Objective To study the effect of platelet-rich plasma (PRP) on the survival and quality of fat grafts in the nude mice so as to provide a method and the experimental basis for clinical practice. Methods Fat tissue was harvested from the lateral thigh of a 25-year-old healthy woman and the fat was purified by using saline. The venous blood was taken from the same donor. PRP was prepared by centrifugation (200 × g for 10 minutes twice) and activated by 10% calcium chloride (10 : 1). Then 24 female nude mice [weighing (20 ± 3) g, 5-week-old] were allocated randomly to the experimental group and the control group (12 mice per group). Each subcutaneous layer of two sides of the back (experimental group) was infiltrated with 0.8 mL fat tissue-activated PRP mixtures (10 : 2); the control group was infiltrated with 0.8 mL fat tissue-saline mixtures (10 : 2); 0.14 mL activated PRP and 0.14 mL saline were injected into the experimental group and the control group respectively at 5 and 10 days after the first operation. At 15, 30, 90, and 180 days after the first operation, the samples were harvested for gross and histological observations. Results All nude mice survived to the end of the experiment. No inflammation and abscess formation of the graft were observed. Experimental group was better than control group in angiogenesis, liquefaction, and necrosis. The grafted fat weight and volume in the experimental group were significantly larger than those in the control group at 15, 30, and 90 days (P lt; 0.05); but there was no significant difference between the 2 groups at 180 days (P gt; 0.05). Histological observation showed good morphological and well-distributed adipocytes, increasing vacuoles, few necrosis and calcification in the experimental group; but disordered distribution, obvious necrosis, and calcification in the control group. The necrosis area ratio of the experimental group was significantly lower than that of the control group (P lt; 0.05), and the number of micro-vessels was significantly higher in the experimental group than in the control group at 15 and 180 days (P lt; 0.05). Conclusion The method of repeatedly using the PRP within 180 days in assisting fat grafts can obviously improve the survival and quality.
ObjectiveTo investigate the effect of circulating estrogen level on the outcome of free fat grafting in nude mice.MethodsEighteen female nude mice aged 6-8 weeks (weighing, 20-25 g) were randomly divided into 3 groups (n=6). The nude mice in the ovariectomized group were treated with ovariectomy. The nude mice in the high estrogen group and the normal estrogen group only made the same incision to enter the peritoneum without ovariectomy. The nude mice in the high estrogen group were given the estradiol (0.2 mg/g) every 3 days for 30 days. The other two groups were given the same amount of PBS every 3 days. At 30 days after operation, the tail vein blood of nude mice in 3 groups were detected by estradiol ELISA kit, and the free fat (0.3 mL) donated by the females was injected into the sub-scalp of nude mice. After 8 weeks of fat grafting, the samples were taken for gross observation and weighing, and the prepared slices were stained with HE staining, CD31-perilipin fluorescence staining, immunohistochemical staining of uncoupling protein 1 (UCP1), and immunofluorescence staining of estrogen receptor α. The diameter of adipocytes and vascular density of adipose tissue were measured. The mRNA expressions of UCP1 and estrogen receptor α were detected by realtime fluorescence quantitative PCR (qRT-PCR).ResultsAll nude mice survived during experiment. ELISA test showed that the concentration of estradiol significantly decreased in the ovariectomized group and increased in the high estrogen group compared with the normal estrogen group (P<0.05). At 8 weeks after fat grafting, the graft volume from large to small was ovariectomized group, normal estrogen group, and high estrogen group. There was significant difference in wet weight between the ovariectomized group and high estrogen group (P<0.05). Section staining showed that compared with the normal estrogen group, the adipocytes in the ovariectomized group were larger, the expression of peri-lipoprotein was weaker, the vascular density decreased, and the expressions of UCP1 was negative, and the estrogen receptor α positive cells reduced. The above observation results in the high estrogen group were contrary to those in the ovariectomized group. There were significant differences in the diameter of adipocytes, the vascular density of adipose tissue, the number of the estrogen receptor α positive cells between groups (P<0.05). The results of qRT-PCR showed that the mRNA expressions of UCP1 and estrogen receptor α significantly increased in the high estrogen group and decreased in the ovariectomized group compared with the normal estrogen group, and the differences were significant (P<0.05).ConclusionThe level of circulating estrogen has a significant effect on the outcome of free fat grafting in nude mice. Low estrogen level leads to hypertrophy of graft adipocytes, while high estrogen level leads to the production of a large amount of beige fat and high vascular density in fat grafts, which may be related to the activation of estrogen receptor α on adipocytes.
