Objective To observe the therapeutic effect of thermosensitive hydrogel containing curcumin-vitamin E (VE) complex (hereinafter referred to as “curcumin-VE hydrogel”) on radiation-induced oral mucositis in mice. Methods Curcumin-VE hydrogel was prepared using the synthesized curcumin-VE complex as the carrier and poloxam as the substrate. The structure of curcumin-VE complex was characterized by Fourier transform infrared spectrometer, the microstructure of curcumin-VE hydrogel was determined by scanning electron microscope, and the gelation temperature was determined by rheometer, gel swelling and degradation were tested and gel adhesion was determined using a universal testing machine. Thirty healthy male BALB/C mice with specific pathogen free grade were randomly divided into three groups, with ten mice in each group. The radiation group and radiation+hydrogel group were modeled by a single high dose of radiation (25 Gy), while the control group had anesthesia but no radiation. The control group and radiation group were given daily feed and water 7 days after radiation. In addition to daily feed and water, the radiation+hydrogel group was given curcumin-VE hydrogel twice a day. The mice were sacreficed on the 8th day after radiation. The weight changes of each group were recorded after radiation. The ulceration area of tongue was measured by toluidine blue. The tongue of mouse were pathologically observed. The activities of superoxide dismutase, catalase (CAT), and glutathione peroxidase and the level of malondialdehyde in tongue tissue were determined. The levels of tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-6 in tongue tissue were determined by enzyme linked immunosorbent assay. The distribution and positive expression of phosphorylated histone H2AX (γ-H2AX) and nuclear factor-erythroid 2-related factor 2 were determined by immunohistochemistry. Results Curcumin-VE hydrogel had a porous network structure and the gelation temperature was 30℃, the swelling rate was close to 300%, the gel degradation rate was up to 95% after 48 h, and the adhesion strength was 12.748 kPa. Compared with the radiation group, the weight of mice in the radiation+hydrogel group increased (P<0.05), the ulcer area decreased (P<0.05); the activity of CAT increased (P<0.05); the levels of TNF-α, IL-1β and IL-6 decreased (P<0.05); the expression of γ-H2AX was down-regulated (P<0.05). Conclusion Curcumin-VE hydrogel can delay or weaken the process of radiation-induced oral mucositis by reducing the DNA damage caused by radiation, inhibiting the production of reactive oxygen species, and effectively reducing the level of inflammation in tongue tissue.
Objective To investigate the effects of R-spondin 2 (Rspo2) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and bone mineral content in ovariectomized mice. Methods BMSCs were extracted from the bone marrow of the long bones of 7 4-week-old female C57BL/6 mice using whole bone marrow culture and passaged. After the cell phenotype was identified by flow cytometry, the 3rd generation cells were co-cultured with 10, 20, 40, 80, and 100 nmol/L Rspo2. Then, the cell activity and proliferative capacity were determined by cell counting kit 8 (CCK-8), and the intervention concentration of Rspo2 was screened for the subsequent experiments. The osteogenic differentiation ability of BMSCs was detected by alkaline phosphatase (ALP) staining, and the mRNA levels of osteogenesis-related genes [RUNX family transcription factor 2 (Runx2), collagen type Ⅰ alpha 1 (Col1), osteocalcin (OCN)] were detected by real-time fluorescence quantitative PCR (RT-qPCR). In addition, 18 10-week-old female C57BL/6 mice were randomly divided into sham operation group (sham group), ovariectomy group (OVX group), and OVX+Rspo2-intervention group (OVX+Rspo2 group), with 6 mice in each group. The sham group only underwent bilateral back incision and suturing, while the other two groups established osteoporosis mouse