声学微泡联合超声靶向介导耐药基因ASON转染逆转肝癌MDR
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摘要
引言
肝癌是全球范围内最常见的恶性肿瘤之一。在我国,肝癌占恶性肿瘤死亡率的第一或第二位,全球每年新发病例约45%在我国大陆地区,发病年龄趋于年轻化。目前肝癌临床以综合治疗为主,但肝癌细胞多药耐药(multidrug resistance,MDR)严重限制了治疗的效果。有资料显示90%以上肝癌病人的死因与MDR有关,因此如何逆转肿瘤MDR,提高肝癌有效治疗是一个亟待解决的问题。肿瘤MDR理想的逆转方法应该具有高效、无毒和靶向性(能特异性地作用于肿瘤而对正常组织无损害)的特点,然而现在的研究与此标准差距尚远。因此进行肝癌MDR系列研究,探讨新的逆转手段成为提高肿瘤控制的关键,具有非常重要的现实意义和应用价值。
本课题共分四部分进行实验研究。建立人肝癌MDR细胞模型和裸鼠皮下移植瘤模型,利用声学微泡联合超声靶向介导mdr1、mrp耐药基因的反义寡核苷酸(antisense oligonucleotide,ASON)转染,探讨对人肝癌细胞和移植瘤MDR的逆转效应和机理,力求寻找一种低毒、高效和靶向的逆转肝癌MDR的新方法,为提高肝癌综合治疗效果,开展临床肝癌基因治疗和高剂量冲击化疗提供理论依据。本研究将分子生物学寡核苷酸反义技术和声学微泡并超声治疗技术结合,借助声学微泡并超声的空化效应,以促进基因在肿瘤细胞的高效和靶向转染及表达,提高对MDR的逆转效率,在策略和技术上是一种创新。
第一部分肝癌MDR细胞模型建立和生物学特性研究
第一节顺铂递增加量-间歇作用法建立QGY肝癌MDR细胞模型及生物学特性鉴定
目的:建立人肝癌QGY多药耐药细胞模型并初步检测其耐药特性。方法:以人肝癌细胞株QGY为亲本细胞,选用化疗药物顺铂(cisplatin,CDDP),以低浓度递增加量-间歇作用法诱导细胞耐药。光镜、电镜观察新建细胞株组织形态学及超微结构特点。测定细胞倍增时间并绘制生长曲线、观察细胞克隆形成和流式细胞仪(Flow Cytometry,FCM)检测细胞周期分布,鉴定新建细胞生物学特性。四甲基偶氮唑盐( 3-4,5-dimethyIthiazol-z-yl-2,5-dipheny tretrazolium bromide , MTT )法测定细胞药物敏感性,免疫组化(immunohistochemistry assay)检测细胞耐药蛋白P-gp、MRP和LRP,凋亡蛋白Bcl-2和Bax的表达。结果:历时15周多,成功建立MDR细胞株QGY/CDDP。QGY/CDDP在组织形态学及超微结构上与亲本细胞QGY无明显差别。一般生物学特性:MDR细胞的群体倍增时间较亲代细胞延长25小时多,克隆形成率明显低于亲本细胞,G0/G1期细胞比例增加,S期和G2/M期细胞比例下降,与亲本细胞QGY比较有显著性差异(P<0.05)。药敏检测显示:QGY/CDDP细胞对CDDP高度耐药,耐药指数(resistance index,RI)为10.35,对ADM(阿霉素)、5-Fu(5-氟脲嘧啶)和AsT(三氧化二砷)也产生耐药性,RI分别为5.50、8.51和9.23。免疫组化结果为MRP染色阳性细胞率最高,为23.89%,P-gp染色阳性细胞率为13.26%;LRP、Bcl-2和Bax染色阳性细胞率与亲本细胞接近。结论:新建细胞株QGY/CDDP具有MDR特性,其耐药机制由MRP和P-gp介导,以MRP介导机制为主,LRP、Bcl-2/Bax可能不参与QGY/CDDP耐药性的形成。
第二节X射线照射法建立HePG2肝癌MDR细胞模型及耐药性鉴定
目的:建立人肝癌HepG2多药耐药细胞模型并初步检测其耐药特性。方法:以人肝癌细胞株HepG2为亲本细胞,给于6MV直线加速器X-射线照射,诱导细胞耐药。HepG2细胞受X射线累积量照射后,接种到含浓度0.4μg/ml阿霉素(adriamycin,ADM)培养基中培养,发现细胞良好稳定增殖生长,提示细胞对抗ADM的细胞毒作用,即将新建细胞株命名为HepG2/ADM。光镜、电镜观察新建细胞株组织形态学及超微结构特点。测定细胞贴壁率、细胞克隆形成率和FCM检测细胞周期分布观察HepG2/ADM生物学特性。MTT法检测HepG2/ADM细胞药物敏感性,免疫组化测定细胞耐药蛋白P-gp、MRP、LRP以及凋亡蛋白Bcl-2/Bax表达,FCM测定细胞Rh123摄入和外排能力,间接检测P-gp和MRP功能。结果:历时45天多,X-射线照射累积剂量为24GY,处理后细胞能在含0.4μg/mlADM培养基中生长,即成功建立MDR细胞株HepG2/ADM。HepG2/ADM组织形态学及超微结构与亲本细胞HepG2无明显差别。一般生物学特性:HepG2/ADM细胞贴壁率、克隆形成率明显降低,G0/G1期细胞比例增加,S期及G2/M期细胞比例减少,与亲本细胞比较有显著性差异(P<0.05)。药敏检测显示:HepG2/ADM细胞对ADM高度耐药,RI为30.31,同时对CDDP、5-Fu和AsT也产生了耐药性,RI分别为6.12、6.94和9.15。免疫组化染色可见P-gp阳性染色细胞率最高,为85.62%,MRP阳性染色细胞率为12.36%,LRP阳性染色细胞率与亲本细胞接近,Bcl-2与Bax阳性染色细胞率分别为27.35%、6.25%,Bcl-2/Bax比值增高,较亲本细胞HepG2有显著性差异(P<0.05)。结论:新建细胞株HepG2/ADM具有MDR特性,其耐药机制由P-gp、MRP介导和凋亡蛋白Bcl-2/Bax共同作用,以P-gp介导机制为主。
第二部分声学微泡联合超声靶向介导耐药基因ASON转染逆转肝癌细胞MDR的效应和机制
目的:初步讨论声学微泡联合超声靶向介导耐药基因ASON转染逆转QGY/CDDP、HepG2/ADM人肝癌细胞株MDR的效应和作用机制。方法:取对数生长期细胞,调整成单细胞悬液,分别给于mdr1-ASON+声学微泡+超声、mrp-ASON+声学微泡+超声实验处理,待细胞生长良好,进行各项指标检测:在QGY/CDDP细胞,MTT法测定细胞耐药性变化,细胞贴壁率测定、细胞周期分布检测(FCM)、细胞凋亡TUNNEL法测定,观察细胞接受实验处理后的生物学特性变化,RT-PCR检测mdr1、mrp基因的mRNA表达,Western-blot测定细胞P-gp、MRP蛋白表达,免疫组化测定细胞凋亡蛋白Bcl-2、Bax表达。在HepG2/ADM细胞,AO/EB荧光法测定细胞凋亡,其它检测方法与QGY/CDDP实验处理后细胞检测一致。结果:QGY/CDDP细胞对CDDP、ADM、5-FU和AsT四种药物的耐药性有一定下调,RI降低:mdr1-ASON处理中,与对照组比较无显著性差异(P>0.05);mrp-ASON处理中,与阴性对照组比较有显著性差异(P<0.05),与阳性对照组比较无显著性差异(P>0.05)。mdr1-ASON转染后,细胞周期分布、P-gp、MRP的相对表达量和凋亡蛋白阳性细胞百分率的变化均较小,但与对照组比较差异无显著性(P>0.05);在细胞贴壁率、凋亡细胞TUNNEL法测定结果和耐药基因mRNA的相对表达量有一定变化(P<0.05)。mrp-ASON转染后,细胞贴壁率、细胞周期分布、TUNNEL法测定凋亡细胞、耐药基因mRNA的相对表达和P-gp、MRP的相对表达六项指标均有一定变化,有显著性差异(P<0.05);凋亡蛋白免疫组化结果一项变化较小。HepG2/ADM细胞实验处理后,细胞药物敏感性、细胞贴壁率、细胞周期分布、凋亡细胞、耐药基因mRNA表达、耐药蛋白的相对表达量和凋亡蛋白阳性细胞百分率等共七项检测指标均显示细胞有一定的变化,且与对照组比较有显著性差异(P<0.05);同样可见mdr1-ASON处理中,细胞的指标变化大于mrp-ASON处理。结论:mdr1-ASON+声学微泡+超声照射与mrp-ASON+声学微泡+超声照射能够部分逆转QGY/CDDP、HepG2/ADM人肝癌细胞的MDR。声学微泡结合超声比阳离子脂质体结合超声有更好的转染基因ASON靶向介导作用,显示对肝癌细胞株MDR更强的逆转效应。在QGY/CDDP细胞,mrp-ASON转染逆转MDR的作用明显强于mdr1-ASON转染;在HepG2/ADM细胞,mdr1-ASON、mrp-ASON转染均有较强逆转肿瘤MDR的作用,mdr1-ASON转染的MDR逆转作用更强。
第三部分裸鼠肝癌MDR移植瘤模型的建立和生物学特性研究
目的:建立人肝癌QGY/CDDP、HepG2/ADM多药耐药的裸鼠移植瘤模型并鉴定其耐药特性。方法:取对数生长期QGY/CDDP、HepG2/ADM细胞,调整为单细胞悬液,按实验分组,采用细胞悬液皮下直接注射法,将MDR肿瘤细胞接种在裸鼠皮下,观察肿瘤生长并记录。待皮下移植瘤生长到一定大小,各组随机取两只裸鼠处死,剥离移植瘤组织块,提取移植瘤细胞培养,待细胞处于对数生长期进行耐药性检测。光镜、电镜观察移植瘤细胞组织形态学和超微结构特点。进行移植瘤细胞倍增时间并绘制生长曲线和细胞克隆形成率测定,观察移植瘤细胞的生物学特性。MTT测定移植瘤细胞药物敏感性,FCM检测移植瘤细胞P-gp、MRP表达,Western-blot测定细胞凋亡蛋白Bcl-2、Bax表达。结果:QGY/CDDP、HepG2/ADM荷瘤裸鼠的平均生存期为40±15d,移植瘤生长体积统计分析结果:两株细胞移植瘤生长均较其对应非耐药移植瘤细胞慢,有显著性差异(P<0.05)。MDR移植瘤细胞的组织形态学及超微结构与对应非耐药移植瘤细胞无明显差异。移植瘤细胞的生物学特性检测:两株细胞均呈现细胞倍增时间延长(P<0.05),细胞克隆形成率降低(P<0.05)。MTT法结果:两株MDR移植瘤细胞均对ADM、CDDP、5-FU和AsT四种药物耐药,QGY/CDDP移植瘤细胞对CDDP的RI最高,HepG2/ADM细胞对ADM的RI最高,与各自非耐药移植瘤细胞比较差异具有显著性差异(P<0.05)。耐药蛋白与凋亡蛋白测定结果:在QGY/CDDP移植瘤细胞,MRP蛋白染色阳性细胞率最高,为23.85%(P<0.05),P-gp染色阳性细胞率为7.44%(P<0.05);Bcl-2和Bax的相对表达与移植瘤QGY细胞接近(P>0.05)。在HepG2/ADM移植瘤细胞,P-gp蛋白染色阳性细胞率最高,为84.97%,MRP染色阳性细胞率为10.26%,Bcl-2和Bax的相对表达与移植瘤HepG2细胞比较有显著性差异(P<0.05)。结论:成功建立人肝癌QGY/CDDP、HepG2/ADM裸鼠皮下移植瘤模型。其耐药机制可能主要与mdr1及蛋白P-gp、mrp及蛋白MRP(P190)和凋亡蛋白Bcl-2/bax的表达有关。本移植瘤模型成功率高,可重复性强,生物学特性稳定。