The model of transplanted colonic SW480 cell line carcinoma in gymnomouse body was set up to observe the effect of octapeptide somatostatin (SMS 201-995,SMS) on the transplanted carcinoma and elucidate its mechanism. Results: the volume, weight, DNA and protein content in carcinoma cell, cell amount and proliferation index of S and G2M phase in SMS group and SMS+PG (pentagastrin) group were markedly lower than those in PG group and control group, those of PG group were markedly higher than those in control group.The cell amount of G0/G1 phase in SMS group and SMS+PG group was markedly higher than that in PG group and control group, and that of PG group was markedly lower than that in control group.All these suggested that somatostatin could not only inhibit the growth of transplanted human colonic SW480 cell line carcinoma directly but also inhibit the growthpromoting effect of gastrin on the transplanted carcinoma.The mechanism might be that somatostatin inhibit the synthesis of cAMP, DNA and protein in carcinoma cells, then inhibit the cell growing from G0/G1 phase to S and G2M phases.Our study might provide experimental basis for the homonotherapy with analogue of somatostatin in patients with large intestine carcinoma.
Objective To investigate the effects of adipose-derived stem cells (ADSCs) and endothelial cells (ECs) on the survival and neovascularization of fat tissue transplants. Methods The ADSCs were isolated by collagenase digestion from the adipose tissues voluntarily donated by the patients undergoing mastectomy, and subcultured. The passage 3 ADSCs were used for subsequent experiments. The residual fat tissues were used to prepare fat particles (FPs). The human umbilical vein endothelial cells (HUVECs) were used as ECs for subsequent experiments. Eighty healthy male nude mice, aged 4-6 weeks, were randomly divided into 4 groups (n=20). The mice were received subcutaneous injection at the dorsum of 1 mL FPs+0.3 mL normal saline (NS) in control group, 1 mL FPs+2×106 ECs+0.3 mL NS in ECs group, 1 mL FPs+2×106 ADSCs+0.3 mL NS in ADSCs group, and 1 mL FPs+1×106 ECs+1×106 ADSCs+0.3 NS in ADSCs+ECs group. General observations of the injection sites were performed, and the survival of the mice was recorded. At 2, 4, 8, and 12 weeks after injection, grafted fat tissues were firstly assessed by ultrasonography, then they were collected for volume measurement (water displacement method) and histology observation (HE staining and immunofluorescence staining). Results All mice survived until the end of experiment. At each time point, no significant difference was noted between groups in ultrasonography assay. There was no significant blood flow signal in the grafted fat tissues, or cysts, calcification, solid occupying in recipient area. Generally, the volume of grafted fat tissues decreased with time in all groups. Specifically, the volumes of grafted fat tissues were larger in ADSCs group and ADSCs+ECs group than that in control group and ECs group (P<0.05) at each time point, and in ADSCs group than in ADSCs+ECs group (P<0.05) at 8 and 12 weeks. HE staining showed that all groups had similar tendencies in general histology changes, and remodeling in ADSCs group was the fastest than in the other groups. By immunofluorescence staining for neovascularization, the new vessels in all groups were increasing with time. The vessel densities were higher in ECs group, ADSCs group, and ADSCs+ECs group than in control group (P<0.05) at each time point, in ADSCs group than in ECs group and ADSCs+ECs group (P<0.05) at 4 weeks, in ADSCs group and ADSCs+ECs group than in ECs group (P<0.05) at 8 and 12 weeks. Conclusion ADSCs can significantly increase the survival of transplanted fat tissue, which may be related to promoting the neovascularization.
