pHGF Porcine

What is pHGF and how does it differ from other growth factors?

pHGF (porcine Hepatocyte Growth Factor) is a multifunctional growth factor extracted from pig liver that regulates both cell growth and cell motility. It consists of a series of polypeptides with molecular weights less than 10,000 Da . Unlike many other growth factors, pHGF exerts a strong mitogenic effect specifically on hepatocytes and primary epithelial cells while having the unique ability to synergize with Interleukin-3 and GM-CSF to stimulate colony formation of hematopoietic progenitor cells . This dual functionality in both liver-specific regeneration and broader hematopoietic modulation distinguishes pHGF from other more narrowly targeted growth factors in research applications.

What are the physical and biochemical properties of commercially available pHGF?

Commercially available pHGF typically presents as a sterile filtered white lyophilized (freeze-dried) powder without additives . The purity typically exceeds 98.0% as determined by both RP-HPLC and SDS-PAGE analysis . When properly extracted and purified, pHGF retains stability at room temperature for approximately 3 weeks, though long-term storage requires temperatures below -18°C . Upon reconstitution, the protein remains stable at 4°C for 2-7 days, but requires carrier proteins such as 0.1% HSA or BSA for extended stability to prevent degradation during freeze-thaw cycles .

What synonyms and alternative nomenclature exist for pHGF in scientific literature?

Researchers should be aware of several synonyms when conducting literature searches on pHGF. The compound is also known as Scatter Factor (SF), Hepatopoietin (HPTA), HGF, HGFB, F-TCF, and pHGF . The multiple nomenclatures reflect the historical discovery path where different research groups identified the same compound independently based on different observed biological activities before unifying these under the current understanding of HGF's multifunctional nature.

What are the established protocols for extracting pHGF from porcine liver tissue?

The established extraction protocol for pHGF begins with freshly harvested pig liver tissue. The extraction process typically employs proprietary chromatographic techniques that separate the desired polypeptides based on molecular weight and chemical properties . While commercial suppliers do not disclose their precise methodologies, research indicates that the process generally involves tissue homogenization, initial crude protein extraction, followed by sequential chromatographic purification steps. For laboratory-scale extraction, researchers should implement tissue homogenization in appropriate buffer systems followed by ammonium sulfate precipitation, then employ a combination of ion-exchange chromatography and gel filtration to isolate the active polypeptide fractions with molecular weights below 10,000 Da .

What purification techniques yield the highest biological activity for pHGF?

The highest biological activity for pHGF is achieved through a multi-step purification process. Initial purification typically employs DEAE-Sephadex A25 ion exchange chromatography to separate active fractions . Subsequent purification uses size exclusion chromatography (such as Superdex 75) followed by reversed-phase chromatography using C18 columns for final purification . This sequential approach has been demonstrated to produce fractions with 95.8% relative purity and specific molecular weights (e.g., 4020.6 Da for Subfraction 4) as determined by HPLC and MALDI-TOF-MS mass spectrometry . Proper fraction collection during each purification step is critical, as different fractions may exhibit distinct biological activities - some promoting hepatocyte growth while others inhibiting neoplasm cell proliferation.

How can researchers validate the purity and identity of extracted pHGF?

Validation of pHGF purity and identity requires multiple analytical approaches. Industry standards include:

• RP-HPLC analysis demonstrating a single predominant peak with purity exceeding 98%

• SDS-PAGE confirmation showing appropriate molecular weight bands

• Mass spectrometry (particularly MALDI-TOF-MS) to determine precise molecular weights of purified fractions

• Functional bioassays measuring the mitogenic activity on primary rat hepatocytes

• Inhibition assays using hepatoma cell lines like BEL-7402 to confirm antiproliferative properties

The most rigorous validation combines both physicochemical characterization and biological activity assessment, as certain fractions of pHGF display distinct biological effects that must be evaluated independently.

What are the documented biological activities of different pHGF fractions?

Research has revealed distinct biological activities across different pHGF fractions. When purified through DEAE-Sephadex A25 chromatography, six major fractions are typically obtained with divergent activities:

This functional dichotomy demonstrates that pHGF contains at least two different bioactive component classes: those promoting normal hepatocyte growth (fractions 1, 2, and 5) and those inhibiting hepatoma cell proliferation (fractions 3 and 4) . This complexity highlights the importance of fraction-specific research approaches when working with pHGF.

