CN110038116B - Use of human liver secreted protein GPNMB or its antagonist or agonist - Google Patents

Abstract

The present invention relates to the use of human liver secreted protein GPNMB or its antagonist or agonist. The present invention finds that GPNMB gene and GPNMB protein can be used as drug targets for treating metabolic diseases such as obesity, diabetes, insulin resistance and hyperlipidemia.

Description

Use of human liver secreted protein GPNMB or its antagonist or agonist

technical field

The invention relates to the technical field of biomedicine, in particular to the application of protein Glycoprotein nonmetastaticmelanoma protein B (GPNMB) in the diagnosis, treatment and medical research and development of metabolic diseases (such as obesity, diabetes, insulin resistance, hyperlipidemia, etc.).

Background technique

In recent years, obesity and related diseases (including: obesity, diabetes, insulin resistance, hyperlipidemia, etc.) have increased substantially, and have gradually become major problems affecting public health and their quality of life. According to the World Health Organization, more than 100 million adults are overweight (BMI>25), of which more than 3 million are severely obese (BMI>30). The number of people with type 2 diabetes has risen sharply in Asian countries, and obesity is a major risk factor for developing type 2 diabetes. In fact, in the past decade alone, the number of people with diabetes in China has nearly doubled to 126 million.

Because obesity is often caused by energy intake exceeding energy consumption, the current method of treating obesity is mainly to reduce energy intake, such as dieting, but the long-term implementation of this method is difficult and the treatment effect is poor; , kidney and heart have strong side effects. At present, the most effective method for the treatment of obesity is surgery, which has not been widely used due to its high cost. With the changes in people's diet structure and living habits in my country, obesity and various metabolic diseases caused by obesity are occurring more and more, and there is an urgent need to strengthen the research on new drug target systems and innovative drugs with independent intellectual property rights.

GPNMB is a type I transmembrane protein whose gene is located on the seventh human chromosome and consists of 572 amino acids. The intact transmembrane protein can be cleaved by extracellular proteases such as ADAM10, releasing the extracellular segment of GPNMB into the blood to play the role of cytokines. GPNMB can be detected in tissues such as normal bone, hematopoietic system, and skin, and can also be detected at high levels in malignant hyperplasia tissues such as breast cancer, melanoma, and hepatocellular carcinoma. Our proprietary study is the first to demonstrate that GPNMB, as a liver-secreted protein, plays an important role in obesity and its associated metabolic diseases.

SUMMARY OF THE INVENTION

The inventors conducted fat metabolism studies and found that L-Scap-/- and L-gp78-/-deficient mice have decreased hepatic fatty acid synthesis, but at the same time increased fatty acid synthesis in white adipose tissue. Serum studies were conducted to confirm that the substances secreted by the mouse liver can upregulate the expression of genes related to fat synthesis. Extracted the up-regulated genes in the deficient mice, a total of 301 genes, and then found 12 protein-coding genes with N-terminal signal peptides, and further quantitative PCR confirmed that the expression levels of 4 were increased by 30 times compared with the wild type. The above genes, namely Gpnmb, Timp1, Ly6d, and Lcn2, were finally constructed with adenovirus expression vector for verification, and it was confirmed that the gene Gpnmb caused the increase of fatty acid synthesis in white adipose tissue.

An object of the present invention is to provide the application of Gpnmb gene in medicine for diagnosis, prevention and treatment of obesity and related metabolic diseases.

Another object of the present invention is to provide the application of GPNMB protein in medicine for diagnosis, prevention and treatment of obesity and its related metabolic diseases.

The present invention identifies that Glycoprotein nonmetastatic melanoma protein B (GPNMB) secreted by mouse liver can significantly enhance the fat synthesis ability of white fat, reduce energy metabolism in mice, and cause obesity, hyperlipidemia and insulin resistance in mice. In obese mice, blood levels of GPNMB were significantly higher than in normal mice. Population analysis showed that GPNMB levels in blood were positively correlated with body weight, obesity, blood pressure, insulin resistance, diabetes, and hyperlipidemia.

This indicates that GPNMB may be used as a drug target for the treatment of obesity, etc., either by reducing the expression of Gpnmb by gene therapy, or by inhibiting the activity of GPNMB protein by using an antagonist or the like.

