ObjectiveTo investigate the effect of natural hirudin combined with hyperbaric oxygen therapy on the survival of transplanted random-pattern skin flap in rats.MethodsA random-pattern skin flap in size of 10.0 cm×2.5 cm was elevated on the dorsum of 72 Sprague Dawley rats. Then the 72 rats were randomly divided into 4 groups (n=18) according to the therapy method. At immediate and within 4 days after operation, the rats were treated with normal saline injection in control group, normal saline injection combined with hyperbaric oxygen treatment in hyperbaric oxygen group, the natural hirudin injection in natural hirudin group, and the natural hirudin injection combined with hyperbaric oxygen treatment in combined group. The flap survival was observed after operation, and survival rate was evaluated at 6 days after operation. The skin samples were collected for histological analysis, microvessel density (MVD) measurement, and evaluation of tumor necrosis factor α (TNF-α) expression level by the immunohistochemical staining at 2 and 4 days after operation.ResultsPartial necrosis occurred in each group after operation, and the flap in combined group had the best survival. The survival rate of flap was significantly higher in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group, and in combined group than in hyperbaric oxygen group and natural hirudin group (P<0.05). There was no significant difference between hyperbaric oxygen group and natural hirudin group (P>0.05). At 2 days, more microvascular structure was observed in hyperbaric oxygen group, natural hirudin group, and combined group in comparison with control group; while plenty of inflammatory cells infiltration in all groups. At 4 days, the hyperbaric oxygen group, natural hirudin group, and the combined group still showed more angiogenesis. Meanwhile, there was still infiltration of inflammatory cells in control group, inflammatory cells in the other groups were significantly reduced when compared with at 2 days. At 2 days, the MVD was significantly higher in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group (P<0.05); the expression of TNF-α was significantly lower in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group (P<0.05). There was no significant difference in above indexes between hyperbaric oxygen group, natural hirudin group, and combined group (P>0.05). At 4 days, the MVD was significantly higher in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group, in natural hirudin group and combined group than in hyperbaric oxygen group (P<0.05). The expression of TNF-α was significantly lower in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group, in combined group than in natural hirudin group and hyperbaric oxygen group (P<0.05).ConclusionHyperbaric oxygen and natural hirudin therapy after random-pattern skin flap transplantation can improve the survival of flaps. Moreover, combined therapy is seen to exhibit significant synergistic effect. This effect maybe related to promotion of angiogenesis and the reduction of inflammation response.
【Abstract】Objective To introduce the current studies of the role of vascular endothelial growth factorC (VEGFC) and VEGFD in lymphangiogenesis and lymph node metastasis of gastrointestinal neoplasma. Methods The related literatures in recent 5 years were reviewed. Results The growth factors VEGFC and VEGFD enhance lymphangiogenic metastasis of gastrointestinal neoplasma with the property of angiogenesis and lymphangiogenesis. In gastric adenocarcinoma, VEGFC mRNA and tissue protein expression correlate with lymphatic invasion, lymph node metastasis, venous invasion and reduced 5year survival rates. The role of VEGFC in esophageal squamous cancer and colorectal cancer and VEGFD in colorectal cancer is not certain, with conflicting reports in the published literatures.Conclusion The VEGFC, VEGFD/VEGFR3 signal pathway may become the ideal target for inhibition of tumor proliferation and metastases, antilymphangiogenesis therapy may be a novel potential strategy in tumor biological therapy.
ObjectiveTo review the research progress on the role and mechanism of matrix stiffness in regulating endothelial cell sprouting. MethodsThe related literature at home and abroad in recent years was extensively reviewed, and the behaviors of matrix stiffness related endothelial cell sprouting in different cell cultivation conditions were analyzed, and the specific molecular mechanism of matrix stiffness regulating related signal pathways in endothelial cell sprouting was elaborated. Results In two-dimensional cell cultivation condition, increase of matrix stiffness stimulates endothelial cell sprouting within a certain range. However, in three-dimensional cell cultivation condition, the detailed function of matrix stiffness in regulating endothelial cell sprouting and angiogenesis are still unclear. At present, the research of the related molecular mechanism mainly focuses on YAP/TAZ, and roles of its upstream and downstream signal molecules. Matrix stiffness can regulate endothelial cell sprouting by activating or inhibiting signal pathways to participate in vascularization. ConclusionMatrix stiffness plays a vital role in regulating endothelial cell sprouting, but its specific role and molecular mechanism in different environments remain ambiguous and need further study.
