| Objective and BackgroundPercutaneous coronary interention (PCI) which were applied widely, improved clinical curative effect and prognosis of the coronary heart disease (CHD) significantly. But restenosis caused by mechanical damage to blood vessels in the course of PCI, was one of the important issues which have not been solved in the clinical study. In the present study, the occurrence of restenosis has to do with vascular remodeling and neointimal hyperplasia which is caused by endothelial injury, the proliferation and migration of vascular smooth muscle cells(VSMCs), etc. Our group’s previous researches showed that mesenchymal stem cells (MSCs) transplantation therapy can accelerate the reendothelialization of injuried artery to reduce the extent of angiostenosis, but was relatively weak to VSMCs. Therefore, if related target gene is to be used to modify MSCs, which not only protects endothelium but also enhances the potency of MSCs to inhibit the proliferation of VSMCs and intimal hyperplasia. Restenosis would possibly decrease further. Many research showed:Calcitonin gene related protein (CGRP) existing widely in cardiovascular system is a vascular active peptide, which has the potency of inhibiting the proliferation of VSMCs signally. So we construct recombinant lentivirus vector, including Enhanced Green Fluorescent Protein(EGFP) and CGRP gene(Lv-EGFP-CGRP), transfect it to MSCs and co-culture with VSMCs in vitro, and transplantate to Rat Model with sacculus damaged atherosclerotic carotid through mimesis the PCI therapy pattern in vivo. To examine the proliferation, apoptosis and phenotypic modulation of VSMCs synchronously, and to explore the therapeutic potency of MSCs modified by CGRP gene on endothelial protection, whether the proliferation of VSMCs and other functions can be inhibited at the same time, which could provide some theoretical and experimental bases for the theatment of restenosis with gene optimization of stem cell transplantation. MethodsPart One:MSCs isolated by centrifugation and adherent culture, were examined by Flow cytometry(FCM) analysis. MSCs were infected by lentivirus encoding recombinant enhanced green fluorescent protein (EGFP) gene and CGRP (Lv-EGFP-CGRP and Lv-EGFP). The transfection efficiency was determined by inverted fluorescence microscope and FCM. The expression of CGRP was assayed in MSCs modified by CGRP using Real-time PCR and tissue immunohistochemical assays, and the CGRP concentration in the culture supernatant of MSCs was measured by ELISA. Simultaneously, to observe the biological characteristics of MSCs transfected by lentivirus, the experiment was divided into three groups:MSCs-CGRP, MSCs-EGFP and MSCs three groups (n=3), to examin the proliferation, aging and differentiation of MSCs transtection with lentivirus by MTT,(3-galactosidase staining, and inducing differentiation respectively.Part Two:VSMCs were isolated from the tissue adherence method and identified by inverted microscope and immunofluorescence. MSCs modified by CGRP were co-cultured with the synchronized VSMCs for48h, and the experiment was divided into four groups: Lv-EGFP-CGRP-MSCs+VSMCs(MSCs-CGRP group), Lv-EGFP-MSCs+VSMCs (MSCs-EGFP group), MSCs+VSMCs(MSCs group) and VSMCs(control group); the migration ability of VSMCs was examined with a modified Boyden-chamber assay; Cell counting and trypan blue staining were used to investigate the proliferation and survival of VSMCs and Flow cytometry to observe the cell cycle and apoptosis. Western Blot were used to analyze the expression of SM-a-action and osteopontin(OPN) in VSMCs.Part Three:Rats model with sacculus damaged carotid were set up. Beforehand MSCs transfected with lentivirus vector encoding recombinant EGFP(lv-EGFP) or with lentivirus vector encoding recombinant EGFP and CGRP(lv-EGFP-CGRP) were transplanted into Rats model immediately. All animals were randomly divided into three groups: Lv-EGFP-CGRP-MSCs transplantation group (MSCs-CGRP group, n=24), and Lv-EGFP-MSCs transplantation group (MSCs group, n=24), and control group (n=24). After cell transplantation the injured carotid arteries were obtained at7d,14d and28d.Homing and differentintion of MSCs were detected by detecting EGFP and CD31. The expression of CGRP, SM-a-action and OPN were measured by Western Blot. Organization morphology was observed by HE stain. Proliferating cell nuclear antigen (PCNA) was detected by tissue immunohistochemical assays.ResultsPart One:MSCs were successfully infected by Lv-EGFP-CGRP or Lv-EGFP, and expressed EGFP stably after48h. When Multiplicity Of Infection(MOI) was30, transfection efficiency was observed by fluorescence microscope and FCM, which was more than80%; The effective expression of CGRP in infected cells has been proved by RT-PCR, Laser Scanning Confocal Microscope(LSCM) and ELISA. Compared with control group, MSCs-CGRP group increased significantly (P<0.01). Lv-EGFP or Lv-EGFP-CGRP transfection did not show a significantly inhibitory and senescent effect on MSCs, and didn’t influence the differentiation to endothelial cell of MSCs.Part Two:Compared with MSCs-EGFP, MSCs and Control group, the migration ability of MSCs-CGRP group was the weakest, and cell count was the least(P<0.01), but there were no differences between other groups. The survival rate of the four groups was more than90%. FCM showed that more VSMCs in MSCs-CGRP group were blocked in the stage of G0/G1and few entered into the S Phase. FCM also showed that the level of VSMCs apoptosis in MSCs-CGRP group was much higher. The results of western blot indicated that the protein expression of SM-a-action was increased, whereas OPN was decreased in MSCs-CGRP group, when compared with other groups.Part Three:Both MSCs-CGRP group and MSCs group, EGFP marker and CD31expression were found in intima of damaged carotid, but CD31expression not found in control group. After cell transplantation28d western blot showed that the expression of CGRP was found in damaged carotid intima of three groups, and MSCs-CGRP group was much higher than other two groups. HE stain indicated that the rate of neointima and media in MSCs-CGRP group was lower than that in MSCs group; and both in groups of MSCs transplantation was much lower than that in control groups (P<0.05). Also, the expression of PCNA was the highest in control group, MSCs group lower, and MSCs-CGRP group the lowest (P<0.05). The phenotypes transformation of VSMCs examined by western blot showed that:the expression of SM-a-action in MSCs-CGRP group increased than MSCs group and control group, but less than normal group; whereas OPN decreased in MSCs-CGRP than MSCs group and control group, but more than normal group.Conclusion:Part One:It is concluded that the expression of CGRP was up-regulated in MSCs, which were transfected successfully by lentiviral vector, MSCs modified by CGRP could secrete CGRP protein to supernate, and the transfection has no significant effects on biological characteristics of MSCs. MSCs are ideal cells for cell-mediated CGRP gene therapy or cell therapy.Part Two:In vitro, MSCs modified by CGRP could inhibit the proliferation and migration of VSMCs, and accelerate apotosis and phenotypes transformation from synthetic to contractile type. These results lay the foundation for gene therapy in vivo.Part Three:In vivo of Rats, compared with natural MSCs, MSCs modified by CGRP not only have the potency on the recovery of damaged endothelium, but also significantly inhibit the proliferation of VSMCs and phenotypes transformation from synthetic to contractile type. The results showed that stem cells modified with genes have the potency of more effective inhibition of hyperplasia intima, and thus further reduce the occurs of restenosis after angioplasty. |
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