【作者】 赵平；

【导师】 李世荣；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 瘢痕疙瘩是皮肤损伤如创伤、烧伤或手术后引发的过度纤维化。病变常蔓延至正常皮肤,严重损害容貌外观,造成功能障碍,不发生退行变化和单纯手术切除后极易复发。临床上治疗方法虽然很多,但疗效不佳。CTGF是一种重要的促纤维化因子,通过SMADs途径参与病理性瘢痕的形成,阻断CTGF的产生及其促纤维化效应可能为今后病理性瘢痕的治疗提供了一个新的靶点。有研究报道,利用CTGF反义寡核苷酸能够抑制HKFs的增殖、CTGF蛋白质和mRNA的表达、胶原合成。RNA干扰是利用与目的mRNA具有同源性的siRNA诱发序列特异性的转录后基因沉默的现象,它产生类似于“基因敲除”的生物表型,能够高效、特异地抑制靶基因。有报道能够代替基因打靶技术,应用于临床治疗相关疾病。目的运用生物信息学方法,构建CTGFsiRNA干扰质粒,转染细胞后诱发RNAi,观察其对人瘢痕疙瘩成纤维细胞CTGF表达和胶原蛋白分泌的影响,并探讨其机制,为瘢痕疙瘩的基因治疗提供了一条新的思路。方法根据RNAi设计原则,运用生物信息学方法,筛选出3条靶序列,分别合成3对64nt寡核苷酸,另以一条同样GC比例的错乱序列作为阴性对照,经与BamHI和HindIII酶切的线性化质粒pGenesil-1定向连接后转化大肠杆菌DH5a,质粒分别命名为pGenesil-CTGF1、pGenesil-CTGF2、pGenesil-CTGF3,pGenesil-CTGF4。质粒扩增后经酶切、测序鉴定。为提高转染效率,使用Roche公司的Dosper脂质体进行转染,转染筛选实验分为六组:对照组(单纯DMEM培养基+脂质体);空质粒组(转染pGenesil-1空质粒);pGenesil-CTGF1组;pGenesil-CTGF2组;pGenesil-CTGF3组;pGenesil-CTGF4组。细胞经G-418筛选后,得到细胞克隆,逐步扩大培养。使用荧光定量PCR检测各组细胞中CTGF mRNA水平、Western blotting半定量检测CTGF蛋白水平、H3-脯氨酸渗入法检测胶原蛋白含量。分别用10ng/ml剂量TGF-β1刺激抑制效率最高的干扰质粒组细胞和未经转染的瘢痕疙瘩成纤维细胞,观察从形态学、CTGF mRNA、CTGF蛋白和胶原蛋白含量方面观察对细胞生物学的影响。结果1.成功构建CTGF基因的siRNA表达质粒,测序证实插入序列正确。2.转染pGenesil-CTGF1、pGenesil-CTGF2干扰质粒的瘢痕疙瘩成纤维细胞CTGF mRNA表达水平的抑制效率分别是59.9%、20.4%(P<0.05),pGenesil-CTGF3干扰质粒的抑制效率明显不佳(9.09%),在前两组干扰质粒组中,尤以pGenesil-CTGF1组抑制效果最佳。pGenesil-CTGF1干扰质粒对CTGF蛋白和胶原蛋白的抑制率分别达33.6%和49.7%(P<0.05)。这说明构建siRNA干扰质粒可有效抑制相应基因和蛋白的表达水平,其抑制效果的差异与靶序列有关。3.经TGF-β1刺激后,发现瘢痕疙瘩成纤维细胞的CTGF mRNA水平明显升高2倍多,但转染了pGenesil-CTGF1干扰质粒的细胞虽经TGF-β1刺激,CTGFmRNA水平也只有未经刺激细胞的56.6%,与刺激后的正常细胞相比明显降低(P<0.05),CTGF蛋白水平、胶原蛋白含量与刺激后相比也明显降低33.6%、49.7%(P<0.05)。结论1.针对CTGF基因的siRNA干扰质粒转染瘢痕疙瘩成纤维细胞后能明显抑制CTGF mRNA表达,降低CTGF蛋白含量,提示该基因可作为基因治疗的靶点; 2.针对CTGF基因不同位点的siRNA干扰质粒转染细胞后,能够不同程度地抑制CTGF基因表达,其抑制效果和靶序列的位置有关; 3.经TGF-β1刺激后,转染pGenesil-CTGF干扰质粒的瘢痕疙瘩成纤维细胞CTGF mRNA、CTGF蛋白和胶原蛋白含量与与未经转染细胞相比明显降低,但与未经刺激的细胞相比,仍未到基线水平,说明TGF-β1是通过CTGF部分调节了细胞胶原代谢。更多还原

【Abstract】 Background Keloid(KD) is benign scar of dermal origin that is the result of intense and abnormal fibroblastic response to trauma、burn and postoperation, particularly in areas of high skin tension. It grows beyond the margins of the original wound and tends to be susceptible to high rate of recurrence, persists without regression unlike normal scar. And how to treat on it is always a challenge.Connective tissue growth factor(CTGF),a important fibrosis factor, can accelerate pathologic scar formation by TGF/SMADs sigal pass.So to block down CTGF may be a pharmacecular target.Some reseaches indicate CTGF AS-ODN can inhibit proliferation of HKFs,expression of CTGFmRNA and the collagen secretion.RNA interference(RNAi) is the process of using specific sequences of dsRNA to knock down the expression levels of complementary genes. SiRNAs are loaded into RISC(RNA-induced silencing complex) and cleave the target mRNA. RNAi has been widely applied in the research of gene function and gene therapy as an efficient tool for