本文選題：基因治療 + 骨髓間充質(zhì)干細胞��； 參考：《浙江大學(xué)》2016年博士論文

【摘要】：作為一種極具前景的新型腫瘤輔助治療手段,基因療法近年來已被證明有著極大的應(yīng)用潛力,然而,如何安全、有效地將這些治療基因傳遞至腫瘤部位,以及跟蹤傳遞至轉(zhuǎn)移瘤卻是當(dāng)前腫瘤基因治療領(lǐng)域一直面臨的一個瓶頸問題。例如,對于抗腫瘤自殺基因療法,就需要有一種載體,它既要能克服體內(nèi)的種種生理障礙將治療基因傳遞至腫瘤細胞,并促使相關(guān)功能蛋白在腫瘤區(qū)域獲得高表達,又要在抵達腫瘤細胞前能有效保護所攜載的治療基因,避免其在血液循環(huán)中被降解,這就對相應(yīng)的載體系統(tǒng)提出了很高的要求。本課題針對這一腫瘤基因治療中的重要問題,基于靶向藥物傳遞系統(tǒng)(Target Drug Delivery System, TDDS)的概念,提出了一種以生物活性細胞構(gòu)建基因靶向傳遞系統(tǒng)的設(shè)想。研究采用非病毒基因轉(zhuǎn)染系統(tǒng)對骨髓間充質(zhì)干細胞(BMSC)進行基因重組以攜載治療基因,進而利用BMSC的腫瘤歸巢性和免疫豁免性等特性,實現(xiàn)將治療基因靶向運輸至腫瘤細胞的目的。并通過體內(nèi)外研究全面考察了這一基于BMSC的基因靶向傳遞系統(tǒng)在小鼠肺腫瘤模型中的抗腫瘤治療效果。同時,對靜脈注射的BMSC向腫瘤組織的遷移和穿透以及BMSC作為基因傳遞載體的安全性進行了探討。為了使BMSC能高效攜載治療基因,本課題首先對BMSC的體外基因轉(zhuǎn)染系統(tǒng)進行了考察。通過對課題前期工作中合成的一些新型非病毒基因載體以及部分常用的商品化轉(zhuǎn)染試劑在BMSC上的基因轉(zhuǎn)染效率和細胞毒性的篩選,發(fā)現(xiàn)精胺修飾的陽離子化普魯蘭多糖(spermine-pullulan, SP)在BMSC上表現(xiàn)了高效的轉(zhuǎn)染效率和較低的細胞毒性。進一步,本課題對SP的合成及轉(zhuǎn)染條件分別進行了優(yōu)化以獲得在大鼠BMSC上最佳的基因轉(zhuǎn)染效率。課題同時還對SP/pDNA復(fù)合物入胞后在細胞內(nèi)的分布和SP的降解過程進行了考察,研究了該載體在干細胞上應(yīng)用的安全性。隨后,本課題分別在體內(nèi)外考察了BMSC對黑色素轉(zhuǎn)移瘤細胞(B16F10)的趨向性(歸巢性),探討利用BMSC作為載體向腫瘤組織靶向傳遞治療基因的可行性。課題首先通過體外Transwell實驗確認了BMSC對B16F10腫瘤細胞的趨向性。進一步在體內(nèi)通過熒光標記的方式觀察BMSC靜脈注射后在小鼠主要臟器中的分布及在荷瘤肺部的局部分布,確認了BMSC向肺部黑色素腫瘤細胞的歸巢特性。為了研究構(gòu)建的基于BMSC的靶向基因傳遞系統(tǒng)在腫瘤基因治療中的療效,本課題選擇了經(jīng)典的基于皰疹病毒胸苷激酶／更昔洛韋(Herpes simplex virus thymidine kinase/Ganciclovir, HSV-TK/GCV)的自殺基因治療系統(tǒng),考察了以BMSC作為HSV-TK的傳遞載體結(jié)合GCV治療后在黑色素瘤肺轉(zhuǎn)移小鼠模型上的治療效果。體外研究結(jié)果顯示,利用本課題構(gòu)建的基于SP的非病毒基因轉(zhuǎn)染系統(tǒng)可以有效重組BMSC表達自殺基因HSV-TK,并引起B(yǎng)MSC的自殺,且這種自殺作用與GCV呈明顯的劑量依賴關(guān)系。體外旁觀者效應(yīng)的考察發(fā)現(xiàn)重組自殺基因后的BMSC在一定GCV濃度下能有效抑制B16F10腫瘤細胞的生長。但這種旁觀者效應(yīng)需要BMSC與腫瘤細胞的良好接觸才能有效發(fā)揮,且對腫瘤細胞的殺傷效率與BMSC和腫瘤細胞的細胞數(shù)目比有關(guān),BMSC所占的細胞數(shù)比例越高,相應(yīng)的旁觀者效應(yīng)就越強。體內(nèi)抑瘤實驗進一步證明以BMSC作為HSV-TK的靶向腫瘤的傳遞載體在GCV存在的條件下可以顯著抑制黑色素轉(zhuǎn)移瘤的發(fā)展,顯示了良好的腫瘤抑制效果。進一步地,為了實現(xiàn)自殺基因和前體藥物對于荷瘤組織的同步靶向,提高這一自殺基因治療策略的抗腫瘤效果,本課題設(shè)計了一種可以將基因和前體藥物分別靶向到腫瘤組織的組合傳遞系統(tǒng)。該系統(tǒng)在采用BMSC傳遞自殺基因HSV-TK靶向至腫瘤細胞的同時利用脂質(zhì)體載體系統(tǒng)將前體藥物GCV共傳遞至荷瘤肺部。通過同時提高基因在腫瘤組織的表達水平和前體藥物在腫瘤負荷臟器的局部濃度來獲得更好的抑瘤效果,并降低潛在的毒副反應(yīng)。課題通過逆向蒸發(fā)法制備了具有肺被動靶向功能的GCV脂質(zhì)體,結(jié)果顯示該脂質(zhì)體靜脈注射后能顯著提高GCV在肺部的藥物濃度。體內(nèi)共靶向研究發(fā)現(xiàn),靜脈注射該脂質(zhì)體后,能將攜載的藥物有效傳遞至BMSC與腫瘤細胞所在的區(qū)域。體外在三維腫瘤球模型上的治療結(jié)果表明,重組自殺基因HSV-TK的BMSC結(jié)合GCV脂質(zhì)體進行治療后,能有效抑制腫瘤球體積的增大。體內(nèi)在黑色素瘤肺轉(zhuǎn)移小鼠模型上的治療結(jié)果也表明,采用該共靶向傳遞策略進行抗腫瘤治療亦顯示了更好地腫瘤殺傷效率,其對肺腫瘤結(jié)節(jié)的抑制效果是單用GCV溶液和重組自殺基因HSV-TK的BMSC的兩倍,同時小鼠的生存時間相比單用GCV溶液和重組BMSC也得到了明顯的延長。TUNEL細胞凋亡染色實驗結(jié)果也顯示使用該共靶向傳遞策略進行治療后能引起更多腫瘤細胞的凋亡。為了考察BMSC作為治療基因的靶向傳遞載體在趨向腫瘤組織后是否還會產(chǎn)生向腫瘤組織深層穿透的能力。本研究構(gòu)建了體外三維腫瘤球模型以模擬體內(nèi)的微小癌巢結(jié)構(gòu),利用激光掃描共聚焦顯微鏡觀察BMSC對三維腫瘤球模型的穿透能力。結(jié)果顯示BMSC具有良好的腫瘤球穿透性。在與腫瘤球共孵育48 h后即可觀察到BMSC向腫瘤球內(nèi)部的穿透,在共孵育72h后,則可發(fā)現(xiàn)BMSC已分布于腫瘤球的核心區(qū)域。而對于其他非干細胞的細胞系,如HEK293細胞,即使孵育72 h后也未觀察到其對腫瘤球的穿透。進一步地,研究采用基因重組綠色熒光蛋白的BMSC (GFP-BMSC)觀察其在體內(nèi)向腫瘤組織的穿透能力。結(jié)果發(fā)現(xiàn),在BMSC注射后第15天,在腫瘤組織的深層可以觀察到明顯的綠色熒光信號。為了更好地示蹤BMSC向腫瘤組織的遷移和穿透,課題在前期研究的基礎(chǔ)上制備了乙二胺-普魯蘭多糖修飾的氧化鐵納米粒,并用該納米粒標記BMSC。標記后的BMSC用普魯士藍(Prussian Blue)染色法特異性顯色。對染色后的肺部腫瘤切片的觀察證明,BMSC通過靜脈注射并被肺部截留后,會逐漸向肺部的腫瘤組織遷移并穿透入腫瘤組織深層。在獲得體內(nèi)外良好的抗腫瘤效果基礎(chǔ)上,本課題對BMSC作為基因靶向傳遞載體對小鼠的潛在毒副作用進行了初步考察。研究結(jié)果顯示,未經(jīng)基因重組的BMSC本身并不具備致瘤性,但是在荷瘤小鼠模型上注射大量未經(jīng)基因重組的BMSC(每只小鼠注射超過1×106個細胞)會一定程度地促進腫瘤生長。對經(jīng)重組自殺基因的TK-BMSC聯(lián)合GCV溶液或GCV脂質(zhì)體治療后的小鼠體重觀察結(jié)果顯示,該療法并未造成小鼠體重的急劇變化。研究還對肝和肺這兩個BMSC靜脈注射后主要的分布臟器在治療后的毒副作用進行了考察。對血液中谷丙轉(zhuǎn)氨酶(ALT)和谷草轉(zhuǎn)氨酶(AST)的檢測結(jié)果顯示自殺基因治療前后小鼠血液中的ALT和AST水平并未發(fā)生顯著變化。但肺和肝的組織切片顯示在治療后會有輕微的炎癥反應(yīng),但未見對正常的組織結(jié)構(gòu)形態(tài)造成破壞。進一步地,本課題對經(jīng)自殺基因重組的BMSC和GCV脂質(zhì)體治療后的小鼠進行了初步地短期和長期的毒副作用觀察。對肺部切片的觀察結(jié)果顯示治療后短期內(nèi)(治療結(jié)束后第2天)正常肺部細胞存在著輕微的損傷。但這種損傷是可逆的,在治療結(jié)束后的第90天肺部細胞已完全恢復(fù)至正常形態(tài),小鼠亦未見有整體毒性反應(yīng)。上述結(jié)果初步證明本課題構(gòu)建的基于BMSC的自殺基因治療策略是相對安全和低毒的。本課題為發(fā)展基于干細胞載體的基因靶向傳遞系統(tǒng)及其在腫瘤基因治療中的研究提供了理論與實驗的基礎(chǔ)。
