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Objective To investigate the changes of B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL) in serum of children with Hen(o)ch-Sch(O)nlein purpura nephritis (HSPN),and to explore their role in the pathogenesis of children HSPN.Methods A total of 28 children with HSPN who were before treatment were selected in Department of Pediatrics Nephrology and Rheumatology,Shengjing Hospital of China Medical University from November 2017 to August 2018.Sixteen children with Hen(O)ch-Sch(O)nlein purpura were selected as HSP group,and 20 healthy children were selected as healthy control group.Followed the HSPN guideline to cure the patients for 6-8 weeks.The clinical data were collected.Serum levels of BAFF and APRIL were measured by adopting enzyme-linked immunosorbent assay (ELISA).Results (1) Changes of serum BAFF level:the serum levels of BAFF in HSPN children were significantly lower than those in the HSP group and the healthy control group [HSPN group (0.652 ± 0.360) μg/L,HSP group (1.276 ± 0.459) μg/L,healthy control group (1.285 ± 0.299) μg/L,F =17.519,P =0.000].Moreover,the serum levels of BAFF in before treatment were significantly lower than those in after treatment [before treatment (0.652 ± 0.360) μg/L,after treatment (0.860 ± 0.262) μg/L,P ＜ 0.05).However,there were no significant di-fferences in the serum levels of BAFF between HSP group and healthy control group (P ＞ 0.05).(2)Changes of serum APRIL level:the serum levels of APRIL in HSPN and HSP children were both significantly higher than those in healthy control group,but there were no marked differences between the 2 groups [HSPN group (2.285 ± 1.015) μg/L,HSP group (2.609 ± 1.264) μg/L,healthy control group (1.677 ±0.118) μg/L,F =3.647,P =0.016].There were no significant differences in the serum levels of APRIL between before treatment and after treatment [before treatment (2.285 ± 1.015) μg/L,after treatment (2.042 ± 0.695) μg/L,P ＞ 0.05].(3) Pearson correlation analysis results showed that the serum levels of BAFF were negatively correlated with 24 h urinary protein,urinary microalbumin,and urine red blood cell count (r =-0.587,-0.608,-0.515,all P ＜ 0.05).The serum levels of APRIL were positively correlated with serum IgA (r =0.588,P ＜ 0.05).Conclusions The level of serum BAFF decreased and APRIL increased in children with HSPN,which was related to the degree of renal involvement.It suggests that BAFF and APRIL may be related to the pathogenesis of HSPN in children.
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Objective@#To investigate the changes of B cell-activating factor (BAFF) and a proliferation- inducing ligand (APRIL) in serum of children with Henöch- Schönlein purpura nephritis (HSPN), and to explore their role in the pathogenesis of children HSPN.@*Methods@#A total of 28 children with HSPN who were before treatment were selected in Department of Pediatrics Nephrology and Rheumatology, Shengjing Hospital of China Medical University from November 2017 to August 2018.Sixteen children with Henöch-Schönlein purpura were selected as HSP group, and 20 healthy children were selected as healthy control group.Followed the HSPN guideline to cure the patients for 6-8 weeks.The clinical data were collected.Serum levels of BAFF and APRIL were measured by adopting enzyme-linked immunosorbent assay (ELISA).@*Results@#(1)Changes of serum BAFF level: the serum levels of BAFF in HSPN children were significantly lower than those in the HSP group and the healthy control group[ HSPN group (0.652±0.360) μg/L, HSP group (1.276±0.459) μg/L, healthy control group (1.285±0.299) μg/L, F=17.519, P=0.000]. Moreover, the serum levels of BAFF in before treatment were significantly lower than those in after treatment [before treatment (0.652±0.360) μg/L, after treatment (0.860±0.262) μg/L, P<0.05). However, there were no significant di-fferences in the serum levels of BAFF between HSP group and healthy control group (P>0.05). (2)Changes of serum APRIL level: the serum levels of APRIL in HSPN and HSP children were both significantly higher than those in healthy control group, but there were no marked differences between the 2 groups [HSPN group (2.285±1.015) μg/L, HSP group (2.609±1.264) μg/L, healthy control group (1.677±0.118) μg/L, F=3.647, P=0.016]. There were no significant differences in the serum levels of APRIL between before treatment and after treatment [ before treatment (2.285±1.015) μg/L, after treatment (2.042±0.695) μg/L, P>0.05]. (3)Pearson correlation analysis results showed that the serum levels of BAFF were negatively correlated with 24 h urinary protein, urinary microalbumin, and urine red blood cell count (r=-0.587, -0.608, -0.515, all P<0.05). The serum levels of APRIL were positively correlated with serum IgA(r=0.588, P<0.05).@*Conclusions@#The level of serum BAFF decreased and APRIL increased in children with HSPN, which was related to the degree of renal involvement.It suggests that BAFF and APRIL may be related to the pathogenesis of HSPN in children.
