AIM: To explore the significance of measuring urinary complement C5b-9 complex in various types of immune complex (IC) nephritis models of rats. METHODS: The four models of rats, namely, passive Heymann nephritis (PHN), anti-thymocyte serum nephritis(ATSN), anti-glomerular basement membrane nephritis (AGBMN) and chronic serum disease nephritis (CSDN) were reproduced. Then, the contents of complement C5b-9 complex in plasma and urine of the rats were detected with sandwich ELISA. And the deposits of C5b-9 complex in glomeruli of the rats were examined by ABC immunohistochemistry staining. RESULTS: The contents of rat plasma C5b-9 were elevated and deposits of C5b-9 in glomeruli could be detected in the four model rats. But the increased urinary excretion of C5b-9 was observed only in PHN rats. Moreover, the time of urinary C5b-9 complex excretion was earlier than that of urinary protein in the rats with PHN. CONCLUSION: Urinary C5b-9 complex excretion could be taken as one of several sensitive immunologic parameters in diagnosing of PHN and in distinguishing PHN from other type of nephritis.

AIM: To investigate the effects of FK506 on anti-glomerular basement membrane(GBM) nephritis in rats. METHODS: Anti-GBM nephritis model was elaborated by rabbit anti-rat GBM antibody injection in SD rats in this study. The rats were divided into three groups: FK506 treated group(0 5 mg?kg -1 ?d -1 , sc), untreated nephritis control group and normal control group. FK506 was administered daily six hours after injection of anti-GBM IgG. All the rats were observed urinary protein at the 4th day, the 14th day and the 21st day. At the same time, the kidney specimens were collected, and T cell transforming function was also monitored. RESULTS: Rats injected with rabbit anti-GBM Ab developed heavy proteinuria by 4 days, and serum creatinine and serum urea appeared which kept on the rising. Glmerular hypercellularity, crescents, and protein casts were observed in nephritic rats. By electron microscopy, the thickening of GBM and loss of foot processes were seen. T cell transforming function was higher than normal. But, all pathological changes obviously turned for the better in FK506 treated group. CONCLUSION: FK506 could inhibit the progression of rat anti-GBM nephritis.

AIM: To explore the localization and semi-quantification o f the glo merular complement C5b-9 complexes and synthesis of some inflammatory mediators or cytokines such as nitric oxide (NO) and tumor necrosis factor ?(TNF?) in the ra ts with anti-thymocyte serum nephritis(ATSN). METHODS: The animal mo del of rat AT SN was reproduced by a single intravenous injection of anti-thymocyte serum (ATS ). Then, the deposits of glomerular C5b-9 complexes were localized and quantifie d by immunohistochemical staining and microscopic image scanning separately. And the glomerular mesangial cells (MC) surrounded by C5b-9 complexes were counted under microscope. In addition, the expression of glomerular MC inducible NO synt hase(iNOS) mRNA and excretion of urinary NO metabolite ( NO - 2/NO - 3 ) and TNF ? in the rats with ATSN were detected. RESULTS: The MC in t h e rats with ATSN emerged necrosis followed by a rapid proliferation. In the early time of MC injury, the C5b-9 complexes were mainly seen in glomerular mesangium and MC sur fac e. But with the progression of ATSN, the MC enclosed by C5b-9 appeared gr adual decrease. Moreover, the expression of MC iNOS mRNA in early stage of ATSN obviously increased and the excretion of urinary NO - 2/NO - 3 and TNF ? also significantly increased. However, the changes of parameters mentioned abov e in ATSN proliferative stage (after 7 days) alleviated gradually. CONCLUSION: The second ary lysis of MC has relation to the deposition of C5b-9 complexes and synthesis and release of NO and TNF ? in rats with ATSN.

