To observe the effect of different dosage of curcumin on expression of MMP-2 and MMP-9 in the tissue of cystiform in air-pouch mouse models after the injection of polyethylene wear particles, and to investigate its mechanism of intervening inflammatory response induced by wear particles. Methods Seventy-two kunming strain mice were used to establ ish air-pouch animal models by referring to the method of Yang et al. and injecting 3 mL suspension of ultrahigh molecular weight polyethylene wear particles (concentration 1 × 108 cells/mL) into dorsal cyst cavity. Then the animals were randomized into 3 groups (n=24 per group): group A (control group), 0.6 mL/day normal sal ine by gavage; group B(low-dosage experimental group), 0.6 mL/day curcumin solution at a concentration of 1.6 mg/mL by gavage; group C (highdosage experimental group), 0.6 mL/day curcumin solution at a concentration of 3.2 mg/mL by gavage. General condition of the animals was observed after operation. The mice were killed 3, 7 and 14 days after operation (8 mice per group at a time), the tissue of cystiform was harvested to receive gross, histology and immunohistochemistry observation, as well as RT-PCR and Western blot detection. Results All mice survived till the end of experiment. White cystiform tissue was evident on the back of mice subcutaneously in each group. For diameter of the cyst cavity at each time point, group A was obviously greater than groups B and C, and group C was significantly less than group B. Microscope observation showed that inflammatory response in group A was ber than that of groups B and C, and group C was obviously less than group B at 7 and 14 days. There was a significant difference between groups B and C and group A in terms of MMP-2 and MMP-9 expression at 7 and 14 days after curcumin del ivery (P lt; 0.05), and no significant differences were evident at 3 days (P gt; 0.05). There was no significant difference between group B and group C in MMP-2 expression at 7 days after curcumin del ivery (P gt; 0.05), and significant difference was evident at 14 days (P lt; 0.05). There was significant difference bewteen group B and group C in MMP-9 expression at 7 and 14 days after curcumin del ivery (P lt; 0.05). Nuclear translocation of NF-κB P65 was inhibited remarkably after curcumin del ivery,and there were significant differences among three groups at 7 and 14 days (P lt; 0.05), and no significant differences were evident at 3 days (P gt; 0.05). Conclusion Ultra-high molecular weight polyethylene wear particles can stimulate expression of MMP-2 and MMP-9 in cystiform tissue. Curcumin can restrain expression of MMP-2 and MMP-9 in cystiform tissue of air-pouch animal models, and expression of MMP-2 and MMP-9 may be regulated by the activation of NF-κB.
Objective
To study the transfection and expression of pleiotrophin (Ptn) gene in mice adipose-derived stem cells (ADSCs) so as to provide a new approach for the treatment of ischemic injury.
Methods
ADSCs from clean inbred C57BL/6W mice (weighing, 15-20 g) were isolated and cultured in vitro. The cell surface markers (CD29 and CD44) of ADSCs were identified by flow cytometry. The ADSCs were transfected with plasmid pIRES2-LEGFPN1 (containing Ptn gene coding sequence) as experimental group (group A) and with plasmid pLEGFP-N1 (containing GFP gene coding sequence) as control group (group B). After ADSCs were transfected by different plasmids respectively, the cells containing Ptn gene were selected by G418 (the best selected concentration was 200 μg/mL), and the immunophenotype of the cells was identified by flow cytometry after transfection. Meanwhile, real-time fluorescence quantitative PCR and Western blot were used to analyse the expression levels of Ptn mRNA and PTN protein in selected cells.
Results
The mice ADSCs were isolated and cultured successfully in vitro. The positive rates of the cell surface markers CD29 and CD44 of ADSCs were 99.5% and 95.8%, respectively; the double positive rate of CD44 and CD29 was 93.6%. The positive rates of the cell surface markers CD29 and CD44 of ADSCs were 99.1% and 95.6%, respectively after transfection of Ptn gene; the double positive rate of CD44 and CD29 was 93.4%. The expression levels of Ptn gene and PTN protein in group A were significantly higher than those in group B (P lt; 0.05).
