Objective To Investigate the effects of lithocholic acid (LCA) on the balance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods Twelve 10-week-old SPF C57BL/6J female mice were randomly divided into an experimental group (undergoing bilateral ovariectomy) and a control group (only removing the same volume of adipose tissue around the ovaries), with 6 mice in each group. The body mass was measured every week after operation. After 4 weeks post-surgery, the weight of mouse uterus was measured, femur specimens of the mice were taken for micro-CT scanning and three-dimensional reconstruction to analyze changes in bone mass. Tibia specimens were taken for HE staining to calculate the number and area of bone marrow adipocytes in the marrow cavity area. ELISA was used to detect the expression of bone turnover markers in the serum. Liver samples were subjected to real-time fluorescence quantitative PCR (RT-qPCR) to detect the expression of key genes related to bile acid metabolism, including cyp7a1, cyp7b1, cyp8b1, and cyp27a1. BMSCs were isolated by centrifugation from 2 C57BL/6J female mice (10-week-old). The third-generation cells were exposed to 0, 1, 10, and 100 μmol/L LCA, following which cell viability was evaluated using the cell counting kit 8 assay. Subsequently, alkaline phosphatase (ALP) staining and oil red O staining were conducted after 7 days of osteogenic and adipogenic induction. RT-qPCR was employed to analyze the expressions of osteogenic-related genes, namely ALP, Runt-related transcription factor 2 (Runx2), and osteocalcin (OCN), as well as adipogenic-related genes including Adiponectin (Adipoq), fatty acid binding protein 4 (FABP4), and peroxisome proliferator-activated receptor γ (PPARγ). Results Compared with the control group, the body mass of the mice in the experimental group increased, the uterus atrophied, the bone mass decreased, the bone marrow fat expanded, and the bone metabolism showed a high bone turnover state. RT-qPCR showed that the expressions of cyp7a1, cyp8b1, and cyp27a1, which were related to the key enzymes of bile acid metabolism in the liver, decreased significantly (P<0.05), while the expression of cyp7b1 had no significant difference (P>0.05). Intervention with LCA at concentrations of 1, 10, and 100 μmol/L did not demonstrate any apparent toxic effects on BMSCs. Furthermore, LCA inhibited the expressions of osteogenic-related genes (ALP, Runx2, and OCN) in a dose-dependent manner, resulting in a reduction in ALP staining positive area. Concurrently, LCA promoted the expressions of adipogenic-related genes (Adipoq, FABP4, and PPARγ), and an increase in oil red O staining positive area. Conclusion After menopause, the metabolism of bile acids is altered, and secondary bile acid LCA interferes with the balance of osteogenic and adipogenic differentiation of BMSCs, thereby affecting bone remodelling.
ObjectiveTo explore the effect and mechanism of directive differentiation of microglia by SN50 on hypoxia-caused neurons injury in mice.MethodsThe microglia were isolated and purified from brain tissue of new-born BALB/c mice through differential velocity adherent and vibration technique. The quantity of the microglia was identified by immunofluorescence staining of inducible nitric oxide synthetase (iNOS) and ionized calcium binding adapter molecule 1 (Iba1) and real-time fluorescence quantitative PCR (qRT-PCR) for special expression genes [iNOS, CD32, and interlenkin 10 (IL-10)]. Then the microglia were cultured with SN50, and the expressions of nuclear factor κB (NF-κB), differentiation-related genes (iNOS, CD11b, IL-10, and CD206), and apoptosis were detected by Western blot, qRT-PCR, and flow cytometry, respectively. The hypoxia model of neuron was established, and the cell apoptosis was evaluated by MTT after 0, 2, 6, 12, 24, and 48 hours of anoxic treatment. The apoptosis related markers (Bcl-2 and Caspase-3) were measured by Western blot and flow cytometry. In addition, the neurons after anoxic treatment were co-cultured with SN50 treated microglia (experimental group) and normal microglia (control group) for 24 hours. And the cell viability and apoptosis related markers (Bcl-2 and Caspase-3) were also measured.ResultsImmunofluorescence staining and qRT-PCR analysis showed that the cells expressed the specific proteins and genes of microglia. Compared with the normal microglia, the relative expressions of NF-κB protein and iNOS and CD11b mRNAs in the microglia treated with SN50 significantly decreased (P<0.05), the relative expressions of IL-10 and CD206 mRNAs significantly increased (P<0.05), and the cell apoptosis rate had no significant change (P>0.05). Compared with the normal neurons, the cell viability, the relative expressions of Bcl-2 and Caspase-3 proteins after anoxic treatment significantly decreased (P<0.05), while the relative expressions of cleaved-Caspase-3 protein and cell apoptosis rate of neurons significantly increased (P<0.05). In the co-culture system, the cell viability, the relative expressions of Bcl-2 and Caspase-3 proteins were significantly higher in experimental group than those in control group (P<0.05), while the relative expressions of cleaved-Caspase-3 protein and cell apoptosis rate were significantly lower in experimental group than those in control group (P<0.05).ConclusionSN50 can induce the microglia differentiation into M2 type through NF-κB pathway. The SN50-induced microglia can protect neurons from hypoxic injury.
