Objective To construct recombinant lentiviral vectors of porcine bone morphogenetic protein 2 (BMP-2) gene and to detect BMP-2 gene activity and bone marrow mesenchymal stem cells (BMSCs) osteogenetic differentiation so as to lay a foundation of the further study of osteochondral tissue engineering. Methods BMSCs were isolated from bone marrow of 2-month-old Bama miniature porcines (weighing, 15 kg), and the 2nd generation of BMSCs were harvested for experiments. The porcine BMP-2 gene lentiviral vector was constructed by recombinant DNA technology and was used to transfect BMSCs at multiplicity of infection (MOI) of 10, 25, 50, 100, and 200, then the optimal value of MOI was determined by fluorescent microscope and inverted phase contrast microscope. BMSCs transfected by BMP-2 recombinant lentiviral vectors served as experimental group (BMP-2 vector group); BMSCs transfected by empty vector (empty vector group), and non-transfected BMSCs (non-transfection group) were used as control groups. RT-PCR, immunohistochemistry staining, and Western blot were performed to detect the expressions of BMP-2 mRNA and protein. Then the BMSCs osteogenesis was detected by alkaline phosphatase (ALP) staining, ALP activities, and Alizarin red staining. Results The recombinant lentiviral vectors of porcine BMP-2 gene was successfully constructed and identified by RT-PCR and gene sequencing, and BMSCs were successfully transfected by BMP-2 recombinant lentiviral vectors. Green fluorescent protein could be seen in the transfected BMSCs, especially at MOI of 100 with best expression. The immunohistochemistry staining and Western blot showed that BMSCs transfected by BMP-2 recombinant lentiviral vectors could express BMP-2 protein continuously and stably at a high level. After cultivation of 2 weeks, the expression of ALP and the form of calcium nodules were observed. Conclusion The porcine BMP- 2 gene lentiviral vector is successfully constructed and transfected into the BMSCs, which can express BMP-2 gene and protein continuously and stably at a high level and induce BMSCs differentiation into osteoblasts.
Objective To construct recombinant lentiviral expression vectors of porcine transforming growth factor β1 (TGF-β1) gene and transfect bone marrow mesenchymal stem cells (BMSCs) so as to provide TGF-β1 gene-modified BMSCs for bone and cartilage tissue engineering. Methods The TGF-β1 cDNA was extracted and packed into lentiviral vector, and positive clones were identified by PCR and gene sequencing, then the virus titer was determined. BMSCs were isolated frombone marrow of the 2-month-old Bama miniature pigs (weighing 15 kg), and the 2nd and 3rd generations of BMSCs wereharvested for experiments. BMSCs were then transfected by TGF-β1 recombinant lentiviral vectors (TGF-β1 vector group)respectively at multi pl icity of infection (MOI) of 10, 50, 70, 100, and 150; then the effects of transfection were detected bylaser confocal microscope and Western blot was used to determine the optimal value of MOI. BMSCs transfected by empty vector (empty vector group) and non-transfected BMSCs (non-transfection group) were used as control group. RT-PCR, immunocytochemistry, and ELISA were performed to detect the expressions of TGF-β1 mRNA, TGF-β1 protein, and collagen type II. Results Successful construction of recombinant lentiviral vectors of porcine TGF-β1 gene was identified by PCR and gene sequencing, and BMSCs were successfully transfected by TGF-β1 recombinant lentiviral vectors. Green fluorescence was observed by laser confocal microscope. Western blot showed the optimal value of MOI was 70. The expression of TGF-β1 mRNA was significantly higher in TGF-β1 vector group than in empty vector group and non-transfection group (P lt; 0.05). Immunocytochemistry results revealed positive expression of TGF-β1 protein and collagen type II in BMSCs of TGF-β1 vector group, but negative expression in empty vector group and non-transfection group. At 21 days after transfection, high expression of TGF-β1 protein still could be detected by ELISA in TGF-β1 vector group. Conclusion TGF-β1 gene can be successfully transfected into BMSCs via lentiviral vectors, and long-term stable expression of TGF-β1 protein can be observed, prompting BMSCs differentiation into chondrocytes.
