阿霉素、miRNA-34a前体药和索拉非尼联合治疗骨肉瘤及肺转移的实验研究

发布时间：2018-06-10 18:07

本文选题：生物工程 + 非编码RNA　；参考：《武汉大学》2017年博士论文

【摘要】：第一部分tRNA/ncRNA前体药生物合成与纯化及治疗骨肉瘤的疗效与机制研究目的:探索大规模高纯度基于tRNA为支架的重组RNA分子生物合成方法,为功能性非编码RNA(ncRNA)的基础研究与临床应用提供经济而可靠的材料来源。在系列生物工程化tRNA/ncRNA分子中筛选出有效的新型前体药,评价tRNA/ncRNA前体药在异种移植肿瘤小鼠模型中对原位骨肉瘤的治疗效果及其效应机制。方法:通过克隆系列ncRNA 分子(miR-34a、miR-124a、miR-144、miR-126、simiR-21)序列,插入 tRNA-pre-miRNA-34a(pre-miR-34a 不含成熟 miR-34a 序列)支架相应位点形成tRNA/ncRNA嵌合体,构建表达质粒,转染大肠杆菌HST08菌株,采用阴离子交换快速蛋白质液相色谱(FPLC)方法分离与纯化在HST08中表达的目标tRNA/ncRNA分子,以变性尿素聚丙烯酰胺凝胶电泳(urea PAGE)估计目标tRNA/ncRNA分子的含量和纯度;进一步通过高效液相色谱法(HPLC)定量分析tRNA/ncRNA分子纯度,采用CleanAll DNA/RNA Clean-up试剂盒去除tRNA/ncRNA纯化物的内毒素并采用Pyrogent-5000动态内毒素比浊法检测tRNA/ncRNA纯化物中内毒素含量,Nanodrop 2000分光光度计测定纯化tRNA/ncRNA分子的浓度并计算其产量。人骨肉瘤143B细胞转染pCCLc-Luc-EGFP慢病毒载体,构建luciferase荧光素酶和eGFP荧光蛋白阳性表达细胞系用于后续试验;系列生物工程合成tRNA/ncRNA分子转染143B细胞、MG-63细胞,72小时后采用Celltiter-Glo发光细胞活力检测法测定药物的抗骨肉瘤增殖活性;随后选择具有极佳抗骨肉瘤细胞增殖效应的tRNA/ncRNA(tRNA/miR-34a)以梯度浓度处理143B细胞,以同样方法检测细胞活力,评价药物剂量-反应关系,计算药效学参数,包括ED50、Hill slope、底值(Bottom);采用qRT-PCR和western blotting分别分析tRNA/miR-34a前体药处理的143B细胞内miR-34a水平及其靶标蛋白c-MET、SIRT1的表达;143B细胞胫骨骨膜下注射构建原位骨肉瘤SCID鼠模型,尾静脉注射tRNA/miR-34a前体药治疗,通过小动物生物发光活体成像系统检测的肿瘤信号强度和游标卡尺测量的肿瘤大小监测肿瘤生长情况;所有试验均设置药物载体及tRNA/MSA作为对照。采用GraphPad Prism进行统计分析,p0.05时差异具有统计学意义。结果:系列生物工程化 tRNA/ncRNA(tRNA/miR-34a、tRNA/miR-124a、tRNA/miR-144、tRNA/miR-126、tRNA/simiR-21)分子可在大肠杆菌HST08菌株中累积至相当高水平。FPLC法能快速分离目标ncRNA分子;培养物经纯化后可获得大约占提取总RNA分子20～30%的tRNA/ncRNA分子。Urea PAGE分析显示tRNA/ncRNA嵌合体具有极高的同质性和纯度;HPLC分析显示目标tRNA/ncRNA纯度高达98%以上;内毒素检测显示1μgtRNA/ncRNA纯化产物内含有内毒素3.0EU;经Nanodrop 2000定量目标RNA分子显示从1L细菌培养物可获得约10～20mg tRNA/ncRNA嵌合体。系列tRNA/ncRNA分子抗骨肉瘤细胞增殖活性检测显示,与药物载体、tRNA/MSA相比,tRNA/ncRNA前体药均可显著抑制骨肉瘤143B细胞、MG-63细胞增殖,其中tRNA/miR-34a前体药对143B细胞生长的抑制率可达50%以下。tRNA/miR-34a前体药抑制骨肉瘤143B细胞生长呈剂量依赖性,且比tRNA/MSA抑制效果更显著,其中tRNA/miR-34a前体药对143B细胞的抑制可达100%,而tRNA/MSA最大抑制率仅达34%。tRNA/miR-34a前体药处理的143B细胞内成熟miR-34a水平比tRNA/MSA或药物载体处理组高约200倍,并且能显著减少143细胞内miR-34a靶蛋白c-MET、SIRT1的水平。无论从肿瘤信号强度和范围还是测量的肿瘤大小上,tRNA/miR-34a前体药治疗可显著抑制SCID鼠原位骨肉瘤的生长。结论:基于tRNA作为支架的重组RNA生物工程合成方法可经济而有效地大规模生产高纯度而均一的生物活性tRNA/ncRNA分子。生物工程合成的tRNA/miRNA-34a嵌合体作为前体药在细胞内以miRNA-34a的形式有效控制骨肉瘤生长。生物工程化RNA分子具有进一步发展为新型大分子治疗药物的价值。第二部分阿霉素、tRNA/miR-34a前体药与索拉非尼联合治疗骨肉瘤及肺转移的疗效及机制研究目的:采用多种高度临床相关的动物模型以更真实而全面地评价新型抗肿瘤药物及治疗方案的可行性及有效性。基于多因素参与骨肉瘤增殖-侵袭-转移过程,采用"饱和攻击"策略,合理联合应用阿霉素、miRNA-34a前体药(tRNA/miR-34a)、索拉非尼共靶向骨肉瘤内DNA、RNA、蛋白激酶,探索药物间的联合效应及其机制,在原位骨肉瘤自发性肺转移和广泛肺转移晚期骨肉瘤小鼠模型中评价联合用药方案对于控制骨肉瘤进展、改善高转移性骨肉瘤预后的有效性和安全性。方法:采用免疫荧光和western blotting分别分析阿霉素、tRNA/miR-34a前体药、索拉非尼单独或联合处理的143B细胞内相应药物效应靶点或标志物γH2A.X、c-MET、p-Erk1/2的表达水平;将梯度浓度的阿霉素、tRNA/miR-34a前体药、索拉非尼单独或联合处理143B细胞,72小时后采用Celltiter-Glo发光法检测骨肉瘤细胞活力,评价药物剂量-反应关系,计算药效学参数,包括ED50、Hillslope;采用Chou-Talalay方法计算联合指数(CI),评价药物之间相互作用;采用8.0mm PET膜且包被基质胶的Transwell小室检测药物处理后143B细胞的侵袭性。