Research advances on the mechanism of non-coding RNA regulated diabetic wound healing / 中华烧伤杂志

Diabetic wounds are a common complication of diabetic patients, and the incidence has been increasing in recent years. In addition, its poor clinical prognosis seriously affects the quality of life of patients, which has become the focus and difficulty of diabetes treatment. As the RNA regulating gene expression, non-coding RNA can regulate the pathophysiological process of diseases, and play an important role in the healing process of diabetic wounds. In this paper, we reviewed the regulatory role, diagnostic value, and therapeutic potential of three common non-coding RNA in diabetic wounds, in order to provide a new solution for the diagnosis and treatment of diabetic wounds at the genetic and molecular level.

Research advances on mesenchymal stem cell-derived extracellular vesicles in promoting angiogenesis of diabetic ulcers / 中华烧伤杂志

Extracellular vesicles are nanoparticles secreted by most eukaryotic cells and play important roles in material transport and information transmission between cells, involved in inflammation, angiogenesis, antigen presentation, cell apoptosis, cell differentiation, and other biological processes. The culture supernatant of mesenchymal stem cells is rich in extracellular vesicles, and the extracellular vesicles can regulate the formation of new blood vessels, a key step in wound healing and tissue repair. The persistence of diabetic ulcers is closely related to the blocked formation of wound vascular network. This article reviews the role of extracellular vesicles derived from mesenchymal stem cells in promoting angiogenesis of diabetic ulcers, in order to provide a new idea for the treatment of diabetic ulcers.

Effect of tetrandine on gene expression of collagen type I, collagen type III and TGF-beta1 in scar tissue's of rabbits ear / 中华整形外科杂志

<p><b>OBJECTIVE</b>To observe the effect of tetrandine on gene expression of collagen type I, collagen type III, transformation growth factor-beta1 and to investigate the inhibitory effect of tetrandine on the scar tissue hyperplasia in rabbits' ears.</p><p><b>METHODS</b>After the scar model was formed on the rabbits' ears, the rabbits were divided into 4 groups to receive intro-lesion injection with saline, or prednisolone (Pre) or tetrandrine in low concentration (L-Tet, 1.0 mg/ml) or tetrandrine in high concentration (H-Tet, 7.5 mg/ml). The morphological changes of scar tissue were observed. The changes of fibroblasts quantity and collagen expression were observed with HE and Masson staining. Immunohistochemical study was used to observe the expression level of collagen type I and collagen type III and TGF-beta1. Collagen type I and collagen type III and TGF-beta1, and signal factor Smad 3 mRNA were detected with RT-PCR.</p><p><b>RESULTS</b>(1) 24 days after injury, all the wounds healed completely with formation of red, tough and hypertrophic scar. HE and Masson staining showed significant increase of fibroblasts and collagen density with irregularly arrangement. (2) Compared with that in saline group, the scar in other groups became softer, lighter and thinner, especially in H-Tet group. (3) HE and Masson staining shows the scar in Tet and Pre groups contained less fibroblasts and lower collagen dentsity with comparatively regular arrangement than that in saline group (P < 0.01), especially in H-Tet group. (4) According to the immunohistochemical study, the expression of collage type I and III and TGF-beta was positive in all the groups, but the positive rate and the ratio of collagen density I to III decreased in the order of saline, L-Tet, H-Tet and Pre groups (P < 0.01). (5) PT-PCR detection results showed that the amplification bands brightness of collagen type I and III and TGF-beta1 and signal molecular Smad 3 mRNA in scar tissue were obviously different. Compared with that in saline group, the expression of collagen type I and III and TGF-beta1 and Smad 3 mRNA decreased in Tet and Pre groups (P < 0.01). H-Tet group showed the most obvious reduce in the expression of type I collagen and TGF-beta1 and Smad 3 mRNA. Conclusions Tetrandine can significantly suppress the expression of collagen type I and collagen type III and TGF-beta1 on hypertrophic scar of rabbit ears, and reduce signal factor Smad 3 mRNA' s expression. It may be one of the important mechanism for its inhibitory effect on scar hyperplasia.</p>

Advances in the research of the role of MicroRNAs in wound healing / 中华烧伤杂志

MicroRNAs are endogenous noncoding RNA molecules with 19-22 nucleotides in length. MicroRNAs can post-transcriptionally regulate gene and (or) protein expression by binding to their target messenger RNAs (mRNAs), leading to mRNA degradation or suppression of translation. As a huge family that regulates gene expression, microRNAs has recently been shown to not only participate in the normal healing processes of wounds but also closely related to pathologic wound healing, and formation of hypertrophic scars and keloids. This review focuses on the biogenesis of microRNA and its role in wound healing.

