【Abstract】Objective To investigate the production and possible significance of plasma trypsinogen activation peptides (TAP) in rat experimental acute pancreatitis. Methods Ninety SD rats were randomly allocated to five groups: group EP with retrograde ductal infusion of 3%sodium taurocholate; group NP with retrograde ductal infusion of 5%sodium taurocholate; group TP with retrograde ductal infusion of 3%sodium taurocholate and ulinastatin(UTI) intravenous infusion half an hour later; group CP with 0.9% NS retrograde ductal infusion; group OP with sham operation. Animals in each group were killed 3h,6h and 24h after infusion. Plasma TAP was determined by EIA.The histological severity of the pancreas were assessed by Schmidt method. Results The pancreatic pathological changes in group NP was significantly severe than in group EP. At 3h and 6h after infusion, plasma TAP concentration of group NP (4.798±0.169)nmol/L and (3.999±0.299)nmol/L were significant higher than that of group EP(2.416±0.148)nmol/L and (3.356±0.211)nmol/L. At 6h after infusion plasma TAP concentration of group TP 〔(1.611±0.113)nmol/L〕 was significant lower than that of group EP(3.356±0.211)nmol/L. The difference of plasma TAP concentration between group EP and group NP appeared prior to the difference of the histopathological changes of pancreas between two groups. Conclusion Plasma TAP concentration is connected with the severity of sodium taurocholate-induced rat pancreatitis. Plasma TAP concentration may be used as a marker for early assessment of the severity of this experimental acute pancreatitis.
Objective To induce embryonic stem cell (ESC) to differentiate into endothel ioid cells using a simple adhesive culture method, and to provide a new cells seed source for vascular tissue engineering or cell therapy. Methods SV129-derived ESC were seeded at 2 × 104/cm2 and maintained undifferentiated on ESC culture medium in the presence of 1 000 U/mL leukaemia inhibitory factor (LIF). Embryoid body (EB) formatted when ESC cultured in suspension in the lack of LIF. At 4 days, EB was transferred to 0.1% gelatin coated dish and cultured with medium supplementary of VEGFto be induced differentiation. The characteristics of differentiated cells were determined by immunohistochemistry staining, flow cytometry (FCM), 1, 1-dioctadecyl-3, 3, 3, 3-tetramethyl indocarbocyanine-labeled acetylated low density l ipoprotein (DiIAc- LDL) takeup test, and TEM detection. Results Differentiated cells were morphologically characterized as endothel ial cells. They could takeup DiI-Ac-LDL, be stained positive by Flk-1 and CD31. The CD31 positive cells reached above 90% when measured by FCM. Furthermore, Weibel-Palade bodies were detected and tight junctions were found when differentiated cells were examined by TEM. Conclusion Using a simple adhesive culture method and by suppl ied with VEGF alone, ESCs can be induced to differentiate into endothel ioid cells. The differentiation method is simple and economic, and can provide seed cells for vascular tissue engineering or cell-therapy.
Objective To observe the changes in the number and function of bone marrow-derived endothel ial progenitor cells (EPCs) after bone-marrow stimulation, and to investigate the possible mechanism of improving ischemicl imb disease after bone-marrow stimulation through autologue bone-marrow stem cell implantation. Methods Twelvemale Lewis rats, weighing 200-250 g, were classified into the bone marrow stimulation group (n=6) and the control group(n=6). In the stimulation group, the bone marrow of each rat was stimulated by injection of recombinant human granulocytemacrophage colony-stimulatory factor. Mononuclear cells were harvested from bone marrow and cultured in EBM-2 medium. After 7-day culture, EPCs were stained by 1, 1-dioctadecyl-3, 3, 3, 3-tetramethyl indocarbocyanine-labbled acetylated low density l ipoprotein/fluorescein isothiocyanate-ulex europaeus agglutinin 1, and the double positive cells were counted by the fluorescent microscope. The adhesive abil ity of EPCs was determined by counting the number of re-cultured EPCs. The unilateral ischemia hindl imb model was made with 12 Lewis rats. Three days later, EPCs were transplanted into the ischemic tissues. According to different sources of EPCs, the 12 rats were divided into 2 groups: the stimulation group (n=6) and the control group (n=6). At 3 weeks after EPCs transplantation, the quantity of the collateral vascular was observed by digital subtraction angiography (DSA). Results After 7-day culture, the number of EPCs in the stimulation and control groups was (145.2 ± 37.0)/HP and (95.2 ± 39.4)/HP, respectively, and there was significant difference between the two groups (P lt; 0.05). Meanwhile, the number of adhesive EPCs in the stimulation and control groups was (21.8 ± 4.3)/HP and (15.0 ± 5.2)/HP, respectively, and the difference between the two groups was significant (P lt; 0.05). At 3 weeks after the EPCs implantation, the number of the collateral vascular was significantly larger in the stimulation group (4.2 ± 1.2) compared with the control group (2.7 ± 0.8), (P lt; 0.05). Conclusion Bone marrow stimulation increases the number of EPCs and improves the function concurrently, which may be the reason why autologue bone-marrow stem cell implantation improves the curative effect of ischemic l imb diseases after bone-marrow stimulation.
