JP2006267125A - Method of screening disease by measuring leptin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a judging method for pregnancy by measuring leptin, that is a hormone known as expression in a fatty tissue, having a secretory signal outside a cell, and secreted in a blood flow, from a nonfatty cell, and a leptin measuring method for determining a cancer or an uterus-related disease. <P>SOLUTION: This method of screening a disease by measuring the leptin derived from a nonfatty tissue in a biosample comprises an immunoassay and an immune tissue assay using an anti-human leptin antibody. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a method for detecting cancer or a pregnancy disease by measuring leptin, an obesity gene product, and a method for determining pregnancy.

   Leptin is a product of the obesity gene (ob gene) cloned positionally as a gene lacking hereditary obese mice (ob / ob mice). F. The cDNA of the human ob gene was cloned from a cDNA library derived from human tissue cells using the mouse ob gene (3). Leptin is known to be expressed in adipose tissue, is a hormone that has a secretion signal outside the cell and is secreted into the bloodstream. Secreted leptin is thought to act on the appetite center in the hypothalamus of the brain, controlling feeding activity and suppressing fat intake.

Recently, mouse and human leptin receptors (ob-R) have been cloned and reported to be a single transmembrane receptor protein belonging to the gp130 family, the cytokine receptor superfamily (11). It was also confirmed that the modified ob-R specifically and strongly bound to leptin. Ob-R is strongly expressed in the kidney and lung, and in the brain and others, it has also been confirmed in the heart, liver, and smooth muscle (1, 13, 14, 15, 23). There are several isoforms of ob-R, and the biologically active ob-Rb isoform is most abundantly expressed in the brain, particularly in the hypothalamus, the central part of satiety (12, 13, 14, 15, 16, 17). Ob-Rb deficiency due to gene point mutations in obese mice (db / db mice), ob-R mutations due to ob-R gene mutations in obese rats (fa / fa rats and fak / fak rats) (15, 24, 25), all of these animals were leptin resistant, developed obesity, and showed an obesity-related phenotype similar to ob / ob mice (26).
These facts indicate that obesity is caused by the fact that it does not normally function as a leptin hormone due to mutation or deletion of the ob gene or its receptor gene.
Ob gene expression and leptin secretion have been reported to increase in proportion to their severity in obese model animals and human obesity (3, 5, 18, 19, 20, 21, 22). Administration of recombinant mouse leptin and human leptin to ob / ob mice and obese mice with normal leptin to investigate the anti-obesity effect caused an increase in body temperature and an increase in energy consumption (basal metabolism), resulting in a decrease in body weight Is considered to be a weight regulating factor (1, 2, 3) and a satiety factor (3, 4, 5, 6, 7, 8, 9, 10).

Leptin is also an important regulator of the neuroendocrine and reproductive systems. Ob / ob mice, db / db mice and fa / fa rats, which are diabetes model animals, are known to be infertile with some endocrine abnormalities (26), and leptin is administered to female ob / ob mice Then, it has been reported that infertility can be treated independently of eating and weight changes (27). It has also been reported that administration of leptin to normal fasted mice increases levels of gonadotropin and thyroxine in plasma and decreases levels of corticotropin and corticosterone (28, 29).

At present, a method for detecting urinary human chorionic gonadotropin (hCG) is widely used as an indicator of pregnancy as an adjunct to pregnancy diagnosis in daily clinical tests. hCG is a glycoprotein hormone having a molecular weight of about 38,000 that is secreted from the villous tissue of the placenta by fertilization and implantation. Urinary hCG rises from the fifth week of pregnancy and remains at a similar level until delivery, although it decreases slightly after reaching the highest value in the ninth to fourteenth weeks. Therefore, hCG in urine is used as an index for pregnancy diagnosis, and the presence of hCG means the presence of chorionic tissue and is also used for diagnosis and management of chorionic diseases. However, hCG has cross-reactivity with human luteinizing hormone (hLH), human follicle stimulating hormone (hFSH), human thyroid stimulating hormone (hTSH) and human prolactin (hPRL), and its specificity is a problem. Since hCG shows cross-reactivity with other hormones in pregnancy diagnosis, it was not perfect as a pregnancy determination marker.