According to one aspect of the present invention, reducing Gpnmb gene expression includes, but is not limited to, inhibiting the expression of the gene encoding the GPNMB protein. For example, an agent that inhibits Gpnmb gene expression or reduces its expression level can be administered, and these agents include: an agent that inhibits the transcriptional activity of Gpnmb gene, an agent that inhibits the transcription level of Gpnmb mRNA, an agent that promotes degradation of Gpnmb mRNA, and siRNA against Gpnmb gene, Reagents that inhibit the translation of Gpnmb mRNA, and reagents that specifically recognize the guide nucleic acid of Gpnmb gene and cut it to reduce its level, and can also be reagents that cause mutation of Gpnmb gene sequence, making the expressed GPNMB protein inactive, such as targeting vector.

According to another aspect of the present invention, there is provided an antagonist that reduces the activity of GPNMB protein, which can be, for example, a specific antibody or a small molecule with inhibitory activity. After neutralizing GPNMB with antibodies, it was found that it can prevent and treat obesity, reduce fat accumulation, reduce blood lipids, enhance energy metabolism, increase insulin sensitivity, and enhance blood glucose clearance.

According to a further aspect of the present invention, Gpnmb gene/GPNMB protein is used as a molecular indicator for clinically diagnosing the progression of metabolic diseases such as insulin resistance, diabetes, hyperlipidemia, and obesity. Reagents for detecting Gpnmb gene or protein include, but are not limited to, various primers and probes for detecting Gpnmb gene, and/or specific antibodies for detecting Gpnmb protein, etc. Such reagents include samples containing Gpnmb gene or protein and Other reagents used in the detection process, such as solvents, etc., include but are not limited to various reagents required for PCR and the like.

The experimental results show that GPNMB protein and gene can be used as indicators for the diagnosis and treatment of metabolic diseases.

Description of drawings

Figure 1: Functional study of adeno-associated virus (AAV) overexpression system on hepatic secretion of GPNMB to upregulate the adipogenic ability of white adipose tissue;

a. Body weight measurement; b. Food intake; c. Metabolic parameters of mice; d. qPCR analysis of lipid synthesis-related genes in mouse white fat; e. mouse oxygen consumption; f. Brown adipose tissue differentiation and thermogenesis-related genes Level change; g. blood glucose level; h. blood insulin level; i. glucose tolerance test; j. insulin tolerance test.

Figure 2: Antibody neutralization of blood GPNMB against high-fat diet-induced obesity;

a. Schematic diagram of antibody treatment of food-induced obesity; b. body weight of mice on day 18; c. food intake; d. white adipose tissue weight; e. white adipose tissue HE staining and cell size statistics; f. white adipose tissue adipogenesis Changes of related genes; g. oxygen consumption in mice; h. respiration entropy in mice; i. blood insulin concentration; j. blood glucose concentration; k. glucose tolerance test; l. insulin tolerance test; m. brown fat Changes in tissue differentiation and thermogenesis-related gene levels.

Figure 3: AAV-specific knockdown of hepatic Gpnmb expression improves diet-induced obesity;

a. Experimental flow chart; b. Gpnmb expression level in liver; c. Western blotting to detect the amount of GPNMB in serum; d. Expression level of adipose synthesis-related genes in white adipose tissue; e. Production of white adipose tissue and brown adipose tissue Expression levels of heat-related genes; f. oxygen consumption of mice in metabolic cages; g. glucose tolerance test.

Figure 4: Study on the relationship between the concentration of GPNMB in blood and obesity and its related metabolic diseases;

a. Serum GPNMB concentrations of WT and diet-induced-obesity (DIO) mice after 4 weeks of chow diet or high-fat diet; b. Serum GPNMB concentrations of 8-week-old WT and OB mice; c . Serum GPNMB concentration in obese population (BMI>=28) and non-obese population (BMI<28); d. Correlation between blood GPNMB and BMI in population.

Specific implementation method

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

It should be noted that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the exemplary embodiments according to the present application.