Objective To study effect of carcinoembryonic antigen (CEA) positive targeted lymphocytes on gastric cancer cells in vitro and in vivo. Methods The peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy volunteers. The recombinant vector anti-CEA-scFv-CD3ζ-pcDNA3.0 was transfected into the PBMCs by lipofectamine 2000, by this means, the CEA special lymphocytes were obtained. Meanwhile, the PBMCs transfected with empty plasmid pcDNA3.0 were used as control (empty vector lymphocytes). The different lymphocytes and gastric cancer cells (CEA positive KATOⅢ gastric cancer cells and CEA negative BGC-823 gastric cancer cells) were co-cultured, then the ability to identify the gastric cancer cells and it’s effect on apoptosis of gastric cancer cells were observed at 24 h or 36 h later respectively. The CEA special lymphocytes and empty vector lymphocytes were injected by the tail vein of nude mice bearing gastric cancer cells, then it’s effect on the tumor was observed. Results ① The CEA special lymphocytes could strongly identify the KATOⅢ gastric cancer cells (identification rate was 72.3%), which could weakly identify the BGC-823 gastric cancer cells (identification rate was 7.8%). ② The apoptosis rate of the co-culture of CEA special lymphocytes and KATOⅢ gastric cancer cells was significantly higher than that of the co-culture of empty vector lymphocytes and KATOⅢ gastric cancer cells (P=0.032), which had no significant difference between the co-culture of CEA special lymphocytes and BGC-823 gastric cancer cells and the co-culture of empty vector lymphocytes and BGC-823 gastric cancer cells (P=0.118). ③ The tumor volume of the co-culture of CEA special lymphocytes and KATOⅢ gastric cancer cells was significantly smaller than that of the co-culture of empty vector lymphocytes and KATOⅢ gastric cancer cells (F=5.010, P<0.01) or the co-culture of CEA special lymphocytes and BGC-823 gastric cancer cells (F=4.982, P<0.01), which had no significant difference between the co-culture of CEA special lymphocytes and BGC-823 gastric cancer cells and the co-culture of empty vector lymphocytes and BGC-823 gastric cancer cells (F=1.210, P>0.05). Conclusion CEA special lymphocytes can promote cell apoptosis and inhabit tumor reproduction of CEA positive gastric cancer cells in vitro and in vivo.
Objective To explore heterotopic chondrogenesis of canine myoblasts induced by cartilage-derived morphogenetic protein 2 (CDMP-2) and transforming growth factor β1 (TGF-β1) which were seeded on poly (lactide-co-glycolide) (PLGA) scaffolds after implantation in a subcutaneous pocket of nude mice. Methods Myoblasts from rectus femoris of 1-year-old Beagle were seeded on PLGA scaffolds and cultured in medium containing CDMP-2 and TGF-β1 for 2 weeks in vitro. Then induced myoblasts-PLGA scaffold, uninduced myoblasts-PLGA scaffold, CDMP-2 and TGF-β1-PLGA scaffold, and simple PLGA scaffold were implanted into 4 zygomorphic back subcutaneous pockets of 24 nude mice in groups A, B, C, and D, respectively. At 8 and 12 weeks, the samples were harvested for general observation, HE staining and toluidine blue staining, immunohistochemical staining for collagen type I and collagen type II; the mRNA expressions of collagen type I, collagen type II, Aggrecan, and Sox9 were determined by RT-PCR, the glycosaminoglycans (GAG) content by Alician blue staining, and the compressive elastic modulus by biomechanics. Results In group A, cartilaginoid tissue was milky white with smooth surface and slight elasticity at 8 weeks, and had similar appearance and elasticity to normal cartilage tissue at 12 weeks. In group B, few residual tissue remained at 8 weeks, and was completely degraded at 12 weeks. In groups C and D, the implants disappeared at 8 weeks. HE staining showed that mature cartilage lacuna formed of group A at 8 and 12 weeks; no cartilage lacuna formed in group B at 8 weeks. Toluidine blue staining confirmed that new cartilage cells were oval and arranged in line, with lacuna and blue-staining positive cytoplasm and extracellular matrix in group A at 8 and 12 weeks; no blue metachromatic extracellular matrix was seen in group B at 8 weeks. Collagen type I and collagen type II expressed positively in group A, did not expressed in group B by immunohistochemical staining. At 8 weeks, the mRNA expressions of collagen type I, collagen type II, Aggrecan, and Sox9 were detected by RT-PCR in group A at 8 and 12 weeks, but negative results were shown in group B. The compressive elastic modulus and GAG content of group A were (90.79 ± 1.78) MPa and (10.20 ± 1.07) μg/mL respectively at 12 weeks, showing significant differences when compared with normal meniscus (P lt; 0.05). Conclusion Induced myoblasts-PLGA scaffolds can stably express chondrogenic phenotype in a heterotopic model of cartilage transplantation and represent a suitable tool for tissue engineering of menisci.