Objective To establish chronic hindlimb ischemia model with suture-occluded method in rats， and then compare the effects of chronic hindlimb ischemia model with acute ischemia model. Methods Models of chronic hindlimb ischemia were established by using suture-occluded femoral artery method. The laser Doppler blood flow analysis and angiography were performed on day 7， 14， 28， 42， and 49 after operation， and then the rats were sacrificed after angiography， respectively， the quadriceps and gastrocnemius of contralateral and ipsilateral （surgical side） were gotten， which were tested by HE staining and α-actin immunohistochemistry staining， and then calculate arteriolar density. Results There were no lameness and limb necrosis after operation in chronic hindlimb ischemia models. Laser Doppler analysis found that chronic hindlimb ischemia models were still maintained in ischemia state on day 49 after operation compared with acute ischemic models. The resluts of HE staining showed no acute necrosis and muscle fibrosis in chronic hindlimb ischemia model group. Chronic hindlimb ischemia models after operation did not appear obvious lameness and limb necrosis. The arteriolar density of quadriceps femoris on day 7 after operation in chronic hindlimb ischemia models were less than that in acute hindlimb ischemia models （0.015 2 vs. 0.036 4）. Conclusions Compared with the commonly used acute ischemic models， the duration of arterial limb ischemia in chronic hindlimb ischemia rats， which were established by suture-occluded method， is longer and less likely to be affected by the compensatory mechanisms. So suture-occluded method can provide a new animal hindlimb ischemia model for further study of ischemia angiogenesis mechanism and treatment of severe lower extremity ischemia.
Objective To investigate the feasibil ity of preparing the porous extracellular matrix (ECM) by use of some chemicals and enzymes to decellularize the porcine carotid artery. Methods The porcine carotid artery was procured, and warm ischemia time was less than 30 minunts. The porcine carotid artery was decellularized with 1% sodium dodecyl sulfate (SDS) for 60 hours to prepare common ECM; then common ECM was treated with 0.25% trypsin (for 6 hours) and 0.3 U/ mL collagenase (for 24 hours) to prepare porous ECM. The common ECM and porous ECM were stained with HE,Masson’s trichrome, and Orcein to evaluate the histological features. Then the mechanical property, cytotoxicity, and pore size of ECMs were determined. After 4 weeks of subcutaneous implantation in dogs, the histological examination was used for the study. Results Histological observation confirmed that 2 kinds of ECMs were decellularized completely and more porous structure was observed in porous ECM. Scanning electron microscope showed the pores in porous ECM were greater and the length of shorter axis in porous ECM ranged from 5 to 30 μm, the length of longer axis from 40 to 100 μm. The porosity of porous ECM (99.25%) was greater than that of common ECM (91.50%). The burst pressure of porous ECM decreased when compared with common ECM, showing significant difference [(0.154 3 ± 0.012 7) MPa vs [0.305 2 ± 0.015 7) MPa, P lt; 0.05]. There was no significant difference in suture retention strength between 2 kinds of ECMs (P gt; 0.05). The cytotoxicity test showed no obvious cytotoxicity in 2 kinds of ECMs. In vivo implantation test showed that the deeper host cells infiltration and more neo-microvessels in porous ECM were observed than in common ECM. Conclusion SDS and some enzymes can be used to prepare porous ECM as the scaffold for tissue engineered blood vessels.
