ObjectiveTo study the possbility of using intranasal instillation of fine particulate matter (PM2.5) combined with inhalation of ozone (O3) to establish mouse model of combined pulmonary fibrosis and emphysema (CPFE), and to provide a reference for the establishment of CPFE model.MethodsMale C57/BL6 mice were divided randomly into phosphate buffer saline (PBS) intranasal instillation+air inhalation group, PBS intranasal instillation+O3 inhalation group and PM2.5 intranasal instillation+O3 inhalation group, with 8 mice in each group. The mice were intranasally instilled with PBS or PM2.5 suspension (7.8 mg/kg) followed by air or ozone inhalation 24 hours later, twice a week over 8 weeks. Lung function, bronchoalveolar lavage fluid (BALF) cell counts and classification were detected, the pathological changes of lung tissues in hematoxylin-eosin staining were observed, including inflammation scores and mean linear intercept (Lm). The thickness of collagen deposition in subepithelium was measured in lung tissues in Masson staining, and simultaneously hydroxyproline contents in lung tissues were determined.ResultsCompared to PBS instillation+air inhalation group, inspiratory capacity (IC), total lung capacity (TLC) and chord compliance (Cchord) were increased, FEV25 (the forced expiratory volume at 25 ms)/FVC (forced vital capacity) was decreased, total cell counts in BALF, Lm and lung inflammatory scores were increased, the thickness of the subepithelial collagen layer (SEc/Pbm) or hydroxyproline contents was not changed in PBS instillation +O3 inhalation group; IC was decreased, functional residual capacity (FRC) was increased, TLC was increased, Cchord was decreased, FEV25/FVC and FEV50 (the forced expiratory volume at 50 ms)/FVC were decreased, total cell counts in BALF, Lm, lung inflammatory scores, SEc/Pbm and hydroxyproline contents were increased in PM2.5 instillation+O3 inhalation group. Compared to PBS instillation+O3 inhalation group, IC was decreased, FRC was increased, Cchord was decreased, FEV25/FVC and FEV50/FVC were decreased, total cell counts in BALF, Lm, lung inflammatory scores, SEc/Pbm and hydroxyproline contents were increased in PM2.5 instillation +O3 inhalation group.ConclusionCPFE mouse model can be successfully established by PM2.5 intranasal instillation combined with ozone inhalation for consecutive 8 weeks.
Objective To investigate the effect of myoblast transplantation on duchenne muscular dystrophy (DMD) and to explore the method and feasibil ity of applying gene therapy to DMD. Methods Myoblast of C57/BL10 mice were cultured using multiple-step enzyme digestion method and differential velocity adherent technique. The morphology of the cells was observed with inverted phase contrast microscope. The cells at passage 4 were labeled with 5-BrdU. Twenty-four DMDmodel mice (mdx mice: aged 4-6 weeks, male, 13.8-24.6 g) were randomly divided into two groups (n=12 per group): group A, 1 × 106/mL labeled myoblast were injected via ven caudal is twice at an interval of 2 weeks; group B: 1 mL DMEM/F12 was injected in the same manner serving as a control group. The mice were killed 4 weeks after operation and the motor abil ity of the mice was detected by one-time exhaustive swimming before their death. HE staining and immunohistochemistry staining observation for 5-BrdU, desmin, and dystrophin (Dys) were preformed, and the imaging analysis was conducted. Results The primary myoblast could be sub-cultured 5-7 days after culture, providing stable passage and sufficient cells. The time of onetime exhaustive swimming was (60.72 ± 5.76) minutes in group A and (47.77 ± 5.40) minutes in group B, there was significant significance between two groups (P lt; 0.01). At 4 weeks after injection, HE staining showed that in group A, there were round and transparent-stained myocytes and the percentage of centrally nucleated fibers (CNF) was 67%; while in group B, there were uneven muscle fiber with such pathological changes as hypertrophia, atrophia, degeneration, and necrosis, and the percentage of CNF was above 80%. Immunohistochemistry staining revealed that the expression of 5-BrdU, desmin, and Dys was positive in group A; while in group B, those expressions were l ittle or negative. Image analysis result displayed that integral absorbency (IA) value of desmin was 489.70 ± 451.83 in group A and 71.15 ± 61.14 in group B (P lt; 0.05) and the ratio of positive area to thetotal vision area was 0.314 3 ± 0.197 3 in group A and 0.102 8 ± 0.062 8 in group B (P lt; 0.05); the Dys IA value was 5 424.64 ± 2 658.01 in group A and 902.12 ± 593.51 in group B (P gt; 0.05) and the ratio of positive area to the total vision area was 0.323 7 ± 0.117 7 in group A and 0.035 2 ± 0.032 9 in group B (P lt; 0.05). Conclusion Myoblast transplantation has certain therapeutic effect on DMD of mice.
