本文選題：生物工程 + 非編碼RNA��； 參考：《武漢大學》2017年博士論文

【摘要】：第一部分tRNA/ncRNA前體藥生物合成與純化及治療骨肉瘤的療效與機制研究目的:探索大規(guī)模高純度基于tRNA為支架的重組RNA分子生物合成方法,為功能性非編碼RNA(ncRNA)的基礎研究與臨床應用提供經(jīng)濟而可靠的材料來源。在系列生物工程化tRNA/ncRNA分子中篩選出有效的新型前體藥,評價tRNA/ncRNA前體藥在異種移植腫瘤小鼠模型中對原位骨肉瘤的治療效果及其效應機制。方法:通過克隆系列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)支架相應位點形成tRNA/ncRNA嵌合體,構建表達質粒,轉染大腸桿菌HST08菌株,采用陰離子交換快速蛋白質液相色譜(FPLC)方法分離與純化在HST08中表達的目標tRNA/ncRNA分子,以變性尿素聚丙烯酰胺凝膠電泳(urea PAGE)估計目標tRNA/ncRNA分子的含量和純度;進一步通過高效液相色譜法(HPLC)定量分析tRNA/ncRNA分子純度,采用CleanAll DNA/RNA Clean-up試劑盒去除tRNA/ncRNA純化物的內(nèi)毒素并采用Pyrogent-5000動態(tài)內(nèi)毒素比濁法檢測tRNA/ncRNA純化物中內(nèi)毒素含量,Nanodrop 2000分光光度計測定純化tRNA/ncRNA分子的濃度并計算其產(chǎn)量。人骨肉瘤143B細胞轉染pCCLc-Luc-EGFP慢病毒載體,構建luciferase熒光素酶和eGFP熒光蛋白陽性表達細胞系用于后續(xù)試驗;系列生物工程合成tRNA/ncRNA分子轉染143B細胞、MG-63細胞,72小時后采用Celltiter-Glo發(fā)光細胞活力檢測法測定藥物的抗骨肉瘤增殖活性;隨后選擇具有極佳抗骨肉瘤細胞增殖效應的tRNA/ncRNA(tRNA/miR-34a)以梯度濃度處理143B細胞,以同樣方法檢測細胞活力,評價藥物劑量-反應關系,計算藥效學參數(shù),包括ED50、Hill slope、底值(Bottom);采用qRT-PCR和western blotting分別分析tRNA/miR-34a前體藥處理的143B細胞內(nèi)miR-34a水平及其靶標蛋白c-MET、SIRT1的表達;143B細胞脛骨骨膜下注射構建原位骨肉瘤SCID鼠模型,尾靜脈注射tRNA/miR-34a前體藥治療,通過小動物生物發(fā)光活體成像系統(tǒng)檢測的腫瘤信號強度和游標卡尺測量的腫瘤大小監(jiān)測腫瘤生長情況;所有試驗均設置藥物載體及tRNA/MSA作為對照。采用GraphPad Prism進行統(tǒng)計分析,p0.05時差異具有統(tǒng)計學意義。結果:系列生物工程化 tRNA/ncRNA(tRNA/miR-34a、tRNA/miR-124a、tRNA/miR-144、tRNA/miR-126、tRNA/simiR-21)分子可在大腸桿菌HST08菌株中累積至相當高水平。FPLC法能快速分離目標ncRNA分子;培養(yǎng)物經(jīng)純化后可獲得大約占提取總RNA分子20～30%的tRNA/ncRNA分子。Urea PAGE分析顯示tRNA/ncRNA嵌合體具有極高的同質性和純度;HPLC分析顯示目標tRNA/ncRNA純度高達98%以上;內(nèi)毒素檢測顯示1μgtRNA/ncRNA純化產(chǎn)物內(nèi)含有內(nèi)毒素3.0EU;經(jīng)Nanodrop 2000定量目標RNA分子顯示從1L細菌培養(yǎng)物可獲得約10～20mg tRNA/ncRNA嵌合體。