协同效应,且不良反应高于单独放疗。     

survivin基因RNA干涉对宫颈癌裸鼠移植瘤生长及凋亡和顺铂化疗敏感性的影响

目的观察survivin基因RNAi对宫颈癌裸鼠移植瘤生长、凋亡和化疗敏感性的影响。方法随机选择雌性BALB/C-nu/nu裸小鼠24只,细胞接种法建立4组人宫颈癌裸鼠皮下移植瘤模型,每天观察裸鼠一般状况及肿瘤生长情况,通过绘制肿瘤生长曲线并计算肿瘤生长抑制率,观察survivin基因RNAi对人宫颈癌裸鼠皮下移植瘤生长的影响;通过免疫组化SP法检测各组移植瘤组织中survivin蛋白表达情况,TUNEL染色观察survivin基因RNAi对人宫颈癌裸鼠皮下移植瘤凋亡的影响;当肿瘤体积达0.2cm3时给予顺铂化疗以观察survivin基因RNAi对人宫颈癌裸鼠皮下移植瘤化疗敏感性的影响。结果成功建立4组人宫颈癌裸鼠皮下移植瘤模型,接种HeLa-s2组裸鼠肿瘤体积在每个检测点均明显小于接种HeLa组;观察结束时,接种HeLa-s2组裸鼠瘤重明显小于接种HeLa组,分别为：（0.369±0.043）g和（1.150±0.136）g（P〈0.05）;接种HeLa-s2组裸鼠肿瘤生长抑制率为67.9%。免疫组化结果显示：接种HeLa-s2组裸鼠survivin蛋白表达显著下降;TUNEL染色结果显示：接种HeLa-s2组裸鼠细胞凋亡明显增多,凋亡指数（AI）值达（22.73±1.37）%。顺铂化疗后不同检测点接种HeLa-s2组裸鼠肿瘤体积明显小于接种HeLa组,肿瘤生长明显受抑;观察结束后,接种HeLa-s2组裸鼠瘤重明显小于接种HeLa组,分别为：（0.323±0.058）g和（1.347±0.173）g（P〈0.05）;接种HeLa-s2组裸鼠肿瘤细胞凋亡明显增多,与接种HeLa组AI比较,接种HeLa-S2组AI明显升高,分别为：（37.38±1.01）%和（5.19±0.61）%（P〈0.05）。结论 survivin基因RNAi可通过下调移植瘤组织survivin蛋白表达抑制移植瘤生长并促进其凋亡,并通过增加顺铂化疗诱导的细胞凋亡,增强顺铂化疗对移植瘤的生长抑制,进而提高移植瘤对顺铂化疗的敏感性。     

沉默ATF5对卵巢癌裸鼠成瘤的影响

目的研究沉默人转录激活因子5（ATF5）后对卵巢癌SKOV-3细胞体外增殖、凋亡及体内成瘤的影响,探讨人类卵巢癌基因治疗的新途径。方法将慢病毒携带的小干扰RNA感染卵巢癌SKOV-3细胞（实验组）,空载体转染的卵巢癌SKOV-3细胞（空载体对照组）和卵巢癌SKOV-3细胞（空白对照组）进行对照,对比观察细胞的生长状态;MTT法检测细胞的增殖生长情况;罗氏凋亡检测试剂盒检测细胞凋亡情况;分别于裸鼠皮下接种3组卵巢癌SKOV-3细胞,观察各组裸鼠的存活状态、种植瘤生长情况;RT-PCR检测瘤组织中ATF5mRNA的表达;Western-Blot方法检测瘤组织中ATF5蛋白的表达;苏木素-伊红染色（HE染色）观察瘤组织生物学形态变化。结果经转染ATF5基因的小干扰RNA后,在相同培养时间内3组细胞的增殖和生物学形态没有明显差异,但实验组细胞凋亡数量较其他两组明显增多。皮下接种成瘤后,3组裸鼠的成瘤率没有明显区别,实验组与空载体对照组和空白对照组相比,肿瘤成瘤时间晚,生长缓慢;移植瘤ATF5mRNA表达及蛋白表达降低;细胞分裂象明显减少,凋亡现象明显。结论沉默ATF5对人上皮性卵巢癌裸鼠移植瘤生长有抑制作用,这可能成为卵巢癌基因治疗的新靶点。     