Objective To investigate the effect of NGF on fracture heal ing, and to study the role of BMP-2 induced osteoblast. Methods Sixty cleaned male Kunming mice (aging 6-8 weeks and weighing 23-25 g) were made fracture models in the middle of femoral shaft and randomly divided into four groups (groups A, B, C and D, n=15). Fracture was treated with NGF/ normal sal ine, BMP-2, BMP-2 /NGF/normal sal ine, and normal sal ine in groups A, B, C and D, respectively. After 14, 21 and 28 days, the specimens were selected from 5 mice each group to do the biochemical and histological analysis. Beforethe mice were killed, the arteriovenous blood was taken from their eye-ball to test the ALP activity. Results After 14 days,21 days and 28 days, the gross observation showed that the size and hardness of bone tissue, and callus tissue growth increased in groups A, B and C order and were higher than those in group D; the X-ray films showed that the calcified area increased in groups A, B and C order and were higher than those in group D; the histological observation showed that the trabecular maturity increased in groups A, B and C order and were higher than those in group D. The osteoblast area, the gray degree value of the radiographs in callus tissue, the ALP contents of serum and callus tissue, calcium content of callus tissue and net weight of callus were higher in groups A, B and C than in group D. There were significant differences (P lt; 0.05) in osteoblast area and gray degree values of the radiographs at 14, 21 and 28 days; in ALP contents of serum at 14 days; in ALP contents of callus tissue at 14 days and 21 days; in calcium content of callus tissue at 21 days and 28 days among 4 groups. There were significant differences in net weight of callus between groups B, C and groups A, D at 14 days (P lt; 0.05). At 21 days and 28 days, the trabecular surface index of osteoblast, the average trabecular volume and the mean trabecular width decreased as time went on, having an increase order of groups A, B, C and was higher in groups A, B, C than in group D, showing significant differences among 4 groups (P lt; 0.05). Conclusion NGF promotes the heal ing of fractures. NGF possesses synergistic effect on ectopic bone formation induced by BMP-2.
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.
Objective To evaluate the effects and the molecular mechanism of Liuwei dihuang pills in preventing steroid-induced osteonecrosis of the femoral head (ONFH) so as to provide an expremental basis for preventing ONFH cl inically. Methods Thirty-six adult Kunming mice (weighing 40-50 g, 46 g on average) were randomly divided into three groups (n=12): group A (control group), group B (model group) and group C (prevention group). In groups B and C, ONFHmice models were produced by intraperitoneal injection of horse serum at first (10 mL/kg) and a second injection of horse serum intraperitoneally (5 mL/kg) and prednisolone intramuscularly [45 mL/(kg?day), for 5 days] 2 weeks later. At the same time, the mice in group C were given Liuwei dihuang pills intragastrically [2 g/(kg?day)] and were given normal sal ine [10 mL/(kg?day)] in group B. In group A, mice were given normal sal ine intramuscularly and intragastrically as controls. The animals were sacrificed 2, 4, and 8 weeks after first treatment with prednisone, and femoral heads and l ivers were harvested to do histopathology analysis and apoptosis assay. Results Other mice survived throughout the experiment period except two death at 7 and 11 days after second injection of horse serum intraperitoneally in group B and one death at 24 hours after second injection of horse serum intraperitoneally in group C. The appearance and shape of the femoral head and the surface of cartilages were all normal. The histological observation showed: normal structures of l iver and femoral head were seen in group A at each time point; swell ing l iver cells with small fat vacuole, unclear structure of hepatic cords and narrower sinus hepaticus were seen, the bone trabeculae of femoral head was thin, sparse and collapsed in some regions and the changes became more obvious with time in group B; group C had similar results to group A. The percentage of empty osteocyte lacunae was significantly higher in group B than in groups A and C (P lt; 0.01). The osteoprotegrin expression significantly decreased and the osteoprotegrin l igand expressionsignificantly increased in group B when compared with groups A and C (P lt; 0.01). Apoptosis analysis showed that the apoptosis index in group B was significantly higher than that in groups A and C (P lt; 0.01). Conclusion Liuwei dihuang pills can prevent steroid-induced ONFH by improving l ipid metabol ism, releiving bone lesion, and protecting against cell death.
