脂肪干细胞转染hEGF基因和应用于创面修复的实验研究

发布时间：2018-07-14 19:30

【摘要】：目的：体外克隆及构建人表皮生长因子(human Epidermal Growth Factor, hEGF)基因真核表达质粒。分离培养大鼠的脂肪干细胞(adipose-derived stem cells, ADSCs),观察其转染效率；并建立大鼠全层皮损模型,进行ADSCs联合纤维蛋白胶进行创面移植治疗,探讨ADSCs对皮损组织是否具有促愈作用,为ADSCs在创伤修复中的应用提供理论基础和实验依据。方法：1.根据Gene Bank提供的已知Human cDNA EGF序列,合成EGF基因的信号肽SP (NM_001963,nt453-nt518)+EGF基因片段(NM_001963, nt3363-nt3521),将sp-hEGF基因装载到puc57载体,并经测序和酶切鉴定正确。用NheI、SalⅠ双酶切puc57载体,取目的基因片段,与同样酶切过的表达载体pYr-ads-6在T4DNA连接酶的作用下连接得到表达质粒pYr-ads-6-sp-hEGF,转化感受态大肠杆菌DH5a,纯化质粒,通过酶切及测序鉴定,确定重组质粒构建成功。2.ADSCs分离、培养、鉴定：SD大鼠脱臼处死,腹股沟处取得脂肪垫,剔除筋膜和血管,剪成直径约为2mm的小块,胶原酶消化法获得原代细胞,观察细胞生长状态和形态变化。传代纯化后,收集生长良好的第3代ADSCs,免疫细胞化学法染色检测CD44表达,以鉴定其抗原标记。3.将表达质粒pYr-ads-6-sp-hEGF质粒采用脂质体法转染第3代ADSCs,荧光显微镜下观测绿色荧光细胞所占的比例,判断转染效率。4.制备大鼠全层皮损模型：大鼠30只,3%戊巴比妥钠腹腔注射麻醉大鼠,在其脊柱两侧制作三个1.0cmx1.0cm全层皮损创面。将大鼠随机分为三组,分ADSCs联合纤维蛋白胶组(A)、纤维蛋白胶组(B)、空白组(C)。将40u1细胞悬液与纤维蛋白胶均匀涂于ADSCs联合纤维蛋白胶组创面内,对照组只涂纤维蛋白胶,空白组不做任何处理,无菌纱布覆盖。术后3、7、14天观察大鼠各组创面变化,第14天取愈合组织,常规石蜡切片、包埋。HE染色法观察各组愈合组织的形态学变化。免疫组织化学法检测愈合组织内CK19、Ⅲ型胶原的表达变化。结果：1.重组质粒pYr-ads-6-sp-hEGF经酶切及测序鉴定证实目的序列插入正确,表明含hEGF基因的真核表达质粒pYr-ads-6-sp-hEGF构建成功。2.大鼠ADSCs贴壁生长,初期呈圆形或三角形,杂质细胞较多。3-5d后,形态呈成纤维细胞样,体外培养增殖速度快。传代后,细胞形态和原代基本一致。第三代ADSCs中,约87%的细胞CD44呈阳性表达。3.质粒转染ADSCs24h后,在荧光显微镜下观察,判断转染效率均达12%左右。4.术后第3天,三组皮损差异不大；第7天,A组创面干燥,其余两组创面略湿润；第14天,A组创面面积明显减小,表面光滑,创面基本闭合。B组创面只有散在皮岛形成,创面粗糙,C组部分有痂状物形成。组织学观察：A组皮肤表皮层较厚,真皮层胶原纤维排列整齐；B组表皮较薄；C组无表皮生成。免疫组化鉴定：A组皮肤表皮层CK19表达明显增多(P0.05),B组表达较A组低(P0.05)；C组几乎无CK19表达(P0.05),三组表达水平有差异；A组Ⅲ型胶原表达增多(P0.05),B组和C组Ⅲ型胶原表达水平无明显差异(P0.05)。结论：1.大鼠ADSCs获取容易,数量足,增殖稳定易体外培养。2.成功构建hEGF基因重组质粒。3.脂质体介导法可将hEGF基因转入ADSCs,但效率较低。4.ADSCs可增加CK19和Ⅲ型胶原表达,促进创面愈合。
[Abstract]:Objective : To clone and construct human epidermal growth factor ( hEGF ) gene eukaryotic expression plasmid in vitro and to isolate adipose - derived stem cells ( ADSCs ) from cultured rat .
Methods : 1 . The recombinant plasmid was isolated , cultured and identified . The results showed that the recombinant plasmid was constructed successfully . The recombinant plasmid was isolated , cultured and identified . The expression vector pY01963 , nt453 - nt518 was used to study the expression of the recombinant plasmid .
On Day 7 , the wounds of group A were dry , and the remaining two groups were slightly wet ;
On the 14th day , the surface area of group A was obviously reduced , the surface was smooth , the wound surface was basically closed . The wound of group B was only scattered on the skin island , the wound was rough , and the C - group had a scab formation . Histological observation showed that the skin epidermis of group A was thicker and the collagen fibers in the dermis layer were arranged neatly ;
The epidermis of group B was thinner ;
The expression of CK19 in skin epidermis of group A was significantly increased ( P0.05 ) , and the expression of CK19 in group B was lower than that in group A ( P0.05 ) .
There was almost no expression of CK19 in group C ( P0.05 ) .
Conclusion : 1 . The expression level of type 鈪，

本文编号：2122715