發(fā)布時(shí)間：2018-01-11 03:16

  本文關(guān)鍵詞：復(fù)合遞送基因修飾干細(xì)胞與藥物的新型凝膠埋植體系的構(gòu)建及其對(duì)脊髓損傷的治療研究　出處：《浙江大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 骨髓間充質(zhì)干細(xì)胞 甲基潑尼松龍 基因轉(zhuǎn)染 脊髓損傷 凝膠支架 明膠微球

【摘要】：干細(xì)胞具有多向分化潛能和豐富的細(xì)胞功能,對(duì)干細(xì)胞的基因修飾和基于干細(xì)胞的神經(jīng)組織修復(fù)策略長(zhǎng)期以來受到廣泛關(guān)注。其中,對(duì)于中樞神經(jīng)系統(tǒng)疾病,如脊髓損傷(SCI),其發(fā)生后呈現(xiàn)復(fù)雜的動(dòng)態(tài)病程和病理狀況,加之中樞神經(jīng)組織自愈力低,對(duì)損傷后神經(jīng)功能的恢復(fù)不但需要進(jìn)行神經(jīng)細(xì)胞的補(bǔ)充,還必須克服細(xì)胞外基質(zhì)缺失以及組織微環(huán)境炎癥反應(yīng)等多方面的不利因素。如何建立能夠在SCI發(fā)生后實(shí)現(xiàn)有效的神經(jīng)功能恢復(fù)的多功能復(fù)合療法,已經(jīng)成為科學(xué)研究及臨床治療中的熱點(diǎn)和亟待解決的難點(diǎn)問題。本文研究針對(duì)嚴(yán)重SCI后的神經(jīng)組織修復(fù)這一難題,結(jié)合基因傳遞、干細(xì)胞療法及局部緩控釋遞藥手段,構(gòu)建新型的多功能復(fù)合埋植體系。研究建立腦源性神經(jīng)營(yíng)養(yǎng)因子(BDNF)基因修飾的骨髓間充質(zhì)干細(xì)胞(MSC),結(jié)合臨床治療脊髓損傷的抗炎藥物甲基潑尼松龍(甲潑尼龍,MP),進(jìn)行脊髓組織的聯(lián)合埋植治療。首先,實(shí)驗(yàn)通過對(duì)MSC基因轉(zhuǎn)染體系的優(yōu)化和系統(tǒng)研究,制備BDNF基因修飾的MSC,另一方面,構(gòu)建甲潑尼龍明膠微球(MPGM)緩釋制劑以克服MP全身用藥的系統(tǒng)毒副作用和脊髓組織利用率低的問題。同時(shí),為實(shí)現(xiàn)干細(xì)胞與藥物制劑的脊髓局部埋植,本研究基于天然細(xì)胞外基質(zhì)成分透明質(zhì)酸(HA)的交聯(lián)反應(yīng)和黏附肽的修飾建立三維凝膠埋植支架載體,利用該載體包載基因修飾的MSC和MPGM進(jìn)行SCI后的神經(jīng)組織修復(fù),通過體內(nèi)外考察,對(duì)這一復(fù)合埋植體系的治療效果進(jìn)行評(píng)價(jià),并對(duì)復(fù)合埋植體系內(nèi)各組分在神經(jīng)恢復(fù)中的功能及各組分的聯(lián)合作用方式進(jìn)行了探討。為建立高效低毒的MSC基因轉(zhuǎn)染體系,本文對(duì)新型脂質(zhì)載體Screenfect A(SF)在MSC中的基因轉(zhuǎn)染進(jìn)行了優(yōu)化和系統(tǒng)研究。基于SF所優(yōu)化的MSC基因轉(zhuǎn)染體系可以實(shí)現(xiàn)較高的基因轉(zhuǎn)染效率,并同時(shí)保持MSC良好的細(xì)胞活性。進(jìn)一步考察發(fā)現(xiàn),SF在質(zhì)粒的細(xì)胞攝取和表達(dá)陽(yáng)性率中的表現(xiàn)與LipofectamineTM 2000(Lipo)相當(dāng),但SF的轉(zhuǎn)染顯示了更高的細(xì)胞平均表達(dá)水平和MSC增殖活性,且血清對(duì)SF轉(zhuǎn)染MSC的效率及細(xì)胞活性的影響皆較小。相比常用脂質(zhì)載體Lipo,基于SF的基因傳遞明顯減少質(zhì)粒用量,在對(duì)基因轉(zhuǎn)染效率及MSC細(xì)胞活性的兼顧中顯示明顯的優(yōu)勢(shì)。進(jìn)一步對(duì)SF所攜載質(zhì)粒的胞內(nèi)轉(zhuǎn)運(yùn)行為的考察發(fā)現(xiàn),質(zhì)粒可于轉(zhuǎn)染0.5h之內(nèi)開始入胞,順利進(jìn)行溶酶體逃逸,進(jìn)而于4h之內(nèi)逐漸進(jìn)入MSC細(xì)胞核。為了補(bǔ)充受損組織中缺失的細(xì)胞外基質(zhì),避免所埋植MSC的流失和細(xì)胞死亡,并為受損組織提供支撐和橋接,本研究基于HA構(gòu)建三維凝膠支架埋植載體。研究通過對(duì)HA分子量和支架制備處方的優(yōu)化篩選,獲得具有三維多孔網(wǎng)狀結(jié)構(gòu)和一定力學(xué)強(qiáng)度的凝膠支架。為了提高支架的細(xì)胞相容性,促進(jìn)MSC在支架內(nèi)的黏附生長(zhǎng),研究引入源于層粘連蛋白鏈的多肽PPFLMLLKGSTR對(duì)凝膠支架進(jìn)行表面修飾。體外實(shí)驗(yàn)結(jié)果顯示,黏附肽的修飾顯著提高了 HA凝膠支架對(duì)MSC的細(xì)胞相容性,MSC可于肽修飾的支架中黏附生長(zhǎng)并長(zhǎng)期存活。實(shí)驗(yàn)進(jìn)一步對(duì)埋植體系的體內(nèi)脊髓組織相容性及在體內(nèi)對(duì)MSC的支持作用進(jìn)行考察,將攜載MSC的支架埋植入脊髓組織,肽修飾的支架本身即顯示了良好的組織橋接功能,同時(shí)可顯著提高M(jìn)SC在組織中的存活。通過對(duì)支架-細(xì)胞復(fù)合埋植及二者單獨(dú)埋植的研究分析發(fā)現(xiàn),支架載體與干細(xì)胞在脊髓修復(fù)過程中具有明顯的協(xié)同作用:支架載體或MSC單獨(dú)埋植時(shí)作用微弱,而二者復(fù)合埋植后顯著提高了脊髓組織完整性,對(duì)大鼠運(yùn)動(dòng)功能恢復(fù)和組織神經(jīng)絲重建發(fā)揮了顯著的促進(jìn)作用。進(jìn)一步對(duì)組織內(nèi)繼發(fā)性損傷初期情況的考察初步揭示了復(fù)合埋植體系發(fā)揮顯著修復(fù)作用的可能原因,即干細(xì)胞與支架載體的埋植對(duì)炎癥細(xì)胞浸潤(rùn)和膠質(zhì)疤痕形成具有協(xié)同抑制作用�；�于對(duì)支架載體體內(nèi)外相容性和與MSC之間協(xié)同作用的探討,本研究進(jìn)一步利用所構(gòu)建的支架載體攜載BDNF基因修飾的MSC進(jìn)行SCI后的神經(jīng)組織修復(fù)治療。體外實(shí)驗(yàn)結(jié)果顯示,支架載體可支持基因修飾的MSC的長(zhǎng)期存活及對(duì)所修飾基因的表達(dá)。體內(nèi)埋植后,對(duì)MSC的BDNF基因修飾及支架的攜載,都顯著增強(qiáng)了 MSC細(xì)胞促進(jìn)脊髓修復(fù)的作用,埋植治療可明顯抑制膠質(zhì)疤痕形成,同時(shí)重建組織中的神經(jīng)絲,在橋接脊髓神經(jīng)組織的基礎(chǔ)上,明顯改善脊髓組織內(nèi)的細(xì)胞形態(tài)。對(duì)于與支架共同埋植的MSC,BDNF的基因修飾可促進(jìn)MSC存活以及對(duì)神經(jīng)組織的修復(fù)作用,但對(duì)于缺少支架攜載的MSC,其BDNF基因修飾對(duì)細(xì)胞功能的增強(qiáng)效果則極其有限,進(jìn)一步證明支架載體對(duì)MSC脊髓埋植的重要作用。為將抗炎藥物MP進(jìn)行局部埋植以克服其全身用藥的毒副作用和利用率低的問題,本研究制備甲潑尼龍明膠微球緩釋制劑。