Construction and characterization of the fourth-generation CAR-T cells targeting fibroblast activation protein (FAP) / 中华微生物学和免疫学杂志

Objective:To construct the second and fourth generations of CAR-T cells targeting fibroblast activation protein (FAP) on the surface of stromal carcinoma-associated fibroblasts and compare their characteristics in vitro and in vivo. Methods:ELISA was used to detect the cytokines secreted by CAR-T cells. Cell proliferation and viability were analyzed by counting. Chemotactic ability was tested by Transwell migration assay. Distribution of T cell subsets was analyzed by flow cytometry. Cytotoxicity was assessed by luciferase bioluminescence. The safety and therapeutic effects were evaluated in a NOG mouse model of metastatic human lung cancer.Results:The expression rates of the second and fourth generations of CAR-T cells (h4BBz CAR-T and h4BBz-7.19 CAR-T) were (74.280±4.384)% and (67.220±4.013)%, respectively. The h4BBz-7.19 CAR-T cells had better in vitro proliferation and chemotactic activity than h4BBz CAR-T cells as they were able to secrete IL-7 and CCL19, while the viability of h4BBz-7.19 CAR-T cells was comparable to that of h4BBz CAR-T cells. There was no significant difference in the expression rate of h4BBz CAR or h4BBz-7.19 CAR between CD4 + T and CD8 + T cells. The proportions of both Naive cells and T memory stem cells (TSCM) in CD4 + and CD8 + T cells were higher in h4BBz-7.19 CAR-T cells than in h4BBz CAR-T cells. Moreover, h4BBz-7.19 CAR-T cells possessed stronger specific cytotoxicity on the target cancer cells than h4BBz CAR-T cells when the ratio of effectors/targets was low ( P1∶1=0.004, P2∶1=0.000 6, P5∶1<0.000 1, P10∶1=0.022, P20∶1=0.116), while the expression of PD-1 on the surface of h4BBz-7.19 CAR-T cells was lower than that on h4BBz CAR-T cells. In the NOG mouse model of metastatic human lung cancer, h4BBz-7.19 CAR-T cells could slow the tumor growth and prolong the survival time of mice without causing weight loss or pathological changes in the organs. Conclusions:The fourth-generation CAR-T cells targeting FAP were shown to have stronger proliferation, better penetration and more potent specific cytotoxicity by secreting IL-7 and CCL19 and could slow the tumor growth and prolong survival by improving tumor immunosuppressive microenvironment. This study provided reference for the clinical application of the fourth generation of CAR-T cells.

A case of inherited afibrinogenemia caused by an IVS7-12A>G splice mutation of FGG gene / 中华医学遗传学杂志

OBJECTIVE@#To explore the genetic basis for a Chinese pedigree affected with inherited afibrinogenemia.@*METHODS@#For the proband and his family members, prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), Fibrin(ogen) degradation products (FDPs), D-dimer (D-D), plasminogen activity (PLG:A) and the TT mixed experiment with protamine sulfate were determined with a STAGO-R automatic coagulation analyzer. The activity and antigen of fibrinogen (Fg) in plasma were measured with the Clauss method and immunonephelometry method, respectively. All exons and flanking regions of the fibrinogen genes (FGA, FGB and FGG) were amplified by PCR and directly sequenced. Human Splicing Finder software was used to predict and score the change of splicing site caused by the mutation.@*RESULTS@#The proband showed normal FDPs and D-D but significantly prolonged TT, PT and APTT. The activity and antigen of fibrinogen in plasma were significantly decreased (G (g.4147A>G) mutations of the FGG gene, for which his parents and young sister were heterozygous. As predicted by Human Splicing Finder and Mutation Taster software, the variant may generate a new splicing site which can extend the sequence of exon 7 by 11 bp, with alteration of the coding sequence. PROVEAN suggested the variant to be deleterious.@*CONCLUSION@#The afibrinogenemia of the proband may be attributed to the FGG IVS7-12A>G variant, which was unreported previously.

