JP2018007619A - Aip knockout growth hormone-producing cell lines - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide: an AIP knockout pituitary growth hormone (GH)-producing cell line (GH3-FTY cell line) in which the function of AIP gene of a pituitary tumor cell line (GH3 cell line) is knocked out using genome editing (CRISPR-Cas9 system); and methods for establishing the same.SOLUTION: The AIP knockout growth hormone-producing cell line is prepared by knocking out the AIP gene of a tumor cell line by genome editing, the cell line having an amino acid sequence and a function different from those of the wild type AIP gene. The truncated AIP protein formed by knocking out the AIP gene has an amino acid sequence represented by SEQ ID NO:2 or SEQ ID NO:3.SELECTED DRAWING: None

Description

  The present invention relates to an AIP knockout growth hormone (GH) producing cell line, and more particularly to a pituitary-derived AIP knockout growth hormone (GH) producing cell line, a method for establishing the same, and a method for using the same.

  Germline mutations in the AIP (Aryl hydrocarbon receptor interacting protein) gene are predisposing to pituitary adenomas, mainly somatotropinoma. These AIP germline mutations occur in 15% to 20% of patients with familial isolated pituitary adenoma (FIPA) and 3% to 5% of patients with sporadic pituitary adenoma Have been identified (Non-Patent Documents 1 to 5).

  The prevalence due to these mutations reaches as much as 40% to 50% in the family of familial somatotropinoma (Non-patent Documents 2 and 4). In addition, sporadic cases of prolactin-producing adenoma or somatotropinoma in macro adenomas younger than 30 years are observed in 10% to 15% of patients (Non-patent Document 6).

  The most common AIP gene modification results in an amino acid substitution or cleavage AIP protein, particularly in the C-terminus, including three tetratricopeptide repeats (TRP) involved in protein-protein interactions (Non-patent Documents 7 and 8). .

  Such tumors containing AIP gene mutations typically develop at an early age, become larger and more invasive (Non-Patent Documents 1-7), and somatostatin, a first-line treatment for acromegaly There is a tendency to become resistant to analogs (Non-Patent Documents 3, 4, 9, 10).

  The AIP gene is considered to be a tumor suppressor gene based on findings from several experiments regarding its in vitro function (Non-patent Documents 3 and 11-14). In other words, in vitro culture experiments using a forced expression system suppressed cell growth of wild-type AIP, but this phenomenon was not observed with mutant AIP, and cell proliferation increased by knockdown of AIP by siRNA. (Non-Patent Documents 12-14).

  Although the molecular mechanism of pituitary tumor development due to AIP inactivation is not yet clear, several mechanisms have been proposed. For example, AIP inactivation impairs the function of suppressing cyclic adenosine monophosphate (cAMP) production via dysfunctional G protein i signaling (Non-patent Document 13), Promotes inhibited interaction with phosphodiesterase and increases cAMP production (Non-Patent Document 14). With respect to the relative insensitivity of certain somatotropinomas to somatostatin analogs, AIP inactivation leads to anti-proliferative genes (Zn finger regulator of apoptosis and cell cycle arrest: ZAC-1; also known as PLAGL1) It has been suggested that the decrease in expression is the mechanism (Non-Patent Documents 15 and 16). ZAC-1 can exert an antiproliferative effect by causing apoptosis and G1 cell cycle arrest (Non-patent Document 17).

  The above hypothesis on AIP action is based on clinical findings, mutational analysis of pituitary tumors and immunohistochemical studies and exogenous expression of wild-type or mutant AIP in pituitary cells or especially in the rat pituitary GH3 cell line It is mainly based on the combination with in vitro experiments using siRNA knockdown of AIP. However, the phenotype of complete knockout of AIP in GH producing cells has not been elucidated so far.

  In Aip knockout (KO) mice, heterozygous mice are extremely likely to develop pituitary adenomas, whereas mice completely deficient in Aip have been reported to be embryonic lethal (Non-patent Document 18). A pituitary cell line that does not endogenously express AIP has not been established so far. If a pituitary cell line completely lacking AIP function can be established, the endogenous function of Aip can be analyzed. A useful research model can be provided.

  On the other hand, the rat pituitary tumor cell line GH3 was originally described as a homogenous clonal cell line that secretes Gh (Non-patent Document 19), but it was later reported to secrete prolactin (Prl). (Non-patent document 20). Therefore, this GH3 cell line is not a homogeneous cell population, but rather functions as a mixture of Gh-producing cells and a subset of Prl-producing cells because the ratio of Gh and Prl changes in response to different stimuli by reverse hemolytic plaque assay This is suggested to be heterogeneous (Non-patent Document 21).

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  Therefore, in order to better understand the function of the AIP gene, the present inventors used an AIP knockout growth hormone (GH) -producing cell line that knocked out the function of the AIP gene from the GH3 cell line using the CRISPR-Cas9 system. The in vitro and in vivo properties of cell lines and GH secretion of the cell lines prepared and obtained were compared with those of the original GH3 cell line. Furthermore, as a result of investigating the mechanism of the effects of AIP inactivation on cell proliferation and GH secretion using this cell line, the AIP knockout pituitary growth hormone (GH) producing cell line in which AIP function was knocked out After confirming that production and cell proliferation were remarkably enhanced, it was found that an AIP knockout pituitary growth hormone (GH) producing cell line was established, and the present invention was completed. Therefore, this cell line is also useful as a research model for analyzing AIP gene function.

  Accordingly, the present invention provides an AIP knockout growth hormone (GH) producing cell line such as an AIP knockout pituitary growth hormone (GH) producing cell line in which the AIP gene of a tumor cell line GH3 such as a pituitary tumor cell is knocked out. For the purpose.

  Another object of the present invention is to provide an AIP knockout growth hormone (GH) producing cell line such as an AIP knockout pituitary growth hormone (GH) producing cell line from a tumor cell line GH3 such as a pituitary tumor using the CRISPR-Cas9 system. To provide a method for producing an AIP knockout growth hormone (GH) producing cell line.

  Still another object of the present invention is to provide a method for using an AIP knockout growth hormone (GH) -producing cell line comprising using an AIP knockout growth hormone (GH) -producing cell line as a tumor model cell line such as a pituitary tumor. It is intended to provide.

  As used herein, the term “knockout” means that a frameshift mutation has occurred in a part of each allelic sequence, and the original function of the gene has been lost.

  To achieve the above object, the present invention knocks out an AIP gene from a tumor cell line including a pituitary tumor cell line such as a rat by genome editing, and has a function different from that of a wild-type AIP gene. AIP knockout growth hormone (GH) producing cell lines such as AIP knockout pituitary growth hormone (GH) producing cell lines are provided.