Furthermore, the hormone can be expected to be useful as an index for tumor diagnosis. A hormone that is produced when a tissue that originally does not produce a hormone becomes a tumor is called an ectopic hormone, and a tumor that produces an ectopic hormone is called an ectopic hormone-producing tumor. Diagnosis of ectopic hormone-producing tumors includes (1) hormonal excess symptoms or abnormal laboratory findings, (2) improvement of hormonal excess symptoms by removal of tumor tissue, (3) higher hormonal activity than normal tissue from tumor tissue Proof is the standard. There are many known ectopic hormones such as adrenocorticotropic hormone (ACTH), antidiuretic hormone (ADH), parathyroid hormone (PTH), pancreatic secretory trypsin inhibitor (PSTI), growth hormone, vasopressin and somastatin. Leptin can be expected to have the same usefulness. Methods for measuring many tumor markers other than ectopic hormones have been developed for tumor diagnosis. Currently useful tumor markers have low specificity to specific diseases, and many of them are extremely low in blood even in cancer patients. For example, α-fetoprotein (AFP) and carcinoembryo Many measurements such as antigen (CEA) and pancreatic carcinoembryonic antigen (POA) require high sensitivity. Although radioimmunoassay (RIA) can be used for highly sensitive measurement, various regulations such as expiration date and waste disposal are problematic. Recent advances in science make it possible to obtain high sensitivity even in enzyme immunoassay (EIA) and fluorescence immunoassay (FIA) by developing antigens and monoclonal antibodies, and developing new enzyme substrates and fluorescent agents. became. However, no tumor marker that can be said to be sufficient in terms of economy and convenience has been reported.

From previous reports, leptin is an obesity factor produced and secreted by adipocytes, and plays an important role in body fat regulation. It is also an important physiological activity regulator in the neuroendocrine and reproductive systems. Guessed. Currently, leptin is an adipocyte-derived circulating obesity factor involved in the regulation of food intake and energy expenditure, and its usefulness in the diagnosis and treatment of obesity and its clinical application are being investigated. However, little has been elucidated regarding leptin production from non-adipocytes and functions of regulating physiological activity in the neuroendocrine and reproductive systems.

Plasma leptin levels in pregnant women are significantly higher than those estimated from body index (BMI) in non-pregnant women, and are almost the same as plasma leptin levels (18, 20, 21) in obese women I found out. In order to examine leptin-expressing cells, immunohistochemical staining and Northern blot analysis of human gestational uterine cells were performed. As a result, placental chorionic tissue, smooth muscle chorion, amniotic membrane, and more specifically, trophoblast cells, which are non-adipocytes They found that leptin was produced in syncytiotrophoblast cell layers and amniotic cells.

The same analysis was also performed on BeWo cells derived from human pregnant choriocarcinoma metastasis. It was confirmed that leptin was produced and that trophoblastic diseases such as hydatidiform mole and choriocarcinoma such as choriocarcinoma were also observed. It has been found that plasma leptin levels are significantly increased in patients and decreased after surgical treatment or chemotherapy. From this, the usefulness of leptin as an ectopic hormone-producing tumor marker can be expected.

The present inventors have determined that measurement of non-adipose tissue-derived leptin in a biological sample is a useful diagnostic index that reflects disease activity and / or therapeutic effect in cancer and villous disease, and a determination index for pregnancy Found useful. That is, the present invention
(1) A screening method for diseases by measuring leptin derived from non-adipose tissue in a biological sample,
(2) The method according to (1) above, which is cancer,
(3) The method according to (2) above, wherein the cancer is choriocarcinoma (4) the method according to (1) above, which is a uterine-related disease,
(5) The method according to (4) above, wherein the uterine-related disease is a trophoblast disease,
(6) The method according to (5) above, wherein the trophoblastic disease is hydatidiform mole or choriocarcinoma,
(7) A method for determining the presence or absence of cancer cells, wherein a biological sample is collected from a patient suspected of having cancer and the amount of leptin is measured. Comparing the amount of leptin in the sample,
(8) A leptin measurement method for determining the presence or absence of cancer cells,
(9) A method for determining pregnancy by measuring leptin derived from non-adipose tissue in a biological sample,
About.

The present invention is based on the result of measuring the presence and amount of leptin expression in a biological sample.
That is, the present invention relates to a disease detection method by measuring the presence or absence of expression in a non-adipose tissue and the amount of leptin derived from the tissue in a biological sample of a patient.
Adipose tissue stores fat in cells and is composed of cells that have lipid biosynthesis from sugar and its degradation function. Thus, “non-adipose tissue” means tissue composed of cells other than such cells derived from adipose tissue.
As the “biological sample”, tissues, plasma, serum, urine, amniotic fluid and the like can be used, but are not limited thereto.
“Diseases” include cancer and pregnancy-related diseases. Here, cancer refers to a general malignant tumor or a disease state caused by it, and the morphology and arrangement of tumor cells are different from the original normal cells in various respects. A tumor that is fatal and includes, but is not limited to, stomach cancer, lung cancer, liver cancer, pancreatic cancer, colon cancer, and choriocarcinoma. In addition, the pregnancy-related disease may be a pregnant woman or a non-pregnant woman, and specifically means a disease found in organs including female-specific tissues such as uterine tissue and placenta, amniotic cells and serosa.

