Objective To explore the effect of age and gene therapyon the differentiation of marrow mesenchymal stem cells （MSCs） of the rats. Methods MSCs from the young (1-month-old), adult (9-month-old), and the aged(24monthold) rats were expanded in culture and infected with adenovirus mediated human bone morphogenetic protein 2 gene (Ad-BMP-2). The expression of BMP-2 and osteoblastic markers such as alkaline phosphatase(ALP), collagen Ⅰ(Col Ⅰ), bone sialoprotein(BSP) and osteopontin(OPN) were assayed during the process of differentiation. Their abilities to induce ectopic bone formation in nude mice were also tested. Results There was no significant difference in the expression of BMP-2 among the 3 groups. ALP activity assay and semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) demonstrated that there were no significant differences in the expression of osteoblastic markers ALP, Col-Ⅰ, OPN and BSP amongthe 3 groups. Histomorphometric analysis indicated that there were no significant differences in the volume of the newly formed ectopic bones in nude mice amongthe 3 groups. Conclusion MSCs obtained from the aged ratscan restore their osteogenic activity following human BMP-2 gene transduction, therefore provides an alternative to treating the aged bone disease.
OBJECTIVE To determine the role of endogenous carbon monoxide(CO) in oxidant-mediated organ injury following limb ischemia-reperfusion (I/R) in rats. METHODS: Sixty-four SD rats were divided into 4 groups: Sham group, Sham + zinc protoporphyrin (ZnPP, an inhibitor of heme oxygenase activity), 2-hour ischemia followed by 4-hour reperfusion (I/R) group and I/R + ZnPP group. Carboxyhemoglobin (COHb) level in the artery blood, malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in the lung, heart, liver and kidney were detected. The 24-hour survival rate of rats was studied. RESULTS: Compared with the sham group, the COHb level and MDA content significantly increased, while the SOD activity and the survival rate significantly decreased in I/R group (P lt; 0.05). Compared with the I/R group, MDA content significantly increased, while the SOD activity, the 24-hour survival rate and COHb level significantly decreased in I/R + ZnPP group (P lt; 0.05, respectively). CONCLUSION: Limb I/R could lead to the oxidant-mediated multiple organ injury accompanied by the increase of CO level which play an important role in the defense against I/R-induced remote multiple organ injury in rats.
目的 探讨不同水温对大鼠鼻黏膜的损伤及恢复情况。 方法 126只健康成年SD大鼠随机分为A、B、C组，每组42只；A组：常温；B组：50℃；C组：70℃。另设健康成年SD大鼠2只，使用不同温度生理盐水滴入实验组大鼠双鼻中隔黏膜30次，每分钟1次。分别于第30分钟，1、3、7、14、28、42 d各取6只大鼠的刺激区鼻中隔黏膜，对照组取相应区域黏膜制作光、电镜标本观察其病理形态学改变。 结果 A组各时间的光镜下结构正常，在电镜下1、3 d有轻微改变，7 d后基本恢复。B组第30分钟、1 d在镜下有明显损伤，尤其在3 d达严重损伤， 14 d后基本恢复。C组第30分钟，1、3 d在镜下表现出严重损伤，28 d后有少数恢复，42 d后仍未能完全恢复。 结论 常温对大鼠鼻黏膜病理形态学改变较轻，恢复时间短。温度越高、刺激时间越长则病理形态学改变越重，恢复时间越长。
【Abstract】ObjectiveTo establish an efficient, effective hepatocyte isolation technique in order to increase cell production and decrease the prime cost. Methods The inferior vena cava below diaphragm was dissected and ligatured, and the inferior vena cava below liver was separated. Subsequently, the liver was perfused with EGTA through the portal vein while the inferior vena cava below liver was opened, and then the liver was harvested. The liver tissue was cut into 1 mm×1 mm×1 mm and digested at 37 ℃ water bath with Ⅳ collagenase for 30-40 minutes, then the hepatocytes were purified and cultured in CO2 incubator. The production and function of hepatocytes were assessed. ResultsThe isolated hepatocytes using this technique were more than 95% among the all isolated cells. No statistic difference was found in cell production and cell function comparing with traditional technique. But this technique was simplified and more economically. ConclusionThis modified hepatocyte isolation technique is efficient and effective. It can ensure the amount of production and purity of hepatocytes.