第四部分声学微泡联合超声靶向介导耐药基因ASON转染逆转裸鼠肝癌移植瘤MDR的效应和机制
目的:初步探讨声学微泡联合超声靶向介导耐药基因ASON转染在体逆转裸鼠肝癌移植瘤MDR的效应及其作用机制。方法:对于肝癌MDR移植瘤裸鼠,按实验分组在体给于实验处理: QGY/CDDP移植瘤给于mrp-ASON+声学微泡+超声, HepG2/ADM移植瘤给于mdr1-ASON+声学微泡+超声,并以相应ASON+阳离子脂质体+超声为阳性对照,相应MDR移植瘤+转染液注入为阴性对照,在体实验结束处死裸鼠,剥离移植瘤块,提取相应移植瘤细胞培养。取对数生长期培养细胞进行MTT药敏检测、RT-PCR测定细胞mdr1及mrp基因mRNA表达、Western-blot检测细胞P-gp及MRP蛋白表达、免疫组化测定细胞凋亡蛋白bcl-2及bax表达和移植瘤细胞膜ATPase活性变化测定。结果:裸鼠肝癌移植瘤在体实验处理后,移植瘤细胞检测发现:mrp-ASON+声学微泡+超声作用QGY/CDDP移植瘤后,与阴性对照组比较,移植瘤细胞对CDDP、ADM、5-FU和AsT四种药物的耐药性明显下调(P<0.05),与阳性组比较,耐药性有一定下调,但差异无显著性意义(P>.05),RI降低。RT-PCR显示:移植瘤细胞mrp基因的mRNA表达下降明显(对mdr1基因的mRNA表达影响较小),较对照组有显著性差异(P<0.05)。Western-blot结果:移植瘤细胞P-gp、MRP表达均有一定下降(MRP绝对值大于P-gp),与对照组比较有显著性差异(P<0.05)。凋亡蛋白的免疫组化结果表明:移植瘤细胞bcl-2、bax有一定变化,与对照组比较差异无显著性意义(P>0.05)。细胞膜ATPase活性变化测定可见:移植瘤细胞的细胞膜ATPase活性有一定上调,但与对照组比较差异无显著性意义(P>0.05)。mdr1-ASON+声学微泡+超声作用HepG2/ADM移植瘤后,移植瘤细胞对ADM、CDDP、5-FU和AsT四种药物耐药性明显下调,与对照组比较有显著性差异(P<0.05),RI降低。RT-PCR显示:移植瘤细胞mdr1基因和mrp基因的mRNA表达明显下降,较对照组有显著性差异(P<0.05)。Western-blot结果表明:移植瘤细胞P-gp、MRP表达明显下降(P-gp绝对值大于MRP),与对照组比较有显著性差异(P<0.05)。凋亡蛋白的免疫组化测定结果显示:移植瘤细胞bcl-2、bax明显变化,较对照组有显著性差异(P<0.05)。移植瘤细胞膜ATPase活性变化检测结果可见:细胞膜ATPase活性有一定上调,与对照组比较有显著性差异(P<0.05)。结论:ASON+声学微泡+超声照射能够在体逆转裸鼠肝癌移植瘤的MDR,声学微泡联合超声比阳离子脂质体结合超声有更好的转染基因ASON的靶向介导作用,显示对肝癌裸鼠移植瘤MDR更强的逆转效应。分析这种现象的可能原因是:超声的照射导致微泡破裂,微泡破裂的空化效应促进了ASON在细胞的转染与表达。裸鼠肝癌移植瘤QGY/CDDP的MDR形成主要是mrp介导机制,与mdr1介导机制和细胞抗凋亡增强机制关系不大,相应逆转细胞MDR的作用是通过下调细胞mrp及其编码蛋白MRP表达来实现的。移植瘤HepG2/ADM的MDR形成是mdr1介导机制、mrp介导机制和细胞抗凋亡增强机制共同作用的结果,以mdr1介导机制为主,相应逆转细胞MDR的作用是通过降低三机制的作用,尤其是下调mdr1及其编码蛋白P-gp表达来实现。
小结
1.采用CDDP低浓度递增加量-间歇作用法、直线加速器X-射线照射法分别建立了两株MDR细胞模型:QGY/CDDP和HepG2/ADM。两种细胞都具有较高的MDR特性,QGY/CDDP细胞对CDDP、ADM、5-FU和AsT的耐药指数分别为10.35、5.50、8.51和9.23;HepG2/ADM细胞对CDDP、ADM、5-FU和AsT的耐药指数分别为6.12、30.31、6.94和9.15。
2. QGY/CDDP细胞的MDR机制主要是由mrp及其编码蛋白MRP介导,与mdr1介导机制和细胞抗凋亡增强机制关系不大。HepG2/ADM细胞的MDR机制是由mdr1及其蛋白P-gp介导、mrp及其蛋白MRP介导和细胞抗凋亡增强共同发挥作用,但以mdr1及其蛋白P-gp介导机制为主。
3.用mdr1、mrp-ASON+声学微泡+超声照射能够部分逆转人肝癌细胞QGY/CDDP、HepG2/ADM的多药耐药,声学微泡联合超声比阳离子脂质体结合超声有更好的转染耐药基因ASON的靶向介导作用,显示出对肝癌细胞株MDR更强的逆转效应。对于QGY/CDDP细胞逆转MDR的作用,mrp-ASON转染明显强于mdr1-ASON转染。对于HepG2/ADM细胞,mdr1-ASON、mrp-ASON转染均有较强逆转肿瘤MDR的作用,mdr1-ASON转染的MDR逆转作用更强。
4. MDR肝癌单细胞悬液直接裸鼠皮下注射法接种,能够建立人肝癌裸鼠QGY/CDDP和HepG2/ADM移植瘤模型。移植瘤细胞有高度耐药特性:QGY/CDDP移植瘤细胞对CDDP、ADM、5-FU和AsT的RI依次为9.28、4.50、7.52和8.36;HepG2/ADM移植瘤细胞对CDDP、ADM、5-FU和AsT的RI依次为6.05、27.94、6.73和8.96。
5.用mrp-ASON+声学微泡+超声照射能够在体部分逆转人肝癌QGY/CDDP细胞裸鼠移植瘤的MDR,用mdr1-ASON+声学微泡+超声照射能够在体部分逆转人肝癌HepG2/ADM细胞裸鼠移植瘤的MDR;其中声学微泡联合超声比阳离子脂质体结合超声有更好的转染基因ASON的靶向介导作用,显示出对移植瘤MDR逆转效应更强。
6.声学微泡联合超声靶向介导耐药基因-ASON转染逆转人肝癌细胞株和移植瘤MDR的可能主要机制是封闭或下调mdr1、mrp及编码蛋白的表达,进而降低细胞耐药性。其它机制可能包括提高细胞对药物的敏感性、提高细胞膜ATPase活性,促进细胞对药物的摄取,上调细胞内药物浓度和改变凋亡基因的表达,使Bcl-2/Bax比例下调等等。声学微泡造影剂联合超声较阳离子脂质体结合超声靶向介导耐药基因ASON转染具有更好的逆转MDR效应的可能原因是:低强度超声照射导致微泡破裂,微泡破裂的空化效应促进了ASON在细胞的转染与表达,进而表现MDR细胞株和移植瘤的耐药性下降。
Introduction
Hepatoma is one of the most common cancer worldwide. In China its mortality rate is the first or second malignancies. Each year about 45% of new cases are in China and it is tendency to onset in younger. The combined treatment is the main method for liver tumors at present. But hepatoma cells MDR (multidrug resistance, MDR) severely limits the effect of the treatment. Some data indicate that more than 90% of patients with liver cancer and the death of the MDR. How to reverse the MDR is an urgent task to improve the treatment of liver cancer. The ideal method of tumor MDR reversal must be efficient, asepsis and target (be specific to tumor while no damage to normal tissue). But recent researches are far from this standard. Therefore a series of studiesof MDR HCC have being carried out to explore new method which is the key to improving the effect on cancer control, and that is of important practical significance and value at present.