143B细胞胫骨内注射构建原位骨肉瘤自发肺转移SCID鼠模型,阿霉素、tRNA/miR-34a前体药、索拉非尼单独或联合治疗,监测SCID鼠的体重,通过小动物生物发光活体成像系统检测的肿瘤信号强度和游标卡尺测量的肿瘤大小反映肿瘤生长情况;收集血液样本行血液生化学分析;解剖分离原位肿瘤和肺组织,原位肿瘤称重比较,肺组织切片HE染色行组织学检查,计量肺内骨肉瘤转移灶数目及转移灶长径之和。143B细胞尾静脉注射构建广泛肺转移晚期骨肉瘤SCI]D鼠模型,阿霉素+索拉非尼、tRNA/miR-34a前体药单独或联合治疗,记录SCID鼠的死亡率及死亡时间进行生存分析;SCID鼠死亡后尸检肺组织验证广泛肺转移的存在。采用GraphPad Prism进行统计分析,p0.05时差异具有统计学意义。结果:γH2A.X免疫荧光分析显示,阿霉素导致143B细胞γH2A.X荧光信号强度显著增高,而索拉非尼和tRNA/miR-34a前体药单独或联合均可增强阿霉素诱导的yH2A.X水平升高效应。Western blotting检测显示,与药物载体相比,tRNA/miR-34a前体药减少了 40%的c-MET蛋白表达,而阿霉素或索拉非尼均可明显上调c-MET,tRNA/miR-34a可阻断阿霉素和索拉非尼诱导c-MET增加的作用;对p-Erk1/2而言,较药物载体,索拉非尼抑制了 80%Erk1/2的磷酸化,阿霉素或tRNA/miR-34a可下调约20%p-Erk1/2水平,三药联合时p-Erk1/2下调水平与索拉非尼单独应用时相当。阿霉素、索拉非尼和tRNA/miR-34a单独抑制143B细胞增殖呈剂量依赖性;二联或三联用药抑制效果要强于单独给药;药物联合效应分析显示,低浓度索拉非尼和tRNA/miR-34a联合时,两者抗细胞增殖效应为相加甚至拮抗作用;而阿霉素与索拉非尼或tRNA/miR-34a或索拉非尼+tRNA/miR-34a的联合在抑制143B细胞的增殖中呈协同效应,其中三联药物组在所有浓度及抗增殖效应下均产生协同作用,且在联合用药时阿霉素和tRNA/miR-34a可实现明显的剂量减低。细胞侵袭性检测显示,tRNA/miR-34a可抑制143B细胞的侵袭达50%,阿霉素联合索拉非尼减少细胞侵袭力达74%,而三药联合应用则抑制其侵袭性超过90%。原位骨肉瘤自发肺转移SCID鼠模型中,无论从生物发光信号强度与范围还是原位骨肉瘤的体积与重量上,阿霉素、索拉非尼、tRNA/miR-34a前体药三联治疗对原位骨肉瘤生长的抑制程度均高于药物载体、二联或单独给药;而肺转移的发生无论是从GFP信号的分布范围还是信号强度上,三联药物治疗显示出对肺转移最大程度的抑制。肺组织切片可见,三联药物疗法一致地显示最小的肺转移范围(15.0-287.5mm,平均116.3mm)和最少的肺转移灶数目(2-4个,平均3.0个),明显少于药物载体治疗(475.0-1212.5mm,平均829.9mm;9-29个,平均18.75个)。在药物治疗结束后,所有SCID鼠显示出5-10%的体重损失,ALT、AST、总胆红素、BUN和肌酐水平均有变化,但在正常范围内,且cTnI水平均在0.156ng/ml以下;药物载体组中1只SCID鼠在最后一周出现了严重的体重减轻和异常BUN和肌酐水平。在广泛肺转移晚期骨肉瘤SCID鼠模型中,生存分析显示,阿霉素+索拉非尼或单独tRNA/miR-34a前体药治疗将SCID鼠的中位生存时间从38天延长至54天,而三药联合治疗将SCID鼠中位生存时间延长至60.5天。在接种后第56天所有载体治疗组的SCID鼠均死亡,而三药联合治疗组仍有50%存活;在研究结束时,三药联合治疗组仍有25%的SCID鼠存活。结论:在原位骨肉瘤SCID鼠模型中,阿霉素、tRNA/miR-34a前体药、索拉非尼联合治疗骨肉瘤、抑制肺转移十分有效而安全。阿霉素、tRNA/miR-34a前体药、索拉非尼联合治疗可延长晚期骨肉瘤的生存时间而改善其预后。共靶向细胞内DNA、RNA和蛋白质分子的新策略为开发治疗致死性肿瘤转移提供了新方向。
[Abstract]:The first part of tRNA/ncRNA prodrug biosynthesis, purification and treatment of osteosarcoma: the purpose of this study: To explore a large-scale and high purity tRNA based recombinant RNA molecular biosynthesis method for the basic research and clinical application of functional non coded RNA (ncRNA). The effective new precursor drugs were screened in the tRNA/ncRNA molecule, and the therapeutic effect and the effect mechanism of tRNA/ncRNA prodrug in the xenograft tumor mice model were evaluated. Methods: the sequences of ncRNA molecules (miR-34a, miR-124a, miR-144, miR-126, simiR-21) were cloned, and tRNA-pre-miRNA-34a (pre-miR-34a) was inserted. Without mature miR-34a sequence) the scaffold of the scaffold formed the tRNA/ncRNA chimeras, constructed the expression plasmid, transfected the Escherichia coli HST08 strain, and separated and purified the target tRNA/ncRNA molecules expressed in HST08 by the anion exchange fast protein liquid chromatography (FPLC) method, and estimated the urea PAGE by denatured urea polyacrylamide gel electrophoresis (urea PAGE). The content and purity of the target tRNA/ncRNA molecules, the