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Henoch-schonlein purpura nephritis(HSPN) is the most common secondary glomerular disease in children.The pathogenesis of HSPN is unclear.In recent years,there have been some reports on B lymphocyte activating factor(BAFF) and a proliferation inducing ligand (APRIL),new members of the tumor necrosis factor family,as well as their association with HSPN.This paper reviews the related research from the following aspects:structure and biological functions of BAFF and APRIL,the role of BAFF/APRIL in the pathogenesis of HSPN,the prospect of BAFF and APRIL targeted biological agents in the treatment of HSPN.It provides a reference for further research on BAFF/APRIL system and HSPN.
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Objective To quantitatively analyze the mRNA and protein expression level of a proliferation-inducing ligand (APRIL) and investigate its clinical significance in peripheral blood mononuclear cells and plasma from newly diagnosed patients with B-cell chronic lymphocytic leukemia (B-CLL).Methods The mRNA of the target gene in 32 B-CLL patients and 15 health controls was quantified with real-time fluorescence quantitative polymerase chain reaction (RFQ-PCR) and protein by enzyme-linked immunosorbent assay (ELISA).Results APRIL mRNA was assayed with RFQ-PCR,the intra-and inter-batch reproducibility showed the coefficient of variation (CV) were 1.69 ％-6.98 ％ and 6.49 ％-10.27 ％,respectively.The expressions of APRIL mRNA and protein in patients with B-CLL were significantly higher than those in control (P ＜ 0.05),and significant difference was noted among the comparable stages in the arms (P ＜ 0.05).The expression of APRIL mRNA and protein in TDI (treatment-demand-indicator) arm was significantly higher than those in non-TDI arm (P ＜ 0.05).Conclusions APRIL may be involved in the formation and development of B-CLL and be an influence factor for disease staging.Thus,APRIL may be a prognostic indicator as well as the therapy target for the disease.
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Objective:To investigate the expression and significance of B cell activating factor (BAFF) and a proliferation-inducing ligand ( APRIL) in children with acute lymphoblastic leukemia ( ALL).Methods:The mRNA and protein expressions in ALL.
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Objective To prove the remarkable inhibitive effect of multiple siRNAs targeting a proliferation-inducing ligand (APRIL) on the human colorectal cancer cell.Methods We constructed a multiple short hairpin RNA(shRNA) expression vector containing four shRNAs (pG4) as well as four single one (pGsh644,pGsh1451,pGsh1938,pGsh2231) against APRIL gene in SW480 cell,and then transfected them into the human colorectal cancer cell line by cationic liposome.Ultimately,SW480 were screened by EGFP to obtain expression cell lines.APRIL expression levels including mRNA and APRIL protein were detected after transfected with all different kinds of vectors.Results A multiple shRNA expression vector containing four shRNAs (pG4) and four single ones were successfully constructed.Four single vectors (pGsh644,pGsh1451,pGsh1938,pGsh2231) and the multiple siRNAs expression vector (pG4) all decreased the APRIL mRNA by 56.2％,49.5％ ;50.9％,49.2％ and 79.3％.And APRIL protein expression was also remarkably reduced,especially by multiple siRNAs expression vector(87.5％).Conclusion Multiple siRNAs expression vector produced a more significant knockdown effect of APRIL than the vectors containing only one APRIL shRNA.What we found suggested us using the vector containing multiple shRNA to silence the expression of APRIL might be exploited as a novel therapeutic strategy for tumors.