The present invention also relates to a method for determining the presence or absence of cancer cells. Specifically, blood, urine, tissue, etc. are collected from a patient suspected of having cancer as a biological sample, and the amount of leptin in the sample is measured. The diagnosis of cancer is made by determining the presence or absence of cancer cells by comparing the amount of leptin in the same sample of the healthy subject with the same age and body index as the results.
Furthermore, the present invention relates to a method for determining pregnancy by measuring leptin derived from non-adipose tissue in a biological sample. Blood, urine, amniotic fluid, and the like can be used as the biological sample.

The leptin detection method provided by the present invention is useful for analyzing the expression state of leptin, and is useful for elucidating the causes of cancer and uterine-related diseases and for newly developing methods for diagnosing and treating uterine-related diseases. In addition, the present invention provides a new means for pregnancy diagnosis and is useful for elucidating the function of placenta, differentiation and maturation of vegetative cells, energy metabolism and regulation of pregnancy by elucidating the function of leptin in non-adipocytes It is useful for providing a new means for analyzing the physiological function of leptin. Furthermore, the screening method for cancer or uterine-related diseases by leptin measurement provided by the present invention can be used as a useful marker for gestational trophoblastic diseases and cancers that reflect disease activity and / or therapeutic effects.

  Next, the steps of the present invention will be described sequentially. In the present invention, unless otherwise indicated, immunological techniques, tissue specimen preparation techniques, cell culture techniques, RNA separation and purification methods and analysis methods known in the art can be employed.

Production of anti-human leptin antibody
For antibodies against human leptin, for example, peptides synthesized by ordinary peptide synthesizers based on part of the amino acid sequence of leptin reported by Masuzaki et al. (4) and genetic recombination techniques known in the art are used. Recombinant leptin prepared in this way is purified by conventional protein chemical methods, and these are used as immunogens to immunize animals such as mice, rats, hamsters, rabbits, etc., and polyclonal antibodies derived from the sera are prepared. obtain. Alternatively, cells are removed from the spleen or lymph nodes of immunized mice and rats, fused with myeloma by the cell fusion method (42) by Köhler and Milstein, and then hybridomas are cultured. An antibody is produced, and a monoclonal antibody is separated and prepared from the culture solution.

Immunological determination of leptin
Examples of the immunoassay include enzyme immunoassays such as a radioisotope, peroxidase, alkaline phosphatase, luciferase, etc., an anti-leptin antibody labeled with a fluorescent substance such as acridinium or cryptate, or a sandwich method using leptin or a competitive method. Quantification can be performed by a method selected from a method (EIA), a fluorescence immunoassay (FIA), a radioimmunoassay (RIA), and the like. The antibody to be used may be a polyclonal antibody or a monoclonal antibody, and a method known per se described in Ishikawa's method (43) and the like can be used. For example, leptin can be quantified according to the Hosoda et al. Leptin radioimmunoassay method (21). By these methods, it is possible to measure leptin contained in trace amounts in samples such as organs, tissues, cells, blood, urine and amniotic fluid, but the samples are not limited thereto.

Preparation of measurement sample
For example, after collecting blood from a subject, the blood sample is dispensed into a glass tube treated with silicon containing 1 mg / ml disodium ethylenediaminetetraacetate (EDTA · 2Na) and centrifuged at 4 ° C. and 1000 × g for 10 minutes. . After centrifugation, the sample is frozen with liquid nitrogen, stored at −20 ° C., and thawed at use. For example, amniotic fluid is collected from a subject, centrifuged, and the supernatant is stored at -20 ° C.

Method for determining the presence or absence of cancer cells
A biological sample is collected from a patient suspected of having cancer and the amount of leptin is measured, and the presence or absence of cancer cells is determined by comparing the amount of leptin in the same sample of the healthy subject with the same age and body index. It is a method to do. The “body index” is called BMI (Body Mass Index) and means an index calculated from the product of “height” and “reciprocal of the square of weight”. Therefore, “the amount of leptin in the same sample of a healthy person having the same age and body type index as the patient” means the amount of leptin expected when cancer cells are not present in the patient. This is because the amount of leptin is regulated within a certain range mainly by age and body type index in a normal state. Note that the expression “equivalent” represents identity with an allowable range that may be recognized by those skilled in the art as it is difficult to obtain mathematical coincidence of valences in the field of diagnosis.