As2O3对人卵巢癌裸鼠皮下移植瘤抑制作用及机制

目的探讨不同剂量的三氧化二砷（As2O3）对人卵巢癌细胞株SKOV3细胞裸鼠皮下移植瘤生长的抑制作用及其机制。方法将4×10^6个SKOV3细胞接种于裸鼠皮下,建立人卵巢癌裸鼠皮下移植瘤模型,随机分为As2O3高、中、低剂量组及生理盐水组（空白对照）、卡铂（CBP）组（阳性对照）,连续给药10d,停药后24h处死裸鼠,计算移植瘤的瘤质量、抑瘤率及caspase-3基因表达水平的变化,并检测用药后裸鼠的肝、肾功能及血常规。结果As2O3对移植瘤生长的抑制作用具有剂量依赖性,各剂量As2O3组及CBP组瘤质量与生理盐水组比较均有统计学意义（F=17．931,P〈0．05）;As2O3作用后caspase-3基因的表达较空白对照组升高,差异有统计学意义（F=31．821,q=2．89～3．84,P〈0．05）。同时,As2O3各组与CBP组相比,未表现出明显的肝、肾及血液学毒性。结论As2O3对人卵巢癌移植瘤的生长具有明显的抑制作用,其机制可能与上调caspase-3基因的表达及诱导细胞凋亡有关。     

替勃龙对去势裸鼠子宫内膜癌移植瘤的影响

目的 通过裸鼠子宫内膜癌移植瘤模型的体内实验,探讨替勃龙应用于子宫内膜癌患者的安全性.方法 (1)体外培养人子宫内膜癌Ishikawa细胞株;建立去势裸鼠子官内膜癌Ishikawa细胞株皮下移植瘸动物实验模型;(2)将成瘤裸鼠随机分为替勃龙组(15只)和对照组(15只),分别予以替勃龙0.375mg·kg-1·d-1和对照治疗35 d;(3)比较给药前后两组肿瘤体积变化、肿瘤重量和肿瘤转移状况(体表、盆腹腔淋巴结、腹水);应用免疫组化SP法测定两组实验动物皮下移植瘤Ki67、P53、P27、bcl-2蛋白表达情况,判断替勃龙对移植瘤的影响和对内膜癌细胞增殖及凋亡的影响.结果 (1)替勃龙组和对照组裸鼠两组子宫内膜癌皮下移植瘤体积变化无统计学差异[ (2.063±1.038) cm3饥 vs.(2.170±0.822) cm3,P=0.791];肿瘤重量无统计学差异[(0.699±2.02)gvs.(0.807±2.83)g,P =0.314];替勃龙治疗35 d后,没有引起子宫内膜癌肿瘤体表、盆腹腔转移和腹水发生;(2)替勃龙组和对照组两组裸鼠人子宫内膜癌Ishikawa细胞株皮下移植瘤Ki67增殖指数、P53、P27、bcl-2蛋白表达情况均无统计学差异.结论 替勃龙不刺激裸鼠子宫内膜癌Ishikawa细胞株皮下移植瘤生长、转移和细胞增殖、凋亡.替勃龙应用于子宫内膜癌患者的术后激素补充治疗可具有一定的安全性.     

miR-92b在神经胶质瘤裸鼠移植瘤模型中的作用

目的建立U251胶质瘤裸鼠皮下移植瘤模型,观察沉默miR-92b对U251神经胶质瘤裸鼠皮下移植瘤生长的影响。方法采用神经胶质瘤U251细胞株建立神经胶质瘤的裸鼠皮下移植瘤动物模型,将shRNA-miR-92b表达的质粒稳定转染U251细胞,筛选稳转细胞采取多点注射的方法注入裸鼠皮下,以阴性质粒和PBS为阴性对照组(shRNA-miR-92b NC组)和空白对照(PBS组);检测裸鼠成瘤体积和重量;real-time PCR检测移植瘤中miR-92b、胰岛素样生长因子结合蛋白-3(IGFBP-3)的表达水平;western blot检测移植瘤中的IGFBP-3、β-链蛋白(β-catenin)和钙黏蛋白(E-cadherin)蛋白水平;免疫组织化学技术检测移植瘤中IGFBP-3、β-catenin和E-cadherin表达水平。结果移植瘤形成过程中裸鼠未出现死亡,全部裸鼠均皮下成瘤,肿瘤体积检测后发现shRNA-miR-92b转入后显著抑制移植瘤的生长(P0.05);转入shRNA-miR-92b后,移植瘤中的miR-92b表达水平降低,IGFBP-3的表达水平增加(P0.05);western blot结果显示IGFBP-3和E-cadherin蛋白表达升高,β-catenin蛋白表达降低(P0.05);免疫组化结果也显示IGFBP-3蛋白表达升高,β-catenin蛋白表达降低(P0.05)。结论沉默miR-92b表达后可能通过上调IGFBP3、E-cadherin蛋白表达,下调β-catenin蛋白表达,从而抑制神经胶质瘤裸鼠皮下移植瘤的生长。     