The present researches were divided four parts. It would try to find a new low toxicity, efficient and targetted method for MDR reversion of HCC, and try to provide a theoretical basis to improve comprehensive treatment effects, clinical gene therapy and high-dose chemotherapy for liver cancer, which had Built the model of MDR human hepatoma cell and subcutaneous transplant tumor, explored the reversal effects and mechanisms on MDR of human hepatoma cells and transplant tumor through using acoustic microbubbles joint ultrasound taget-mediating for the antisense oligonucleotides transfection of mdr1 and mrp resistance gene. In this study, it had improved the efficiency of the MDR reversal by using acoustic microbubbles and ultrasonic cavitation effect to facilitate the efficient of targeted transfection and expression of gene in tumor cells for antisense oligonucleotide technologies of molecular biology in combintion with acoustics microbubbles and ultrasonic treatment. It is a strategy and technical innovation.
Part 1 Establishment of HCC model for MDR cells and biological characterization
Section 1 Establishment of QGY Multidrug-resistant cell line induced by exposure of increasing concentration by degrees and intermission affect approach of CDDP and biological characterization
Objective: To establish human hepatoma cell multidrug resistance model QGY and preliminary testing their resistance. Methods : The human hepatoma cell line for the pro-inhibiting cell, the use of chemotherapy drug cisplatin (cisplatin. CDDP), a low concentration increases delivery-resistant cells induced by intermittent role. Light microscopy, electron microscopy observation of morphological and ultrastructural characteristics of the new cell lines. Determination of cell doubling time and the mapping of the growth curve, observation of cell colony formation and flow cytometry (Flow Cytometry, FCM) detection of cell cycle distribution, identification of new biological characteristics. MTT assay of cell drug sensitivity, immunohistochemistry detecion of the expression of cells resistant Protein P-gp, MRP and LRP, apoptotic protein Bcl-2 and Bax. Results: During the 15 weeks, the MDR cell lines QGY/CDDP successful. QGY/CDDP in morphology and ultrastructure with the parental cells QGY is no different. General characterazation: MDR cell population doubling time than the parent cells extended 21-hour, colony formation was significantly lower than the parental cells, the cells in G0/G1 phase increased, the cell ratio in S and G2/M phase decreased, and the difference that with the parental cell QGY was statistically significant (P <0.05). Susceptibility testing revealed: QGY/CDDP cells were highly resistant to CDDP, RI (resistance index, RI) 10.35; of ADM (ADM). 5-FU (5-fluorouracil) and AST (arsenic trioxide) also resistance, RI 5.50, 8.51 and 9.23 respectively. The results of immunohistochemical were that staining positive for cell MRP highest rate of 23.89%, P-gp cells staining positive for 13.26%; the rate of LRP, Bcl-2 and Bax-positive cells was close to the parental cells. Conclusion: The new cell lines have MDR QGY/CDDP characteristics, resistance mechanisms mediating by the MRP and P-gp. MRP-mediating mechanism, while LRP, QGY/CDDP Bcl-2/Bax may not be involved in the formation of resistance.
Section 2 Establishment of HePG2 Multidrug-resistant cell line induced by X-radiation exposure approach and biological characterization
Objective: To establish human hepatoma HepG2 cell MDR model and preliminary testing their resistance. Methods: The human hepatoma cell line HepG2 cells for parent was to X-ray irradiation at 6 MV linear accelerator, induce cell resistance. HepG2 cells after irradiation by X-ray accumulation, was inoculated with the 0.4μg/ml concentration of ADM (adriamycin, ADM) was cultured, good stability proliferation and growth of cells, suggesting that cell cytotoxicity against ADM. HepG2/ADM named for the upcoming new cell lines. Light microscopy, electron microscopy observation of morphological and ultrastructural characteristic of the new cell lines. Determination of cell adhesion rate, cell colony formation rate, and FCM detection of cell cycle distribution, observation HepG2/ADM biological characteristics. MTT assay sensitivity in HepG2/ADM cells, immunohistochemistry detecion of the expression of cells resistant Protein P-gp, MRP and LRP, apoptotic protein Bcl-2 and Bax, FCM in cell Rh123 intake and discharge capacity, indirect detection of P-gp and MRP functions. Results: During the 45 days, the cumulative dose of X-ray irradiation 24GY, treated cells in culture medium containing 0.4μg/ml ADM growth, HepG2/ADM MDR cell lines successfully established. HepG2/ADM in morphology and ultrastructure with the parental cells HepG2 was no significant different. General characterazation: HepG2 cells adherent rate, rate of colony formation significantly reduced, the proportion of cells in G0/G1 phase increased, the proportion of cells in S and G2/M phase decreased, that difference with the parental cells was statistically significant (P <0.05). Susceptibility testing revealed: HepG2/ADM cells were highly resistant to ADM, RI to 30.31; meanwhile to CDDP, 5-Fu and AsT also produced resistance, RI 6.12, 6.94 and 9.15. Immunohistochemical staining shows that the highest rate of P-gp positive staining cells to 85.62%, MRP rate of 12.36% positive staining cells, LRP positive staining cells with the parental cells close, Bcl-2 and Bax were 27.35%, 6.25% positive staining cells, Bcl-2/Bax ratio increased, all the differences with the parental HepG2 cells had statistically significance (P <0.05). Conclusion: new cell line HepG2 with MDR characteristics its resistance was combined by P-gp, MRP-mediating and apoptotic protein Bcl-2, the main mechanism mediating by P-gp.
Part 2 Effects and mechanisms of hepatoma cells MDR reversed by acoustic microbubbles joint ultrasound target-mediating ASON of drug resistance gene transfection
Objective: To discuss the effect and mechanism of QGY/CDDP and HepG2/ADM human hepatoma cell line MDR reversed by ASON + ultrasound +contrast microbubbles. Methods: Log phase cells to adjust as a single cell suspension were to mdr1-ASON+ microbubbles+ ultrasound and mrp-ASON+ contrast microbubbles + ultrasound treatment, cell growth to be good, detection of the various indicators: In QGY/CDDP cells, MTT assay to detect changes in cell resistance, cell adhesion rate determination, detection of cell cycle distribution (FCM), apoptosis; TUNNEL determination to observe the biological characteristics after cells receiving the experimental treatment; RT-PCR assay to detect the mRNA expression of mdr1and mrp gene; Western-blot assay to detect the expression changes of protein P-gp and MRP; immunohistochemical staining assay to detect the expression of cell apoptotic protein bcl-2 and bax. In HepG2 cells, fluorescence AO/EB determination to detect apoptosis, other detection methods agreed to experimental treatment QGY/CDDP cell line testing. Results: The drug resistance of QGY/CDDP cells to CDDP, ADM, 5-FU and AsT was down to a certain extent, RI reduced: mdr1-ASON processed, compared with the control group the difference was no statistically significant (P> 0.05); mrp-ASON processed, compared with the negative control group the difference was statistically significant (P <0.05), and compared with the control group, the difference was no statistically significant (P> 0.05). mdr1-ASON after transfection, cell cycle distribution, relative expressing amonts of P-gp, MRP protein, and percentage of positive cells of apoptosis protein in the cells were smaller changes, compared with the control group the difference was not statistically significant (P> 0.05); and the rate of cells adherent, TUNNEL detection of apoptosis resulted, relative expression amonts of resistance gene mRNA to a certain extent for statistically significant (p <0.05). mrp-ASON after transfection, the six indicators of cells adherent rate, cell cycle distribution, TUNNEL determination of apoptotic cells, relative expression amonts of resistance gene mRNA, relative expressing amonts of P-gp, MRP protein were certain changes, the differences were statistically significant (P<0.05); immunohistochemical results of apoptotic protein was a small change. HepG2 cells after experimental treatment, the seven indicators of drug sensitivity of cells, cell adhesion rate, cell cycle distribution, apoptosis, mRNA expression of resistance gene, percentage of positive cells of resistance protein and apoptotic protein, showed certain cells changes, and compared with the control group the difference was statistically significant (P <0.05); also seen colony processing, changes of indicators were larger than that after mrp-ASON treatment. Conclusion: mdr1-ASON+ acoustic contrast microbubbles + ultrasound treatment and mrp-ASON+ contrast microbubbles + ultrasound exposure QGY/CDDP could partly reverse human hepatoma HepG2 cells MDR. Combination of ultrasound treatment, Ultrasound contrast microbubbles have a better target-mediating roles for ASON gene transfection than cationic liposomes, which showed a stronger role reversal to MDR of hepatoma cell lines. In QGY/CDDP cells, mrp-ASON transfection had a significantly stronger role in the reversal of MDR colony transfection than mdr1-ASON transfection; In HepG2 cells, mdr1-ASON and mrp-ASON transfection have greater role in the reversal of the MDR, transfection of MDR reversal colony stronger.
