【Abstract】 Objective To investigate the feasibil ity of applying enzymatic method to prepare decellularizedporcine aorta and to evaluate its biomechanical properties, immunogenicity and cell compatibil ity. Methods 0.1% trypsin- 0.01% EDTA was appl ied to extract cells from porcine aorta under 37 continuously vibrating condition and its histology and microstructure were observed. The thickness, stress-strain curve, ultimate tension stress (UTS) and strain of failure (SOF) were compared before and after decellularization for 48, 96 and 120 hours under uniaxial tensile tests, respectively. The histological change was observed at 1, 3 and 6 weeks after the decellularized tissue was implanted subcutaneously in 3 dogs. According to the HE stains and a semi-quantitative Wakitani grading method, gross changes, category and amounts of infiltrated cells and neo-capillaries were compared between pre- and post-decellularization of porcine aortae. Endothel ial cells from canine external jugular vein were seeded onto the decellularized patches to observe the cell compatibil ity. Results Microscopy and electron microscopies examination identified that cell components was completely removed from the fresh porcine aorta and Masson’ strichrome showed that the structure of matrix (fibrins) was maintained intact at 96 hours using the decellularization method. There were no significant differences in the thickness, UTS and SOF between before and after decellularization (P gt; 0.05). However, The UTS values showed a decrease tendency and SOF showed an increase tendency. The stress-strain curve also verified a decrease tendency in mechanical intensity and an increase one in ductil ity after decellularization. After implanting the acellularized matrix subcutaneously in canine, moderately lymphocyte infiltration was seen at the 1st week and the infiltration was replaced by fibroblasts accompanied by neocapillary formation at the 6th week. A semi-quantity histological evaluation showed that there were differences in gross observation, category and the numbers of the infiltrated cells between decellularized and non-decellularized tissues（P lt; 0.05). A cell monolayer was identified by HE staining and scanning electron microscopywhen the endothel ial cells were seeded onto the inner luminal surface of the scaffold, al igned at the same direction on the whole. Conclusion The decellularized porcine aortic scaffold, prepared by trypsin-EDTA extraction under continuously vibrating condition, could meet the requirements of tissue-engineering graft in biomechanical properties, immunogenicity and cell compatibil ity.
Objective To investigate the effect of adenovirus vector mediated transfer of human herpes simplex virus thymidine kinase (HSVtk) gene inhibits intimal hyperplasia of vein grafts. Methods Auto vein graft models of Wistar rats were established. Adenovirus vector dwelled in cervical veins which were transplanted into inferior renal abdominal aorta. The combination of HSVtk (4×109 plaque forming units) and ganciclovir (GCV) was applied to test the inhibition effect. GCV was infused 〔60 mg/(kg·d), IP, Bid〕 from day 3 to day 21 after transplantation. Vein samples were harvested and the existence of HSVtk DNA was measured by PCR and the mRNA of it was studied by in situ hybridization. Van gieson (VG) and proliferating cell nuclear antigen (PCNA) stains were carried out in paraffin sections to study the thickness of neointima and smooth muscle cells (SMCs) proliferation with a computer-assisted analysis system. The apoptosis of SMCs also was detected by TUNEL. Results The existence of HSVtk gene in veins and its transcription were demonstrated. Morphometric analysis demonstrated a reduced intima thickness in the group receiving combination therapy (HSVtk/GCV) compared with HSVtk alone 〔(17.2±3.2) μm versus (31.1±2.5) μm, P＜0.05〕. GCV per se had no effect on intimal hyperplasia after vein transplantation. The apoptosis of SMCs increased significantly and expression of PCNA decreased in HSVtk/GCV gene therapy group versus blank control group 〔(9.1±2.3)% vs (28.7±3.6)%, P＜0.05; (38.7±5.6)%vs (18.5±2.6)%, P＜0.05〕. Conclusion GCV conditions reduction of intimal hyperplasia after intraluminal delivery of HSVtk in transplanting vena veins involving SMCs apoptosis.