Pregnancy determination
This is a method for determining pregnancy by collecting a biological sample from a woman suspected of being pregnant and measuring leptin derived from non-adipose tissue. Normal non-pregnant leptin levels are defined within a certain range from age and body type index. Since leptin is produced only from fat cells in normal individuals, it is considered that leptin exceeding the range estimated from the body type index is produced from non-adipocytes. Thus, “measuring non-adipose tissue-derived leptin” means measuring the amount of leptin in a pregnant person minus the amount of leptin in the same sample of a normal non-pregnant woman with the same age and body index. means.

EXAMPLES Next, although an Example demonstrates this invention further in detail, the scope of the present invention is not limited only to these.

Measurement of plasma leptin levels in pregnant women (1) Anti-leptin antibody: Rabbit anti-human leptin antiserum prepared by Hosoda et al. (21) using full-length leptin as an immunogen was used.
Immunization was carried out by subcutaneously injecting male Japanese rabbits with a suspension of 500 μl of a solution containing 250 μg of recombinant human leptin mixed with an equal volume of Freund's complete adjuvant.
Booster immunization was performed every 3 weeks, and leptin antiserum (No. 95-1-4) was obtained from 10 to 14 days after each immunization.

(2) Labeling of human leptin with ¹²⁵ I: Similarly to the method of Hosoda et al. (21), human leptin was labeled with ¹²⁵ I by the chloramine T method and purified using a Sephadex G-50 column 1 × 30 cm (Pharmacia).
5 μg of human leptin was labeled with ¹²⁵ I by the chloramine T method, and the reaction solution containing the standard solution was applied to a Sephadex G-50 column 1 × 30 cm. ¹²⁵ I-labeled human leptin was eluted with 0.1 M phosphate buffer, pH 7.4 (containing 0.5% BSA, 0.1% NaN ₃ ) to obtain a ¹²⁵ I-labeled leptin fraction.

(3) Leptin RIA: The reagent used in leptin RIA was prepared with RIA buffer; 0.1 M phosphate buffer, pH 7.4 (containing 0.5% BSA, 0.1% NaN3). The RIA antibody solution used was prepared by diluting the polyclonal antibody (No. 95-1-4) prepared in Example 1 (1) 24,000 times with an RIA buffer. 100 μl of RIA antibody solution and 200 μl of RIA buffer were added to 100 μl of various standard human leptins or samples, and left at 4 ° C. for 24 hours. Thereafter, 100 μl (10,000 cpm) of ¹²⁵ I-labeled leptin was added, and the mixture was further left at 4 ° C. for 24 hours. B / F separation was performed by the two-antibody method, and the radioactivity of the precipitate was measured with a γ-counter.

(4) Collection of samples from subjects: 73 normal pregnant women (30.1 ± 3.9 years old) and 87 non-pregnant women (31.2 ± 5.1 years old) from 8 to 36 weeks of gestation After overnight fasting, blood was collected from the antecubital vein in the recumbent position at 6 am. Pregnant women continuously collected blood during pregnancy and after delivery. Four cesarean sections before parting took blood samples 1 hour before delivery and 1, 4, 24 hours after surgery. The collected blood sample was immediately dispensed into a chilled silicon-treated glass tube containing EDTA · 2Na (final concentration 1 mg / ml), and centrifuged at 4 ° C. and 1,000 × g for 10 minutes. After separation, samples were frozen in liquid nitrogen and stored at -20 ° C until use.

(5) Quantification of leptin in plasma: The amount of leptin in plasma samples collected from pregnant and non-pregnant women was measured with leptin RIA, and the amount of leptin in the sample was calculated from the standard curve.
The obtained results are shown in FIG. When compared with the body type index (BMI) of the subjects, it was found that 37 non-pregnant women (FIG. 1A, lane 1) at 18 ≦ BMI <22 versus 13 pregnant women (FIG. 1A, FIG. 1A, lane 1). In lane 2), 41.4 ± 5.5 ng / ml (about 4.7 times; P <0.001), and 22 ≦ BMI <25, 19 non-pregnant women (FIG. 1A, lane 3) 17.2 ± Nine pregnant women (FIG. 1A, lane 4) 44.6 ± 6.3 ng / ml (about 2.4 times; P <0.001) vs. 4.6 ≦ ng / ml, 25 ≦ BMI <32 In 31 women (FIG. 1A, lane 5), 24.2 ± 2.9 ng / ml, whereas in 7 pregnant women (FIG. 1A, lane 6), 55.1 ± 11.7 ng / ml (approximately 2.3 times; Both P <0.05) showed significantly higher values in pregnant women.
In addition, in non-pregnant women (FIG. 1B, ○), a positive correlation was observed between plasma leptin levels and BMI (correlation coefficient r = 0.46), as reported by Considine et al. And Hosoda et al. (20, 21). P <0.001). However, there was no correlation between plasma leptin levels and BMI in pregnant women (Fig. 1B, ●).

