| The ability to study the effects of mechanical stimuli in vitro is important for understanding how they contribute to both normal and pathologic states at present. With the enormous progress of study on mechanobiological conditioning of various kinds of cells (including osteocytes, intervertebral disc cell, vascular smooth muscle cell, mesangial cell and stem cells, etc), most accumulating evidences suggested that mechanical forces are one of the important factors that induce a variety of cellular responses including morphological changes, proliferation, differentiation, apoptosis, protein synthesis, and gene expression and which involve tissue remodelling. Hyperplastic scars (HS) occur following cutaneous wounding and result in severe functional and esthetic defects. However, current therapies for HS are ineffective because of the limited understanding into the pathogenesis of the underlying fibroproliferative process. At the cellular level, it is widely accepted that the histological composition of HS is consisted of a great number of fibroblasts with functional disorder, by which excess deposition of extracellular matrix. Therefore, hyperplastic scar fibroblasts become a focus on the field of research in prevention and cure of HS.Although the pressure therapy, one of commonly used treatment for early HS has been widely recognized in clinical practice, but how to definite the pressure size, therapic opportunity, duration and to understand the related molecular mechanism by which remains a problem. Most early investigations had been shown that mechanical pressure may act by interfering with blood supply on HS tissue, leading to the atrophy of HS due to ischemia or congestion. However, detailed knowledge is not available to assess the effects of biological features of hyperplastic scar fibroblasts under such a mechanical load at present, despite extensive studies. Thereby, in-depth studies on the proliferating, differentiating and apoptosis characteristics of hyperplastic scar fibroblasts under the influence of the pressure might be helpful to provide significant rationale during the clinical practice. According to the above views, in the present study, we have designed a new air controlled mechanical pressure system based upon the following design variables: minimal size, stability of pressure and humidity, maximal accessibility. To biologically validate our system, we tested the response of human hyperplastic scar fibroblasts (HHSFb) to an unremitting pressure stimulation of 5～150 mm Hg, for 2～8 days. Cellular morphology, proliferation and apoptosis were measured using multidisciplinary technique respectively. Meanwhile protein extracts were analyzed by Western blot for p38MAPK and Caspase-3. In addition, based on the above results, we investigated the dose-effect relationship and related molecule mechanisms of these cells respond to mechanical pressure by alterations in cellular viability.The major research contents and conclusions were as follows:1. HHSFb obtained from the patient who suffered from hyperplastic scar in our hospital were isolated and cultured in vitro, then they were randomly divided into 8 groups as follows: control group (no pressure) , continuous pressure groups with 5, 10, 15, 25, 50, 100, 150mmHg pressure for 4 days respectively. The inhibition ratio of cellular growth was determined by MTT assay, cellular cycle was detected on the flow cytometer, and the cellular apoptosis was measured with Annexin V binding with PI labeling methods. It was found that in group 5mmHg and 10mmHg pressure, there was not statistical significant to be found in cellular A value and rate of G1 stage compared with control group (P>0.05). However, in group 15, 25, 50, 100, 150mmHg pressure, the cellular A value was 0.213±0.005, 0.180±0.005, 0.172±0.007 and the rates of G1 stage were 74.56±1.01%, 82.82±2.76%, 86.77±2.06%, 88.23±1.27%, 89.11±1.74% respectively, while those in control group were 0.230±0.005, 71.46±0.49% and 4.00±0.36%, showing significant difference (P<0.05). Furthermore, among groups 10, 15, 25, 50 mmHg pressure, the effects of proliferation inhibition in HHSFb were presented the enhancing tendency with pressure increasing, but this phenomenon was not occured in groups 50, 100, 150mmHg pressure yet (P>0.05) . Cellular A value and ratio of G1 stage didn't enhance noticeable with pressure time. In group 5mmHg and 10mmHg pressure, there was not statistical significant to be found in cellular apoptosis compared with control group (P>0.05). However, in group 15, 25, 50, 100, 150mmHg pressure, cellular apoptosis rates were 8.58±0.79%, 19.28±1.40%, 25.60±1.21%, 35.80±2.39%, 36.18±2.38% respectively, while those in control group were 0.230±0.005, 71.46±0.49% and 4.00±0.36%, showing significant difference (P<0.05). Furthermore, among groups 10, 15, 25, 50, 100mmHg pressure, the effects of the apoptosis promotion in HHSFb were presented the enhancing tendency with pressure increasing, but this phenomenon was not occured in groups 100, 150mmHg pressure yet (P>0.05). These findings suggested that there were combined effects of proliferation inhibition and apoptosis promotion on HHSFb under the stimulus of over 10 mmHg continuous pressure.2. HHSFb were randomly divided into 3 groups as follows: control group (no pressure), continuous pressure group ( with 25 mmHg, for 6-8 days) and the cancelled load after pressure group. The cellular proliferation was determined by MTT assay, cell cycle was detected on the flow cytometer, and PCNA expression in HSFb was confirmed by immunohistochemistry method. The apoptosis of scar fibroblasts was measured with Annexin V binding with PI labeling. It was found that both the inhibiting rate of HHSFb growth and the apoptosis were markedly increased in continuous pressure group with pressure time. Compared with control group, the PCNA expression and cell ratio within proliferative phase(S+G2+M) was decreased. However, the inhibiting ratio of HHSFb growth, PCNA expression, cell ratio within proliferative phase(S+G2+M) and apoptosis ratio had gradually returned in the cancelled load after pressure group. These results suggested that combined effects of the proliferation inhibition and apoptosis promotion induced by continuous pressure in HHSFb may potentially be the important reason of well obtained therapeutic effect with pressure therapy for needing early stage and long time maintenance during the clinical practice.3. After exposing HHSFb to a steady continuous pressure of 25 mmHg for 12 hours, protein extracts were analyzed by Western blot for p-p38MAPK and Caspase-3. As control, these cells underwent sham pressure with identical technique but no stress. It was founded that p-p38MAPK and cleaved Caspase-3 expression enhance in HHSFb of continuous pressure compared with the control group. These findings suggested that combined effects of the proliferation inhibition and apoptosis promotion under the mechanical pressure stimuli may be via p38MAPK and Caspase-3 activation. |
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