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Objective To investigate the effects of a proliferation-inducing ligand(APRIL) on migration and invasion of colorectal cancer (CRC) and matrix metalloproteinases (MMPs) expression in order to observe the role of APRIL in CRC metastasis.Methods The siRNA plasmid vector targeting APRIL gene (siRNA-APRIL) was transfected into SW480 cells and recombinant human APRIL(rhAPRIL) was used to stimulate HCT-116 cells.Tumor cell migration and invasion were measured by Transwell chambers.RT-PCR and ELISA were applied to examine the expression level of MMPs.Results Metastatic and invasive capacities of siRNA-APRIL transfected SW480 were significantly inhibited,and these capacities of APRIL stimulated HCT-116 cells were significantly enhanced compared with their respective controls( all P＜0.05 ),accompanied with the alterations of MMPs mRNA and secreted protein expression( P＜0.05).The number of invading cells of SW480 control and rhAPRIL stimulated HCT-116 was significantly decreased by a MMP inhibitor GM6001 ( P＜0.05 ).Conclusion APRIL facilitates migration and invasion of CRC via regulation of MMPs,which suggests that APRIL might be used as a new target for the intervention and treatment of CRC metastasis.
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BACKGROUND: BAFF (B cell-activating factor) and APRIL (a proliferation-inducing ligand) are members of the tumor necrosis factor family and promote B cell survival and proliferation. We evaluated the correlation between serum concentration of BAFF or APRIL and severity of acute graft-versus-host disease (GVHD). METHODS: Fifteen patients who received allogeneic hematopoietic stem transplantation for leukemia and developed acute GVHD were enrolled. We determined serum concentrations of BAFF and APRIL at the onset of the first clinical manifestation of GVHD by enzyme-linked immunosorbent assay. RESULTS: Nine patients had grade 2 acute GVHD, and 6 had grade 3-4 acute GVHD. The BAFF serum concentration was higher in patients with grade 3-4 acute GVHD (1,093.42 in grade 2 vs. 2,171.99 pg/mL in grade 3-4), although the difference was not significant (P=0.077). However, the ratio of BAFF serum concentration to absolute lymphocyte count (ALC) (BAFF/ALC) was significantly higher in patients with grade 3-4 acute GVHD (P=0.045). The APRIL serum concentration and APRIL/ALC ratio showed similar results (P=0.077 and P=0.013, respectively). CONCLUSION: Patients with grade 3-4 acute GVHD had higher BAFF/ALC and APRIL/ALC ratios than patients with grade 2 acute GVHD. These findings suggest that B cells might play an important role in the development of acute GVHD, and that the BAFF and APRIL concentrations in serum might be significant predictive factors for estimating the severity of acute GVHD. Their clinical significance should be further evaluated in a larger patient population.
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Humanos , Linfocitos B , Supervivencia Celular , Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas , Leucemia , Recuento de Linfocitos , Trasplantes , Factor de Necrosis Tumoral alfaRESUMEN
Objective To construct and screen siRNA targeting a proliferation-inducing ligand (APRIL) gene in a mouse colorectal cancer celline, CT-26. To investigate the effects to the cell growth and migrant capacity of CT-26 after knockdown APRIL gene, lay the foundation for molecular targeted therapy to colorectal cancer. Methods Four pairs of APRIL siRNA were designed and chemically synthesized. And disorder sequences were synthesized as a negative control. These sequences were transfected with LipofectAMINE 2000 into CT-26 cells, which high-expressed APRIL gene. The transfection efficency rate of 6-FAM labelled control siRNA was detected by fluorescence microscope. The inhibition effectiveness of APRIL mRNA and protein was analyzed by FQ-RT-PCR and Western blot, respectively. Cell proliferation activity was analyzed by cell counting kit-8, cell migration capacity was detected by the repair of cell damage, and MMP-2 together with TIMP-1, two important regulatory genes in cell metastasis, were measured by RT-PCR.Results The different kinds of APRIL siRNA effectively suppressed the level of APRIL mRNA and the protein expression in CT-26 (P ＜ 0.05 ). Cell proliferation and metastasis ability were repressed after APRIL siRNA transfection( P ＜ 0.05 ), compared with random siRNA control and nontransfected control. The mRNA levels of MMP-2 and TIMP-1 genes wre significantly altered among APRIL siRNA groups and two control groups ( P ＜ 0.05). Conclusion We have constructed and screened a kind of siRNA (APsi737) targeting APRIL gene in a mouse colorectal cancer cell line, CT-26. APRIL siRNA can effectively inhibit the cell growth and migration capacity, maybe be regulated by MMP-2 and TIMP-1.