Example 1 GPNMB secreted by the liver can up-regulate the adipogenic capacity of white adipose tissue

1.1 Quantitative PCR assay method:

Quantitative-PCR was used to determine the effect of GPNMB secreted by mouse liver on the adipogenic ability of white adipose tissue. After taking the mice for experimental treatment, the mice were killed, and the tissues were transferred to the tissue crushing tube, which was then used for mRNA extraction, Synthesis of cDNA by reverse transcription and real-time quantitative PCR, the specific steps are as follows:

1.1.1. mRNA extraction and quantification

1) Add 200 μl of chloroform to each 1 ml of TRIzol, shake vigorously on a vortexer to mix, and let stand at room temperature for 15 minutes.

2) Centrifuge at 13200rpm for 10 minutes at 4°C, transfer 500μl of the upper aqueous phase to a new 1.5ml Eppendorf tube.

3) Add 600 μl of isoamyl alcohol to each sample and mix by inversion.

4) Centrifuge at 13,200 rpm for 10 minutes at 4°C, discard the supernatant, and a milky white RNA precipitate can be seen at the bottom of the tube. Add 1 ml of 70% ethanol (diluted with DEPC-treated deionized water) to each sample and mix by inversion.

5) Centrifuge at 13,200 rpm for 10 minutes at 4°C, discard the supernatant, dry the pellet at room temperature, add 30 μl of DEPC-treated deionized water, and dissolve it fully.

6) Take 2 μl of RNA into 98 μl of water, and use a spectrophotometer (Eppendorf) to measure the light absorption value at 260 nm after mixing, calculate the sample concentration, measure the ratio of 260 nm and 280 nm light absorption value, calculate the sample purity, and finally adjust the sample concentration to be 1 μg/μl.

1.1.2. Synthesis of cDNA by reverse transcription

cDNA synthesis was performed using the M-MLV reverse transcriptase kit from Promega. Each 50 µl of the system contained 4 µg of RNA, 1 µg of OligodT, and dNTPs at a final concentration of 0.4 mM each.

1) In each 0.2 ml PCR tube, add 4 μl RNA at a concentration of 1 μg/μl, 1 μl OligodT at 1 μg/μl, and 15 μl DEPC water, and mix well.

2) Denaturation at 70°C for 5 minutes, then quenched on ice.

3) Add 17 μl DEPC water, 10 μl 5×MLV buffer, 1 μl dNTP (10 mM each), 2 μl M-MLV reverse transcriptase to each reaction system, and mix well.

4) React at 37°C for 1 hour.

5) Denaturation at 70°C for 10 minutes.

6) Cool down at 10°C for 5 minutes, and add 200 μl of water to dilute the cDNA.

1.1.3. Fluorescence real-time quantitative PCR

Realtime PCR uses Shengyuan Bio's Sharpvue 2x Universal qPCR Master Mix reagent. In each 20 μl system, Sharpvue 2x Mix: 10 μl, primers with a final concentration of 0.5 μM, 2 μl template cDNA (for 4-fold dilution of reverse transcription products), and make up to 20 μl with water. After the reaction system is configured, mix well, and each sample has 3 replicate wells. On a Stratagene Mx30005P real-time PCR instrument, the following procedures were followed: a. 95°C heat activation for 5 minutes, b. 95°C denaturation for 30 seconds, c. 60°C annealing for 30 seconds, d. 72°C extension for 30 seconds, after the extension Collect fluorescence signal, e.b-d cycle 40 times, f. Dissolution curve analysis: 95℃ for 15 seconds, 60℃ for 1 minute, rise to 95℃ at 1% rate, 95℃ for 15 seconds, 60℃ to 95℃ Fluorescence signals were collected during the process. Relative mRNA levels (relative to Cyclophilin) were calculated using the comparative Ct method and normalized to 1 for the WT mouse group. The primer sequences used in fluorescence quantitative PCR in this example are shown in Table 1.

Table 1 Information on primers used in fluorescence quantitative PCR in this example

1.2 Measurement methods of glucose tolerance and insulin tolerance test:

Mice were preconditioned by fasting before experiments were performed. Glucose tolerance test (GTT) fasting overnight, insulin tolerance test (Insulin tolerance test, ITT) fasting for 4 hours. In GTT experiments, 2 g/kg of glucose was intraperitoneally injected; for ITT experiments, 0.75 U/kg of insulin (Sigma) was intraperitoneally injected. Tail tip blood was collected at 15, 30, 60 and 120 minutes, respectively. Onetouch Ultra blood glucose monitoring systerm-LifeScan was used to measure blood glucose concentration.

