ObjectiveTo evaluate the application of portosystemic shunt after subcutaneous transposition of the spleen (STS) to orthotopic liver transplantation (OLT) in the rat. MethodsOne hundred and eighty Wistar rats were randomly divided into the group of orthotopic liver transplantation after portosystemic shunts by subcutaneous transposition of the spleen (STS+OLT group) and the group of orthotopic liver transplantation (OLT group). The two groups were further divided into A, B, C subgroups in the light of duration of anhepatic phase (phases were respectively less than 25 min, around 35 min and 45 min).There were fifteen rats in each subgroup. At the described intervals, blood samples were collected from the peripheral and portal vein for testing ALT, pH and endotoxin levels. The survival rates were also observed. Results The ALT value of all animals basically returned to normal levels on the 7th postoperative day in the STS+OLT group and the OLT A subgroup, but in OLT B subgroup, ALT was still remarkably elevated on the 7th postoperative day (P＜0.01), and returned to normal levels on the 30th postoperative day. The pH values and endotoxin levels from the portal vein of all animals in STS+OLT groups and OLT A subgroup had no significant difference (Pgt;0.05) at the beginning, the end of the anhepatic phase and at the time of reperfusion for 30 min. But in the OLT B and C groups, the pH values and endotoxin levels were significantly higher at the end of anhepatic phase and reperfusion for 30 min than those in the beginning of anhepatic phase (P＜0.01). The survival rates at postoperative different time points in both B and C subgroup of the OLT group were significantly lower than those in STS+OLT group animals (P＜0.05).Conclusion The portosystemic shunt by subcutaneous transposition of the spleen can notably improve both the success rate of the OLT procedure and the postoperative survival rate in the rat.
To explore the role of cell apoptosis in denervated skeletal muscle atrophy in rats and the effect of losartan on it. Methods Forty-two Sprague Dawley rats were randomly divided into 3 groups: group I (n =14, normal control group), group II (n =14, denervated group) and group III (n =14, losartan group). The rats were not treated in group I, and were made denervated gastrocnemius models in groups II and III. In group III, the rats were treated with losartan 10 mg /kg• d by gavage and with normal sal ine in groups I and II. After 4 weeks, gastrocnemius mass to body mass ratio (GAS/BM) served as the degree of muscle atrophy. Apoptotic cells in gastrocnemius were stained in situ by using TUNEL. Gastrocnemius Bcl-2 and Bax protein were quantified by immunohistochemistry and Western blot. Bax /Bcl-2 served as the degree of apoptosis. Results The ratio of apoptosis was higher in group II than that in group I (11.32% ± 4.51% vs 0.56% ± 0.21%, P lt; 0.05). The ratio of apoptosis was lower in group III than that in group II (7.21% ± 2.05% vs 11.32% ± 4.51%, P lt; 0.05). The atrophy of skeletal muscle（GAS/BM） in group II was more serious than that in group I (11.68 ± 1.98 vs 12.86 ±0.74, P lt; 0.05), there was no significant difference between group III and group II (12.11 ± 0.65 vs 11.68 ± 1.98, P gt; 0.05). The expression of Bcl-2 in group II (18.3% ± 4.9%) was significantly lower than that in group I (27.5% ± 2.8%) and group III (25.5% ± 3.5%); there was no significant difference between group III and group I (P gt; 0.05). The expression of Bax in group II (24.1% ± 3.1%) was significantly higher than that in group I (22.1% ± 3.6%) and group III (21.7% ± 2.3%); there was no significant difference between group III and group I (P gt; 0.05). Western blot results showed that: the expressions of Bcl-2 were 122.5 ± 14.6 in group II, 135.3 ± 6.2 in group I and 139.2 ± 16.2 in group III; showing significant diffeerences between group II and group I, between group III and group II (P lt;0.05). The expressions of Bax were 107.1 ± 15.8 in group II, 89.3 ± 8.4 in group I, and 94.2 ± 9.5 in group III; showing significant diffeerences between group II and group I, between group III and group II (P lt; 0.05). There was no significant difference in the expression of Bcl-2 and Bax between group Ⅲ and group I (P gt; 0.05). Conclusion Cell apoptosis plays an important role in denervated skeletal muscle atrophy in rats and may be one of the factors causing skeletal muscle atrophy. Losarton can decrease skeletal muscle cell apoptosis through regulating the ratio of Bax / Bcl-2.
