BACKGROUND:Diabetic foot ulcers threaten the patients’ health and even survival seriously. It is an international difficult problem and lacks an effective treatment. But gene therapy and stem celltherapy possess special advantages and potential in wound healing. OBJECTIVE:To assess the therapeutic effect of transplantation of bone marrow mesenchymal stem cells transfected by human vascular endothelial growth factor 165 (hVEGF165) gene on foot wound healing in diabetic rats. METHODS:Recombinant adenovirus was established in vitro which expressed hVEGF165 gene and transfected into the third generation of bone marrow mesenchymal stem cells. Total y 120 male Wistar rats were divided into five groups:group A (non-diabetic controls), group B (diabetic controls), group C (Ad-hVEGF165 therapy), group D (stem celltherapy) and group E (transplantation of bone marrow mesenchymal stem cells transfected by Ad-hVEGF165 gene). Rats in the latter four groups were intraperitoneal y injected with streptozotocin to induce diabetic models. In al rats, a 3 mm×7 mm rectangular ful-thickness skin sample was cut from the instep of the hind foot to make a model of foot wound. The rats were subcutaneously injected at equidistant six points 5 mm distal to the wound edge on the dorsum of the foot:50μL PBS per point for group A, 50μL adenovirus suspension (1×1013 pfu/L) per point for group C, 50μL stem cellsuspension (1×1010/L) per point for group D, and 50μL adenovirus suspension+50μL stem cellsuspension per point for group E. RESULTS AND CONCLUSION:After injection, the rate of wound healing, the expression of VEGF and the qualities of capil aries in group E were higher when compared with groups B, C, D (P<0.05), but were lower than those in group A (P<0.05). Transplantation of bone marrow mesenchymal stem cells transfected by hVEGF165 gene can promote foot wound healing, angiogenesis and expression of VEGF in diabetic rats.

Objective To analyze the expression feature and function of microRNAs in exosomes secreted by leukemia cells (LCEX). Methods The mice leukemia cell line L1210 was taken as the example, and LCEXL1210 was obtained by isolating supernate of L1210 cells through density gradient centrifugation. MicroRNAs isolated from LCEXL1210 were analyzed by microarray analysis, compared with miRNA from L1210 cell line, and then some of miRNAs with different expression were verified by real-time PCR and were analyzed by Gene Ontology (GO) database. Results The number of miRNAs identified in LCEXL1210 was 1 044, and that in L1210 cell line was 872. The number of shared miRNAs between LCEXL1210 and L1210 cell line was 732, accounting for 70.1 % of LCEXL1210 and 83.9 % of L1210 cell line, respectively, which indicated that 70 % of LCEXL1210 was derived from the parental cells. Interestingly, 312 miRNAs in LCEXL1210 were found to be underrepresented in the parental cells, indicating their specificity in LCEXL1210. Some miRNAs were significantly highly expressed in LCEXL1210 compared with those in L1210 cell line, including miR-16-1, miR-210, miR-195 and so on, which showed that miRNAs isolated from LCEXL1210 were differentially expressed with those from the parental cells. Some differentially expressed miRNAs from LCEXL1210 were verified by real-time PCR, and then were analyzed by GO database, which demonstrated that these highly expressed miRNAs participated in the processes of various biological function and signal transduction. Conclusions MiRNAs isolated from LCEXL1210 show a high similarity to miRNAs isolated from L1210 cells, whereas of which one-third are specific. The highly expressed miRNAs participate in the processes of various biological function and signal transduction.

