特异抑制TIMP-1基因表达促进胶原降解的实验研究

To develop new vectors abundant expressing the U6 driven ribozymes anti-TIMP-1 mRNA in hypertrophic scar and keloid, study the cleavage activity of the ribozymes. Anti-TIMP-1 ribozyme genes named Rz182, Rz358, Rz412 and corresponding mutant ribozyme genes were designed and cloned into pBSKneorU6, a vector for abundant expression of ribozymes. TIMP-1 cDNA gene fragments were acquired by RT-PCR and cloned into T-vector. 32P-labeled TIMP-1 transcripts as targeted RNAs and 32P-labeled ribozyme transcripts were transcribed in vitro, incubated together at different conditions for cleavage reactions and autoradiographed after denaturing gel-electrophoresis. Both U6Rz182 ( Km=29.7 nmol/L，kcat=0.32min-1)and U6Rz358 （Km=39.6 nmol/L, Kcat=0.21 min-1）cleaved the targeted mRNA successfully at 37oC , while U6Rz412 and mutant ribozymes failed to cleave the targeted mRNA. The cleavage efficiencies (CE) of U6Rz182 and U6Rz358 were up to 49.23% and 55.21％at 37 oC. The designed ribozymes possessed perfect specific cleavage activity anti-TIMP-1 in vitro. The ribozymes ere cloned into pEGFPC1 and the vectors were introduced into fibroblasts derived from hypertrophic scar and selected by G418.In the ribozyme expressing cells, RT-PCR and westernblot showed that the TIMP-1 expression were significantly depressed, Westernblot analysis showed less collagen I/III in the cytoplasm.The growth of the Rz expressing fibroblasts were suppressed. The ribozymes were than introduced into hypertrophic scars in nude mouse by intradermal injection of the plsmid DNA. The ribozyme were expressed in vivo and depressed the TIMP-1 expession.

瘢痕过度增生源于胶原等细胞外基质的异常代谢和过度沉积。胶原酶活性的改变是导致胶原分解代谢异常的关键因素之一，而组织金属蛋白酶抑制因子TIMP-1能特异性抑制胶原酶活性。本课题运用生物新技术，探讨核酶牧民性抑制，间接提高胶原酶活性，从而达到促进胶原降解的可能性，为开辟控制胶原代谢，防治瘢痕过度增生的新途径提供理论依据。