Objective To prepare collagen-chitosan /nano-hydroxyapatite-collagen-polylactic acid (Col-CS/ nHAC-PLA) biomimetic scaffold and to examine its biocompatibility so as to lay the foundation for its application on the treatment of osteochondral defect. Methods PLA was dissolved in dioxane for getting final concentration of 8%, and the nHAC power was added at a weight ratio of nHAC to PLA, 1 ∶ 1. The solution was poured into a mold and frozen. CS and Col were dissolved in 2% acetum for getting the final concentrations of 2% and 1% respectively, then compounded at a weight ratio of CS to Col, 20 ∶ 1. The solution was poured into the frozen mold containing nHAC-PLA, and then biomimetic osteochondral scaffold of Col-CS/nHAC-PLA was prepared by freeze-drying. Acute systemic toxicity test, intracutaneous stimulation test, pyrogen test, hemolysis test, cytotoxicity test, and bone implant test were performed to evaluate its biocompatibility. Results Col-CS/nHAC-PLA had no acute systemic toxicity. Primary irritation index was 0, indicating that Col-CS/nHAC-PLA had very slight skin irritation. In pyrogen test, the increasing temperature of each rabbit was less than 0.6℃, and the increasing temperature sum of 3 rabbits was less than 1.3℃, which was consistent with the evaluation criteria. Hemolytic rate of Col-CS/nHAC-PLA was 1.38% (far less than 5%). The toxicity grade of Col-CS/nHAC-PLA was classified as grade I. Bone implant test showed that Col-CS/nHAC-PLA had good biocompatibility with the surrounding tissue. Conclusion Col-CS/ nHAC-PLA scaffold has good biocompatibility, which can be used as an alternative osteochondral scaffold.
目的 对尿液特征组分与糖尿病早期肾损害的关系进行了初步探索。 方法 对2011年12月－2012年5月间28例2型糖尿病组、33例2型糖尿病肾病组及26例健康对照组尿液中尿蛋白含量和几种常见非蛋白氮物质，包括肌酸、尿囊素、肌酐、尿酸和假尿嘧啶核苷的浓度进行测定，采用多种归一化方法对数据进行对比分析，并通过t检验减少高效液相色谱测定的变量信息，保留P＜0.05的检出峰进行主成分分析，获得分类结果。 结果 采用体积归一化方法，发现健康对照组尿液中肌酸、尿囊素和尿酸的含量与2型糖尿病组和糖尿病肾病组相比，差异均有统计学意义（P＜0.05），2型糖尿病组尿液中尿蛋白的浓度与糖尿病肾病组相比，差异有统计学意义（P＜0.05）。 结论 通过肌酸、尿囊素、尿酸和尿蛋白的联合测定为肾脏损伤程度的监测及疗效观察提供依据，为2型糖尿病患者肾功能损坏的早期预防与诊断进行初步判断提供了新的方法。
ObjectiveTo summarize the experience in the treatment of infection after limb salvage surgery for malignant tumor around knee joint, and explore the risk factor related to infection after limb salvage surgery.MethodsA clinical data of 212 patients with malignant tumor around the knee joint underwent limb salvage surgery between January 2008 and December 2017 were retrospectively analyzed. Among them, 14 cases had infection after limb salvage surgery. Two cases of acute infection were treated with sensitive antibiotics; 12 cases of chronic infection were treated with debridement and antibiotic bone cement occupying device implantation in the first stage, and prosthesis revision (8 cases), knee joint fusion (2 cases), or amputation (2 cases) in the second stage after infection control. The age, gender, preoperative chemotherapy cycle, bone marrow suppression, serum albumin, hemoglobin, operation time, postoperative drainage time, and blood transfusion volume were analyzed to screen the risk factors related to infection after limb salvage surgery. The infection and tumor recurrence were observed, and the limb function was evaluated by Enneking scoring system.ResultsThe univariate analysis showed that the preoperative chemotherapy cycle, bone marrow suppression, operation time, and postoperative drainage time were the influencing factors of postoperative infection (P<0.05). Multivariate analysis showed that the operation time, preoperative chemotherapy cycle, and postoperative drainage time were risk factors of postoperative infection (P<0.05). Among the 14 patients, 1 patient died of traffic accident at 6 months after the second stage operation, and 13 patients were followed up 12.2-48.0 months (mean, 19.9 months). Two cases of acute infection cured. Among the 11 patients with chronic infection, 2 cases of subluxation of the antibiotic bone cement occupying device after the first stage operation occurred; 9 cases of infection cured and 2 cases recurred. At 12 months after operation, except 1 case died by accident, the Enneking scores of the other 13 patients were 12-26, with an average of 20. At last follow-up, 1 case of lung metastasis was still alive, and no tumor metastasis or recurrence was found in the rest.ConclusionThe time of limb salvage surgery, preoperative chemotherapy cycle, and drainage time after limb salvage surgery are the risk factors of infection after limb salvage surgery. Early etiological examination and drug sensitivity test is the key to the treatment of infection. One-stage debridement combined with antibiotic bone cement occupying device can effectively cure infection and save patients’ limbs.