[Abstract]:As a promising new type of cancer adjuvant therapy, gene therapy has been proved to have great potential in recent years. However, how to safely, effectively transfer these therapeutic genes to tumor sites, and track the transfer to metastatic tumor is a bottleneck problem in the field of tumor gene therapy. For example, In the case of antitumor suicide gene therapy, a carrier is needed. It can overcome the physiological obstacles in the body, transfer the gene to the tumor cells, promote the high expression of the related functional proteins in the tumor area, and protect the carrying therapeutic genes effectively before reaching the tumor cells, and avoid it in the blood circulation. Degradation, this is a very high requirement for the corresponding carrier system. This subject is based on the concept of Target Drug Delivery System (TDDS), which is an important problem in this tumor gene therapy, and proposes an idea of constructing a gene targeting transfer system with bioactive cells. The transfection system recombines the gene of bone marrow mesenchymal stem cells (BMSC) to carry the gene, and then uses the characteristics of the homing and immunity immunity of BMSC to target the target of the gene delivery to the tumor cells. The BMSC based gene targeting transmission system in the lung of mice is thoroughly investigated through the study in vitro and in vivo. The effect of antitumor therapy in the tumor model is also discussed. The migration and penetration of intravenous BMSC to the tumor tissue and the safety of BMSC as a gene delivery carrier are discussed. In order to enable BMSC to carry the therapeutic gene efficiently, this topic first examined the gene transfection system of BMSC in vitro. The gene transfection efficiency and cytotoxicity of some new non viral gene vectors and some commonly used commercialized reagents on BMSC showed that the spermine modified cationic pullulan polysaccharide (spermine-pullulan, SP) showed high efficient transfer efficiency and low cytotoxicity on BMSC. The synthesis and transfection conditions of SP were optimized to obtain the best gene transfection efficiency on rat BMSC. The subject also investigated the distribution of SP/pDNA complex in cell and the degradation process of SP, and studied the safety of the application of the carrier on the stem cells. Then, the subject investigated the B in vivo and in vitro, respectively. MSC's tendency (homing) to melanin metastatic tumor cells (B16F10), and to explore the feasibility of using BMSC as a carrier to target the tumor tissue targeting gene delivery. The subject first confirmed the tendency of BMSC to B16F10 tumor cells by in vitro Transwell experiment. Further observation of BMSC intravenous injection by fluorescence labeling in vivo was carried out. The distribution of the main organs in the mice and the local distribution of the tumor bearing lungs confirmed the homing characteristics of BMSC to the lung melanoma cells. In order to study the