本文選題：骨骼肌成肌細(xì)胞 + 純化��； 參考：《復(fù)旦大學(xué)》2012年博士論文

【摘要】：第一部分 小鼠成體骨骼肌成肌細(xì)胞提取及純化方法的改進(jìn) 研究背景 作為成體種子細(xì)胞及基因治療工程細(xì)胞,高純度骨骼肌成肌細(xì)胞(skeletal myoblasts, SkMs)在缺血性心臟病的治療中展示了良好的臨床應(yīng)用前景。如何快速、高效、經(jīng)濟(jì)地獲取足量的高純度SkMs一直在不斷地進(jìn)行摸索、改進(jìn)。 研究目的建立一種快速、高效的小鼠成體SkMs提取、純化的實(shí)驗(yàn)方法。 研究方法 通過(guò)布比卡因預(yù)處理激活肌骨骼肌中的SkMs,48h后,配制優(yōu)化混合酶,通過(guò)一步消化分離法及改良純化法,從成年骨骼肌中分離高純度SkMs,并與普通差速貼壁法、反復(fù)差速貼壁法及Percoll非連續(xù)密度梯度離心法等傳統(tǒng)純化方法分離的SkMs的純度相比較。 研究結(jié)果 通過(guò)改良提取、純化法所得到的SkMs純度可達(dá)到92.59±1.29%,明顯高于普通差速貼壁法、反復(fù)差速貼壁法及Percoll非連續(xù)密度梯度離心法(P0.05),并具有很好的增殖及分化能力。 研究結(jié)論 通過(guò)采用改良成體SkMs提取及純化法,能快速高效地獲取高純度SkMs。 研究背景 基因治療正逐步被應(yīng)用于多種疾病的防治,前景十分廣闊。而在基因轉(zhuǎn)染的過(guò)程中,最關(guān)鍵的核心問(wèn)題是如何選擇理想的基因載體。 研究目的 探討一種新型的非病毒基因載體——超支化聚酰胺胺(hyperbranched polyamidoamine, h-PAMAM)納米顆粒在基因轉(zhuǎn)染中的效率、毒性及轉(zhuǎn)染后的基因表達(dá)情況。 研究方法 以“改良一鍋法”合成h-PAMAM納米顆粒,將核酸(deoxyribonucleic acid, DNA)及h-PAMAM以不同的質(zhì)量比復(fù)合構(gòu)建h-PAMAM-DNA復(fù)合物,觀察其粒徑大小及Zeta電位情況；在核酸內(nèi)切酶的作用下,觀察納米顆粒對(duì)質(zhì)粒的保護(hù)作用；以h-PAMAM-DNA復(fù)合物轉(zhuǎn)染COS7及HEK293細(xì)胞系,測(cè)定在不同質(zhì)量比情況下的轉(zhuǎn)染效率及細(xì)胞存活率,得出最優(yōu)化的轉(zhuǎn)染方案：h-PAMAM介導(dǎo)的人血管內(nèi)皮生長(zhǎng)因子165(human vascular endothelial growth factor165, hVEG165)基因轉(zhuǎn)染后,測(cè)定hVEG165基因在轉(zhuǎn)染后的表達(dá)情況。 研究結(jié)果 h-PAMAM-DNA復(fù)合物的粒徑及zeta電位隨著h-PAMAM/DNA質(zhì)量比的增加而提高,h-PAMAM能保護(hù)質(zhì)粒不受核酸內(nèi)切酶的消化,保持其完整性和穩(wěn)定性長(zhǎng)達(dá)2小時(shí)。h-PAMAM介導(dǎo)質(zhì)粒轉(zhuǎn)染時(shí),最佳轉(zhuǎn)染條件下,轉(zhuǎn)染效率可達(dá)47A7±1.42%(COS7細(xì)胞)及40.8±0.98%(HEK293細(xì)胞),此時(shí)細(xì)胞毒性較輕微,細(xì)胞存活率較高(COS7:91.38±0.46%；HEk293:92.38±O.61％)；h-PAMAM介導(dǎo)的hVEG165基因轉(zhuǎn)染后,hVEG165能表達(dá)14天之久,峰值出現(xiàn)在第2天。 研究結(jié)論 作為一種新型非病毒基因載體,h-PAMAM介導(dǎo)了一種經(jīng)濟(jì)、高效、安全的基因治療方法。 研究背景 骨骼肌成肌細(xì)胞(skeletal myoblasts, SkMs)移植協(xié)同血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor, VEGF)基因治療是一種有潛力的治療缺血性心臟病的方案。然而,有待優(yōu)化的基因載體及不可調(diào)控的血管內(nèi)皮生長(zhǎng)因子的表達(dá)阻礙了基因治療的應(yīng)用。因此,尋求一種經(jīng)濟(jì)、高效、可控的基因轉(zhuǎn)染平臺(tái)非常是有必要的。 研究目的 本實(shí)驗(yàn)中,我們探討了超支化聚酰胺胺(hyperbranched polyamidoamine, h-PAMAM)納米顆粒介導(dǎo)的低氧可調(diào)控性人血管內(nèi)皮生長(zhǎng)因子165(hypoxia regulated human vascular endothelial growth factor, HRE-hVEGF165)基因轉(zhuǎn)染協(xié)同SkMs移植治療缺血性心臟病的可行性及有效性。 