1.3 Determination method of metabolic cage test

Mice in the control group and mice in the experimental group were transferred to a comprehensive experimental animal monitoring system (Columbus Instruments, Columbus, OH). Accommodate in the metabolic cage and monitor continuously according to the instrument operating guidelines until the experimental data show a steady state. This process takes about 24 hours, and this period is called the animal acclimation period. Thereafter, the instrument monitored the changes in the volume of O ₂ consumption, the volume of CO ₂ production, and the movement of the mice for 24–48 hours. Statistics and calculation of respiratory entropy (volume of O consumption _: volume of _CO production).

2. The effect of expressing GPNMB exocrine segment in the liver by adenovirus vector on mice

An adeno-associated virus overexpression system was used. AAV has the advantages of high infection efficiency, long expression time (up to 6 months) and small immune response. Mouse GPNMB belongs to type I secretory protein, including 574 amino acid residues, of which 1-502 amino acids belong to the extracellular secretion segment (ectodomain, ECD) of GPNMB. Entrusted Obio Technology Co., Ltd. (Shanghai) to construct an adenovirus vector plasmid, using the pAOV-CMV-3×Flag vector, constructed the AAV-CMV-Gpnmb-ECD-3×Flag plasmid, and selected and packaged it to be mainly expressed in the liver Adeno-associated virus type 8 (AAV8-Gpnmb-ECD). Mice were injected with AAV8-Gpnmb-ECD at a dose of ⁵ x 1011 pfu per mouse.

One week after the injection, the mice were fed a 60% high-fat diet (all mice were C57BL/6 unless otherwise specified), and the weight and food intake of the mice were monitored at different time points during the feeding process. It was found that with prolonged feeding time, mice overexpressing GPNMB-ECD gained faster year-on-year body weight than control mice (injected with AAV8-control virus), and there was a significant difference at the checkpoint 4 weeks after injection, while between the two groups The food intake did not change significantly (Fig. 1a,b). Decreased metabolic rate is an important indicator of obese mice, and a functional analysis of metabolic cages was performed. The results showed that oxygen consumption (VO ₂ ) in mice overexpressing GPNMB-ECD decreased, indicating that the metabolic rate of the mice was indeed decreased. (Fig. 1e). Next, quantitative-PCR analyzed the expression of adipogenic differentiation genes (422/ap2) and genes (Ucp-1) that promote energy consumption and heat production in brown adipose tissue. Heat genes were significantly downregulated (Fig. 1f), which may account for the decreased metabolic rate. Similarly, lipid synthesis genes (Srebp1c, Acs, Acc, Acl, and Fasn) in mouse white adipose tissue were analyzed, and the results showed that GPNMB-ECD promoted significant upregulation of lipid synthesis-related genes in WAT (Fig. 1d). .

The results of analyzing the metabolic indicators of the mice showed (Figure 1c) that there was no obvious inflammation (alanine aminotransferase (ALT), aspartate aminotransferase (AST)) in the two groups of mice. The weight of the mice in the experimental group increased significantly, and the weight of the liver As well as no change in liver specific gravity, the weight of adipose tissue and the proportion of adipose tissue and body weight were significantly up-regulated and statistically different, serum cholesterol and triglyceride levels did not change significantly, but blood glucose and insulin levels were significantly up-regulated. Elevated blood glucose and insulin levels suggested that GPNMB-ECD-overexpressing mice may have insulin resistance (Fig. 1g,h). The subsequent GTT and ITT experiments also further confirmed our conjecture (Fig. 1i,j).

Therefore, during high-fat-induced obesity, GPNMB-ECD-overexpressing mice gained weight, decreased metabolic rate, decreased thermogenesis, and aggravated insulin resistance.

Example 2 Antibodies neutralize blood GPNMB against high-fat diet-induced obesity

The measurement method is the same as in Example 1

2.1 HE staining of mouse white adipose tissue

(1) The mouse white adipose tissue was taken out, rinsed with cold PBS, and then fixed in 4% PFA for 4 hours;

(2) Wash 3 times with PBS;

(3) Dehydration with gradient ethanol, and embedding in xylene after transparent;

(4) Use a Lycra paraffin microtome to manually slice, with a slice thickness of 4-10m;

(5) Place the patch on a polylysine-treated glass slide, and bake the slide at 37°C overnight;

(6) HE staining was performed.