specific gene silencing.Objective Vectors expressing CTGF siRNA were designed, constructed and transfected into HKFs. Their effects onCTGFmRNA level and collagen secretion were observed. And at last the mechanisms were researched in order to explore a new way for gene treatment on keloids. Methods Three 64nt oligonucleotides forming double strands after annealing were designed by three 21nt specific sequences of CTGF siRNA respectively. Then the double strands mentioned above were inserted into pGenesil-1 vector to construct recombined expression vectors which can express shRNA, named pGenesil -CTGF1,2,3. And a mixed sequence was named pGenesil-CTGF4 as control. Recombinant plasmids were verified by enzyme cut and sequencing analysis.To increase transfection efficiency, lipsome Dosper from Co.Roche was selected. The experiment included six groups: control , plasmids, pGenesil-CTGF1, pGenesil-CTGF2, pGenesil- CTGF3 and pGenesil-CTGF4. After transfection,every group except control was cultured in DMEM medium added with 350μg/ml G-418. About three weeks later, cell clones were obtained. Clones of each group were selected and transfered to fresh dish and then to culture bottles.The mRNA levels of CTGF were measured by The real-time quantitative PCR(RQ-PCR). The protein expression was determined by western blotting. The collagen secretion was assessed by 3H-proline incorporation method.HKFs and HKFs transfected by pGenesil-CTGF1 were stimulated by TGF-β1 with the dosage of 10ng/ml.Results 1.Vectors expressing CTGF siRNA were successfully constructed and proved to be correct by enzyme cut and sequencing.2.The CTGFmRNA expression levels of pGenesil-CTGF1, pGenesil-CTGF2 were significantly lower than that of control(59.9%、20.4%,both P<0.01).Both of them, pGenesil- CTGF1’s was lower. And CTGF protein and collagen secretion were 33.6% and 49.7% separately (P<0.05). We can conclude that Vectors expressing CTGF siRNA can suppress target gene and suppressive efficiency correlate with sequence of siRNA. The CTGFmRNA expression level of HKFs was higher as 2 times after instimulated by TGF-β1. But if transfected by pGenesil-CTGF1, the CTGFmRNA expression level decreased significantly(P<0.05).CTGF protein and collagen secretion also markedly reduced(P<0.05).Conclusions 1.CTGF protein and collagen secretion were markedly decreased after CTGFmRNA expression level was inhibited by RNAi. This indicated CTGF could be a candidate target for gene therapy.2. For CTGF RNAi, inhibiting effect varies for different target site on mRNA. 3. Contrasted to those in HKFs, the CTGFmRNA expression level、CTGF protein and collagen secretion in HKFs transfected by vector expressing siRNA were markedly decreased after stimulated by TGF-β1. This reveals TGF-β1 can partly adjust collagen secretion through CTGF.更多还原