models by bilateral ovarian castration. Then, the mice were given a weekly intraperitoneal Rspo2 (1 mg/kg) treatment in OVX+Rspo2 group and saline at the same dosage in sham group and OVX group. After 12 weeks of treatment, the body mass and uterus mass of the mice were weighed in the 3 groups to assess whether the OVX model was successfully prepared; the tibia bones were stained with HE and immunohistochemistry staining to observe the changes in tibial bone mass and the expression level of Runx2 protein in the bone tissues. Blood was collected to detect the expressions of bone metabolism markers [ALP, OCN, type Ⅰ procollagen amino-terminal peptide (PINP)] and bone resorption marker [β-collagen degradation product (β-CTX)] by ELISA assay. Micro-CT was used to detect the bone microstructure changes in the tibia, and three-dimensional histomorphometric analyses were performed to analyze the trabeculae thickness (Tb.Th), trabeculae number (Tb.N), trabeculae separation (Tb.Sp), and bone volume fraction (BV/TV). Results CCK-8 assay showed that Rspo2 concentrations below 80 nmol/L were not cytotoxic (P>0.05), and the cell viability of 20 nmol/L Rspo2 group was significantly higher than that of the control group (P<0.05). Based on the above results, 10, 20, and 40 nmol/L Rspo2 were selected for subsequent experiments. ALP staining showed that the positive cell area of each concentration of Rspo2 group was significantly larger than that of the control group (P<0.05), with the highest showed in the 20 nmol/L Rspo2 group. The expression levels of the osteogenesis-related genes (Runx2, Col1, OCN) significantly increased, and the differences were significant between Rspo2 groups and control group (P<0.05) except for Runx2 in the 40 nmol/L Rspo2 group. In animal experiments, all groups of mice survived until the completion of the experiment, and the results of the body mass and uterus mass after 12 weeks of treatment showed that the OVX model was successfully prepared. Histological and immunohistochemical staining showed that the sparseness and connectivity of bone trabecula and the expression of Runx2 in the OVX group were lower than those in the sham group, whereas they were reversed in the OVX+Rspo2 group after treatment with Rspo2, and the differences were significant (P<0.05). ELISA assay showed that compared with the sham group, the serum bone metabolism markers in OVX group had an increase in ALP and a decrease in PINP (P<0.05). After Rspo2 intervention, PINP expression significantly reversed and increased, with significant differences compared to the sham group and OVX group (P<0.05). The bone resorption marker (β-CTX) was significantly higher in the OVX group than in the sham group (P<0.05), and it was significantly decreased in the OVX+Rspo2 group when compared with the OVX group (P<0.05). Compared with the sham group, Tb.Th, Tb.N, and BV/TV significantly decreased in the OVX group, while Tb.Sp significantly increased (P<0.05); after Rspo2 intervention, all of the above indexes significantly improved in the OVX+Rspo2 group (P<0.05) except Tb.Th. Conclusion Rspo2 promotes differentiation of BMSCs to osteoblasts, ameliorates osteoporosis due to estrogen deficiency, and promotes bone formation in mice.
In this study, the role of newcastle disease virus (NDV) combined thermic solidified tumor vaccine in inhibiting growth of tumor and immune control was investigated, and rate of inhibiting tumor and cellular immunity were measured. The results showed that rate of inhibiting tumor in experimental group Ⅰ and Ⅱ were 24.8% and 41.1% respectively; average weight of tumor was significantly lower in both experimental groups than in control group, and activity of natural killing (NK) cells in experimental groups was higher than that in control group (Ｐ＜0.01). This suggests that NDV combined thermic solidified tumor vaccine can inhibit growth of tumor and improve activity of NK cells, and their effects are better than that of NDV.