therapeutic effect of the constructed BMSC based gene delivery system on the tumor gene therapy, the subject selected the classic Herpes s based on the herpes virus thymidine kinase / Guecy Lowe (Herpes s) Implex virus thymidine kinase/Ganciclovir, HSV-TK/GCV) system of suicide gene therapy, which examined the therapeutic effect of BMSC as a carrier of HSV-TK and GCV treated mice model of melanoma lung metastasis. The results of the study in vitro showed that the system based on this topic was effective for the non viral gene transfection system based on SP. The recombinant BMSC expressed the suicide gene HSV-TK and caused the suicide of BMSC, and the suicide effect was in a dose-dependent manner with GCV. In vitro bystander effect found that the BMSC of the recombinant suicide gene could effectively inhibit the growth of B16F10 tumor cells in a certain GCV concentration. However, this bystander effect requires the good of BMSC and tumor cells. Good contact can be used effectively, and the killing efficiency of the tumor cells is related to the ratio of BMSC to the cell number of the tumor cells. The higher the number of cells in BMSC is, the stronger the bystander effect is. In vivo tumor inhibition experiment further proves that the carrier of BMSC as the target tumor of HSV-TK can be significantly suppressed under the presence of GCV. The development of melanin metastases shows a good tumor inhibition effect. Further, in order to achieve the synchronization target of the suicide gene and precursor drug to the tumor bearing tissue and improve the antitumor effect of this suicide gene therapy strategy, a combination of the base and the precursor drugs is designed to be targeted to the tumor tissue, respectively. The system uses the BMSC to transfer the suicide gene HSV-TK to the tumor cells and simultaneously uses the liposome carrier system to transfer the precursor drug GCV to the tumor bearing lung. GCV liposomes with passive targeting of the lung were prepared by reverse evaporation. The results showed that the liposomes could significantly increase the drug concentration of GCV in the lungs after intravenous injection. In vivo co targeting study found that after intravenous injection of the liposome, the drug could be effectively transferred to BMSC and tumor finely after the injection of the liposome. The treatment results on the three-dimensional tumor ball model in vitro show that the BMSC combined with the GCV liposome of the recombinant suicide gene HSV-TK can effectively inhibit the increase of the tumor ball volume. The treatment results on the mice model of melanoma lung metastasis also show that the common targeting transfer strategy is used to treat the tumor. The treatment also showed a better tumor killing efficiency, and its inhibitory effect on lung tumor nodules was two times as much as the BMSC of the GCV solution and the recombinant suicide gene HSV-TK. At the same time, the survival time of the mice was significantly longer than the GCV solution and the recombinant BMSC. The result of the apoptosis staining experiment of.TUNEL cells also showed the use of the co targeting delivery. The strategy can induce more tumor cells to apoptosis. In order to investigate the ability of BMSC as a target delivery carrier to the tumor tissue to penetrate the tumor tissue deep through the tumor tissue, this study constructed a three-dimensional tumor ball model in