两种方法建立人胃癌裸鼠移植瘤模型的比较

目的采用两种不同方法建立人胃癌裸鼠皮下移植瘤模型。方法人胃癌细胞悬液直接注射法:将处于对数生长期的人胃癌细胞悬液接种于裸鼠皮下;胃癌裸鼠瘤转瘤法:将胃癌细胞悬液接种入裸鼠,待肿瘤生长直径在8 mm左右时,处死裸鼠并取其肿瘤组织边缘新鲜的组织,接种于裸鼠皮下,形成皮下移植瘤。观察并比较两种方法所建立的模型肿瘤移植成功率及瘤体生长速度,实验结束后MSP法检测移植瘤组织中Reprimo基因的甲基化水平并采用免疫组织化学法检测移植瘤的Ki-67和CD31分子表达。结果细胞悬液直接注射组的瘤体成瘤速度较瘤转瘤组慢(P<0.05);瘤转瘤组的Reprimo基因甲基化水平及Ki-67和CD31的表达均高于细胞悬液直接注射组(P<0.05)。结论胃癌裸鼠皮下瘤转瘤法的瘤体生长状况优于胃癌细胞悬液直接注射法。     

蜂毒肽对卵巢癌生长抑制作用的实验研究

目的 研究蜂毒肽对卵巢癌细胞株及卵巢癌皮下移植瘤动物模型的生长抑制作用。方法 选取卵巢癌细胞株SKOV3,以四甲基偶氮唑盐（MTT）比色法观察蜂毒肽对SKOV3的生长抑制作用。通过建立卵巢癌皮下移植瘤动物模型来观察蜂毒肽对移植瘤的生长抑制作用。结果 体外实验观察到蜂毒肽对卵巢癌细胞株生长有抑制作用,并且随着其浓度的增加抑制作用增强。在卵巢癌皮下移植瘤动物模型中,注射蜂毒肽各组瘤重明显低于对照组（P〈0.05）。结论 蜂毒肽可明显抑制卵巢癌细胞株及卵巢癌皮下移植瘤的生长并且随着蜂毒肽的浓度增加抑制作用增强。     

High-intensity focused ultrasound in ovarian cancer xenografts

INTRODUCTION: The purpose of this study was to determine whether highintensity focused ultrasound (HIFU) is an effective treatment for ovarian carcinoma in an athymic nude mouse model. METHODS: Thirty-nine female athymic nude mice were inoculated subcutaneously with 5-7xx10(6) SKOV3 human ovarian cancer cells. Thirty-two animals developed tumours and were randomly divided into three groups: HIFU (n=18), sham treatment (n=8) and control (n=6). The sonographic appearance of the tumours during therapy was recorded. After therapy, the tumours were examined transcutaneously by ultrasound every 4 days for 4 weeks. Tissue samples were taken from the treatment sites and histopathologically examined by light or electron microscopy. RESULTS: Three weeks after HIFU treatment, a 100% reduction in tumour volume was observed in all animals in the HIFU group, whereas tumours in the sham-treated and control groups continued to grow. Pathological examination of HIFU-treated tumour tissue samples showed complete coagulation necrosis of the tumour. CONCLUSION: Our results indicate that HIFU appears to be an effective therapy for ovarian carcinoma tumours in the athymic nude mouse model. We suggest that it may hold promise for the clinical treatment of late-stage and recurrent ovarian cancer.     