Effect of substance P combined with epidermal stem cells on wound healing and nerve regeneration in rats with diabetes mellitus / 中华烧伤杂志

<p><b>OBJECTIVE</b>To observe the effect of sensory neuropeptide substance P combined with epidermal stem cells (ESC) on wound healing and nerve regeneration in diabetic rats.</p><p><b>METHODS</b>ESC that had been isolated from SD rats were identified and cultured in vitro, and they were inoculated onto nourishing layer of amniotic membrane to construct amniotic membrane-ESC. Four full-thickness skin wounds were produced on the back of each of 48 diabetic rats. The resulted 192 wounds were randomly divided into ESC + substance P group, ESC group, substance P group, and control group according to the lottery method, with 48 wounds in each group. Wounds in ESC + substance P group and ESC group were transplanted with amniotic membrane-ESC, and those in substance P group and control group were transplanted with amniotic membrane. After transplantation, 250 µL substance P in the concentration of 1 × 10(-7) mol/L was injected around and into the middle of the wounds in ESC + substance P group and substance P group, 2 times a day, and continued for 4 days, while 250 µL PBS solution was injected in the above-mentioned position in ESC group and control group as control, 2 times a day, and continued for 4 days. On post injury day (PID) 4, 7, 10, 14, 17, and 23, the wound healing rate (with 8 wounds at each time point) was observed and determined, and changes in wound tissue structure were observed with HE staining. On PID 4, 7, and 10, collagen distribution in wound tissue was observed with Masson staining, and type I and type III collagen deposition in wound tissue was respectively observed after immunohistochemical staining. The distribution of protein gene product 9.5 (PGP 9.5) and regeneration of substance P positive nerve fibers in wound tissue were observed with immunohistochemical staining on PID 14 and 23. Data were processed with one-way analysis of variance and t test.</p><p><b>RESULTS</b>(1) The wound healing rate in ESC + substance P group reached 100.0% on PID 14, which was obviously earlier than that in ESC group, substance P group, and control group, healing was respectively observed on PID 17, 17, and 23. The wound healing quality in ESC + substance P group was better than that in the other three groups as shown by HE staining. (2) On PID 10, collagen that was darkly stained and widely distributed was observed in wound tissue of ESC + substance P group and substance P group, while collagen in the other two groups was lightly stained and narrowly distributed. Deposition quantity of type I collagen gradually increased, and that of type III collagen gradually decreased in the wounds of each group over time. On PID 4, 7, and 10, distribution amount of type I collagen in wound tissue of ESC + substance P group was significantly higher than that in ESC group (with t value respectively 32.72, 118.21, 26.71, P values all below 0.01) and control group (with t value respectively 44.37, 22.76, 30.32, P values all below 0.01), while there was no significance between ESC + substance P group and substance P group. On PID 4, 7, and 10, distribution amount of type III collagen in wound tissue of ESC + substance P group was significantly higher than that in ESC group (with t value respectively 32.27, 28.68, 14.51, P values all below 0.01) and control group (with t value respectively 35.68, 22.52, 22.24, P values all below 0.01). (3) A large amount of PGP 9.5 and regeneration of substance P positive nerve fibers, and some peripheral nerve fibers in deep wound extending to epidermis were observed in wound tissue of ESC + substance P group and substance P group. A small amount of PGP 9.5 and regeneration of substance P positive nerve fibers without peripheral nerve fibers extending to epidermis were observed in deep wound tissue of ESC group and control group. On PID 14, 23, ratios of area of PGP 9.5 positive nerve fiber in the wounds of ESC + substance P group were (3.86 ± 0.25)% and (7.03 ± 0.28)%, and they were significantly higher than those of ESC group [(1.48 ± 0.30)%, (3.01 ± 0.43)%, with t value respectively 23.95, 30.27, P values all below 0.01] and control group [(1.46 ± 0.23)%, (2.84 ± 0.29)%, with t value respectively 27.35, 40.32, P values all below 0.01]. On PID 14, 23, ratios of substance P positive nerve fiber area in the wounds of ESC + substance P group were (2.01 ± 0.14)% and (1.19 ± 0.11)%, which were obviously higher than those of ESC group [(0.85 ± 0.17)%, (1.34 ± 0.21)%, with t value respectively 20.50, 2.60, P < 0.05 or P < 0.01] and control group [(0.74 ± 0.15)%, (1.30 ± 0.17)%, with t value respectively 23.98, 2.41, P < 0.05 or P < 0.01].</p><p><b>CONCLUSIONS</b>Joint application of substance P and ESC can effectively promote healing of wound and nerve regeneration in diabetic rats.</p>