Clinical and genetic analysis of a pedigree affected with type I hereditary antithrombin deficiency due to a g.2736dupT variant of the AT gene / 中华医学遗传学杂志

OBJECTIVE@#To analyze the phenotype and genotype of a patient affected with inherited antithrombin deficiency.@*METHODS@#All exons and exon-intron boundaries of the AT genes were subjected to PCR amplification and Sanger sequencing. The influence of variants on the disease was predicted using bioinformatic software (MutationTaster).@*RESULTS@#The results of all coagulation tests were normal, though the antithrombin activity and antigen content of the proband and his father have decreased significantly (34%, 48% and 12.97 mg/dL, 15.60 mg/dL, respectively). His mother was normal. Genetic analysis revealed that the proband and his father both carried a heterozygous g.2736dupT variant of the AT gene. Bioinformatic analysis suggested that the variant may be pathogenic.@*CONCLUSION@#The proband and his father both had type I hereditary antithrombin deficiency caused by a g.2736dupT variant of the AT gene. The variant was unreported previously.

Short-term clinical efficacy and analysis of quality of life of anlotinib in third-line and above treatment for advanced non-small cell lung cancer / 国际肿瘤学杂志

Objective To evaluate the short-term efficacy,safety and impact on the quality of life of anlotinib in third-line and above treatment for advanced non-small cell lung cancer (NSCLC) patients.Methods All the patients received alotinib 12 mg/d.One cycle was defined as 2 weeks on-treatment followed by 1 week off-treatment until disease progression or treatment intolerance.Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 was used to assess tumor responses.Common Terminology Criteria for Adverse Events (CTCAE) 4.02 was used to assess the adverse events.The European Organization for Research on Treatment of Cancer (EORTC) QLQ-C30 and QLQ-LC13 were used to assess quality of life.Results Among 27 patients in study,no complete response (CR) was found,2 patients (7.4％) achieved partial response (PR),16 patients (59.3％) achieved stable disease (SD),9 patients (33.3％) achieved progressive disease (PD),objective response rate (ORR) was 7.4％,and disease control rate (DCR) was 66.7％.The scores of physical functioning (76.00 ± 10.55 vs.64.44 ± 11.59),emotional functioning (81.67 ± 8.71 vs.76.11 ±6.71) and global health status (48.87 ±7.97 vs.40.56 ± 12.49) of the QLQ-C30 scale after treatment were higher than those before treatment,with statistically significant differences (t =-4.516,P ＜0.001;t=-2.646,P=0.019;t=-3.872,P=0.002).Fatigue (50.37±8.95 vs.40.74±13.86),nausea and vomiting (26.54 ± 16.18 vs.14.20 ± 11.97),loss of appetite [M(QR):33.33 (33.33) vs.33(33.33)] were better than before (t =-2.476,P =0.027;t =-5.036,P ＜0.001;Z =-2.923,P =0.003);pain (28.88 ± 14.23 vs.33.33 ± 13.60) and dyspnea [33.33 (33.33) vs.33.33 (66.67)] scores were lower than before (t =3.674,P =0.003;Z =-3.266,P =0.001).The scores of cough (24.44 ±19.12 vs.45.24 ±20.34),shortness of breath [11.11(22.22) vs.33.33(22.22)] and chest pain [0.00(33.33)vs.33.33 (33.33)] in the QLQ-LC13 scale after treatment were lower than those before treatment,with statistically significant differences (t =4.000,P =0.001;Z =-4.125,P ＜0.001;Z =-1.890,P =0.034);the scores of sore mouth or tongue [0.00(33.33) vs.0.00(0.00)] and hands and feet tingling [33.33(33.33) vs.0.00(0.00)] were higher than before (Z=-2.000,P=0.046;Z=-2.264,P=0.024).Common adverse reactions included hypertension,fatigue,elevated thyroid stimulating hormone,proteinuria,hand-foot syndrome,oral mucositis,hemoptysis,etc,mainly grade 1-2,and they were all improved after the treatments.Conclusion Anlotinib as a third-line and further therapy is positive effected and well tolerated.It can alleviate the clinical symptoms and significantly improve the quality of life of NSCLC patients.

