本文選題：真核表達質(zhì)粒 + pCMV-脂聯(lián)素��； 參考：《中國糖尿病雜志》2017年07期

【摘要】：目的構(gòu)建pCMV-脂聯(lián)素(APN)真核表達質(zhì)粒,觀察轉(zhuǎn)染小鼠腎系膜細(xì)胞后APN的表達以及對高糖環(huán)境下腎系膜細(xì)胞增殖的影響。方法通過RT-PCR擴增小鼠腎系膜細(xì)胞中APN基因片段,經(jīng)酶切、純化后與pcDNA3.1質(zhì)粒連接構(gòu)建pCMV-APN重組質(zhì)粒。pCMV-APN重組質(zhì)粒經(jīng)酶切鑒定及DNA測序驗證后,由脂質(zhì)體轉(zhuǎn)染將pCMV-APN真核表達質(zhì)粒轉(zhuǎn)導(dǎo)入腎系膜細(xì)胞中,Western blot檢測轉(zhuǎn)染后細(xì)胞中APN蛋白表達,MTT檢測轉(zhuǎn)染后細(xì)胞在高糖環(huán)境下增殖活性的變化。結(jié)果真核表達質(zhì)粒pCMV-APN經(jīng)酶切、測序鑒定后提示構(gòu)建成功。轉(zhuǎn)染后腎系膜細(xì)胞APN蛋白表達明顯增高,且在高糖環(huán)境下的增殖活性較空載體轉(zhuǎn)染組明顯下降[(0.87±0.06)vs(0.60±0.01),P0.05]。結(jié)論成功構(gòu)建pCMV-APN真核表達質(zhì)粒,能穩(wěn)定表達于小鼠腎系膜細(xì)胞及抑制高糖的誘導(dǎo)增殖作用,為進一步研究APN的生物學(xué)功能奠定理論基礎(chǔ)。
[Abstract]:Objective to construct the eukaryotic expression plasmid of pCMV- adiponectin (APN) and observe the expression of APN and its effect on the proliferation of Mesangial cells in high glucose environment. Methods the APN gene fragment of mouse Mesangial cells was amplified by RT-PCR. The recombinant plasmid pCMV-APN was ligated with pcDNA3.1 plasmid and digested by enzyme. The recombinant plasmid pCMV-APN was identified by restriction endonuclease digestion and confirmed by DNA sequencing. The eukaryotic expression plasmid of pCMV-APN was transfected into Mesangial cells by liposome transfection. Western blot was used to detect the expression of APN protein in the transfected cells. Results the eukaryotic expression plasmid pCMV-APN was digested by enzyme and identified by sequencing. The expression of APN protein in renal Mesangial cells was significantly increased after transfection, and the proliferative activity in high glucose environment was significantly lower than that in empty vector transfection group [0.87 鹵0.06)vs(0.60 鹵0.01 p0.05]. Conclusion the successful construction of pCMV-APN eukaryotic expression plasmid can stably express in mouse Mesangial cells and inhibit the proliferation induced by high glucose, which lays a theoretical foundation for the further study of the biological function of APN.
【作者單位】： 福建省老年醫(yī)院內(nèi)分泌科;福建省立醫(yī)院內(nèi)分泌科;福建醫(yī)科大學(xué)附屬協(xié)和醫(yī)院內(nèi)分泌科;
【基金】：福建省醫(yī)學(xué)創(chuàng)新課題(2015-CX-13)
【分類號】：R587.2;R692.9

【參考文獻】

相關(guān)期刊論文 前1條

1 邊芳;楊秦予;郭桂智;;脂聯(lián)素與2型糖尿病腎病患者內(nèi)皮功能及炎癥狀態(tài)的關(guān)系[J];中國實驗診斷學(xué);2013年07期

【共引文獻】

相關(guān)期刊論文 前6條

1 馮敏;溫俊平;呂琦;黃國良;;真核表達質(zhì)粒pCMV-脂聯(lián)素的構(gòu)建及其轉(zhuǎn)染對高糖環(huán)境下腎系膜細(xì)胞增殖影響的研究[J];中國糖尿病雜志;2017年07期

2 李雯妮;王俊琳;張春炳;葛麗娜;山潔慧;劉建平;;血生長停滯特異性蛋白6、胱抑素C與老年2型糖尿病腎病相關(guān)性的研究[J];現(xiàn)代生物醫(yī)學(xué)進展;2016年36期

3 黃亞珍;程曉霞;;脂聯(lián)素與糖尿病腎病[J];浙江中西醫(yī)結(jié)合雜志;2015年12期

4 周宗愛;湯智越;王新紅;;新診斷2型糖尿病老年患者血清脂聯(lián)素、視黃醇結(jié)合蛋白4水平與代謝綜合征的關(guān)系研究[J];中國醫(yī)師雜志;2015年06期

5 趙濤;畢會民;;2型糖尿病患者脂聯(lián)素與炎癥因子相關(guān)性研究[J];檢驗醫(yī)學(xué)與臨床;2015年09期

6 聶明明;馬小茹;;2型糖尿病腎病與血脂聯(lián)素、Gas6、血清膽紅素相關(guān)性研究[J];繼續(xù)醫(yī)學(xué)教育;2015年02期

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本文編號：1968810