Experimental study on the differentiation of human induced pluripotent stem cells into epidermal-like stem cells / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of differentiation of human induced pluripotent stem cells (iPSCs) into epidermal-like stem cells.</p><p><b>METHODS</b>(1) Human strain of iPSCs were plated on-to trophoblast of inactivated Fb strain of mouse embryos and cultured in complete medium of embryonic stem cells, iPSCs were subcultured by collagenase IV digestion method. The morphology and growth of iPSCs were observed under inverted phase contrast microscope, and the cells were stained with alkaline phosphatase (AKP). iPSCs were cultured in incomplete medium of embryonic stem cells to observe the ability of embryoid body formation. (2) Human iPSCs were inoculated onto 6-well plate covered with human amniotic membrane to culture as induction group. Other iPSCs were cultured on 6-well plate without human amniotic membrane as control group. Morphological changes in iPSCs in two groups were observed. Expressions of integrin beta1 and CK19 of iPSCs in two groups were determined by immunocytochemical staining.</p><p><b>RESULTS</b>Human iPSCs showed a typical stem cell clone-like growth with a clear boundary, and they proliferated vigorously in complete medium of embryonic stem cells. These cells were AKP-positive. iPSCs formed embryoid body in trophoblast-free and suspension culture conditions. After 4 days of co-culture, stem cell clones were formed on the surface of amniotic membrane in induction group, and part of the cells were integrin beta1 and CK19 positive. Most of the cells died, and no integrin beta1 and CK19 positive cells were found in control group.</p><p><b>CONCLUSIONS</b>Human iPSCs can be differentiated into epidermal-like stem cells by amniotic membrane induction, and it lays an experimental basis for providing new source of seed cells of skin tissue engineering.</p>

Screening of differential expression genes of human skin epidermal stem cells at different development stages by cDNA microarray technique / 中华烧伤杂志

<p><b>OBJECTIVE</b>To analyze expression characteristics of human skin epidermal stem cell at different developmental stages, and to explore its biological significance.</p><p><b>METHODS</b>Health skin samples from 28-32 w fetuses (F group), 4-12 y children (C group), and 35-55 y adult (A group) were harvested, with 10 cases in each group. Epidermis were separated using trypsin digestion and EDTA, and human epidermal stem cells were isolated and purified with type IV collagen attachment method. The monoclonal antibody of integrin beta1 and keratin 19 were used for detection and identification of epidermal stem cells by immunohistochemical staining. Total RNA was extracted from above cells by Trizol one-step method, and were detected by formaldehyde denaturing agarose gel electrophoresis. Probes were prepared and hybridized into cDNA microarray for scanning fluorescent signals and analysis of images, with two-fold differential expression value for screening. Significantly up/down-regulated genes were selected for verification by real time RT-PCR.</p><p><b>RESULTS</b>By comparing expression profile between A and C groups, a total of 1808 genes with differential expression were detected, including 1089 up-regulated genes and 719 down-regulated genes, and they were classified into 128 categories. Among them, 1462 genes were known (found in GeneBank), 346 genes were unknown. A total of 4534 genes with differential expression were detected between C and F groups, in which 1783 genes were up-regulated and 2751 genes were down-regulated, and they were classified into 216 categories. Among them, 3577 genes were known (found in GeneBank), and 957 genes were unknown. There were 1104 genes with differential expression consistently detected in F, C and A groups, which were classified into 32 categories according to gene function. Among them, 94 genes were consistently up-regulated and 75 genes consistently down-regulated. Test results of real time RT-PCR were in accordance with above-mentioned results.</p><p><b>CONCLUSIONS</b>Gene expression profiles of epidermal stem cells cultured in vitro, harvested from fetuses, children, and adult, exhibit obvious difference. This may be closely related to different stages of proliferation and differentiation of human epidermal stem cell and self-repair ability of wound at different developmental stages.</p>