Analysis of a pedigree affected with congenital dysfibrinogenemia due to a novel Gly31Glu mutation of FGA gene / 中华医学遗传学杂志

Objective@#To analyze the phenotype and genotype of a pedigree affected with congenital dysfibrinogenemia.@*Methods@#Liver and kidney functions of the proband and her relatives were determined. Coagulation tests including prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time(TT), fibrin(ogen) degradation products (FDPs), D-dimer(D-D) and the calibration experiment of protamine sulfate of against plasma TT were detected in the proband and her predigree members. The activity and antigen of fibrinogen (Fg) in plasma were measured by Clauss method and immunonephelometry method, respectively. All of the exons and exons-intron boundaries of the three fibrinogen genes (FGA, FGB and FGG) were subjected to PCR amplification and Sanger sequencing. Potential influence of the suspected mutations were analyzed with bioinformatics software including PolyPhen-2, SIFT and Mutation Taster.@*Results@#The proband had normal PT, APTT, FDPs, D-D and prolonged TT (31.8 s). The activity of fibrinogen (Fg) in plasma was significantly decreased but the antigen was normal. Genetic analysis revealed a heterozygous c. 92G>A (p.Gly31Glu) mutation in exon 2 of the FGA gene. Family studies revealed that the mother carried the same mutation. Bioinformatic analysis suggested that the mutation may affect the function of Fg Protein.@*Conclusion@#The dysfibrinogenemia was probably caused by the novel Gly31Glu mutation of the FGA gene.

Clinical and genotypic analysis of two Chinese pedigrees affected with hereditary coagulable factor VII deficiency / 中华医学遗传学杂志

OBJECTIVE@#To explore molecular etiology and clinical characteristics of two pedigrees affected with hereditary factor VII(FVII) deficiency.@*METHODS@#The nine exons and flanking sequences of the F7 gene of the probands were amplified by PCR. The amplicons were analyzed by direct sequencing. Suspected mutations were subjected to SWISS-MODEL modeling and analysis of protein structure change by Pymol software and conservation of amino acids across various species.@*RESULTS@#For proband of pedigree 1, the prothrombin time (PT), FVII activity (FVII:C) and FVII antigen (FVII:Ag) were 36.3 s, 3%, 53.56%, respectively. Sequencing revealed a compound heterozygous variants of c.80_81delCT and c.1371G>T(p.Arg439Ser). His son carried a heterozygous c.1371G>T (p.Arg439Ser) variant. For proband of pedigree 2, the PT, FVII:C and FVII:Ag were 22.3 s, 4%, 1.58%, respectively. Sequencing has revealed a compound heterozygous c.278G>T(p.Arg75Met) missense variant in exon 3 and c.1278T>G (p.His408Gln) in exon 9 of the F7 gene. His mother and son both carried a heterozygous c.278G>T(p.Arg75Met) variant. Three-dimensional simulation and homology analysis revealed that the p.Arg439Ser and p.Arg75Met can respectively alter part of hydrogen bonds and two highly conserved amino acids.@*CONCLUSION@#Two novel heterozygous missense variants of the F7 gene [c.1371G>T(p.Arg439Ser) and c.278G>T(p.Arg75Met)] probably account for the decrease of factor VII in the two pedigrees.