ObjectiveTo investigate the relationship between vascular endothelial growth factor receptor-3 (VEGFR-3) and clinical pathology of gastric carcinoma(GC).MethodsThe expression of VEGFR-3 in 80 GCs and 20 gastric benign tissues (GBT) was detected by immunohistochemistry(SP), by which the density of lymphatic vessels (DLV) was calculated. ResultsThe DLV in GC was （5.800 0±2.318 9）/×200, in GBT （2.380 0±0.462 9）/×200(P=0.000); in GC with lymph node metastasis （6.948 3±1.583 1）/×200, without lymph node metastasis （2.772 7±0.428 9）/×200 （P=0.000）. In poorly differentiated type group, DLV was （7.681 8±0.982 9）/×200, higher than that in moderately and highly differentiated type group 〔(3.500 0±1.028 2）/×200, P=0.000〕. DLV in pTNM Ⅰ+Ⅱ was （4.291 7±1.688 0）/×200, in Ⅲ+Ⅳ （8.062 5±0.759 4）/×200 （P=0.000）.ConclusionDLV shows positive relations with pTNM stage, differentiation and lymph node metastasis of GC.
ObjectiveTo investigate the effect of adipose-derived stem cell derived exosomes (ADSC-Exos) on angiogenesis after skin flap transplantation in rats.MethodsADSCs were isolated and cultured by enzymatic digestion from voluntary donated adipose tissue of patients undergoing liposuction. The 3rd generation cells were observed under microscopy and identified by flow cytometry and oil red O staining at 14 days after induction of adipogenesis. After cells were identified as ADSCs, ADSC-Exos was extracted by density gradient centrifugation. And the morphology was observed by transmission electron microscopy, the surface marker proteins (CD63, TSG101) were detected by Western blot, and particle size distribution was measured by nanoparticle size tracking analyzer. Twenty male Sprague Dawley rats, weighing 250-300 g, were randomly divided into ADSC-Exos group and PBS group with 10 rats in each group. ADSC-Exos (ADSC-Exos group) and PBS (PBS group) were injected into the proximal, middle, and distal regions of the dorsal free flaps with an area of 9 cm×3 cm along the long axis in the two groups. The survival rate of the flap was measured on the 7th day, and then the flap tissue was harvested. The tissue morphology was observed by HE staining, and mean blood vessel density (MVD) was measured by CD31 immunohistochemical staining.ResultsADSCs were identified by microscopy, flow cytometry, and adipogenic induction culture. ADSC-Exos was a round or elliptical membrane vesicle with clear edge and uniform size. It has high expression of CD63 and TSG101, and its size distribution was 30-200 nm, which was in accordance with the size range of Exos. The distal necrosis of the flaps in the ADSC-Exos group was milder than that in the PBS group. On the 7th day, the survival rate of the flaps in the ADSC-Exos group was 64.2%±11.5%, which was significantly higher than that in the PBS group (31.0%±6.6%; t=7.945, P=0.000); the skin appendages in the middle region of the flap in the ADSC-Exos group were more complete, the edema in the proximal region was lighter and the vasodilation was more extensive. MVD of the ADSC-Exos group was (103.3±27.0) /field, which was significantly higher than that of the PBS group [(45.3±16.2)/field; t=3.190, P=0.011].ConclusionADSC-Exos can improve the blood supply of skin flaps by promoting the formation of neovascularization after skin flap transplantation, thereby improve the survival rate of skin flaps in rats.