  In this specific embodiment, the present invention provides that the truncated AIP protein generated by the frameshift mutation of the AIP gene is completely different from the amino acid sequence of the wild-type AIP protein (SEQ ID NO: 1 SEQ ID NO: 1) An AIP knockout growth hormone (GH) producing cell line having an amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 is provided.

  Further, according to the present invention, the AIP knockout growth hormone (GH) -producing cell line has a higher growth hormone (GH) production and cell proliferation than the tumor cell line. (GH) Producing cell lines.

  Furthermore, the present invention provides, as a specific embodiment, an AIP knockout growth hormone (GH) -producing cell line in which the expression of somatostatin receptor type 2 (Sstr 2) or ZAC1 is reduced compared to a tumor cell line. A knockout growth hormone (GH) producing cell line is provided.

  The present invention also provides an AIP knockout pituitary growth hormone (GH) producing cell line obtained by knocking out an AIP gene from a tumor cell line GH3 including a pituitary tumor cell line such as a rat pituitary tumor cell line using the CRISPR-Cas9 system. Provided is a method for producing an AIP knockout growth hormone (GH) producing cell line, comprising producing an AIP knockout pituitary growth hormone (GH) producing cell line.

  Furthermore, the present invention provides a method for using an AIP knockout growth hormone (GH) producing cell line, comprising using the AIP knockout growth hormone (GH) producing cell line as a model cell line for tumors such as pituitary tumors. .

  Since the AIP knockout growth hormone (GH) -producing cell line according to the present invention has an inactive AIP function, the molecular mechanism involved in the development of pituitary tumors due to the AIP gene mutation has been investigated. Useful for prevention and treatment.

  Moreover, since homozygous Aip knockout of mice is known to be fatal, the GH3-FTY cell line of the present invention is useful even if it is the first model of pituitary-derived Aip knockout cell line. is there.

The nucleotide sequences of normal Aip (wild type) (A) and mutant Aip (B, C) in exon 4 of the Aip gene of the GH3-FTY cell line are shown. In addition, the GH3-FTY cell line contains a compound heterozygous mutation of insertion of adenine at c496 bp and deletion of two bases of c496-497, respectively, and codon 173 (TGA) of both Aip alleles and Codon 172 (TGA) each predisposes to an immature stop codon. The figure which shows the screening result of the Aip destruction GH3 clone produced with the CRISPR-Cas9 system. A shows Western blots of Aip protein of GH3 cell line and mutant GH3 clones 1 and 2. B and C show the relative expression levels of Gh mRNA and Prl mRNA with respect to Actb mRNA, respectively. D shows the relative expression level of clone 1 Aip mRNA (GH3-FTY cell line) to Actb mRNA. Note that ** P <0.01 vs. GH3. The figure which shows the amount of Gh secretion of cultured GH3 cell line (A) and GH3-FTY cell line, the Gh mRNA expression level (B, D) before and behind exogenous Aip expression, and intracellular cAMP amount (C). The figure which shows the comparison about the cell growth and mitotic mechanism of GH3 cell line and GH3-FTY cell line. In the figure, * P <0.05, ** P <0.01, and ns = no significant difference. A shows the cell numbers of the GH3 cell line and the GH3-FTY cell line after 24 hours and 48 hours, respectively. B shows the 24-hour as well as 48-hour BrdU assay results. C shows the effect of exogenous Aip (LV-Aip) or Gfp (LV-Gfp) lentivirus (LV) -mediated forced expression as a control on cell proliferation of cultured GH3-FTY cell lines. In the figure, LV (-) indicates an LV-uninfected GH3-FTY cell line. There was no statistical difference between LV (-) and LV-Gfp, and there was a statistical difference only between LV-Gfp and LV-Aip. Western blot analysis of Stat and p-Stat3 (Tyr705) of cultured GH3 and GH3-FTY cell lines. FIG. 4A is a Western blot analysis chart for Stat and p-Stat3 (Tyr705), and FIGS. 4B and 4C show statistical evaluation of the results. FIG. 4D shows the effect of LV-mediated forced expression of exogenous Gfp (LV-Gfp) or Aip (LV-Aip) as a control on p-Stat3 expression. FIG. 4E shows a statistical evaluation of the results. A, C, and F are diagrams showing respective mRNA expression labels for the GH3 cell line and GH3-FTY cell line Il6 receptor (Il6r), Sstr2 and Actb mRNA of Zac1. FIG. 5D is a Western blot analysis diagram for Sstr2. FIG. 3D shows a comparison of sensitivity to somatostatin. B, E, and G show the effect of LV-mediated forced expression of exogenous Gfp (LV-Gfp) or Aip (LV-Aip) as a control on Il6 receptor (Il6r), Sstr2 and Zac1 expression. In the figure, ND is not detected, ** p <0.01, ns = no significant difference. The figure which analyzed the tumor of the mouse | mouth (GH3-FTY mouse | mouth) which transplanted the mouse | mouth (GH3 mouse) or GH3-FTY cell line which transplanted GH3 cell line. A shows the tumor volume 8 weeks after transplantation, and B shows the tumor weight 8 weeks after transplantation. C is a typical cell histology of somatotrophic tumor of GH3 and GH3-FTY mice by HE staining 4 weeks after inoculation and D 8 weeks after inoculation. The lower panel shows an observation by immunofluorescence after staining a somatotroph cell tumor of GH3 mouse and GH3-FTY mouse 4 weeks after transplantation with Gh staining or DAPI. E is a statistical comparison of Ki67 scores. In the figure, * p <0.05, ns = no significant difference. The figure which shows the plasma GH level (A), plasma IGF-1 level (B), and actual phenotype (C) of a control mouse, GH3 mouse, and GH3-FTY mouse. The figure which shows the body weight (A), body length (B), and liver weight (C) of a control mouse | mouth, GH3 mouse | mouth, and GH3-FTY mouse | mouth. In A, body weight was measured over time. There was a significant difference in body weight between control mice and GH3-FTY mice from the 46th day of transplantation, but there was no significant difference between control mice and GH3 mice. . In the figure, * p <0.05, ** p <0.01, ns = no significant difference. 8B and 8C are comparisons of body length and liver weight 8 weeks after transplantation into control mice, GH3 mice, and GH3-FTY mice. The figure which shows the glucose metabolism after the transplantation of a control mouse, a GH3 mouse, and a GH3-FTY mouse. A is a blood glucose level, B is a blood glucose AUC, C is a plasma insulin level, and D is a plasma insulin level AUC. FIG. E shows the change in blood glucose after ITT. The plasma insulin level AUC after ipGTT was measured 6 weeks after transplantation, and the change in blood glucose level after ITT was measured 7 weeks after transplantation. In the figure, * p <0.05, ** p <0.01 between GH3-FTY mice and control mice, and #p <0.05 between GH3-FTY mice and GH3 mice. There is no significant difference. FIG. 10 is a schematic diagram showing the nucleotide sequence of exon 4 of the GH3-FTY cell line and the Aip protein. A is the nucleotide sequence of the normal (wild type) Aip gene and the mutant Aip gene of exon 4 of the GH3-FTY cell line. Both alleles of the GH3-FTY cell line contain complex heterozygous mutations with an adenine insertion at c496 and a two base deletion at c496-497, respectively, immature stop codon 173 (TGA) and codon 172 (TGA) is formed. B is a schematic diagram of the wild-type Aip structure of the GH3 cell line and the mutant Aip structure of the GH3-FTY cell line.