分別對(duì)甲潑尼龍琥珀酸鈉明膠微球(MPSS-GM)、琥珀酸甲潑尼龍明膠微球(MPH-GM)和甲潑尼龍明膠微球(MPGM)三種微球進(jìn)行藥物釋放性質(zhì)的研究,考察支架的肽修飾體系對(duì)微球最終包載藥量和釋藥性質(zhì)的影響,結(jié)果發(fā)現(xiàn),經(jīng)過支架肽修飾體系的處理后,MPSS-GM只保留極少量藥物,MPH-GM保留部分藥物且釋放較快速,而MPGM保留了最多的藥物且釋藥時(shí)間最長(zhǎng)。實(shí)驗(yàn)建立MP的高效液相分析方法學(xué),對(duì)MPGM釋藥性質(zhì)的進(jìn)一步研究顯示其可實(shí)現(xiàn)400小時(shí)以上的持續(xù)釋藥。為了實(shí)現(xiàn)MPGM在脊髓組織中的埋植,實(shí)驗(yàn)進(jìn)一步利用肽修飾支架包載MPGM。體外考察發(fā)現(xiàn),包載MPGM的肽修飾支架仍具有藥物緩釋功能和良好的MSC細(xì)胞相容性。利用肽修飾的支架攜載MPGM和基因修飾MSC進(jìn)行聯(lián)合埋植,體內(nèi)試驗(yàn)結(jié)果顯示,MP和BDNF-MSC在脊髓組織中的聯(lián)合埋植可有效調(diào)節(jié)組織中炎癥反應(yīng),埋植7天時(shí)對(duì)促炎因子的下調(diào)作用和抑炎因子的上調(diào)作用最為明顯,14天和28天時(shí),聯(lián)合埋植仍顯示明顯的抑炎作用,并顯著促進(jìn)了脊髓組織再生。對(duì)包載空白GM的支架攜載BDNF-MSC進(jìn)行埋植的研究,顯示其也具有一定的炎癥緩解作用,與前期對(duì)支架和MSC埋植的結(jié)果一致,也揭示了復(fù)合埋植體系中微球和MP的作用。本課題為了實(shí)現(xiàn)基因修飾的干細(xì)胞和甲潑尼龍藥物在脊髓組織局部的聯(lián)合遞送,對(duì)MSC的非病毒基因轉(zhuǎn)染體系和甲潑尼龍的緩釋制劑進(jìn)行研究,并構(gòu)建一種對(duì)干細(xì)胞和神經(jīng)組織相容性良好的肽修飾的HA埋植支架載體。研究通過對(duì)攜載埋植后的脊髓修復(fù)情況的研究,逐步揭示了干細(xì)胞、BNDF基因修飾、甲潑尼龍緩釋給藥和埋植支架載體在神經(jīng)組織修復(fù)中的功能,本研究為共載干細(xì)胞與藥物的埋植體系的構(gòu)建提供了理論與實(shí)驗(yàn)依據(jù)。
[Abstract]:Stem cells have the potential of multi-directional differentiation and abundant cell function, gene modification on stem cells and stem cells based on neural tissue repair strategy has long attracted widespread attention. Among them, the central nervous system diseases, such as spinal cord injury (SCI), the present course and pathological status of the complex, and the healing power the central nervous tissue is low, supplement to the restoration of neural function after injury is not only the need for nerve cells, must also overcome the disadvantage of extracellular matrix and lack of tissue microenvironment inflammation and other aspects. How to establish a multi function composite therapy can effectively realize the recovery of nerve function after SCI, has become a hot topic of scientific research and in the clinical treatment and the problems to be solved. For repair of severe nerve tissue after SCI this problem in this paper, combined with the gene transfer, stem cell Local therapy and controlled-release drug delivery methods, construction of multifunctional composite implant system. A novel study on establishment of brain-derived neurotrophic factor (BDNF) gene modified bone marrow mesenchymal stem cells (MSC) combined with anti-inflammatory drugs, Kakip Nixonn Ron clinical treatment of spinal cord injury (methyl prednisolone, MP), combined with implant treatment the spinal cord tissue. First, through research and system optimization of MSC gene transfection system, preparation of BDNF gene modified MSC, on the other hand, methylprednisolone gelatin microspheres (MPGM) sustained-release preparation construction to overcome the problem of low system toxicity and spinal cord tissue by MP systemic administration. At the same time, in order to achieve dry spinal cord cells and local implantation of pharmaceutical preparations, this research is based on the natural extracellular matrix of hyaluronic acid (HA) modified by cross-linking reaction and adhesion peptides to establish three-dimensional gel implant scaffolds, Zaiji package by using the carrier By the modified MSC and MPGM SCI after the repair of nerve tissue, through in vivo study, to evaluate the therapeutic effect of compound implant system, and the composite implant system components in the combined effects of each nerve function and recovery of the points are discussed. For the