系列tRNA/ncRNA分子抗骨肉瘤細胞增殖活性檢測顯示,與藥物載體、tRNA/MSA相比,tRNA/ncRNA前體藥均可顯著抑制骨肉瘤143B細胞、MG-63細胞增殖,其中tRNA/miR-34a前體藥對143B細胞生長的抑制率可達50%以下。tRNA/miR-34a前體藥抑制骨肉瘤143B細胞生長呈劑量依賴性,且比tRNA/MSA抑制效果更顯著,其中tRNA/miR-34a前體藥對143B細胞的抑制可達100%,而tRNA/MSA最大抑制率僅達34%。tRNA/miR-34a前體藥處理的143B細胞內(nèi)成熟miR-34a水平比tRNA/MSA或藥物載體處理組高約200倍,并且能顯著減少143細胞內(nèi)miR-34a靶蛋白c-MET、SIRT1的水平。無論從腫瘤信號強度和范圍還是測量的腫瘤大小上,tRNA/miR-34a前體藥治療可顯著抑制SCID鼠原位骨肉瘤的生長。結論:基于tRNA作為支架的重組RNA生物工程合成方法可經(jīng)濟而有效地大規(guī)模生產(chǎn)高純度而均一的生物活性tRNA/ncRNA分子。生物工程合成的tRNA/miRNA-34a嵌合體作為前體藥在細胞內(nèi)以miRNA-34a的形式有效控制骨肉瘤生長。生物工程化RNA分子具有進一步發(fā)展為新型大分子治療藥物的價值。第二部分阿霉素、tRNA/miR-34a前體藥與索拉非尼聯(lián)合治療骨肉瘤及肺轉移的療效及機制研究目的:采用多種高度臨床相關的動物模型以更真實而全面地評價新型抗腫瘤藥物及治療方案的可行性及有效性�；诙嘁蛩貐⑴c骨肉瘤增殖-侵襲-轉移過程,采用"飽和攻擊"策略,合理聯(lián)合應用阿霉素、miRNA-34a前體藥(tRNA/miR-34a)、索拉非尼共靶向骨肉瘤內(nèi)DNA、RNA、蛋白激酶,探索藥物間的聯(lián)合效應及其機制,在原位骨肉瘤自發(fā)性肺轉移和廣泛肺轉移晚期骨肉瘤小鼠模型中評價聯(lián)合用藥方案對于控制骨肉瘤進展、改善高轉移性骨肉瘤預后的有效性和安全性。方法:采用免疫熒光和western blotting分別分析阿霉素、tRNA/miR-34a前體藥、索拉非尼單獨或聯(lián)合處理的143B細胞內(nèi)相應藥物效應靶點或標志物γH2A.X、c-MET、p-Erk1/2的表達水平;將梯度濃度的阿霉素、tRNA/miR-34a前體藥、索拉非尼單獨或聯(lián)合處理143B細胞,72小時后采用Celltiter-Glo發(fā)光法檢測骨肉瘤細胞活力,評價藥物劑量-反應關系,計算藥效學參數(shù),包括ED50、Hillslope;采用Chou-Talalay方法計算聯(lián)合指數(shù)(CI),評價藥物之間相互作用;采用8.0mm PET膜且包被基質膠的Transwell小室檢測藥物處理后143B細胞的侵襲性。143B細胞脛骨內(nèi)注射構建原位骨肉瘤自發(fā)肺轉移SCID鼠模型,阿霉素、tRNA/miR-34a前體藥、索拉非尼單獨或聯(lián)合治療,監(jiān)測SCID鼠的體重,通過小動物生物發(fā)光活體成像系統(tǒng)檢測的腫瘤信號強度和游標卡尺測量的腫瘤大小反映腫瘤生長情況;收集血液樣本行血液生化學分析;解剖分離原位腫瘤和肺組織,原位腫瘤稱重比較,肺組織切片HE染色行組織學檢查,計量肺內(nèi)骨肉瘤轉移灶數(shù)目及轉移灶長徑之和。143B細胞尾靜脈注射構建廣泛肺轉移晚期骨肉瘤SCI]D鼠模型,阿霉素+索拉非尼、tRNA/miR-34a前體藥單獨或聯(lián)合治療,記錄SCID鼠的死亡率及死亡時間進行生存分析;SCID鼠死亡后尸檢肺組織驗證廣泛肺轉移的存在。采用GraphPad Prism進行統(tǒng)計分析,p0.05時差異具有統(tǒng)計學意義。結果:γH2A.X免疫熒光分析顯示,阿霉素導致143B細胞γH2A.X熒光信號強度顯著增高,而索拉非尼和tRNA/miR-34a前體藥單獨或聯(lián)合均可增強阿霉素誘導的yH2A.X水平升高效應。