Part 3 Establishment and characterization MDR model of hepatocellular carcinoma in nude mice
Objective: To establish a human hepatoma QGY/CDDP, HepG2/ADM nude mouse model of multi-drug resistance and identified their resistance. Methods: Log phase QGY/CDDP, HepG2 cells to adjust as the single cell suspension, by the experimental groups, were directly injected subcutaneously, so MDR tumor cells were inoculated in nude mice subcutaneously, to observe and record the growth of tumors. When the implanted tumors grew to a certain size, two nude mice were randomly killed in each group, dissecting transplanted tumor tissue, extracting tumor cells to cultivate, when cells were in the logarithmic growth phase for resistance testing. Light microscopy, electron microscopy detection of morphological and ultrastructural characteristic of tumor cells. Observation of transplant tumor doubling time and the mapping of the growth curve, determination of cell colony formation rate and cell cycle distribution, to observe the biological characteristics of transplanted cells. MTT to detect the sensitivity of tumor cells to drug. FCM to detect the expression of P-gp and MRP. Western blot to determine the expression of cell apoptosis protein Bcl-2 and Bax. Results: In QGY/CDDP, HepG2/ADM nude mice, the average survival period was 40±15 days. Growth Statistical analysis of transplanted tumor size: the growth of the two cell tumors was slower than that of the parental cells transplanted tumor, and the difference was statistically significant (P <0.05). MDR tumor cells in morphology and ultrastructure of the cells had no significant difference to pro counterparts. Detection of biological characteristics of the transplanted tumor cells: two strains of cell doubling time were longer (P <0.05), the rate of cell colony formation decreased (P <0.05), the proportion of cells in G0/G1 phase of the cell cycle distribution increased, the proportion of cells in S and G2/M phases declined, which the difference was statistically significant compared with parental implanted tumor cells (P <0.05). Results of MTT assay: Two MDR tumor cells were resistant to ADM, CDDP, 5-FU and AsT, and the RI of QGY/CDDP tumor cells to CDDP was the highest, he RI of HepG2/ADM tumor cells to ADM was the highest, compared with each parental implanted tumor cells, which the difference had statistically significant (P <0.05). The results of detection of resistance protein and apoptosis protein: In implanted tumor cells QGY/CDDP, the rate of positive cells of MRP protein staining was the highest to 23.85% (P <0.05), the rate of positive cells of P-gp cells staining was 7.44% (P <0.05); the rate of positive cells of Bcl-2 and Bax cells staining was close to that of the parental tumor cells. In HepG2/ADM transplanted tumor cells, the rate of positive cells of P-gp cells staining was highest to 84.97%, 10.26% rate of cells staining positive MRP, and relative expressing amonts of Bcl-2 and Bax protein, which the difference was statistically significant (P <0.05) compared with the parental transplanted tumor cells. Conclusion: To successfully establish the human hepatoma QGY/CDDP, HepG2/ADM nude mice model. The main resistant machanism may be of the expression of mdr1, protein P-gp, mrp, protein MRP (P190) and apoptotic proteins Bcl-2/Bax. The implanted model had the high success rate and had repeatable, stable biological characteristics.
Part 4 Effect and mechanisms of reversal of hepatocellular carcinoma MDR in nude mice by acoustic microbubbles joint ultrasound target-mediating ASON of drug resistance gene transfection
Objective: To preliminary study the effect and mechanism of reversal human hepatocellular carcinoma MDR in nude mice in vivo by ASON+ ultrasound contrast microbubbles+ ultrasound treatment. Method: The nude mice implanted tumor in vivo, by the lab groups, were given experimental treatment: mrp-ASON+ ultrasound contrast microbubbles+ ultrasound treatment to QGY/CDDP transplanted tumor, mdr1-ASON+ ultrasound contrast microbubbles+ ultrasound treatment to HepG2/ADM transplanted tumor, corresponding ASON+ cationic liposome+ ultrasound as a positive control, corresponding MDR tumor + switch dye injected as a negative control. At the end of death in nude mice in vivo, transplanted tumor was stripped, the corresponding tumor cells were extracted and cultivated. Cells were in the logarithmic growth phase for MTT susceptibility testing, mRNA expression of mdr1 and mrp gene measured by RT-PCR, and expression of P-gp cells and protein MRP was detected by Western blot, expression of apoptosis protein of bcl-2 and bax was determined by immunohistochemical staining, the implanted tumor cell membrane ATPase activity was determined. Results: After in vivo treatment of the human hepatocellular carcinoma xenograft in nude mice, the implanted tumor cells were detected to find: after mrp-ASON+ultrasound contrast microbubbles+ultrasound treatment to QGY/CDDP transplanted tumor, compared with the negative control group, the drug resistance of transplanted tumor cells to CDDP, ADM, 5-FU and AsT significantly reduced (P <0.05); compared with the positive control group, the resistance reduced to a certain extent, but the difference was not statistically significant (p>0.05), RI reduced. RT-PCR detection showed: the expression of mrp gene of transplanted tumor cell was significantly decreased (to mRNA expression of mdr1 gene have a relatively small impact), and compared with the control group, the difference was significant (P <0.05). Western-blot analysis showed: the expression of MRP of transplanted tumor cells declined to a certain (absolute value of MRP greater than that of P-gp), and compared with the control group, the difference was statistically significant (P <0.05). The immunohistochemistry results of apoptotic protein showed: bcl-2 and bax of transplanted tumor cells had changed to some degree, and compared with the control group, the difference was statistically significant (P> 0.05). Determination of cell membrane ATPase activity can be found that: the cell membrane ATPase activity of transplanted tumor cells increased to some degree, however, no significant difference compared with the control group was significant (P <0.05). After Mdr1-ASON+ ultrasound contrast microbubbles + ultrasound treatment to HepG2/ADM transplanted tumor, the drug-resistance of transplanted tumor cells to ADM, CDDP, 5-FU and AsT significantly reduced, and compared with the control group, the difference was statistically significant (P <0.05), RI reduced. RT-PCR detection showed: the mRNA expression of mdr1 gene and mrp gene of transplanted tumor cells decreased significantly higher than that of the control group (P <0.05). Western blot results showed that: the expression of P-gp and MRP gene of transplanted tumor cells was significantly decreased (absolute value of P-gp greater than that of MRP), and compared with the control group, the difference was statistically significant (P <0.05). The immunohistochemistry results of apoptotic protein showed: the bcl-2 and bax of transplanted tumors noticeable changed significantly higher than the control group (P <0.05). Determination of cell membrane ATPase activity can be seen that: the cell membrane ATPase activity of transplanted tumor cells was up to a certain extent, and compared with the control group, the difference was statistically significant (P <0.05). Conclusion: ASON+ ultrasound acoustic microbubbles+ ultrasound treatment in vivo could reverse the human hepatocellular carcinoma MDR xenograft in nude mice, and ultrasound acoustic microbubbles had a better target-mediating roles for ASON gene transfection than cationic liposomes combination of ultrasound, which showed a stronger role reversal to hepatocellular carcinoma MDR in nude mice. Analysis of the possible reasons for this phenomenon: the ultrasonic irradiation led to the breakdown of microbubbles, cavitation effect of micro-bubble breakdown promoted the transfection and expression of ASON in the cells. The formation of MDR of the transplanted hepatocellular carcinoma QGY/CDDP in nude mice is mainly mediating by mrp, little relations with mdr1 mediating mechanisms and anti-apoptotic mechanisms, accordingly the effect to reverse cells MDR was by down the expression of mrp and its encoded protein. The formation of MDR of the transplanted hepatocellular carcinoma HepG2/ADM was of mdr1-mediating mechanisms, mrp-mediating mechanisms and the anti-apoptotic cell mechanisms to enhance the role of a common mechanism, the main mechanism mediating by mdr1, accordingly the effect to reverse cells MDR was by reducing the role of the three mechanisms, in particular, achieved by down the expression of mdr1and its encoded protein P-gp.
Summaries
1. CDDP delivery using low concentration increases-intermittent role and Linac X-ray irradiation method to establish two MDR cell lines model: QGY/CDDP and HepG2/ADM. The two cell lines have a high level of MDR character . QGY/CDDP to CDDP, ADM, 5-FU and AsT resistance index were 10.35, 5.50, 8.51 and 9.23. HepG2/ADM to CDDP, ADM, 5-FU and AsT, RI were 6.12, 30.31 ,6.94 and 9.15.
2. QGY/CDDP’s main mechanism of MDR is mediating by the mrp and its encoded protein MRP. There is little mdr1 and cell-mediating anti-apoptotic mechanisms and strengthen mechanisms. HepG2 cells, it’s MDR mechanism mediating by mdr1and protein P-gp,mrp and protein MRP and the anti-apoptotic ,But the main mechanism ismdr1 and protein P-gp mediating.
3.Irradiation of mdr1, mrp-ASON+acoustic microbubbless+ultrasound can partially reverse the multidrug resistance of QGY/CDDP and HepG2/ADM human hepatoma cells. acoustic microbubbles and ultrasound combined have a better target-mediating roles for MDR gene ASON transfection than cationic liposomes and ultrasound combined, it’s showed a stronger reversed effect in MDR of hepatoma cell lines. In the role of QGY/CDDP reversed MDR ,the mrp-ASON transfection significantly stronger than mdr1-ASON transfection. For HepG2/ADM, mdr1-ASON and mrp-ASON transfection have great effect in the reversal of the MDR, mdr1-ASON Transfection more stronger.
4. MDR hepatoma cell suspension were inoculated subcutaneously in nude mice directly. we established human hepatoma QGY/CDDP and HepG2/ADM xenograft model in nude mice. The transplanted tumor cells are highly tolerance tumor cells :the RI of QGY/CDDP transplant tumor cells to CDDP,ADM,5-FU and AsT is 9.28, 4.50, 7.52,8.36; the RI of HepG2/ADM transplanted tumor cells to ADM, CDDP, 5-FU and AsT is 6.05, 27.94, 6.73 , 8.96.
5. Irradiation of mrp-ASON+ acoustic microbubbles + ultrasound can reverse partial the resistance of MDR QGY/CDDP human hepatoma cells that transplanted in nude mice. mdr1-ASON+ acoustic microbubbless + ultrasound irradiation can reverse partial the resistance of MDR HepG2/ADM human hepatoma cells that transplanted in nude mice. acoustic microbubbles and ultrasound combined have a better target- mediating for MDR gene ASON transfection than cationic liposomes and ultrasound combined,it’s showed a stronger reversed effect in MDR of transplanted tumor cells.
6. Acoustic microbubbles joint ultrasound target-mediating for MDR gene ASON transfection reversed the resistance of MDR human hepatoma cell lines and transplanted tumor, maybe major mechanisms is closed down or blocking mdr1 mrp and the encoded protein expression, thus lowering the resistance of cells. other mechanisms may include to improve the cell sensitivity of drug , improve cell membrane ATPase activity,promote cell uptake of the drug , increase intracellular drug concentration and changes in the expression of apoptosis-related gene,lowered Bcl-2/Bax ratio and so on. Maybe the reasons that acoustic microbubbles agent and ultrasound combined have a better taget- mediating for MDR gene ASON transfection and reversed effect in MDR than cationic liposomes and ultrasound combined: low-intensity ultrasound exposure may lead to micro-bubble bursting and micro-bubble cavitation effect for the bursting can accelerate ASON transfection and expression in the cells ,then the MDR cell lines and transplanted tumor cells resistance decreased.