establishment of high efficiency and low toxicity of MSC gene transfection this new type of lipid carrier system, Screenfect A (SF) gene transfection in MSC were studied. MSC gene transfection system optimization and SF optimization system can achieve higher gene transfection efficiency based on the activity of MSC cells and maintain good. Further study found that SF and LipofectamineTM positive expression in cells and the rate of uptake of plasmid in 2000 (Lipo), but SF transfection showed higher mean cell expression and MSC proliferation, and the efficiency of transfection of MSC and serum on SF cell activity Effect are small. Compared with common lipid carrier Lipo, SF gene transfer significantly reduced the amount of the plasmid based shows obvious advantages in both the efficiency of gene transfection and MSC cell activity in the study of SF. Further carrying the plasmid intracellular transport behavior that can begin to enter the cell transfected with plasmid 0.5h, smooth for lysosomal escape, and then gradually into the MSC within 4H in the nucleus. In order to supplement the extracellular matrix in the damaged tissue loss, avoid implanting MSC loss and cell death, and provide support for bridging and damaged tissue, this study based on the HA 3D gel scaffold implant carrier. By optimizing the research on the molecular weight of HA and support system the preparation prescription screening, obtain a gel scaffold with three-dimensional porous structure and good mechanical strength. In order to improve the compatibility of the scaffold cells, promote MSC adhesion in stent grow, research The introduction of PPFLMLLKGSTR polypeptide from laminin chain for surface modification of the scaffold. In vitro experiment results show that the modified adhesion peptide significantly increased cell HA gel scaffold on the compatibility of MSC and MSC to support peptide modified adhesion growth and long-term survival. Further experiments on implant system in spinal cord tissue and inspect the support effect in vivo of MSC, with MSC stent implanted spinal cord tissue scaffold peptide modified itself shows a good bridging function, and can significantly improve the MSC in the organization's survival. Through the study of stent implants and two single cells composite implantation analysis found that the carrier and the stem cells have obvious synergistic effect on spinal cord repair process: support vector or MSC alone when implanted mild effect, and the two composite after implantation significantly improves the spinal cord Tissue integrity, played a significant role in promoting the recovery of motor function of rats and nerve tissue reconstruction. Further investigation on silk tissue secondary injury early situation reveals the possible causes of composite implant system play a significant role in the repair, the stem cells and the scaffold implant has a synergistic inhibitory effect on inflammatory cell infiltration and study on the formation of glial scar. Between the scaffold in vitro and in vivo biocompatibility and synergy with MSC based on this study, further support using the constructed vector carrying BDNF gene modified MSC SCI after nerve tissue repair treatment in vitro. The experimental results show that the support vector can support the modified MSC and long term survival to modify gene expression in vivo. After BDNF gene modification and support of MSC carrier, significantly