摘要
目的①构建pSUPER-shRNA／mrp1的表达载体，为探讨体内、体外抑制肝细胞癌(Hepatocelluar carcinoma，HCC)多药耐药基因(Multidrug resitance，MDR)表达的方法提供实验基础。②观察pSUPER-shRNA／mrp1对HepG2／mrp1细胞生长的影响作用，研究mrp1基因的功能。⑧探讨pSUPER-shRNA／mrp1对HepG2／mrp1细胞MDR抑制作用及其作用的机理。④观察pSUPER-shRNA／mrp1对活体HepG2／mrp1细胞移植瘤的抑制作用及初步探讨其毒副作用。
方法从GenBank查mrp1基因序列，L05628，利用InvivoGen公司提供的计算机设计软件，根据siRNA的设计原则，寻找合适的siRNA靶序列，采用BLAST软件，对选择的靶序列进行同源分析，排除非特异性地抑制其他基因片段的可能，从mrp1基因中选择5段各21个碱基的特异性靶序列，分别设计5组寡核苷酸链，第5组是对照组。每条寡核苷酸包括：两端用于形成酶切位点的序列，两个反向互补排列的21nt mrp1特异性序列，中央被一个9nt的间区隔开，用于形成茎环结果，每条链64nt退火后形成两端各带Bg1Ⅱ、HindⅢ位点的dsRNA。将5组dsRNA分别重组到pSUPER载体构建P1、P2、P3、P4、P5表达载体，PCR初步筛选重组子，质粒测序确定碱基序列正确的重组载体。构建成功后的表达载体转染HepG2、HepG2／mrp1细胞株和皮下接种于裸鼠的移植瘤。进行一系列的细胞和动物实验：①观察转染pSUPER-shRNA／mrp1对HepG2／mrp1细胞生长的作用；②用RT-PCR、Western blot杂交测定细胞mrp1基因转录后其mRNA的水平和MRP1蛋白表达水平；③流式细胞术分别检测MRP1蛋白的表达水平和细胞内柔红霉素(DNR)积累量；④MTT法检测转染后细胞的耐药性；⑤裸鼠成瘤实验，观察HepG2、HepG2／mrp1细胞成瘤情况；⑥肿瘤治疗实验，瘤内注射pSUPER-shRNA／mrp1、pSUPER-shRNA／mrp1negative vectors，质粒载体0.75mg／kg，和腹腔内注射ADM1.5mg／kg，观察裸鼠及肿瘤生长及肿瘤抑制率情况；⑦药物毒副作用试验，检测并比较转染前后裸鼠肝肾功能及白细胞变化，并做心、肝、肾、肺组织解剖病理学检查。
结果①成功合成重组质粒pSUPER-shRNA／mrp1载体P1、P2、P3、P4、P5并可以有效地进行体外、动物体内转染，说明pSUPER-shRNA／mrp1能直接细胞内合成、表达siRNA，有好的稳定性，以P4效果最好；②转染pSUPER-shRNA／mrp1的HepG2／mrp1细胞，P4组mrp1 mRNA转录水平下调(88.03±2.04)％，多药耐药相关蛋白MRP1的表达量下降至(10.2±1.3)％，相对逆转效率89.2％，细胞内DNR积累量明显增加；③成功建立裸鼠肝癌种植瘤模型，成瘤率均为100％；④裸鼠肝癌种植瘤经转染pSUPER-shRNA／mrp1，ADM化疗前后实验组肿瘤体积差613.24±28.58mm~3，肿瘤生长率(10.35±1.26)％，与对照组比较差异均有统计学意义[1120.04±90.76＆(17.90±4.58)％，P＜0.05]，肿瘤抑制率达45.0％；⑤D、E、F三组裸鼠均行肝肾功能及血液常规检测，及心、肝、肾、肺病理学检查。结果显示，组间均无明显差异，显示pSUPER-shRNA／mrp1体内转染抑制HepG2／mrp1特定基因mrp1的表达，无明显的毒副作用，用于逆转肿瘤细胞的多药耐药具有良好的安全性。
结论①在HCC细胞中mrp1基因是介导多药耐药的基因，在HCC耐药过程中具有重要作用。②pSUPER载体介导的siRNA／mrp1能下调mrp1 mRNA的水平，抑制MRP1的表达。③经瘤内注射可显著逆转肝细胞癌的耐药性，且初步探讨其毒副作用较小，具有很好的安全性。④为RNA干扰技术逆转肝细胞癌多药耐药应用于临床提供了较为可靠的理论依据。
Objective(1)To construct a expression vector plasmid pSUPER-shRNA/mrpl carrying small interfering RNA, provide theoretical and experimental evidence for inhibitory of hepatocellular carcinoma(HCC) multidrug resistance; (2) To observe the effect of pSUPER-shRNA/mrp1 on HepG2/mrp1, and investigate the function of mrp1; (3)To explore the inhibitory effect of pSUPER-shRNA/mrp1 on HepG2 multidrug resistance mediated by mrp1; (4) To test the application of constructed plasmid pSUPER-shRNA/mrp1 in vivo transfection for reversing the MDR of HCC, initiatory explore the side effect of pSUPER-shRNA/mrp1.