肝癌是全球范围内最常见的恶性肿瘤之一。在我国,肝癌占恶性肿瘤死亡率的第一或第二位,全球每年新发病例约45%在我国大陆地区,发病年龄趋于年轻化。目前肝癌临床以综合治疗为主,但肝癌细胞多药耐药(multidrug resistance,MDR)严重限制了治疗的效果。有资料显示90%以上肝癌病人的死因与MDR有关,因此如何逆转肿瘤MDR,提高肝癌有效治疗是一个亟待解决的问题。肿瘤MDR理想的逆转方法应该具有高效、无毒和靶向性(能特异性地作用于肿瘤而对正常组织无损害)的特点,然而现在的研究与此标准差距尚远。因此进行肝癌MDR系列研究,探讨新的逆转手段成为提高肿瘤控制的关键,具有非常重要的现实意义和应用价值。
本课题共分四部分进行实验研究。建立人肝癌MDR细胞模型和裸鼠皮下移植瘤模型,利用声学微泡联合超声靶向介导mdr1、mrp耐药基因的反义寡核苷酸(antisense oligonucleotide,ASON)转染,探讨对人肝癌细胞和移植瘤MDR的逆转效应和机理,力求寻找一种低毒、高效和靶向的逆转肝癌MDR的新方法,为提高肝癌综合治疗效果,开展临床肝癌基因治疗和高剂量冲击化疗提供理论依据。本研究将分子生物学寡核苷酸反义技术和声学微泡并超声治疗技术结合,借助声学微泡并超声的空化效应,以促进基因在肿瘤细胞的高效和靶向转染及表达,提高对MDR的逆转效率,在策略和技术上是一种创新。
第一部分肝癌MDR细胞模型建立和生物学特性研究
第一节顺铂递增加量-间歇作用法建立QGY肝癌MDR细胞模型及生物学特性鉴定
目的:建立人肝癌QGY多药耐药细胞模型并初步检测其耐药特性。方法:以人肝癌细胞株QGY为亲本细胞,选用化疗药物顺铂(cisplatin,CDDP),以低浓度递增加量-间歇作用法诱导细胞耐药。光镜、电镜观察新建细胞株组织形态学及超微结构特点。测定细胞倍增时间并绘制生长曲线、观察细胞克隆形成和流式细胞仪(Flow Cytometry,FCM)检测细胞周期分布,鉴定新建细胞生物学特性。四甲基偶氮唑盐( 3-4,5-dimethyIthiazol-z-yl-2,5-dipheny tretrazolium bromide , MTT )法测定细胞药物敏感性,免疫组化(immunohistochemistry assay)检测细胞耐药蛋白P-gp、MRP和LRP,凋亡蛋白Bcl-2和Bax的表达。结果:历时15周多,成功建立MDR细胞株QGY/CDDP。QGY/CDDP在组织形态学及超微结构上与亲本细胞QGY无明显差别。一般生物学特性:MDR细胞的群体倍增时间较亲代细胞延长25小时多,克隆形成率明显低于亲本细胞,G0/G1期细胞比例增加,S期和G2/M期细胞比例下降,与亲本细胞QGY比较有显著性差异(P<0.05)。药敏检测显示:QGY/CDDP细胞对CDDP高度耐药,耐药指数(resistance index,RI)为10.35,对ADM(阿霉素)、5-Fu(5-氟脲嘧啶)和AsT(三氧化二砷)也产生耐药性,RI分别为5.50、8.51和9.23。免疫组化结果为MRP染色阳性细胞率最高,为23.89%,P-gp染色阳性细胞率为13.26%;LRP、Bcl-2和Bax染色阳性细胞率与亲本细胞接近。结论:新建细胞株QGY/CDDP具有MDR特性,其耐药机制由MRP和P-gp介导,以MRP介导机制为主,LRP、Bcl-2/Bax可能不参与QGY/CDDP耐药性的形成。
第二节X射线照射法建立HePG2肝癌MDR细胞模型及耐药性鉴定
目的:建立人肝癌HepG2多药耐药细胞模型并初步检测其耐药特性。方法:以人肝癌细胞株HepG2为亲本细胞,给于6MV直线加速器X-射线照射,诱导细胞耐药。HepG2细胞受X射线累积量照射后,接种到含浓度0.4μg/ml阿霉素(adriamycin,ADM)培养基中培养,发现细胞良好稳定增殖生长,提示细胞对抗ADM的细胞毒作用,即将新建细胞株命名为HepG2/ADM。光镜、电镜观察新建细胞株组织形态学及超微结构特点。测定细胞贴壁率、细胞克隆形成率和FCM检测细胞周期分布观察HepG2/ADM生物学特性。MTT法检测HepG2/ADM细胞药物敏感性,免疫组化测定细胞耐药蛋白P-gp、MRP、LRP以及凋亡蛋白Bcl-2/Bax表达,FCM测定细胞Rh123摄入和外排能力,间接检测P-gp和MRP功能。结果:历时45天多,X-射线照射累积剂量为24GY,处理后细胞能在含0.4μg/mlADM培养基中生长,即成功建立MDR细胞株HepG2/ADM。HepG2/ADM组织形态学及超微结构与亲本细胞HepG2无明显差别。一般生物学特性:HepG2/ADM细胞贴壁率、克隆形成率明显降低,G0/G1期细胞比例增加,S期及G2/M期细胞比例减少,与亲本细胞比较有显著性差异(P<0.05)。药敏检测显示:HepG2/ADM细胞对ADM高度耐药,RI为30.31,同时对CDDP、5-Fu和AsT也产生了耐药性,RI分别为6.12、6.94和9.15。免疫组化染色可见P-gp阳性染色细胞率最高,为85.62%,MRP阳性染色细胞率为12.36%,LRP阳性染色细胞率与亲本细胞接近,Bcl-2与Bax阳性染色细胞率分别为27.35%、6.25%,Bcl-2/Bax比值增高,较亲本细胞HepG2有显著性差异(P<0.05)。结论:新建细胞株HepG2/ADM具有MDR特性,其耐药机制由P-gp、MRP介导和凋亡蛋白Bcl-2/Bax共同作用,以P-gp介导机制为主。
第二部分声学微泡联合超声靶向介导耐药基因ASON转染逆转肝癌细胞MDR的效应和机制
目的:初步讨论声学微泡联合超声靶向介导耐药基因ASON转染逆转QGY/CDDP、HepG2/ADM人肝癌细胞株MDR的效应和作用机制。方法:取对数生长期细胞,调整成单细胞悬液,分别给于mdr1-ASON+声学微泡+超声、mrp-ASON+声学微泡+超声实验处理,待细胞生长良好,进行各项指标检测:在QGY/CDDP细胞,MTT法测定细胞耐药性变化,细胞贴壁率测定、细胞周期分布检测(FCM)、细胞凋亡TUNNEL法测定,观察细胞接受实验处理后的生物学特性变化,RT-PCR检测mdr1、mrp基因的mRNA表达,Western-blot测定细胞P-gp、MRP蛋白表达,免疫组化测定细胞凋亡蛋白Bcl-2、Bax表达。在HepG2/ADM细胞,AO/EB荧光法测定细胞凋亡,其它检测方法与QGY/CDDP实验处理后细胞检测一致。结果:QGY/CDDP细胞对CDDP、ADM、5-FU和AsT四种药物的耐药性有一定下调,RI降低:mdr1-ASON处理中,与对照组比较无显著性差异(P>0.05);mrp-ASON处理中,与阴性对照组比较有显著性差异(P<0.05),与阳性对照组比较无显著性差异(P>0.05)。mdr1-ASON转染后,细胞周期分布、P-gp、MRP的相对表达量和凋亡蛋白阳性细胞百分率的变化均较小,但与对照组比较差异无显著性(P>0.05);在细胞贴壁率、凋亡细胞TUNNEL法测定结果和耐药基因mRNA的相对表达量有一定变化(P<0.05)。mrp-ASON转染后,细胞贴壁率、细胞周期分布、TUNNEL法测定凋亡细胞、耐药基因mRNA的相对表达和P-gp、MRP的相对表达六项指标均有一定变化,有显著性差异(P<0.05);凋亡蛋白免疫组化结果一项变化较小。HepG2/ADM细胞实验处理后,细胞药物敏感性、细胞贴壁率、细胞周期分布、凋亡细胞、耐药基因mRNA表达、耐药蛋白的相对表达量和凋亡蛋白阳性细胞百分率等共七项检测指标均显示细胞有一定的变化,且与对照组比较有显著性差异(P<0.05);同样可见mdr1-ASON处理中,细胞的指标变化大于mrp-ASON处理。结论:mdr1-ASON+声学微泡+超声照射与mrp-ASON+声学微泡+超声照射能够部分逆转QGY/CDDP、HepG2/ADM人肝癌细胞的MDR。声学微泡结合超声比阳离子脂质体结合超声有更好的转染基因ASON靶向介导作用,显示对肝癌细胞株MDR更强的逆转效应。在QGY/CDDP细胞,mrp-ASON转染逆转MDR的作用明显强于mdr1-ASON转染;在HepG2/ADM细胞,mdr1-ASON、mrp-ASON转染均有较强逆转肿瘤MDR的作用,mdr1-ASON转染的MDR逆转作用更强。
第三部分裸鼠肝癌MDR移植瘤模型的建立和生物学特性研究
目的:建立人肝癌QGY/CDDP、HepG2/ADM多药耐药的裸鼠移植瘤模型并鉴定其耐药特性。方法:取对数生长期QGY/CDDP、HepG2/ADM细胞,调整为单细胞悬液,按实验分组,采用细胞悬液皮下直接注射法,将MDR肿瘤细胞接种在裸鼠皮下,观察肿瘤生长并记录。待皮下移植瘤生长到一定大小,各组随机取两只裸鼠处死,剥离移植瘤组织块,提取移植瘤细胞培养,待细胞处于对数生长期进行耐药性检测。光镜、电镜观察移植瘤细胞组织形态学和超微结构特点。进行移植瘤细胞倍增时间并绘制生长曲线和细胞克隆形成率测定,观察移植瘤细胞的生物学特性。MTT测定移植瘤细胞药物敏感性,FCM检测移植瘤细胞P-gp、MRP表达,Western-blot测定细胞凋亡蛋白Bcl-2、Bax表达。结果:QGY/CDDP、HepG2/ADM荷瘤裸鼠的平均生存期为40±15d,移植瘤生长体积统计分析结果:两株细胞移植瘤生长均较其对应非耐药移植瘤细胞慢,有显著性差异(P<0.05)。MDR移植瘤细胞的组织形态学及超微结构与对应非耐药移植瘤细胞无明显差异。移植瘤细胞的生物学特性检测:两株细胞均呈现细胞倍增时间延长(P<0.05),细胞克隆形成率降低(P<0.05)。MTT法结果:两株MDR移植瘤细胞均对ADM、CDDP、5-FU和AsT四种药物耐药,QGY/CDDP移植瘤细胞对CDDP的RI最高,HepG2/ADM细胞对ADM的RI最高,与各自非耐药移植瘤细胞比较差异具有显著性差异(P<0.05)。耐药蛋白与凋亡蛋白测定结果:在QGY/CDDP移植瘤细胞,MRP蛋白染色阳性细胞率最高,为23.85%(P<0.05),P-gp染色阳性细胞率为7.44%(P<0.05);Bcl-2和Bax的相对表达与移植瘤QGY细胞接近(P>0.05)。在HepG2/ADM移植瘤细胞,P-gp蛋白染色阳性细胞率最高,为84.97%,MRP染色阳性细胞率为10.26%,Bcl-2和Bax的相对表达与移植瘤HepG2细胞比较有显著性差异(P<0.05)。结论:成功建立人肝癌QGY/CDDP、HepG2/ADM裸鼠皮下移植瘤模型。其耐药机制可能主要与mdr1及蛋白P-gp、mrp及蛋白MRP(P190)和凋亡蛋白Bcl-2/bax的表达有关。本移植瘤模型成功率高,可重复性强,生物学特性稳定。