Objective To observe the clinical efficiency of the implantation of the autologous bone marrow mononuclear cells for treatment of lower limb ischemia after the bone marrow stimulation. Methods From May to December 2005, 43 ischemic limbs in 35 patients (23 males,12 females; aged 3490 years,averaged 71.3 year) were treated. Of the 35 patients, 30 had diabetic lowerlimb ischemia with 38 lower ischemic limbs, 2 had atherosclerosis obliterans with 2 ischemic lower limbs, and 3 had thromboangiitis obliterans with 3 ischemic lower limbs. Five patients with 5 ischemic limbs were in stage Ⅰ lower limb ischemia (intermittentclaudication), 15 patients with ischemic 19 limbs were in stage Ⅱ (rest pain),9 patients with 12 ischemic limbs were in stage Ⅲa(ulceration), and 6 patients with 7 ischemic lower limbs in stage Ⅲb (gangrene); 88.4% of all the ischemic lower limbs （38/43）had a pain, 79.1%（34/43） had coldness, and 69.8%（30/43）had limb numbness. The bone marrow of each patient was stimulated by an injection of the recombinant human granulocyte-macrophage colony-stimulatory factor 300 μg/d for 2-3 days. The bone marrow 130-200 ml was drawn from the iliac spine and the mononuclear cells were obtained. Each patient received implantation of the autologous bone marrow mononuclear cells by an intramuscular injection, an arterial intraluminal injection or a combined injection of the two routes.Results The pain relief was found in 94.7% of theischemic lower limbs, and pain improvement in 97.1% . Relived numbness was found in 93.3%. The distance of the claudication was increased by all the ischemic limbs. An increase in the ankle/ brachial index (ABI)was found in 47.9%. The transcutaneous oxygen pressure (TcPO2) increased in 92.3%. The ulcer heal rate was 9.1% (1/11). Markedlyreduced ulcer wound was found in 27.3% (3/11). The amputation rate was 6.3% (3/48). Arterial angiography revealed that there was a new collateral vessel formationin 91.2%. Complications were as follows: fever and mild fatigue-developed respectively in 1 patient after the bone marrow stimulation, but relieved by themselves. Acute but mild myocardial infarction was found in 1 patient with a slight precordial pain and elevation of myocardial enzymes 1 week after transplantation of the bone marrow mononuclear cells, but recovered after medical treatment. The follow-up averaged 5 months. According to the subjective criteria, the overall efficacy was90%. ABI increased in 62.5% of the patients after operation and the value of TcPO2 was higher in 90% of the patients after this kind of therapy. Arterial angiography revealed a new collateral vessel formation in 90.5% of the 21 ischemic limbs. The foot ulcer healed in 7 and obviously improved in 3. Three of the foot ulcer patients were discharged 2-3 months after the amputation was performed on the diseased toes. Conclusion Implantation of the autologous bone marrow mononuclear cells after the bone marrow stimulation of treatment of the lower limb ischemia has advantages of less marrow aspiration, more mononuclear cell content, satisfactory shortterm effect, and relatively high safety. Itis a new method of treating the lower limb ischemia besides the autologous bone marrow and peripheral blood mononuclear cell implantation. The longterm effect of this method needs a further study.
【Abstract】ObjectiveSome studies have demonstrated that recombinant adenoviruses are efficient vectors for gene transfer to the venous wall and AdCMV.tk encoded thymidine kinase can be used to reduce restenosis. In this study AdCMV.tk was apply to human vein smooth muscle cells (SMC) and organ cultured saphenous veins to study its effects on proliferation of SMCs and reduction of intimal hyperplasia. MethodsThe adenovirus vector transferred tk gene and mark gene lacZ to the SMC of human saphenous veins and organ cultured vein segments. Various concentrations ganciclovir (GCV) were contained in culture media. The efficiency of gene transfer was studied by using Xgal staining. The proliferation of SMC was monitored by the method of trypan blue exclusion. The bystander effect was observed by mixed cell culture. After vein segments treated by AdCMV.tk+GCV and cultured for 14 days, HE and VG staining were carried out and intimal thickness was analysis by computer image system. ResultsAdenovirus vector could infect saphenous vein SMC efficiently both in cultured SMCs and organ cultured vein segments. Gene expression sustained 14 d at least. The inhibition of SMCs proliferation in vitro was a positive correlation in GCV concentrations and the levels of tk expression. The proliferation of SMCs transfectered lacZ wasn’t restrained by GCV (P＜0.05). In mixed cell experiment there was at least 55% reduction in total cell number when as few as 10% of the cells express tk. Assessment of this “suicide gene strategy” in saphenous vein organ culture model demonstrated that veins treated with AdCMV.tk+GCV had a significant reduction at 14 days in the intimal thickness compared to control group (P＜0.01). ConclusionThe results suggest that adenovirusmediated gene transfer of tk along with GCV administration may be a useful strategy to treat the proliferation of intimal hyperplasia of transplanting saphenous veins. Bystander effects are amplified by AdCMV.tk/GCV gene therapy system.