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.
摘要:目的:探讨表达吲哚胺2,3二氧化酶(IDO)的KC对同种异体小鼠移植皮片存活时间的影响及其机制。方法:构建BABL/c →C57BL/6的皮肤移植模型,分别于移植术后第2、7、14天输注KC,于移植术后第7天每组各取2只皮瓣行HE染色和TUNEL以检测淋巴细胞浸润和凋亡情况。KaplanMeier对数秩检验对各组进行生存分析。结果:输入表达IDO和FasL的KC能明显延长BABL/c →C57BL/6皮肤移植模型中皮肤移植物的存活时间,1－甲基色氨酸能阻断此效应。IFNγ组皮瓣浸润淋巴细胞的凋亡率较高(Plt;0.05)。结论:表达IDO和FasL的KC在体内能明显延长同种异体小鼠皮片的存活时间,IDO在KC维持外周免疫耐受中发挥重要作用。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.
In the development of radio frequency (RF) coils for better quality of the mini-type permanent magnetic resonance imager for using in the small animal imaging, the solenoid RF coil has a special advantage for permanent magnetic system based on analyses of various types of RF coils. However, it is not satisfied for imaging if the RF coils are directly used. By theoretical analyses of the magnetic field properties produced from the solenoid coil, the research direction was determined by careful studies to raise further the uniformity of the magnetic field coil, receiving coil sensitivity for signals and signal-to-noise ratio (SNR). The method had certain advantages and avoided some shortcomings of the other different coil types, such as, birdcage coil, saddle shaped coil and phased array coil by using the alloy materials (from our own patent). The RF coils were designed, developed and made for keeled applicable to permanent magnet-type magnetic resonance imager, multi-coil combination-type, single-channel overall RF receiving coil, and applied for a patent. Mounted on three instruments (25 mm aperture, with main magnetic field strength of 0.5 T or 1.5 T, and 50 mm aperture, with main magnetic field strength of 0.48 T), we performed experiments with mice, rats, and nude mice bearing tumors. The experimental results indicated that the RF receiving coil was fully applicable to the permanent magnet-type imaging system.
Objective To study the effects of morroniside (MOR) on the proliferation and osteogenic differentiation of mouse MC3T3-E1 cells. MethodsThe 4th generation MC3T3-E1 cells were randomly divided into 6 groups: control group (group A), MOR low dose group (10 μmol/L, group B), MOR medium-low dose group (20 μmol/L, group C), MOR medium dose group (40 μmol/L, group D), MOR medium-high dose group (80 μmol/L, group E), and MOR high dose group (100 μmol/L, group F). The proliferation activity of each group was detected by cell counting kit 8 (CCK-8) assay; the bone differentiation and mineralized nodule formation of each group were detected by alizarin red staining; real-time fluorescence quantitative PCR (RT-qPCR) was performed to detect cyclin-dependent kinase inhibitor 1A (P21), recombinant Cyclin D1 (CCND1), proliferating cell nuclear antigen (PCNA), alkaline phosphatase (ALP), collagen type Ⅰ (COL-1), bone morphogenetic protein 2 (BMP-2), and adenosine A2A receptor (A2AR) mRNA expressions; Western blot was used to detecte the expressions of osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), and adenosine A2AR protein. ResultsThe CCK-8 assay showed that the absorbance (A) values of groups B to F were significantly higher than that of group A at 24 hours of culture, with group C significantly higher than the rest of the groups (P<0.05). The MOR concentration (20 μmol/L) of group C was selected for the subsequent CCK-8 assay; the results showed that the A values of group C were significantly higher than those of group A at 24, 48, and 72 hours of culture (P<0.05). Alizarin red staining showed that orange-red mineralized nodules were visible in all groups and the number of mineralized nodules was significantly higher in groups B and C than in group A (P<0.05). RT-qPCR showed that the relative expressions of P21, CCND1, and PCNA mRNAs were significantly higher in group C than in group A (P<0.05). The expressions of ALP, BMP-2, COL-1, and adenosine A2AR mRNAs in groups B to E were significantly higher than those in group A, with the expressions of ALP, BMP-2, COL-1 mRNAs in group C significantly higher than the rest of the groups (P<0.05). Compared with group A, the expressions of OPN and RUNX2 proteins in groups B and C were significantly increased, while those in group D and E were significantly inhibited (P<0.05). There was no significant difference between groups B and C and between groups D and E (P>0.05). The relative expression of adenosine A2AR protein in groups B to E was significantly higher than that in group A, with group C significantly higher than the rest of the groups (P<0.05). Conclusion MOR can promote the proliferation and osteogenic differentiation of MC3T3-E1 cells; the mechanism of MOR may be achieved by interacting with adenosine A2AR.