Objective To transfect bone marrow mesenchymal stem cells (BMSCs) of rats by recombinant adenovirus Ad-human matrix metalloproteinase 1 (hMMP-1) in vitro so as to lay the experimental foundation for the treatment of liver fibrosis with a combination of BMSCs and hMMP-1 gene transplantation. Methods BMSCs were isolated from bone marrow of 2-3 weeks old Sprague Dawley rats by whole bone marrow adherence method and identified, then transfected by recombinant adenovirus Ad-hMMP-1 carrying enhanced green fluorescent protein (EGFP) marker in vitro. The green fluorescent expression was observed by fluorescence microscope and the transfection efficiency was detected by flow cytometry to determine the optimum multiplicity of infection (MOI). BMSCs at passage 3 were divided into 3 groups: untransfected BMSCs group (group A), Ad-EGFP transfected BMSCs group (group B), and Ad-hMMP-1-EGFP transfected BMSCs group (group C); the gene and intracellular protein of hMMP-1 were detected by RT-PCR and Western blot; the ELISA assay was used to detect the supernatant protein expression, and the hMMP-1 activity was measured by fluorescent quantification kit. Results The green fluorescent was observed in BMSCs transfected by recombinant adenovirus at 24 hours after transfection; the fluorescence intensity was highest at 72 hours; and the optimum MOI was 200. The cells of 3 groups entered the logarithmic growth phase on the 3rd day and reached plateau phase on the 6th day by MTT assay; no significant difference was found in the cell proliferation rate among 3 groups (P gt; 0.05). RT-PCR, Western blot, and ELISA assay showed high expressions of the hMMP-1 gene and protein in group C, but no expression in groups A and B. The hMMP-1 activity was 1.24 nmol/(mg · min) in group C, but hMMP-1 activity was not detectable in groups A and B. Conclusion The exogenous hMMP-1 gene is successfully transfected into BMSCs of rats via recombinant adenovirus and can highly express, which lays the experimental foundation for the treatment of liver fibrosis with a combination of BMSCs and hMMP-1 gene transplantation.
Objective To study the distribution of P2 Y2 receptor in spine cord, dorsal root ganglia and sciatic nerve in rat, and to provide the basis for clarifying the mechanism of the effect of adenosine triphosphate(ATP) on the peripheral nerve regeneration. Methods Six specimens of the spine cord, dorsal root ganglia and sciatic nerve from SD rats were fixed rapidly in 4% paraformaldehyde which included DEPC, imbedded by paraffin and made into ultrathin section. According to the sequence of P2 Y2 receptor’s gene, DNA needle was adopted to detect the distribution of P2 Y2 receptor by hybridization technique in section under the light microscope after theyhad been stained in NBT liquid（50 mg/ml） and BCIP liquid （75 mg/ml）. In thecontrol group, the ultrathin section was only covered with hybridism buffer solution. The result of staining was observed. ResultsHybridization in section showed that P2 Y2 receptor was distributed mainly in the anterior horn cell of spine cordgray matter and Schwann cell of the dorsal root ganglia. No P2 Y2 receptor was observed in the sciatic nerve of both groups. Conclusion P2 Y2 receptor is located mainly in the spine cord and the dorsal root ganglia. Extracellular ATP can affect the cell of spine cord, dorsal root ganglia through P2 Y2 receptor.
Objective To study the role of hydrogen sulfide （H2S） in prophase of acute peritoneal cavity infection. Methods NaHS was taken as a donor of H2S. Seventy-two Sprague-Dawley rats were divided into 4 groups randomly：control group， cecal ligation and puncture （CLP） and treated with natural saline group，CLP and treated with NAHS group， and CLP and treated with DL-propargylglycine （PAG， an inhibitor of H2S formation） group. Selected 6 rats at 2h， 6h， and 12h after treatment in each group. The contents of TNF-αand H2S in serum and the content of MPO in intestinal tissue were measured， respectively. The histopathological change of ileum tissues were observed at 6 h after treatment in each group. Results The H2S could alleviate CLP-induced inflammation obviously， decrease the content of TNF-α in serum when inflammation，and attenuate the infiltration of neutrophilic granulocyte in small intestine. Conclusion The H2S has anti-inflammation effect in prophase of acute peritoneal cavity infection.
【摘要】 目的 探讨慢性缺氧对大鼠岩神经节神经元酸敏感离子通道（acid-sensing ion channels，ASICs）亚型3（ASIC3）和亚型2a（ASIC2a）表达的影响。 方法 将12只健康成年SD大鼠随机分为正常组和缺氧组。用免疫组织化学法（PV）观察正常和慢性缺氧大鼠岩神经节神经元ASIC3和ASIC2a的表达。 结果 给予慢性缺氧刺激后，岩神经节ASIC3阳性表达神经元数目增多（Plt;0.05），灰度值降低（Plt;0.05）；而ASIC2a阳性表达神经元数目和灰度值无明显变化（Pgt;0.05）。 结论 慢性缺氧可上调大鼠岩神经节神经元ASIC3的表达，而对ASIC2a的表达无明显影响，提示ASIC3和ASIC2a可能在岩神经节对缺氧的反应中起着不同的作用。【Abstract】 Objective To investigate the effects of chronic hypoxia on expression of acid-sensing ion channels (ASIC) 3 and ASIC2a in neurons of petrosal ganglions of rats. Methods A total of 12 SD rats were randomly assigned to control group and hypoxia group. The expressions of ASIC3 and ASIC2a of the neurons in the petrosal ganglions in the two groups were investigated with the immunohistochemical technique. Results The level of positive ASIC3 expression in the petrosal ganglions was higher in the hypoxia group than that in the control group (Plt;0.05); the difference of positive ASIC2a expression levels between the control group and the hypoxia group was not statistically significant (Pgt;0.05). Conclusion Chronic hypoxia can significantly increase the expression of ASIC3, but not that of ASIC2a, of the neurons in the petrosal ganglions, suggesting their different roles in mediating a cellular response to chronic hypoxia.