Experimental study on the transfection of human vascular endothelial growth factor 165 gene into human marrow mesenchymal stem cells / 中华烧伤杂志

<p><b>OBJECTIVE</b>To establish an effective method of transfecting human marrow mesenchymal stem cells (MSC) with human vascular endothelial growth factor 165 (VEGF 165) gene.</p><p><b>METHODS</b>MSCs isolated and cultured in vitro were divided into transfection group (pShuttle-CMV/VEGF 165 plasmid was transfected into MSCs through liposome-mediating method), empty plasmid group (pShuttle-CMV vehicle was transfected into MSCs as control), liposome group (liposome was transfected into MSCs as control) and control group (normal culture). Expressions of mRNA and protein of MSCs were determined by RT-PCR, enzyme-linked immunosorbent assay and Western Blot. Sensitivity to MSCs on VEGF plasmid transfection was detected by MTT test.</p><p><b>RESULTS</b>Expression level of VEGF 165 gene mRNA in transfection group, empty plasmid group, liposome group, and control group was respectively 0.89 +/- 0.03, 0.34 +/- 0.04, 0.40 +/- 0.03, and 0.30 +/- 0.03, and the difference between transfection group and the other three groups was statistically significant (P < 0.01). Content of VEGF protein in transfection group, empty plasmid group, liposome group, and control group was respectively (778 +/- 35), (543 +/- 24), (561 +/- 28), (571 +/- 23) pg/mL, and the difference between transfection group and the other three groups was statistically significant (P < 0.01). In the transfection group, expression level of VEGF protein peaked on 7(th) day after transfection, which was decreased gradually later. In transfection group, expression level of VEGF 165 protein was obviously higher than that of the other three groups (P < 0.01), and no inhibitory effect of VEGF plasmid transfection on MSCs proliferation was found.</p><p><b>CONCLUSIONS</b>The method for transfecting human VEGF 165 gene into MSCs is established in this research, through which target gene and protein can express effectively.</p>

Effect of transfection of recombinant human endothelial nitric oxide synthase gene on hypertrophic scar fibroblasts in vitro / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of transfection of recombinant human endothelial nitric oxide synthase (eNOS) into human hypertrophic scar fibroblasts (HSFbs), and to observe NO secretion and the synthesis of collagen I and III.</p><p><b>METHODS</b>Recombinant human eNOS with karyocyte expressive vector was constructed in vitro, then was transfected into HSFbs which was isolated from hypertrophic scar tissues and cultured in vitro (T group). The HSFbs untransfected (normal culture) or transfected with empty-vector was used as control group and empty-vector group respectively. The mRNA expression of eNOS, collagen I and III was determined by Realtime PCR. The content of NO was determined by NO assay kit.</p><p><b>RESULTS</b>The expression of eNOS mRNA in T group was 5.92 +/- 0.21, which was obviously higher than that in empty-vector group (0.98 +/- 0.13, P < 0.05). The expression of collagen I mRNA (0.76 +/- 0.15), and collagen III (0.79 +/- 0.08) in T group was significantly lower than those in empty-vector group (0.98 +/- 0.15, 1.02 +/- 0.12, P < 0.05, respectively). The content of NO in T group (36.1 +/- 0.8 micromol/L) was obviously higher than that in empty-vector group (28.4 +/- 1.0 micromol/L, P < 0.01) and control group (27.7 +/- 1.3 micromol/L, P < 0.01).</p><p><b>CONCLUSION</b>HSFbs can be the target cells for eNOS gene transfection. The transfected cells can express eNOS and produce NO, which inhibit the synthesis of collagen.</p>

