本文選題：腫瘤歸巢穿膜肽　切入點(diǎn)：固相合成　出處：《重慶醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：第一部分腫瘤歸巢穿膜肽合成及體外活性研究目的采用固相法人工合成異硫氰酸熒光素(fluorescein isothiocyanate,FITC)標(biāo)記的新型腫瘤歸巢穿膜肽t Ly P-1(CGNKRTR),并于體外檢測(cè)其腫瘤靶向性及穿膜特性。方法采用Na-芴甲氧羰基(Fmoc)法固相合成t Ly P-1,并用FITC標(biāo)記N端。高效液相色譜及質(zhì)譜儀檢測(cè)其純度及分子量;激光共聚焦顯微鏡及流式細(xì)胞儀觀察其腫瘤靶向性及穿膜能力;建立三維腫瘤球模型檢測(cè)其靶向穿膜性;CCK8(cell counting kit-8)評(píng)價(jià)其細(xì)胞毒性。結(jié)果合成的FITC-t Ly P-1純度為99.14%,分子量為1334.7;激光共聚焦顯微鏡可見FITC-t Ly P-1能靶向聚集到MDA-MB-231細(xì)胞膜周圍并部分穿膜進(jìn)入到細(xì)胞內(nèi),而在HUVEC細(xì)胞膜周圍未見明顯的聚集和穿膜現(xiàn)象;流式細(xì)胞儀檢測(cè)MDA-MB-231細(xì)胞內(nèi)熒光強(qiáng)度值高于HUVEC;FITC-t Ly P-1可穿入MDA-MB-231三維腫瘤球模型內(nèi)部約120μm;CCK8檢測(cè)不同濃度的FITC-t Ly P-1對(duì)細(xì)胞活性沒有明顯影響(P0.05)。結(jié)論采用固相法成功合成FITC標(biāo)記的新型腫瘤歸巢穿膜肽t Ly P-1,具有乳腺癌MDA-MB-231腫瘤靶向性和穿膜性,能作為一種潛在的腫瘤靶向藥物運(yùn)輸載體。第二部分腫瘤歸巢穿膜肽介導(dǎo)的靶向載藥相變納米粒制備及體外靶向性、穿膜性研究目的制備一種新型超聲分子探針--腫瘤歸巢穿膜肽t Ly P-1介導(dǎo)的靶向載10-羥基喜樹堿(10-HCPT)的液-氣相變型氟碳(PFP)納米粒(t Ly P-1-10-HCPT-PFP NPs),檢測(cè)其基本表征,并評(píng)價(jià)其體外靶向性及穿膜性。方法薄膜水化-聲振法制備新型超聲分子探針,檢測(cè)其形態(tài)、分布、粒徑及表面電位;激光共聚焦顯微鏡定性觀察、流式細(xì)胞儀定量分析其體外靶向性和細(xì)胞穿膜性;建立三維腫瘤球模型檢測(cè)納米粒的靶向穿膜性。結(jié)果t Ly P-1-10-HCPT-PFP NPs大小均一、分散性好,粒徑為(366.30±11.50)nm,表面電位為(5.70±3.70)m V;激光共聚焦顯微鏡結(jié)果顯示t Ly P-1-10-HCPT-PFP NPs可靶向聚集到MDA-MB-231細(xì)胞膜周圍且部分穿膜進(jìn)入到細(xì)胞質(zhì)內(nèi),而HUVEC細(xì)胞膜周圍未見明顯聚集和穿膜現(xiàn)象;無t Ly P-1肽介導(dǎo)的非靶向載藥相變納米粒(10-HCPT-PFP NPs)在兩種細(xì)胞周圍也未見明顯聚集和穿膜現(xiàn)象;流式細(xì)胞儀結(jié)果顯示MDA-MB-231細(xì)胞內(nèi)t Ly P-1-10-HCPT-PFP NPs的熒光強(qiáng)度明顯高于其余各組(P0.05);MDA-MB-231三維腫瘤球模型的激光共聚焦顯微鏡結(jié)果顯示t Ly P-1-10-HCPT-PFP NPs可穿入MDA-MB-231三維腫瘤球內(nèi)部約80μm,而10-HCPT-PFP NPs在腫瘤球模型表面未見明顯聚集,同時(shí)僅見極少量的納米粒進(jìn)入到腫瘤球內(nèi)部,且距離僅約25μm。結(jié)論成功制備了腫瘤歸巢穿膜肽t Ly P-1介導(dǎo)的靶向載10-羥基喜樹堿(10-HCPT)的液-氣相變型氟碳(PFP)納米粒(t Ly P-1-10-HCPT-PFP NPs),其能有效地靶向乳腺癌MDA-MB-231細(xì)胞并穿膜進(jìn)入細(xì)胞質(zhì)內(nèi)及腫瘤球內(nèi)部,為后續(xù)實(shí)驗(yàn)奠定了基礎(chǔ)。第三部分腫瘤歸巢穿膜肽介導(dǎo)的靶向載藥相變納米粒體外超聲成像及靶向治療研究目的制備一種新型超聲分子探針--腫瘤歸巢穿膜肽t Ly P-1介導(dǎo)的靶向載10-羥基喜樹堿(10-HCPT)的液-氣相變型氟碳(PFP)納米粒(t Ly P-1-10-HCPT-PFP NPs),評(píng)價(jià)其體外超聲成像及對(duì)乳腺癌MDA-MB-231細(xì)胞的體外治療效果。方法高效液相色譜儀檢測(cè)所制備納米粒10-HCPT的包封率及載藥率;加熱板加熱和低強(qiáng)度聚焦超聲儀(low intensity focused ultrasound,LIFU)輻照后觀察納米粒液氣相變及體外超聲成像效果;流式細(xì)胞儀檢測(cè)納米粒對(duì)MDA-MB-231細(xì)胞早晚期凋亡影響,CCK8檢測(cè)納米粒對(duì)MDA-MB-231細(xì)胞毒性作用。結(jié)果本實(shí)驗(yàn)制備的t Ly P-1-10-HCPT-PFP NPs中10HCPT的包封率為86.04±4.27%,載藥率為7.82±0.38%。t Ly P-1-10-HCPT-PFP NPs加熱到45℃可致相變,此外一定功率的LIFU輻照后也可致相變并能增強(qiáng)超聲成像(P0.05);t Ly P-1-10-HCPT-PFP NPs聯(lián)合LIFU輻照后,MDA-MB-231早晚期凋亡率明顯增加,細(xì)胞毒性也明顯增加,且高于其他各組(P0.05)。結(jié)論本實(shí)驗(yàn)所制備的t Ly P-1-10-HCPT-PFP NPs在一定功率的LIFU輻照后能增強(qiáng)超聲成像,靶向破壞微泡后釋放藥物可實(shí)現(xiàn)更佳的治療效果。