quantitative analysis of the purity of tRNA/ncRNA molecules by high performance liquid chromatography (HPLC), the removal of endotoxin from tRNA/ncRNA purify by CleanAll DNA/RNA Clean-up kit and the determination of endotoxin in tRNA/ncRNA purify by Pyrogent-5000 dynamic endogenous turbidimetry, Nanodrop 2000 The concentration of purified tRNA/ncRNA molecules was measured by spectrophotometer and its yield was calculated. Human osteosarcoma 143B cells were transfected with pCCLc-Luc-EGFP lentivirus vector, and luciferase luciferase and eGFP fluorescent protein positive expression cell lines were used for follow-up test. A series of bioengineered tRNA/ncRNA molecules transfected to 143B cells, MG-63 cells, after 72 hours extraction The proliferation activity of anti osteosarcoma anti osteosarcoma was measured by Celltiter-Glo luminescent cell activity assay. Then tRNA/ncRNA (tRNA/miR-34a) with excellent osteosarcoma cell proliferation effect was selected to treat 143B cells in gradient concentration, the cell viability was detected by the same method, the dose response relationship was evaluated, and the pharmacodynamic parameters, including ED50, Hill, were calculated. Slope, bottom value (Bottom); qRT-PCR and Western blotting were used to analyze the miR-34a level in 143B cells treated with tRNA/miR-34a precursor drugs and the expression of c-MET, SIRT1; 143B cells subperiosteal injection of subperiosteum to construct an orthotopic osteosarcoma rat model, the tail vein injection of precursor medicine, through the bioluminescence of small animals. The tumor signal intensity measured by the living imaging system and the size of the tumor size measured by the vernier caliper were used to monitor the tumor growth. All the trials set the drug carrier and tRNA/MSA as the control. GraphPad Prism was used for statistical analysis, and the difference in P0.05 was statistically significant. Results: bioengineered tRNA/ncRNA (tRNA/miR-34a, tRNA/miR). The molecules of -124a, tRNA/miR-144, tRNA/miR-126, tRNA/simiR-21) can be accumulated to the HST08 strain of Escherichia coli to a fairly high level of.FPLC method to quickly separate the target ncRNA molecules. After purification, the culture can obtain a tRNA/ncRNA molecule about 20 to 30% of the total RNA molecule, and.Urea PAGE analysis shows that the tRNA/ncRNA chimeras have high homogeneity. HPLC analysis showed that the purity of target tRNA/ncRNA was as high as 98%, and endotoxin test showed that 1 micron gtRNA/ncRNA contained endotoxin 3.0EU, and 10 to 20mg tRNA/ncRNA chimeras were obtained from 1L bacterial cultures by Nanodrop 2000 quantitative target RNA molecules. A series of tRNA/ncRNA molecules were detected to detect the proliferation activity of osteosarcoma cells. Compared with the