Objective To study the expression of inducible nitric oxide synthase (iNOS)，endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) in human gastric cancer and their relationship with tumor angiogenesis and to investigate the interaction of NOS and VEGF in gastric cancer. Methods The expression and distribution of VEGF, iNOS and eNOS in 34 gastric cancer specimens were detected with immunohistochemistry. Microvessel density (MVD) was counted with FⅧRAg immune specific staining. Results The expression rates of iNOS, eNOS and VEGF in 34 gastric cancers were 73.5%, 82.4% and 91.2% respectively. The expression of VEGF had a significant positive relation with iNOS, but not with eNOS. The MVDs of VEGF or iNOS positive gastric cancers were obviously higher than those of VEGF or iNOS negative gastric cancers. There was no significant difference between the MVDs of eNOS positive gastric cancers and eNOS negative ones. Conclusion MVD increases with increase of expression of VEGF and iNOS in gastric cancer. It is indicated that VEGF and iNOS can promote gastric cancer angiogenesis. VEGF and iNOS have a significant positive correlation, which suggests that in human gastric cancer, iNOS plays an important role in the production and action of VEGF.
Objective To study the expression of thymidine phosporylase (TP) and the counts of lymph vessels in pancreatic cancer and chronic pancreatitis tissues, and to explore their clinicopathologic significances and correlation in the course of pancreatic cancer. Methods SP immunohistochemical method was used to detetct the expression of TP and the locations of lymph vessels on the routinely paraffin-embedded sections of the specimens from 51 cases pancreatic cancer and 10 cases of chronic pancreatitis. Results The positive rate of TP and the counts of lymph vessels were significantly higher (P＜0.05 and P＜0.01 respectively) in pancreatic cancer 〔54.9%, (12.5±4.3)/HP〕 than those in chronic pancreatitis 〔20.0%，(5.2±2.4)/HP〕. The positive rate of TP and the counts of lymph vessels were significantly lower （P＜0.05, P＜0.01） in well-differentiated adenocarcinoma cases and cases without metastasis compared with poor-differentiated adenocarcinoma cases and cases with metastasis. The counts of lymph vessels were significantly higher in the positive cases of TP than those in the negative ones in pancreatic cancer 〔(13.8±3.4)/HP vs (10.9±3.2)/HP〕, P＜0.01.Conclusion The expression of TP and counts of lymph vessels might be important markers reflecting the progression, biological behaviors, metastatic status and prognosis of pancreatic cancer. TP might promote lympoangiogenesis in pancreatic cancer tissues.
ObjectiveTo investigate the effects of adipose-derived stem cell released exosomes (ADSC-Exos) on wound healing in diabetic mice.MethodsThe ADSCs were isolated from the adipose tissue donated by the patients and cultured by enzymatic digestion. The supernatant of the 3rd generation ADSCs was used to extract Exos (ADSC-Exos). The morphology of ADSC-Exos was observed by transmission electron microscopy. The membrane-labeled proteins (Alix and CD63) were detected by Western blot, and the particle size distribution was detected by nanoparticle tracking analyzer. The fibroblasts were isolated from the skin tissue donated by the patients and cultured by enzymatic digestion. The 5th generation fibroblasts were cultured with PKH26-labeled ADSC-Exos, and observed by confocal fluorescence microscopy. The effects of ADSC-Exos on proliferation and migration of fibroblasts were observed with cell counting kit 8 (CCK-8) and scratch method. Twenty-four 8-week-old Balb/c male mice were used to prepare a diabetic model. A full-thickness skin defect of 8 mm in diameter was prepared on the back. And 0.2 mL of ADSC-Exos and PBS were injected into the dermis of the experimental group (n=12) and the control group (n=12), respectively. On the 1st, 4th, 7th, 11th, 16th, and 21st days, the wound healing was observed and the wound healing rate was calculated. On the 7th, 14th, and 21st days, the histology (HE and Masson) and CD31 immunohistochemical staining were performed to observe the wound structure, collagen fibers, and neovascularization.ResultsADSC-Exos were the membranous vesicles with clear edges and uniform size; the particle size was 40-200 nm with an average of 102.1 nm; the membrane-labeled proteins (Alix and CD63) were positive. The composite culture observation showed that ADSC-Exos could enter the fibroblasts and promote the proliferation and migration of fibroblasts. Animal experiments showed that the wound healing of the experimental group was significantly faster than that of the control group, and the wound healing rate was significantly different at each time point (P<0.05). Compared with the control group, the wound healing of the experimental group was better. There were more microvessels in the early healing stage, and more deposited collagen fibers in the late healing stage. There were significant differences in the length of wound on the 7th, 14th, and 21st days, the number of microvessels on the 7th and 14th days, and the rate of deposited collagen fibers on the 14th and 21st days between the two groups (P<0.05).ConclusionADSC-Exos can promote the wound healing in diabetic mice by promoting angiogenesis and proliferation and migration of fibroblasts and collagen synthesis.