ObjectiveTo explore the endothelial cells from human peripheral blood and islet of rat co-transplantation under the renal capsule of diabetic nude mice to improve the survival and function of transplanted islet. MethodsThe endothelial cells from human peripheral blood(5×105)and freshly isolated rat islet cells were co-transplanted under the renal capsule of diabetic nude mice model, then the fasting blood glucose, body weight, peripheral blood C-peptide level, and intraperitoneal glucose tolerance test(IPGTT) were measured to evaluated the islet graft survival and function. ResultsCompared with the control group, the fasting blood glucose level significantly decreased(P < 0.01), peripheral blood C-peptide level rised(P < 0.01), and body weight increased(P < 0.01) of receptor nude mice in experience group, the IPGTT also improved. ConclusionThe endothelial cells from human peripheral blood and islet of rat co-transplan-tation can obviously improve the survival and function of transplanted islet of nude mice.
ObjectiveTo investigate the survival rate of core fat tissue with different diameters by advanced fat harvesting instrument. MethodsBased on core fat transfer by 1 mL syringe, the fat harvesting instrument was modified with different diameters, including 4, 6, 8, and 10 mm respectively. Between May 2014 and April 2015, the fat harvesting instrument with diameters of 4, 6, 8, and 10 mm was respectively used to harvest abdominal fat in 3 of 12 patients undergoing autologous fat transplantation. The glucose transportation quantities and the fat cell viability were measured. Then 64 nude mice at the age of 3-4 weeks were randomly divided into 4 groups (groups A, B, C, and D, n=16). And 0.5 mL fat harvested with diameters of 4, 6, 8, and 10 mm was implanted into the dorsal subcutaneous space. After fat transplantation, the mice survival and the appearance at the recipient site were observed. At 1, 2, 4, and 8 weeks after fat transplantation, the grafted fat was harvested for gross, histological and immunohistochemical observations; the intact adipocytes and capillary were counted. ResultsThe glucose transportation quantities gradually increased with increased diameter, showing significant difference among groups (P<0.05). And the fat cell viability had a rising tendency, showing significant differences when comparing groups A and B with group D (P<0.05). With the time passing by, the protuberant appearance became flat at the recipient site, but the appearance of groups C and D was better than groups A and B. Normal shape of the fat and capillary were found in groups C and D. At immediate and 1 week after fat transplantation, there was no significant difference in fat weight among 4 groups (P>0.05); the fat weight of group A was significantly less than that of groups B, C, and D (P<0.05) at 2, 4, and 8 weeks after fat transplantation, and it was significantly less in group B than groups C and D (P<0.05), but no significant difference was found between groups C and D (P>0.05). Histological and immunohistochemical observations showed better integrity of the cells, less necrosis, and higher vascular density in group D than groups A, B, and C as time extension. The adipocyte integrity of group A was significantly worse than that of other 3 groups at other time points (P<0.05) except at 1 week (P>0.05). At each time point, the capillary counting had an increasing trend with increased diameter in all groups, showing significant difference among groups (P<0.05). ConclusionWith diameters within 10 mm, the thicker the core fat is transferred, the better integrlity, higher vessel density, and quicker revascularization time can be predicted. So the postoperative appearance could be maintained longer.