drug carrier, tRNA/MSA, tRNA/ncRNA prodrugs could significantly inhibit the proliferation of osteosarcoma 143B cells and MG-63 cells, in which the inhibition rate of tRNA/miR-34a precursor to 143B cell growth was less than 50%,.TRNA/miR-34a prodrugs inhibited the growth of osteosarcoma 143B cells, and was more significant than tRNA/MSA inhibition. The inhibitory effect of tRNA/miR-34a prodrugs on 143B cells was up to 100%, while the maximum inhibitory rate of tRNA/MSA was only 200 times higher than that of tRNA/MSA or drug carrier treated 143B cells treated with 34%.tRNA/miR-34a prodrugs, and the level of miR-34a target egg white c-MET and SIRT1 was significantly reduced in 143 cells. TRNA/miR-34a prodrug therapy can significantly inhibit the growth of orthotopic osteosarcoma in SCID rats. Conclusion: a recombinant RNA biosynthesis method based on tRNA as a scaffold can economically and effectively produce high purity and homogeneous bioactive tRNA/ncRNA molecules. Bioengineered tRNA/miRNA -34a chimerism as a precursor drug in the form of miRNA-34a in the form of effective control of osteosarcoma growth. Bioengineered RNA molecules have the value of further development of new macromolecular therapy. The second part of adriamycin, tRNA/miR-34a prodrugs and sorafenib in the treatment of osteosarcoma and lung metastasis Use a variety of highly clinically related animal models to evaluate the feasibility and effectiveness of new antitumor drugs and treatments in a more true and comprehensive way. Based on multiple factors involved in osteosarcoma proliferation invasion and metastasis process, the "saturated attack" strategy is used, and the combined use of adriamycin, miRNA-34a precursor drug (tRNA/miR-34a) and sorafeni are targeted. DNA, RNA, protein kinase in osteosarcoma, explore the combined effect of drug and its mechanism, evaluate the effectiveness and safety of combination regimen in the in situ osteosarcoma spontaneous pulmonary metastasis and extensive pulmonary metastatic advanced osteosarcoma mice model to control the progression of osteosarcoma and improve the prognosis of high metastatic osteosarcoma. Methods: immunofluorescence and W Estern blotting analysis of adriamycin, tRNA/miR-34a prodrugs, the corresponding drug effect targets or markers of H2A.X, c-MET, p-Erk1/2 in 143B cells treated by Sola Fini alone or in combination with 143B cells; the gradient concentration of adriamycin, tRNA/miR-34a precursor, sorafeni alone or combined with 143B cells, 72 hours later used Cellti. Ter-Glo luminescence assay was used to detect the activity of osteosarcoma cells, to evaluate the dose response relationship and to calculate the pharmacodynamic parameters, including ED50, Hillslope, and Chou-Talalay method to calculate the combined