Analysis of a pedigree affected with congenital dysfibrinogenemia due to a novel Gly31Glu mutation of FGA gene / 中华医学遗传学杂志

OBJECTIVE@#To analyze the phenotype and genotype of a pedigree affected with congenital dysfibrinogenemia.@*METHODS@#Liver and kidney functions of the proband and her relatives were determined. Coagulation tests including prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time(TT), fibrin(ogen) degradation products (FDPs), D-dimer(D-D) and the calibration experiment of protamine sulfate of against plasma TT were detected in the proband and her predigree members. The activity and antigen of fibrinogen (Fg) in plasma were measured by Clauss method and immunonephelometry method, respectively. All of the exons and exons-intron boundaries of the three fibrinogen genes (FGA, FGB and FGG) were subjected to PCR amplification and Sanger sequencing. Potential influence of the suspected mutations were analyzed with bioinformatics software including PolyPhen-2, SIFT and Mutation Taster.@*RESULTS@#The proband had normal PT, APTT, FDPs, D-D and prolonged TT (31.8 s). The activity of fibrinogen (Fg) in plasma was significantly decreased but the antigen was normal. Genetic analysis revealed a heterozygous c.92G>A (p.Gly31Glu) mutation in exon 2 of the FGA gene. Family studies revealed that the mother carried the same mutation. Bioinformatic analysis suggested that the mutation may affect the function of Fg Protein.@*CONCLUSION@#The dysfibrinogenemia was probably caused by the novel Gly31Glu mutation of the FGA gene.

Identification of compound heterozygous mutations p.Gly400Val and p.Arg532Ter of the F11 gene in a Chinese patient with hereditary factor XI deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the phenotype and genotype defect characteristics of a Chinese patient with hereditary factor XI deficiency.</p><p><b>METHODS</b>The activated partial thromboplastin time (APTT), prothrombin time (PT), FXI activity (FXI:C) of the proband and his relatives were measured by a clotting method using automatic coagulation analyzer. FXI antigen (FXI:Ag) was assayed by enzyme-linked immunosorbent assay (ELISA). Fifteen exons of the F11 gene were amplified by PCR and sequenced. Pymol software was used to analyze the novel mutations.</p><p><b>RESULTS</b>The APTT of the proband was significantly prolonged (70.3 s, reference 34.5 s) with decreased FXI activity (6%, reference 50%-150%) and FXI antigen (1.9%, reference 50%-150%). The FXI activity and FXI antigen of his son was 31% and 39%, respectively. Two heterozygous F11 mutations were identified in the proband, which included a G to T substitution at nucleotide 1296 in exon 11 resulting in substitution of glycine by valine at codon 400 (p.Gly400Val) and a A to T substitution at nucleotide 1691 in exon 14 resulting in substitution of arginine (AGA) by a termination codon (TGA) at codon 532 (p.Arg532Ter). Analysis using Pymol indicated that the number of hydrogen bonds has changed, which led to a transformation of the structure of the FXI protein. The son of the proband was found to be heterozygous for the c.1296G to T (p.Gly400Val) mutation. NM_13142 c.1691A to T (p.Arg532Ter) is a novel mutation based on HGMD professional 2016.4. Based on 2015 Guidelines of ACMG, it is PVS1 (very strong pathogenicity).</p><p><b>CONCLUSION</b>The compound heterozygous mutations of F11 NM_13142 c.1296G to T (p.Gly400Val) and F11 NM_13142 c.1691A to T(p.Arg532Ter) probably underlies the FXI deficiency in the proband.</p>