Objective To investigate the effectiveness of percutaneous transluminal renal artery stenting (PTRAS) in treating atherosclerotic renal artery stenosis (ARAS). Methods A total of 69 patients with severe ARAS were treated with PTRAS between January 2002 and December 2008. There were 47 males and 22 females with an average age of 66.2 years(range, 42-88 years), including 66 cases of unilateral ARAS (single functional kidney, 1 case) and 3 cases of bilateral ARAS. Renal angiography revealed that the degree of renal artery stenosis was 70%-99%. Concomitant diseases included hypertension (67 cases), atherosclerosis obl iterans (69 cases), coronary heart disease (34 cases), diabetes (44 cases), and hyperl ipidemia (36 cases). Blood pressure, serum creatinine (sCr), and patency of the renal artery were measured to assess the effectiveness of PTRAS after 12 months. Results The renal artery stent was successfully implanted in 68 patients and the technical success rate was 98.6%. One patient was converted to il io-renal bypass because of intra-operative acute renal artery occlusion. One patient died of heart failure at 6 months after PTRAS, and 1 patient was lost at 3 months after PTRAS. The other 66 patients were followed up 32 months on average (range, 13-60 months). The blood pressure decreased significantly at 1 month and gained a further decrease at 12 months after PTRAS when compared with the preoperative ones [systol ic blood pressure: (132 ± 24) mm Hg vs (163 ± 34) mm Hg, P lt; 0.05; diastol ic blood pressure: (78 ± 11) mm Hg vs (89 ± 17) mm Hg, P lt; 0.05; 1 mm Hg=0.133 kPa]. Hypertension was cured in 4 cases (6.3%), improved in 52 cases (81.2%), failure in 8 cases (12.5%), and the overall benefit rate was87.5%. The sCr level was stable after 12 months of PTRAS, showing no significant difference when compared with preoperative basel ine [(107.8 ± 35.4) μmol/L vs (104.1 ± 33.8) μmol/L, P gt; 0.05]. Renal function was improved in 9 cases (13.6%), stable in 48 cases (72.8%), deterioration in 9 cases (13.6%), and the overall benefit rate was 86.4%. Instent restenosis found in 2 patients (3.0%) at 12 months after operation. Conclusion PTRAS is a safe and effective method to treat ARAS. It can control the blood pressure and stabil ize the renal function in most ARAS patients. Long-term efficacy needs further investigation.