Methods Five duplex stranded short RNA(dsRNA) targeting mrp1 gene(dsRNA/mdr1) including one negative control were designed and synthesized. By using BLAST software, homologization sequences were excluded. The five dsRNA were recombinated with pSUPER vector who named P1, P2, P3, P4 and P5 respectively, the recon were preliminary screen by PCR, base sequence were checked by plasmid sequencing. HepG2 and HepG2/mrp1 cloned strain were transfected expression vector pSUPER-shRNA/mrp1, the nude mice were inoculated the cloned strain subcutaneous. (1)To observe the effect of transfection of pSUPER-shRNA/mrp1 on HepG2/mrpl cell; (2) Mrp1 mRNA and MRP1 protein were detected by RT-PCR and Western blot hybridization respectively; (3) The expression of MRP1 protein and the accumulation of daunorubicin(DNR) were dectected by flow cytometry(FCM); (4)Drug tolerance of transfected HepG2/mrp1 was analyzed by MTT method; (5)HepG2/mrp1 and HepG2 were transplanted to nude mice, and the transplanted tumors were observed every day. (6).pSUPER-shRNA/mrp1 vectors and pSUPER-shRNA/mrp1 negative vectors were injected into the tumor respectively when the tumor sized achieved 5 mm in diameter. Seventy-two hours after injection, adriamycin of 1.5mg/kg were injected into the tumor, the information about the nude mice and the transplanted tumor were collected; (7) Blood examination and histological examination of group D, E and F nude mice were conducted, compare the data of the three groups, and evaluate the side-effect of pSUPER-shRNA/mrp1 on using in reversal of MDR.
Results(1)Five duplex stranded RNAs targeting mrp1 gene(dsRNA/mrp1) were synthesized. Plasmid vector of pSUPER-shRNA/mrp1 was constructed successfully by duplex incision enzyme technique. Transfected by the recombinant plasmid, HepG2/mrp1 showed its MDR was reversed notablely in vivo and in vitro. Compared with dsRNA, pSUPER-shRNA/mrp1 could synthesize and express shRNA intra-cellular, its stability was better than the former. The effect of group of P4 was the best of the five group; (2)Transfected by pSUPER-shRNA/mrp1, the transcription of mrp1 mRNA decreased, it could be obsevered especially in group P4. the transcription of mrp1 mRNA decreased(88.03±2.04)％. The expression of MRP1 protein was(10.2±1.3)％, the accumulation of DNR was increased evidently. (3)HCC model of nude mice carried HepG2/mrp1 was established; (4) In HCC model of nude mice, the tumodgenes of both groups was identified. Transfected by pSUPER-shRNA/mrp1, pre-chemotherapy and post-chemotherapy, tumor volume difference of the experimental group was 613.24±28.58mm~3, it was significantly smaller than that of the control group(1120.04±90.76, P＜0.05). The tumor growth rate of the experimental group was(10.35±1.26)％, it was significantly lower than that of the control group(17.90±4.58)％. The tumor inhibition ratio was 45.0％; (5)Blood and pathologic examination were conducted on group D, E and F. No significant damage about liver and kidney function were detected, and no pathologic damage were detected in the three group. The inhibitory effect of the expression of mrp1 by pSUPER-shRNA/mrp1 transfection in vivo will be an safe method.
Conclusions(1) Mrp1 gene was a multidrug resistance gene, it has important effect on the multidrug resistance of HCC. (2) pSUPER-siRNA/mrp1 could down-regulate mrp1 mRNA, suppress the expression of MRP1 protein. (3)Injection pSUPER-siRNA/mrp1 in vivo could reverse multidrug resistance of HCC effectively, and it has better security. (4)The experiment provide a good theoretical evidence for the use of RNAi technology on reverseing multidrug resistance of HCC on clinic.
引文
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