本文選題：hela細胞 + hTfR　； 參考：《中南大學》2011年碩士論文

【摘要】：研究目的人類轉鐵蛋白受體(human Transferrin receptor, hTfR)是一種廣泛分布于細胞膜的跨膜糖蛋白,通過與轉鐵蛋白的特異性結合來介導鐵的轉運,與細胞的生長、增殖、分化等有密切聯(lián)系。它在大多數(shù)成熟的正常細胞中表達水平較低,而在多種腫瘤細胞中過量表達,結合分子探針技術,使得hTfR可以作為比較理想的MR分子影像報告基因。本研究運用hTfR作為MR報告基因,制作包含hTfR蛋白編碼區(qū)的表達質粒,并對其進行檢測鑒定,為下一步體外、體內MR分子影像研究打下基礎。 方法于Yr基因庫中購買包含hTfR基因cDNA的表達質粒,參考Gene-Bank (No. BC001188)的信息合成引物,運用RT-PCR方法對hTfR基因的蛋白編碼區(qū)進行擴增,經酶切、連接之后構建pcDNA3.1(+)-hTfR的表達質粒,經鑒定及測序正確后按脂質體轉染方法瞬時轉染Hela細胞,使該細胞過量表達hTfR。運用PCR及Westen-Blot的方法在基因水平和蛋白水平檢測hTfR在轉染前后的表達水平,鑒定轉染效果。 結果成功構建了pCDNA3.1(+)-hTfR過表達質粒,測序鑒定正確；通過脂質體法轉染Hela細胞,RT-PCR顯示轉染pCDNA3.1(+)-hTfR質粒組較對照組生成的hTfR mRNA明顯增高(P0.05)；Western-Bolt顯示轉染組較對照組編碼的TfR蛋白明顯增高(P0.05)。 結論成功構建了pCDNA3.1(+)-hTfR表達質粒；質粒轉染Hela細胞后表達的hTfR mRNA及hTfR蛋白均明顯增高；為下一步分子影像學的實驗奠定了基礎。
[Abstract]:Objective Human transferrin receptor human Transferrin receptor (hTfR) is a transmembrane glycoprotein widely distributed in cell membrane. It is closely related to cell growth, proliferation and differentiation through specific binding with transferrin.The expression level of hTfR is low in most mature normal cells, but overexpressed in many kinds of tumor cells. Combined with molecular probe technique, hTfR can be used as an ideal molecular imaging reporter gene.In this study, hTfR was used as Mr reporter gene to produce expression plasmid containing coding region of hTfR protein, and it was detected and identified, which laid a foundation for further study of Mr molecular imaging in vitro and in vivo.Methods the expression plasmid containing hTfR gene cDNA was purchased from Yr gene bank.BC001188 primer was used to amplify the protein coding region of hTfR gene by RT-PCR method. After restriction endonuclease digestion, the expression plasmid of pcDNA3.1 (hTfR) was constructed. After identification and sequencing, Hela cells were transiently transfected by liposome transfection method.Overexpression of hTfR.PCR and Westen-Blot were used to detect the expression of hTfR before and after transfection at the gene and protein levels, and to evaluate the transfection effect.Results the pCDNA3.1 (-hTfR) overexpression plasmid was successfully constructed and identified by sequencing, and the pCDNA3.1 (pCDNA3.1- hTfR plasmid group) was transfected with Hela cells by reverse transcription-polymerase chain reaction (pCDNA3.1- hTfR plasmid group was significantly higher than the hTfR mRNA generated in the control group (P0.05) Western-Bolt display showed that the TfR protein encoded by the transfection group was significantly higher than that in the control group (P0.05).Conclusion the pCDNA3.1 (hTfR) expression plasmid was successfully constructed, and the expression of hTfR mRNA and hTfR protein increased significantly after transfection of pCDNA3.1hTfR into Hela cells, which laid a foundation for further molecular imaging experiments.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R346

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