Effects of Short-Term Mechanical Stretch on the Proliferation and Osteogenic Differentiation of Mesenchymal Stem Cell and TRPC1 Expression
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摘要
Objective Mechanical stretch regulates mesenchymal stem cell(MSC)function,which much more emphasis has been placed its prolonged effect on lineage differentiation,especially osteogenic differentiation.In contrast,there are few reports about its short term effect on MSC proliferation.In the present study,effects of short-term mechanical stretch on the proliferation and osteogenic differentiation of mesenchymal stem cell proliferation were investigated.In addition,the stretchinfluenced expression of transient receptor potential cation channel,subfamily C,member 1(TRPC1)was also investigated due to its mechanosensitivity and positive correlation with MSC proliferation.Methods MSCs,harvested from rat bone marrow,were seeded on collagen l-coated silicone chamber and exposed to mechanical stretch with various magnitude(0%,5%,10%and 15%)or various duration(2 h,6 h,12 h and 24 h).Cell proliferation was examined by cell counting kit-8(CCK-8)assay and cell cycle analysis.The gene and protein expression of two makers for osteogenic differentiation,collagen I and Cbfα1,and TRPC1 were determined by RT-PCR and western blotting,respectively.BMSC were harvested,and total RNA was isolated with Trizol reagent.A 2μg portion of total RNA was synthesized to cDNA according to the manufacturer s instructions.cDNA was used as a template for each PCR amplification.BMSC were solubilized in RIPA lysis buffer on ice for 30 min.Phenylmethane sulfonyl fluoride(PMSF)was added to avoid proteolysis.Equal portions of the cell lysates were separated on 10%sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and transferred to polyvinylidene fluoride(PVDF)membranes.The membranes were incubated with primary antibodies to TRPC1 and GAPDH at4℃overnight to identify the specific proteins.The PVDF membranes were washed with TBST three times and incubated with a horseradish peroxidase(HRP)-conjugated secondary antibody.Immunoreactive bands were visualized using an enhanced chemiluminescent(ECL)system.Results The OD value for the three stretch cases(5%-15%)was increased～1.4-fold compared with that for control(0%).There is no significantly difference among the three stretch cases.The percentage of cells for three stretch cases were more in the S phase but less in the G0/G1 phase compared to those for control.The cell cycle distribution still had no significant difference among the three stretch cases.In addition,the stretch application for 24 h didn't affect the gene or protein level for collagen I and Cbfα1 compared with those of control.Application of 10%stretch for 2 h didn't affect TRPC1 gene or protein expression,but that application for 6-24 h significantly up-regulated TRPCl gene and protein level.That increase exhibited a stretch duration-independent manner.Conclusions Short-term mechanical stretch promoted MSC proliferation in a magnitude-independent manner,whereas had no effect on its oesteogenic differentiation.Paralleled to which,TRPC1 was up-regulated by stretch,implying that TRPCl may be implicated in that proliferation courses.Future work is still needed to confirm whether TRPC1 participates in that stretch-induced MSC proliferation using RPC1 blockade or knockout.
Objective Mechanical stretch regulates mesenchymal stem cell(MSC)function,which much more emphasis has been placed its prolonged effect on lineage differentiation,especially osteogenic differentiation.In contrast,there are few reports about its short term effect on MSC proliferation.In the present study,effects of short-term mechanical stretch on the proliferation and osteogenic differentiation of mesenchymal stem cell proliferation were investigated.In addition,the stretchinfluenced expression of transient receptor potential cation channel,subfamily C,member 1(TRPC1)was also investigated due to its mechanosensitivity and positive correlation with MSC proliferation.Methods MSCs,harvested from rat bone marrow,were seeded on collagen l-coated silicone chamber and exposed to mechanical stretch with various magnitude(0%,5%,10%and 15%)or various duration(2 h,6 h,12 h and 24 h).Cell proliferation was examined by cell counting kit-8(CCK-8)assay and cell cycle analysis.The gene and protein expression of two makers for osteogenic differentiation,collagen I and Cbfα1,and TRPC1 were determined by RT-PCR and western blotting,respectively.BMSC were harvested,and total RNA was isolated with Trizol reagent.A 2μg portion of total RNA was synthesized to cDNA according to the manufacturer s instructions.cDNA was used as a template for each PCR amplification.BMSC were solubilized in RIPA lysis buffer on ice for 30 min.Phenylmethane sulfonyl fluoride(PMSF)was added to avoid proteolysis.Equal portions of the cell lysates