【Abstract】Objective To investigate the effect of donor blood transfusion on inducing pancreatic allograft tolerance in outbred rat model. Methods Wistar male rats were used as blood and pancreas donor, and diabetic recipients. One ml of donor blood injected into abdomen of diabetic recipients on the day of transplantation and azathioprine given 2 days pretransplant and continued for three days. Results Pancreas allograft survival was significantly prolonged (28 to 112 days, media survival time 64.2 days). One ml of donor blood alone injected into the abdomen and azathioprine given alone 2 days pretransplant did not improve allograft survival (media survival time 9.8 vs 10.2 days). Conclusion Donor blood injected on the day of transplantation and a 3 days course of azathioprine started 2 days pretransplant have b synergism in inducing long term graft survival in this rat model.
Objective To study the effect of recombinant lentiviral vector mediated human hepatocyte growth factor (hHGF) gene-modified bone marrow mesenchymal stem cells (BMSCs) on the immunological rejection after allograft l iver transplantation in rats, and to reveal the mechanism of immune tolerance. Methods Eight male Sprague Dawley (SD)rats of clean grade (aged 3 to 4 weeks, weighing 75-85 g) were selected for the isolation and culture of BMSCs; 64 adult male SD rats of clean grade (weighing 200-250 g) were used as donors; and 64 adult male Wistar rats of clean grade (weighing 230-280 g) were used as receptors. After establ ishing a stable model of rat allogeneic l iver transplantation, 1 mL sal ine, 2 ×106/mL of BMSCs 1 mL, 2 × 106/mL of BMSCs/green fluorescent protein 1 mL, and 2 × 106/mL of BMSCs/hHGF 1 mL were injected via the portal vein in groups A, B, C, and D respectively. Then the survival time of the rats was observed. The hepatic function was determined and the histological observation of the l iver was performed. The hHGF mRNA expression was detected by RT-PCR, the level of cytokine including hHGF, interleukin 2 (IL-2), IL-4, IL-10, and interferon γ (IFN-γ) by ELISA assay, the level of apoptosis by TUNEL method, and the expression level of prol iferating cell nuclear antigen (PCNA) by immunohistochemical method. Results The survival time of group D was significantly higher than that of groups A, B, and C (P lt; 0.01); the survival time of groups B and C was significantly higher than that of group A (P lt; 0.01), but there was no significant difference between group B and group C (P gt; 0.05). RT-PCR demonstrated the transcription of hHGF mRNA in the grafts of group D; the serum cytokine hHGF reached to (6.2 ± 1.0) ng/mL. Compared with groups B and C, group D exhibited significant inhibitory effect, significantly improved l iver function, and showed mild acute rejection. In addition, the levels of cytokine IL-2 and IFN-γ decreased; the levels of cytokine IL-4 and IL-10 increased; the level of apoptosis reduced; and the expression level of PCNA increased. Except for the expression of IL-4 (P gt; 0.05), there were significant differences in the other indexes between group D and groups B, C (P lt; 0.05). Conclusion BMSCs/hHGF implanting to rat l iver allograft via portal vein can induce immune tolerance. Compared with injection of BMSCs alone, BMSCs/hHGF treatment can alleviate acute rejection and prolong the survival time significantly. The immunosuppressive effect of BMSCs/hHGF is correlated with Th2 shifts up of Th1/Th2 shift, reduced apoptosis, promoted l iver regeneration.
