本文選題：hela細(xì)胞 + hTfR　； 參考：《中南大學(xué)》2011年碩士論文

【摘要】：研究目的人類轉(zhuǎn)鐵蛋白受體(human Transferrin receptor, hTfR)是一種廣泛分布于細(xì)胞膜的跨膜糖蛋白,通過(guò)與轉(zhuǎn)鐵蛋白的特異性結(jié)合來(lái)介導(dǎo)鐵的轉(zhuǎn)運(yùn),與細(xì)胞的生長(zhǎng)、增殖、分化等有密切聯(lián)系。它在大多數(shù)成熟的正常細(xì)胞中表達(dá)水平較低,而在多種腫瘤細(xì)胞中過(guò)量表達(dá),結(jié)合分子探針技術(shù),使得hTfR可以作為比較理想的MR分子影像報(bào)告基因。本研究運(yùn)用hTfR作為MR報(bào)告基因,制作包含hTfR蛋白編碼區(qū)的表達(dá)質(zhì)粒,并對(duì)其進(jìn)行檢測(cè)鑒定,為下一步體外、體內(nèi)MR分子影像研究打下基礎(chǔ)。 方法于Yr基因庫(kù)中購(gòu)買(mǎi)包含hTfR基因cDNA的表達(dá)質(zhì)粒,參考Gene-Bank (No. BC001188)的信息合成引物,運(yùn)用RT-PCR方法對(duì)hTfR基因的蛋白編碼區(qū)進(jìn)行擴(kuò)增,經(jīng)酶切、連接之后構(gòu)建pcDNA3.1(+)-hTfR的表達(dá)質(zhì)粒,經(jīng)鑒定及測(cè)序正確后按脂質(zhì)體轉(zhuǎn)染方法瞬時(shí)轉(zhuǎn)染Hela細(xì)胞,使該細(xì)胞過(guò)量表達(dá)hTfR。運(yùn)用PCR及Westen-Blot的方法在基因水平和蛋白水平檢測(cè)hTfR在轉(zhuǎn)染前后的表達(dá)水平,鑒定轉(zhuǎn)染效果。 結(jié)果成功構(gòu)建了pCDNA3.1(+)-hTfR過(guò)表達(dá)質(zhì)粒,測(cè)序鑒定正確；通過(guò)脂質(zhì)體法轉(zhuǎn)染Hela細(xì)胞,RT-PCR顯示轉(zhuǎn)染pCDNA3.1(+)-hTfR質(zhì)粒組較對(duì)照組生成的hTfR mRNA明顯增高(P0.05)；Western-Bolt顯示轉(zhuǎn)染組較對(duì)照組編碼的TfR蛋白明顯增高(P0.05)。 結(jié)論成功構(gòu)建了pCDNA3.1(+)-hTfR表達(dá)質(zhì)粒；質(zhì)粒轉(zhuǎn)染Hela細(xì)胞后表達(dá)的hTfR mRNA及hTfR蛋白均明顯增高；為下一步分子影像學(xué)的實(shí)驗(yàn)奠定了基礎(chǔ)。
[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(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346

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