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Hepatocyte growth factor incorporated chitosan nanoparticles augment the differentiation of stem cell into hepatocytes for the recovery of liver cirrhosis in mice.

Pulavendran S, Rose C, Mandal AB - J Nanobiotechnology (2011)

Bottom Line: Immunostaining for alpha smooth muscle actin (αSMA) and type I collagen showed decreased expression in the MSC+HGF-CNP treatment.These results indicated that HGF-CNP enhanced the differentiation of stem cells into hepatocytes and supported the reversal of fibrolysis of extracellular matrix (ECM).Biodegradable biopolymeric nanoparticles were prepared with the pleotrophic protein molecule and it worked well for the differentiation of stem cells, especially mesenchymal phenotypic cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Central Leather Research Institute, Adyar, Chennai-600020, India.

ABSTRACT

Background: Short half-life and low levels of growth factors in the niche of injured microenvironment necessitates the exogenous and sustainable delivery of growth factors along with stem cells to augment the regeneration of injured tissues.

Methods: Here, recombinant human hepatocyte growth factor (HGF) was incorporated into chitosan nanoparticles (CNP) by ionic gelation method and studied for its morphological and physiological characteristics. Cirrhotic mice received either hematopoietic stem cells (HSC) or mesenchymal stemcells (MSC) with or without HGF incorporated chitosan nanoparticles (HGF-CNP) and saline as control. Biochemical, histological, immunostaining and gene expression assays were carried out using serum and liver tissue samples. One way analysis of variance was used for statics application

Results: Serum levels of selected liver protein and enzymes were significantly increased in the combination of MSC and HGF-CNP (MSC+HGF-CNP) treated group. Immunopositive staining for albumin (Alb) and cytokeratin 18 (CK18), and reverse transcription-polymerase chain reaction (RT-PCR) for Alb, alpha fetoprotein (AFP), CK18, cytokeratin 19 (CK19) ascertained that MSC-HGF-CNP treatment could be an effective combination to repopulate liver parenchymal cells in the liver cirrhosis. Zymogram and western blotting for matrix metalloproteinases 2 and 9 (MMP2 and MMP9) revealed that MMP2 actively involved in the fibrolysis of cirrhotic tissue. Immunostaining for alpha smooth muscle actin (αSMA) and type I collagen showed decreased expression in the MSC+HGF-CNP treatment. These results indicated that HGF-CNP enhanced the differentiation of stem cells into hepatocytes and supported the reversal of fibrolysis of extracellular matrix (ECM).

Conclusion: Bone marrow stem cells were isolated, characterized and transplanted in mice model. Biodegradable biopolymeric nanoparticles were prepared with the pleotrophic protein molecule and it worked well for the differentiation of stem cells, especially mesenchymal phenotypic cells. Transplantation of bone marrow MSC in combination with HGF-CNP could be an ideal approach for the treatment of liver cirrhosis.

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Expression of MMPs: (A) The activity of MMPs was analysed by zymography. The liver homogenate was resolved in 10% SDS-PAGE containing 0.1% gelatin without reducing agent. The gel was then stained and washed. MMPs were well expressed in MSC and MSC+HGF-CNP groups; particularly, MMP2 was increasingly expressed in these groups compared to control, HSC and HSC+HGF-CNP treated groups. This indicated the activation of MMPs by MSC. (B) Expression of MMPs was analyzed by western blotting. Electrophoretically resolved proteins were transferred and then immunostained against mouse MMP2 and MMP9. Control group 1, HSC 2, MSC 3, HSC+HGF-CNP 4, MSC+HGF-CNP 5 treated mice showed increased expression of MMPs, particularly MMP2. Semiquantitative analysis of MMP2 and MMP9 was carried out and significant level of expression of MMP2 and MMP9 was found in MSC and MSC+HGF-CNP treated groups (P < 0.05) compared to control, HSC and HSC+HGF-CNP treated groups. Significant level of expression of MMP2 appeared compared to MMP9. It explored the reason for the decrease of ECM.
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Figure 6: Expression of MMPs: (A) The activity of MMPs was analysed by zymography. The liver homogenate was resolved in 10% SDS-PAGE containing 0.1% gelatin without reducing agent. The gel was then stained and washed. MMPs were well expressed in MSC and MSC+HGF-CNP groups; particularly, MMP2 was increasingly expressed in these groups compared to control, HSC and HSC+HGF-CNP treated groups. This indicated the activation of MMPs by MSC. (B) Expression of MMPs was analyzed by western blotting. Electrophoretically resolved proteins were transferred and then immunostained against mouse MMP2 and MMP9. Control group 1, HSC 2, MSC 3, HSC+HGF-CNP 4, MSC+HGF-CNP 5 treated mice showed increased expression of MMPs, particularly MMP2. Semiquantitative analysis of MMP2 and MMP9 was carried out and significant level of expression of MMP2 and MMP9 was found in MSC and MSC+HGF-CNP treated groups (P < 0.05) compared to control, HSC and HSC+HGF-CNP treated groups. Significant level of expression of MMP2 appeared compared to MMP9. It explored the reason for the decrease of ECM.

Mentions: The fibrolytic activity of MMPs and their expression are depicted in Figure 6A and 6B respectively. The zymography assay revealed a band of gelatin degradation at 68 kDa, representing active MMP-2 and another band at 97 kDA representing MMP9 (Figure 6A). The MSC groups, particularly MSC+HGF-CNP, showed increased expression of MMPs, more especially MMP2. The overall data on the quantitative analysis of MMP9 (Figure 6C) and MMP2 (Figure 6D) indicated that MMP9 was relatively less expressed and active compared to MMP2.


