Bcl-2反义寡核苷酸诱导增生性瘢痕细胞凋亡的研究

目的：探讨反义寡核苷酸(ASODN)对bcl-2基因的表达调控及诱导增生性瘢痕成纤维细胞凋亡的作用。方法：采用RT-PCR法和流式细胞术检测ASODN对bcl-2蛋白和mRNA表达量的调控；通过MTT法比较两条人工合成ASODN直接作用及脂质体D0’FAt，转染对增生性瘢痕成纤维细胞的抑制效果；用相差显微镜、电子显微镜观察bcl-2ASODN诱导增生性瘢痕成纤维细胞凋亡效果。结果：ASODN降低bcl-2蛋白和mRNA表达；两条ASODN抑制增生性瘢痕成纤维细胞增殖的作用呈浓度和时间依赖性，且靶位点于mRNA蛋白编码区(ASODN2)和以DOTAP为载体的ASODN对成纤维细胞的增殖抑制效果最佳。Bcl-2ASODN作用于成纤维细胞后，成纤维细胞缩小、凋亡小体和染色质浓缩等特征性改变出现。结论：bcl-2ASODN能抑制bcl-2mRNA和蛋白表达，诱导成纤维细胞凋亡。

Apoptosis of hypertrophic scar fibroblasts induced by bcl -2 antisense oligodeoxynucleotides

Objective: To investigate the regulation bcl -2 gene expression and induction of apoptosis by bcl -2 antisense oligodeoxynucleotides(ASODN) on hypertrophic scar fibroblasts in vitro. Methods: The expression of bcl -2 at mRNA and protein levels was detected by RT -PCR and flow cytometry. Two bcl -2 ASODNs were synthesized, one covering the initiation sequence of translation of bcl -2 mRNA(ASODN1) and the other covering the protein coding region(ASODN2). Fibroblasts in logarithmic phase of growth were cultured in the presence of free or liposome(DOTAP) -encapsulated ASODN; cell growth was assessed by MTT method. Apoptosis changes were observed by electron microscopy and flow cytometry in ASODN treated cells. Results: Both ASODNs reduced bcl -2 expression of fibroblasts at mRNA and protein levels, both ASODNs inhibited proliferation of fibroblasts. The inhibitory activity of ASODN2 was stronger than that of ASODN1, ASODNs encapsulated in liposome led to more marked inhibition of cell growth than free ASODNs. Apoptosis of fibroblasts were the appearance of apoptotic bodies, chromatin condensation. Conclusion: Antisense oligodeoxynucleotide of bcl-2 decreases bcl -2 gene expression and induces apoptosis of hypertrophic scar fibroblasts in vitro.