Genotypic and phenotypic analysis of a case with inherited coagulation factor X deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the correlation between F10 gene mutation and its phenotype in a Chinese pedigree affected with FX deficiency.</p><p><b>METHODS</b>Prothrombin time(PT), activated partial thromboplastin time(APTT), fibrinogen, FII activity(FII:C), FVII activity(FVII:C), FIX activity (FIX:C), FX activity(FX:C) were determined with a one-stage clotting assay. The FX antigen(FX:Ag) was detected with an enzyme linked immunosorbent assay(ELISA). The 8 exons, introns and 5' and 3' untranslated regions(UTR) of the F10 gene of the proband and her family members were subjected to PCR amplification and Sanger sequencing. Suspected mutation was confirmed by reverse sequencing. Polymorphisms were excluded by direct sequencing of 100 healthy individuals.</p><p><b>RESULTS</b>The PT and APTT of the proband have prolonged to 16.1 s and 49.0 s, respectively. Her FX:C and FX:Ag were reduced by 27% and 56%, and her mother's PT, APTT, FX:C and FX:Ag were 14.8 s, 37.4 s, 44%, 34%, respectively. Her grandmother's PT, APTT, FX:C and FX:Ag were 15.8 s, 42.2 s, 31%, 45%, respectively. The results of her father and other family members were all within the normal range. Genetic analysis has revealed a heterozygous G to A mutation in the proband at position 28076 in exon 8 of the F10 gene, which resulted in a p.Gly363Ser substitution. The same mutation was also found in her mother and grandmother. No mutation of the F10 gene was found in her father. Gly363Ser may result in changes in the secondary structure of the FX protein and reduction of its activity.</p><p><b>CONCLUSION</b>The g.28076G to A(p.Gly363Ser) mutation of the F10 gene probably underlies the FX deficiency in this pedigree. The mutation was discovered for the first time in Chinese patients.</p>

Alteration of heat shock protein 20 expression in preeclamptic patients and its effect in vascular and coagulation function / 医学前沿

Preeclampsia (PE) is a pregnancy-specific, multi-system disorder and the leading cause of maternal and perinatal morbidity and mortality in obstetrics worldwide. Excessive vasoconstriction and dysregulated coagulation function are closely associated with PE. Heat shock protein 20 (HSP20) is ubiquitously expressed under normal physiological conditions and has important roles in vascular dilatation and suppression of platelet aggregation. However, the role of HSP20 in the pathogenesis of PE remains unclear. In this study, we collected chorionic plate resistance arteries (CPAs) and serum from 118 healthy pregnant women and 80 women with PE and detected the levels of HSP20 and its phosphorylated form. Both HSP20 and phosphorylated HSP20 were downregulated in CPAs from women with PE. Comparison of the vasodilative ability of CPAs from the two groups showed impaired relaxation responses to acetyl choline in preeclamptic vessels. In addition to the reduced HSP20 in serum from women with PE, the platelet distribution width and mean platelet volume were also decreased, and the activated partial thromboplastin time and thromboplastin time were elevated.With regard to the vital roles of HSP20 in mediating vasorelaxation and coagulation function, the decreased HSP20 might contribute to the pathogenesis of PE.

Phenotypic and genetic analysis of two pedigrees affected with hereditary coagulation FXII deficiency / 中华医学遗传学杂志

OBJECTIVE@#To carry out phenotypic and genotypic analysis for two Chinese pedigrees affected with coagulation factor XII (F XII) deficiency.@*METHODS@#Plasma prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), thrombin time (TT), and blood coagulation factor VIII, IX, XI, XII activity (FVIII:C, FIX:C, FXI:C, FXII:C) were determined with one stage clotting assay on a STAGO coagulation analyzer. FXII antigen was determined with an enzyme linked immunosorbent assay (ELISA). The 14 exons and their flanking sequences of the F12 gene were subjected to PCR amplification and Sanger sequencing. The conservation and structure of mutant protein were analyzed with MegAlign software and PYMOL software.@*RESULTS@#The APTT of the probands was significantly prolonged, while their FXII:C and FXII:Ag were significantly reduced. Genetic analysis of the proband has revealed three novel mutations in the F12 gene, including g.5972G>A splice site mutation in intron 5, g.8810_8814delGTCTA in exon 14, and g.6259G>A (p.Pro182Leu) in exon 7. In addition, a previously known mutation IVS13-1G>A has been found.@*CONCLUSION@#Four mutations have been identified in the two Chinese pedigrees, among which three were novel. Above mutations probably played a role in the defect of FXII in the two pedigrees.

