本文選題：腺病毒載體 + 骨髓間充質(zhì)干細胞�。� 參考：《遵義醫(yī)學院》2010年碩士論文

【摘要】： 目的：構建重組腺病毒載體pAdxsi-EGFP-hRAMP1,轉染兔骨髓間充質(zhì)干細胞(BMSCs),觀察其轉染率及hRAMP1的mRNA及蛋白表達。方法：質(zhì)粒pOTB7-hRAMP1用EcoRI+XhoI雙酶切后回收hRAMP1片段,亞克隆至pShuttle-EGFP-CMV中,得到重組穿梭質(zhì)粒；I-CeuI+I-Sce1雙酶切處理pShuttle-EGFP-CMV-hRAMP1,回收EGFP-CMV-EGFP-hRAMP 1片段,亞克隆至腺病毒骨架載體pAdxsi,得到重組腺病毒質(zhì)粒；重組腺病毒質(zhì)粒酶切線性化后應用脂質(zhì)體法轉染293細胞,觀察細胞出毒跡象并進行包裝、收毒、凍融、擴增、再收毒并作毒種鑒定后以離心,透析方法純化腺病毒進行滴度測定及檢測。提取新鮮骨髓,體外分離、培養(yǎng)并鑒定MSCs。將帶有hRAMP1和EGFP基因的腺病毒載體,感染第3代MSCs,熒光倒置顯微鏡下計算感染率。收集病毒感染后的MSCs行RT-PCR檢測表明存在hRAMP1基因的mRNA表達,WesternBlot亦證實hRAMP1基因的蛋白表達。結果：構建的重組穿梭質(zhì)pShuttle-EGFP-CMV-hRAMP1用EcoRI+XhoI雙酶切,得到大小為0.8kbp (hRAMP1)和5.1kbp (pShuttle-EGFP-CMV)兩個片段；重組腺病毒質(zhì)粒pAdxsi-EGFP-CMV-hRAMP 1用XhoI酶切得到7個片段,而作為對照的空腺病毒質(zhì)粒只得到6個片段；重組腺病毒質(zhì)粒在293細胞中包裝后產(chǎn)生的重組腺病毒對293細胞有致病作用；重組腺病毒Ad-EGFP-hRAMP1 PCR鑒定可見164bp的陽性擴增條帶；經(jīng)多次重復感染后,病毒滴度檢測達2.5×1011PFU/ml.成功轉染Ad-EGFP-hRAMP1的兔MSCs可表達綠色熒光,當MOI值為800,轉染效率為80%。RT-PCR檢測轉染后的MSCs有hRAMP1基因mRNA表達;Wester-blot檢測轉染后的MSCs有bRAMP1蛋白表達。結論：(1)成功構建了攜帶增強型綠色熒光蛋白基因和hRAMP1基因的重組腺病毒載體；(2)MSCs是一種理想的基因載體細胞,可用于hRAMP1的基因治療。
[Abstract]:Aim: to construct recombinant adenovirus vector pAdxsi-EGFP-hRAMP1 and transfect it into rabbit bone marrow mesenchymal stem cells (BMSCs) and observe its transfection rate, mRNA and protein expression of hRAMP1. Methods: the recombinant shuttle plasmid pShuttle-EGFP-CMV-hRAMP1 was obtained by double enzyme digestion of plasmid pOTB7-hRAMP1, which was digested with EcoRI XhoI and subcloned into pShuttle-EGFP-CMV. The recombinant adenovirus plasmid was subcloned into adenovirus skeleton vector pAdxsi. the recombinant adenovirus plasmid was obtained by double digestion of pShuttle-EGFP-CMV-hRAMP1. The recombinant adenovirus plasmid was linearized by enzyme digestion and transfected into 293 cells by liposome method. The virulence of the cells was observed and packaged, poisoned, freeze-thawed, amplified, then poisoned and identified by centrifugation. The titer of adenovirus purified by dialysis was determined and detected. Fresh bone marrow was extracted, isolated, cultured and identified in vitro. The adenovirus vector containing hRAMP1 and EGFP genes was infected with MSCs of the third generation and the infection rate was calculated by fluorescence inverted microscope. The mRNA expression of hRAMP1 gene was detected by RT-PCR analysis after MSCs infection. Western Blot also confirmed the protein expression of hRAMP1 gene. Results: the recombinant shuttle pShuttle-EGFP-CMV-hRAMP1 was digested with EcoRI XhoI to obtain two fragments, 0.8kbp hRAMP1) and 5.1kbp pShuttle-EGFP-CMV, and the recombinant adenovirus plasmid pAdxsi-EGFP-CMV-hRAMP 1 was digested with XhoI to obtain 7 fragments, while the empty adenovirus plasmid as control only got 6 fragments. The recombinant adenovirus produced by the recombinant adenovirus plasmid packaged in 293 cells had pathogenicity to 293 cells, and the positive amplified bands of 164bp were identified by Ad-EGFP-hRAMP1 PCR of recombinant adenovirus. After repeated infection for many times, the titer of the recombinant adenovirus was 2.5 脳 10 11 PFU / ml. When the MOI value was 800, the transfection efficiency was 80%.RT-PCR to detect the mRNA expression of hRAMP1 gene in the transfected MSCs. Western blot was used to detect the bRAMP1 protein expression in the transfected MSCs. ConclusionThe recombinant adenovirus vector hRAMP1 carrying enhanced green fluorescent protein gene and hRAMP1 gene is an ideal gene vector cell and can be used for gene therapy of hRAMP1.
【學位授予單位】：遵義醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R329

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