Objective: To investigate the morphological and pathological changes of the larynx after severe laryngeal burn in dogs and their relationship with laryngostenosis. Methods: Eighteen healthy, male beagle dogs were assigned into control group, immediately after injury group, and 2, 4, 6, and 8 weeks after injury groups according to the random number table, with 3 dogs in each group. Dogs of injury group inhaled saturated steam through mouth for 5 seconds to reproduce severe laryngeal burn. Tracheotomy and intubation were performed immediately after injury, and 400 000 U/d penicillin was intravenously infused for 1 week. The feeding, activity, and vocalization of dogs in each group after injury were observed until they were sacrificed. Immediately after injury and 2, 4, 6, and 8 weeks after injury, the laryngeal morphology of the dogs in corresponding time point groups were observed by endoscope. After the observation, the dogs in each injury group were sacrificed, and the laryngeal tissue was taken. The epiglottis, glottis, and cricoid cartilage were collected to make full-thickness tissue slice, respectively, and their pathological changes were observed with hematoxylin and eosin staining. The dogs of control group were not specially treated, and their life activities, laryngeal morphological and pathological changes were observed. Results: (1) The dogs of control group had normal feeding, activities, and vocalization. All the dogs in injury group survived until they were sacrificed, and their feeding, activities, and vocalization were obviously reduced after injury compared with those of control group. The dogs of 2, 4, 6 and 8 weeks after injury groups ate and moved normally 2 weeks after injury but vocalized abnormally in frequency and volume compared with those of control group, which lasted until they were sacrificed. (2) The dog′s laryngeal mucosa in control group was complete and pink, without obvious exudation. The laryngeal mucosa of the dog in immediately after injury group was pale and edematous, with obvious exudation, local ulceration, necrosis, and exfoliation, and dilated microvessels on the surface. The laryngeal mucosa of the dogs in 2 weeks after injury group was pale, edematous, and oozed less than that of immediately after injury group, and the glottis was blocked by an obviously extruding mass. The paleness and edema of laryngeal mucosa were significantly reduced in the dogs of 4 weeks after injury group compared with those of 2 weeks after injury group, without dilated microvessel, and the glottic extruding mass was obviously smaller than that of 2 weeks after injury group. The sizes of glottic mass were similar between the dogs of 6 and 8 weeks after injury groups, which were obviously smaller than that in 4 weeks after injury group. (3) In the dogs of control group, the epithelial cells of epiglottis, glottis, and cricoid cartilage were normal in morphology, the proper glands were visible in the intrinsic layer, and the muscle fibers and the chondrocytes were normal in morphology. In the dogs of immediately after injury group, large sheets of epiglottis epidermis exfoliated, the epithelial cells were swollen and necrotic, the intrinsic glands were atrophic and necrotic, and the chondrocytes were degenerated and necrotic. The epidermis of the glottis partially exfoliated, the epithelial cells were swollen and necrotic, the intrinsic glands were atrophic and necrotic, the muscle fibers were partially atrophic and fractured, and the vacuolar chondrocytes were visible. The cricoid cartilage epidermis was ablated, the epithelial cells were swollen, the intrinsic layer and submucosal layer were slightly edematous, and the morphological structure of glands, chondrocytes, and muscle fibers were normal. In the dogs of 2 weeks after injury group, the epiglottis epidermis was completely restored, a small amount of glands in the intrinsic layer were repaired, and obsolete necrotic chondrocytes and new chondrocytes could be seen. A large number of fibroblasts, new capillaries, and inflammatory cells infiltration were observed in the epidermis of glottis, and intrinsic layer glands were repaired. The cricoid cartilage epidermis was repaired intactly, and there was no edema in the intrinsic layer. In the dogs of 4 weeks after injury group, the epiglottis intrinsic layer glands were further repaired compared with those of 2 weeks after injury group, and new chondrocytes were seen in the submucosa of the glottis. The condition of cricoid cartilage was consistent with that of control group. The dog′s epiglottis, glottis, and cricoid cartilage were similar between the 6 and 8 weeks after injury groups, and no significant change was observed compared with those of 4 weeks after injury group. Conclusions The morphological changes of larynx after severe laryngeal burn in dogs include mucosa detachment and necrosis, and mass blocking glottis. Pathological changes include epidermis shedding and necrosis, gland atrophy and necrosis, vascular congestion and embolism, chondrocytes degeneration, necrosis and proliferation, even local granulation tissue formation and cartilaginous metaplasia. These results may be the cause of laryngostenosis after laryngeal burn.