BACKGROUND:Nano-hydroxyapatite has excellent biocompatibility, biological activity, and modifiability, but the synthesized nano-hydroxyapatite has a high specific surface energy, which makes it agglomerate in solution. OBJECTIVE: To compare the effects of ultrasound and different dispersants on the aggregation, cytotoxicity and intracellular distribution of nano-hydroxyapatite. METHODS: Needle like nano-hydroxyapatite was prepared by chemical precipitation method, and kept for further use after high pressure sterilization. Different concentrations of dispersant sodium hexametaphosphate (0, 0.25, 0.5, 1, 2 mmol/L), sodium citrate (0, 0.25, 0.5, 1, 2 mmol/L), sodium polymethacrylate (0%, 0.0625%, 0.125%, 0.25%, 0.5%) were co-cultured with MC3T3E1 cells. Cell proliferation was detected by CCK-8 assay. The appropriate concentration of dispersant was screened for subsequent experiments. The nano-hydroxyapatite solution was divided into five groups: the control group was not dispersed; and the remaining four groups were subjected to ultrasonic dispersant, 1 mmol/L sodium hexametaphosphate dispersant, 1 mmol/L sodium citrate dispersant, and 0.125% sodium polymethacrylate dispersant. The aggregate size of nano-hydroxyapatite was detected. The hydroxyapatite was treated with ultrasound, 1 mmol/L sodium hexametaphosphate, 1 mmol/L sodium citrate, and 0.125% sodium polyacrylate dispersants and then co-cultured with MC3T3E1 cells. Undispersed nano-hydroxyapatite co-cultured with MC3T3E1 cells was considered as control. Cell proliferation was detected by CCK-8 assay. At 1 day after culture, the distribution of nano-hydroxyapatite was observed by transmission electron microscope. RESULTS AND CONCLUSION: (1) Sodium hexametaphosphate at a concentration of 1 mmol/L and lower had no obvious cytotoxicity. Sodium citrate at 0.25-2 mmol/L had no obvious cytotoxicity. Sodium polymethacrylate at different concentrations had certain cytotoxicity in time-and concentration-dependent manners. Subsequent experiments selected 1 mmol/L sodium hexametaphosphate, 1 mmol/L sodium citrate, and 0.125% sodium polyacrylate for dispersion treatment. (2) All three dispersants significantly reduced the agglomeration size of nano-hydroxyapatite, and sodium hexametaphosphate had the best dispersion effect. (3) The dispersion method and the addition of dispersant could significantly affect the biological function of nano-hydroxyapatite. Sodium citrate promoted the proliferation of cells co-cultured with nano-hydroxyapatite. Ultrasound and sodium polymethacrylate inhibited proliferation of cells co-cultured with nano-hydroxyapatite. (4) Transmission electron microscopy showed that in the control group and the ultrasound group, large agglomerates of nano-hydroxyapatite existed inside and outside the cell; the nano-hydroxyapatite dispersed by the three dispersants also had agglomeration, but the size of the agglomeration was significantly smaller. Among them, the nano-aggregation size of the sodium hexametaphosphate group was the smallest. Some nanoparticles were wrapped in a double-layer membrane in the cell to form a structure similar to “small vacuoles”. (5) The results show that the commonly used dispersants themselves have certain cytotoxicity, and the addition of dispersants will significantly reduce the agglomeration size of nano-hydroxyapatite and its intracellular distribution, and affect cell proliferation.

AIM:To study the effect of human complement C 5b～9 complex on nitric oxide(NO) synthesis of glomerular mesangial cells (MC). METHODS: First, the human complement C 5b～9 complexes were isolated and glomerular MC of rats were cultured. Second, the MC were stimulated with C 5b～9 complex and changes of metabolism products of NO(NO 3 and NO 2) in MC culture supernatant at 6,24 and 48 hours after C 5b～9 stimulating were detected. Moreover, cGMP levels in cultured MC were also measured. RESULTS: NO 3/NO 2 contents from culture supernatant and cGMP levels in MC were increased parallelly after C 5b～9 complex stimulation. Further, NO synthesis was inhibited by L-NG-nitro-arginine-methylester(L-NAME). CONCLUSION: NO synthesis of rat glomerular MC was incerased by human complement C 5b～9 stimulation.

BACKGROUND: Tumor necrosis factor-α (TNF-α) is one of important cytokines to promote the maturation of dendritic cells. Blockage of TNF-α action by binding with soluble tumor necrosis factor receptor 1 (sTNFR1) may arrest dendritic cells in an immature state and induce stable, long-term tolerance. OBJECTIVE: To construct the lentiviral vectors carrying sTNFR1 gene and investigate sTNFR1 expression in immature dendritic cells. METHODS: Total RNA of human peripheral blood mononuclear cells was taken as a template. The sTNFR1 gene fragment was amplified by RT-PCR, subcloned to the lentiviral vectors pXZ208, and ligated to the enhanced green fluorescent protein (eGFP) reporter gene to establish lentiviral vector, called pXZ9-sTNFR1. DNA sequencing was performed for lentiviral vector identification. Lentivirus was prepared by transfection of 293 FT cells with pXZ9-sTNFR1. Viral titer was determined by eGFP expression. C57BL/6 mouse bone marrow-derived dendritic cells were in vitro cultured with low-dose granulocyte-macrophage colony stimulating factors and interleukin 4. On day 5 of culture, immature dendritic cells were transfected with pXZ9-sTNFR1 recombinant lentiviral supernatant, sTNFR1 transcription was detected by RT-PCR, sTNFR1 protein expression by Western blot analysis. Following sTNFR1 gene modification and lipopolysaccharide stimulation, the phenotype characteristics of dendritic cells were observed. RESULTS AND CONCLUSION: Recombinant plasmid pXZ9-sTNFR1 was successfully constructed. Twenty-four hours after 293 FT cell transfection, eGFP expression was observed and viral titer was over 10<'6> U/L. RT-PCR demonstrated that pXZ9-sTNFRl-transfected immature dendritic cells showed sTNFR1 positive expression. Western blot analysis revealed that sTNFR1 protein appeared in the immature dendritic cells and supernatant following 293 FT cell transfection. On day 5 of culture, dendritic cells expressed low level of class Ⅱ major histocompatibility complex (MHC Ⅱ), as well as CD40, CD86, CD80, molecules. However, following lipopolysaccharide stimulation, dendritic cells expressed high level of MHC Ⅱ, as well as CD40, CD80, and CD86, molecules, exhibiting the phenotype characteristics of mature dendritic cells. But after sTNFR modification, the expression level of MHC Ⅱ, as well as CD40, CD80, and CD86, molecules was not altered obviously. Lentiviral vectors carrying sTNFR1 gene and eGFP reporter gene were successfully constructed, and recombinant lentiviral plasmids with high titer were acquired. Following high efficacy of lentiviral gene transfection, immature dendritic cells stably express sTNFR1 mRNA and protein, which prevents immature dendritic cells from activation by exogenous lipopolysaccharide and maintains the immature state.