  The AIP knockout growth hormone (GH) producing cell line according to the present invention (hereinafter also referred to as “GH3-FTY cell line”) is a genome of a tumor cell line such as a pituitary tumor (hereinafter also referred to as “GH3 cell line”). The AIP gene can be knocked out by editing (CRISPR-Cas9 system).

  As the tumor cell line GH3 used in the present invention, a rat pituitary-derived GH3 cell line was used, but a pituitary-derived GH3 cell line such as a mouse other than a rat can also be used. Tumors other than pituitary derived tumors can also be used. The rat GH3 cell line used in the experiment of the present invention can be obtained from a public depository such as American Type Culture Collection (ATCC).

  In the present invention, as a method for knocking out the AIP gene from the rat pituitary tumor cell line GH3, genome editing by the CRISPR-Cas9 system is preferable, but other genome editing and gene modification methods can also be used.

  The GH3-FTY cell line according to the present invention is an Aip knockout growth hormone (GH) -producing cell line obtained by knocking out the Aip gene function from the GH3 cell line using the CRISPR-Cas9 system. This GH3-FTY cell line contains a complex heterozygous mutation that predisposes to frameshift mutations and forms immature stop codons at positions 173 and 172, and the truncated Aip protein produced is wild The amino acid sequence is completely different from that of type Aip, and the TPR domain and the last five carboxyl groups reported to be necessary for the biological activity of Aip (Non-patent Document 27) are deleted. In addition, the GH3-FTY cell line of the present invention hardly expresses Aip mRNA as compared with the GH3 cell line. Moreover, this protein was not detected immunologically by Western blot analysis using an anti-AIP antibody that mainly recognizes the FKBP domain of the N-terminal region of Aip. From these results, it is suggested that these mutant genes have a low transcription rate and the stability of the obtained cleaved protein even if translated.

  In the GH3-FTY cell line of the present invention, Gh secretion is dramatically increased from 20 to 43 times compared to the GH3 cell line. It has already been reported that the GH3 cell line is unstable due to fluctuations in the basal Gh secretion and Gh / Prl secretion ratio (Non-patent Document 26). This can be explained in part because the GH3 cell line is not a homogeneous cell clone (Non-Patent Documents 21 and 26). However, even though such unstable elements of the GH3 cell line are reflected to some extent in the results of the present invention, the strong Gh secretion capacity of this GH3-FTY cell line is not related to the previously reported secretion of the GH3 cell line. It far surpasses the ability (approximately 2 to 6 times) (Non-Patent Document 26), and is actually the basis of a very powerful and stable biological activity in cancer-bearing animal models. Note that the GH3-FTY cell line is actually produced from a single cell of the GH3 cell line by genome editing using the CRISPR-Cas9 system, and thus is actually considered to be a homogeneous cell clone.

  The cell growth capacity of the GH3-FTY cell line of the present invention is not significantly different from that of the GH3 cell line, but the GH3-FTY cell line significantly increases DNA synthesis in the BrdU assay compared to the GH3 cell line. doing. This is also supported by the finding that the S phase relative to the G1 phase increases in the cell cycle analysis of the GH3-FTY cell line. In addition, the fact that the sub-G1 phase is not changed indicates that inhibition of cell apoptosis is not involved in the cell growth mechanism. Importantly, forced expression with lentivirus on the GH3-FTY cell line partially reverses the increase in Gh mRNA expression and almost completely reverses cell proliferation and cell cycle profile. The exact reason why this increase in GH synthesis is partially reversed is not yet clear, but is due to insufficient levels of exogenous Aip expression or heterogeneous or variable GH secretion in the GH3 cell line there is a possibility. However, in the present invention, GH secretion and cell proliferation are increased by completely losing the Aip function of GH-producing cells.

  By the way, since homozygous Aip knockout of mice is known to be fatal, it has been difficult to confirm endogenous Aip function in vivo. Although Aip knockout mouse embryo fibroblasts (MEF) can be obtained for experiments (Non-patent Document 13), the GH3-FTY cell line of the present invention is useful as the first model of the Aip knockout pituitary cell line.

  In addition, it is known that the increase in GH secretion is associated with an increase in cAMP production by the GH3-FTY cell line of the present invention compared to the GH3 cell line. This finding is supported by the fact that cAMP production increases in Aip knockout MEF or Aip expression-suppressed (silence) GH3 cells, as previously reported (Non-patent Document 13).

  The type I cytokine receptor family such as Il-6 receptor binds to a ligand to activate Janus kinase 2 (Jak2) and activates Stat3 by phosphorylation at Tyr705. Activated phosphorylated Stat3 (p-Stat3) undergoes homodimerization and nuclear translocation, and regulates cell proliferation, survival and differentiation under the transcriptional regulation of target genes (Non-patent Document 28). There is a recent report that STAT3 upregulation in pituitary somatotroph tumors is associated with GH hypersecretion (Non-patent Document 29). In the present invention, in the GH3-FTY cell line, GH induces Stat3 phosphorylation as well as nuclear translocation, suggesting the existence of a self-regulating positive feedback loop between Stat3 and GH in somatotroph tumor cells ( Non-patent document 29). However, it is not clear whether Aip is involved in this phenomenon, but it is interesting to note that according to the present invention, the GH3-FTY cell line deficient in Aip has a stable Stat3 expression level. Regardless, p-Stat3 is significantly increased compared to the GH3 cell line. In addition, lentivirus-mediated Aip forced expression partially reverses this phenomenon, but clearly. These results suggest that Stat3 activation in the GH3 cell line is mediated, at least in part, by Aip. Thus, the autoregulatory positive feedback loop between Stat3 and GH can be enhanced more in the GH3-FTY cell line than in the GH3 cell line.