Objective To study morphological and biological senescence changes induced by D-galactose in the cultured rat mesenchymal stem cells. Methods After 3rd generations cultured in the DMEM-F12, MSCs were changed into DMEMF12 medium containing 8 g/L D-galatose and cultured to the 6th generations as the inducement group. The comparison were the 6thgenerations which was cultured in the DMEM-F12 medium all along, and then indentified by surface wave. Using flow cytometer to check the comparisons cell cycle change after swing in with 8 g/L D-galatose within the 4 days. In the first 7daysto draw the growth curve to the two groups. Optical and electronic microscope were used to identify the influences of characteristic morphological of mesenchymal stem cells of the two groups, the influences of biological markers were identified by single cell gel electrophoresis and β-galactose dye. Results After treatment with D-galactose, the mesenchymal stem cells displayed morphological and biological changes in the cell senescence with the senescent characteristic morphological markers; 85% of the cells were X-gal dye masculine, and the singal cell gel electrophoresis showed DNA damnification. The flow cytometry showed that 90% of the cells stayed in G 0/G 1, but the cells in S and G 2/M almost disappeared.However, the cells in the control group had no such DNA damages. Conclusion D-galactose can induce senescence of the mesenchymal stem cells, and 8 g/L is the best concentration to do so. This study has provided a good model forthe research of the mesenchymal stem cells senescence.
Objective To develop a technique that can directly demonstrate collateral sprouting of intact nerve fibers at endtoside neurorrhaphy site. Methods Five Wistar adult rats were used in this study. The common peroneal nerves at one side were sectioned at the level of knee joint, and their distal ends were sutured to the tibial nerves after removal of a 1 mm-diameter window in the epineurium. Three months after the operation, the nerve segments at neurorrhaphy site and the normal tibial nerves at the contralateral site were harvested. The specimens were fixed in 10% formaldehyde and postfixed in 1% osmium tetroxide, thenmacerated in glycerol. Single fiber was teased out in pure glycerol under an operative microscope, then transferred to a slide and observed under light microscope. The nerve segments at neurorrhaphy site and distal peroneal nerves were alsoharvested for histological evaluation. Results At the neurorrhaphy site, small nerve fibers sprouted from a donor nerve fiber near node’s of Ranvier. While such phenomena were not found in normal tibial nerve. From the longitudinal sectionof neurorrhaphy site, bundles of nerve fibers ranged from tibial nerve to peroneal nerve were observed. Lots of regenerative nerve fibers emerged in distal peroneal nerve. Conclusion The phenomena of collateral sprouting at end-to-side neurorrhaphy site can be demonstrated directly by nerve fiber micro-tease technique.
Objective To investigate the quantity and distribution of motor fiber of rat’s C7 nerve root. Methods Motor fiber quantity and section area in the main nerves of the upper extremity and the fascicles of C7 in 30 SD rats were analyzed.Results Fascicles and certain amount (207) of motor fibers from the anterior division of C7 were distributed to musculocutaneous nerve and median nerve, the orientation of these fibers were not clear. The ones (323) from posterior division were to the axillary, radial, and dorsal thoracic nerves, thus the orientation of these fascicles was relatively definite. Conclusion Thedistribution of the motor fibers and fascicles in the divisions of C7 in rat is similar to human beings, so rat is a relatively good model for the study of selective C7 nerve root transfer.
【Abstract】 Objective The seed cells source is a research focus in tissue engineered cartilage. To observe whether the post-RNA interference (RNAi) chondrocytes could be used as the seed cells of tissue engineered cartilage. Methods Chondrocytes were separated from Sprague Dawley rats. The first passage chondrocytes were used and divided into 2 groups: normal chondrocytes (control group) and post-RNAi (experimental group). Normal and post-RNAi chondrocytes were seeded into chitosan/gelatin material and cultured in vitro to prepare tissue engineered cartilage. The contents of Aggrecan and Aggrecanase-1, 2 were measured by HE and Masson staining, scanning electron microscope (SEM), and RT-PCR. Results The histological results: no obvious difference was observed in cell number and extracellular matrix (ECM) between 2 groups at 2 weeks; when compared with control group, the secretion of ECM and the cell number increased in experimental group with time. The RT-PCR results: the expression of Aggrecan mRNA in experimental group was significantly higher than that in control group (P lt; 0.05); but the expressions of Aggrecanase-1, 2 mRNA in experimental group were significantly lower than those in control group (P lt; 0.05). The SEM results: the cell number in experimental group was obviously more than that in control group, and the cells in experimental group were conjugated closely. Conclusion The post-RNAi chondrocytes can be used as the seed cells for tissue engineered cartilage, which can secrete more Aggrecan than normal chondrocytes. But their biological activities need studying further.