index (CI) and evaluate the interaction between drugs. The 8.0mm PET membrane and the Transwell chamber wrapped with matrix glue were used to detect the invasive.143B of the 143B cells after the drug treatment. SCID rat model of spontaneous pulmonary metastases in situ osteosarcoma, adriamycin, tRNA/miR-34a prodrug, Sola Fini alone or combined therapy, were injected into the tibia of the tibia of the tibia to monitor the weight of SCID rats. The tumor growth was reflected by the tumor signal intensity and the size of the vernier caliper measured by the bioluminescent living imaging system of small animals. Blood samples were analyzed by hematological analysis; dissecting in situ tumor and lung tissue, in situ tumor weighing comparison, tissue biopsy of lung tissue section HE staining, measuring the number of metastatic foci of bone sarcoma in the lung and the length of metastatic foci, and using.143B cell tail vein to construct a wide range of advanced osteosarcoma SCI]D rat model of pulmonary metastases, adriamycin + sorafer Nei, tRNA/miR-34a prodrug alone or combined therapy, recorded the mortality and death time of SCID mice for survival analysis; SCID mice died after death to verify the existence of extensive lung metastasis. GraphPad Prism was used for statistical analysis. The difference in P0.05 was statistically significant. Results: gamma H2A.X immunofluorescence analysis showed that adriamycin led to 14 The intensity of 3B cell gamma H2A.X fluorescence signal increased significantly, while both Sola Fini and tRNA/miR-34a precursor drugs alone or combined can enhance the yH2A.X level induced by adriamycin induced.Western blotting detection. Compared with drug carrier, tRNA/miR-34a precursor drugs reduced the expression of c-MET protein by 40%, but Adriamycin or Sola Fini could be identified. Up regulation of c-MET, tRNA/miR-34a can block the effect of adriamycin and sorafeni induced c-MET increase; for p-Erk1/2, Sola Fini inhibited the phosphorylation of 80%Erk1/2 compared with drug carrier, Adriamycin or tRNA/miR-34a could down regulate the level of 20%p-Erk1/2. The down regulation of p-Erk1/2 was equivalent to sorafenib when three drugs were combined. The inhibitory effect of Sola Fini and tRNA/miR-34a on the proliferation of 143B cells was dose-dependent; the inhibitory effect of two or triple drugs was stronger than that of individual administration; the combination effect analysis showed that the combination of low concentration sorafenib and tRNA/miR-34a was a combination of anti cell proliferation and even antagonism; and adriamycin and Sola Fini or tRNA The combination of /miR-34a or sorafenib +tRNA/miR-34a was synergistic in inhibiting the proliferation of 143B cells, in which the triple drug group had synergistic