Preliminary Study on the Effect of Adipocytes on the Biological Behaviors of Lung Adenocarcinoma A549 Cells in Tumor Microenvironment / 中国肺癌杂志

BACKGROUND@#Adipocytes in the tumor microenvironment may provide the metabolic fuel or signal transduction through media and other means to promote a variety of malignant proliferation and invasion, of tumor cells, but their role in lung cancer progression is still unclear. The purpose of this study was to investigate the effect of adipocytes on lung cancer cell biology.@*METHODS@#3T3-L1 pre-adipocytes were induced into mature adipocytes. The cell morphology was observed by microscopy and Oil Red O staining. MTT assay, colony formation assay, wound-healing and Transwell methods were used to detect lung cancer cell proliferation, migration and invasion ability. The content of triglyceride in cells was determined by colorimetry.@*RESULTS@#The morphology of lung adenocarcinoma A549 cells became more slender after co-culture with mature adipocytes, and the proliferation and cloning ability were significantly enhanced (P<0.05). In addition, mature adipocytes can also promote the migration ability (P<0.05), invasion ability (P<0.01) and accumulation of intracellular lipid (P<0.05) of A549 cells.@*CONCLUSIONS@#These findings suggested that adipocytes in tumor microenvironment can promote the proliferation, migration and invasion of lung adenocarcinoma A549 cells, which may be related to lipid metabolism.

Study on blood lead of rats in long-term toxicity test with goupi gao / 中国中药杂志

<p><b>OBJECTIVE</b>To observe dynamic changes of blood lead concentration in rats with long-term toxicity test with Goupi Gao by the flame atomic absorption spectrometry, in order to provide reference for safe administration of Goupi Gao.</p><p><b>METHOD</b>The rats were administered with Goupi Gao by high-dose (7 g x kg(-1)), medium-dose (3.5 g x kg(-1)), low-dose (1.75 g x kg(-1)) by external use for consecutively 90 days. Then, the blood samples were collected from the rats before the administration and at 10, 30, 45, 52, 60, 90 d after the administration respectively, as well as 16 d and 28 d after the drug withdrawal. The samples were dispelled with microwave digestion system and then were determined by graphite furnace atomic absorption spectrometry for blood lead levels.</p><p><b>RESULT</b>According to methodological study, the standard curve regression equation in this experiment was A = 0.004 9X + 0. 017, r = 0.999 5, with the detection limit up to 0. 380 microg x L(-1). The RSD was 1.4% by precision checks. Blood lead level of mixed blood samples was 175.77 microg x L(-1), whose RSD was 6. 0%. Blood lead concentration gradually increased after low-dose and medium-dose administration to rats and became stable at the 10th day and the 30th day by high-dose. Dose is directly related to blood lead concentration. Meanwhile, the blood lead concentration decreases after the drug withdrawal.</p><p><b>CONCLUSION</b>The method of determination in this experiment is so accurate and reliable that it can be used for the determination of blood lead. Long-term and high-dose external use of Goupi Gao can increase blood lead.</p>

Effects of long-term external use of goupi gao on renal functions and lead accumulation in rats / 中国中药杂志

<p><b>OBJECTIVE</b>To observe the effect of long-term external use of Goupi Gao on renal function and lead accumulation in rats.</p><p><b>METHOD</b>Rats were externally administered with Goupi Gao at different doses (7, 3.5 and 1.75 g x kg(-1)) for 90 d. At 45 days and 90 days after administration, the renal indicator, levels of blood urea nitrogen (BU) and creatinine (Cr) in serum, beta2-microglobulin (beta2-MG) and N-acetyl-beta-D-glucosaminidase (NAG) in urine were determined. Lead content in kidneys was detected by atomic absorption spectrometry.</p><p><b>RESULT</b>A 90-day administration with Goupi Gao significantly enhanced the renal indicator, levels of NAG in urine and lead content in renal, when compared with the normal rats. However, the levels of BUN and beta2-MG as well as renal pathology in Goupi Gao treated rats were not obviously changed.</p><p><b>CONCLUSION</b>Consecutive administration of Goupi Gao for 90 days can increase the renal indicator and levels of NAG in urine, enhance the accumulation of lead in renal, but with no effect on excretory function of kidneys and organic changes.</p>