Objective To study effects of enteral immunonutrition and econutrition on intestinal mucosa barrier function in wounded rats. Methods Forty Wistar rats were randomly divided into four groups, with ten rats in each group 〔ie.control group, enteral nutrition (EN) group, enteral immunonutrition (EIN) group and enteral econutrition (EEN) group〕. After gastrostomy, rats in each group were treated with the isocaloric and isonitrogenous nutritional formulas for 7 days, respectively. The morphology of ileum membrane was studied, and the quantities of IgA+, CD3+, CD4+ and CD8+ cells (each HP) of ileum membrane were determined. Results The villus height, crypt depth, mucosal thickness (except EN group) and villus surface area of ileum were increased in EN, EIN and EEN group compared with control group (P＜0.05), but there was no significant difference among the former three groups (Pgt;0.05). The numbers of IgA+, CD3+, CD4+ and CD8+ cells were increased in EN, EIN and EEN group compared with control group (P＜0.05), and those numbers in EN group were lower than those in EIN and EEN group (P＜0.05). Conclusion EIN and EEN may improve intestine mechanical barrier function and promote restoration of small intestine mucous membrane barrier function in rats. EIN and EEN also improve intestine immune barrier function and strengthen its immune function.
ObjectiveTo compare the effect of ileal transposition (IT) and Roux-en-Y gastric bypass (RYGBP) on blood glucose and expression of glucagon-like peptide-1 (GLP-1) in Goto-Kakizaki (GK) rats with non-obese type 2 diabetes mellitus (T2DM). MethodsThirty male GK rats were randomized divided into three groups:IT group (n=10), RYGBP group (n=10), and Sham group (n=10). The mortality and complication were observed after surgery. The levels of fasting blood glucose (FBG), fasting insulin (FINS), glycosylated hemoglobin (HbA1c), and GLP-1 were determined before operation, and 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months after operation in the GK rats of 3 groups. Results① Mortality and morbility. There was no death and complication occurred in IT group and Sham group, only 5 rats of RYGBP group suffered from complication, and 2 of them died. The mortality and morbility were higher in RYGBP group than those of IT group and Sham group (P < 0.05). ② FBG. Compared with before operation in the same group, the FBG levels of IT group and RYGBP group in 1 week, 2 weeks, 1 month, 2 months, 3 months, and 6 months after operation were all lower (P < 0.05). In 1 week, 2 weeks, 1 month, 2 months, 3 months, and 6 months after operation, FBG levels of IT group and RYGBP group were all lower than those of Sham group at the same time point (P < 0.05), but there was no significant difference between IT group and RYGBP group at the 6 time points (P > 0.05). ③ FINS and HbA1c. Compared with before operation in the same group, the FINS levels of IT group and RYGBP group in 3 months and 6 months after operation were higher than those of Sham group (P < 0.05), HbA1c levels of IT group and RYGBP group were both lower at the 2 time points (P < 0.05). In 3 months and 6 months after operation, FINS levels of IT group and RYGBP group were both higher, and HbA1c levels were both lower than corresponding indexes of Sham group at the same time point (P < 0.05), but there was no significant difference between IT group and RYGBP group at the 2 time points (P > 0.05). ④ GLP-1. Compared with before operation in the same group, the GLP-1 levels of IT group and RYGBP group in 1 week, 2 weeks, 1 month, 2 months, 3 months, and 6 months after operation were all higher (P < 0.05). In 1 week, 2 weeks, 1 month, 2 months, 3 months, and 6 months after operation, GLP-1 levels of IT group and RYGBP group were both higher than those of Sham group at the same time point (P < 0.05), but there was no significant difference between IT group and RYGBP group at the 6 time points (P > 0.05). ConclusionIT and RYGBP have a significant hypoglycemic effect on non-obese T2DM GK rats, but IT has lower mortality and morbility, which is more effective and safer, comparing with RYGBP.