Conclusion
The ADSCs can be stablely transfected by Ptn gene, the transfected ADSCs can express PTN protein highly, which is a new idea for tissue engineering of vascular reconstruction.
ObjectiveTo study microRNA (miRNA) involved in the regulation of sinus cell differentiation by comparing sinus node, atrial myocardium, and ventricular myocardium specific miRNA expression profile differences in Kunming mice.
MethodsA total of 180 Kunming mice, aged 60-90 days and weighing 35-45 g, were selected without gender differences after the method of anatomical localization for sinus node had been confirmed by preliminary experiments in another 10 Kunming mice. All the sinus node, atrial myocardium, and ventricular myocardium tissue from 180 mice were dissected and frozen by liquid nitrogen. The structure of tissue was observed by HE staining. Total RNA were extracted and quality-controlled before hybridize with miRNA chip. The chips with miRNA were used to screen specific miRNAs; and correlation analysis of gene function was done.
ResultsThe area of mice sinus node located at juncture of the superior vena cava and the right atrium junction with crista as its longitudinal axis, ranged 2.0 mm×1.5 mm×1.0 mm. HE staining showed the sinus cells were less, with no stripes, lightly stained cytoplasm, large and round nucleus, and there were much fibrous connective tissue around cells with a visible sinus node artery. The miRNA microarray results showed that compared with atrial myocardium and ventricular myocardium, there were 39 differentially expressed miRNAs in sinus node, including 12 up-regulated miRNAs and 27 down-regulated miRNAs. Based on the regulatory networks of differential miRNA and target gene, the regulatory miRNA was obtained.
ConclusionThe differentially expressed miRNA in mice sinus node possibly may be involved in the regulation of sinus cell differentiation.
As a relatively novel technique for drug delivery, the needle-free injection technique is characterized by transporting the drug liquid to the designated subcutaneous position through a high-speed micro-jet. Although this technique has been applied in many fields, the research on its drug dispersion mechanism and injection performance is insufficient. The presented study aims to identify critical parameters during the injection process and describe their influence on the injection effect. The injection completion rate and performance of a needle-free injector under various operating conditions were compared based on mouse experiments. The results show that the nozzle diameter imposes a more significant influence on jet characteristics than other injection parameters. Moreover, the injection completion rate increases with the nozzle diameter. The nozzle diameters of 0.14 mm and 0.25 mm correspond to injection completion rates of 89.7% and 95.8%, respectively. Furthermore, by analyzing the rate of blood glucose change in the tested mice, it is found that insulin administration through the needle-free injection can achieve a drug effect duration longer than 120 min, which is better than that obtained using conventional needle-syringe technique. In summary, the obtained conclusions can provide an important reference for the optimal design and extending application of the air-powered needle-free injector.
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 explore a new method for the pre-degeneration of peripheral nerve in vitro for obtaining many effective Schwann cells so as to provide a large number of seed cells for the research and application of tissue engineered nerves.
Methods
The bone marrow derived cells (BMDCs) from transgenic green fluorescent protein C57BL/6 mouse and the sciatic nerve segments from the C57BL/6 mouse were co-cultured to prepare the pre-degeneration of sciatic nerve in vitro (experimental group, group A), and only sciatic nerve was cultured (control group, group B). At 7 days after culture, whether BMDCs can permeate into the sciatic nerve in vitro for pre-degeneration was observed by gross and immunohistofluorescence staining. And then Schwann cells were obtained from the sciatic nerves by enzymic digestion and cultured. The cell number was counted, and then the purity of primary Schwann cells was determined using immunohistofluorescence staining and flow cytometer analysis.
Results
At 7 days after pre-degeneration, gross observation showed that enlargement was observed at nerve stumps, and neuroma-like structure formed; the group A was more obvious than group B. Immunohistofluorescence staining showed many BMDCs permeated into the nerve segments, with positive F4/80 staining in group A. After culture, the yield of Schwann cells was (5.59 ± 0.19) × 104 /mg in group A and (3.20 ± 0.21) × 104/mg in group B, showing significant difference (t=2.14, P=0.03). At 48 hours after inoculation, the cells had blue bipolar or tripolar cell nuclei with small size and red soma by immunohistofluorescence staining; fibroblasts were flat polygonal with clear nucleus and nucleolus, showing negative p75NTR staining; and there were few of fibroblasts in group A. The purity of Schwann cells was 88.4% ± 5.8% in group A and 76.1% ± 3.7% in group B, showing significant difference (t=2.38, P=0.04). And the flow cytometer analysis showed that the purity was 89.6% in group A and 74.9% in group B.