ObjectiveTo establish an animal model of anaplastic thyroid cancer with high metastatic activity as in human body. MethodsHuman anaplastic thyroid cancer cell line TAK was injected into one of the lateral lobes of the thyroid gland, as well as in the subcuitis in a series of nude mice. Mice were sacrificed when found moribund, and autopsy and histology were performed subsequently.ResultsThe implantation of human anaplastic thyroid cancer cells in an ectopic enviroment did not permit expression of metastasis potential. In contrast, intrathyroid implantation did. Lymph node (5/10), lung (3/10) and one metastasis (1/10) were noted upon histological examination. ConclusionAn animal model with high metastatic activity is established when human anaplastic thyroid cancer cell line TAK is implanted orthotopically into nude mice.
ObjectiveTo observe the effect of conditional knocking out (KO) vascular endothelial growth factor (VEGF) gene on the mouse model of oxygen induced retinopathy (OIR).MethodsThe conditional VEGF KO mice were generated using Cre-Loxp technology, resulting in the deletion of VEGF in a portion of Müller cells permanently in mouse retina. Cre positive was CKO mice, Cre negative was NKO mice. OIR was induced by keeping mice in 75% oxygen at postnatal 7 days (P7) to P12 and in room air from P12 to P17 (each 20 mice for CKO and NKO, respectively). The mice mortality was analyzed. At day P17, the percentage of retinal avascular area was calculated using retinal flat-mounting with fluorescence angiography, the number of vascular endothelial cell nucleus breaking through retinal inner limiting membrane was counted with hematoxylin eosin (HE) staining of retinal sections, and the expression of hypoxia-inducible factor-1α (HIF-1α) was detected by immunofluorescence analysis. ResultsDuring the development of OIR, the mortality rate of CKO mice (65.00%) was higher than that of NKO mice (30.00%) with the significant difference (x2=4.912, P=0.027). At day P17, all the mice retinas were harvested. The retinal fluorescence angiography displayed that the normal retinal vascularization of CKO mice was delayed, and large avascular areas were observed. Meanwhile, rare new vascular plexus was found in CKO mice and the thickness of whole retina decreased dramatically. In contrast, NKO mice developed larger area of normal retinal vascular network structure with higher blood vessel density and more new vascular plexus with obvious fluorescein leakage. The percentage of avascular area in CKO mice [(28.31±11.15)%] was higher than NKO mice [(16.82±7.23)%] with the significant difference (t=2.734, P=0.014). The HE staining of retinal sections indicated smaller counts of vascular endothelial cell nucleus breaking through retinal inner limiting membrane in CKO mice (26.10±6.37) when compared to NKO mice (28.80±7.59) , the difference was significant (t=2.437, P=0.016). The immunofluorescence analysis showed stronger expression of HIF-1α in CKO mice than NKO mice, which was mainly located in the retinal ganglion cell layer.ConclusionsThe local VEGF gene knockout partially inhibits retinal neovascularization in OIR mice. However, it also suppresses the normal retinal blood vascular development with a decrease of OIR mice survival ability.
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.
