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Background/Aims@#Although the conversion from tacrolimus (TAC) to cytotoxic T-lymphocyte-associated antigen 4-immunoglobulin (CTLA4-Ig) is effective in reducing TAC-induced nephrotoxicity, it remains unclear whether CTLA4-Ig has a direct effect on TAC-induced renal injury. In this study, we evaluated the effects of CTLA4-Ig on TAC-induced renal injury in terms of oxidative stress. @*Methods@#In vitro study was performed to assess the effect of CTLA4-Ig on TAC-induced cell death, reactive oxygen species (ROS), apoptosis, and the protein kinase B (AKT)/forkhead transcription factor (FOXO) 3 pathway in human kidney 2 cells. In the in vivo study, the effect of CTLA4-Ig on TAC-induced renal injury was evaluated using renal function, histopathology, markers of oxidative stress (8-hydroxy-2’-deoxyguanosine) and metabolites (4-hydroxy-2-hexenal, catalase, glutathione S-transferase, and glutathione reductase), and activation of the AKT/FOXO3 pathway with insulin-like growth factor 1 (IGF-1). @*Results@#CTLA4-Ig significantly decreased cell death, ROS, and apoptosis caused by TAC. TAC treatment increased apoptotic cell death and apoptosis-related proteins (increased Bcl-2-associated X protein and caspase-3 and decreased Bcl-2), but it was reversed by CTLA4-Ig treatment. The activation of p-AKT and p-FOXO3 by TAC decreased with CTLA4-Ig treatment. TAC-induced renal dysfunction and oxidative marker levels were significantly improved by CTLA4-Ig in vivo. Concomitant IGF-1 treatment abolished the effects of CTLA4-Ig. @*Conclusions@#CTLA4-Ig has a direct protective effect on TAC-induced renal injury via the inhibition of AKT/FOXO3 pathway.
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Objective:To establish an inductively coupled plasma mass spectrometry (ICP-MS) based immunoassay method for the quantitative detection of human chorionic gonadotropin (HCG), and evaluate the clinical applicability of this method.Methods:Sm was selected as element tags, and the HCG quantitative detection system was established by double antibody sandwich method. The dosage of biotinylated antibody and reaction time were optimized. According to EP documents of Clinical and Laboratory Standards Institute (CLSI) and related standards, the analytical performance was evaluated after the establishment of the assay, including the limit of blank (LOB), linearity, precision, recovery, cross reactivity and interference test. And 88 clinical samples were measured using the new method compared to the electrochemical immunoassay (ECLIA) method.Results:Total process completed within 30 min after optimization, and the optimal biotinylated antibody dosage was 0.5 μl. The LOB was 0.29 mIU/ml. The linearity was good within the range of 1.16-8 365.62 mIU/ml with the linear correlation coefficient greater than 0.995 ( R2=0.998 0), the recovery was 97.53%-102.01%. Both intra-and inter-assay coefficients of variation of the high-value sample and the low-value sample were less than 10%. And there was no significant cross-reaction with Luteinizing hormone (LH), follicular stimulating hormone (FSH), and thyroid-stimulating hormone (TSH). The interference bias caused by different concentrations of interference substances was less than 10%. When compared with the ECLIA method for clinical sample detection, the proposed method showed a significant correlation( R2=0.960 0). Conclusion:The proposed ICP-MS base immunoassay for HCG detection has good accuracy, high sensitivity and specificity, and the results of analytical performance verification meet the clinical requirements, which provides experimental basis for the clinical application of this method.
