| 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.
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