Conclusion
BMDCs can promote the pre-degeneration of peripheral nerve in vitro, and it is a new method to effectively obtain Schwann cells for tissue engineered nerve.
Objective To study the immunological tolerance induced by blocking the second signal of T cell with extrinsic cytotoxic T lymphocyteassociated antigen 4 immuno globlin(CTLA4-Ig). Methods Fifty-four BALB/C mice, inbred strains, were employed as recipients of bone allografts, using a model of heterotopic muscle pouch. The 54 mice were divided into 3 groups and18 for each group. The first group, in which the donor was C57BL/6 with intraperitoneal injection ofL6(as a control), was named AL group. The second group,also C57BL/6 with injection CTLA4-Ig, was named AC group. The third group,homologous BALB/C with injection of PBS buffer solution, was named AB group.The serum antibody, lymphocyte proliferation of the second stimulation by splenic cell and bone supernatant of donor, the analysis of lymphocyte subsets, a regraft experiment and histology were determined 2, 4 and 6 weeks after transplantation. The second transplantation was to regraft C57BL/6(BC group) and C3H(BHgroup) mice respectively after first 12 mice being transplantated with C57BL/6 and injected with CTLA4-Ig as to detect donor-specificity of immunological tolerance. Results Compared with AB group, AL group created more intensive immune rejection: CD4 T cell subsets(Plt;0.05), the serum antibody(Plt;0.05) and lymphocyteproliferation of the second stimulation by splenic cell and bone supernatant ofdonor (Plt;0.01 and 0.05) were significantly increased. However, the results of AC group showed that CTLA4-Ig significantly inhibited the immune rejection: CD4T cell subsets(Pgt;0.05), the serum antibody (Pgt;0.05), and lymphocyte proliferation of the second stimulation(Pgt;0.05) were similar to those of AB group. Histological observation of AC group showed that lymphocyte infiltration disappeared,cartilage and new bone formed, and bone marrow cavities emerged. A regraft experiment showed that CD4 T cell subsets (Plt;0.05) and lymphocyte proliferation of the second stimulation by splenic cell and bone supernatant of donor(Plt;0.05), BC group was significantly lower than those of BH group. So theimmunological tolerance induced by CTLA4-Ig was of donor-specificity. Conclusion The immunological tolerance induced by CTLA4-Ig was prolonged for 6 weeks. This study provides a brand-new path for bone transplantation, which can be helpful to other organ transplantation.
【Abstract】ObjectiveTo investigate the effect of RNA editing enzyme ADAR1 on the function of lymphocyte immune by transferring mouse lymphocytes with plasmid of sense siRNA and by suppressing the expression of ADAR1. MethodsThe cell strains of human hepatic cellular carcinoma （HCC） were frozen and thawed repeatedly to prepare for tumor soluble antigen. The isolated mouse lymphocytes, which were transferred with antisense siRNA plasmid of ADAR1 and were sensitized with soluble tumor antigen were used as the study group; those which were not transferred but were sensitized were used as the control group. The 3HTdR adulteration experiment was used to test the sensitivity of lymphocytes. The effect of ADAR1 on lymphocyte immunity was detected by lymphocytotoxicity tests. ResultsThe observation of the isolated lymphocytes implied that the growth cycle of lymphocyte was 10-14 days. The 3HTdR adulteration experiment showed the result was optimal. The number of HCCs decreased significantly for both of the groups compared with those in the blank holes, but the amplitude was much larger in the control group. The expression of ADAR1 in lymphocytes of the study group was significantly lower than that of the control group, which demonstrated that the RNA plasmid of ADAR1 suppressed the expression of ADAR1 in sensitized lymphocytes and the suppressing rate of the control group (87.47±4.62)% was significantly higher than that of study group （53.19±3.95）%. The function of lymphocytes killing targetcells in the study group was significantly inferior to that of control group (P＜0.05). ConclusionRNA editing enzyme ADAR1 may play an important role in mouse cellullar immunologic response and it is possible to attenuate the cellimmune response by depressing the expression of ADAR1.
