發(fā)布時(shí)間：2018-02-05 04:19

  本文關(guān)鍵詞： 干細(xì)胞白血病基因 慢病毒載體 轉(zhuǎn)染 Cajal樣間質(zhì)細(xì)胞　出處：《中國(guó)現(xiàn)代醫(yī)學(xué)雜志》2017年06期 　論文類(lèi)型：期刊論文

【摘要】：目的構(gòu)建含人干細(xì)胞白血病(SCL)基因的重組慢病毒載體,并轉(zhuǎn)染體外培養(yǎng)的Cajal樣間質(zhì)細(xì)胞(ICC),為進(jìn)一步利用慢病毒表達(dá)載體行體內(nèi)實(shí)驗(yàn)奠定基礎(chǔ)。方法通過(guò)聚合酶鏈反應(yīng)(PCR)將人SCL基質(zhì)粒內(nèi)的遺傳物質(zhì)擴(kuò)增,與慢病毒載體質(zhì)粒GV287-EGFP結(jié)合,構(gòu)建重組質(zhì)粒GV287-EGFP/SCL,通過(guò)Western blot檢測(cè)及基因測(cè)序?qū)﹃?yáng)性克隆進(jìn)行鑒定,并測(cè)定病毒滴度。體外分離、培養(yǎng)及鑒定ICC,重組慢病毒載體以感染復(fù)數(shù)(MOI)值為0.5、1.0、5.0、10.0、50.0和100.0時(shí),分別轉(zhuǎn)染ICC,通過(guò)激光共聚焦顯微鏡觀察,計(jì)算不同MOI值的轉(zhuǎn)染效果,確定最佳MOI值。重組慢病毒載體以最佳MOI值感染ICC作為實(shí)驗(yàn)組,以空載體轉(zhuǎn)染的ICC(空載體組)及未轉(zhuǎn)染的ICC(空白組)作為對(duì)照,通過(guò)逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)檢測(cè)SCL基因在ICC中的表達(dá)。結(jié)果經(jīng)Western blot檢測(cè)及基因測(cè)序鑒定SCL重組慢病毒表達(dá)載體構(gòu)建成功,并成功轉(zhuǎn)染ICC,激光共聚焦顯微鏡下可觀察到強(qiáng)綠色熒光。MOI為10時(shí)轉(zhuǎn)染效果最佳,轉(zhuǎn)染效率85%;RT-PCR結(jié)果表明,實(shí)驗(yàn)組SCL基因的表達(dá)量高于空載體組和空白組。結(jié)論成功制備人SCL基因重組慢病毒載體,并能高效轉(zhuǎn)染ICC,介導(dǎo)SCL基因在ICC中表達(dá)。
[Abstract]:Objective to construct a recombinant lentivirus vector containing human stem cell leukemia (hSCL) gene and transfect it into Cajal like stromal cells in vitro. Methods Polymerase chain reaction (PCR) was used to amplify the genetic material in human SCL matrix. The recombinant plasmid GV287-EGFP/SCL was constructed by binding with lentivirus vector plasmid GV287-EGFP. The positive clones were identified by Western blot detection and gene sequencing, and virus titer was determined. ICC was isolated, cultured and identified in vitro. The recombinant lentivirus vector was transfected with ICC by laser confocal microscopy when the moi value of the recombinant lentivirus vector was 0.5 ~ 1.0 ~ 5.0 ~ 10.0 ~ 50.0 and 100.0 respectively. The transfection effect of different MOI values was calculated and the optimal MOI value was determined. The recombinant lentivirus vector infected ICC with the best MOI value was used as the experimental group. ICC- (empty vector group) and untransfected ICC- (blank group) were used as control group. Reverse transcriptase polymerase chain reaction (RT-PCR). Results the recombinant lentivirus expression vector of SCL was successfully constructed by Western blot detection and gene sequencing. The transfection efficiency was 85% under laser confocal microscope. RT-PCR results showed that the expression of SCL gene in experimental group was higher than that in blank vector group and blank group. Conclusion Recombinant lentivirus vector of human SCL gene was successfully prepared and highly transfected into ICC. To mediate the expression of SCL gene in ICC.
【作者單位】： 石河子大學(xué)醫(yī)學(xué)院附屬第一醫(yī)院泌尿外科;
【基金】：國(guó)家自然科學(xué)基金(No:81360120)
【分類(lèi)號(hào)】：R587.2;R694.5
【正文快照】： 糖尿病膀胱異常病癥(diabetic cystopathy,DCP)1材料與方法屬于糖尿病末期出現(xiàn)頻率最高的并發(fā)癥,其在糖尿病1.1實(shí)驗(yàn)動(dòng)物患者中的發(fā)病率可達(dá)19%~84%[1-2],嚴(yán)重影響患者的成年豚鼠4只,雌雄不限,體重250~400 g,由生活質(zhì)量,其病因復(fù)雜,截止目前尚未完全闡明糖尿新疆醫(yī)科大學(xué)動(dòng)物實(shí)

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 張永革;何曉偉;張偉;馬文強(qiáng);吳永勝;吳群;李偉文;方強(qiáng);宋波;;逼尿肌反射亢進(jìn)大鼠膀胱ICC細(xì)胞興奮性變化及意義[J];臨床泌尿外科雜志;2015年02期

2 尹焯;楊金瑞;;膀胱間質(zhì)細(xì)胞與膀胱病理生理[J];中南大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2014年06期

3 徐禮臻;王勤章;韓濤;丁國(guó)富;馬克濤;王江平;;高糖環(huán)境對(duì)豚鼠膀胱Cajal樣間質(zhì)細(xì)胞內(nèi)向電流的影響[J];山東醫(yī)藥;2011年34期

4 周建民;丁國(guó)富;王勤章;劉新軍;劉永國(guó);;應(yīng)用AdMax載體系統(tǒng)構(gòu)建SCL基因重組腺病毒表達(dá)載體[J];中國(guó)現(xiàn)代醫(yī)學(xué)雜志;2011年17期

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王子雄;王勤章;錢(qián)彪;;經(jīng)尿道灌注SCL基因重組慢病毒的DCP豚鼠膀胱Cajal樣間質(zhì)細(xì)胞數(shù)量、分布及超微結(jié)構(gòu)觀察[J];山東醫(yī)藥;2017年15期

2 于惠，

本文編號(hào)：1492099