2.2 Preparation of GPNMB neutralizing antibody

1) Eukaryotic expression and purification of Gb1 protein, filtration with a 0.22 μm filter, to obtain no less than 50 mg of eukaryotic protein.

2) On the first day, for the first immunization, 1 mg of protein (2 mg/mL) was taken from each rabbit and mixed with 0.5 mL of complete Freund adjuvant (Freundadjuvant), emulsified, and injected subcutaneously on the back of the rabbit;

3) On the third day, the second immunization. Take 1mg protein (2mg/mL) from each rabbit and mix well with 0.5mL complete Freund's adjuvant, emulsify, and inject subcutaneously on the back of the rabbit;

4) On the 28th day, the third immunization. Take 1 mg of protein (2 mg/mL) from each rabbit and mix well with 0.5 mL of incomplete Freund's adjuvant, emulsify, and inject subcutaneously on the back of the rabbit;

5) After 7 days, blood was collected from the ear vein, and the specificity of the antibody was detected by western blot;

6) After 7 days, the fourth immunization. Take 1 mg of protein (2 mg/mL) from each rabbit and mix well with 0.5 mL of incomplete Freund's adjuvant, emulsify, and inject subcutaneously on the back of the rabbit;

7) After immunization once every 14 days, the time to sacrifice the rabbit for blood collection is determined according to the needs, usually after 5-7 times of immunization;

8) When 7-10 days after the last immunization, carotid artery blood collection, each rabbit can collect about 50-100mL plasma;

9) Place whole blood at 4°C overnight, centrifuge at 1500g for 15 minutes at 4°C, and separate serum;

10) Western Blot detection, select a better antiserum for use.

2.3 The resistance of GPNMB neutralizing antibody to high-fat diet-induced obesity

Wild-type mice fed a high-fat diet for 4 weeks were intraperitoneally injected with GPNMB neutralizing antibody. The mice were injected every 3 days, and the body weight and other physiological indicators of the mice were monitored at different time points (Fig. 2a). The results showed that, on the premise of no significant change in food intake, the weight gain of GPNMB-neutralized mice was slower than that of control mice, and showed a difference on the 6th day after injection. With the prolongation of injection time, the difference was significant. increased (Fig. 2b,c). On the 15th day from the start of the neutralizing antibody injection, the mice were subjected to metabolic cage testing, and the results showed that the metabolic rate (oxygen consumption VO2) of the mice in the treatment group increased significantly (Fig. 2g,h). Blood glucose levels and insulin concentrations were analyzed and both were downregulated, suggesting that insulin resistance may have been improved (Fig. 2i,j). Consistently, the results of GTT and ITT assays showed that the insulin resistance symptoms of mice after neutralization of GPNMB were significantly improved (Fig. 2k,l).

The expression of lipid synthesis genes in white adipose tissue was detected, and the results showed that lipid synthesis-related genes (Srebp1c, Acs, Acc, Acl, and Fasn) were down-regulated to varying degrees, and Fasn was the most significantly down-regulated (Fig. 2f). The weight of adipose tissue was also significantly reduced (Fig. 2d), and the results of HE staining of WAT paraffin sections showed that adipocytes were significantly smaller (Fig. 2e). In brown adipose tissue, the thermogenic gene (Ucp-1) was significantly increased under the premise of no obvious change in adipocyte differentiation efficiency, which may be the reason for the increased metabolic rate in the metabolic cage experiment (Fig. 2m).

Taken together, antibody neutralization of GPNMB can enhance the metabolic rate of diet-induced obese mice, increase mouse thermogenesis, decrease the rate of lipid synthesis in white adipose tissue, improve insulin resistance, and ultimately resist high-fat diet-induced obesity.

Example 3 AAV-specific knockdown of hepatic Gpnmb expression can improve diet-induced obesity

The measurement method is the same as that of Example 1

Entrusted Obio Technology Co., Ltd. (Shanghai) to construct adenovirus interference vector, using pAKD-CMV-bGlobin-EGFP-H1-shRNA, constructed Gpnmb-shRNA (also written as AAV8-Gpnmb-shRNA, AAV8-Gpnmb-shRNA, AAV-shGpnmb).