磁共振成像在高强度聚焦超声治疗乳腺癌术后随访中的作用

目的 探讨磁共振成像(MRI)技术在高强度聚焦超声(HIFU)治疗乳腺癌术后随访中的作用.方法 对26例乳腺癌在HIFU治疗后行MRI平扫及动态增强磁共振(DCE-MRI)检查,观察其影像学信号改变及组织病理学变化.结果 HIFU治疗乳腺癌术后MRI表现为等中心T1WI信号和等(短)混杂T2WI信号.光镜检查见HIFU...     

Pulsed High-Intensity Focused Ultrasound Therapy Enhances Targeted Delivery of Cetuximab to Colon Cancer Xenograft Model in Mice

Our aim was to evaluate whether pulsed high-intensity focused ultrasound (HIFU) therapy enhances the effect of an epidermal growth factor receptor–targeted chemotherapeutic drug, cetuximab, in treating human colon cancer xenografts in a mouse model. Balb/c nude mice with subcutaneous xenografts of HT-29 cells were randomly categorized into control (n = 9), pulsed HIFU alone (n = 10), cetuximab monotherapy (n = 8) or combined pulsed HIFU and cetuximab therapy (n = 9) group. Cetuximab, pulsed HIFU therapy, or both were administered three times per week starting from day 8 after tumor cell injection. Based on tumor growth curves up to 34 days, the combination therapy group showed more suppressed tumor growth than all other groups (p < 0.05). The final relative tumor volumes were 5.4 ± 2.1, 5.2 ± 1.3, 4.8 ± 1.8, and 3.1 ± 0.9 for control, pulsed HIFU alone, cetuximab monotherapy, and combination therapy groups, respectively. In conclusion, pulsed HIFU therapy appears to enhance the anti-tumor effect of epidermal growth factor receptor–targeted cetuximab on human colon cancer xenograft models in mice.     

高强度聚焦超声体外治疗恶性实体肿瘤的临床安全性研究

目的：研究高强度聚焦超声（HIFU）体外治疗恶性实体肿瘤的临床安全性。方法：HIFU体外治疗恶性实体肿瘤患者164例,观察治疗过程中,治疗后的全身和局部的变化。结果：治疗中,治疗后患者生命体征稳定,主要器官功能基本正常;局部瘤灶无破裂或出血,无黄疸或胆汁漏溢,未发现瘤灶邻近脏器或血管损伤,结论：HIFU体外治疗恶性实体肿瘤是安全的。     

Pathological changes in human malignant carcinoma treated with high-intensity focused ultrasound

The purpose of this study was to investigate the pathologic changes of extracorporeal ablation of human malignant tumors with high-intensity focused ultrasound (HIFU). HIFU treatment was performed in the 164 patients with liver cancer, breast cancer, malignant bone tumor, soft tissue sarcoma and other malignant tumors at focal peak intensities from 5000 W x cm(-2) to 20,000 W x cm(-2), with operating frequencies of 0.8 to 3.2 MHz. To explore the pathologic impact of extracorporeal HIFU, 30 patients with malignant carcinoma underwent surgical removal after HIFU treatment. Pathologic findings showed that the treated tissues demonstrated homogeneous coagulative necrosis with an irreversible tumor cell death and severe damage to tumor blood vessels at the level of microsvasculature within the HIFU-targeted region. Thermolesions to intervening tissue were never observed. The treated region had a sharp border comprising only several cell layers between the treated and untreated areas. The repair of lesions had the processes of necrotic tissue absorption and granulation tissue replacement. It is concluded that extracorporeal treatment of human solid malignancies with HIFU could be safe, effective and feasible. As a noninvasive therapy, HIFU would be used clinically to treat patients with solid malignancies.     

