Experiaiental-research On Human Hypertrophic Scar Fibroblasts With RNAi-PTN

Pathologic scar is one of the most difficult clinical problems during plastic surgerydue to lacking of effective treatment. The formation mechanism of Hypertrophic scarhowever is not very clear. Generally speaking, it is caused by the abnormal proliferation offibroblast, making large number of collagen deposited in the extracellular matrix.Therefore, it is useful to study the expression of factors associating with proliferation offibroblast in elucidating the formation mechanism of hypertrophic scar and meaningful toexplore new methods on clinical treatment to hypertrophic scar.Pleiotrophin (PTN) is a heparin-binding protein which was purified from the perinatalrat brain tissues by Milner in1989. Some studies indicated that PTN was also present inseveral non-neural tissues, such as uterine, heart, cartilage tissues. The main physiologicalfunctions are simulating the cell proliferation, migration, angiogenesis and promoting thedevelopment of bone and nervous system. Recently, Paddock found that PTN had a highexpression in the hypertrophic scar by Microarray profiling expression technology. But theeffect on the proliferation and apoptosis of fibroblast cells is still unclear.Based on the results of these studies above, this study is to further validate whetherPTN had an expression difference between normal skins and hypertrophic scar tissues. Inaddition, fibroblast cells of scars were cultured in vitro and then down–regulated theexpression of PTN by RNA interference technology. At last, we detected the influences ofthe low expression of PTN on the proliferation and apoptosis of fibroblast cells, and makesure whether PTN was participate in the formation of Hypertrophic scars. Part I experiment: Pathological Detection of ClinicalSamplesObjective：The formation mechanism of Hypertrophic scar is not very clear. Generallyspeaking, it is caused by the joint efforts of the abnormal proliferation of fibroblast anddeposition of collagen in the extracellular matrix. PTN is a highly conserved extracellularmatrix secretion protein, and has multiple biological functions as an oncogene. The aim ofthis study was to discuss whether PTN has an effect on the formation of hypertrophic scarbased on the different PTN expression between normal skin tissues and hypertrophic scars.Methods：Patients with hypertrophic scars after surgery operation were randomlyselected, normal skin tissues were corresponded as control, HE and Masson staining wereused to observe the changes of morphology, RT-PCR, Western Blot andimmunohistochemistry were used to evaluate the expression and location of PTN in thehypertrophic scars.Results：RT-PCR and Western Blot results showed that the expression of PTN in thehypertrophic scars were more higher than that in normal skin tissues, and had a significantdifference. The immunohistochemistry results showed that PTN expressed both in thedermal and epidermal cells, and had a positive expression in hypertrophic scars.Conclusion：According to the results, we could infer that PTN may participate in theformation of hypertrophic scars. As it showed in the immunohistochemistry and stainingresults, PTN is likely to take part in the abnormal proliferation of fibroblast and depositionof collagen in the extracellular matrix. PTN could be expected as the therapeutic targets ofhyperplastic scar during the treatment. Part II experiment PTN siRNA recombinant plasmidconstruction, preparation, identification and screening ofinterfering fragmentsObjective：To screen the possible interfering targets of PTN, constructing andpreparing the recombinant plasmid of PTN. Fibroblast cells of scars were separated andcultured in vitro, then transfected the siRNA recombinant plasmid of PTN into the cells.The one had the most maximum efficiency was selected to do following research.Methods：According to the designing principles of siRNA, selected the specialinterfering targets to design and construct the recombinant plasmid (verification bysequencing), then transfected into E. coli DH5α for culture. High purity recombinantplasmid was extracted by endotoxin-free plasmid extracting kit. Liposome method wasused to transfect the fibroblast cells. RT-PCR and Western Blot were used to test theexpression of PTN, choosing the most maximum interfering efficiency one to do followingcell research.Results：Recombinant plasmid was successful constructed by sequencing verification.The extracted concentration of plasmid is enough for transfecting. PTN-sh3was conferredto had inhibition rate of70%to the expression of PTN in the experiment, which wasselected to do the following cell research..Conclusion：PTN siRNA recombinant plasmid was successfully constructed in vitro,and the concentration and purity of the plasmid were enough for transfecting. Theinterfering effect of siRNA is consistent with expected and could be used for the followingresearch. Part III experiment Effects of Down-regulation of PTNexpression by RNAi on hypertrophic scar fibroblastsObjective：Transfect the screening recombinant plasmid into the fibroblast cells fromscars. To analysis the effect of down-regulated expression of PTN on fibroblastproliferation and the relationship with other associated genes at the level of cell, molecularand protein.Methods：Evaluate the effect of down-regulated expression of PTN on cell growth byMTT, cell cycle and apoptosis experiment. Detect the interfering efficiency of siRNA tothe target gene and the changes of expression of ColⅠand ColⅢ by RT-PCR and WesternBlot.Results：The selected recombinant plasmid by detection of RT-PCR and Western Blotcould inhibit the expression of PTN effectively, as well as the growth of fibroblast cellscomparing to the cell without transfection (P＜0.05). The results of RT-PCR and WesternBlot showed that the expression of ColⅠand ColⅢ had a significant decrease after theexpression of PTN being down regulated.Conclusion：As a secreted growth factor, PTN had a higher expression in variouskinds of cancers. It could stimulate cell proliferation, migration and angiogenesis. Scarfibroblasts tansfected by PTN siRNA recombinant plasmid had a low expression of PTN,and cell proliferation was inhibited as well as the expression of ColⅠand ColⅢ. Theresults indicated that the PTN may be a candidate target for inhibiting the formation ofhypertrophic scars, and provide theoretical basis of gene therapy.