第四部分声学微泡联合超声靶向介导耐药基因ASON转染逆转裸鼠肝癌移植瘤MDR的效应和机制
目的:初步探讨声学微泡联合超声靶向介导耐药基因ASON转染在体逆转裸鼠肝癌移植瘤MDR的效应及其作用机制。方法:对于肝癌MDR移植瘤裸鼠,按实验分组在体给于实验处理: QGY/CDDP移植瘤给于mrp-ASON+声学微泡+超声, HepG2/ADM移植瘤给于mdr1-ASON+声学微泡+超声,并以相应ASON+阳离子脂质体+超声为阳性对照,相应MDR移植瘤+转染液注入为阴性对照,在体实验结束处死裸鼠,剥离移植瘤块,提取相应移植瘤细胞培养。取对数生长期培养细胞进行MTT药敏检测、RT-PCR测定细胞mdr1及mrp基因mRNA表达、Western-blot检测细胞P-gp及MRP蛋白表达、免疫组化测定细胞凋亡蛋白bcl-2及bax表达和移植瘤细胞膜ATPase活性变化测定。结果:裸鼠肝癌移植瘤在体实验处理后,移植瘤细胞检测发现:mrp-ASON+声学微泡+超声作用QGY/CDDP移植瘤后,与阴性对照组比较,移植瘤细胞对CDDP、ADM、5-FU和AsT四种药物的耐药性明显下调(P<0.05),与阳性组比较,耐药性有一定下调,但差异无显著性意义(P>.05),RI降低。RT-PCR显示:移植瘤细胞mrp基因的mRNA表达下降明显(对mdr1基因的mRNA表达影响较小),较对照组有显著性差异(P<0.05)。Western-blot结果:移植瘤细胞P-gp、MRP表达均有一定下降(MRP绝对值大于P-gp),与对照组比较有显著性差异(P<0.05)。凋亡蛋白的免疫组化结果表明:移植瘤细胞bcl-2、bax有一定变化,与对照组比较差异无显著性意义(P>0.05)。细胞膜ATPase活性变化测定可见:移植瘤细胞的细胞膜ATPase活性有一定上调,但与对照组比较差异无显著性意义(P>0.05)。mdr1-ASON+声学微泡+超声作用HepG2/ADM移植瘤后,移植瘤细胞对ADM、CDDP、5-FU和AsT四种药物耐药性明显下调,与对照组比较有显著性差异(P<0.05),RI降低。RT-PCR显示:移植瘤细胞mdr1基因和mrp基因的mRNA表达明显下降,较对照组有显著性差异(P<0.05)。Western-blot结果表明:移植瘤细胞P-gp、MRP表达明显下降(P-gp绝对值大于MRP),与对照组比较有显著性差异(P<0.05)。凋亡蛋白的免疫组化测定结果显示:移植瘤细胞bcl-2、bax明显变化,较对照组有显著性差异(P<0.05)。移植瘤细胞膜ATPase活性变化检测结果可见:细胞膜ATPase活性有一定上调,与对照组比较有显著性差异(P<0.05)。结论:ASON+声学微泡+超声照射能够在体逆转裸鼠肝癌移植瘤的MDR,声学微泡联合超声比阳离子脂质体结合超声有更好的转染基因ASON的靶向介导作用,显示对肝癌裸鼠移植瘤MDR更强的逆转效应。分析这种现象的可能原因是:超声的照射导致微泡破裂,微泡破裂的空化效应促进了ASON在细胞的转染与表达。裸鼠肝癌移植瘤QGY/CDDP的MDR形成主要是mrp介导机制,与mdr1介导机制和细胞抗凋亡增强机制关系不大,相应逆转细胞MDR的作用是通过下调细胞mrp及其编码蛋白MRP表达来实现的。移植瘤HepG2/ADM的MDR形成是mdr1介导机制、mrp介导机制和细胞抗凋亡增强机制共同作用的结果,以mdr1介导机制为主,相应逆转细胞MDR的作用是通过降低三机制的作用,尤其是下调mdr1及其编码蛋白P-gp表达来实现。
小结
1.采用CDDP低浓度递增加量-间歇作用法、直线加速器X-射线照射法分别建立了两株MDR细胞模型:QGY/CDDP和HepG2/ADM。两种细胞都具有较高的MDR特性,QGY/CDDP细胞对CDDP、ADM、5-FU和AsT的耐药指数分别为10.35、5.50、8.51和9.23;HepG2/ADM细胞对CDDP、ADM、5-FU和AsT的耐药指数分别为6.12、30.31、6.94和9.15。
2. QGY/CDDP细胞的MDR机制主要是由mrp及其编码蛋白MRP介导,与mdr1介导机制和细胞抗凋亡增强机制关系不大。HepG2/ADM细胞的MDR机制是由mdr1及其蛋白P-gp介导、mrp及其蛋白MRP介导和细胞抗凋亡增强共同发挥作用,但以mdr1及其蛋白P-gp介导机制为主。
3.用mdr1、mrp-ASON+声学微泡+超声照射能够部分逆转人肝癌细胞QGY/CDDP、HepG2/ADM的多药耐药,声学微泡联合超声比阳离子脂质体结合超声有更好的转染耐药基因ASON的靶向介导作用,显示出对肝癌细胞株MDR更强的逆转效应。对于QGY/CDDP细胞逆转MDR的作用,mrp-ASON转染明显强于mdr1-ASON转染。对于HepG2/ADM细胞,mdr1-ASON、mrp-ASON转染均有较强逆转肿瘤MDR的作用,mdr1-ASON转染的MDR逆转作用更强。
4. MDR肝癌单细胞悬液直接裸鼠皮下注射法接种,能够建立人肝癌裸鼠QGY/CDDP和HepG2/ADM移植瘤模型。移植瘤细胞有高度耐药特性:QGY/CDDP移植瘤细胞对CDDP、ADM、5-FU和AsT的RI依次为9.28、4.50、7.52和8.36;HepG2/ADM移植瘤细胞对CDDP、ADM、5-FU和AsT的RI依次为6.05、27.94、6.73和8.96。
5.用mrp-ASON+声学微泡+超声照射能够在体部分逆转人肝癌QGY/CDDP细胞裸鼠移植瘤的MDR,用mdr1-ASON+声学微泡+超声照射能够在体部分逆转人肝癌HepG2/ADM细胞裸鼠移植瘤的MDR;其中声学微泡联合超声比阳离子脂质体结合超声有更好的转染基因ASON的靶向介导作用,显示出对移植瘤MDR逆转效应更强。
6.声学微泡联合超声靶向介导耐药基因-ASON转染逆转人肝癌细胞株和移植瘤MDR的可能主要机制是封闭或下调mdr1、mrp及编码蛋白的表达,进而降低细胞耐药性。其它机制可能包括提高细胞对药物的敏感性、提高细胞膜ATPase活性,促进细胞对药物的摄取,上调细胞内药物浓度和改变凋亡基因的表达,使Bcl-2/Bax比例下调等等。声学微泡造影剂联合超声较阳离子脂质体结合超声靶向介导耐药基因ASON转染具有更好的逆转MDR效应的可能原因是:低强度超声照射导致微泡破裂,微泡破裂的空化效应促进了ASON在细胞的转染与表达,进而表现MDR细胞株和移植瘤的耐药性下降。
Introduction
Hepatoma is one of the most common cancer worldwide. In China its mortality rate is the first or second malignancies. Each year about 45% of new cases are in China and it is tendency to onset in younger. The combined treatment is the main method for liver tumors at present. But hepatoma cells MDR (multidrug resistance, MDR) severely limits the effect of the treatment. Some data indicate that more than 90% of patients with liver cancer and the death of the MDR. How to reverse the MDR is an urgent task to improve the treatment of liver cancer. The ideal method of tumor MDR reversal must be efficient, asepsis and target (be specific to tumor while no damage to normal tissue). But recent researches are far from this standard. Therefore a series of studiesof MDR HCC have being carried out to explore new method which is the key to improving the effect on cancer control, and that is of important practical significance and value at present.
