本文關鍵詞： 干細胞白血病基因 慢病毒載體 轉染 Cajal樣間質細胞　出處：《中國現(xiàn)代醫(yī)學雜志》2017年06期 　論文類型：期刊論文

【摘要】：目的構建含人干細胞白血病(SCL)基因的重組慢病毒載體,并轉染體外培養(yǎng)的Cajal樣間質細胞(ICC),為進一步利用慢病毒表達載體行體內實驗奠定基礎。方法通過聚合酶鏈反應(PCR)將人SCL基質粒內的遺傳物質擴增,與慢病毒載體質粒GV287-EGFP結合,構建重組質粒GV287-EGFP/SCL,通過Western blot檢測及基因測序對陽性克隆進行鑒定,并測定病毒滴度。體外分離、培養(yǎng)及鑒定ICC,重組慢病毒載體以感染復數(shù)(MOI)值為0.5、1.0、5.0、10.0、50.0和100.0時,分別轉染ICC,通過激光共聚焦顯微鏡觀察,計算不同MOI值的轉染效果,確定最佳MOI值。重組慢病毒載體以最佳MOI值感染ICC作為實驗組,以空載體轉染的ICC(空載體組)及未轉染的ICC(空白組)作為對照,通過逆轉錄聚合酶鏈反應(RT-PCR)檢測SCL基因在ICC中的表達。結果經Western blot檢測及基因測序鑒定SCL重組慢病毒表達載體構建成功,并成功轉染ICC,激光共聚焦顯微鏡下可觀察到強綠色熒光。MOI為10時轉染效果最佳,轉染效率85%;RT-PCR結果表明,實驗組SCL基因的表達量高于空載體組和空白組。結論成功制備人SCL基因重組慢病毒載體,并能高效轉染ICC,介導SCL基因在ICC中表達。
[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】： 石河子大學醫(yī)學院附屬第一醫(yī)院泌尿外科;
【基金】：國家自然科學基金(No:81360120)
【分類號】：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實驗動物患者中的發(fā)病率可達19%~84%[1-2],嚴重影響患者的成年豚鼠4只,雌雄不限,體重250~400 g,由生活質量,其病因復雜,截止目前尚未完全闡明糖尿新疆醫(yī)科大學動物實

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