WO2007007706A1 - Procede de criblage d'une substance capable d'induire ou d'inhiber la proteinurie, plasmide et cellule transfectee a utiliser dans le procede et biocapteur mettant en oeuvre le plasmide ou la cellules transfectee - Google Patents

Procede de criblage d'une substance capable d'induire ou d'inhiber la proteinurie, plasmide et cellule transfectee a utiliser dans le procede et biocapteur mettant en oeuvre le plasmide ou la cellules transfectee Download PDF

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WO2007007706A1
WO2007007706A1 PCT/JP2006/313674 JP2006313674W WO2007007706A1 WO 2007007706 A1 WO2007007706 A1 WO 2007007706A1 JP 2006313674 W JP2006313674 W JP 2006313674W WO 2007007706 A1 WO2007007706 A1 WO 2007007706A1
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gene
proteinuria
cell
plasmid
nephrin
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Japanese (ja)
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Masanori Kitamura
Kozue Yamauchi
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University of Yamanashi NUC
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University of Yamanashi NUC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • Proteinuria is one of the typical clinical findings in renal diseases, particularly renal glomerular diseases. Leakage of protein into the urine results in hypoproteinemia and hypoalbuminemia due to decreased protein levels in the serum, systemic edema due to fluid that has moved between cytoplasms and body cavities due to decreased colloid osmotic pressure, Causes edema of the organ, pleural effusion, ascites, etc., resulting in circulatory failure due to intravascular dehydration It is also known that renal tubule and interstitial damage and fibrosis progress due to proteins leaked into the urinary cavity, leading to decreased kidney function and promotion of transition to chronic kidney failure. . Therefore, control of protein urine is an important target in the treatment of kidney disease.
  • Non-Patent Document 1 Non-Patent Document 1
  • Non-Patent Document 2 nephrin gene abnormalities in renal diseases that cause proteinuria.
  • Non-Patent Documents 2 and 3 decreased expression of nephrin.
  • drugs compounds, natural extracts, etc.
  • the present invention has been made in view of the above circumstances, and provides an in vitro screening 'evaluation method capable of quickly and inexpensively screening for a substance causing or suppressing proteinuria and evaluating a pharmacological effect. This is the issue.
  • the present invention is characterized by having a gene promoter whose expression level is changed when proteinuria is induced or suppressed, and a secretory reporter gene incorporated downstream of the promoter.
  • a plasmid is provided.
  • the reporter gene is expressed according to the activity of the promoter of the gene whose expression level changes upon induction or suppression of the proteinuria.
  • the gene whose expression level is changed when proteinuria is induced or suppressed is preferably a nephrin gene whose expression is decreased in kidney diseases that cause proteinuria.
  • the secretory reporter gene is preferably a secreted alkaline phosphatase gene (SEAP gene).
  • SEAP gene secreted alkaline phosphatase gene
  • the gene-transferred cell is in the presence of a test drug!
  • the present invention provides a method for evaluating pharmacological effects or side effects on proteinuria, which comprises culturing in vitro and measuring the expression level of the reporter gene.
  • the amount of reporter protein in the culture supernatant is measured, whereby the pharmacological effect of the test drug on proteinuria, and proteinuria as a side effect. Can be evaluated.
  • the present invention provides a method for separating glomerular epithelial cells, characterized by culturing the above-described gene-transfected cell expressing a nephrin gene in the presence of a test substance or a factor, and measuring the expression level of the nephrin gene.
  • the present invention is characterized by using the above-described gene-transfected cell that expresses a nephrin gene, and analysis of glomerular epithelial cell intracellular communication related to the expression control of nephrin gene and the differentiation or dedifferentiation of glomerular epithelial cells Provide a method.
  • proteinuria is rapidly and inexpensively measured by a simple in vitro technique for measuring the expression level of a reporter protein in a culture supernatant after culturing a transgenic cell in the presence of a test substance / drug. It is possible to screen for substances involved in the induction or suppression of protein, and to evaluate pharmacological effects or side effects of drugs on proteinuria.
  • the present invention is excellent as a tool for analyzing and discovering the molecular mechanism that causes proteinuria, and the search and development of therapeutic agents useful for renal diseases such as nephrotic syndrome that is difficult to cure by medication. /!