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Objective:To study the non-target metabolomics analysis and to analyze the metabolomic changesof cerebrospinal fluid (CSF) in patients with tuberculous meningitis.Methods:Case-control study. From July 2018 to July 2019, 20 cerebrospinal fluid specimens of diagnosed patients with tuberculous meningitis were collectedin the department of neurology from the first medical center of the PLA general hospital and the eighth medical center of the PLA general hospital and 20 CSF without tuberculous meningitis as the control. Among them, there were 12 males and 8 femalesin the tuberculous meningitis group, aged (37.9±16.1) years; there were 13 males and 7 femalesin the control group, aged (34.7±14.8) years. Using ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) technology with three different mode, namely reverse phase chromatography positive ion mode, reverse phase chromatography negative ion mode and hydrophilic chromatography positive ion mode,to detectthe metabolic fingerprints of patients′CSF and analyzed by SIMCA software for orthogonal partial least squares discriminant analysis (OPLS-DA). The variable importance projection value of OPLS-DA model (threshold value>1) plus the P value of t-test (P<0.05) was applied to find the differential metabolites in the cerebrospinal fluid of the two groups of patients.Results:Ten differential metabolites were found in CSF, including L-isoleucine, L-phenylalanine, L-kynurenine, L-methionine, L-tyrosine acid, dimethylglycine, L-alanine, L-threonine, L-histidine and L-lysine, and all of them were up-regulated in the tuberculous meningitis group.Conclusion:Changesof the amino acid metabolism found in the cerebrospinal fluid of tuberculous meningitis patients can provide basis for differential diagnosis and basic molecular research of tuberculous meningitis.
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Objective:To investigate the clinical efficacy and safety of oral glucocorticoid combined with intravenous cyclophosphamide or oral cyclophosphamide in the treatment of idiopathic membranous nephropathy.Methods:According to the random number table method, 50 patients in our hospital were divided into oral cyclophosphamide group (oral group) and intravenous cyclophosphamide group (intravenous group). The oral group was treated with prednisone and compound cyclophosphamide tablets, while the intravenous group was given prednisone and intravenous injection of cyclophosphamide. The 24-hour urinary protein, serum albumin, liver and kidney function and adverse events were observed before treatment and 1, 3, 6 and 12 months after treatment. The remission rate and drug safety of the two groups were compared.Results:After 3 months of treatment, the effective rate of oral group was 40%, which was significantly higher than that of intravenous group (4%). After 6 months of treatment, the effective rate of oral group (52%) was higher than that of intravenous group (44%). After 12 months of treatment, the effective rate of intravenous group (72%) was higher than that of oral group (64%), with no statistically significant difference ( P>0.05). Before treatment, there were no significant differences in albumin, urea nitrogen, creatinine, Cystatin C, glomerular filtration rate and 24-hour urine protein between the oral group and the intravenous group ( P>0.05). After 12 months of treatment, the level of albumin in the oral group and the intravenous group was significantly higher than that before treatment ( P<0.05), and the 24-hour urine protein level was significantly lower than that before treatment ( P<0.05). The total incidence of adverse reactions in the intravenous group was lower than that in the oral group ( P<0.05). Conclusions:Hormone combined with intravenous infusion of cyclophosphamide has better long-term efficacy and higher safety in the treatment of idiopathic membranous nephropathy.
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Idiopathic membranous nephropathy (IMN) is one of the pathological types of nephrotic syndrome.Besides secondary factors,such as immune system diseases,hepatitis and tumors,the etiology is still unclear.Recent studies have shown that the incidence of IMN is increasing year by year,and the age of onset is gradually younger.Adolescents have become a representative group of new IMN patients.Researches have showed that adolescent idiopathic membrane patients have mild clinical and pathological manifestations,good renal function,and good prognosis.Most patients have fertility requirements,and there are clinical concerns about the application of hormones and immunosuppressive agents.This paper reviews the progress on the clinicopathological manifestation,treatment and prognosis of adolescent IMN patients.
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Objective Toinvestigatestatistically significant peptide peaks as biomarkersto diagnose osteoarticular tuberculosis, matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF MS) was applied to identify the characteristic fingerprint among the serum of patients with osteoarticular tuberculosis, rheumatoid arthritis and healthy adults.Methods Clinical Study. Serum samples of untreatedpatients with osteoarticular tuberculosis and rheumatoid arthritis were collected from August 2018 to December 2018, and serum samples of healthy adults from physical examination were collected as control. After analysis with MALDI-TOF MS, the serum peptide fingerprint datawas imported into software, and protein polypeptide peaks with obvious differences were screened to establish diagnostic models.Results Established the diagnostic model of osteoarticular tuberculosis and healthy adults with m/z 2943.9, 5929.6, 7615.4 and 9033.8 as differential protein polypeptides, the diagnostic model of osteoarticular tuberculosis and rheumatoid arthritis with m/z 4195.6, 5847.6, 5929.6 and 7748.6 as differential protein polypeptides. To these two models, the sensitivity were 95.00％ and 97.50％, respectively. The specificity were 85.71％ and 88.46％, respectively. The accuracy rates were 89.58％ and 92.39％, respectively. The AUC value of ROC curves were 0.8859 and 0.8709, respectively. Conclusions By mass spectrometry and software analysis, the serum protein polypeptides with statistical difference were found successfully. The related diagnostic modelsarealso established, which has certain reference value for auxiliary diagnosis of osteoarticular tuberculosis.