were separated on 10%sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and transferred to polyvinylidene fluoride(PVDF)membranes.The membranes were incubated with primary antibodies to TRPC1 and GAPDH at4℃overnight to identify the specific proteins.The PVDF membranes were washed with TBST three times and incubated with a horseradish peroxidase(HRP)-conjugated secondary antibody.Immunoreactive bands were visualized using an enhanced chemiluminescent(ECL)system.Results The OD value for the three stretch cases(5%-15%)was increased～1.4-fold compared with that for control(0%).There is no significantly difference among the three stretch cases.The percentage of cells for three stretch cases were more in the S phase but less in the G0/G1 phase compared to those for control.The cell cycle distribution still had no significant difference among the three stretch cases.In addition,the stretch application for 24 h didn't affect the gene or protein level for collagen I and Cbfα1 compared with those of control.Application of 10%stretch for 2 h didn't affect TRPC1 gene or protein expression,but that application for 6-24 h significantly up-regulated TRPCl gene and protein level.That increase exhibited a stretch duration-independent manner.Conclusions Short-term mechanical stretch promoted MSC proliferation in a magnitude-independent manner,whereas had no effect on its oesteogenic differentiation.Paralleled to which,TRPC1 was up-regulated by stretch,implying that TRPCl may be implicated in that proliferation courses.Future work is still needed to confirm whether TRPC1 participates in that stretch-induced MSC proliferation using RPC1 blockade or knockout.
Objective Mechanical stretch regulates mesenchymal stem cell(MSC)function,which much more emphasis has been placed its prolonged effect on lineage differentiation,especially osteogenic differentiation.In contrast,there are few reports about its short term effect on MSC proliferation.In the present study,effects of short-term mechanical stretch on the proliferation and osteogenic differentiation of mesenchymal stem cell proliferation were investigated.In addition,the stretchinfluenced expression of transient receptor potential cation channel,subfamily C,member 1(TRPC1)was also investigated due to its mechanosensitivity and positive correlation with MSC proliferation.Methods MSCs,harvested from rat bone marrow,were seeded on collagen l-coated silicone chamber and exposed to mechanical stretch with various magnitude(0%,5%,10%and 15%)or various duration(2 h,6 h,12 h and 24 h).Cell proliferation was examined by cell counting kit-8(CCK-8)assay and cell cycle analysis.The gene and protein expression of two makers for osteogenic differentiation,collagen I and Cbfα1,and TRPC1 were determined by RT-PCR and western blotting,respectively.BMSC were harvested,and total RNA was isolated with Trizol reagent.A 2μg portion of total RNA was synthesized to cDNA according to the manufacturer s instructions.cDNA was used as a template for each PCR amplification.BMSC were solubilized in RIPA lysis buffer on ice for 30 min.Phenylmethane sulfonyl fluoride(PMSF)was added to avoid proteolysis.Equal portions of the cell lysates were separated on 10%sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and transferred to polyvinylidene fluoride(PVDF)membranes.The membranes were incubated with primary antibodies to TRPC1 and GAPDH at4℃overnight to identify the specific proteins.The PVDF membranes were washed with TBST three times and incubated with a horseradish peroxidase(HRP)-conjugated secondary antibody.Immunoreactive bands were visualized using an enhanced chemiluminescent(ECL)system.Results The OD value for the three stretch cases(5%-15%)was increased～1.4-fold compared with that for control(0%).There is no significantly difference among the three stretch cases.The percentage of cells for three stretch cases were more in the S phase but less in the G0/G1 phase compared to those for control.The cell cycle distribution still had no significant difference among the three stretch cases.In addition,the stretch application for 24 h didn't affect the gene or protein level for collagen I and Cbfα1 compared with those of control.Application of 10%stretch for 2 h didn't affect TRPC1 gene or protein expression,but that application for 6-24 h significantly up-regulated TRPCl gene and protein level.That increase exhibited a stretch duration-independent manner.Conclusions Short-term mechanical stretch promoted MSC proliferation in a magnitude-independent manner,whereas had no effect on its oesteogenic differentiation.Paralleled to which,TRPC1 was up-regulated by stretch,implying that TRPCl may be implicated in that proliferation courses.Future work is still needed to confirm whether TRPC1 participates in that stretch-induced MSC proliferation using RPC1 blockade or knockout.
引文
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