Objective To investigate the effects of 14-3-3 phosphorylation (p-14-3-3) induced by C-Jun N-termi-nal kinase (JNK) on ischemic brain injury in rats. Methods Twenty rats were divided into 4 groups:sham operation group, ischemia-reperfusion group, SP600125 group and solvent control group. The rat model of cerebral ischemia was established. The p-14-3-3, the binding of 14-3-3 and Bax and the protein expression of Bax in cytoplasm and mitochondria in hippo-campal CA1 region were detected by immunoprecipitation (IP) and immunoblotting 12-hour after ischemia-reperfusion in four groups. Results Compared with the sham operation group, protein expression levels of p-14-3-3 in cytoplasm and Bax in mitochondria were significantly increased, the binding of 14-3-3 and Bax was significantly decreased in ischemia-re-perfusion group, solvent control group and SP600125 group. The protein expressions of p-14-3-3 and Bax were significantly lower in SP600125 group than those of ischemia-reperfusion group and solvent control group. The binding of 14-3-3 and Bax was significantly higher in SP600125 group than that of ischemia-reperfusion group and solvent control group (P <0.05). Conclusion 14-3-3 phosphorylation induced by JNK plays important effects on ischemic brain injury in rats.

Objective:To investigate the oncological efficacy and functional evaluation of total elbow arthroplasty (TEA) for the reconstruction of tumor around elbow joint.Methods:A retrospective case series study was made on the clinical data of 26 patients who underwent total elbow joint replacement after tumor resection in Beijing Jishuitan Hospital from June 1988 to June 2019. According to the inclusion and exclusion criteria, 23 patients were enrolled in the final study, there were 14 males and 9 females, the mean and median age was 37.6±19.9 and 35.0 years respectively. 23 patients included 3 cases of giant cell tumor, 4 cases of metastatic cancer, 4 cases of Ewing's sarcoma, 2 cases of osteosarcoma, 2 cases of aneurysmal bone cyst, 1 angiosarcoma, 1 primary malignacy in giant cell tumor, 1 low-grade central osteosarcoma, 1 parosteosarcoma, 1 synovial sarcoma, 1 plasma cell myeloma, 1 tendon sheath giant cell tumor and 1 case of mixed tumor. There were 6 cases of benign tumor, 4 cases of low grade sarcoma and 13 cases of high grade malignancy. With 19 cases of distal humerus, 3 cases of proximal ulna and 1 case of elbow. Each patient underwent tumor resection followed by restrictive tumor prosthesis and semi-restrictive of coonrad-morrey prosthesis were used for reconstruction.The duration of the operation, the amount of blood loss, epidemiological data, reconstruction length, oncology parameter, complications and functional evaluation were enrolled and statistical analyzed.Results:The mean length of the osteotomy followed by reconstruction was 12.5±3.9 cm, the mean operative time was 154.1±50.1 minutes, and the mean bleeding was 262.2±100.9 ml. Thirteen patients were treated with customized tumor limited prosthesis while 10 patients with Coonrad-Morrey semi-limited prosthesis. The 5-year survival rates of 23 patients was 64.3%, benign tumors, low-grade and high-grade malignancies were 100%, 100% and 39.7%, respectively. Three cases of lung cancer and three cases of Ewing's sarcoma died during the follow-up period (6/23, 26.1%), one case of giant cell tumor and one case of synovial sarcoma developed local recurrence (2/23, 8.7%). The median range of motion for the elbow increased from 35 to 85 degrees ( t=-13.787, P<0.05), the median NRS score decreased from 5.0 to 0.5 ( t=14.391, P<0.05). Postoperative complications occurred in 9 cases (9/23, 39.1%), the recent complications were nerve injury in 4 cases and infection in 1 case, late complications were prosthesis loosening and failure in 4 cases, the 5 year survival rate of prosthesis was 82.0%. The mean and median MSTS 93 score was 84.5%±11.0% and 88.3% respectively. Conclusion:The local control around the elbow is satisfactory after tumor resection. Total elbow arthroplasty can relieve pain and significantly improve function.