  The sensitivity to somatostatin is lower in the GH3-FTY cell line than in the GH3 cell line. It has been suggested that the anti-proliferative effect of octreotide, a somatostatin analog, is associated with up-regulation of ZAC1 expression (Non-Patent Document 16), which is associated with IGF-I response and tumor regression. (Non-patent Document 30). Pituitary tumors with reduced AIP expression are often reported to be resistant to the first generation somatostatin analogs (Non-patent Documents 10 and 15). However, it is not clear whether reduced AIP expression is actually associated with AIP inactivity. Furthermore, it is not clear whether AIP inactivity is associated with reduced SSTR2 expression. In addition, it has been reported that SSTR2 staining was not decreased in pituitary somatotroph tumors regardless of the presence or absence of AIP mutation (Non-patent Documents 3 and 16). However, in the present invention, the GH3-FTY cell line has decreased Sstr2 expression and Zacl expression compared to the GH3 cell line. In other words, inactivation of Aip can down-regulate Zac1 (Non-patent Document 16), but can also regulate Sstr2. The antisecretory and antiproliferative effects of somatostatin occur through several circuits. The direct effect of somatostatin via Sstr2 is to modulate the MAPK cascade, activate tyrosine / serine phosphatase, and inhibit adenylate cyclase (Non-Patent Documents 31-33). As an indirect effect, release of growth promoting factors such as IGF-I, IGF-II, and IGF-binding protein 3 can be mentioned (Non-patent Document 34). Therefore, it should be noted that there may be other possibilities for the sensitivity of the GH3-FTY cell line to somatostatin.

  The biological function of the GH3-FTY cell line of the present invention was examined in vivo by inoculating nude mice with the GH3-FTY cell line. Growth hormone (GH) promotes the growth of the body including the liver via STAT5b-mediated signaling, and this effect has been reported to be mediated by STAT5b (Non-patent Document 35). GH secretion from GH3-FTY-inoculated mice (hereinafter also referred to as “GH3-FTY mice”) is five times higher than that from GH3-inoculated mice (hereinafter also referred to as “GH3 mice”) Nevertheless, the body weight of GH3-FTY mice increased from day 46 of inoculation (P <0.05 vs control or GH3 mice). The various organs are also likely to increase over time. However, the body weight of GH3 mice has not increased much compared to control mice during the observation period. The body length and liver weight of GH3-FTY mice increased from GH3 mice 8 weeks after inoculation.

  It was confirmed by evaluation of glucose metabolism that GH3-FTY mice have higher biological in vivo functions than GH3 mice. According to the glucose tolerance test (GTT) conducted 6 weeks after the inoculation, the glucose tolerance of the GH3-FTY mouse model was almost the same as that of the GH3 mouse model. However, hyperinsulinemia in GH3-FTY mice was clearly more severe than in GH3 mice. However, it was unexpected that the glucose tolerance of GH3-FTY and GH3 mice was better than that of control mice. However, the insulin resistance test (ITT) conducted 7 weeks after the inoculation revealed that insulin resistance of GH3-FTY mice was clearly higher than that of GH3 mice and control mice. These results may reflect the time-dependent changes in glucose tolerance associated with insulin resistance caused by Gh hypersecretion. Igf-1 simultaneous hypersecretion can also regulate glucose tolerance by improving insulin sensitivity. These two types of mouse models are very useful for examining developmental processes from insulin resistance against diabetes due to GH hypersecretion (Non-patent Documents 35 and 36).

  Moreover, the average tumor size of GH3-FTY mice is larger than that of GH3 mice. This can be attributed to the increased cell proliferation ability of the GH3-FTY cell line. Another possibility is that the tumor is stimulated autocrinely by Gh and Igf-1, which are secreted and circulated in large quantities from the tumor. The GH3-FTY cell line of cultured and inoculated tumors has a higher N / C ratio, higher frequency of polymorphic nuclei, and increased Ki67 score than the GH3 cell line It shows more mitotic properties of.

  Such a change promotes cell growth that is preferentially mediated by the mitogen-activated protein (MAP) kinase signaling circuit as well as the anti-apoptotic circuit, thus continuing the GH-IGF-1 system. It seems to be somewhat related to the effect of hypersecretion on tumor development (Non-patent Document 37). However, it is considered that the decay of Aip is highly related to the findings according to the present invention determined from the function of AIP as a tumor suppressor gene (Non-patent Documents 3 and 11-14).

  That is, the present invention established a GH3-FTY cell line in which endogenous Aip is completely destroyed by the CRISPR-Cas9 system. In the GH3-FTY cell line of the present invention, Aip inactivation increases the Stat3 activation of the GH3-FTY cell line over the parent GH3 cell line, thereby promoting the canceration function of somatotroph cells. This partially explains that suppression of Sstr2 expression reduces the sensitivity of Aip-inactivated somatotroph cells to somatostatin. The GH3-FTY cell line of the present invention is a useful model for explaining whether Aip inactivation promotes somatotroph tumorigenesis as well as autonomous GH secretion. Moreover, since the GH3-FTY cell line of the present invention is useful for screening innovative drug discovery for acromegaly, it is very interesting to study GH3-FTY mice in detail for the characteristics of acromegaly. Is about to lean on.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, it should be understood that the following examples only illustrate the present invention more specifically and do not intend to limit the present invention at all. It is.

  First, the gene sequence was analyzed for the region containing each exon and splicing site of the Aip gene of the rat pituitary tumor cell line GH3. The GH3 cell line was purchased from ATCC and cultured in F-12K medium (Life Technologies) containing 15% horse serum and 2.5% fetal calf serum. Genomic DNA was extracted from the GH3 cell line using Wizard genomic DNA purification kit (Promega), and each exon containing the splicing site of the adjacent intron of the rat Aip gene was amplified by PCR using KOD FX (TOYOBO). The sequence shown in Table 1 was used to determine the sequence, and the sequence was compared with rat Aip cDNA (NM_172327.2). As a result, the sequence of the Aip gene of the GH3 cell line coincided with the rat Aip cDNA (NM_172327.2), and no gene mutation was observed.

  In this example, the AIP gene of the GH3 cell line was knocked out from the rat pituitary tumor cell line GH3 using the genome-edited CRISPR-Cas9 system to produce an AIP knockout GH-producing cell line (GH3-FTY).

  In order to knock out the Aip gene from the GH3 cell line, GeneArt CRISPR Nuclease Vector Kit (Life technologies), an all-in-one plasmid for Aip gene knockout, was used according to the protocol of the kit. This plasmid contains a gene encoding the gene cleavage enzyme Cas9 and a marker CD4 gene for selecting a transgenic cell. Proto-spacer adjacent motif (PAM) was rat Aip cDNA (NM_172327.2) c.490-492, and the target sequence was 20 bases c.493-512. Further, 5′-TGCCCATGGGTCCTGCTGTTTT-3 ′ as the top strand and 5′-AGCAGGACCCATGGGCACGGTG-3 ′ as the bottom strand were synthesized and annealed. The annealed double-stranded DNA was incorporated into a CRISPR Nuclease Vector, and the prepared CRISPR Nuclease Vector plasmid construct was introduced into the GH3 cell line using Amaxa Cell Line Nucleofector (Lonza). Two days after the transfection, CD4 positive cells were selected using an anti-CD4 antibody (Milteni Biotec), and the selected CD4 positive cells were seeded in each well of a 96-well plate and cultured. After cell growth, genomic DNA was extracted from each cell clone, and the exon 4 region of Aip was amplified by PCR and directly sequenced for analysis. Other exon sequences, including the exon-intron region of the GH3 cell line used in the present invention, have been completely homologous to the rat Aip cDNA (NM172327.2) and have been subjected to genomic sequencing of the target clone Aip. It was confirmed that there was no mutation.