Observation of the effect of early fluid resuscitation on hepatic fatty degeneration in rats after severe scald / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the efficacy of early fluid resuscitation on hepatic steatosis in rats after severe scald.</p><p><b>METHODS</b>One hundred and forty-four Sprague-Dawley rats were enrolled in the study. In thirty-six rats skin of 30% TBSA was treated with cold water to serve as sham injury group. All other rats were inflicted with 30% full-thickness scald, and they were subdivided into 3 groups, i. e. scald group(S, without resuscitation), delayed resuscitation group ( DR, with Ringer's solution at 6 post-scald hour(PSH) ) and early resuscitation group( ER, with Ringer's solution immediately after scald). The hepatic tissues of the rats were harvested at 0.5, 1.0,2.0,3.0,7.0 post-scald hour( PSH) and on 21.0 PSD for the observation of pathological changes with light-microscope and transmission electron microscope. The serum contents of TC, TG, HDL, ALP were determined at the same time-points. Body weight of each rat was measured before blood sampling, and total liver weight after blood sampling. Liver weight/body weight ratio was recorded.</p><p><b>RESULTS</b>Compared with sham injury group, the fat denaturation degree of hepatic tissue in ER group was obviously less than that in S and DR group . The serum level of high density lipoprotein (TC) , triglyceride ( TG) , and alkaline phosphatase (ALP) after scald increased ranking as S > DR > ER, while the level of HDL decreased in that order. The liver weight/body weight ratio of the rats in DR group on 1.0 PSD was obviously elevated compared with that in ER group( P <0. 05) , and there exhibited significant difference of liver weight/body weight ratio between DR and ER groups on 7. 0 PSD ( P < 0. 01). The liver steatosis had obvious negative correlation with HDL content after scald( r = -0. 37, P <0.01) , but it had positive correlation with the ALP content( r = 0. 45, P <0. 01), TG content( r = 0. 25, P <0. 01) and liver weight/body weight ratio( r = 0. 440, P <0. 01). The remaining parameters showed no correlation with the liver steatosis.</p><p><b>CONCLUSION</b>Fluid resuscitation immediately after scald can ameliorate hepatic fatty degeneration, reduce its incidence, and beneficial to recovery of liver damage to a certain extent.</p>

Experimental study on effects of endothelin in the proliferation and collagen synthesis of human scar-derived fibroblasts / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the role of endothelin (ET) in the proliferation and collagen synthesis of human scar-derived fibroblasts and the modulation of its antagonists such as nitric oxide (NO), tetrandrine (Tet).</p><p><b>METHODS</b>With the cultured fibroblasts from the scarring tissue, the cell proliferation was determined by [3H]-TdR incorporation, while the collagen synthesis was evaluated by [3H]-proline incorporation.</p><p><b>RESULTS</b>The ET-1 was significantly increasing the proliferation and collagen synthesis of human scar-derived fibroblasts. The values of [3H]-TdR absorption in the 2.5 ng/ml, 25 ng/ml and 100 ng/ml of ET-1 groups were 1.8 times, 4 times and 4.9 times more than in the control group, respectively (P < 0.01), while the values of the [3H]-proline incorporation were 1.1 times, 3.1 times and 3.8 times respectively (P < 0.01). The fibroblasts, treated with 50 micrograms/ml of S-nitroso-N-acetyl penicillamine(SNAP), were no detectable effect on the basal level of DNA synthesis, but produced decreasing effect on the [3H]-TdR absorption (the rate of inhibition was 22.89%, P < 0.05). It was found that the SNAP inhibited the [3H]-proline incorporation in cultured fibroblasts, but the rate of [3H]-proline incorporation induced by ET-1 was unaltered. The Tet with 3 micrograms/ml, in which does not inhibit the basal level of DNA synthesis, was significantly decreasing the collagen synthesis and decreasing the ET-mediated DNA synthesis (the rate of inhibition was 33.21% (P < 0.01).</p><p><b>CONCLUSION</b>These results indicate that the ET can obviously increase the proliferation and collagen synthesis of human scar-derived fibroblasts, but it can be partially antagonized by NO and Tet.</p>