vitro to simulate the microcarcinoma nest structure in the body and use laser scanning copolymerization. The penetration ability of BMSC to the three-dimensional tumor ball model was observed by the focal microscope. The results showed that BMSC had good tumor penetration. After incubating 48 h with the tumor ball, the penetration of BMSC into the tumor sphere was observed. After incubation of 72h, it was found that BMSC was distributed in the core area of the tumor ball. HEK293 cells, such as HEK293 cells, did not observe the penetration of the tumor cells even after incubation for 72 h. Further, we studied the penetration ability of the recombinant green fluorescent protein (GFP-BMSC) to the tumor tissue in the body. The results showed that the obvious green fluorescence could be observed at the deep level of the tumor tissue at the fifteenth day after BMSC injection. Signal. In order to better trace the migration and penetration of BMSC into the tumor tissue, we prepared the ethylenediamine prulonide modified iron oxide nanoparticles on the basis of previous studies, and the BMSC was marked with the BMSC. staining method after the labeling of the nanoparticles with Prussian blue (Prussian Blue) staining. It has been shown that after intravenous injection and being intercepted by the lungs, BMSC will gradually migrate into the tumor tissue of the lungs and penetrate into the deep tumor tissue. On the basis of good antitumor effect in vivo and in vivo, the potential toxic and side effects of BMSC as a gene targeting carrier in mice are preliminarily investigated. The recombinant BMSC itself does not have a tumorigenicity, but a large number of ungenetically modified BMSC (injected more than 1 * 106 cells per mouse) in the tumor bearing mice model will promote tumor growth to a certain extent. The observation of the body weight of the mice after the combined GCV solution or GCV liposome treated by the recombinant suicidal gene and the GCV liposomes The treatment did not cause a sharp change in the weight of the mice. The study also examined the toxic and side effects of the main distributed organs after the two BMSC intravenous injection of the liver and lung. The results of the detection of ALT and AST in the blood showed the level of ALT and AST in the blood of mice before and after the gene therapy. There was no significant change in the lung and liver tissue sections, but there was a slight inflammatory response after treatment, but no damage to normal tissue structure was not found. Further, this subject was preliminary to the short-term and long-term side effects of the BMSC and GCV liposomes treated by suicide gene recombination. The observation of the section showed that there was a slight injury in the normal lung cells in the short term after treatment (second days after the end of the treatment). But the damage was reversible. The lung cells were completely restored to the normal form on the ninetieth day after the end of the treatment, and there was no overall toxicity in the mice. The strategy of suicide gene therapy at BMSC is relatively safe and low toxic. This topic provides a theoretical and experimental basis for the development of gene targeting delivery system based on stem cell carriers and its research in tumor gene therapy.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R730.5
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本文編號：2068732