  Two GH3 cell clones were identified by genome-editing the GH3 cell line using the CRISPR-Cas9 system. These clones contain compound heterozygous mutations of both alleles and introduce immature terminal codons to produce truncated Aip proteins. In the obtained two cell clones, that is, clone 1 and clone 2, no band corresponding to the estimated molecular weight of 37 kDa of the GH3 cell line containing wild type Aip was detected (FIG. 1A). Thus, in the obtained cell clone, the Aip gene was genetically disrupted, and the Aip protein was completely lost.

  When both clones were quantitatively PCR, they did not show the same phenotype for GH synthesis as well as PRL synthesis (FIGS. 1B, 1C). This is probably because the cells are not uniform in the GH3 cell line (Non-patent Documents 21 and 26). Furthermore, of the two cell clones, only one clone (clone 1) increased GH synthesis by 7.3 times and PRL synthesis by 1.3 times compared to the GH3 cell line. Although this clone 1 (hereinafter also referred to as “GH3-FTY cell line”) is a somatotroph cell line, the other cell clone (clone 2) showed a PRL synthesis level 3.2 times higher than that of the GH3 cell line. The GH synthesis was only 0.2 times. This indicates that clone 2 is a more Lactotropic cell line.

  In the present invention, the Aip gene sequence and amino acid sequence of clone 1 (GH3-FTY cell line) are as shown in FIGS. Clone 1 (GH3-FTY cell line) contains a complex heteroframeshift mutation. That is, in both alleles, codon 173 (TGA) is formed by insertion of adenine at c496, and codon 172 (TGA) is formed by deletion of 2 bases at c.496-497 (FIG. 1, 11A).

  The truncated Aip protein of clone 1 (GH3-FTY cell line) has a completely different amino acid sequence (165 amino acids (AA) to 172AA and 164AA to 171AA) from the wild-type Aip of the GH3 cell line. (FIG. 1, 11A). In addition, the TPR site is completely absent in both proteins (FIG. 11B). Interestingly, the mutant Aip of clone 1 (GH3-FTY cell line) had an extremely low relative mRNA expression level compared to the GH3 cell line (p <0.01) (FIG. 2D). This suggests that the stability of mRNA from these genes is also reduced.

  On the other hand, clone 2 has a complex heterozygous mutation with an adenine insertion at c496 of one allele and a guanine deletion at c497 of the other allele, and finally a stop codon is formed at position 174. .

  As described above, the GH3-FTY cell line of the present invention (clone 1) lacks the function of Aip and contains a complex heterozygous mutation that predisposes to a frameshift mutation, and positions 173 and 172. Form an immature stop codon. The truncated Aip protein is completely different in amino acid sequence from wild-type Aip, and also has a TPR domain and the last five carboxyl groups reported in Non-Patent Document 27 that are necessary for the biological activity of Aip. Is missing.

  Further, as described above, the GH3-FTY cell line (clone 1) hardly expresses Aip mRNA as compared with the GH3 cell line (FIG. 2D). Moreover, this cleaved protein was not detected immunologically by Western blot analysis using an anti-Aip antibody that mainly recognizes the FKBP domain of the N-terminal region of Aip (FIG. 2A). From these results, it is suggested that these mutant genes have a low transcription rate and the stability of the cleaved protein obtained even if translated.

  In this example, Aip, Gh, Prl, somatostatin receptor type 2 (Sstr2), Zac-1, Il-6 receptor (Il6r) and glycoprotein 130 (gp130) of GH3-FTY cell line and GH3 cell line mRNA levels were assessed by quantitative PCR.

GH3-FTY cell line and GH3 cell line were seeded 2 × 10 ⁵ cells in each well of a 24-well plate (n = 3), and total RNA was prepared 24 hours later using RNeasy Kit (QIAGEN) . CDNA was prepared from total RNA (1μg) using QuantiTect Reverse Transcription Kit (QIAGEN), and quantitative RT-PCR was performed using Light Cycler 2.0 (Roche) and SYBR Premix Ex Taq II (TAKARA BIO) according to conventional methods. It was. PCR conditions were appropriately adjusted according to the experiment.

  The relative expression level of each gene mRNA relative to the internal control Actb (−actin) was calculated by the ΔCt method. The forced expression of Aip was performed using the lentivirus-mediated method. In this method, a synthetic rat Aip cDNA (Eurofins Genomics Co., Ltd.) was constructed in a lentiviral vector plasmid (CSII-EF-IRES2-Venus) (RIKEN BRC), and the pCAG-HIV-g / p-plasmid and It was transfected into 293T cells with the pCMV-VSV-G-RSV-Rev plasmid. It was confirmed by sequencing that the rat Aip cDNA retained the same sequence.

  To examine whether exogenous Aip reverses the phenomenon observed in GH3-FTY cells, GH3 cells and GH3-FTY cells were cultured and rat Aip cDNA (LV-Aip) or Gfp cDNA (LV- Infected with lentivirus containing Gfp). Viral infection was performed at a multiplicity of infection (MOI) of 25.

  The growth hormone (GH) secretion amount of the GH3-FTY cell line of the present invention was extremely large, 20 to 43 times that of the GH3 cell line (FIG. 3A; P <0.01). The Gh mRNA level of the GH3-FTY cell line was 7.3 to 25 times that of the GH3 cell line (FIG. 3B; P <0.01). The cAMP level of the GH3-FTY cell line was higher than the GH3 cell line with a statistically significant difference (FIG. 3C). Importantly, the Gh mRNA increase in the GH3-FTY cell line was inhibited by lentivirus (LV) -mediated exogenous Aip (P <0.01), whereas the GH3 cell line had no change in Gh mRNA. is there. This is because it is suggested that the endogenous Aip function is disrupted in the GH3-FTY cell line (FIG. 3D).

To measure cAMP concentration, inoculate a GH3 cell line or GH3-FTY cell line in each well of a 24-well plate to a cell density of 2x10 ⁵ cells, incubate for 24 hours, aspirate the medium, and add lysis buffer The intracellular cAMP concentration ([cAMP] i) was measured using Cyclic AMP Select EIA Kit (Cayman Chemical) according to the manufacturer's protocol.