The present researches were divided four parts. It would try to find a new low toxicity, efficient and targetted method for MDR reversion of HCC, and try to provide a theoretical basis to improve comprehensive treatment effects, clinical gene therapy and high-dose chemotherapy for liver cancer, which had Built the model of MDR human hepatoma cell and subcutaneous transplant tumor, explored the reversal effects and mechanisms on MDR of human hepatoma cells and transplant tumor through using acoustic microbubbles joint ultrasound taget-mediating for the antisense oligonucleotides transfection of mdr1 and mrp resistance gene. In this study, it had improved the efficiency of the MDR reversal by using acoustic microbubbles and ultrasonic cavitation effect to facilitate the efficient of targeted transfection and expression of gene in tumor cells for antisense oligonucleotide technologies of molecular biology in combintion with acoustics microbubbles and ultrasonic treatment. It is a strategy and technical innovation.
Part 1 Establishment of HCC model for MDR cells and biological characterization
Section 1 Establishment of QGY Multidrug-resistant cell line induced by exposure of increasing concentration by degrees and intermission affect approach of CDDP and biological characterization
Objective: To establish human hepatoma cell multidrug resistance model QGY and preliminary testing their resistance. Methods : The human hepatoma cell line for the pro-inhibiting cell, the use of chemotherapy drug cisplatin (cisplatin. CDDP), a low concentration increases delivery-resistant cells induced by intermittent role. Light microscopy, electron microscopy observation of morphological and ultrastructural characteristics of the new cell lines. Determination of cell doubling time and the mapping of the growth curve, observation of cell colony formation and flow cytometry (Flow Cytometry, FCM) detection of cell cycle distribution, identification of new biological characteristics. MTT assay of cell drug sensitivity, immunohistochemistry detecion of the expression of cells resistant Protein P-gp, MRP and LRP, apoptotic protein Bcl-2 and Bax. Results: During the 15 weeks, the MDR cell lines QGY/CDDP successful. QGY/CDDP in morphology and ultrastructure with the parental cells QGY is no different. General characterazation: MDR cell population doubling time than the parent cells extended 21-hour, colony formation was significantly lower than the parental cells, the cells in G0/G1 phase increased, the cell ratio in S and G2/M phase decreased, and the difference that with the parental cell QGY was statistically significant (P <0.05). Susceptibility testing revealed: QGY/CDDP cells were highly resistant to CDDP, RI (resistance index, RI) 10.35; of ADM (ADM). 5-FU (5-fluorouracil) and AST (arsenic trioxide) also resistance, RI 5.50, 8.51 and 9.23 respectively. The results of immunohistochemical were that staining positive for cell MRP highest rate of 23.89%, P-gp cells staining positive for 13.26%; the rate of LRP, Bcl-2 and Bax-positive cells was close to the parental cells. Conclusion: The new cell lines have MDR QGY/CDDP characteristics, resistance mechanisms mediating by the MRP and P-gp. MRP-mediating mechanism, while LRP, QGY/CDDP Bcl-2/Bax may not be involved in the formation of resistance.
Section 2 Establishment of HePG2 Multidrug-resistant cell line induced by X-radiation exposure approach and biological characterization
Objective: To establish human hepatoma HepG2 cell MDR model and preliminary testing their resistance. Methods: The human hepatoma cell line HepG2 cells for parent was to X-ray irradiation at 6 MV linear accelerator, induce cell resistance. HepG2 cells after irradiation by X-ray accumulation, was inoculated with the 0.4μg/ml concentration of ADM (adriamycin, ADM) was cultured, good stability proliferation and growth of cells, suggesting that cell cytotoxicity against ADM. HepG2/ADM named for the upcoming new cell lines. Light microscopy, electron microscopy observation of morphological and ultrastructural characteristic of the new cell lines. Determination of cell adhesion rate, cell colony formation rate, and FCM detection of cell cycle distribution, observation HepG2/ADM biological characteristics. MTT assay sensitivity in HepG2/ADM cells, immunohistochemistry detecion of the expression of cells resistant Protein P-gp, MRP and LRP, apoptotic protein Bcl-2 and Bax, FCM in cell Rh123 intake and discharge capacity, indirect detection of P-gp and MRP functions. Results: During the 45 days, the cumulative dose of X-ray irradiation 24GY, treated cells in culture medium containing 0.4μg/ml ADM growth, HepG2/ADM MDR cell lines successfully established. HepG2/ADM in morphology and ultrastructure with the parental cells HepG2 was no significant different. General characterazation: HepG2 cells adherent rate, rate of colony formation significantly reduced, the proportion of cells in G0/G1 phase increased, the proportion of cells in S and G2/M phase decreased, that difference with the parental cells was statistically significant (P <0.05). Susceptibility testing revealed: HepG2/ADM cells were highly resistant to ADM, RI to 30.31; meanwhile to CDDP, 5-Fu and AsT also produced resistance, RI 6.12, 6.94 and 9.15. Immunohistochemical staining shows that the highest rate of P-gp positive staining cells to 85.62%, MRP rate of 12.36% positive staining cells, LRP positive staining cells with the parental cells close, Bcl-2 and Bax were 27.35%, 6.25% positive staining cells, Bcl-2/Bax ratio increased, all the differences with the parental HepG2 cells had statistically significance (P <0.05). Conclusion: new cell line HepG2 with MDR characteristics its resistance was combined by P-gp, MRP-mediating and apoptotic protein Bcl-2, the main mechanism mediating by P-gp.
Part 2 Effects and mechanisms of hepatoma cells MDR reversed by acoustic microbubbles joint ultrasound target-mediating ASON of drug resistance gene transfection
Objective: To discuss the effect and mechanism of QGY/CDDP and HepG2/ADM human hepatoma cell line MDR reversed by ASON + ultrasound +contrast microbubbles. Methods: Log phase cells to adjust as a single cell suspension were to mdr1-ASON+ microbubbles+ ultrasound and mrp-ASON+ contrast microbubbles + ultrasound treatment, cell growth to be good, detection of the various indicators: In QGY/CDDP cells, MTT assay to detect changes in cell resistance, cell adhesion rate determination, detection of cell cycle distribution (FCM), apoptosis; TUNNEL determination to observe the biological characteristics after cells receiving the experimental treatment; RT-PCR assay to detect the mRNA expression of mdr1and mrp gene; Western-blot assay to detect the expression changes of protein P-gp and MRP; immunohistochemical staining assay to detect the expression of cell apoptotic protein bcl-2 and bax. In HepG2 cells, fluorescence AO/EB determination to detect apoptosis, other detection methods agreed to experimental treatment QGY/CDDP cell line testing. Results: The drug resistance of QGY/CDDP cells to CDDP, ADM, 5-FU and AsT was down to a certain extent, RI reduced: mdr1-ASON processed, compared with the control group the difference was no statistically significant (P> 0.05); mrp-ASON processed, compared with the negative control group the difference was statistically significant (P <0.05), and compared with the control group, the difference was no statistically significant (P> 0.05). mdr1-ASON after transfection, cell cycle distribution, relative expressing amonts of P-gp, MRP protein, and percentage of positive cells of apoptosis protein in the cells were smaller changes, compared with the control group the difference was not statistically significant (P> 0.05); and the rate of cells adherent, TUNNEL detection of apoptosis resulted, relative expression amonts of resistance gene mRNA to a certain extent for statistically significant (p <0.05). mrp-ASON after transfection, the six indicators of cells adherent rate, cell cycle distribution, TUNNEL determination of apoptotic cells, relative expression amonts of resistance gene mRNA, relative expressing amonts of P-gp, MRP protein were certain changes, the differences were statistically significant (P<0.05); immunohistochemical results of apoptotic protein was a small change. HepG2 cells after experimental treatment, the seven indicators of drug sensitivity of cells, cell adhesion rate, cell cycle distribution, apoptosis, mRNA expression of resistance gene, percentage of positive cells of resistance protein and apoptotic protein, showed certain cells changes, and compared with the control group the difference was statistically significant (P <0.05); also seen colony processing, changes of indicators were larger than that after mrp-ASON treatment. Conclusion: mdr1-ASON+ acoustic contrast microbubbles + ultrasound treatment and mrp-ASON+ contrast microbubbles + ultrasound exposure QGY/CDDP could partly reverse human hepatoma HepG2 cells MDR. Combination of ultrasound treatment, Ultrasound contrast microbubbles have a better target-mediating roles for ASON gene transfection than cationic liposomes, which showed a stronger role reversal to MDR of hepatoma cell lines. In QGY/CDDP cells, mrp-ASON transfection had a significantly stronger role in the reversal of MDR colony transfection than mdr1-ASON transfection; In HepG2 cells, mdr1-ASON and mrp-ASON transfection have greater role in the reversal of the MDR, transfection of MDR reversal colony stronger.