Hepatocyte growth factor incorporated chitosan nanoparticles augment the differentiation of stem cell into hepatocytes for the recovery of liver cirrhosis in mice.

Pulavendran S, Rose C, Mandal AB - J Nanobiotechnology (2011)

Expression of MMPs: (A) The activity of MMPs was analysed by zymography. The liver homogenate was resolved in 10% SDS-PAGE containing 0.1% gelatin without reducing agent. The gel was then stained and washed. MMPs were well expressed in MSC and MSC+HGF-CNP groups; particularly, MMP2 was increasingly expressed in these groups compared to control, HSC and HSC+HGF-CNP treated groups. This indicated the activation of MMPs by MSC. (B) Expression of MMPs was analyzed by western blotting. Electrophoretically resolved proteins were transferred and then immunostained against mouse MMP2 and MMP9. Control group 1, HSC 2, MSC 3, HSC+HGF-CNP 4, MSC+HGF-CNP 5 treated mice showed increased expression of MMPs, particularly MMP2. Semiquantitative analysis of MMP2 and MMP9 was carried out and significant level of expression of MMP2 and MMP9 was found in MSC and MSC+HGF-CNP treated groups (P < 0.05) compared to control, HSC and HSC+HGF-CNP treated groups. Significant level of expression of MMP2 appeared compared to MMP9. It explored the reason for the decrease of ECM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3108285&req=5

Figure 6: Expression of MMPs: (A) The activity of MMPs was analysed by zymography. The liver homogenate was resolved in 10% SDS-PAGE containing 0.1% gelatin without reducing agent. The gel was then stained and washed. MMPs were well expressed in MSC and MSC+HGF-CNP groups; particularly, MMP2 was increasingly expressed in these groups compared to control, HSC and HSC+HGF-CNP treated groups. This indicated the activation of MMPs by MSC. (B) Expression of MMPs was analyzed by western blotting. Electrophoretically resolved proteins were transferred and then immunostained against mouse MMP2 and MMP9. Control group 1, HSC 2, MSC 3, HSC+HGF-CNP 4, MSC+HGF-CNP 5 treated mice showed increased expression of MMPs, particularly MMP2. Semiquantitative analysis of MMP2 and MMP9 was carried out and significant level of expression of MMP2 and MMP9 was found in MSC and MSC+HGF-CNP treated groups (P < 0.05) compared to control, HSC and HSC+HGF-CNP treated groups. Significant level of expression of MMP2 appeared compared to MMP9. It explored the reason for the decrease of ECM.
Mentions: The fibrolytic activity of MMPs and their expression are depicted in Figure 6A and 6B respectively. The zymography assay revealed a band of gelatin degradation at 68 kDa, representing active MMP-2 and another band at 97 kDA representing MMP9 (Figure 6A). The MSC groups, particularly MSC+HGF-CNP, showed increased expression of MMPs, more especially MMP2. The overall data on the quantitative analysis of MMP9 (Figure 6C) and MMP2 (Figure 6D) indicated that MMP9 was relatively less expressed and active compared to MMP2.

Bottom Line: Immunostaining for alpha smooth muscle actin (αSMA) and type I collagen showed decreased expression in the MSC+HGF-CNP treatment.These results indicated that HGF-CNP enhanced the differentiation of stem cells into hepatocytes and supported the reversal of fibrolysis of extracellular matrix (ECM).Biodegradable biopolymeric nanoparticles were prepared with the pleotrophic protein molecule and it worked well for the differentiation of stem cells, especially mesenchymal phenotypic cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Central Leather Research Institute, Adyar, Chennai-600020, India.

ABSTRACT

Background: Short half-life and low levels of growth factors in the niche of injured microenvironment necessitates the exogenous and sustainable delivery of growth factors along with stem cells to augment the regeneration of injured tissues.

Methods: Here, recombinant human hepatocyte growth factor (HGF) was incorporated into chitosan nanoparticles (CNP) by ionic gelation method and studied for its morphological and physiological characteristics. Cirrhotic mice received either hematopoietic stem cells (HSC) or mesenchymal stemcells (MSC) with or without HGF incorporated chitosan nanoparticles (HGF-CNP) and saline as control. Biochemical, histological, immunostaining and gene expression assays were carried out using serum and liver tissue samples. One way analysis of variance was used for statics application

Results: Serum levels of selected liver protein and enzymes were significantly increased in the combination of MSC and HGF-CNP (MSC+HGF-CNP) treated group. Immunopositive staining for albumin (Alb) and cytokeratin 18 (CK18), and reverse transcription-polymerase chain reaction (RT-PCR) for Alb, alpha fetoprotein (AFP), CK18, cytokeratin 19 (CK19) ascertained that MSC-HGF-CNP treatment could be an effective combination to repopulate liver parenchymal cells in the liver cirrhosis. Zymogram and western blotting for matrix metalloproteinases 2 and 9 (MMP2 and MMP9) revealed that MMP2 actively involved in the fibrolysis of cirrhotic tissue. Immunostaining for alpha smooth muscle actin (αSMA) and type I collagen showed decreased expression in the MSC+HGF-CNP treatment. These results indicated that HGF-CNP enhanced the differentiation of stem cells into hepatocytes and supported the reversal of fibrolysis of extracellular matrix (ECM).

Conclusion: Bone marrow stem cells were isolated, characterized and transplanted in mice model. Biodegradable biopolymeric nanoparticles were prepared with the pleotrophic protein molecule and it worked well for the differentiation of stem cells, especially mesenchymal phenotypic cells. Transplantation of bone marrow MSC in combination with HGF-CNP could be an ideal approach for the treatment of liver cirrhosis.

Show MeSH
Related in: MedlinePlus