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>

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>