Downregulation of human type III collagen gene expression by antisense oligodeoxynucleotide.
The amount of type III collagen is increased during the early healing stage following ligament and tendon injury. Concomitantly, the mechanical properties of the healing tissues are abnormal and the fibril diameters are homogeneously small. It is therefore thought that downregulating type III collagen gene expression after injury may be helpful in improving the quality of healing tissue. In the current study, the efficacy of using antisense oligodeoxynucleotides (ODNs) to downregulate type III collagen gene expression in human patellar tendon fibroblasts (HPTFs) was tested, with Lipofectamine reagent used to deliver the ODN. It was shown that the majority of HPTFs can efficiently uptake antisense ODN from as early as 1 h to as long as 3 days after delivery; also, one selected ODN can consistently inhibit human type III collagen gene expression at both the mRNA and protein levels. Reverse transcriptase-polymerase chain reaction results showed that the inhibitory effects by this ODN were significant at 1 day, as the type III collagen mRNA level was 38.9 +/- 19.6 and 42.8 +/- 28.1% of missense and sense controls, respectively (p < 0.05). At 3 days, these differences could no longer be observed (p >0.05), but the amount of type III collagen protein was significantly less than for missense and sense controls (31.7 +/- 5.5 and 25.3 +/- 5.3%, respectively; p < 0.05). At 5 days after the delivery, these differences in protein were no longer observed (p > 0.05). Immunohistochemical staining of the type III collagen confirmed these results. The findings of this study demonstrate that antisense ODN can downregulate type III collagen gene expression of tendon fibroblasts. Therefore, this approach offers the potential to explore the effect of the reduction of type III collagen in healing ligaments and tendons as a means to improve their mechanical properties.
Jia
Fengyan
F
Musculoskeletal Research Center, Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania 15219, USA.
Shimomura
Takatoshi
T
Niyibizi
Christopher
C
Woo
Savio L-Y
SL
D015153:Blotting, Western; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D024061:Collagen Type III / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D005240:Feasibility Studies; D005347:Fibroblasts / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology*; D017847:Patellar Ligament / Q000166:cytology / Q000378:metabolism; D011347:Procollagen / Q000096:biosynthesis / Q000235:genetics; D011506:Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D024061:Collagen Type III; D016376:Oligonucleotides, Antisense; D011347:Procollagen; D011506:Proteins; D012333:RNA, Messenger