Expression of Autocrine Motility Factor in Keloid
|School||Chongqing Medical University|
|Keywords||Keloid Autocrine motility factor Western blot Realtime PCR Tumor invasion related factors|
ObjectiveKeloid belongs to pathological scars and can be frequently seen in usual life. The biological characteristics of keloid are similar to malignant tumors. Autocrine motility factor (AMF) is a cytokine, secreted by tumor cells and over-expressed in malignant cells. After specificly bind to its receptor AMFR, AMF plays an important role in various aspects of tumor progression including neoplastic transformation, cell motility, tumorigenicity, angiogenesis and metastasis. The above biological effects are consistent with the biological characteristics of the keloid; which indicated that the AMF may have a special relationship with the pathogenesis of keloid. In our study, we intended to detect the AMF expression in keloid, hypertrophic scar and normal scar tissues, with the intention of providing an experimental evidence for possible mechanism of AMF in keloid.MethodsSixty cases which include normal scar (n=20), hypertrophic scar (n=20), keloid (n=20) were studied. The distribution and expression of AMF in tissues were assessed using immunofluorescen. Their protein level and mRNA level of AMF expressions were analyzed using western blotting and Real Time PCR. Immunohistochemical staining:the organization removed from the liquid nitrogen, frozen section, air-dried slices and fixed by acetone, PBS washed, blocked, dropping the first antibody and incubated at4℃overnight, washed several times by PBS, dropping fluorescent secondary antibody,37℃incubation and PBS washing, mounted by glycerol, observed under fluorescence microscope and taking photos, with PBS instead of primary antibody as negative control. Western blotting:extracting total protein,the concentration were measured, adding buffer and boiled, gel loading, electrophoresis, transferred to a membrane, blocked, and dropping the first antibody, incubating overnight at4℃washing by TBST, secondary antibody labeled HRP incubating at37℃for1h, washing by TBST, imaging, taking photos. Quantitative PCR: extracting total RNA, determination of total RNA concentration and purity, reverse transcription of total RNA, adding samples, knitting board, PCR reaction. Set the group of normal scar as the control group.The results for the three types of scar tissue is the relative expression of the AMF.ResultsThe immunohistochemical staining results:negative results showed no color. Positive results can be seen in three kinds of scar tissue from the epidermis to the deeper layer of cells widely colored (green). The epidennis mainly located in the cytoplasm of cells, and the deeper layer are widely distributed along the collagen fibers. Normal scars and hypertrophic scars were weakly colored. And the tissues of keloid were colored stronger than the other two. Western blotting results showed that the expression of AMF in the normal scar and hypertrophic scar was weak and no significant difference was found between them (P>0.05), while the expression in the keloid was significantly higher than the normal scars and hypertrophic scars (P<0.05). PCR results showed that AMF expression can be observed in three types of scar, and the mRNA of AMF in keloid was significantly higher than other two group(P<0.01).ConclusionAMF overexpression was found in human keloid, but not in normal scar and hypertrophic scar. This indicates that the AMF may play an important role in the progression of keloid.