OBJECTIVE: To assess the therapeutic effect of treating diabetic foot ulcers (DFUs) in rats by subcutaneously transplanting around the wounds and intramuscularly into the leg with bone marrow derived mesenchymal stem cells (BM-MSCs). METHODS: BM-MSCs from male Wistar rats were cultured by the whole bone marrow adherence method until the third generation. The BM-MSCs were labeled by 4,6-diamino-2-phenylindole (DAPI) in vitro. Forty-eight male Wistar rats were randomly divided into 4 groups: group A (n=12), rats with DFUs receiving BM-MSCs subcutaneous transplantation; group B (n=12), rats with DFUs receiving BM-MSCs intramuscular transplantation; group C (n=12), nondiabetic rats with foot ulcers; and group D (n=12), rats with DFUs receiving no BM-MSCs. A diabetic rat model was induced by intraperitoneally injecting streptozotocin (STZ). Then DFU model was established by removing a 3 mm × 7 mm rectangular full-thickness skin on the 2 back dorsum pedis surfaces. On day 2, 5, 8 and 11 after the transplantation, the rate of wound closure was raised; trace of DAPI labeled-BM-MSCs in the wound tissues was observed on frozen sections and the thickness of granulation tissues was detected by HE stain. Immunohistochemistry was performed to detect the expression of CD31 and Ki-67. The expression of vascular endothelial growth factor (VEGF) in the wound tissues was detected by ELISA and RT-PCR. RESULTS: The wound closure in group C was faster than in other groups (P<0.05). The rate of wound healing in group B was higher than group A on day 11 (P<0.05). The intensity and area of the fluorescence in group B were higher than group A on day 2 and 5 on the frozen sections. HE stain showed that the granulation tissue formation in group B was thicker than group A on day 5. Immunohistochemistry of CD31 demonstrated that the mean number of small blood vessels in group B was more than in group A on day 5 and 8 (P<0.05). Immunohistochemistry of Ki-67 showed it had no difference between group A and group B. ELISA and RT-PCR revealed that the expression level of VEGF in the wound tissues in group B was higher than in group A on day 8 and 11 (P<0.05, P<0.001, respectively). CONCLUSION Both transplantations promote the wound healing of DFUs in rats. The intramuscular transplantation into the leg shows a better persistence and a higher expression level of VEGF in the wound tissues at later stages.
Animals ; Bone Marrow Cells ; cytology ; Diabetes Mellitus, Experimental ; complications ; Diabetic Foot ; therapy ; Male ; Mesenchymal Stem Cell Transplantation ; methods ; Rats ; Rats, Wistar ; Vascular Endothelial Growth Factor A ; metabolism ; Wound Healing

Objective To explore the expression of miRNA-181a (miR-181a) in patients with multiple myeloma (MM) and its effect on biological features of MM cells. Methods CD138+cells of bone marrow from 25 MM patients and 10 patients with hematological non-malignancies were purified by using immunomagnetic separation, and the expression of miR-181a in CD138+cells and MM cell lines including RPMI 8226, H929 and U266 were detected by real-time quantitative PCR. The effects of down-regulation and up-regulation of miR-181a expression on the biological characteristics of MM cells were studied with miR-181a antagomir and agomir. Results Compared with patients with hematological non-malignant diseases, the expression of miR-181a in CD138+ cells was upregulated in MM patients. Compared with CD138+ cells in hematological non-malignancies, high expressions of miR-181a were observed in RPMI 8226 and U266 myeloma cell line, while low expressions of miR-181a were observed in H929 cells. Down-regulation of miR-181a with 100 nmol/L miR-181a antagomir could inhibit the proliferation of U266 cells at 24,48 and 72 h [(67.1 ± 3.3) %vs. (50.5 ± 4.1) %, (71.5 ± 3.6) % vs. (52.3 ± 2.2) %, (78.1 ± 5.4) % vs. (69.5 ± 4.3) %, P < 0.05 respectively], whereas up-regulation of miR-181a with 100 nmol/L miR-181a agomir could significantly promote the proliferation of H929 cells at 24 h and 48 h [(21.2 ± 2.4) %vs. (38.5 ± 3.6) %, ( 61.3 ± 5.4) %vs. (82.2 ±6.9)%, P<0.01 respectively]. Cell cycle analysis showed that miR-181a antagomir made U266 cell cycle arrest in the G0/G1 phase. Meanwhile, susceptibility test results indicated that the apoptosis of U266 cells induced by doxorubicin, paclitaxel and 5-fluorouracil was increased when the proliferation of miR-181a expression was down-regulated with miR-181a antagomir. In migration assay, the data showed that down-regulation of miR-181a with miR-181a antagomir could inhibit the migration of U266 cells, and the proportion of migrated cells in the experimental group (62 ± 10) %was lower than that in the control group (89 ± 12) %(P< 0.05), whereas up-regulation of miR-181a with miR-181a agomir could improve the migration of H929 cells, and the proportion of migrated cells in the experimental group (242 ± 9) % was higher than that in the control group (98 ± 8)%(P<0.01). Conclusions The high expression of miR-181a expressed highly by MM cells may promote the proliferation, migration and drug resistance of myeloma cells, indicating that miR-181a could be an important prognostic biomarker candidate, and the application of gene silencing may improve the prognosis of MM.