  • FIG. L (a) An explanatory diagram showing the structures of recombinant plasmids p5N-SEAP and p8N-SEAP that are relevant to an embodiment of the present invention. (B) A graph showing the SEA P activity of NePo5N5 cells into which a recombinant plasmid has been introduced. (C) A photograph showing the results of electrophoresis for confirming a PCR product in which the mRNA expressed by ATRA stimulation is in a vertical shape.
  • FIG. 2 is a graph showing the ATRA concentration dependence of SEAP activity in NePo5N5 cells.
  • FIG. 3 is a graph showing the dependence of the SEAP activity of NePo5N5 cells on the culture time after ATRA stimulation.
  • FIG. 4 is a graph showing the SEAP activity of other NePo cells.
  • FIG. 5 is a graph showing the SEAP activity when various test substances are added to NePo5N5 cells.
  • FIG. 6 is a graph showing the vitamin D concentration dependency of SEAP activity of NePo cells.
  • FIG. 7 is a graph showing the dependency of SEAP activity of NePo cells on dexamethasone concentration. Explanation of symbols
  • promoter that controls nephrin gene expression A reporter gene consisting of a gene and an alkaline phosphatase gene (SEAP gene) as a secreted reporter gene connected downstream is used.
  • SEAP gene alkaline phosphatase gene
  • An expression vector suitable for introduction into cultured cells is cleaved with a restriction enzyme, and the nephrin promoter and alkaline phosphatase gene (SEAP gene) are inserted, whereby the alkaline phosphatase gene (SEAP) is activated by the activity of the nephrin promoter.
  • SEAP alkaline phosphatase gene
  • a method for constructing a recombinant plasmid by incorporating a reporter gene to which the nephrin promoter and alkaline phosphatase gene (SEAP gene) are connected into an expression vector the nephrin promoter or alkaline phosphatase gene (SEAP gene) is used.
  • SEAP gene the nephrin promoter or alkaline phosphatase gene
  • an alkaline phosphatase is produced according to the activity of the nephrin promoter and secreted into the culture solution (transformant).
  • Gene transfer can be performed by a general method used for gene transfer, such as a calcium phosphate method or a lipofussion method.
  • a substance that induces nephrin gene expression can be quantitatively detected as the expression of alkaline phosphatase (SEAP) secreted into the culture medium.
  • FIG. 1 (a) shows the structure of plasmids p5N-SEAP and p8N-SEAP (1).
  • a recombinant plasmid pSEAP-Basic2 (BD Biosciences) containing a secreted alkaline phosphatase gene (SEAP gene) (3), which functions as a reporter gene, is inserted into the multicloning site (upstream of the SEAP gene).
  • MCS restriction enzyme site: After cutting at Xhol, a nephrin promoter 5N (2) consisting of 5.4 kb upstream of the nephrin gene was inserted to construct a recombinant plasmid p5N-SEAP (l).
  • a recombinant plasmid p8N-SEAP (l) was constructed by inserting a nephrin promoter 8N (2) consisting of 8.3 kb upstream of the nephrin gene into the recombinant plasmid pSEAP-Basic2 (BD Biosciences).
  • the above recombinant plasmid p5N- SEAP (1) 4 g of the plasmid pcDNA3.1 (Invitrogen) that constantly expresses the neomycin resistance gene: 0.4 ⁇ g and culture immortalized with the temperature-sensitive SV40 larg e T antigen Genes were introduced into mouse glomerular epithelial cells (provided by Dr. Karlhans Erium) using Lipofectamine 2000 (Invitrogen): 6 ⁇ 1 by the lipofusion method.
  • the recombinant plasmid p8N-SEAP (l) was similarly transfected into the cultured mouse glomerular epithelial cells.
  • NePo nephrin reporting podocyte
  • the nephrin promoter is known to be activated by all-trans retinoic acid (ATRA) in cultured human cervical cancer-derived cells (HeLa cells). Using this, the responsiveness of NePo cells established by the above method was verified.
  • ATRA all-trans retinoic acid
  • NePo cells were seeded in a 96-well culture plate coated with type I collagen (lxlO 4 cells / 100 / zl), cultured for 48 hours in the absence of interferon ⁇ (IFN y), and then 10 M Of ATRA (in the presence of 1% fetal calf serum) and further cultured for 24 hours. NePo cell culture supernatant was collected and SEAP activity was measured.
  • type I collagen lxlO 4 cells / 100 / zl
  • IFN y interferon ⁇
  • ATRA in the presence of 1% fetal calf serum
  • SEAP activity was measured using the Great EscAPe SEAP measurement kit (BD Bioscience) and a luminometer. As a result, NePo cells that were responsive to ATRA were selected.
  • Figure 1 (b) shows the measurement results of the SEAP activity of NePo5N5 cells that have been confirmed to respond to ATRA (No.5 of NePo cell clone into which recombinant plasmid p5N-SEAP has been introduced).
  • NePo5N5 cells showed a constant SEAP activity (20,000RLU) even in the absence of ATRA stimulation (control), confirming that they retain the trait of glomerular epithelial cells before recombinant plasmid introduction. It was done.
  • stimulation of these cells with 10 M ATRA increased the SEAP activity 2.3 times (46,000 RLU).