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Objective To construct a simple method for the measurement of activity of S-homocysteine methyltransferase (HMT),and explore the best processing condition for HMT and the preservation of HMT.Methods HMT was expressed in pro-karyotic system by using genetic engineering technology,then was purified by using affinity and Sephadex G1 5 chromatography. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)was performed to identify the physicochemical and bio-logical properties of target protein.Based on the principle of 5 ,5′-disulfide-double (2- nitro benzoic acid)(DTNB)could react with sulfydryl compounds rapidly,the reduction of homocysteine was detect to evaluate the activity of enzyme,then the best processing conditions of HTM were determined.Activity of enzyme,preserved in preservation solution with or without glycerol and preserved under different temperatures,was detected.Activity remaining ratios were detected and compared between HMT preserved in pres-ervation solution with different protective agents of different concentration and preserved by cryodesiccation.Results The purity of recombined HMT was above 95%,with molecular weight of 36 000 and excellent catalytic activity,and the catalytic activity was 2 000 U/mg.The optimum condition for the detection of biological activity was using HEPES buffer of pH 7.4 at 37 ℃ and reac-tion for 25 min.Glycerol could significantly prolong the preserving time of HMT,and half activity of HMT could be remained for six months.The reservation rates of activity of HMT,preserved in preservation solution with mannitol and trehalose,were 104%and 100%,respectively.Conclusion HMT could be obtained through genetic engineering.A simple test method of HMT was es-tablished,and the best processing conditions and preserving methods of HMT were determined,which laid a foundation for clinical application.
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Objective To compare the two kinds of purification method for purifying recombinant human cardiac troponin I(cT-nI)to obtain the stable cTnI and promote the study of cTnI diagnosis standardization.Methods The cTnI inclusion body was ob-tained by the ultrasonic broken engineering,after washing by 2% Tritonx-100,2M urea,dissolved in 8M urea,then purified by the column refolding on CM-FF and the dilution refolding respectively.The cTnI yields were compared between the two kinds of meth-od and the stability at 4 ℃,20 ℃,-80 ℃ and on the freeze-dried condition was compared.Then the purification method to effi-ciently obtain the stable cTnI was established.Results The protein about 2 mg and 1.4 mg could be obtained by CM-FF on the col-umn refolding and the dilution refolding from 0.1 g of wet inclusion body,respectively.The former method had the short cycle and high efficiency.The cTnI purified by the column refolding on CM-FF was more stable at 4 ℃,20 ℃,-80 ℃ and on the freeze-dried condition.Conclusion The column refolding on CM-FF is more stable and highly efficient in purification of cTnI than the dilution refolding.
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Objective To prepare and identify the monoclonal antibody against asymmetric dimethylarginine (ADMA) and to do further research on clinical application .Methods The short immune peptide antigen was synthetized by solid phase technology . Splenocytes from the Balb /c mice immunized by synthetized antigen were fused with myeloma cells SP 2/0 for producing hybrido-ma .Hybridoma cell line secreting antibodies against ADMA was sifted by indirect ELISA and limiting dilution assay .The specificity of monoclonal antibodies against human ADMA were evaluated with Western blot and ELISA .Results Two hybridoma cell lines stably secreting monoclonal antibodies against ADMA were developed and named 22-1-1and 38-1-6 respectively .By applying West-ern blot and ELISA ,the results indicated that all monoclonal antibodies raised could specifically react with ADMA .Conclusion The success in the production of ADMA monoclonal antibody establishes foundation for application in the practice of clinical labora-to ry .