(6) Plasma leptin levels over time in pregnant women: Plasma leptin levels in 40 pregnant women (28.2 ± 4.5 years) were measured over time with leptin RIA, and 41 non-pregnant women (26.26). 7 ± 3.3 years old; 9.0 ± 1.9 ng / ml) and BMI. The obtained results are shown in FIGS.

It was significantly increased to 26.7 ± 3.2 ng / ml (P <0.001) in the first trimester of pregnancy (1 to 12 weeks of gestation) (FIG. 2, lane 1). 42.7 ± 4.3 ng / ml (P <0.001) in the second trimester (13 to 26 weeks of gestation) (FIG. 2, lane 2), third trimester (27 to 40 weeks of gestation) (FIG. 2, lane 3) also showed a significantly high value of 40.6 ± 4.5 ng / ml (P <0.001). The plasma leptin level measured 24 hours after placental delivery after delivery (Fig. 2, lane 4) was 21.4 ± 2.7 ng / ml, which was lower than the first trimester of pregnancy. For 4 cesarean section (29.3 ± 3.2 years old) (FIG. 3), it was 34.8 ± 5.2 ng / ml 1 hour before delivery, whereas 19.4 ± 3 after 1 hour delivery. .2 ng / ml, tending to decrease to 10.4 ± 1.5 ng / ml after 24 hours, and decreased to a value estimated from BMI.
Despite the absence of significance in the age of non-pregnant women for pregnant women, there is a significant increase compared with the plasma leptin level estimated from BMI, and the plasma leptin level (18, 20 and 21). This suggests that leptin is produced in the gestation uterus, and that a significant amount enters the maternal circulation. During pregnancy, the humoral signal from the placenta acts on the mother and plays a different functional role than weight control. I can guess that it plays.

Expression of leptin in tissue (1) Expression analysis of leptin mRNA by Northern flot analysis: mature adipocyte of abdominal subcutaneous fat, placental chorionic tissue (36 weeks of gestation), first trimester chorionic villus (8 weeks of gestation), Total RNA from the third trimester smooth chorion (36 weeks gestation), amniotic membrane (16 and 36 weeks gestation), true decidua (8 weeks gestation) and myometrium (14 weeks gestation) according to the method of Masuzaki et al. (4) Extracted. 20 μg of the isolated RNA was electrophoresed in an agarose gel and transferred to a nylon membrane GeneScreenPlus (DuPont). What all human ob cDNA of these films Masuzaki et al. Reported (4) was labeled with ^{32 P} as a probe was carried out the hybridization. The membrane subjected to hybridization was exposed to an X-ray film for 24 hours at −70 ° C. using an enhancement screen, developed, and quantified with a densitometer P-45 (Pharmacia). The obtained results are shown in FIG.
Ob mRNA is abundantly expressed in the first trimester chorionic villus (FIG. 4, lanes 3 and 4), and a small amount in the third trimester smooth chorion (FIG. 4, lane 5) and amnion (FIG. 4, lane 6). Expression is observed. Little was detected in the true decidua (FIG. 4, lanes 9, 10) and myometrium (FIG. 4, lane 11).

(2) Analysis of leptin production by immunohistochemical staining: the first trimester (gestation 6, 8, 9 weeks), the second trimester (gestations 14, 16, 19 weeks) and the third trimester (gestations 31, 36, 38, 39 weeks) placental chorionic tissue, smooth chorionic membrane, true decidua and amniotic membrane, and uterine fibroid layer tissue from pregnancy uterus. C. After embedding with T-Tissue Tech (Miles), it was rapidly frozen in dry ice-acetone. A frozen tissue of 4 μm was prepared with a microtome CRYOCUT 1800 (Leica), placed on a glass slide Superfrost-Plus slide (Fisher Scientific), and dried at room temperature for 20 minutes. The dried slice sample was washed 3 times with PBS 5 ml for 15 minutes, and the rabbit anti-human leptin antiserum No. 1 prepared in Example 1 (1) was used. A 95-1-4 PBS (containing 1% BSA) 100-fold diluted solution was added and reacted at room temperature for 30 minutes. The sample was washed with PBS (containing 1% BSA) three times for 15 minutes, and a 250-fold diluted solution of fluorescein isothiocyanate-labeled goat anti-rabbit IgG antiserum (Sirenas Laboratories) in PBS (containing 1% BSA) was added at room temperature. For 30 minutes. The sample was washed with PBS (containing 1% BSA) three times for 15 minutes, and then fluorescence was detected with an immunofluorescence microscope BH2-RFCA (Olympus). To verify specificity, adjacent tissue sections were processed in a similar manner. However, pre-immune rabbit serum was used instead of rabbit anti-human leptin antiserum. The obtained results are shown in FIG.
Leptin is expressed in the trophoblast of the first trimester chorionic villus tissue (FIGS. 5 and 1), in the syncytiotrophoblast layer (FIGS. 5 and 2) and in the amniotic cells (FIGS. 5 and 3) of the third trimester chorionic tissue. In particular, the chorionic villus tissue stained more positively in the syncytiotrophoblast layer than in the trophoblast cells. Staining with fibroblasts and Hofbauer cells and preimmune rabbit serum (FIGS. 5, 4, 5, 6) was leptin negative.