  • NePoN5N cells are seeded in a 6-well culture plate (lxl0 5 cells / 100 ⁇ 1) After further stimulation for 24 hours, total RNA was extracted using TrisoKlnvitrogen), and reverse transcription reaction was performed using Omniscript RT kit (Qiagen) using this as a template.
  • FIG. 1 (c) shows the result of electrophoresis of the PCR product obtained by the PCR method.
  • Figure 2 shows changes in SEAP activity dependent on ATRA concentration in NePo5N5 cells.
  • NePo5N5 cells As the concentration of ATRA to stimulate increases, the SEAP activity of NePo5N5 cells increases! This shows that the expression of the nephrin gene is enhanced in NePo5N5 cells depending on the concentration of ATRA.
  • NePo5N17 cells and NePo8N2 cells have the same responsiveness to ATRA as NePoN5N cells described above. It was confirmed.
  • NePo5N5 cells were seeded in a 96-well culture plate coated with type I collagen (lxlO 4 cells / 100 ⁇ 1) and cultured for 48 hours in the absence of interferon ⁇ (IFN ⁇ ). 1% fetal bovine serum was added). Furthermore, after culturing for 24 hours, the culture supernatant was collected and SEAP activity was measured.
  • the above test substances include (1) ATRA: 1 ⁇ , (2) ATRA: 10 ⁇ M, (3) Dexamethasone: 0.1 M, (4) Dexamethasone: 1 ⁇ , (5) Active type Vitamin D : 10— 8 ⁇ , (6) Active B
  • FIG. 5 shows changes in SEAP activity when the test substance is added.
  • NePo5N5 cells added with substances that give inflammatory stimuli such as interleukin-1 ⁇ (IL-1 ⁇ ), tumor necrosis factor (TNFa), and growth stimulators such as hepatocyte growth factor (HGF) A significant decrease in SEAP activity was observed.
  • IL-1 ⁇ interleukin-1 ⁇
  • TNFa tumor necrosis factor
  • HGF hepatocyte growth factor
  • dexamethasone, active vitamin D, etc. are mediated through increased expression of the nephrin gene.
  • Inflammation-related stimuli such as -1 ⁇ (IL-1 ⁇ ), tumor necrosis factor (TNF a), and hepatocyte growth factor (HGF) may decrease nephrin gene expression, resulting in proteinuria. Performance is suggested.
  • Fig. 6 and Fig. 7 show the S of NePo cells depending on the concentrations of active vitamin D and dexamethasone.
  • the SEAP activity of NePoN5N cells is increased in proportion to the increase in the number of NePoN5N cells.
  • the SEAP activity and the expression of the nephrin gene are correlated with each other by RT-PCR.
  • NePo cells which are transgenic cells, are cultured for a predetermined time in the presence of a test substance, and then the culture supernatant is collected and SEAP activity is measured in
  • a simple procedure in vitro it is possible to screen a large number of test substances at once at a low cost for the ability to induce or suppress proteinuria. Therefore, it is useful for the development of therapeutic agents for glomerular diseases that cause proteinuria such as nephrotic syndrome.
  • NePo cells of the present invention By using the NePo cells of the present invention, a pharmacological effect on proteinuria in vitro by adding a therapeutic agent to NePo cells and culturing and measuring SEAP activity before the start of the trial. An assessment of the presence or absence of side effects can be obtained. Similarly, it can also be evaluated whether or not a drug administered as a therapeutic agent for other diseases can cause proteinuria as a side effect.
  • Nephrin is a major component of the slit membrane formed between glomerular foot processes, It is known as a marker of glomerular epithelial cells.
  • NePo cells were added with substances or factors that may be involved in glomerular epithelial cell sorting or detachment, cultured, and measured for SEAP activity. By doing so, it is possible to identify a substance that induces differentiation of glomerular epithelial cells or a substance that induces dedifferentiation.
  • substances necessary for maintaining homeostasis such as retinoic acid, steroid hormones, vitamin D, and the like are endogenous differentiation factors of glomerular epithelial cells.
  • Inflammation such as interleukin 1 j8 (IL-1 ⁇ ) and tumor necrosis factor- a (TNF a) produced locally only during inflammation It is suggested that sex-site force-in acts as a detachment factor for glomerular epithelial cells.
  • NePo cells of the present invention are exposed to various substances, and the SEAP activity in the culture supernatant is measured, thereby easily screening for substances and factors involved in inducing or suppressing nephrin gene expression. Can do.
  • Signal transduction pathways via protein kinase A and protein kinase C are representative of intracellular signal pathways that control various cell functions and cell dynamics.
  • the NePo cell of the present invention is an intracellular mechanism involved in the regulation of nephrin gene expression, glomerular epithelial cell sorting or de-folding, and the biological activity of glomerular epithelial cells. It can also be used as a useful analysis tool for analyzing various characteristics.