effects under all concentrations and anti proliferation effects, and adriamycin and tRNA/miR-34a were significantly reduced in dose when combined with drug use. Cell invasiveness detection showed that tRNA/miR-34a could inhibit 143. The invasion of B cells was 50%, and doxorubicin combined with sorafenib reduced cell invasion by 74%, while the combination of three drugs inhibited its invasive activity over 90%. in situ osteosarcoma spontaneous lung metastasis SCID rat model, regardless of the intensity and scope of the bioluminescence signal or the volume and weight of the orthotopic osteosarcoma, adriamycin, Sola Fini, tRNA/miR-34a The inhibition of the prodrug triple therapy on the growth of orthotopic osteosarcoma was higher than that of the drug carrier, two or separate, and the occurrence of the pulmonary metastases from the distribution of the GFP signal or the intensity of the signal showed the maximum inhibition of pulmonary metastasis. The minimum lung metastases (15.0-287.5mm, mean 116.3mm) and the least number of lung metastases (2-4, averaging 3) were significantly less than drug carrier therapy (475.0-1212.5mm, mean 829.9mm; 9-29, averaging 18.75). After the end of the drug treatment, all SCID mice showed 5-10% weight loss, ALT, AST, total bilirubin, BUN, and creatinine levels There was a change, but in the normal range, and the cTnI level was below 0.156ng/ml; 1 SCID mice in the drug carrier group had severe weight loss and abnormal BUN and creatinine levels in the last week. In the extensive SCID rat model of advanced osteosarcoma of the lung, the survival analysis showed that amamycin + sorafeni or a separate tRNA/miR-34a precursor drug treatment The median survival time of the SCID rats was extended from 38 days to 54 days, and the median survival time of the SCID mice was prolonged to 60.5 days by the combination of three drugs. All the SCID mice in the carrier treatment group died fifty-sixth days after the inoculation, while 50% of the three drug combined treatment groups were still alive; and at the end of the study, 25% of the SCID mice were still alive in the combined treatment group of three drugs. Conclusion: at the end of the study: In the SCID rat model of orthotopic osteosarcoma, adriamycin, tRNA/miR-34a prodrug, Sola Fini combined with osteosarcoma and inhibition of lung metastasis are very effective and safe. Adriamycin, tRNA/miR-34a prodrug, and Sola Fini combined therapy can prolong the survival time of advanced osteosarcoma and improve its prognosis. A new target to intracellular DNA, RNA and protein molecules The strategy provides a new direction for the development of lethal tumor metastasis.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R738.1

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