These results indicate that leptin is an adipocyte-derived circulating satiety factor involved in the regulation of feeding and energy expenditure (1, 2, 3, 7, 8, 9, 10), and its gene expression and its secretion is mature fat It was thought that it was expressed only in cells (3, 4, 5), but it was revealed that it was expressed in human pregnant placenta and amniotic cells, which are non-adipose tissues. Because leptin is produced in placental trophoblast and amniotic cells derived from human gestation uterus, and because the placenta is an endocrine organ producing various hormones that play an important role in pregnancy (32), leptin Is considered to be a placental hormone. In non-pregnant women, leptin circulating in the body is produced in adipose tissue, whereas in pregnant women several times the amount is produced in the placenta, and when leptin is administered in normal mice, metabolic rate, body temperature, Whereas motility decreases, ob / ob mice increase metabolic rate, body temperature, and motility (7), so that leptin produced from placenta is involved in the high metabolic state characteristic of pregnancy. It is thought that there is.
In addition, leptin receptors (ob-R) are classified into cytokine receptor families based on their forms (1, 13, 14, 15, 23), and include not only brain and peripheral tissues, but also trophoblasts, decidual cells, and amniotic cells. (Data not yet published), cytokine interleukin-6 and its homolog receptor receptors are expressed in trophoblasts (34, 35), human chorionic gonadotropin (hCG) and human placental lactogen Leptin promotes secretion of (hPL) (35, 36) and constitutes a local signaling pathway that suppresses secretion of growth hormone releasing factor (37, 38), so leptin is secreted in the placenta or / and It is suggested that it acts as a paracrine regulator and regulates placental hormone production during pregnancy.

Expression of leptin in BeWo cells (1) Culture of BeWo cells: In order to clarify whether placental trophoblasts produce leptin in vitro, many biological and biochemical characteristics characteristic of placental trophoblasts remain. Ob gene expression and leptin secretion were measured using BeWo cells (30, 31) derived from human pregnancy choriocarcinoma metastasis.
BeWo in cells (American Type Culture Collection) the added RPMI 1640 medium with 10% fetal calf serum (FCS) (Difco), 37 ° C., cell density under 5% carbon dioxide equilibrium air 2 × 10 ⁵ cell After culturing to 10 ml / ml, 10 ml of cultured cells were dispensed into a 10 cm culture dish, maintained in a single layer, and subjected to experiments. The preferred phenotype of proliferating BeWo cells is similar to that of undifferentiated trophoblast cells, uterine trophoblast cells, and differentiates into syncytiotrophoblast layers by treating forskolin, an adenyl cyclase activator. Is known (31). Therefore, after BeWo cells were cultured until the cells became confluent, the RPMI 1640 medium (containing 10% FCS) containing 20 μM or 2 μM forskolin (Sigma) in the forskolin-treated section of the cells after removing the medium. In the untreated group, the same medium containing no forskolin was added and cultured.

(2) Quantification of leptin in medium by RIA: Secreted leptin in the medium was quantified by leptin RIA.
To 100 μl of cell culture solution, 100 μl of RIA antibody solution and 200 μl of RIA buffer were added, and left at 4 ° C. for 24 hours. Thereafter, measurement was carried out in the same manner as in Example 1 (2), and the amount of leptin was calculated from a standard curve.

(3) Measurement of leptin in medium by gel filtration chromatography: Leptin in the medium was fractionated by high performance gel filtration chromatography according to the method of Hosoda et al. (21) and quantified by leptin RIA. Apply 0.2 ml of cell culture solution to Sephadex G-50 column (1 × 30 cm) (Pharmacia) equilibrated with RIA buffer, elute at a flow rate of 30 ml / ml using the same buffer, and dispense 0.5 ml each. did. The amount of leptin in each fraction was measured by leptin RIA and quantified. The flow rate was calculated from blue dextran (Vo) and ¹²⁵ I (Vt).

(4) Quantification of leptin mRNA in cultured cells by Northern blot analysis: Total RNA was extracted from BeWo cells cultured for 48 hours after forskolin treatment according to the method of Masuzaki et al. (4), and the same as in Example 2 (1). Northern blot analysis was performed.