As shown in Figure 3a, mice injected with AAV-Gpnmb-shRNA knocked down Gpnmb expression in the liver by mediating expressed Gpnmb-shRNA (Figure 3a). Studies have shown that AAV8-Gpnmb-shRNA can significantly knock down the expression of Gpnmb in the liver (Fig. 3b), further reducing the concentration of GPNMB in the blood.

After the second week from the start of AAV injection, the metabolic cage assay of the mice showed that the metabolic rate of the mice in the treatment group increased significantly (Fig. 3f). The results of GTT assay showed that the insulin resistance symptoms of mice after knocking down the expression of Gpnmb in the liver were significantly improved (Fig. 3g).

The expression levels of lipid synthesis-related genes (Srebp1c, Acs, Acc, Acl, and Fasn) were significantly decreased in the white adipose tissue of mice with knockdown of intrahepatic Gpnmb expression (Fig. 3d). However, in brown adipose tissue and inguinal fat, the expression of a thermogenesis-related gene (Ucp-1) was significantly increased, which is consistent with the previous results of the metabolic cage experiments, indicating that the energy expenditure of the mice increased (Fig. 3e).

This example demonstrates that AAV8-specific knockdown of hepatic Gpnmb expression can also enhance the metabolic rate of diet-induced obese mice, increase mouse thermogenesis, decrease the rate of lipid synthesis in white adipose tissue, improve insulin resistance, and ultimately resist high-fat diet-induced obesity. obesity.

Example 4 Analysis of the correlation between blood GPNMB concentration and obesity and related metabolic diseases in the population

To investigate the relationship between GPNMB and obesity, we analyzed the concentration of GPNMB in the serum of DIO (diet-induced obesity) and OB (obesity) mice (Fig. 4a,b), and the results showed that diet-induced obesity and OB mice serum GPNMB concentrations were significantly higher than the respective control WT mouse concentrations.

Similarly, we collected 318 human serum samples and divided the population into obese group and non-obese group according to BMI value. The GPNMB content in obese patients was significantly higher than that in non-obese group by ELISA analysis (Fig. 4c, d). We comprehensively analyzed the relationship between serum GPNMB concentrations and obesity-related indicators in humans. In the collected 318 serum samples (human serum samples of different BMIs were collected without any drug and surgical treatment before serum collection), they were divided into three quintiles according to the concentration of GPNMB. Statistical analysis was performed (Table 2). Through analysis, we found that many obesity-related indicators (especially BMI and HOMA-IR) were positively and closely correlated with GPNMB concentrations.

Therefore, GPNMB is present in mice and humans under normal physiological conditions, and the concentration of GPNMB in blood is positively correlated with obesity and its associated metabolic diseases.

Table 2. Comparison of metabolite components of Gpnmb (ng/ml) three groups of subjects with different BMI index

The data shown is the median (range).

*p<0.05vs.Low group(Gpnmb<10.16ng/mL)

BMI body mass index, WHR waist-to-hip ratio, WH waist-to-height ratio, SBP systolic blood pressure, DBP diastolic blood pressure, TG triglycerides, TC cholesterol, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol Ogtt Oral glucose tolerance test, HbA1C hemoglobin a1c, HOMA-IR homeostasis model to evaluate insulin resistance. BMI>30 was diagnosed as obesity.

Note: The amino acid sequence of human GPNMB is shown in SEQ ID NO: 1, that is, as follows:

Homo sapiens glycoprotein nmb (GPNMB)

NM_001005340.1

SEQUENCE LISTING

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Claims (1)

1. Use of a substance inhibiting the secretion of protein GPNMB from human liver, said GPNMB protein having an amino acid sequence shown in SEQ ID NO. 1, in the manufacture of a medicament for the treatment and/or prevention of metabolic disorders selected from the group consisting of obesity, diabetes, insulin resistance and hyperlipidemia; the substance for inhibiting human liver secretion protein GPNMB is selected from:

1) antibodies that neutralize GPNMB protein;

2) an adenovirus-associated virus expression vector capable of knocking down Gpnmb gene expression.

Images