GH3-FTY cell line or GH3 cell line was seeded in each well of a 24-well plate to a cell density of 2X10 ⁵ cells, and 15% horse serum and 2.5% fetal bovine serum treated with dextran coated charcoal, respectively. Incubated with 1 ml of F-12K medium. After 24 hours of culture, the culture medium was collected, and the GH concentration was measured with Growth Hormone Rat EIA Kit (Bertin Pharma), and calculated with the RNA content. Total RNA was extracted and the absorbance was measured at 260 nm by spectrophotometry. GH secretion and PRL secretion from the GH3-FTY cell line were expressed in multiples compared to the GH3 cell line. In addition, the expression levels of GhmDNA and Prl mRNA against Actb (-actin) in the GH3-FTY cell line were measured by quantitative PCR and compared with those in the GH3 cell line.

In this example, cell growth of GH3-FTY cell line and GH3 cell line was examined. For cell growth, GH3-FTY cell line and GH3 cell line were seeded in a 96-well plate so that the cell density of each well was 1 × 10 ⁴ cells (n = 3), and 0, 24 and 48 hours after seeding. The number of cells after was measured with Cell Counting Kit-8 (Dojindo). The measured value at each time point was calculated based on the number of cells at the start of the experiment (0 hour).

In order to evaluate the growth of GH3 cell line and GH3-FTY cell line, BrdU assay was performed using BrdU Cell Proliferation ELISA Kit (Abcam) according to the protocol of the kit. In this BrdU assay, GH3-FTY cell line or GH3 cell line is injected into each well of a 96-well plate containing 1x10 ⁴ cells, and BrdU solution (10 mmol / L) is injected in the final 2 hours of stimulation. Added. The post-treatment after the culture was performed according to the manufacturer's protocol, and the absorbance of the sample was measured with a microplate reader.

  The cell growth rate of the GH3-FTY cell line of the present invention after 24 hours and 48 hours of culture was significantly higher than that of the GH3 cell line (FIG. 4A). It was confirmed by the BrdU assay that the cell growth rate of the GH3-FTY cell line was significantly higher than that of the GH3 cell line (FIG. 4B; P <0.01).

In this example, the cell cycle profile and apoptosis of GH3-FTY and GH3 cell lines were evaluated. The cell cycle profile was measured by flow cytometry (Non-patent Document 24). The GH3 cell line and the GH3-FTY cell line were cultured in a serum-free medium for 24 hours, and then further cultured in a serum-containing medium for 24 hours, or cultured in a serum-free medium for 48 hours. Cell nuclei were stained with Cycletest Plus DNA Reagent Kit (BD Biosciences, Tokyo), and fluorescence of probidium iodide was analyzed with FACSVerse (BD Biosciences). Each sample contained 2 × 10 ⁴ cells for GO / G1, S and G2 / M fraction measurements. Apoptosis was determined based on the presence of sub-G1 fraction.

  From the cell cycle analysis described above, in GH3-FTY cells, even in the absence of serum stimulation, the S phase corresponding to GH3 cells increased, and this trend was more evident in the presence of serum stimulation. In the absence of serum, the GH3 cell line was in a state where the G1 phase was stopped (Table 2). In addition, the sub-G1 fraction, which is an apoptotic cell fraction, was not observed in either GH3-FTY cells or GH3 cells.

  In the same manner as in Example 3, increase in cell proliferation of GH3-FTY cells was examined by a lentivirus (LV) mediated method. As a result, the increase in cell proliferation of GH3-FTY cells was significantly reversed by lentivirus (LV) -mediated expression of Aip, whereas it was not changed by LV-mediated expression of control Gfp ( FIG. 4C).

  In this example, the relationship between the cell growth of the GH3-FTY cell line and the GH3 cell line and the sensitivity to somatostatin was examined. As a result, somatostatin inhibited the cell growth of GH3-FTY cell line and GH3 cell line in a dose-dependent manner (FIG. 4D). The GH3-FTY cell line was less sensitive to somatostatin related to cell growth suppression than the GH3 cell line (FIG. 4D).

  In this example, in order to investigate the cell growth mechanism of the GH3-FTY cell line, Aip, Signal transducer / activator of transcription 3: Stat3, phosphorylated Stat3 Y705 (p- Stat3 Y705) and -actin protein expression levels were examined by Western blot analysis (see FIG. 5). The GH3 cell line and GH3-FTY cell line were each cultured for 48 hours, and the cell lysate was added to RIPA buffer (Sigma-Aldrich) and precipitated. Total protein (15-20 g) was electrophoresed by SDS-PAGE and transferred to a PVDF immunoblot membrane (BIO-RAD). Subsequently, this membrane was cultured with the primary antibody described below, and then cultured with an HRP-conjugated anti-mouse IgG secondary antibody (Cell Signaling Technology Japan KK). Aip protein was detected using ECL Prime Western Blotting Detection Reagent (GE Healthcare). Primary antibodies against Aip were obtained from Novus Biological, p-Stat3 Y705 and primary antibodies against Stat3 from Cell Signaling Technology Japan KK. Antibodies against anti-GHR were obtained from Santa Cruz Biotechnology and antibodies against anti-actin were obtained from Sigma-Aldrich Japan. These antibodies were used after appropriate dilution according to the kit protocol.

  Western blot analysis of the cell growth mechanism of the GH3-FTY cell line showed no change in Stat3 expression between the GH3 and GH3-FTY cell lines, but the GH3-FTY cell p- Stat3 (Y705) showed a significant increase compared to GH3 cells (FIG. 5A). Importantly, up-regulated p-Stat3 in GH3-FTY cells is reversed by LV-mediated forced expression of exogenous Aip (FIG. 5B).

  As a result of quantitative PCR of the mRNA expression of Il6 receptor (Il6r), which is one of the upstream receptors of Stat3, and its assembly component, it is interesting that GH3-FTY cells have a higher mRNA level than that of GH3 cells. Upregulated (FIG. 6A) and their increase is reversed by LV-mediated forced expression of exogenous Aip (FIG. 6B).

  In addition, in the GH3-FTY cell line, the Sstr2 mRNA expression level was dramatically reduced by quantitative PCR compared to the GH3 cell line (FIG. 6E). This finding also supports the results shown in FIG. 4D that the GH3-FTY cell line is less sensitive to somatostatin in the expression of cell proliferation than the GH3 cell line.

  Further, in the GH3-FTY cell line, the mRNA expression level of Zac1, which is probably a factor (Non-patent Document 16) involved in suppression of growth of GH-producing cells and suppression of GH secretion, was significantly reduced compared to the GH3 cell line ( FIG. 6F).