Research of on-line vision inspection technology for width of NC membrane / 生物医学工程学杂志

This paper presents a vision-based method for the width of NC membrane online inspection. In the production of bio-test strip, the number of antigen or antibody which is coated on the membrane depends on the width and the uniformity of test line T and control line C. People should control the width and the uniformity strictly to ensure the accuracy of lines in order to achieve quantitative inspection with high sensitivity. And online inspection must be done, it cannot be processed when the solution has been dry up. This paper gives a design of online inspection system based on linear charge-coupled device (linear CCD), it makes such advantages in terms of speed, accuracy, and the operation to achieve real-time, online inspection.

The therapeutic effect of Arsenic Trioxide to the lupus nephritis and the effect of the abnormal expression of transforming growth factor-β1 in the nephridial tissue of MRL/lpr mice / 中国医师杂志

Objective To investigate the roles of Arsenic Trioxide to the lupus nephritis in MRL/ lpr mice and the effect on the expression of TGF-pl in the renal tubule. Methods 45 MRL/lpr mice were chosen for such experiment, then separated in 3 different groups, including arsenic trioxide ( ATO) group, cyclophosphamide (CYC) group and sodium chloride (NS) group. Urine of these mice was kept after the treatment to detect the urine protein levels. The pathological changes and the expression of IgG were observed , and the complement 3 of the nephridial tissue as well as the TGF-pl were detected by immunohisto-chemistry. Result The levels of urine protein in ATO group and CTX group were lower than NS group after the treatment ( P <0.05). The expression of IgG along the glomerular mesangium and capillary loop in ATO and CTX groups was less than NS group ( P <0.05). The expression of C3 had no significant difference within three groups ( P > 0.05). The glomcrulus spherulous cells and the integral of activity in ATO group and CTX group were less than that in NS group ( P <0.05). TGF-β1 wildly presented in the epithelial cells endochylema of the nephric tubule, but it was rarely seen in glomcrulus in NS group. The expression in the nephric tubule of ATO group was less than NS group (20.28 ± 1. 90 vs 68. 23 ± 2. 87, P < 0.01) , and the expression in the nephric tubule in CTX group was less than that in NS group (23. 26 ± 1.71 vs 68. 23 ±2. 87, P <0. 01). Conclusion TGF-pl may take part in the immunopathogenesis of lupus nephritis in MRI/lpr mice. ATO can relieve the inflammation of nephric tubule by down regulation the expression of TGF-β1. ATO can also relieve the tissue damage of kidney in lupus nephritis through reducing the inflammation of glomcrulus and interstitial substance,as well as the expression of the immunocomplex.

The cloning and construction of eukaryotic expression vector of human CIDE3 and its proapoptotic effect on HeLa cells / 医学研究生学报

Objective:To clone the human cell death-inducing DFF45-like effectors 3(CIDE3) full length cDNA for construction the eukaryotic expression vector pcDNA3.l(+)-CIDE3 and to study its bioactivity.Methods:① Total RNA was extracted from human white adipose tissues and the desired cDNA fragment was obtained by RT-PCR.After the fragment had being inserted into an eukaryotic expression vector pcDNA3.l(+),the resulted recombinant plasmid pcDNA3.l(+)-CIDE3 was transformed into DH5?.The positive clone was selected and confirmed to contain full length of CIDE3 cDNA by agarose gel and DNA sequence analysis.②After the pcDNA3.l(+)-CIDE3 plasmid was transfected into HeLa cells under lipofectamine mediation,the effect of target gene expression on growth of HeLa cells was analysed by TUNEL staining. Results:① The recombinant eukaryotic expression plasmid pcDNA3.l(+)-CIDE3 was constructed successfully and the sequence of CIDE3 was consistent with that of genebank.②After transfected pcDNA3.l(+)-CIDE3,HeLa cells presented distinguished apoptosis(about 15%),compared with that of transfected plasmid pcDNA3.l(+)(