| Percutaneous transluminal coronary angioplasty(PTCA), an effective method to treating obstructed atherosclerotic vessels, have been widely used. However, its overall benefits are interrupted by inducing local arterial restenosis. Intimal hyperplasia resulted from a series of ectopic expression of gene.Krüppel-like factors(KLFs) belong to the transcription factors family containing zinc-finger, mainly involved in regulating cell growth, differentiation, proliferation and apoptosis process. It is reported that KLF4 inhibits cell proliferation. In present study, KLF4 adenovirus expression system was established to investigate the effect of KLF4 on neointimal hyperplasia after balloon injury.Methods:1 Construction of the recombinant adenovirus pAd-KLF4Recombinant shuttle plasmid (pTOPO-KLF4) was constructed, and then homologous recombination was performed between attL1-attL2 sequence in TOPO-KLF4 and attR1-attR2 sequence in pAd. The recombinant adenovirus named as pAd-KLF4. The pAd-KLF4 and pAd linearized by PacI was transfected into 293 cells, and packaged , respectively.2 Establishment of the animal model SD rats were anesthetized with urethane. The thoracic- abdominal aorta and carotid artery were de-endothelialized. Briefly, the catheter was pushed from left carotid artery into the aorta down to the level of the renal arteries three times with a 2F Fogarty catheter inserted through the external carotid artery. 20 ul of pAd-KLF4 suspension was injected into the carotid artery after blockade of blood stream, which maintained for 20 min, and then recoveried the blood stream. SD rats were randomly divided into four groups, including sham group, injured group, pAd group and pAd-KLF4 group.3 Preparation of experimental specimenAll the rats were killed at 14 days after de-endothelium. The aortas were separated for preparation of sections, and RNA extract. Sections were stained with hematoxylin and eosin to detect the neointimal hyperplasia. The microscopical pictures were analyzed by using a computer assisted image analyzer, and the thickness of neointima was measured. The effect of KLF4 on neointimal hyperplasia and the expression of c-Jun, PCNA and SM22αwere analyzed by immuno- histochemistry and RT-PCR.Results:1 Construction and identification of pAd-KLF4DNA sequence analysis showed that KLF4 cDNA sequence inserted into pAd vector was correct.2 The amplification of pAd-KLF4 and pAdThen the pAd-KLF4 and pAd were digested with PacI and transfected into 293A cells to be packaged. The two viruses were normalized to an identical titer (1×109 pfu/ml).3 The expression of pAd-KLF4 in vivoImmunohistochemical staining showed that the expression of KLF4 was rarely detected in the normal vascular of rats. However, the number of KLF4 positive staining in pAd-KLF4 group were significantly increased, compared to the injured group, suggesting that the infection was successful.4 The overexpression of KLF4 inhibits neointimal hyperplasiaAt 14 days after balloon injury, the injured vessel (injured group) showed neointimal hyperplasia. (I/M ratio, injured versus sham, 2.30±0.40 versus 0.12±0.03, p < 0.05). There was no significant difference between pAd-transfected group (I / M ratio, 2.48±0.38) and the injured group. However, KLF4 over- expression significantly reduced neointimal hyperplasia and I/M ratio (I/M ratio, pAd versus pAd-KLF4, 2.48±0.38 versus 0.52±0.15, p < 0.05) compared with the pAd group.5 The overexpression of KLF4 inhibits c-Jun and PCNA expression induced by balloon injuryImmunohistochemistry showed that c-Jun and PCNA expression was not detected in sham group. Balloon injury resulted in significant increase in c-Jun and PCNA expression, compared with sham-operated rats. RT-PCR results showed that the expression of c-Jun and PCNA induced by balloon injury was significantly reduced by 48.7% and 59.2% (P<0.01) at 14 days in pAd-KLF4-treated group. 6 The overexpression of KLF4 upregulates SM22αexpressive levelImmunohistochemistry and RT-PCR results showed that SM22αgene expression in vascular tissues was significationly reduced by 8% after balloon injury, compared with sham-operated rats. After infection of pAd-KLF4, SM22αgene expression was increased by 3.7 folds, which suggest that overexpression of KLF4 abolished the inhibition of SM22αgene expression by ballon injury.Conclusion:1 Adenovirus expression system of pAd-KLF4 is successfully constructed.2 pAd-KLF4 was successfully infected into the carotid artery after balloon injury in rats.3 Overexpression of KLF4 significantly inhibited neointimal hyperplasia induced by balloon injury via inhibiting c-Jun, PCNA expression and upregulation of SM22αgene expression. |
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