What molecular pathways does pHGF activate in hepatocytes?

pHGF activates multiple molecular pathways in hepatocytes through binding to its receptor c-Met, a receptor tyrosine kinase. The primary signaling cascades include:

• The PI3K/Akt pathway, promoting cell survival and anti-apoptotic effects

• The Ras/MAPK pathway, stimulating cellular proliferation

• The STAT3 pathway, influencing gene expression related to regeneration

• The Wnt/β-catenin pathway, regulating hepatocyte proliferation

The Subfraction 4 (S4) of pHGF has been shown to influence the expression of specific oncogenes including p53, Bcl-2, Fas, and c-myc in neoplasm cell lines, suggesting modulation of apoptotic pathways . These interactions explain how pHGF can simultaneously promote normal hepatocyte function while exhibiting inhibitory effects on neoplastic cells.

How does pHGF induce apoptosis in neoplasm cell lines?

pHGF, particularly its Subfraction 4 (S4), induces apoptosis in neoplasm cell lines through multiple mechanisms. Research using the in situ cell death detection kit (TUNEL assay) has demonstrated that S4 induces characteristic apoptotic changes including DNA fragmentation and nuclear condensation . The apoptosis-inducing effect is concentration-dependent and time-dependent, with 10-30% of cells showing positive staining after treatment.

Mechanistically, S4 modulates the expression of key apoptosis regulators:

• Upregulation of pro-apoptotic factors (p53 and Fas)

• Downregulation of anti-apoptotic proteins (Bcl-2)

• Alteration of c-myc expression patterns

The differential response between normal hepatocytes and neoplasm cells suggests that S4 selectively targets cancer-specific vulnerabilities in apoptotic pathways, making it a valuable research tool for understanding hepatocellular carcinoma biology and potential therapeutic approaches .

What are the optimal reconstitution and storage conditions for maintaining pHGF activity?

The optimal reconstitution protocol for maintaining pHGF activity involves dissolving the lyophilized powder in sterile 18 MΩ-cm H₂O at a concentration not less than 100 μg/ml . This stock solution can be further diluted in other aqueous buffers depending on experimental requirements. For storage:

• Lyophilized pHGF: Store desiccated below -18°C (stable at room temperature for up to 3 weeks)

• Reconstituted pHGF: Store at 4°C if using within 2-7 days

• Long-term storage: Keep below -18°C with the addition of carrier protein (0.1% HSA or BSA)

To maintain maximum activity, researchers must strictly minimize freeze-thaw cycles as these significantly reduce biological potency . Aliquoting the reconstituted protein into single-use volumes is recommended for experiments requiring repeated access to the reagent.

What cell culture models are most appropriate for studying pHGF effects?

The most appropriate cell culture models for studying pHGF effects depend on the specific research questions but generally include:

• Primary rat hepatocytes - Ideal for studying normal mitogenic and regenerative effects

  • Protocol: Isolation from male Sprague Dawley rats (250g ± 30g) via liver perfusion with 0.25% trypsin, followed by gentle homogenization and centrifugation at 1000 × g for 10 minutes

  • Culture conditions: RPMI-1640 medium with 10% FCS, 100 IU/mL penicillin and 100 μg/mL streptomycin at 37°C, 5% CO₂

• Hepatoma cell lines (e.g., BEL-7402) - Appropriate for studying anti-proliferative and apoptotic effects

  • Culture conditions: DME/F12 or RPMI-1640 medium containing 10% FCS at 37°C, 5% CO₂

  • Seeding density: 2.5-5.0 × 10⁴ cells/cm³ for optimal response assessment

• Co-culture systems combining primary hepatocytes with non-parenchymal cells - For studying complex liver microenvironment interactions

The choice between these models should reflect the specific pHGF fraction being studied and the biological effect under investigation.

What are the recommended assays for measuring pHGF activity in vitro?

Several complementary assays are recommended for comprehensive measurement of pHGF activity in vitro:

• Proliferation Assays:

  • MTT assay: Adding MTT solution (1.5 g/L in PBS) to treated cells, followed by DMSO solubilization and spectrophotometric measurement at 570nm (reference: 630nm)

  • BrdU incorporation assay: For direct measurement of DNA synthesis

  • Cell counting assays: For direct quantification of proliferation effects

• Apoptosis Assays:

  • TUNEL assay: Using in situ cell death detection kits to identify DNA fragmentation

  • Annexin V/PI staining: For flow cytometry detection of early and late apoptotic cells

  • Immunohistochemical staining: For detection of apoptosis-related proteins (p53, Bcl-2, Fas, c-myc)

• Functional Assays:

  • Scratch wound healing assay: For measuring motogenic effects

  • Transwell migration assay: For quantifying cell motility responses

  • Colony formation assay: For hematopoietic progenitor cell studies when combined with IL-3 and GM-CSF

The integration of multiple assay types provides the most comprehensive characterization of pHGF activities across different experimental models.