Part 3 Establishment and characterization MDR model of hepatocellular carcinoma in nude mice
Objective: To establish a human hepatoma QGY/CDDP, HepG2/ADM nude mouse model of multi-drug resistance and identified their resistance. Methods: Log phase QGY/CDDP, HepG2 cells to adjust as the single cell suspension, by the experimental groups, were directly injected subcutaneously, so MDR tumor cells were inoculated in nude mice subcutaneously, to observe and record the growth of tumors. When the implanted tumors grew to a certain size, two nude mice were randomly killed in each group, dissecting transplanted tumor tissue, extracting tumor cells to cultivate, when cells were in the logarithmic growth phase for resistance testing. Light microscopy, electron microscopy detection of morphological and ultrastructural characteristic of tumor cells. Observation of transplant tumor doubling time and the mapping of the growth curve, determination of cell colony formation rate and cell cycle distribution, to observe the biological characteristics of transplanted cells. MTT to detect the sensitivity of tumor cells to drug. FCM to detect the expression of P-gp and MRP. Western blot to determine the expression of cell apoptosis protein Bcl-2 and Bax. Results: In QGY/CDDP, HepG2/ADM nude mice, the average survival period was 40±15 days. Growth Statistical analysis of transplanted tumor size: the growth of the two cell tumors was slower than that of the parental cells transplanted tumor, and the difference was statistically significant (P <0.05). MDR tumor cells in morphology and ultrastructure of the cells had no significant difference to pro counterparts. Detection of biological characteristics of the transplanted tumor cells: two strains of cell doubling time were longer (P <0.05), the rate of cell colony formation decreased (P <0.05), the proportion of cells in G0/G1 phase of the cell cycle distribution increased, the proportion of cells in S and G2/M phases declined, which the difference was statistically significant compared with parental implanted tumor cells (P <0.05). Results of MTT assay: Two MDR tumor cells were resistant to ADM, CDDP, 5-FU and AsT, and the RI of QGY/CDDP tumor cells to CDDP was the highest, he RI of HepG2/ADM tumor cells to ADM was the highest, compared with each parental implanted tumor cells, which the difference had statistically significant (P <0.05). The results of detection of resistance protein and apoptosis protein: In implanted tumor cells QGY/CDDP, the rate of positive cells of MRP protein staining was the highest to 23.85% (P <0.05), the rate of positive cells of P-gp cells staining was 7.44% (P <0.05); the rate of positive cells of Bcl-2 and Bax cells staining was close to that of the parental tumor cells. In HepG2/ADM transplanted tumor cells, the rate of positive cells of P-gp cells staining was highest to 84.97%, 10.26% rate of cells staining positive MRP, and relative expressing amonts of Bcl-2 and Bax protein, which the difference was statistically significant (P <0.05) compared with the parental transplanted tumor cells. Conclusion: To successfully establish the human hepatoma QGY/CDDP, HepG2/ADM nude mice model. The main resistant machanism may be of the expression of mdr1, protein P-gp, mrp, protein MRP (P190) and apoptotic proteins Bcl-2/Bax. The implanted model had the high success rate and had repeatable, stable biological characteristics.
Part 4 Effect and mechanisms of reversal of hepatocellular carcinoma MDR in nude mice by acoustic microbubbles joint ultrasound target-mediating ASON of drug resistance gene transfection
Objective: To preliminary study the effect and mechanism of reversal human hepatocellular carcinoma MDR in nude mice in vivo by ASON+ ultrasound contrast microbubbles+ ultrasound treatment. Method: The nude mice implanted tumor in vivo, by the lab groups, were given experimental treatment: mrp-ASON+ ultrasound contrast microbubbles+ ultrasound treatment to QGY/CDDP transplanted tumor, mdr1-ASON+ ultrasound contrast microbubbles+ ultrasound treatment to HepG2/ADM transplanted tumor, corresponding ASON+ cationic liposome+ ultrasound as a positive control, corresponding MDR tumor + switch dye injected as a negative control. At the end of death in nude mice in vivo, transplanted tumor was stripped, the corresponding tumor cells were extracted and cultivated. Cells were in the logarithmic growth phase for MTT susceptibility testing, mRNA expression of mdr1 and mrp gene measured by RT-PCR, and expression of P-gp cells and protein MRP was detected by Western blot, expression of apoptosis protein of bcl-2 and bax was determined by immunohistochemical staining, the implanted tumor cell membrane ATPase activity was determined. Results: After in vivo treatment of the human hepatocellular carcinoma xenograft in nude mice, the implanted tumor cells were detected to find: after mrp-ASON+ultrasound contrast microbubbles+ultrasound treatment to QGY/CDDP transplanted tumor, compared with the negative control group, the drug resistance of transplanted tumor cells to CDDP, ADM, 5-FU and AsT significantly reduced (P <0.05); compared with the positive control group, the resistance reduced to a certain extent, but the difference was not statistically significant (p>0.05), RI reduced. RT-PCR detection showed: the expression of mrp gene of transplanted tumor cell was significantly decreased (to mRNA expression of mdr1 gene have a relatively small impact), and compared with the control group, the difference was significant (P <0.05). Western-blot analysis showed: the expression of MRP of transplanted tumor cells declined to a certain (absolute value of MRP greater than that of P-gp), and compared with the control group, the difference was statistically significant (P <0.05). The immunohistochemistry results of apoptotic protein showed: bcl-2 and bax of transplanted tumor cells had changed to some degree, and compared with the control group, the difference was statistically significant (P> 0.05). Determination of cell membrane ATPase activity can be found that: the cell membrane ATPase activity of transplanted tumor cells increased to some degree, however, no significant difference compared with the control group was significant (P <0.05). After Mdr1-ASON+ ultrasound contrast microbubbles + ultrasound treatment to HepG2/ADM transplanted tumor, the drug-resistance of transplanted tumor cells to ADM, CDDP, 5-FU and AsT significantly reduced, and compared with the control group, the difference was statistically significant (P <0.05), RI reduced. RT-PCR detection showed: the mRNA expression of mdr1 gene and mrp gene of transplanted tumor cells decreased significantly higher than that of the control group (P <0.05). Western blot results showed that: the expression of P-gp and MRP gene of transplanted tumor cells was significantly decreased (absolute value of P-gp greater than that of MRP), and compared with the control group, the difference was statistically significant (P <0.05). The immunohistochemistry results of apoptotic protein showed: the bcl-2 and bax of transplanted tumors noticeable changed significantly higher than the control group (P <0.05). Determination of cell membrane ATPase activity can be seen that: the cell membrane ATPase activity of transplanted tumor cells was up to a certain extent, and compared with the control group, the difference was statistically significant (P <0.05). Conclusion: ASON+ ultrasound acoustic microbubbles+ ultrasound treatment in vivo could reverse the human hepatocellular carcinoma MDR xenograft in nude mice, and ultrasound acoustic microbubbles had a better target-mediating roles for ASON gene transfection than cationic liposomes combination of ultrasound, which showed a stronger role reversal to hepatocellular carcinoma MDR in nude mice. Analysis of the possible reasons for this phenomenon: the ultrasonic irradiation led to the breakdown of microbubbles, cavitation effect of micro-bubble breakdown promoted the transfection and expression of ASON in the cells. The formation of MDR of the transplanted hepatocellular carcinoma QGY/CDDP in nude mice is mainly mediating by mrp, little relations with mdr1 mediating mechanisms and anti-apoptotic mechanisms, accordingly the effect to reverse cells MDR was by down the expression of mrp and its encoded protein. The formation of MDR of the transplanted hepatocellular carcinoma HepG2/ADM was of mdr1-mediating mechanisms, mrp-mediating mechanisms and the anti-apoptotic cell mechanisms to enhance the role of a common mechanism, the main mechanism mediating by mdr1, accordingly the effect to reverse cells MDR was by reducing the role of the three mechanisms, in particular, achieved by down the expression of mdr1and its encoded protein P-gp.
Summaries
1. CDDP delivery using low concentration increases-intermittent role and Linac X-ray irradiation method to establish two MDR cell lines model: QGY/CDDP and HepG2/ADM. The two cell lines have a high level of MDR character . QGY/CDDP to CDDP, ADM, 5-FU and AsT resistance index were 10.35, 5.50, 8.51 and 9.23. HepG2/ADM to CDDP, ADM, 5-FU and AsT, RI were 6.12, 30.31 ,6.94 and 9.15.
2. QGY/CDDP’s main mechanism of MDR is mediating by the mrp and its encoded protein MRP. There is little mdr1 and cell-mediating anti-apoptotic mechanisms and strengthen mechanisms. HepG2 cells, it’s MDR mechanism mediating by mdr1and protein P-gp,mrp and protein MRP and the anti-apoptotic ,But the main mechanism ismdr1 and protein P-gp mediating.
3.Irradiation of mdr1, mrp-ASON+acoustic microbubbless+ultrasound can partially reverse the multidrug resistance of QGY/CDDP and HepG2/ADM human hepatoma cells. acoustic microbubbles and ultrasound combined have a better target-mediating roles for MDR gene ASON transfection than cationic liposomes and ultrasound combined, it’s showed a stronger reversed effect in MDR of hepatoma cell lines. In the role of QGY/CDDP reversed MDR ,the mrp-ASON transfection significantly stronger than mdr1-ASON transfection. For HepG2/ADM, mdr1-ASON and mrp-ASON transfection have great effect in the reversal of the MDR, mdr1-ASON Transfection more stronger.
4. MDR hepatoma cell suspension were inoculated subcutaneously in nude mice directly. we established human hepatoma QGY/CDDP and HepG2/ADM xenograft model in nude mice. The transplanted tumor cells are highly tolerance tumor cells :the RI of QGY/CDDP transplant tumor cells to CDDP,ADM,5-FU and AsT is 9.28, 4.50, 7.52,8.36; the RI of HepG2/ADM transplanted tumor cells to ADM, CDDP, 5-FU and AsT is 6.05, 27.94, 6.73 , 8.96.
5. Irradiation of mrp-ASON+ acoustic microbubbles + ultrasound can reverse partial the resistance of MDR QGY/CDDP human hepatoma cells that transplanted in nude mice. mdr1-ASON+ acoustic microbubbless + ultrasound irradiation can reverse partial the resistance of MDR HepG2/ADM human hepatoma cells that transplanted in nude mice. acoustic microbubbles and ultrasound combined have a better target- mediating for MDR gene ASON transfection than cationic liposomes and ultrasound combined,it’s showed a stronger reversed effect in MDR of transplanted tumor cells.
6. Acoustic microbubbles joint ultrasound target-mediating for MDR gene ASON transfection reversed the resistance of MDR human hepatoma cell lines and transplanted tumor, maybe major mechanisms is closed down or blocking mdr1 mrp and the encoded protein expression, thus lowering the resistance of cells. other mechanisms may include to improve the cell sensitivity of drug , improve cell membrane ATPase activity,promote cell uptake of the drug , increase intracellular drug concentration and changes in the expression of apoptosis-related gene,lowered Bcl-2/Bax ratio and so on. Maybe the reasons that acoustic microbubbles agent and ultrasound combined have a better taget- mediating for MDR gene ASON transfection and reversed effect in MDR than cationic liposomes and ultrasound combined: low-intensity ultrasound exposure may lead to micro-bubble bursting and micro-bubble cavitation effect for the bursting can accelerate ASON transfection and expression in the cells ,then the MDR cell lines and transplanted tumor cells resistance decreased.
引文
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