【Abstract】Objective To understand the features of lymphatic vessel, and to summarize the foundation and mechanism of the promotion and inhibition of tumor lymphangiogenesis recorded on the current studies of animal experiments and clinical researches. Methods The related literatures of the structural features of lymphatic vessel, lymphatic endothelial molecular markers, the origin of lymphatic tumors, the molecular mechanisms and regulatory factors were reviewed, and the relationship between tumor lymphangiogenesis and lymphatic metastasis, the treatment targeting at the formation of the anti-tumor lymphatic vessel and its existing problems were also analyzed. Results Hyperplasia of lymphatic vessels occurred during the process of tumor formation and progression. The structural features of the lymphatic vessels in the tumor were conducive to tumor lymphatic metastasis. In recent years, methods of anti-lymphangiogenesis and inhibition of tumor lymphatic metastasis had achieved considerable success in animal experiments. However, there were still a lot of problems need to be solved. Conclusion Tumor lymphangiogenesis has a significantly positive correlation between tumor lymphatic metastasis and patients’ prognosis, which may indicate that treatment against the formation of tumor lymphatic vessel maybe effective.
Objective To explore the effect of natural hirudin on proliferation of human microvascular endothelial cells (HMVECs) and its preliminary mechanism of promoting angiogenesis. Methods Three-dimensional culture models of HMVECs were established in vitro and observed by inverted phase contrast microscopy after 24 hours of culturing. Then, the three-dimensional culture models of HMVECs were treated with different concentrations (1, 4, and 7 ATU/mL) of the natural hirudin, respectively, and Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum as control. The cell proliferations of 4 groups were detected by cell counting kit 8 (CCK-8) method at 24, 48, and 72 hours; the angiogenesis of 4 groups were observed by tube formation assay at 24 hours; the expressions of vascular endothelial growth factor (VEGF) and Notch1 of HMVECs in 4 groups were observed by immunofluorescence staining at 24 hours. Results The observation of cells in three-dimensional culture models showed that HMVECs attached to Matrigel well, and the cells formed tube structure completely after 24 hours. The results of CCK-8 test showed that the absorbance (A) value of 1 and 4 ATU/mL groups were higher than that of control group at each time point (P<0.05), andA value of 4 ATU/mL group was the highest. The A value of 7 ATU/mL group was significantly lower than those of 1 and 4 ATU/mL groups and control group (P<0.05). The tube formation assay showed that the tube structure was more in 1 and 4 ATU/mL groups than in 7 ATU/mL group and control group, and in 4 ATU/mL group than in 1 ATU/mL group, showing significant differences (P<0.05). There was no significant difference between 7 ATU/mL group and control group (P>0.05). The results of immunofluorescence staining showed that compared with control group, the Notch1 expression was higher in 1 and 4 ATU/mL groups and lower in 7 ATU/mL group; and there was significant difference between 4 and 7 ATU/mL groups and control group (P<0.05). The VEGF expression was higher in 1, 4, and 7 ATU/mL groups than in control group, in 4 ATU/mL group than in 1 and 7 ATU/mL groups, showing significant differences (P<0.05). Conclusion Natural hirudin can promote angiogenesis at low and medium concentrations, but suppress angiogenesis at high concentrations. Its mechanism may be related to the VEGF-Notch signal pathway.