ObjectiveTo investigate the mechanism of muscle-derived cells (MDCs) in repairing sciatic nerve defects in mice by observing the early growth of damaged peripheral nerves.MethodsThe hind limb skeletal muscles of mice carrying enhanced green fluorescent protein (EGFP) was collected to extract and culture EGFP-MDCs to P1 generation for later experiments. Five-mm-long nerve defects were created in the right sciatic nerves of C57BL/6 mice to establish a peripheral nerve defect model. The two stumps of sciatic nerve were bridged with 7-mm-long polyurethane (PUR) conduit. For the MDC group, EGFP-MDCs were injected into the PUR conduit. The PUR group without EGFP-MDCs was used as the negative control group. At 1 and 2 weeks after operation, the proximal and distal nerve stumps of the surgical side were collected to generally observe the early growth of nerve. Immunofluorescence staining of S100β, the marker of Schwann cells, was performed on longitudinal frozen sections of nerve tissues to calculate the maximum migration distance of Schwann cells, and observe the source of the Schwann cells expressing S100β. Immunofluorescence staining of phosphorylated erb-b2 receptor tyrosine kinase 2 (p-ErbB2) and phosphorylated focal adhesion kinase (p-FAK) in transverse frozen sections of nerve tissue was performed to calculate the positive rates of both proteins.ResultsThe general observation showed that the proximal and distal stumps of the surgical side in PUR group were not connected at 1 and 2 weeks after operation, while the bilateral nerve stumps in the MDC group were connected at 2 weeks after operation. Immunofluorescence staining showed that the Schwann cells expressing S100β in proximal and distal nerve stumps of PUR group and MDC group was not connected at 1 week after operation. At 2 weeks after operation, the Schwann cells expressing S100β in the two nerve stumps of the MDC group were connected, but not in the PUR group. At 2 weeks after operation, the sum of the maximum migration distance of Schwann cells in the regenerated nerve in both two groups was significantly increased when compared with that in each group at 1 week after operation, and that of MDC group was significantly higher than that in the PUR group at both 1 and 2 weeks after operation, the differences were all significant (P<0.05). At 1 week after operation, the positive rates of p-ErbB2 and p-FAK in the proximal nerve stump of MDC group were significantly higher than those in PUR group (P<0.05). There was no significant difference in the positive rate of p-ErbB2 of proximal stump between the two groups at 2 weeks after operation (t=0.327, P=0.747), while the positive rate of p-FAK of MDC group was significantly higher than that of PUR group (t=4.470, P=0.000). At 1 and 2 weeks after operation, the positive rates of p-ErbB2 and p-FAK in the distal stump of MDC group were significantly higher than those in PUR group (P<0.05). At 1 and 2 weeks after operation, part of Schwann cells expressing S100β, which were derived from EGFP-MDCs, could be observed in the regenerated nerves of MDC group.ConclusionMDCs can promote the phosphorylation of ErbB2 and FAK in the nerve stumps of mice, and promote the migration of Schwann cells. MDCs can be differentiated into cells expressing the Schwann cell marker S100β, or as other cellular components, to involve in the early repair of peripheral nerves.
Objective Aseptic loosening of prosthesis is associated with peri prosthetical osteolysis caused by osteoclast activation. Receptor activator of nuclear factor kappa B (NF-κB) l igand (RANKL)/receptor activator of NF-κB (RANK) signalpathway is fundamental in osteoclast activation. To determine whether RANKL antibody can inhibit inflammatory osteolysis in a osteolysis model of mouse. Methods Sixty female BALB/c mice (aged 8-10 weeks, weighing 18-20 g) were selected. The skull bone piece was harvested from 20 mice as the donor of bone graft; the subcutaneous air pouches (2 cm × 2 cm) models were established on the back of the other 40 mice and the skull bone piece was inserted into the air pouches. The 40 mice were equally divided into groups A (negative control group), B (positive control group), C (low-dose RANKL antibody group), and D (high-dose RANKL antibody group). At 1 day after bone graft, 0.5 mL PBS was injected into the pouch of group A, 0.5 mL PBS containing titanium particle into groups B, C, and D. At 2 days before the titanium particle was injected, RANKL antibody (0.1 mL) were injected into the pouch of group C (50 μg/mL) and group D (500 μg/mL), respectively every day for 2 days, and 0.1 mL PBS into groups A and B. At 14 days after bone implantation, the pouchmembranes containing implanted bone were harvested for gross observation and histological analyse. Results All mice survived to the end of experiment, and incisions healed well. The gross observation showed that inflammatory responses, exudation, and vascular proliferation were obvious in group B, and were inconspicuous in groups A, C, and D. The histological analysis showed that significantly more infiltration of inflammatory cells, more obvious bone resorption, more bone collagen loss, and more positive staining area were observed in group B than in groups A, C, and D. There were significant differences in inflammatory cell number, pouch membrane thickness, bone collagen loss, and osteoclast content between group B and groups A, C, and D (P lt; 0.05). Conclusion RANKL antibody can directly blockRANKL/RANK signal pathway, which is an efficient therapy to inhibit bone absorption associated with implant wearing particles.