| Objective To study the relationship among the transformation of phenotype and the proliferation of vascular smooth muscle cells (VSMCs) and cytoskeleton of VSMCs, providing theoretical evidence for the study of the role of cytoskeletal proteins in the reconstructive diseases. VSMCs of rat were cultured in different conditions(serum cultured and serum-free cultured)and at different passages, change of expression of cytoskeletal proteins was observed in the proccess of the transform of phenotype and vascular smooth muscle cell proliferation .Methods The VSMCs were cultured in vitro through the explant-attached method. The 2th,4th,6th passage cells were used in the experiment. cells per passage were cultured in DMEM culture medium with 0.5% newborn calf blood serum to keep pace with each other before expriment,and then were divided into serum culture group and serum-free culture group.serum culture group: cells per passage were kept pace with each other, and then continued to incubate for 72 h in culture flask containing 12% new-born calf serum(NCS) DMEM culture medium, and accomplished samples. Serum-free culture group: cells per passage were kept pace with each other, and then continued to incubate for 72h in culture flask containing 12% new-born calf serum(NCS) DMEM culture medium( the cells were in synthesis stage),and then,the cells were cultured with serum-free DMEM culture medium, and accomplished samples. The microcosmic structure of VSMCs was observed through transmission electron microscope(TEM); The cytoskeleton was also observed through flourescence-immu nohistochemical staining; The proliferative cells were labeled by 5-bromodeoxyuridine (5-BrDU) with immunocytochemical method;and the mRNA expression of smooth muscle 22 alpha (SM22α) was determined and detected by reverse transcription- polymerase chain reaction method (RT-PCR).Immunocytochemical stainingTwo slice of each group were taken for immunocytochemical 5-BrDU. The procedure was done as follow: The sections were treated with 0.3%H2O2 methanol to block endogenous peroxidase,then put into 2N HCl at 37℃for 1 h,and then incubated in normal sheep serum for 40 min,the mouse-anti-BrDU(1:400) antibody over night at 4℃,with biotinglated sheep antimouse IgG for 30 min at 37℃,and with SABC for 30 min at 37℃,lastly incubated with DAB at RT for 1 min and observed under microscope until desired staining intensity has appeared, then being dehydrated, covered and dried naturally.Rinse the section in 0.1M PBS for 15min×3 at RT between every two of the above steps.The positive cells of each view field of each section were counted and expressed as X±s . t-test was used for testing the significant probability.Flourescence -immunohistochemical stainingTwo slice of each group were taken for immunohistochemical staining. The precedure was done as follow: The sections were treated with 0.3% Triton-100 to block.. Incubated in normal sheep serum for 40 min, ,the frist antibody over night at 4℃,then the second antibody FITC-IgG 37℃60 min. Rinse the section in 0.1M PBS for 15 min×3 at RT between every two of the above steps.Then covered with paraffin liquid, and observed under fluorescence microscope. Each group was expressed as AO( Average optical).t-test was used for testing the significant probability.RT-PCR of SM22αmRNATotal mRNA was extracted from the cultured VSMCs according to one-step method.The frist atrand of cDNA was synthedized by reverse transcription in 50ul reactive system (42℃50min, 70℃15min).The product of reverse transcription was subjected to PCR analysis(35 cycles,94℃denaturing for 50s, 57℃annealing for 50s, 72℃expanding for 1min)in a 50ul reactive system.The PCR reaction products were subjected to 1.7% agarose gel electrophoresis, stained with ethidium bromide and the intensity of the specific bands was qualified by image analysis(n=3,independent expriments).Results1.The change of VSMC proliferation Anti-Bromodeoxyuridine(BrDU) immuno- histochemical staining: most of proliferous cells were labeled by 5-BrDU in all groups under the microscope of the cultured VSMC in vitro.But with the increase of passage generation number of cells labled by BrDU increased,and reaching the maximum at the 6 passages(p6). Number of cells labled decreased,the serum-free cultured groups competed with the serum cultured groups.2.The change of expression of SM22αThe result of RT-PCR: the mRNA ex- pression of SM22αdecrease gradually with the increase of passage in cultured VSMC in vitro,and at 6 passage the mRNA expression of SM22αwas hardly detected , indicated that the phenotype of VSMC can transfer from contractile to synthesis with the increase of passage in cultured VSMC in vitro. The mRNA expression of SM22αcannot be detected in serum groups after being cultured with serum medium, but when being cultured with serum-free medium, the mRNA expression of SM22αupregulated quikly.3.The change of expression of cytoskeletal proteins Flourescence-Immunohisto- chemical staining: With the increase of cell passage the expression of SMα-actin decreased cultured with serum medium, when serum-free cultured the expression of SMα-actin increased, With the increase of cell passage the expression of SMα-actin decreased and at high passage(6th passage), there was part of cell expressed. With the increase of cell passage the expression ofβ–Tublin and Desmin decreased cultured with serum medium, when serum-free cultured up-regulation of the expression ofβ–Tublin and Desmin but not obvious and at 6 passages failed to express. After being cultured with serum-free medium, cytoskeletal proteins were expressed increasingly, but at high passage (p6) serum-free cultured cannot reverse the expression of cytoskeletal associated proteins.4. The change of ultrastructure Transmission electron microscope (TEM):in high passage and serum cultured groups, increased endoplasmic reticulums(ER) and abundant secretory vesicles were found in cytoplasm of VSMCs; and in low passage and serum-free cultured groups myofilament increased while cellular organs decreased in cytoplasm of VSMCs.Conclusions1.With the increase of cell passage, the mRNA expression of SM22αand the expression of SM-a-actin,β–Tubulin and Desmin decreased cultured with serum medium, and the ability of cell proliferation enhanced. But at 6 passages the mRNA of SM22α,β–Tubulin and Desmin failed to express. Suggestion: the 6th passage cell is patho- secretion type proliferated cell.2. Serum-free cultured can reverse the expression of mRNA of SM22α, SM-a-actin,β–Tubulin and Desmin, but at the 6th passage cell serum-free cultured can reverse.3. At high passage (p6) serum-free cultured cannot reverse the expression ofβ–Tubulin and Desmin, suggestion:β–Tubulin and Desmin can be regarded as the marker of the patho-secretion type proliferated VSMC.4.There was internal relationship among the transformation of phenotype and the proliferation of VSMC and cytoskeleton of VSMC.They play important role in the maintaining morphous and construction of VSMC, contractile function and vascular remodeling.But the causal relation among them need further study. |
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