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  • Chemical & Material Sciences (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract

L'invention concerne un procédé destiné au criblage/évaluation in vitro d'une substance capable d'induire ou d'inhiber la protéinurie et pouvant cribler la substance ou évaluer l'effet pharmacologique de la substance rapidement à un faible coût. L'invention concerne également un plasmide possédant un promoteur destiné à un gène dont la quantité d'expression varie quand la protéinurie est induite ou inhibée et un gène rapporteur sécrété intégré en aval du promoteur; et une cellule transfectée comprenant le plasmide introduit dans le gène de celle-ci.
PCT/JP2006/313674 2005-07-14 2006-07-10 Procede de criblage d'une substance capable d'induire ou d'inhiber la proteinurie, plasmide et cellule transfectee a utiliser dans le procede et biocapteur mettant en oeuvre le plasmide ou la cellules transfectee Ceased WO2007007706A1 (fr)

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JP2007524638A JPWO2007007706A1 (ja) 2005-07-14 2006-07-10 タンパク尿を惹起又は抑制する物質のスクリーニング方法、これに用いるプラスミド及び遺伝子導入細胞、並びにこれを用いたバイオセンサー

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008185536A (ja) * 2007-01-31 2008-08-14 Niigata Univ 腎障害の判定方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002506883A (ja) * 1998-03-18 2002-03-05 バイオストレイタム，インコーポレイテッド ネフリン遺伝子とタンパク質

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002506883A (ja) * 1998-03-18 2002-03-05 バイオストレイタム，インコーポレイテッド ネフリン遺伝子とタンパク質

Non-Patent Citations (9)

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Title
BAO R. ET AL.: "Activation of cancer-specific gene expression by the survivin promoter", J. NATL. CANCER INST., vol. 94, no. 7, 3 April 2002 (2002-04-03), pages 522 - 528, XP002367672 *
BELTCHEVA O. ET AL.: "Alternatively used promoters and distinct elements direct tissue-specific expression of nephrin", J. AM. SOC. NEPHROL., vol. 14, no. 2, February 2003 (2003-02-01), pages 352 - 358, XP003003046 *
GREGORY B. ET AL.: "A fast and sensitive colorimetric assay for IL-6 in hepatoma cells based on the production of a secreted form of alkaline phosphatase (SEAP)", J. IMMUNOL. METHODS, vol. 170, no. 1, 29 March 1994 (1994-03-29), pages 47 - 56, XP000601567 *
MOELLER M.J. ET AL.: "Evaluation of a new tool for exploring podocyte biology: mouse Nphs1 5' flanking region drives LacZ expression in podocytes", J. AM. SOC. NEPHROL., vol. 11, no. 12, December 2000 (2000-12-01), pages 2306 - 2314, XP003003044 *
PLUMPTON M. ET AL.: "A high capacity assay for inhibitors of human papillomavirus DNA replication", BIOTECHNOLOGY (N Y), vol. 13, no. 11, 13 November 1995 (1995-11-13), pages 1210 - 1214, XP003003041 *
PUTAALA H. ET AL.: "The murine nephrin gene is specifically expressed in kidney, brain and pancreas: inactivation of the gene leads to massive proteinuria and neonatal death", HUM. MOL. GENET., vol. 10, no. 1, January 2001 (2001-01-01), pages 1 - 8, XP003003045 *
QUAGGIN S.E.: "Transcriptional regulation of podocyte specification and differentiation", MICROSC. RES. TECH., vol. 57, no. 4, May 2002 (2002-05-01), pages 208 - 211, XP003003042 *
SUZUKI A. ET AL.: "Retinoids regulate the repairing process of the podocytes in puromycin aminonucleoside-induced nephrotic rats", J. AM. SOC. NEPHROL., vol. 14, no. 4, April 2003 (2003-04-01), pages 981 - 991, XP003003039 *
WAGNER N. ET AL.: "The major podocyte protein nephrin is transcriptionally activated by the Wilms' tumor suppressor WT1", J. AM. SOC. NEPHROL., vol. 15, no. 12, December 2004 (2004-12-01), pages 3044 - 3051, XP003003043 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008185536A (ja) * 2007-01-31 2008-08-14 Niigata Univ 腎障害の判定方法

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