How do the various fractions of pHGF differ in their molecular mechanisms?

The various fractions of pHGF exhibit distinct molecular mechanisms that explain their divergent biological effects. Research has identified at least six major fractions with different activities:

• Growth-promoting fractions (1, 2, and 5) enhance hepatocyte DNA synthesis and metabolic activity through:

  • Activation of the MAPK/ERK pathway leading to increased cyclin D expression

  • Enhanced PI3K/Akt signaling promoting cell survival

  • Mobilization of intracellular calcium stores

• Antiproliferative fractions (3 and 4) inhibit neoplasm cell growth through:

  • Upregulation of p53 and Fas expression inducing apoptotic pathways

  • Downregulation of anti-apoptotic Bcl-2 proteins

  • Modulation of c-myc expression affecting cell cycle progression

These mechanistic differences are not merely quantitative variations but represent qualitatively distinct biological activities, suggesting that pHGF contains multiple bioactive peptides with different target specificities and signaling outcomes.

What challenges exist in reproducibly studying pHGF across different experimental systems?

Several challenges complicate the reproducible study of pHGF across different experimental systems:

• Source variability: Differences in pig liver sources (age, breed, diet) may affect the composition and activity of extracted pHGF.

• Extraction and purification inconsistencies: Subtle variations in chromatographic conditions can alter the ratio of bioactive fractions in the final preparation.

• Fraction heterogeneity: The multiple peptide components of pHGF with molecular weights ranging from 0.40–323.56 kDa create inherent complexity .

• Receptor variations: Species-specific differences in c-Met receptor structure and downstream signaling pathways can affect responses to pHGF.

• Methodology standardization: Differences in cell culture conditions, assay endpoints, and data analysis approaches complicate cross-laboratory comparisons.

Researchers can address these challenges by implementing standardized procurement, extraction, and characterization protocols; working with well-defined fractions rather than crude extracts; and including appropriate positive and negative controls in all experimental systems.

How does pHGF compare to enzymatically produced porcine placenta hydrolysates in research applications?

pHGF and enzymatically produced porcine placenta hydrolysates (PPH) represent distinct but complementary research tools with different properties and applications:

While pHGF has more specific effects on hepatic tissue and defined cell signaling pathways, PPH demonstrates broader antioxidant and antibacterial properties that vary with the enzyme used for hydrolysis . PPH produced with Alcalase shows superior reducing capacity and metal chelating ability, while Flavourzyme and Papain-derived PPH demonstrate higher DPPH- and ABTS- + inhibitory activities . This makes PPH potentially valuable for different research applications, particularly in functional food ingredient development and antimicrobial studies.

What are the future research directions for pHGF in liver disease and regeneration studies?

Future research directions for pHGF in liver disease and regeneration studies should focus on several promising areas:

• Mechanistic delineation: Further characterization of the specific signaling pathways activated by different pHGF fractions will enhance understanding of their selective effects on normal versus neoplastic cells.

• Therapeutic development: Investigation of pHGF-derived peptides as potential therapeutic agents for acute liver failure and chronic liver diseases, building on existing evidence of clinical efficacy .

• Synergistic approaches: Exploration of combined treatments integrating pHGF with other growth factors or conventional therapies to enhance hepatic regeneration.

• Biomarker identification: Development of pHGF-response biomarkers to predict and monitor therapeutic efficacy in liver disease models.

• Delivery optimization: Creation of targeted delivery systems to enhance pHGF stability in vivo and improve hepatocyte-specific targeting.

These research directions could significantly advance our understanding of liver regeneration mechanisms and potentially lead to novel therapeutic approaches for challenging liver conditions.

What methodological improvements would enhance the isolation and characterization of bioactive pHGF components?

Several methodological improvements would enhance the isolation and characterization of bioactive pHGF components:

• Advanced chromatographic techniques: Implementation of multi-dimensional chromatography and affinity-based separations to achieve higher resolution between closely related peptide fractions.

• Proteomic approaches: Application of high-throughput proteomics to comprehensively identify and characterize all bioactive peptides within pHGF preparations.

• Structural biology tools: Utilization of X-ray crystallography and NMR spectroscopy to determine the three-dimensional structures of key pHGF peptides and their receptor interactions.

• Synthetic peptide validation: Development of synthetic peptide libraries based on identified pHGF sequences to confirm structure-activity relationships.

• Standardized bioassay panels: Creation of consistent bioassay platforms that can be applied across laboratories to enable direct comparison of results.