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 investigate the effect of tranilast on wound healing and the mechanism of inhibiting scar hyperplasia in mice, and to study the relationship between the inhibiting ability of tranilast on scar hyperplasia and administration time. Methods Sixty-six Kunming mice were selected to build deep II degree burn model, and were randomly divided into the control group (18 mice), the early intervention group (18 mice), the medium intervention group (18 mice), and the late intervention group (12 mice). The mice in the early intervention group, the medium-term intervention group, and the late intervention group were given tranilast 200 mg/(kg·d) by gastrogavage at immediate, 7 days, and 14 days after burn respectively, and the mice in the control group were managed with same amount of normal saline every day. The wound healing was observed regularly. At 14, 28, and 42 days in the early and medium intervention groups and at 28 and 42 days in the late intervention group, fresh tissues were taken from 6 mice to observe the shape of mast cells by toluidine blue staining, collagen content by Masson staining; the collagen type I and collagen type III content were measured to calculate the I/III collagen content ratio by immunohistochemistry method, the contents of transforming growth factor β1 (TGF-β1) and histamine were detected by ELISA; and the ultrastructure of fibroblasts was observed under transmission electron microscope. Results There was no significant difference in wound healing time between groups (F=1.105,P=0.371). The mast cells number, collagen content, TGF-β1 content, histamine content, and the I/III collagen content ratio in the early intervention group were significantly less than those in the other groups (P<0.05). Significant difference was found in mast cells number, collagen content, and histamine content between control group and medium or late intervention group at the other time points (P<0.05) except between control group and late intervention group at 42 days (P>0.05). Compared with the control group, the activity of fibroblasts in the early intervention group was obviously inhibited, and the arrangement of the fibers was more regular; the fibroblast activity in the medium and late intervention groups was also inhibited obviously. Conclusion Tranilast has no obvious effect on the wound healing time in mice. Tranilast intervention shows the inhibitory effect on the scar hyperplasia which can significantly reduce the number of mast cells, the content of histamine and TGF-β1, inhibit the ability of fibroblasts synthetic collagen and adjust the proportion of collagen synthesis. The immediate tranilast intervention may have the best inhibitory effect on scar hyperplasia.
ObjectiveTo investigate the effect of all-trans retinoic acid (ATRA) and vascular endothelial growth factor (VEGF) on the osteogenic differentiation of mouse embryonic fibroblasts (MEFs).MethodsThe fetal mice in the uterus of NIH pregnant mice (pregnancy 12-15 days) were collected, and the heads and hearts etc. were removed. Then MEFs were separated from the rest tissues of the fetal mice and cultured by trypsin digestion and adherent culture. HEK-293 cells were used to obtain recombinant adenovirus-red fluorescent protein (Ad-RFP) and Ad-VEGF by repeatedly freezing and thawing. Alkaline phosphatase (ALP) staining and quantitative detection were used to detect the changes of ALP activity in MEFs applied with ATRA or VEGF alone or combined use of ATRA and VEGF on the 3rd and 5th days. The cultured 3rd to 4th generation MEFs were divided into groups A, B, C, and D, and were cultured with DMSO plus Ad-RFP, ATRA, Ad-VEGF, ATRA plus Ad-VEGF, respectively. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the mRNA expressions of osteogenic markers including ALP, collagen type Ⅰ, osteopontin (OPN), osteocalcin (OCN), and angiogenic markers including VEGF, angiopoietin 1 (ANGPT1), and endomucin (EMCN) on the 3rd and 7th days. Immunohistochemical staining was used to detect the protein expressions of OPN and VEGF on the 3rd, 5th, and 7th days in each group. Alizarin red staining was used to detect calcium salt deposition levels in each group at 14 and 21 days after osteogenic induction. Fifteen athymic female nude mice aged 4 to 6 weeks were randomly divided into 3 groups and 5 mice in each group. Then MEFs treated with ATRA, Ad-VEGF, and ATRA plus Ad-VEGF were injected subcutaneously into the dorsal and ventral sides, respectively. X-ray observation, gross observation, and histological staining (Masson, HE, and Safranin O-fast green stainings) were performed at 5 weeks after implantation to observe the ectopic bone formation in nude mice in each group.ResultsMEFs were successfully isolated and cultured. The acquired Ad-RFP and Ad-VEGF were successfully transfected into MEFs with approximately 50% and 20% transfection rates. ALP activity tests showed that ATRA or Ad-VEGF could enhance ALP activity in MEFs (P<0.05), and ATRA had a stronger effect than Ad-VEGF; and the combined use of ATRA and Ad-VEGF significantly enhanced the ALP activity in MEFs (P<0.05). qRT-PCR test showed that the combined use of ATRA and Ad-VEGF also increased the relative mRNA expressions of early-stage osteogenesis-related markers ALP, OPN, and collagen type I (P<0.05); the relative mRNA expressions of angiogenesis-related markers VEGF, EMCN, and ANGPT1 increased at 7 days (P<0.05). Immunohistochemical staining showed that ATRA combined with Ad-VEGF not only enhanced OPN protein expression, but also increased VEGF protein expression on 7th day. Alizarin red staining showed that the application of ATRA or Ad-VEGF induced weak calcium salt deposition, and the combined use of ATRA and Ad-VEGF significantly enhanced the effect of calcium salt deposition in MEFs. The results of implantation experiments in nude mice showed that X-ray films observation revealed obvious bone mass in the ATRA plus Ad-VEGF group, and the bone was larger than that in other groups. Histological staining showed a large amount of collagen and mature bone trabeculae, bone matrix formation, and gray-green collagen bone tissue, indicating that the combined use of ATRA and Ad-VEGF significantly enhanced the osteogenic effect of MEFs in vivo.ConclusionThe combined use of ATRA and VEGF can induce the osteogenic differentiation of MEFs.
ObjectiveTo investigate the co-transplantation of C57-green fluorescent protein (GFP) mouse epidermis and dermis cells subcutaneously to induce the hair follicle regeneration. MethodC57-GFP mouse epidermis and dermis were harvested for isolation the mouse epidermis and dermis cells. The morphology of epidermis and dermis mixed cells at ratio of 1:1 of adult mouse, dermis cells of adult mouse, cultured 3rd generation dermis cells were observed by fluorescence microscope. Immunocytochemistry staining was used to detect hair follicle stem cells markers in cultured 3rd generation dermis cells from new born C57-GFP mouse. And then the epidermis and dermis mixed cells of adult mouse (group A), dermis cells of adult mouse (group B), cultured 3rd generation dermis cells of new born mouse (group C), and saline (group D) were transplanted subcutaneously into Balb/c nude mice. The skin surface of nude mice were observed at 4, 5, 6 weeks of transplantation and hair follicle formation were detected at 6 weeks by immunohistochemistry staining. ResultsThe isolated C57-GFP mouse epidermis and dermis cells strongly expressed the GFP under the fluorescence microscope. Immunocytochemistry staining for hair follicle stem cells markers in cultured 3rd generation dermis cells showed strong expression of Vimentin and α-smooth muscle actin, indicating that the cells were dermal sheath cells; some cells expressed CD133, Versican, and cytokeratin 15. After transplanted for 4-6 weeks, the skin became black at the injection site in group A, indicating new hair follicle formation. However, no color change was observed in groups B, C, and D. Immunohistochemical staining showed that new complete hair follicles structures formed in group A. GFP expression could be only observed in the hair follicle dermal sheath and outer root sheath in group B, and it could also be observed in the hair follicle dermal sheath, outer root sheath, dermal papilla cells, and sweat gland in group C. The expression of GFP was negative in group D. ConclusionsCo-transplantation of mouse epidermis and dermis cells can induce the hair follicle regeneration by means of interaction of each other. And transplantation of isolated dermis cells or cultured dermis cells individually only partly involved in the hair follicles formation.