enhanced MSC cells promote spinal cord repair function, Implant treatment can obviously inhibit the formation of glial scar, and reconstruction of neurofilament in the organization, based on spinal cord bridging, improve spinal cord cell morphology. The common implants and support MSC gene modified BDNF can promote the survival of MSC and nerve tissue repair function, but for lack of support carrying the MSC, the BDNF gene modified enhancement effect on cell function is extremely limited, further demonstrated the important role of MSC in the spinal cord scaffold implantation. The anti-inflammatory drug MP for local implantation to overcome the systemic drug toxicity and low utilization rate, the study on Preparation of methylprednisolone sustained-release gelatin microspheres respectively. Methylprednisolone sodium succinate gelatin microspheres (MPSS-GM), methylprednisolone succinate gelatin microspheres (MPH-GM) and methylprednisolone (MPGM) gelatin microspheres three kinds of microspheres drug release The qualitative research system of the peptide modified stent up influence, drug loading and release properties of the microspheres were found after treatment of stent peptide modified system, MPSS-GM only very small amounts of drugs, drug release and retention of some MPH-GM is fast, while MPGM retained the drug most and drug release time long. The establishment of HPLC analysis of MP method, it can achieve more than 400 hours of sustained release drug release characteristics of further research on the MPGM display. In order to realize the MPGM in spinal cord were further experiments with peptide modified stent loaded MPGM. in vitro study showed that peptide modified stent MPGM package is still with slow-release function and good biocompatibility. The use of MSC peptide modified stents carrying MPGM and MSC gene modified joint implants, in vivo experiments showed that MP and BDNF-MSC in spinal cord implantation can be combined The effective regulation of tissue inflammation, after 7 days of proinflammatory cytokine downregulation and upregulation of anti-inflammatory cytokines is the most obvious, 14 days and 28 days, the joint implants still showed significantly inhibitory effect on inflammation, and promote spinal cord regeneration. Articles on BDNF-MSC loaded on the support package for implantation carrying a blank GM, the display also has a certain role in relieving inflammation, consistent with previous results of stent and MSC implantation, also reveals the microspheres and MP composite implant system. The effect of methylprednisolone cells and drug delivery in the spinal cord tissues combined in order to achieve this subject stem gene modified, sustained-release preparation for MSC non viral gene delivery system and methylprednisolone were studied, and to construct a compatible peptide modified good for stem cells and neural tissue scaffold. HA implantation through the study on the repair of spinal cord after implantation of the carrying situation The status of the research, gradually reveals the stem cells, BNDF gene modified, methylprednisolone sustained-release carrier and implanted in the nerve tissue repair of the function, this study provides a theoretical and experimental basis for the construction of stem cells and drug implant system is loaded.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R943

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