研究方法 將核酸(deoxyribonucleic acid, DNA)及h-PAMAM納米顆粒以不同的質(zhì)量比復(fù)合構(gòu)建h-PAMAM-DNA復(fù)合物,以復(fù)合物轉(zhuǎn)染SkMs,測(cè)定在不同質(zhì)量比情況下的轉(zhuǎn)染效率及細(xì)胞存活率,得出最優(yōu)化的轉(zhuǎn)染方案；免疫染色、Realtime-PCR及Elisa法測(cè)定其基因轉(zhuǎn)染后的表達(dá)情況。將h-PAMAM-HRE-hVEGF165復(fù)合物修飾的SkMs移植至C57/BL6小鼠的缺血心肌中,探討心肌細(xì)胞凋亡情況,移植干細(xì)胞存活率,心梗面積及膠原沉積,血管密度及心功能。 研究結(jié)果 h-PAMAM介導(dǎo)質(zhì)粒轉(zhuǎn)染SkMs時(shí),最佳轉(zhuǎn)染條件下,轉(zhuǎn)染效率可達(dá)43.47士2.22%,此時(shí)細(xì)胞毒性較輕微,細(xì)胞存活率較高(91.38±0.48%)。在低氧情況下,h-PAMAM介導(dǎo)的HRE-hVEG165基因轉(zhuǎn)染時(shí)能表達(dá)hVEGF165基因18天之久。h-PAMAM-pHRE-hVEG165修飾的SkMs移植至缺血心肌后,過(guò)表達(dá)的VEGF可導(dǎo)致心肌凋亡的減少,移植成肌細(xì)胞存活率的增加,梗死面積及膠原沉積的減少,血管密度的增加,從而抑制左室的重構(gòu),提高了心功能。 研究結(jié)論 h-PAMAM介導(dǎo)的HRE-hVEGF165基因轉(zhuǎn)染SkMs是可行、有效的,并且是一種新型的有前景的缺血性心臟病的基因治療策略。
[Abstract]:Part one
Improvement of extraction and purification methods of adult skeletal muscle myoblasts in mice
Research background
As the adult seed cell and gene therapy engineering cells, the high purity skeletal myoblasts (SkMs) shows a good clinical prospect in the treatment of ischemic heart disease. How to quickly, efficiently and economically obtain a full and high purity SkMs has been constantly groping and improving.
Objective: to establish a rapid and efficient method for the extraction and purification of SkMs from adult mice.
research method
After pretreatment with bupivacaine to activate SkMs and 48h in musculoskeletal muscles, the mixed enzyme was optimized, the high purity SkMs was separated from the adult skeletal muscle by one step digestion and purification method, and the SkMs was separated from the conventional differential adherence method, the repeated differential adherence method and the Percoll discontinuous gradient centrifugation method. The purity is compared.
Research results
The purity of SkMs obtained by improved extraction and purification can reach 92.59 + 1.29%, which is obviously higher than that of ordinary differential adhesion method, repeated differential adherence method and Percoll discontinuous density gradient centrifugation (P0.05), and has a good ability of proliferation and differentiation.
research conclusion
By means of improved adult SkMs extraction and purification, high purity SkMs. can be obtained quickly and efficiently.