Objective To investigate the effect of M2 microglia (M2-MG) transplantation on spinal cord injury (SCI) repair in mice. Methods Primary MG were obtained from the cerebral cortex of 15 C57BL/6 mice born 2-3 days old by pancreatic enzyme digestion and identified by immunofluorescence staining of Iba1. Then the primary MG were co-cultured with interleukin 4 for 48 hours (experimental group) to induce into M2 phenotype and identified by immunofluorescence staining of Arginase 1 (Arg-1) and Iba1. The normal MG were harvested as control (control group). The dorsal root ganglion (DRG) of 5 C57BL/6 mice born 1 week old were co-cultured with M2-MG for 5 days to observe the axon length, the DRG alone was used as control. Forty-two 6-week-old female C57BL/6 mice were randomly divided into sham group (n=6), SCI group (n=18), and SCI+M2-MG group (n=18). In sham group, only the laminae of T10 level were removed; SCI group and SCI+M2-MG group underwent SCI modeling, and SCI+M2-MG group was simultaneously injected with M2-MG. The survival of mice in each group was observed after operation. At immediate (0), 3, 7, 14, 21, and 28 days after operation, the motor function of mice was evaluated by Basso Mouse Scale (BMS) score, and the gait was evaluated by footprint experiment at 28 days. The spinal cord tissue was taken after operation for immunofluorescence staining, in which glial fibrillary acidic protein (GFAP) staining at 7, 14, and 28 days was used to observe the injured area of the spinal cord, neuronal nuclei antigen staining at 28 days was used to observe the survival of neurons, and GFAP/C3 double staining at 7 and 14 days was used to observe the changes in the number of A1 astrocytes. Results The purity of MG in vitro reached 90%, and the most of the cells were polarized into M2 phenotype identified by Arg-1 immunofluorescence staining. M2-MG promoted the axon growth when co-cultured with DRGs in vitro (P<0.05). All groups of mice survived until the experiment was completed. The hind limb motor function of SCI group and SCI+M2-MG group gradually recovered over time. Among them, the SCI+M2-MG group had significantly higher BMS scores than the SCI group at 21 and 28 days (P<0.05), and the dragging gait significantly improved at 28 days, but it did not reach the level of the sham group. Immunofluorescence staining showed that compared with the SCI group, the SCI+M2-MG group had a smaller injury area at 7, 14, and 28 days, an increase in neuronal survival at 28 days, and a decrease in the number of A1 astrocytes at 7 and 14 days, with significant differences (P<0.05). ConclusionM2-MG transplantation improves the motor function of the hind limbs of SCI mice by promoting neuron survival and axon regeneration. This neuroprotective effect is related to the inhibition of A1 astrocytes polarization.
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.
摘要:目的:探討表達吲哚胺2,3二氧化酶(IDO)的KC對同種異體小鼠移植皮片存活時間的影響及其機制。方法:構建BABL/c →C57BL/6的皮膚移植模型,分別于移植術后第2、7、14天輸注KC,于移植術后第7天每組各取2只皮瓣行HE染色和TUNEL以檢測淋巴細胞浸潤和凋亡情況。KaplanMeier對數秩檢驗對各組進行生存分析。結果:輸入表達IDO和FasL的KC能明顯延長BABL/c →C57BL/6皮膚移植模型中皮膚移植物的存活時間,1－甲基色氨酸能阻斷此效應。IFNγ組皮瓣浸潤淋巴細胞的凋亡率較高(Plt;0.05)。結論:表達IDO和FasL的KC在體內能明顯延長同種異體小鼠皮片的存活時間,IDO在KC維持外周免疫耐受中發揮重要作用。Abstract: Objective: To investigate kupffer cells(KC) expressing indoleamine 2,3dioxygenase(IDO) on the survival of grafted skin in mouse and its underlying mechanism. Methods: BABL/c skin was transplanted to C57BL/6. Donor KC were injected i.v. at days 2,7, 14 before transplantation. HE and TUNELAP were used to identify infiltrating cells and apoptotic cells in section of skin allografts from 7 days posttransplantation respectively. The survival rate of recipients among groups were analyzed by Logrank test. Results: Injection of KC expressing IDO and FasL from BABL/c mice into C57BL/6 could prolong a skin graft survival from the donor, but 1methyltryptophan could block the effect in vivo. The apoptosis rate of lymphocyte among skin graft in IFNγ group is more than other group(Plt;0.05). Conclusion: IDO and FasLexpressing KC from the donor of mouse can significantly prolong the skin graft survival. IDO may play an important role in KC to induce immune tolerance.