HIFU治疗胰腺癌的初步临床研究

目的探讨高强度聚焦超声（HIFU）治疗胰腺癌的安全性和有效性。 方法7例不能手术的胰腺癌患者进行了HIFU治疗，通过临床症状、实验室检查、影像学变化、并发症情况等观察HIFU的治疗效果。 结果治疗后部分患者临床疼痛症状减轻、Ca 19-9降低、MRI检查显示HIFU治疗区内组织呈凝固性坏死表现，无胃肠穿孔等严重并发症出现。 结论HIFU治疗是一种较为安全、有效和非侵入性治疗胰腺癌的方法。     

Tumor vessel destruction resulting from high-intensity focused ultrasound in patients with solid malignancies

The purpose of this study was to explore the sequential imaging and histologic alterations of tumor blood vessels in the patient with solid malignancies after extracorporeal treatment of high-intensity focused ultrasound (HIFU). A total of 164 patients underwent extracorporeal HIFU ablation of malignant solid tumors. After HIFU treatment, enhanced magnetic resonance imaging (MRI), color Doppler ultrasound (US) imaging, dynamic radionuclide scanning, digital subtraction angiography, and histologic study were performed to monitor the response of tumor vessels to HIFU ablation. Compared with tumor images in the patients before HIFU, clinical images showed an abrupt interruption, followed by the cessation of blood flow within the tumor vessels after HIFU treatment. The histologic examination indicated that not only the treated tumor cells showed coagulative necrosis, but also small tumor vessels were severely damaged by the HIFU treatment. The results strongly imply that the damaged tumor vessels might play a critical role in secondary tumor cell death, and then indirectly strengthen the destructive force of focused US beams on tumor tissue. It is concluded that tumor vessel damage can be induced by HIFU, which may be a promising strategy in the treatment of patients with solid malignancies.     

高强度聚焦超声治疗原发性肝癌术后10年门诊随访现状

目的分析高强度聚焦超声(HIFU)治疗原发性肝癌术后10年的随访现状,探讨HIFU无创治疗技术的术后随访新医学模式。方法对1999~2009年HIFU治疗的原发性肝癌患者进行术后定期随访,通过术后临床症状、实验室检查、影像学检查观察HIFU治疗的效果。结果治疗后2周经磁共振复查提示病灶已凝固坏死,肝癌标志物甲胎蛋白(AFP)下降率为83.12%(192/231),1年生存率为78.16%,2年生存率为59.54%,3年生存率为38.95%,随访率84.30%,失访率15.70%。结论持续开展术后定期门诊随访,对于观察HIFU的治疗效果具有非常重要的意义,可以及时、定期了解肝癌病情的发展变化,从而有效地延长患者生存期限。     

拉帕替尼对人卵巢癌SKOV3裸鼠移植瘤的影响

【目的】探讨新型靶向治疗药物拉帕替尼对人卵巢癌SKOV3裸鼠移植瘤生长的影响。【方法】将人SKVO3细胞株接种于裸小鼠胸壁皮下建立卵巢癌裸小鼠移植瘤模型。随机将其分为空白对照组（A组）和实验组（B组）,实验组根据拉帕替尼药物浓度的不同分为两个亚组：100 mg/kg （B1）和200 mg/kg （B2）,检测各组拉帕替尼用药前后荷瘤体积及裸鼠体质量的变化。【结果】拉帕替尼可显著抑制SKOV3裸小鼠移植瘤的生长,呈剂量依赖性,B2组抑瘤率显著高于B1组,其差异有统计学意义（P＜0．05）,但小鼠体质量用药前后比较无显著性差异（P＞0．05）。【结论】拉帕替尼可显著缩小人卵巢癌细胞株SKVO3裸鼠移植瘤的体积,为卵巢癌新型靶向治疗提供了理论基础。     

Photoacoustic computed tomography of mechanical HIFU-induced vascular injury

Mechanical high-intensity focused ultrasound (HIFU) has been used for cancer treatment and drug delivery. Existing monitoring methods for mechanical HIFU therapies such as MRI and ultrasound imaging often suffer from high cost, poor spatial-temporal resolution, and/or low sensitivity to tissue’s hemodynamic changes. Evaluating vascular injury during mechanical HIFU treatment, therefore, remains challenging. Photoacoustic computed tomography (PACT) is a promising tool to meet this need. Intrinsically sensitive to optical absorption, PACT provides high-resolution imaging of blood vessels using hemoglobin as the endogenous contrast. In this study, we have developed an integrated HIFU-PACT system for detecting vascular rupture in mechanical HIFU treatment. We have demonstrated singular value decomposition for enhancing hemorrhage detection. We have validated the HIFU-PACT performance on phantoms and in vivo animal tumor models. We expect that PACT-HIFU will find practical applications in oncology research using small animal models.