Research background
Gene therapy is being gradually applied to the prevention and control of various diseases, and the prospect is very broad. In the process of gene transfection, the key problem is how to choose the ideal gene carrier.
research objective
To investigate the efficiency, toxicity and gene expression of a new non viral gene vector, hyperbranched polyamidoamine (h-PAMAM) nanoparticles, in gene transfection.
research method
A modified one pot method was used to synthesize h-PAMAM nanoparticles, and nucleic acid (deoxyribonucleic acid, DNA) and h-PAMAM were constructed with different mass ratio compound to construct h-PAMAM-DNA complex, and the size of particle size and Zeta potential were observed. Under the action of nucleic acid endonuclease, the protective effect of nanoparticles on plasmids was observed and transfected with h-PAMAM-DNA complex. COS7 and HEK293 cell lines were used to determine the transfection efficiency and cell survival rate under different mass ratio conditions. The optimal transfection scheme was obtained: h-PAMAM mediated human vascular endothelial growth factor 165 (human vascular endothelial growth factor165, hVEG165) gene was transfected to determine the expression of hVEG165 gene after transfection.
Research results
The particle size and zeta potential of the h-PAMAM-DNA complex increased with the increase of the mass ratio of h-PAMAM/DNA. H-PAMAM could protect the plasmid from the digestion of endonuclease, and maintain its integrity and stability for 2 hours.H-PAMAM mediated plasmid transfection. Under the optimal transfection condition, the transfection efficiency was 47A7 + 1.42% (COS7 cells) and 40.8 + 0.98% (HEK293). At this time, the cell viability was slight and the cell survival rate was higher (COS7:91.38 + 0.46%; HEk293:92.38 + O.61%). After transfection of hVEG165 gene mediated by h-PAMAM, hVEG165 could be expressed for a long time and the peak value appeared in second days.
research conclusion
As a new non viral gene vector, h-PAMAM mediates an economical, efficient and safe gene therapy.
Research background
Skeletal myoblasts (SkMs) transplantation and vascular endothelial growth factor (vascular endothelial growth factor, VEGF) gene therapy is a potential treatment for ischemic heart disease. However, the expression of the genes to be optimized and the expression of the non regulated vascular endothelial growth factor hinders gene therapy. Therefore, it is necessary to find an economical, efficient and controllable gene transfection platform.
research objective
In this experiment, we explored the feasibility and feasibility of hyperbranched hyperbranched polyamidoamine (h-PAMAM) nanoparticles mediated hypoxia regulated human vascular endothelial growth factor 165 (hypoxia regulated human vascular endothelial growth factor, HRE-hVEGF165) gene transfer and SkMs transplantation in the treatment of ischemic heart disease. Validity.
research method
Deoxyribonucleic acid (DNA) and h-PAMAM nanoparticles were combined to construct h-PAMAM-DNA complex with different mass ratio. Transfection efficiency and cell survival rate under different mass ratio were measured by transfection of h-PAMAM-DNA complex. The optimal transfection scheme was obtained. Immunostaining, Realtime-PCR and Elisa method were used to determine the gene transfection. The expression of h-PAMAM-HRE-hVEGF165 complex modified SkMs was transplanted into the ischemic myocardium of C57/BL6 mice. The apoptosis of myocardial cells, the survival rate of transplanted stem cells, the area of myocardial infarction and the deposition of collagen, blood vessel density and cardiac function were investigated.
Research results
When h-PAMAM mediated plasmid transfection to SkMs, the transfection efficiency of the transfection was 43.47, 2.22%, and the cytotoxicity was mild and the cell survival rate was higher (91.38 + 0.48%). Under the condition of hypoxia, h-PAMAM mediated HRE-hVEG165 gene transfection could express the 18 day.H-PAMAM-pHRE-hVEG165 modified SkMs transplantation to the ischemic heart. After muscle, overexpression of VEGF can lead to the decrease of myocardial apoptosis, increase the survival rate of myocytes, decrease of infarct area and collagen deposition, increase the density of blood vessels, inhibit the reconstruction of left ventricle and improve cardiac function.
research conclusion
H-PAMAM mediated HRE-hVEGF165 gene transfection of SkMs is feasible and effective, and is a new promising gene therapy strategy for ischemic heart disease.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

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