[Abstract]:The first part in tumor homing peptide to synthesis and in vitro activity of the solid phase synthesis of fluorescein isothiocyanate (fluorescein isothiocyanate FITC) new tumor marker homing penetrating peptide t Ly P-1 (CGNKRTR), and to detect the in vitro tumor targeting and membrane properties. Methods Na- fluorenylmethoxycarbonyl (Fmoc) t Ly P-1 solid synthesis method, and identified by FITC N. The high performance liquid chromatography and mass spectrometry to detect the purity and molecular weight; confocal laser scanning microscopy and flow cytometry were used to observe the tumor targeting and detection of its transmembrane ability; targeting transmembrane three-dimensional tumor sphere model; CCK8 (cell counting kit-8) to evaluate the cytotoxicity of FITC-t Ly P-1. The purity is 99.14%, molecular weight is 1334.7; laser scanning confocal microscopy showed FITC-t Ly P-1 targeting MDA-MB-231 gathered around the cell membrane and membrane into fine wear part Intracellular and around the membrane of HUVEC cells had no obvious aggregation and penetrating phenomenon; flow cytometry MDA-MB-231 intracellular fluorescence intensity was higher than that of HUVEC; FITC-t Ly P-1 MDA-MB-231 into the internal 3D tumor sphere model is about 120 m; FITC-t Ly P-1 CCK8 to detect different concentrations had no obvious effect on cell activity (P0.05). New tumor homing conclusion by solid phase synthesis of FITC successfully labeled membrane penetrating peptide t Ly P-1, with MDA-MB-231 breast cancer tumor targeting and transmembrane, can be used as a potential tumor to drug carriers. The second part tumor homing penetrating peptide mediated targeted drug loaded nanoparticles transformation preparation and in vitro targeting, targeting 10- hydroxycamptothecin through research on membrane to prepare a new ultrasound molecular probe: tumor homing penetrating peptide t Ly mediated P-1 (10-HCPT) of the liquid gas phase transition of fluorocarbon (PFP) nanoparticles (t Ly P- 1-10-HCPT-PFP NPs), to detect the basic characterization, and evaluate its in vitro targeting and membrane. Method of thin film hydration - Acoustic preparation of new ultrasound molecular probe, detection of its morphology, particle size distribution, and surface potential; laser scanning confocal microscope, flow cytometry, quantitative analysis of the target in vitro the film and to establish three-dimensional model of tumor cells; ball detection nanoparticles targeting transmembrane t Ly P-1-10-HCPT-PFP NPs. The results of uniform size, good dispersion, particle size (366.30 + 11.50) nm, the surface potential is (5.70 + 3.70) m V; laser confocal microscope showed that t Ly P-1-10-HCPT-PFP NPs target gathered around MDA-MB-231 cell membrane and some in membrane into the cytoplasm, while HUVEC cell membrane around no obvious aggregation and penetrating phenomenon; no non target T Ly P-1 peptide mediated drug loaded nanoparticles to phase (10-HCPT-PFP NPs) in around two cells