| Objective To investigate the feasibility of transferring p21 gene into lung tissue with recombinant replication-deficient adenovirus with full-length cDNA of p21 inserted in the wistar rat models of pulmonary hypertension induced by left-to-right shunt, study the expression of the desired gene in vivo, find if overexpression of desired gene can inhibit pulmonary hypertension, observe the change of proliferation and apoptosis of vascular smoothmuscle cells (VSMCs).Methods After the full-length p21 cDNA obtained via PCR amplification using human p21 plasmid (pCEP-waf1) as model, it was then digested by restriction endonuclease (XbaI and KpnI) and the pShuttle was digested by the same enzyme, and then ligates them together. The shuttle and type 5 adenovirus with the E1 and E3 depleted were digested each other by the same restriction endonuclease (PI-SceI and I-CeuI).The recombinant replication-deficient adenovirus harboring the desired gene (Adeno-p21) was harvested by ligating reaction between the desired gene and the adenoviral vector. Lineated after digestion by restriction endonuclease (PacI), it transfected the human embryonic kidney cell (HEK293) with Clonfectin, and was packed, amplified in order to obtain the high-titer recombinant adenovirus. The Adp21 was authenticated by PCR and its infection titer was determined by End-Point dilution method. The control virus Adeno-lacZ was constructed in the same way. 48 wistar rats were divided randomly into 4 groups. Group I (n=10) was control group. Group II(n=13) was model group, which were established PH models using intubation tube between common carotid artery and external jugular vein. Group III(n=14) was Adeno-p21 transfection group, which were established PH models and transfected Adeno-p21 using windpipe Inhalation. Group IV(n=11) was Adeno-lacZ transfection group, which were established PH models and transfected Adeno-lacZ using windpipe inhalation. After 16 weeks, every group were mearsured mRVP, mPAP by right-heart catheterize- tionand and caculated RVHI and RV/BW. HE stained were used to observe the architecture changes in lung arteriole and measured the external diameter and thickness of vessel wall of lung arteriole to calculate WT%.Western blot was used to assessment the expression of p21 gene product in group III and IV. Labelled streptavidin-biotin (LSAB) was used to investigate the expression of PCNA of VSMCs, and TUNEL was used to detect the apoptosis of VSMCs. Calculate PI(proliferation index), AI(apoptosis index) and PI/AI.Results 1. The specific segment of p21 (489bp) and adenovirus (370bp) were obtained by PCR amplification, which proved that the required recombinant adenovirus carrying the desired gene was created. By End-Point dilution method, the titers of Adeno-p21 and Adeno-lacZ were known to be 2×10~8 pfu/L and 1.2×10~8pfu/L respectively.2. The mRVP and mPAP in Group II , III and IV were markedly higher than those in Group I (P<0.05) and the RVHI and RV/BW were also demostrated statistically different (P<0.05). The mPAP,mPVP and WT% in Group III were markedly lower than those in Group II and IV (P<0.05), but higher than those in Group I (P<0.05). In Group II ,III and IV, muscular hypertrophy in the media of pulmonary artery and arteriole was evident, and cavity of the vessels became strictured.3. 21kd specific segment was showed by Western blot in group III, which means the desired gene expressed successfully.4. In Group III, PI was markedly lower than PI in Group II and IV(P<0.05),and AI was higher than any other group(P<0.05), and PI/AI was markedky lower than group II and IV(P<0.05).Conclusion 1. Type 5 adenovirus can successfully carry p21 and transfect the lungtissue of PH model in rats, and full expression of p21.2. p21 gene can inhibit the development of pulmonary hypertensive through inducingthe apoptosis of VSMCs.
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