Study Of The Mechanism Of Organic Model Of VSMCs Proliferation

Objective Study the developing mechanism of the organic model of VSMCs proliferation cultured in vitro made in our laboratory,providing a theoretical evidence for the use of this model.Methods The aorta segments of 1.5cm length obtained from rats were divided into the destroyed endothelium,destroyed endothelium plus BQ123,without destroyed and without destroyed plus BQ123 four experimental groups.Each of them was subdivided into cultured with serum and serum-free DMEM two groups,then cultured in vitro for 10 days;half of their supernatant was changed every two days.There were ten vascular segments in each group,half of which were labled by 5-Brdu( 8×10-4mol/l ) from the 5th day of their culture,then fixed in 4% paraformaldehyde,for HE and Immunohistochemical staining;the other half were cultured for RT-PCR.Their supernatant was gathered and stored at -80℃,for the examination of ET-1.The vascular segments without being cultured in vitro was taken as the control group.Immunohistochemical stainingFor immunolabelling of 5-Brdu,two sections of each vascular segments were taken.The procedure was done as follows:The sections were treated with 0.3%H2O2 methanol to block endogenous peroxidase,then put into 2N HCl at 37℃for 1h,Incubated in normal sheep serum for 20 min;with mouse- anti-Brdu(1:500) 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 3-5 min and observed under microscope until desired staining intensity has developed,then being dehydrated,covered and dried naturally.Rinse the sections in 0.1M PBS for 15 min*3 at RT between each two of the above steps.The positive cells of each view field of each section were counted and expressed as X±.T-test was used for testing the significant probability.Detection of ET-1 in the supernatantThe content of ET-1 in the medium of each group collected above was measured following the manual in the kit of 125I-endothelin Radioimmunoassay Kit,using SN695B type intelligent radioimmunoassay apparatus.(n=3 independent experiments).The result was expressed as X±, T-test was used for testing the significant probability.RT-PCR of HRG-1and SM22αmRNA in cultured aorta segmentTotal RNA was extracted from the cultured aorta segments according to one-step method (Trizol agent).The first strand of cDNA was synthesized by reverse transcription in a 20μl reactive system(42℃50 min,70℃15 min). The product of reverse transcription was subjected to PCR analysis (32 cycles,94℃denaturing for 50s,56℃annealing for 50s,72℃expanding for 1 min) in a 50μl 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 experiments)Statistical analysisStatistical analysis was performed using an unpaired t-test.Values are cited as mean±standard deviation(S.D.).P<0.05 is taken as a significant probability.Results1.The proliferation of VSMC:(1)HE staining:The destroyed aortas cultured in vitro for 10 days have various proliferation of VSMCs and indiscriminate muscle fibers.(2)Immunahistochemical staining:The aortas cultured in vitro for 10 days developed various degree of SMC proliferation,but the numbers of cells labled by Brdu was visibly more in the destroyed and serum cultured groups competed with the intact and serum-free groups.BQ123 can inhibitor this proliferation obviously.no labeled cells were seen on control groups.(3)RT-PCR examination demostrated that the mRNA expressions of HRG-1–a negtive regulatory gene of VSMC proliferation -were all detected on normal aortas,but decreased normally when the aortas were destroyed and cultured in vitro for 10 days,especially in the serum groups.However they increased after being added into BQ123.This implyed that serum culture and ET-1 secretion play an important role in the proliferation of VSMC of this destroyed aortas cultured in vitro.2.The transform of phenotype of VSMC:The mRNA expressions of SM22α-a notate gene of contractile VSM-were all detected on normal aortas,but decreased normally when the aortas were destroyed and cultured in vitro for 10 days,especially in the serum groups,whose SM22αmRNA could not be detected by RT-PCR.Here,BQ123 can increased their expressions.This demonstrated that serum culture and the secretion of ET-1 can produce the transform of phenotype from contractile to synthesize type.3.The content of ET-1 in the supernatant: The content of ET-1 increasedsignificantly when the endothelium of the aortas was destroyed and was even more when cultured with serum(P<0.01),but decreased when cultured with BQ123.This showed that the secretion of ET-1 could not only be accelerated by destroying the vascular endothelium and serum culture, but inhibited by BQ123.Conclusion The results demonstrated that the culturing of rat aorta segments in vitro can induce the proliferation of SMC and the transform of phenotype from contractile to synthesize type. ET-1 and serum is the main factors in the proliferation of VSMC here. This organic model will supply a good experimental platform for us to research the mechanism of proliferous vascular diseases as well as its prevention and treatment.