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Human growth hormone (GH) is a peptide hormone secreted by the anterior pituitary gland. It regulates postnatal growth and metabolism and exhibits multifunctionality in various tissues of the human body. Chronic hypersecretion of growth hormone into the circulation, typically resulting from a GH-secreting pituitary adenoma, is commonly associated with acromegaly, a debilitating condition characterized by excessive bone growth, soft tissue enlargement, insulin resistance, and cardiovascular and gastrointestinal morbidity.

      GHR is a type I cytokine receptor that lacks intrinsic kinase activity and requires the recruitment of the non-receptor tyrosine kinase JAK2 (Janus kinase 2) for activation. The GHR dimer interacts with the GH ligand through two binding sites, which have different affinities for the receptor. Ligand binding causes a conformational change in the receptor’s transmembrane domain, leading to the phosphorylation and activation of two JAK2 molecules associated with the receptor’s cytoplasmic domain. Phosphorylated JAK2 subsequently phosphorylates tyrosine residues in the cytoplasmic region of GHR, which facilitates the recruitment of signaling molecules to the receptor. The primary signaling pathway for GH is the JAK-STAT (signal transduction and transcription activator) pathway; other signaling pathways include the MEK/MAPK, PI3K/AKT/mTOR, and PLC/DAG/PKC pathways.

The GHR Effector Reporter Cell model effectively mimics the in vivo GHR signaling pathway; the underlying mechanism is illustrated in the figure below.

Figure 1. Schematic diagram of the GHR Effector Reporter Cell model

Figure 2. Recombinant hGHR Effector Reporter Cell constitutively expressing GHR.

Figure 3. Dose Response of Human GH1 Protein in GHR Effector Reporter Cell.

Cell Resuscitation
1)Rapidly thaw the frozen cells in a 37 °C water bath for approximately 60 seconds. Once thawed (which may take slightly less or more than 60 seconds), immediately transfer the cell suspension from the cryovial into a 15 mL centrifuge tube containing 10 mL of pre-warmed  HEK293 Human GHR Effector Reporter Cell complete culture medium.
2)Centrifuge cells at 1000 rpm for 5 min to remove medium, then resuspend cells in 5 mL of pre-warmed complete medium.
3)Transfer the cell suspension into a T25 culture flask and incubate at 37 °C with 5% CO₂.
4)After approximately 24–36 hours, replace the medium or passage the cells to remove non-adherent dead cells.

Subculturing procedure
1)When the cell density reaches the appropriate confluency for passaging, wash the cells with PBS, then add 1 mL trypsin to detach the cells. When more than 80% of the cells detach upon gently tapping the culture flask, add complete culture medium to terminate digestion. Gently pipette to obtain a single-cell suspension, transfer to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes.

2)Discard supernatant after centrifugation. Resuspend cells in fresh medium to a single-cell suspension and transfer to a new culture flask for continued growth.

Cell Freezing
After trypsinization and centrifugation of cells from each T75 flask or 10 cm culture dish, discard the supernatant. Add 2 mL of cryopreservation medium (90% FBS + 10% DMSO), gently resuspend thoroughly, and aliquot into two cryovials. Immediately place the cryovials into a controlled-rate freezing container (e.g., Nalgene 5100-0001), fill with isopropanol to the indicated level, and store at −80 °C. After 24 hours, transfer the cryovials to liquid nitrogen for long-term storage.