ObjectiveTo establish the system of isolation, cultivation, and identification of the neural stem cells (NSCs) from subventricular zone (SVZ) of neonatal mice so as to seek for the appropriate seed cells for potential therapeutic interventions of neurological disorders.
MethodsNSCs were isolated enzymatically and mechanically from SVZ of neonatal mice and cultured. The cellular morphology was observed by inverted microscopy. Immunocytochemical stainings of anti-Nestin and anti-SOX-2 were used to identify NSCs of passage 3. To study the differentiation of NSCs, NSCs were plated into 24-wells in the medium supplemented without epidermal growth factor (EGF) and basic fibroblastic growth factor (bFGF) for 3 or 7 days. To compare the differentiation and proliferation potential of NSCs with different cultivation time, the BrdU pulse-labeling method and MTT test were used. To identify neurons and astrocytes, the anti-β-tubulin Ⅲ (Tuj-1) and anti-glial fibrillary acidic protein (GFAP) staining were used.
ResultsThe cells of the SVZ can be isolated and cultured in vitro, and these cells began to form neurospheres after cultured for 3 days at primary passage. While cultured for 7 days, these cells formed more neurospheres, and the volume of the neurospheres became bigger than neurospheres cultured for 3 days. In addition, after cultured for 7 days, the phenomena of fusion of neurospheres and adherent differentiation of neurospheres were observed under inverted microscope. These cells were provided with the typical phenotype of NSCs. The immunofluorescence staining results revealed that these cells showed positive immunoreactivity to Nestin and SOX-2. During the 4 hours BrdU pulse, the number of proliferated NSCs cultured for 3 days (75.817±2.961) was significantly higher than that of NSCs cultured for 7 days (56.600±4.881) (t=3.366, P=0.028). The results of MTT assay revealed that the absorbance (A) value of NSCs cultured for 3 days (0.478±0.025) was significantly higher than that of NSCs which were cultured for 7 days (0.366±0.032)(t=2.752, P=0.011). After cultivated without EGF and bFGF, the percentage of Tuj-1 and GFAP positive cells in NSCs was 23.1%±3.7% and 23.7%±3.8% for 3 days and was 40.1%±3.6% and 37.1%±4.5% for 7 days, respectively, all showing significant differences (t=3.285, P=0.030; t=3.930, P=0.017).
ConclusionThe NSCs from SVZ of neonatal mice have potentials of self-renewal and multipotential differentiation in vitro. With different cultivation time, the potentials of proliferation and differentiation of NSCs are different.
Objective To investigate the effects of hypertonic saline (HTS) treatment on the function and susceptibility to sepsis of reticuloendothelial system (RES) in mice with hemorrhagic shock. Methods Forty percent of total blood volume of male Balb/c mice was withdrawn by cardiac puncture. Two hours later, the mice were treated with blood infusion and normal saline (10 ml/kg) or 7.5% NaCl (10 ml/kg).The survival rate of the mice was observed after cecal ligation and puncture (CLP). The phagocytosis function of the RES was measured by carbon clearance rate(α) and carbon amount ingested by the macrophages of liver and spleen. In vitro, the peritoneal phagocyte function in solutions of different osmotic pressor was measured by assaying neutral red amount taken in. Results The survival rate after CLP in HTS treated group was 70%, whereas all the mice in the normal saline group died. At the third hour after hemorrhagic shock, the RES carbon clearance rate(α) and carbon amount ingested by the macrophages of liver in the HTS treated mice were 5.61±0.42 and 0.59±0.19 respectively, significantly higher than those in the normal saline treated mice (4.15±0.62， 0.42±0.16). In vitro, hyperosmolarity below 40 mmol/L had no significant effects on the phagocytosis activity of peritoneal macrophages in mice. Conclusion Treating hemorrhagic shock with HTS can decrease the susceptibility to sepsis and improve the RES phagocytosis function indirectly.