(5) hCG measurement: It is known that BeWo cells increase the process hCG of differentiation by forskolin treatment. Therefore, in order to confirm whether BeWo cells could be cultured, hCG in the culture solution was measured using an hCG immunoassay kit Amerlite hCG-60 (Johnson & Johnson Clinical Diagnostics).

The results with BeWo cells are shown in FIG. 5, FIG. 6, FIG. 7 and FIG. The serial dilution curve (Fig. 5, ●) of BeWo cell medium was parallel to the standard curve of human leptin (Fig. 5, ○).
The results of gel filtration chromatography elution of cell culture fluid (Fig. 6) were detected at the same elution peak of human leptin-like immunoreactive substance as recombinant human leptin, as reported in human plasma and primary cultured human adipocyte medium (21). It was done.

When the amount of leptin production after forskolin treatment was measured over time, the amount of leptin production increased depending on the forskolin treatment concentration and time, and there was no 20 μM forskolin treatment group (FIG. 7A, ●) 48 hours after treatment. In the treatment group (FIG. 7A, ◯), the amount was increased to about 3 times the amount in the 2 μM forskolin treatment group (FIG. 7A, ▲). Moreover, leptin was accumulated in a time-dependent manner even in the untreated section.

The amount of hCG produced after treatment with forskolin was measured over time. As reported by Burres et al. (30) and Wice et al. (31), 20 μM forskolin treatment group (FIG. 7B, ●), 2 μM forskolin treatment group ( In FIG. 7B, (▲), in the untreated section (FIG. 7B, ○), the forskolin treatment concentration increased in a time-dependent manner.
In Northern blot analysis, a large amount of the ob gene was expressed in the 20 μM forskolin-treated group (FIG. 8, lane 3) and 2 μM forskolin-treated group (FIG. 8, lane 4), and the untreated group (FIG. 8, lane 1). In 2), it was expressed in a small amount.

These results revealed that ob gene and leptin secretion are enhanced during forskolin-induced differentiation from trophoblast cells to syncytial trophoblast cell layers. The placental trophoblast is composed of two types of cells: trophoblast cells, which are undifferentiated cells, and syncytiotrophoblast layers that secrete placenta-derived hormones such as hCG and hPL. This is consistent with the membrane tissue showing a stronger leptin response in the syncytiotrophoblast layer than in the vegetative cell. Furthermore, Sliker et al. (39) and Kosaki et al. (40) revealed that ob gene expression was down-regulated by cAMP in mature adipocytes. From these, it is speculated that there is differential regulation of ob gene expression and leptin secretion between trophoblast and adipocytes.

Leptin production in amniotic cells.
As a result of leptin immunohistochemical staining in Example 2, amniotic cells were positive. Therefore, in order to measure leptin secretion in amniotic cells in vitro and in vivo, the amount of leptin in the amniotic fluid including the cultured cells of amniotic cells and the secretion of amniotic cells was measured.
(1) Collection of amniotic fluid: Amniotic fluid was collected at the time of cesarean section and pregnant women at various stages from 20 to 39 weeks of pregnancy. The collected sample was centrifuged at 1,000 ° C. at 4 ° C., and the supernatant was stored at −20 ° C. until analysis.

(2) Culture of amniotic cells: Amniotic cells were collected at the time of spontaneous delivery at 38 weeks of gestation, and primary cultured human amniotic cells were prepared in 10% FCS-DMEM medium according to the method of Itoh et al. (41). The primary culture of amniotic cells was dispensed and further cultured and maintained until the cells were confluent.

(3) Quantification of the amount of leptin: The amount of leptin in the amniotic cell culture medium and amniotic fluid was quantified. Amniotic cells were added with 100 μl of RIA antibody solution and 200 μl of RIA buffer to 100 μl of culture medium or amniotic fluid cultured for 5 days from the confluent state and left at 4 ° C. for 24 hours. Thereafter, the measurement was performed in the same manner as in Example 1 (2), and the amount of leptin was calculated from the standard curve. DMEM (containing 0.5% FCS, 5 mg / ml BSA) was used for dilution of the culture solution.

(4) Measurement of leptin in the medium by gel filtration chromatography: 0.2 ml of amniotic cell culture solution equilibrated with RIA buffer was applied to a Sephadex G-50 column, 1 × 30 cm (Pharmacia), Example 3 (3) In the same manner as above, elution and fractionation were carried out, and the amount of leptin in each fraction was quantified by RIA.