  From the above findings, the sensitivity of the GH3-FTY cell line to somatostatin decreased in relation to the decreased expression of Sstr2 and Zac-1.

  In this example, the GH3-FTY cell line prepared in Example 1 was inoculated subcutaneously into nude mice to produce GH3-FTY cancer-bearing mice (hereinafter also referred to as “GH3-FTY mice”). Comparison was made with inoculated cancer-bearing mice (hereinafter also referred to as “GH3 mice”). All animal experiment protocols used in the present invention are approved by the Fukuoka University Animal Handling and Use Committee.

For tumor-bearing mice, 5 x 10 ⁵ GH3-FTY cells or GH3 cells are suspended in 0.5 ml of F-12K medium, and nude mice (5 weeks old, male, BALB / c-nu, Charles River, Japan) are used. Inoculated subcutaneously (n = 8). For control mice, the nude mice were inoculated subcutaneously with 0.5 ml of F-12K medium (n = 5). Mice were bred with normal food and normal drinking water, and body weight was measured twice a week. In the tumor-bearing mice, a somatotroph cell tumor was formed at the site of inoculation. Eight weeks after the inoculation, the mouse body length, liver weight, tumor weight and tumor volume were examined.

  There was no difference in body weight between the control group and the GH3 group throughout the breeding period, but the GH3-FTY group showed a significant increase in body weight compared to the control group and the GH3 group from 46 days after inoculation. The average body weights of control mice, GH3 mice and GH3-FTY mice were 28.8 +/- 2.3 g, 30.8 +/- 3.0 g and 35.3 +/- 5.3 g, respectively (FIG. 9A; P <0.05; GH3-FTY vs. control or GH3).

  The length of the mouse was calculated from the CT image using a small animal X-ray CT scanner Skyscan 1178 (Brunker Corporation). The body length of GH3-FTY group mice 8 weeks after inoculation increased significantly compared to the control group and GH3 group. The average body lengths of control mice, GH3 mice, and GH3-FTY mice 8 weeks after inoculation were 87.9 +/- 4.8 mm, 89.3 +/- 3.1 mm, and 95.8 +/- 2.4 mm, respectively (FIG. 9B; ** P <0.01; ns = no significant difference).

  As for the liver weight of the mouse, the whole liver of the mouse and the inoculated tumor were extracted and their weight was measured. Total liver weight was calculated based on body weight. At 8 weeks after inoculation, the liver weight in the GH3 group and GH3-FTY group was significantly increased compared to the control group (P <0.01), and the liver weight in the GH3-FTY group was significantly higher than that in the GH3 group. Increased (FIG. 9C; P <0.05).

Tumor weight and tumor volume of mice were measured by excising tumor tissue. The tumor was measured for major axis, minor axis and depth, and the tumor volume was calculated by the following modified ellipsoid formula (Non-patent Document 25).
Tumor volume = length x square of width x 0.52

  Both tumor volume and tumor weight 8 weeks after inoculation were significantly increased in GH3-FTY mice compared to GH3 mice. Tumor volume and tumor weight were measured 8 weeks after inoculation. The average tumor volume of GH3-FTY mice was 4.3 times greater than that of GH3 mice (FIG. 7A; P <0.05). The average tumor weight of GH3-FTY mice was 3.87 times heavier than that of GH3 mice (FIG. 7B; P <0.05).

  Histological analysis was performed by staining tissue sections of GH3-FTY mice or GH3 mice by the hematoxylin-eosin (HE) staining method (Non-patent Document 23). In addition, immunohistochemical staining of Ki67 positive cells or growth hormone (GH) was performed according to a previous report (Non-patent Document 22). The prepared paraffin sections were cultured with anti-Ki97 antibody (ab66155; Abcam) or anti-rat GH antibody (R & D Systems, Inc.), and then inoculated with Alexa Fluor 546 goat anti-rabbit IgG (A-11010; Life Technologies). Sections were then counterstained with DAPI and visualized with a confocal microscope.

  FIG. 7C is a HE-stained diagram of somatotroph tumors of GH3 mice and GF3-FTY mice (left side shows GH3 mice, right side shows GF3-FTY mice). Moreover, the upper figure shows a HE-stained figure 4 weeks after inoculation, and the lower figure shows 8 hours after inoculation. Tumor HE staining revealed that tumors in GF3-FTY mice contained necrotic tissue and exhibited more mitotic features than GH3 mice. That is, GF3-FTY mice showed a relatively large nucleus / cytoplasm (N / C) ratio 4 weeks after inoculation, and the presence of polymorphic nuclei 8 weeks after inoculation, as compared with GF3 mice.

  FIG. 7D is an immunofluorescence diagram 4 weeks after inoculation with cell tissues of somatotroph tumors of GH3 mice and GF3-FTY mice. As a result, secretory granules on the cell surface were stained with Gh (green), and nuclei were stained with DAPI (blue).

  FIG. 7E shows that tumors in GF3-FTY mice have significantly increased Ki67 scores (* P <0.05). This finding further confirms the results of FIG. 7C.

  In this example, blood Gh and Igf-1 values of GH3 mice and GH3-FTY mice were evaluated. The GH3 cell line and GH3-FTY cell line were suspended in 0.5 ml of F-12K medium, and the suspension was inoculated into nude mice (BALB / c-nu) in the same manner as in Example 10 (each groove: n = 8). The control group of mice received medium alone. Plasma was collected from the peripheral blood of each mouse, and blood Gh and Igf-1 concentrations were measured. Igf-1 concentration was measured by Quantikine ELISA mouse / rat IGF-1 (R & D systems).

  The plasma Gh value and Igf-1 value at 8 weeks after inoculation are shown in FIGS. 8A and 8B, respectively. As shown in FIG. 8A, Gh of GH3 mice and GH3-FTY mice increased significantly compared to the control group, and Gh values of GH3-FTY mice increased significantly compared to GH3 mice. Plasma Gh levels 8 weeks after inoculation of GH3 and GH3-FTY mice were 554 +/- 456 ng / mL and 2764 +/- 1456 ng / mL, respectively (P <0.01). Moreover, as shown in FIG. 8B, Igf-1 of GH3 mice and GH3-FTY mice significantly increased compared to the control group. Its plasma Igf-1 levels were 549 ng / mL and 707 ng / mL in GH3 and GH3-FTY mice, respectively (P <0.13). However, this plasma Igf-1 level was not statistically significant.

  As a result of examining the phenotype of the tumor-bearing mice obtained in Example 12, the GH3-FTY mice were clearly larger than the control mice and GH3 mice (FIG. 8C).