No obvious aggregation and penetrating phenomenon; flow cytometry results showed that the fluorescence intensity of MDA-MB-231 cells in t Ly P-1-10-HCPT-PFP NPs was significantly higher than other groups (P0.05); MDA-MB-231 laser 3D tumor sphere model confocal microscopy showed that t Ly P-1-10-HCPT-PFP NPs MDA-MB-231 can be inserted into the three-dimensional tumor inside the ball about 80 m, and 10-HCPT-PFP NPs in the model of tumor sphere surface no obvious aggregation, and only a very small amount of nanoparticles into the tumor ball inside, and the distance is only about 25 M. conclusion the successful preparation of tumor homing peptide t Ly mediated P-1 targeting 10- Hydroxycamptothecin (10-HCPT) of the liquid gas phase transition of fluorocarbon (PFP) nanoparticles (t Ly P-1-10-HCPT-PFP NPs), which can effectively target the MDA-MB-231 breast cancer cells and penetrating into the internal cytoplasm and tumor sphere, laid the foundation for subsequent experiments. The third part in tumor homing Targeting hydroxycamptothecin loaded 10- peptide mediated targeting of drug loaded nanoparticles in vitro transformation of ultrasound imaging and targeted therapy of the purpose of the preparation of a new ultrasound molecular probe: tumor homing penetrating peptide t Ly mediated P-1 (10-HCPT) of the liquid gas phase transition of fluorocarbon (PFP) nanoparticles (t Ly P-1-10-HCPT-PFP NPs), to evaluate the ultrasound imaging in vitro and in vitro treatment of breast cancer MDA-MB-231 cells. Methods the HPLC detection of 10-HCPT nanoparticles prepared by encapsulation efficiency and drug loading rate; heating board and low intensity focused ultrasound (low intensity, focused ultrasound, LIFU) were observed in vitro ultrasound and liquid gas phase transition the imaging effect after irradiation; flow cytometry nanoparticles on MDA-MB-231 cells of early and late apoptosis, CCK8 were detected on MDA-MB-231 cells. The results of this experiment prepared t Ly P-1-10-HCPT-PFP NPs 10H The encapsulation rate of CPT was 86.04 + 4.27%, the drug loading rate of 7.82 + 0.38%.t Ly P-1-10-HCPT-PFP NPs is heated to 45 DEG C can induce phase transition, in addition to some power after LIFU irradiation can cause phase change and can enhance ultrasound imaging (P0.05); t Ly P-1-10-HCPT-PFP NPs combined with LIFU irradiation, MDA-MB-231 and delayed apoptosis, cell the toxicity also increased significantly, and higher than the other groups (P0.05). Conclusion the preparation of t Ly P-1-10-HCPT-PFP NPs in power LIFU irradiation can enhance ultrasound imaging, targeted microbubble destruction after the release of the drug can achieve better therapeutic effect.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.9;R445.1

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