The results with amniotic cells are shown in FIG. 9 and FIG. The amniotic fluid serial dilution curve (FIG. 9, ●) was parallel to the standard curve of human leptin (FIG. 9, ○). The result of gel filtration chromatography of the amniotic cell culture solution (FIG. 10) shows essentially the same elution pattern as the measurement result of BeWo cell culture solution (FIG. 6), and human leptin has the same elution time as recombinant human leptin. The immunoreactive material for use eluted in one peak. The amount of human leptin in the amniotic fluid in the second and third trimesters was 5.0 ± 2.7 ng / ml (n = 5). -The amount of leptin secreted from the subcultured amniotic cells was about 5 ng / 106 cells / 5 days.

Measurement of plasma leptin level in chorionic disease
The BeWo cells are cells derived from human choriocarcinoma metastasis, suggesting the possibility that leptin is produced and circulated in the blood in chorionic disease. Therefore, plasma is collected from patients with trophoblastic diseases such as hydatidiform mole and choriocarcinoma, and the presence or absence of leptin secretion is confirmed.
(1) Plasma collection: Blood was collected from the antecubital vein in a recumbent position at 6 am after treatment and fasting overnight from 3 patients with hydatidiform mole and 1 patient with choriocarcinoma. The collected blood sample is immediately dispensed into a chilled silicon-treated glass tube containing EDTA · 2Na (1 mg / ml), centrifuged at 4 ° C. and 1,000 × g for 10 minutes, and frozen in liquid nitrogen for use. Until -20 ° C.

(2) Measurement of plasma leptin level: Leptin level in patient samples was quantified by RIA.
After thawing the frozen patient plasma, 100 μl of RIA antibody solution and 200 μl of RIA buffer were added to 100 μl of plasma, and left at 4 ° C. for 24 hours. Thereafter, the measurement was carried out in the same manner as in Example 1 (2), and the amount of leptin was quantified from the standard curve. The obtained results are shown in Table 1.

The plasma leptin level before treatment of hydatidiform mole patients was determined by using the regression line obtained by statistically processing the BMI and leptin levels of normal subjects shown in FIG. 1b (26.7, 29.1 and The value was significantly higher than the amount of leptin calculated from 19.1 kg / m ² ) (23.4, 28.4, and 7.4 ng / ml). The plasma leptin level (47.9 ng / ml) in patients with giant hydatidiform moles reached the level expected from the leptin level of 10.2 ng / ml and BMI (26.7 kg / m ² ) 7 days after the operation of deciduous tissue removal. The hCG amount also decreased at the same time. There was no significant change in body weight during the measurement period (case 1).

Plasma leptin levels (49.0 ng / ml) in choriocarcinoma patients were significantly increased compared to leptin levels (18.1 ng / ml) predicted from BMI (24.2 kg / m ² ). Combined chemotherapy with methotoxalate, actinomycin D and cyclophosphamide reduced plasma leptin levels to the predicted BMI value 25 days after treatment. In addition, the amount of hCG in plasma and urine decreased as reported by Sanfilippo et al. (32). There were no significant changes in body weight and serum creatinine during the measurement period (case 2). In patients with gestational trophoblastic disease such as hydatidiform mole or choriocarcinoma, plasma leptin levels are markedly elevated and are comparable to those in obese individuals (18, 20, 21) circulating in the blood. It is suggested to play a pathophysiological role in membrane diseases.

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A is a graph showing the results of comparing plasma leptin levels in pregnant women and non-pregnant women by means of body type index averages. B is a graph showing the relationship between plasma leptin level and body type index in pregnant and non-pregnant women. FIG. 3 is a graph showing the stage of pregnancy change in plasma leptin levels in pregnant women. FIG. 4 is a graph showing changes in plasma leptin amount over time before and after cesarean section. FIG. 4 is an electrophoresis drawing instead of a drawing showing the results of Northern blot analysis of ob mRNA expression in various human uterine tissues. FIG. 3 is a graph showing a standard curve of human leptin and an RIA dilution curve of human leptin in BeWo cell culture medium. These are the graphs which show the elution pattern from the Sephadex G-50 column of BeWo cell culture medium. A is a graph showing changes over time in the amount of leptin in the BeWo cell culture solution after forskolin treatment and no treatment. B is a graph showing changes over time in the amount of hCG in the BeWo cell culture solution after forskolin treatment and no treatment. It is the drawing of the electrophoresis replaced with drawing which shows the result of the Northern blot analysis of the expression level of obmRNA in the BeWo cell 48 hours after forskolin treatment. FIG. 3 is a graph showing a standard curve of human leptin and an RIA dilution curve of human leptin in an amniotic cell culture medium. These are the graphs which show the elution pattern from the Sephadex G-50 column of amniotic fluid of a pregnant woman (39 weeks).

Claims (1)

A screening method for diseases by measuring leptin derived from non-adipose tissue in a biological sample.

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