OBJECTIVETo assess the therapeutic effect of intramuscular transplantation of bone-marrow derived mesenchymal stem cells (BM-MSCs) in the leg for treatment of diabetic foot ulcers (DFUs) in rats.

METHODSThirty-six male Wistar rats were randomly allocated into 3 equal groups, namely group A with DFUs on the bilateral hindlimb dorsum pedis and intramuscular transplantation of 4,6-diamino-2-phenylindole (DAPI)-labeled third-passage BM-MSCs from male Wistar rats into the leg, group B with nondiabetic foot ulcers, and group C with DFUs but without BM-MSC transplantation. On days 2, 5, 8 and 11 posttransplantation, the rate of wound healing was evaluated, the labeled BM-MSCs in the wound tissues were traced on frozen sections, and the thickness of granulation tissues was measured with HE staining. Immunohistochemistry was performed to detect the expression of CD31 and Ki-67, and the expression of vascular endothelial growth factor (VEGF) in the wound tissues was detected by ELISA and RT-PCR.

RESULTSThe rats in group B showed significantly faster wound closure than those in the other two groups (P<0.05). The rate of wound healing was greater in group A than in group C on days 8 and 11 (P<0.05). The fluorescence intensity and area on the frozen sections were the highest on day 5 posttransplantation in group A, which had a granulation tissue thickness comparable with group B but greater than group C. The mean numbers of small blood vessels and cells positive for CD31 and Ki-67 expressions were similar between groups A and B, which showed significant differences from group C (P<0.05). On day 11, group A showed the highest VEGF expression in the wound tissues (P<0.001).

CONCLUSIONIntramuscular transplantation of BM-MSCs can significantly promote wound healing of DFUs in rats possibly as a result of increased expression of VEGF in the wound tissues.

Animals ; Bone Marrow Cells ; Diabetic Foot ; surgery ; Lower Extremity ; Male ; Mesenchymal Stem Cell Transplantation ; Muscle, Skeletal ; transplantation ; Rats ; Rats, Wistar ; Treatment Outcome ; Wound Healing

OBJECTIVES: In recent years, it has been reported that the anti-shock effect of plasma substitutes in adult patients with major burn in shock stage is not good. However, due to the shortage of clinical frozen plasma supply, it is impossible to guarantee that frozen plasma is used as colloidal solution for anti-shock treatment. The purpose of this study is to explore the effect of the infusion ration between frozen plasma and plasma substitutes on the prognosis of adult patients with major burn in shock stage. METHODS: This study enrolled 586 adult patients with major burn by selecting the hospitalization burn patients, who had been hospitalized at the Jiangxi province burn center from September 2014 to April 2019. The patients with the infusion ratio of frozen plasma to plasma substitutes ≥2꞉1 at 48 hours after admission were included in the experimental group, otherwise they were included in the control group. The basic clinical data and clinical prognosis indicator in the 2 groups were compared. Logistic univariate regression analysis was used to screen the influential factors of 30-day mortality in adult patients with major burn, and logistic multivariate regression analysis was used to obtain independent risk and protective factors; Kaplan-Meier method was used to draw the survival curve of the 2 groups, and log-rank test was used to compare the 30-day survival rate of the 2 groups. RESULTS: There were significant differences in the infusion volume of frozen plasma and plasma substitutes between the 2 groups at 48 hours after admission (both CONCLUSIONS Infusion ration between frozen plasma to plasma substitutes at 48 hours after admission is an independent protective factor for 30-day mortality of adult patients with major burn. In the early stage of adult patients with major burn, frozen plasma should be used as the anti-shock therapy as far as possible (frozen plasma꞉plasma substitute ≥2꞉1) to improve the prognosis and reduce the of 30-day mortality.
Adult ; Hospitalization ; Humans ; Plasma Substitutes ; Prognosis ; Retrospective Studies ; Shock