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Objective: To prepare and purify siRNA targeting a proliferation-inducing ligand targeted (APRIL-siRNA), so as to provide a basis for studying the role of APRIL in human pancreatic cancer. Methods: pET-22b-APRIL was constructed to express APRIL dsRNA of human pancreatic cancer cell line CFPAC-1 in E. coli and the product was purified by chromatography using CF-11 column. APRIL dsRNA was digested by RNase III to prepare APRIL siRNA, then the reaction mixture was loaded onto a DEAE ion exchange chromatography to remove RNase III from oligonucleotides, and size exclusion chromatography was used to purify 21 bp siRNA. The purified APRIL siRNA was used to transfect Chinese hamster ovary (CHO) cells and the expression of APRIL in CHO cells was observed under fluorescence microscope. Results: APRIL dsRNA was successfully expressed in E. coli after IPTG induction and was purified by CF-11 column. dsRNA was hydrolyzed with RNase III and was purified by DEAE ion exchange chromatography and size exclusion chromatography. 15% nondenaturing PAGE and 12% SDS-PAGE confirmed that RNase III was removed from oligonucleotides and 21 bp siRNA was purified with size exclusion chromatography. It was also found that APRIL siRNA obviously depressed APRIL expression in CHO cells. Conclusion: We have successfully constructed APRIL siRNA targeting APRIL gene of CFPAC-1 cells with in vitro transcription, which provides a basis for knock-down of APRIL gene in CFPAC-1 cells.
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Objective: To observe the influence of lentiviral vector-mediated RNA interference on expression of human APRIL (a proliferation-inducing ligand) gene in human pancreatic cancer cell line CFPAC-1, so as to pave a way for APRIL gene-targeted gene therapy of pancreatic cancer. Methods: Gene engineering technique was used to screen 3 RNA interference sequences targeting APRIL gene; the sequences were separately cloned into the pGCL-GFP vector to construct LV-APRIL shRNA1, LV-APRIL shRNA2 and LV-APRIL shRNA3, which were subsequently confirmed by PCR and DNA sequencing analysis. The titer of lentivirus was determined after 293T cells were cotransfected with LV-APRIL shRNA, pHelper 1.0 and pHelper 2.0. The 3 kinds of recombinant lentiviruses were injected into CFPAC-1 cells and the APRIL mRNA and protein expression were examined by real-time RT-PCR and Western blotting, respectively, and the result was compared with those of the non-transfected and blank vector transfected CFPAC-1 cells. Results: PCR analysis and DNA sequencing confirmed that the 3 APRILshRNA sequences were successfully inserted into the lentiviral vectors. The titers of concentrated virus were 5×107 TU/ml, 6 × 107 TU/ml and 4 × 107 TU/ml, respectively. APRIL expression in CFPAC-1 cells was significantly inhibited at both mRNA and protein levels compared with the non-transfected and empty vector transfected CFPAC-1 cells (P<0.05). After transfection with LV-APRIL shRNA1 and LV APRIL shRNA2, APRIL mRNA expression decreased by 73% and 68%, APRIL protein expression decreased by 66% and 59% (P<0.05), respectively; there was no significantly difference between the nontransfected and empty vector transfected CFPAC-1 cells. Conclusion: Three lentiviral RNAi vectors of APRIL gene have been successfully constructed, and they can effectively inhibit the expression of APRIL gene in CFPAC-1 cells in vitro.
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Objective To investigate the mRNA expression of a proliferation inducing ligand (APRIL) and its receptors including B cell maturation antigen (BCMA),transmembrane activator.calcium modulator and cyclophilin ligand interactor (TACI) in the peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SEE).Methods APRIL mRNA、BCMA mRNA and TACI mRNA in PBMCs were detected by real-time quantitative PCR in 66 SLE patients and 25 normal controls.Gene expression level was measured as 2-AACT.Results The expression levels of APRIL mRNA、BCMA mRNA and TACI-mRNA were significantly increased in both active SLE group and stable SLE group compared with those in the normal controls(P<0.01 for all).The expression levels of APRIL mRNA and TACI mRNA in active SLE group were significantly higher than those in stable SLE group(P<0.01,P<0.05,respectively).But there was no significant difierence in the expression levels of BCMA mRNA between the SLE stable and active groups-Beside,the expression levels of APRIL mRNA and TACI mRNA were significantly increased in patients with lupus nephritis (LN) compared to patients with non-LN (P<0.01 for all).Conclusion The expression levels of APRIL and its receptors are significantly elevated in SLE patients.It may suggest that APRIL and its receptors play an important role in the pathogenesis of SLE.
