【摘要】：目的：體外克隆及構(gòu)建人表皮生長因子(human Epidermal Growth Factor, hEGF)基因真核表達(dá)質(zhì)粒。分離培養(yǎng)大鼠的脂肪干細(xì)胞(adipose-derived stem cells, ADSCs),觀察其轉(zhuǎn)染效率；并建立大鼠全層皮損模型,進(jìn)行ADSCs聯(lián)合纖維蛋白膠進(jìn)行創(chuàng)面移植治療,探討ADSCs對皮損組織是否具有促愈作用,為ADSCs在創(chuàng)傷修復(fù)中的應(yīng)用提供理論基礎(chǔ)和實(shí)驗(yàn)依據(jù)。方法：1.根據(jù)Gene Bank提供的已知Human cDNA EGF序列,合成EGF基因的信號肽SP (NM_001963,nt453-nt518)+EGF基因片段(NM_001963, nt3363-nt3521),將sp-hEGF基因裝載到puc57載體,并經(jīng)測序和酶切鑒定正確。用NheI、SalⅠ雙酶切puc57載體,取目的基因片段,與同樣酶切過的表達(dá)載體pYr-ads-6在T4DNA連接酶的作用下連接得到表達(dá)質(zhì)粒pYr-ads-6-sp-hEGF,轉(zhuǎn)化感受態(tài)大腸桿菌DH5a,純化質(zhì)粒,通過酶切及測序鑒定,確定重組質(zhì)粒構(gòu)建成功。2.ADSCs分離、培養(yǎng)、鑒定：SD大鼠脫臼處死,腹股溝處取得脂肪墊,剔除筋膜和血管,剪成直徑約為2mm的小塊,膠原酶消化法獲得原代細(xì)胞,觀察細(xì)胞生長狀態(tài)和形態(tài)變化。傳代純化后,收集生長良好的第3代ADSCs,免疫細(xì)胞化學(xué)法染色檢測CD44表達(dá),以鑒定其抗原標(biāo)記。3.將表達(dá)質(zhì)粒pYr-ads-6-sp-hEGF質(zhì)粒采用脂質(zhì)體法轉(zhuǎn)染第3代ADSCs,熒光顯微鏡下觀測綠色熒光細(xì)胞所占的比例,判斷轉(zhuǎn)染效率。4.制備大鼠全層皮損模型：大鼠30只,3%戊巴比妥鈉腹腔注射麻醉大鼠,在其脊柱兩側(cè)制作三個1.0cmx1.0cm全層皮損創(chuàng)面。將大鼠隨機(jī)分為三組,分ADSCs聯(lián)合纖維蛋白膠組(A)、纖維蛋白膠組(B)、空白組(C)。將40u1細(xì)胞懸液與纖維蛋白膠均勻涂于ADSCs聯(lián)合纖維蛋白膠組創(chuàng)面內(nèi),對照組只涂纖維蛋白膠,空白組不做任何處理,無菌紗布覆蓋。術(shù)后3、7、14天觀察大鼠各組創(chuàng)面變化,第14天取愈合組織,常規(guī)石蠟切片、包埋。HE染色法觀察各組愈合組織的形態(tài)學(xué)變化。免疫組織化學(xué)法檢測愈合組織內(nèi)CK19、Ⅲ型膠原的表達(dá)變化。結(jié)果：1.重組質(zhì)粒pYr-ads-6-sp-hEGF經(jīng)酶切及測序鑒定證實(shí)目的序列插入正確,表明含hEGF基因的真核表達(dá)質(zhì)粒pYr-ads-6-sp-hEGF構(gòu)建成功。2.大鼠ADSCs貼壁生長,初期呈圓形或三角形,雜質(zhì)細(xì)胞較多。3-5d后,形態(tài)呈成纖維細(xì)胞樣,體外培養(yǎng)增殖速度快。傳代后,細(xì)胞形態(tài)和原代基本一致。第三代ADSCs中,約87%的細(xì)胞CD44呈陽性表達(dá)。3.質(zhì)粒轉(zhuǎn)染ADSCs24h后,在熒光顯微鏡下觀察,判斷轉(zhuǎn)染效率均達(dá)12%左右。4.術(shù)后第3天,三組皮損差異不大；第7天,A組創(chuàng)面干燥,其余兩組創(chuàng)面略濕潤；第14天,A組創(chuàng)面面積明顯減小,表面光滑,創(chuàng)面基本閉合。B組創(chuàng)面只有散在皮島形成,創(chuàng)面粗糙,C組部分有痂狀物形成。組織學(xué)觀察：A組皮膚表皮層較厚,真皮層膠原纖維排列整齊；B組表皮較�。籆組無表皮生成。免疫組化鑒定：A組皮膚表皮層CK19表達(dá)明顯增多(P0.05),B組表達(dá)較A組低(P0.05)；C組幾乎無CK19表達(dá)(P0.05),三組表達(dá)水平有差異；A組Ⅲ型膠原表達(dá)增多(P0.05),B組和C組Ⅲ型膠原表達(dá)水平無明顯差異(P0.05)。結(jié)論：1.大鼠ADSCs獲取容易,數(shù)量足,增殖穩(wěn)定易體外培養(yǎng)。2.成功構(gòu)建hEGF基因重組質(zhì)粒。3.脂質(zhì)體介導(dǎo)法可將hEGF基因轉(zhuǎn)入ADSCs,但效率較低。4.ADSCs可增加CK19和Ⅲ型膠原表達(dá),促進(jìn)創(chuàng)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，

本文編號：2122715