Objective To explore the mechanism of mesenchymal stem cells （MSCs） transplantation for chronic hindlimb ischemia in Lewis rats by using cell tracer technique. Methods MSCs were isolated and cultured by using density gradient centrifugation and adherence method respectively， then labeled by 5-bromo-2-deoxyuridine （BrdU）. Eight chronic hindlimb ischemia models of Lewis rats were prepared by using suture-occluded method and then divided randomly to MSCs transplantation group and control group， each group enrolled 4 rats， accepting MSCs transplantation and saline respectively. Then on 7 days and 14 days after transplantation， clinical observation， determination of blood flow， and angiography were performed on rats of the 2 groups. At the same time points after previous tests， rats of the 2 groups were sacrificed to get quadriceps tissues and gastrocnemius tissues to perform HE staining and BrdU immunohis-tochemistry. Results The 8 rats were all survived on 14 days after transplantation， with no tumor happened and necroses in the transplanted area. On 14 days after transplantation， the blood flow ratio of operated side to un-operated side in the hindlimb （1.773 vs. 1.279） of rats in MSCs transplantation group and control group increased， and the angiography results showed that there were no much increase in ratio of collateral vessels number （0.908 vs. 0.835）. There were no significant change in the quadriceps tissues and gastrocnemius tissues by HE staining. The BrdU positive kernels located in the inter-stitial substance cells and vascular endothelia cells， and divided differently in different parts of hindlimb at different time points， that the ratio of positive cells in gastrocnemius tissue was higher than those of quadriceps tissue on 7 days after transplantation， but lower on 14 days. Conclusions MSCs transplantation can increases the blood perfusion of hindlimb in the early stage of chronic hindlimb ischemia model， and the possible mechanism may be the paracrine effect of MSCs but not the number increase of collateral vessels.
ObjectiveTo evaluate the dynamic changes of blood flow and blood pressure of acute hindlimb ischemia of rats by laser Doppler flowmetry (LDF) and laser Doppler perfusion imaging (LDPI). MethodsThe acute hindlimb ischemia model of rats was established by resection of rats femoral arteries of left hindlimb. The blood flow and blood pressure between operated and nonoperated hindlimbs were examined by LDF on 2, 7, 14, 28, and 49 d after operation. And the blood flow was evaluated by LDPI on 7 d after operation. ResultsAll rats survived after operation and no hindlimb necrosis occurred. The mean score was 2 on 14 d after operation and 1 on 49 d after operation. The ratio of blood flow between operated and nonoperated hindlimbs on 2 d after operation significantly increased from 1 to 1.31±0.439 (P=0.021). The ratio of blood flow on 7 d (0.82±0.538) and 14 d (0.93±0.294) after operation was significantly lower than that on 2 d after operation (P=0.032 and P=0.019), although the difference between the two former was not significant (P=0.502). Furthermore, the ratio of blood flow on 28 d after operation reached the bottom (0.41±1.970), which was obviously lower than that on 2, 7, and 14 d after operation (P=0.004, P=0.007, and P=0.006). The blood flow of operated hindlimbs recovered approximately the value before operation (0.98±0.093), which was significantly lower than that on 2 d (P=0.010), higher than that on 28 d (P=0.005), but not different from that on 7 d and 14 d after operation (P=0.126 and P=0.382). The ratio of blood pressure between operated and nonoperated hindlimbs on 2 d after operation significantly increased from 1 to 0.47±0.375 (P=0.031). The ratio of blood pressure decreased on 7 d after operation (0.44±0.118), which was not different from that on 2 d after operation (P=0.203). Furthermore, the ratio of blood pressure on 14 d after operation reached the bottom (0.35±0.115), which was obviously lower than that on 2 d and 7 d after operation (P=0.001 and P=0.036). On 28 d after operation, the ratio of blood pressure increased (0.54±0.146), which was significantly higher than that on 14 d after operation (P=0.008), while not different from that on 2 d (P=0.493) and 7 d after operation (P=0.551). The ratio of blood pressure recovered approximately the value before operation (0.97±0.094), which was significantly higher than that on 2, 7, 14, and 28 d (P=0.013, P=0.021, P=0.002, and P=0.031). ConclusionAcute hindlimb ischemia model of rats can be established by resection of rats femoral arteries of left hindlimb and the most serious stage of hindlimb ischemia is on 14-28 d after operation. LDF and LDPI are of importance for monitoring the dynamic changes of rats hindlimb ischemia after operation.