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Objective To study the effect of matrine on the expression of a proliferation-inducing ligand (APRIL) in colorectal cancer cell line (SW480 cell). Methods MTT assay was used to evaluate the inhibitory effect of matrine on SW480 cells. The protein and mRNA levels of APRIL in SW480 cells were determined by immunohistochemistry and real-time fluorescence quantitative PCR (RFQ-PCR). SW480 cells were treated with 0.5,1.0,2.0 mg/ml of matrine for 24 h, 48 h and 72 h. FU and blank were served as drug control and blank control groups, respectively. Results Matrine had obviously inhibitory effect on proliferation of SW480 cells in a time- and dose-dependant manner. The expression of APRIL was strong in SW480 cells. When treated with 50,100,200 ug/ml of FU, the APRIL mRNA levels in SW480 cells raised gradually and reached the highest levels at 72 h after treatment, which were significantly higher than those in blank control group (all P value<0.001). When treated with 0. 5,1.0, 2.0 mg/ml of matrine, the APRIL mRNA levels in SW480 cells increased at 24 h after treatment, which were significantly higher than those in blank control group (all P value<0. 001), and then decreased gradually and almost equal to level of blank control group at 72 h. Conclusion In treatment with FU, the survival cells.may have stronger ability of proliferation due to higher expression of APRIL in SW480 cells. Anti-APRIL therapy might be an important assistant treatment to counter the impact of APRIL. Matrine will not cause persistent increase of APRIL mRNA levels in SW480 cells, so it might be a helpful drug in anti-tumor theraphy.
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Objective To study the expression and significance of B-cell activating factor(BAFF) and a proliferation-inducing ligand(APRIL) in patients with idiopathic thrombocytopenic purpur(ITP). Methods The serum levels of BAFF and APRIL in 27 cases with ITP were tested by ELISA.Results ①The serum levels of BAFF in ITP were higher than those of control group (3.92?1.88?g/ml vs 2.90?0.52?g/ml,P
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Objective To establish real-time fluorescence quantitative polymerase chain reaction(RFQ-PCR) for measurement of the expression level of a proliferation-inducing ligand(APRIL)and its receptors in children with non-Hodgkin′s lymphoma(NHL).Met-hods Specific primers and TaqMan probes of APRIL and its receptors had been designed,and fluorescence of the PCR products were detected continuously during amplification.According to the standard curves created by plasmid DNA,the expression level of target genes in clinical samples had been determined using software,and these results were presented as the ratios of target genes′ mRNA to ?2 microg-luobulin(?2M)′s mRNA.Results The detection range of RFQ-PCR was between 101-109 ng/L,the coefficient of variation values for both intra-experimental and inter-experimental reproducibility ranged from 1.68% to 5.97% and 6.40% to 10.58%,respectively.The results from 22 samples showed that the expression level of APRIL,B cell maturation antigen(BCMA) and transmembrane activator and calcium modulator and cyclophilin ligand(TACI) in peripheral blood of NHL were significantly higher than those in normal children(P
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Objective To investigate the role of the expression of a proliferation-inducing ligand (APRIL) and its receptors in the gastric carcinogenesis. Methods The real-time fluorescence quantitative polymerase chain reaction (RFQ-PCR) was used to detect expression of APRIL and its receptors. Based on the standard curves, the quantitative levels of target genes in tissue samples were determined by using software, and the results were presented as the ratios of mRNA levels of target genes to ?2-microgluobulin(?2M). Results The detection linear range of RFQ-PCR was 101-109 pg/ml and the coefficient of variation values for both intra-experimental and inter-experimental reproducibility ranged 6.52% - 12.02% and 8.76% - 14.16%, respectively.The expression levels of APRIL in the tissue of intestinal metaplasia , dysplasia and gastric cancer were significantly higher than those in normal tissue(P0.05 , respectively). Conclusions The present study indicated that RFQ-PCR had satisfied sensitivity and reproducibility in quantitative measurement of APRIL and its receptors. APRIL may play an important role in the development and progress of gastric cancer and could be established as a target molecule for early diagnosis and anti-cancer therapy. Besides, there maybe some unknown receptors of APRIL expressed on tumor tissue.