ObjectiveTo investigate MUC1 over-expression on chemotherapy of 5-fluorouracil and cisplatin for esophageal cancer cells. MethodsMUC1 over-expression and stable silencing of MUC1 expression esophageal cancer cell lines were constructed. Xenograft model of esophageal cancer was established in nude mice. Cisplatin (8 mg/kg, day 1 and day 7)and 5-fluorouracil (20 mg/kg, day 1 to 6)were injected intraperitoneally. Tumor volume and body weight of nude mice were measured. Tumor growth curve and body weight curve were drawn, and tumor inhibitory rate was calculated. ResultsBoth cisplatin and 5-fluorouracil suppressed tumor growth of MUC1 over-expression esophageal cancer nude mice. Body weight and tumor volume of nude mice of cisplatin and 5-fluorouracil groups were significantly smaller than those of the control group (P < 0.05), and the inhibitory effects of cisplatin were significantly greater than those of 5-fluorouracil (P < 0.05). There was no significant inhibitory effect in stable silencing of MUC1 expression esophageal cancer nude mice. ConclusionBoth cisplatin and paclitaxel can suppress the growth of MUC1 over-expression esophageal cancer, and cisplatin has greater inhibitory effects than 5-fluorouracil in tumor volume and body weight of nude mice.
ObjectiveTo investigate the effectiveness of human placental decidua basalis derived mesenchymal stem cells (PDB-MSCs) in repairing full-thickness skin defect of nude mice. MethodsHuman placenta samples were obtained from healthy donor mothers with written informed consent. PDB-MSCs were isolated through enzymic digestion and density gradient centrifugation; the 4th passage cells were identified by cellular morphology, cell adipogenic and osteogenic differentiation, and phenotype evaluation. Forty-two 4-5-week-old BALB/c female nude mice were randomly divided into experimental group (n=21) and control group (n=21). The 4th passage PDB-MSCs solution (200 μL, 5×106/mL) was injected into the mice of experimental group via caudal vein; the mice of control group were given equal volume of PBS. The full-thickness skin defect model of 1.5 cm×1.5 cm in size was made after 3 days. The wound healing was observed generally at 1, 2, 4, 7, 14, 18, 21, 25, and 30 days after operation, and the wound healing rate was calculated after wound decrustation. HE staining was used to observe the wound repair at 1, 7, 14, 21, and 31 days; immunofluorescent staining was used for cellular localization at 7, 14, and 31 days after operation. ResultsCells isolated from human placenta were MSCs which had multipotential differentiation ability and expressed MSCs phenotype. Animals survived to the end of the experiment. The general observation showed that the experimental group had a faster skin repairing speed than the control group; the time for decrustation was 12-14 days in experimental group and was 14-17 days after operation in the control group. The wound healing rate of experimental group was significantly higher than that of control group at 14, 18, and 21 days (t=4.001, P=0.016; t=3.380, P=0.028; t=3.888, P=0.018), but no significance was found at 25 and 30 days (t=1.565, P=0.193; t=1.000, P=0.423). HE staining showed lower inflammatory reaction, and better regeneration of the whole skin and glands with time in the experimental group. The immunofluorescent staining was positive in skin defect area of experimental group at different time points which displayed that human PDB-MSCs existed. ConclusionThrough enzymic digestion and density gradient centrifugation, PDB-MSCs can be obtained. Pre-stored PDB-MSCs can mobilize to the defect area and participate in repair of nude mice skin.