Objective To explore the middle-term outcome of autologous bone marrow mononuclear cells transplantation in the treatment of lower l imb ischemia. Methods From March 2003 to June 2005, 65 patients with lower l imb ischemia were treated by autologous bone marrow mononuclear cells transplantation. Of the patients, there were 50 males and 15 females, with a mean age of 66.5 years (range 36-89 years), including 4 cases of simple arteriosclerotic occlusion,5 cases of thromboangiitis obl iterans and 56 cases diabetic lower l imb ischemia. A total of 400 mL bone-marrow blood were extracted from the posterior superior il iac crest. And then the mononuclear cells were isolated from the bone-marrow blood in the laboratory. The amount of transplantation bone marrow mononuclear cells was (0.60-1.80) × 109 (mean 1.05 × 109). Twelve patients received cell transplantation from two to four times and the other patients one time. According to the improvement of cl inical finding, the outcome was evaluated. Results All the patients were followed up for 8-56 months (mean 21.5 months). There were 8 deaths, and the mortal ity was 12.3%; 5 were due to myocardial infarction and heart failure and 3 were due to cerebral infarction. The general effective rate was 70.8% (46/65) and the recurrent rate was 10.7% (7/65). Of them, the response to treatment lasted over 12 months in 42 cases, accounting for 91.3% (42/46); over 24 months in 24 cases, accounting for 52.2% (24/46); and over 37 months in 12 cases, accounting for 26.1% (12/46). The effective rates were 100% in 12 patients who received 2-4 times transplantation and 64.2% in 53 patients who received 1 time transplantation, showing statistically significant difference between them (P lt; 0.001). Conclusion The middle-term outcome of autologous bone marrow mononuclear cells transplantation show that it is a feasible and simple method for treatment of lower l imb ischemia.
ObjectivesTo systematically review the efficacy and safety of intravascular ultrasound (IVUS) and coronary angiography-guided percutaneous coronary intervention (PCI) in left main coronary artery disease. MethodsPubMed, EMbase, The Cochrane Library, CBM, CNKI, VIP and WanFang Data databases were electronically searched to collect randomized controlled trials (RCTs) and cohort studies on the efficacy and safety of IVUS and coronary angiography-guided PCI in left main coronary artery disease from inception to March, 2019. Two reviewers independently screened literature, extracted data and assessed risk of bias of included studies; then, meta-analysis was performed using RevMan 5.3 software.ResultsA total of 7 studies involving 7 777 patients were included. The results of meta-analysis showed that: compared with PCI guided by coronary angiography, the incidence of cardiac death (OR=0.45, 95%CI 0.34 to 0.61, P<0.000 01), myocardial infarction (OR=0.67, 95%CI 0.53 to 0.84, P=0.004), major adverse cardiovascular events (OR=0.46, 95%CI 0.34 to 0.61, P<0.000 01), total deaths (OR=0.54, 95%CI 0.44 to 0.67, P<0.000 01), and in-stent thrombosis (OR=0.28, 95%CI 0.18 to 0.45, P<0.000 01) occurred in PCI guided by IVUS were lower. The differences were statistically significant. However, there were no statistical significance in the incidence of target revascularization in PCI (OR=0.80, 95%CI 0.40 to 1.61, P=0.54) and revascularization of target lesions (OR=0.68, 95%CI 0.36 to 1.27, P=0.23) between two groups.ConclusionsCurrent evidence shows that the IVUS-guided PCI can decrease the incidence of cardiac death, myocardial infarction, MACEs, stent thrombosis, total death and has no effect on target lesion revascularization and target vessel revascularization. Due to limited quality and quantity of the included studies, more high-quality studies are required to verify the above conclusion.