Objective To investigate the regulatory effects of miR-26a-5p on the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) by regulating cAMP response element binding protein 1 (CREB1). Methods The adipose tissues of four 3-4 weeks old female C57BL/6 mice were collected and the cells were isolated and cultured by digestion separation method. After morphological observation and identification by flow cytometry, the 3rd-generation cells were subjected to osteogenic differentiation induction. At 0, 3, 7, and 14 days after osteogenic differentiation induction, the calcium deposition was observed by alizarin red staining, ALP activity was detected, miR- 26a-5p and CREB1 mRNA expressions were examined by real-time fluorescence quantitative PCR, and CREB1 protein and its phosphorylation (phospho-CREB1, p-CREB1) level were measured by Western blot. After the binding sites between miR-26a-5p and CREB1 was predicted by the starBase database, HEK-293T cells were used to conduct a dual-luciferase reporter gene experiment to verify the targeting relationship (represented as luciferase activity after 48 hours of culture). Finally, miR-26a-p inhibitor (experimental group) and the corresponding negative control (control group) were transfected into ADSCs. Alizarin red staining, ALP activity, real-time fluorescent quantitative PCR (miR-26a-5p) and Western blot [CREB1, p-CREB1, Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN)] were performed at 7 and 14 days after osteogenic induction culture. Results The cultured cells were identified as ADSCs. With the prolongation of osteogenic induction culture, the number of calcified nodules and ALP activity significantly increased (P<0.05). The relative expression of miR-26a-5p in the cells gradually decreased, while the relative expressions of CREB1 mRNA and protein, as well as the relative expression of p-CREB1 protein were increased. The differences were significant between 7, 14 days and 0 day (P<0.05). There was no significant difference in p-CREB1/CREB1 between different time points (P>0.05). The starBase database predicted that miR-26a-5p and CREB1 had targeted binding sequences, and the dual-luciferase reporter gene experiment revealed that overexpression of miR-26a-5p significantly suppressed CREB1 wild-type luciferase activity (P<0.05). After 7 and 14 days of osteogenic induction, compared with the control group, the number of calcified nodules, ALP activity, and relative expressions of CREB1, p-CREB1, OCN, and RUNX2 proteins in the experimental group significantly increased (P<0.05). There was no significant difference in p-CREB1/CREB1 between the two groups (P>0.05). Conclusion Knocking down miR-26a-5p promoted the osteogenic differentiation of ADSCs by up-regulating CREB1 and its phosphorylation.
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.
ObjectiveTo investigate the effects of icariin (ICA) on serum bone turnover markers expressions and histological changes of cartilage and subchondral bone in mouse osteoarthritis (OA) model.MethodsEighty 8-week-old male C57BL/6J mouse were randomly divided into 8 groups (n=10). The OA model was established by anterior cruciate ligament transaction (ACLT). Group A: sham operation/early-stage normal saline administration; group B: sham operation/early-stage ICA administration; group C: ACLT/early-stage normal saline administration; group D: ACLT/early-stage ICA administration; group E: sham operation/late-stage normal saline administration; group F: sham operation/late-stage ICA administration; group G: ACLT/late-stage normal saline administration; group H: ACLT/late-stage ICA administration. Each animal received either ACLT or simply opening joint capsule, respectively. For groups B and D, ICA was given by gavage [10 mg/(kg·day)] on the first day after ACLT. For groups F and H, ICA was given with the same volume at 4 weeks after operation. The blood serum of the mouse was collected and prepared at 8 weeks after operation. Serum bone turnover markers and cytokines, including C-telopeptide of type I collagen (CTX), osteocalcin (OC), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and IL-1β, were measured by ELISA. Tissue samples from the knee were stained by alcian blue/hematoxylin & orange G (AB/H&OG). Histological changes of cartilage and subchondral bone were observed and evaluated by Osteoarthritis Research Society International (OARSI) scoring system.ResultsComparison between each group with early-stage administration (groups A, B, C, and D): Compared with groups A and B, the levels of CTX and OC in group C were significantly reduced (P<0.05); the levels of IL-6, TNF-α, and IL-1β and OARSI score was significantly increased (P<0.05). Compared with group C, the levels of CTX and OC in group D were significantly increased (P<0.05); the level of IL-6 was significantly reduced (P<0.05); the levels of TNF-α and IL-1β were not changed (P>0.05), and OARSI score was significantly reduced (P<0.05). Histological observation showed that the tibial cartilage loss was significantly improved. Comparison between each group with late-stage administration (groups E, F, G, and H): Compared with groups E and F, the levels of CTX and OC in group G were significantly reduced (P<0.05); the levels of IL-6, TNF-α, and IL-1β and OARSI score were significantly increased (P<0.05). Compared with group G, the level of CTX in group H were increased (P<0.05); the levels of OC, IL-6, TNF-α, and IL-1β and OARSI score were not changed (P>0.05). Histological observation showed that the tibial cartilage loss had no changes after late-stage ICA administration.ConclusionICA plays protective effects on subchondral bone, hyaline, and calcified cartilage. Meanwhile, ICA can improve bone remodeling in subchondral bone of OA to some extent. The consistent changes of serum bone markers and pathological morphology suggest that early intervention of ICA on OA is more effective.
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.