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Objective:To prepare and purify siRNA targeting a proliferation-inducing ligand targeted(APRIL-siRNA),so as to provxde a basis for studying the role of APRIL in human pancreatic cancer.Methods:pET-22b-APRIL was constructed to express APRIL dsRNA of human pancreatic cancer cell line CFPAC-1 in E.coli and the product was purified by chromatography using CF-11 column.APRIL dsRNA was digested by RNaseⅢto prepare APRIL siRNA,then the reaction mixture was loaded onto a DEAE ion exchange chromatography to remove RNaseⅢfrom oligonucleotides,and size exclusion chromatography was used to purify 21 bp siRNA.The purified APRIL siRNA was used to transfect Chinese hamster ovary(CHO)cells and the expression of APRIL in CHO cells was observed under fluorescence microscope Results:APRIL dsRNA was successfully expressed in E.coli after IPTG induction and was purified by CF-11 column.dsRNA was hydrolyzed with RNaseⅢand was purified by DEAE ion exchange chromatography and size exclusion chromatography.15% nondenaturing PAGE and 12% SDS- PAGE confirmed that RNaseⅢwas removed from oligonucleotides and 21 bp siRNA was purified with size exclusion chromatography.It was also found that APRIL siRNA obviously depressed APRIL expression in CHO cells.Conclusion:We have successfully constructed APRIL siRNA targeting APRIL gene of CFPAC-1 cells with in vitro transcription,which provides a basis for knock-down of APRIL gene in CFPAC-1 cells.
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Objective:To investigate the expression of APRIL in NSCLC and to analyze the relationship between expression of APRIL and clinicalpathological characteristics of NSCLC.Methods:RTQ-PCR and Western blot were used to detect the expression of APRIL mRNA and protein in 68 pair of tumor tissues and corresponding adjacent normal tissues of NSCLC.50 Formalin-fixed,paraffin-embedded NSCLC samples were tested for APRIL protein location and expression by IHC,with results compared to the clinical and pathological parameters of NSCLC to determine significance.Results:The results showed that the expression level of APRIL mRNA and protein in tumor tissues was significantly higher than in normal tissues(both P
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Objective To establish a SYBR Green Ⅰ quantitative real-time PCR method for detecting the expression of APRIL gene(a proliferation-inducing ligand,APRIL) in peripheral blood of patients with auto-immune diseases,e.g.,systemic lupus erythematosus(SLE) and rheumatoid arthritis(RA),and investigate the relationship of APRIL mRNA expression with pathogenesis and prognosis of auto-immune diseases.Methods Plasmid PGEM-T easy-APRIL was cloned as the standard template.SYBR Green Ⅰ quantitative real-time PCR was set up to examine the expression of APRIL mRNA in peripheral blood of 58 patients with auto-immune diseases and 20 healthy controls using Line Gene FQD-33A Detection System.Results The obtained data were normalized by dividing the copy number of target cDNA by those of GAPDH(glyceraldehycle-3-phosphate dehydrogenase,GAPDH).APRIL expression levels ranges from 3.95 to 192 and mean value was 29.68?4.5.APRIL expression of twenty healthy controls showed range from 3.1 to 18.7 and mean value of 10.56?2.0.APRIL expression levels in patients with auto-immune diseases were higher than those in healthy controls.In auto-immune diseases group APRIL expression levels of untreated patients were higher than those of the other patients,and a statistical significance was found.Conclusions APRIL mRNA was successfully detected by SYBR Green Ⅰ quantitative real-time PCR and the method was accurate and reliable.The expression of APRIL mRNA of auto-immune disease patients was higher than those of healthy controls,and the expression of untreated patients was the highest.This method may be used for further study on the high-level expression of APRIL mRNA in mechanism of auto-immune diseases as well as the development and prognosis of diseases.