  In this example, a glucose tolerance test (GTT) was performed to examine the effects of glucose metabolism on GH3 mice and GH3-FTY mice. The glucose tolerance test was performed 6 weeks after inoculating nude mice with the GH3-FTY cell line. Nude mice were fasted for 15 hours before the glucose tolerance test, then 2 g / kg body weight of glucose was intraperitoneally administered, and blood glucose levels were measured at 0 minutes, 15 minutes, 30 minutes, 60 minutes and 120 minutes after administration. The blood glucose level was measured with a blood glucose measuring device Glutest Mint (Sanwa Chemical). In addition, plasma insulin levels at 0 minutes, 15 minutes, 30 minutes and 60 minutes after administration were measured with the Morinaga ultrasensitive mouse insulin measurement kit (Morinaga Institute of Science).

  As a result, in both the GH3 group and the GH3-FTY group, the blood glucose level at 15 minutes and 30 minutes after glucose load significantly decreased at the time of fasting compared to the control group. There was no significant difference in blood glucose level at any time point between the GH3 group and the GH3-FTY group (FIG. 10A; * P <0.01; ** P <0.01). Similarly, in both the GH3 group and the GH3-FTY group, the area under the curve (AUC) of the blood glucose level was significantly smaller than that in the control group. In addition, there was no significant difference in the area under the curve (AUC) of the blood glucose level between the GH3 group and the GH3-FTY group (FIG. 10B; * P <0.01; ** P <0.01; ns = no significant difference) ).

  In this example, an insulin tolerance test (ITT) was performed in order to examine the effect of glucose metabolism on GH3 mice and GH3-FTY mice. The insulin tolerance test was performed 7 weeks after inoculating nude mice with the GH3-FTY cell line. In the insulin tolerance test, after 3 hours of fasting, Novolin R injection (Novo Nordisk Pharma) 0.75 unit / kg body weight was intraperitoneally administered, and insulin values at 15 minutes, 30 minutes and 60 minutes were measured at the time of fasting.

  Insulin levels at 15 minutes, 30 minutes, and 60 minutes after fasting were increased in the GH3 and GH3-FTY groups compared to the control group, and insulin resistance was exacerbated. . Furthermore, in the GH3-FTY group, insulin values at 15 minutes after sugar load and after 30 minutes at the time of fasting significantly increased compared to the GH3 group (FIG. 10C). There was no significant difference between the GH3 group and the control group. Similarly, both the GH3 group and the GH3-FTY group showed significantly higher values of the area under the curve (AUC) of the insulin value than the control group. In addition, there was no significant difference in the area under the curve (AUC) of the insulin value between the GH3 group and the GH3-FTY group (FIG. 10D; * P <0.01; ** P <0.01).

  As a result of ITT 7 weeks after the inoculation, the GH3-FTY group showed significantly higher blood glucose level than the GH3 group and the control group. This suggests that GH3-FTY mice have insulin resistance (FIG. 10E).

  The AIP knockout growth hormone (GH) -producing cell line (GH3-FTY cell line) according to the present invention can be used as a research model cell line for pituitary tumors in particular, and pituitary tumors can be obtained using this model cell line. It can greatly contribute to research and development of drug discovery and treatment methods effective for prevention or treatment.

  In addition, according to the present invention, a GH3-FTY cell line in which the Aip gene is genetically disrupted by genome editing (CRISPR-Cas9 system) is obtained from the GH3 cell line. Compared to the strain, growth hormone (Gh) synthesis was significantly increased, cell proliferation was slightly increased, and cell phase analysis increased S phase profile. However, all these phenomena are reversed by exogenous expression of Aip. In GH3 cell lines, Gh-induced Stat3 phosphorylation is known to cause GH hypersecretion, but interestingly, phosphorylated Stat3 expression is increased in GH3-FTY cell lines compared to GH3 cell lines. This is a powerful driving force for this mechanism in the GH3-FTY cell line. In addition, the GH3-FTY cell line is less sensitive to somatostatin in suppressing cell growth than the GH3 cell line, but this is associated with decreased expression of somatostatin receptor type 2 and the antiproliferative gene Zac1 / Plag11. .

  In GH3-FTY mice in which the GH3-FTY cell line of the present invention was transplanted into Balb / c mice, mitotic somatotroph tumors were formed more than in GH3 mice in which the GH3 cell line was transplanted. In GH3-FTY mice, tumor size was much larger than in GH3 mice, and serum Gh levels were significantly higher. Moreover, in the insulin resistance test, GH3-FTY mice showed relatively higher insulin resistance than GH3 mice.

Claims (9)

  An AIP knockout growth hormone (GH) -producing cell line having an amino acid sequence and function different from those of the wild-type AIP gene by knocking out the AIP (Aryl Hydrocarbon Receptor Interacting Protein) gene of the tumor cell line by genome editing.   The AIP knockout growth hormone (GH) -producing cell line according to claim 1, wherein the cleaved AIP protein produced by knockout of the AIP gene is SEQ ID NO: 2 (SEQ ID NO: 2) or SEQ ID NO: 3 (SEQ ID NO: 3). An AIP knockout growth hormone (GH) -producing cell line characterized by having an amino acid sequence represented by NO: 3).   The AIP knockout growth hormone (GH) producing cell line according to claim 1 or 2, wherein the growth hormone (GH) producing cell line is derived from a rat pituitary tumor. ) Production cell line.   The AIP knockout growth hormone (GH) producing cell line according to any one of claims 1 to 3, wherein the AIP knockout growth hormone (GH) producing cell line is a pituitary-derived growth hormone (GH) producing cell. An AIP knockout growth hormone (GH) -producing cell line characterized in that growth hormone (GH) production and cell proliferation are increased compared to the cell line.   The AIP knockout growth hormone (GH) producing cell line according to any one of claims 1 to 4, wherein the AIP knockout growth hormone (GH) producing cell line is somatostatin receptor type 2 (Sstr 2) or An AIP knockout growth hormone (GH) -producing cell line characterized by reduced expression of ZAC1.   AIP knockout growth hormone (GH) producing cell line, AIP knockout growth hormone (GH) by knocking out the AIP gene of the pituitary derived growth hormone (GH) producing cell by CRISPR-Cas9 system A method for establishing an AIP knockout growth hormone (GH) producing cell line, characterized by establishing a producing cell line.   A method for establishing an AIP knockout growth hormone (GH) -producing cell line according to claim 6, wherein the pituitary gland is a rat pituitary gland. .   An AIP knockout growth hormone (GH) characterized in that the AIP knockout growth hormone (GH) producing cell line according to any one of claims 1 to 5 is used as a model cell line of a growth hormone (GH) producing tumor. How to use production cell lines.   A method of using the AIP knockout growth hormone (GH) producing cell line according to claim 8, wherein the growth hormone (GH) producing tumor is a growth hormone (GH) producing pituitary tumor. How to use growth hormone (GH) producing cell lines.