Western blotting檢測顯示,與藥物載體相比,tRNA/miR-34a前體藥減少了 40%的c-MET蛋白表達,而阿霉素或索拉非尼均可明顯上調(diào)c-MET,tRNA/miR-34a可阻斷阿霉素和索拉非尼誘導c-MET增加的作用;對p-Erk1/2而言,較藥物載體,索拉非尼抑制了 80%Erk1/2的磷酸化,阿霉素或tRNA/miR-34a可下調(diào)約20%p-Erk1/2水平,三藥聯(lián)合時p-Erk1/2下調(diào)水平與索拉非尼單獨應用時相當。阿霉素、索拉非尼和tRNA/miR-34a單獨抑制143B細胞增殖呈劑量依賴性;二聯(lián)或三聯(lián)用藥抑制效果要強于單獨給藥;藥物聯(lián)合效應分析顯示,低濃度索拉非尼和tRNA/miR-34a聯(lián)合時,兩者抗細胞增殖效應為相加甚至拮抗作用;而阿霉素與索拉非尼或tRNA/miR-34a或索拉非尼+tRNA/miR-34a的聯(lián)合在抑制143B細胞的增殖中呈協(xié)同效應,其中三聯(lián)藥物組在所有濃度及抗增殖效應下均產(chǎn)生協(xié)同作用,且在聯(lián)合用藥時阿霉素和tRNA/miR-34a可實現(xiàn)明顯的劑量減低。細胞侵襲性檢測顯示,tRNA/miR-34a可抑制143B細胞的侵襲達50%,阿霉素聯(lián)合索拉非尼減少細胞侵襲力達74%,而三藥聯(lián)合應用則抑制其侵襲性超過90%。原位骨肉瘤自發(fā)肺轉移SCID鼠模型中,無論從生物發(fā)光信號強度與范圍還是原位骨肉瘤的體積與重量上,阿霉素、索拉非尼、tRNA/miR-34a前體藥三聯(lián)治療對原位骨肉瘤生長的抑制程度均高于藥物載體、二聯(lián)或單獨給藥;而肺轉移的發(fā)生無論是從GFP信號的分布范圍還是信號強度上,三聯(lián)藥物治療顯示出對肺轉移最大程度的抑制。肺組織切片可見,三聯(lián)藥物療法一致地顯示最小的肺轉移范圍(15.0-287.5mm,平均116.3mm)和最少的肺轉移灶數(shù)目(2-4個,平均3.0個),明顯少于藥物載體治療(475.0-1212.5mm,平均829.9mm;9-29個,平均18.75個)。在藥物治療結束后,所有SCID鼠顯示出5-10%的體重損失,ALT、AST、總膽紅素、BUN和肌酐水平均有變化,但在正常范圍內(nèi),且cTnI水平均在0.156ng/ml以下;藥物載體組中1只SCID鼠在最后一周出現(xiàn)了嚴重的體重減輕和異常BUN和肌酐水平。在廣泛肺轉移晚期骨肉瘤SCID鼠模型中,生存分析顯示,阿霉素+索拉非尼或單獨tRNA/miR-34a前體藥治療將SCID鼠的中位生存時間從38天延長至54天,而三藥聯(lián)合治療將SCID鼠中位生存時間延長至60.5天。在接種后第56天所有載體治療組的SCID鼠均死亡,而三藥聯(lián)合治療組仍有50%存活;在研究結束時,三藥聯(lián)合治療組仍有25%的SCID鼠存活。結論:在原位骨肉瘤SCID鼠模型中,阿霉素、tRNA/miR-34a前體藥、索拉非尼聯(lián)合治療骨肉瘤、抑制肺轉移十分有效而安全。阿霉素、tRNA/miR-34a前體藥、索拉非尼聯(lián)合治療可延長晚期骨肉瘤的生存時間而改善其預后。共靶向細胞內(nèi)DNA、RNA和蛋白質分子的新策略為開發(fā)治療致死性腫瘤轉移提供了新方向。
[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.
【學位授予單位】：武漢大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R738.1

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