TGFβ1反义基因及表达荧光素酶的重组腺病毒对放射性肺纤维化体内基因治疗初探

Preliminary studies on gene therapy with TGF β1 antisense gene/liposome complexes and adenoviral transfer vector in RPF rats

目的 探索TGFβ1反义基因和脂质体混合物以及重组腺病毒转移载体进行放射性肺纤维化体内基因治疗的可行性。方法 通过支气管插管与滴注的方式进行。结果 检测分析表明 ,转基因后 1 5d ,肺组织DNA用neo基因特异引物PCR扩增阳性 ;第 5 5天 ,PCR扩增 6 7% (2 3)阳性。RNAdotblot分析表明 ,转染TGFβ1反义基因的肺组织TGFβ1mRNA含量降低。转染 35d后取动物肺组织测定其羟脯氨酸含量表明 ,尽管转染TGFβ1反义基因的动物其肺组织羟脯氨酸含量比正常对照组高 (P <0 0 1) ,但它比生理盐水治疗对照组动物和转染TGFβ1正义基因的动物其肺内羟脯氨酸含量低 (均为P <0 .0 1)。用含有荧光素酶基因的复制缺陷型腺病毒感染大鼠 ,其肺脏均检测到了有荧光素酶活性蛋白的表达。结论 由聚阳离子脂质体介导的TGFβ1反义基因进行的肺纤维化体内基因治疗是一个简单易行的方法 ,它可以减缓肺纤维化的发生 ;用复制缺陷的重组腺病毒进行肺脏的基因治疗是一种较为理想的方法 ,有望成为治疗肺纤维化的有效的新途径。

Objective\ To observed the efficiency of gene therapy with TGFβ1 antisense gene/liposome complexes and adenoviral transfer vector in RPF rats. Methods\ TGFβ1 sense and antisense gene expression vectors and adenovirus transfer vector were introduced into rat bronchus by way of intratracheal instillation. Results At day 1.5 after TGFβ1 sense and antisense gene transfer,PCR amplification using neo gene specific primer from lung tissue DNA was all positive.After day 5 5,67%(2/3) of lung tissue DNA was positive.RNA dot blot hybridization indicated that TGFβ1 mRNA content of lung tissue transfected with pMAMneo AntiTGFβ1 gene decreased.Detection of lung hydroxyproline (Hyp) content after day 35 of gene transfer showed that even in lung of rats received pMAMneo AntiTGFβ1 lipid complexes it raised remarkably ( P <0 01) compared with that of normal animals;it was lower singnificantly ( P <0 01) than that of the rats received pMAMneo TGFβ1 lipid complexes and of the rats received saline treatment.The replication deficient recombinant adenovirus bearing firefly luciferase gene were purified by cesium chloride density centrifugation.The virus of 2 5×10 9 pfu/ml were instilled into bronchus at 0.5 ml per rat.After day 2 and day 6,the lung tissues of all six rats (three per each group) expressed the transfected luciferase gene by luminometer. Conclusion\ Cationic lipid mediated TGFβ1 antisense gene therapy was a simple and easy method.It can slow down the course of pathogenesis of lung fibrosis.Replication deficient recombinant adenovirus mediated gene therapy of lung diseases is a good and efficient method.\;