Objective To investigate the effect of pre-infusion with allogeneic lymphocytes treated by 5-FU on inducting immune tolerance of liver transplantation in rats. Methods Wistar and SD rats were used as liver transplantation donors and recipients, respectively. They were divided into 4 groups as following: control group: liver was transplanted from Wistar to SD rats without any other treatment; lymphocytes group: recipient was pre-infused lymphocytes （5×106 cell/ml, 1 ml） from Wistar rat 7 d and 4 d separately before transplantation; low concentration of 5-FU with lymphocytes group: lymphocytes were treated by 5-FU (7.5 μg) before pre-infusion; high concentration of 5-FU with lymphocytes group: lymphocytes were treated by 5-FU (15 μg) before pre-infusion. Pathological changes were observed on day 7 after liver transplantation. Results Acute slight rejection was observed in low concentration of 5-FU with lymphocytes group: liver cell cords were well-arranged basically, hepatic lobules structures could be observed, a few inflammatory cells infiltrated around central veins, and a few lymphocytes infiltrated around portal area. Acute severe rejection was observed in control group, and acute moderate rejection was observed in high concentration of 5-FU with lymphocytes group and lymphocytes group. Conclusion Pre-infusion of lymphocytes treated with low level 5-FU can induce immune tolerance better in recipients after liver transplantation.
Objective To investigate the effect of N-acetylcysteine (NAC) on the apoptosis during myocardial ischemia reperfusion injury in rats’ heart transplantation, and to explore the possible role of NAC in myocardial apoptosis. Methods Sixty healthy male Lewis rats (weighing, 200-220 g) were randomly divided into 3 groups, 20 rats each group (10 donors and 10 recipients). In control group, 1 mL normal saline was infused via inferior vena cava at 30 minutes before donor harvesting; in donor preconditioning group, NAC (300 mg/kg) was infused via inferior vena cava at 30 minutes before donor harvesting, but no treatment in recipients; and in recipient preconditioning group, NAC (300 mg/kg) was infused via inferior vena cava at 30 minutes before recipient transplantation, but no treatment in donors. Heart transplantation was established in each group. Blood was drawn at 6 and 24 hours after reperfusion for analysis of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) as markers of graft injury; myocardial tissue was harvested to determine the superoxide dismutase (SOD) and lipid hydroperoxide (LPO) activity at 24 hours after reperfusion and to observe the histology and ultrastructural changes. Graft active Caspase-3 protein expression was measured by immunohistochemistry staining, and apoptosis index (AI) was calculated by TUNEL. Results The heart transplantation operation was successfully completed in all groups, and the rats survived to the end of the experiment. The serum levels of AST, ALT, and LDH in donor and recipient preconditioning groups were significantly lower than those in control group at 6 hours after reperfusion (P lt; 0.05); the levels of AST and ALT in donor preconditioning group and the levels of AST and LDH in recipient preconditioning group were significantly lower than those in control group at 24 hours (P lt; 0.05); and no significant difference was found between donor and recipient perconditioning groups (P gt; 0.05). The levels of AST, ALT, and LDH at 24 hours were significantly lower than those at 6 hours in each group (P lt; 0.05) except the level of ALT in recipient preconditioning group (P gt; 0.05). SOD activity and SOD/LPO in donor and recipient preconditioning groups were significantly higher than those in control group (P lt; 0.05), but no significant difference between donor and recipient preconditioning groups (P gt; 0.05); there was no significant difference in LPO activity among 3 groups (P gt; 0.05). Histological staining and transmission electron microscope showed that myocardial injury in recipient preconditioning group was obviously lighter than that in donor preconditioning group and control group. Active Caspase-3 in recipient pretreatment group was significantly higher than that in donor preconditioning group and control group (P lt; 0.05). AI of donor and recipient preconditioning groups was significantly lower than that of control group (P lt; 0.05), but no significant difference was found between donor and recipient preconditioning groups (P gt; 0.05). Conclusion NAC can relieve ischemia reperfusion injury in rats’ heart transplantation by improving myocardial SOD content, and reducing active Caspase-3 activity and AI, which has a protective effect on myocardial cell of donor heart.