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OBJECTIVE: To assess the therapeutic effects of Sisheng Decoction, a compound traditional Chinese herbal medicine, on a mouse model of yin deficiency syndrome induced by thyroid hormone, and to make the preliminary study on its mechanisms. METHODS: Simultaneous modeling and treatment were carried out. Sixty mice were randomly divided into six groups: normal group, yin deficiency model group, low-, medium- and high-dose Sisheng Decoction group and Shengmai oral liquid group. Normal group and yin deficiency model group were administered with double distilled water. Spontaneous activity and serum concentration of malondialdehyde in different groups were detected. RESULTS: The symptoms of yin deficiency syndrome such as xerostomia, dysphoria and fervescence were improved in the Sisheng Decoction groups. Compared with the yin deficiency model group, the spontaneous activity was increased and the serum concentration of malondialdehyde was decreased in the Sisheng Decoction groups (P<0.05). There was no significant difference between the Sisheng Decoction groups and the Shengmai oral liquid group (P<0.05). CONCLUSION: Medium- or high-dose Sisheng Decoction is effective for nourishing yin, clearing heat, engendering liquid and allaying thirst. The above effects of Sisheng Decoction may be realised by improving the spontaneous activity and resisting oxidative damage.
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Objective:To clone and express the full length cDNA of human CD38. Methods:The full length cDNA of the human CD38 antigen was amplified from total RNA of Daudi cell by RT-PCR, and it was inserted into pGEM-T. The validity on the sequences was confirmed by automatic DNA sequencing. Inserting the valid CD38 gene into pcDNA3.1(+) plasmid to obtain recombinant mammalian expression vector pcDNA3.1(+)/CD38Z; Using lipofectin gene transfer technique system, recombinant expression vector containing CD38 gene was transfected into COS7 cells. The expression of CD38 molecules on the surface of COS7 cells was detected by FACS and immunohistochemical technique. Results:DNA sequencing showed that the cloned full length cDNA sequence was identical with reported. The result of FACS and immunohistochemical technique indicated that CD38 molecules were expressed on the surface of COS7 cells. Conclusion:The full length cDNA of human CD38 is obtained, recombinant mammalian expression vector pcDNA3.1(+)/CD38Z is successfully constructed, and the CD38 molecules is expressed on the surface of COS7 cells,this may facilitate studies on the biochemistry and function of CD38 antigen.
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Objective To observe a recombinant mutant of HBV core protein for dominant negative gene therapy against HBV encapsidation in vitro. Methods C gene and S gene of HBV were acquired through PCR and subcloned into pGEM T to construct pGEM T C and pGEM T S respectively. After digestion and ligation of these two plasmids, pGEM T CS was constructed. The cloned gene was inserted into pcDNA3.1 + to construct pcDNA3.1 + CS, which was identified by DNA sequencing. The recombinant plasmids were transformed into HepG2 cells, and screened with G418. The resistant HepG2 cell clones were chosen to test the expression of core surface protein by RT PCR, and the expressing HepG2 clones were cultured with 10% HBV DNA positive human serum for 72 hours. The intracellular HBV particles were extracted and the DNA was subjected to dot hybridization. Results The analysis showed that the HepG2 cells expressing mutant C protein had capabilities to resist HBV invasion in varied degrees. The mutant C protein had a dominant negative role in the encapsidation of HBV compared with the naive part of core protein. Conclusions The production of recombinant mutant core protein has a potential value for gene therapy against HBV infection.
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Objective To establish a method for high throughput screening single nucleotide polymorphism(SNP) by tag microarray,and then apply the method to study the gene SNP which is related to the motor function of normal people.Methods The genes related to motor function were firstly defined,and then 48 SNP loci were determined.The rs numbers of these SNP loci were fingered out from PubMed,and the primers were designed with the software in web site "www.autoprimer.com".The primer sequences were then downloaded and sent to the biologic corporation for synthesis.After being synthesized and purified by HPLC these primers were used in the experiments according to the instruction of Bakeman's SNPstream machine.The key techniques of SNPstream machine were tag microarray and single nucleotide extension assay.Once the determination was finished,both the gene frequency and allele frequency of every locus could be statistically analyzed.Results The information of the 48 SNP loci that related to motor function had been determined simultaneously by tag microarray,regardless the number of samples to be detected at the same time.The number of the samples was variable to meet the need.The data of gene frequency and allele frequency of these 48 SNP loci may be used in the subsequent studies.Conclusions Tag microarray used to high throughput screening SNP has the advantages of accuracy,speed,efficiency and reasonable cost.Therefore it can be applied to study the relationship between the SNP and many kinds of diseases.