ObjectiveTo summarize the effectiveness and safety of antiplatelet combined with anticoagulant therapy for peripheral arterial disease (PAD). MethodUsing the search strategy developed by Cochrane Collaborative Network, the relevant literature from domestic and foreign databases as of November 1, 2023 was searched and a meta-analysis of outcome indicators was conducted using Stata 14.0 software and Review Manager 5.4.1 software provided by Cochrane Collaboration Network. ResultsA total of 15 eligible literature and 15 383 patients were included, including 7 692 in the antiplatelet combined with anticoagulant therapy group (study group) and 7 691 in the control group (only antiplatelet drug therapy). The meta-analysis results showed that: ① Symptoms: The ankle brachial index [mean difference (MD) and 95% confidence interval (95%CI)=0.04(0.02, 0.06)] and the minimum lumen diameter [MD (95%CI)=0.48(0.40, 0.55)] of the study group were greater than those of the control group; The plasma D-2 dimer level of the study group was lower than that of the control group [MD (95%CI)=–0.55(–0.57, –0.52)], and the probability of the limb ischemia risk of the study group was lower than that of the control group [risk ratio (RR) and 95%CI=0.67(0.56, 0.80)]. ② Vascular patency: The probability of the vascular patency of the study group was higher than that of the control group [RR (95%CI)=1.13(1.08, 1.17)]; The subgroup analysis results: the vascular patency rate of the two antiplatelet drugs combined with anticoagulation therapy was highest among the different treatment regimens [effect size (ES) and 95%CI=0.90(0.86, 0.94)], which of the other measures in descending order was only one antiplatelet drug combined with anticoagulation therapy [ES(95%CI)=0.82(0.76, 0.89)], two antiplatelet drugs therapy [ES(95%CI)=0.79(0.72, 0.85)], and only one antiplatelet drug therapy [ES(95%CI)=0.71(0.54, 0.87)]; The probability of the vascular patency using vitamin K antagonists in the study group was higher than that in the control group [RR(95%CI)=1.15(1.10, 1.20)], which had no statistical difference using Ⅹa inhibitor between the study group and the control group [RR(95%CI)=1.04 (0.95, 1.15)]. ③ Bleeding risk: The risk of bleeding of the study group was higher than that of the control group [RR(95%CI)=1.55(1.27, 1.89)]; The subgroup analysis results: The bleeding rate of the only one antiplatelet drug therapy among the different intervention measures was the lowest [ES(95%CI)=0.02(0.01, 0.02)], which of the other measures in ascending order was only one antiplatelet drug combined with anticoagulant therapy [ES(95%CI)=0.04(0.03, 0.06)], two antiplatelet drugs therapy [ES(95%CI)= 0.08(0.06, 0.10)], and two antiplatelet drugs combined with anticoagulant [ES(95%CI)=0.12(0.06, 0.18)]; The probabilities of the bleeding occurring using the vitamin K antagonists and Ⅹa inhibitor in the study group were higher than those in the control group [RR(95%CI)=1.76(1.28, 2.42); RR(95%CI)=1.44(1.12, 1.84)]. ConclusionsFrom the results of this meta-analysis, it can be seen that the combination of antiplatelet and anticoagulant therapy can effectively improve symptoms of patients with PAD, increase vascular patency rate, but it has a certain risk of bleeding. The combination of only one antiplatelet drug combined with anticoagulant therapy might achieve an optimum clinical effect and lower bleeding risk.
ObjectiveTo explore the relationship between the oxygen partial pressure of mice hindlimb muscles with normal blood supply or ischemia and expression of HIF-1αprotein, and to provide a theoretical basis for the study of angiogenesis in vitro hypoxia. MethodsMice hind limb ischemia model were established, tissue oxygen tension of gastrocnemius muscle and bone marrow were measured by micro electrode at different time points of ischemia (24 hours, 1 week, 2 weeks, 3 weeks, and unoperated as control). Protein level of hypoxia inducible factor-1αand histological examination were performed on gastrocnemius muscle as well. ResultsThe oxygen tension baselines of gastrocnemius muscle and femoral bone marrow was (47.78±4.37) mm Hg and (21±3.40) mm Hg, respectively. Muscle oxygen tension decreased significantly at all time points after modeling (P < 0.05), and reached lowest level in 1 week of ischemia. The inflammatory reaction was most serious and HIF-1αprotein reached highest level at the same time point. With the extension of ischemic time, the tissue oxygen tension recovered while HIF-1αlevel was down-regulated, however, There was no statistical correlation(r=-0.86, P > 0.05). Oxygen tension in bone marrow didn't show any significant change at all time points. ConclusionsThe expression level of HIF-1αprotein in ischemic tissue can reflect the degree of ischemic limb. The concept that physiological oxygen level differs in different tissue is highlighted, and may provide basis for ex vivo hypoxic research.