Objective To establish a simple, valid rat orthotopic left lung transplantation model with the improved operation technique. Methods One hundred and thirty-six male SD rats were randomly divided into donor （n = 68） and recipient （n = 68）, transplantation were performed by using the improved cuff anastomosis technique. Results Time of donor lung perfusion-picking, donor lung vessel cuff anastomosis and recipient vessel anastomosis was 13±2 min, 9±1 min, 10±1 min respectively, the operative time was 60±3 min. In 68 rats of operations, successful rate was 88%（60/68）, anastomotic stoma leak in one rat, lung congestion 3 rats, lung atelectasis 4 rats. The shortest survival time was 1 day, there were 53 rats whose survival time was longer than 12 days. The chest computed tomography showed no atelectasis and blood gas analysis manifested good respiratory function. Conclusion The improved three cuff anastomosis technique offers a simple, valid, cheap and useful method,it can establish rat orthotopic left lung transplantation model successfully.
Objective To investigate the effect of growth differentiation factor 7 (GDF-7) on the tenogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro, to provide evidence for improving the efficacy of BMSCs on tendon repair. Methods BMSCs were isolated from bone marrow tissue of green fluorescent protein rats by density gradient centrifugation method. Chondrogenic, osteogenic, and adipogenic differentiation assays were used to demonstrate the multi-differentiation potential of the BMSCs. BMSCs at passage 3 were cultured and divided into 4 groups according to different concentrations of GDF-7 (0, 12.5, 25.0, and 50.0 ng/mL): group A, B, C, and D, respectively. After cultured for 2 weeks in vitro, the mRNA expressions of scleraxis, tenomodulin, tenascin C, and collagen type I were detected by real-time fluorescent quantitative PCR method, the protein expressions of tenomodulin, tenascin C, and collagen type I by immunocytochemistry staining in 4 groups, and the protein expressions of tenomodulin by Western blot in groups A and C. Results BMSCs had osteogenic, chondrogenic, and adipogenic differentiation potentials. The mRNA expressions of tenomodulin in groups B, C, and D were 2.85, 3.41, and 3.07 times higher than that in group A, respectively; the mRNA expressions of scleraxis in groups B, C, and D were 2.13, 1.50, and 2.56 times higher than that in group A, respectively; and the mRNA expressions of tenascin C in groups B, C, and D were 2.45, 2.86, and 1.88 times higher than that in group A, respectively. There were significant differences between groups B, C, D and group A (P lt; 0.05), while there was no significant difference among groups B, C, and D (P gt; 0.05). The mRNA expressions of collagen type I in groups B and C were 1.92 and 2.45 times higher than that in group A, showing significant differences between groups B, C and group A (P lt; 0.05), but no significant difference between groups A and D (P gt; 0.05). Immunocytochemistry staining showed that the protein expressions of tenomodulin, tenascin C, and collagen type I were detected in groups B, C, and D but not in group A. The results were further confirmed by Western blot results which showed higher protein expression of tenomodulin in group C than in group A. Conclusion GDF-7 can be used to promote tenogenic differentiation of rat BMSCs in vitro.
Objective To investigate the preventive and therapeutic effects and the mechanisms of pyrrol idine dithiocarbamate (PDTC) on the atrophy of denervated skeletal muscle. Methods Thirty adult Wistar rats of either gender, weighing (200 ± 10) g were randomly divided into 3 groups: group A (n=6, control group), group B (n=12, denervation group), and group C (n=12, PDTC treatment group). The sciatic nerves of the rats were only exposed without cutting off in group A, and the rats were made denervated gastrocnemius models in groups B and C. PDTC of 100 mg/(kg•d) was injected peritoneally in group C and an intraperitoneal injection of the same amount normal sal ine was given in group B. After 14 and 28 days, the gastrocnemius was harvested to measure the ratio of muscle wet weight; the levels of nuclear factor of κB (NF-κB)p65 protein and the opening of the mitochondrial permeabil ity transition pore (MPTP) in the gastrocnemius were detectedrespectively by Western blot and laser confocal scanning microscope; and the apoptotic cells in atrophic muscle were measured with TUNEL. Results The ratio of muscle wet weight in group A was 1.039 ± 0.115, and it significantly decreased in groups B and C (P lt; 0.05); after 14 and 28 days of operation, the ratio of muscle wet weight in group C significantly increased when compared with those in group B (P lt; 0.05). The expression of NF-κB p65 protein in group A was 0.224 ± 0.041; the expressions of NF-κB p65 in groups B and C significantly increased when compared with that in group A (P lt; 0.05); however, the expression of NF-κB p65 in group C was significantly lower than that in group B (P lt; 0.05). The MPTP fluorescence intensity in group A was 31.582 ± 1.754; the MPTP fluorescence intensity was significantly lower in groups B and C than in group A (P lt; 0.05), and the MPTP fluorescence intensity in group C was significantly higher than that in group B (P lt; 0.05). The rate of apoptosis in group A was 4.542% ± 0.722%; after 14 and 28 days of operation, the rates of apoptosis significantly increased when compared groups B and C with group A, and signiticantly decreased when compared group C with group B (P lt; 0.05). Conclusion PDTC can retard denervated skeletal muscle atrophy, and the effect may have a relationship with its inhibition on NF-κB, the opening of the MPTP, and the ratio of apoptosis.
Objective To examine an effect of the locally-used platelet derived growth factor-BB (PDGF-BB) on the healing of the medial collateral ligament (MCL) in the knee joints of rats. Methods Forty-eight rats were equally randomly divided into 2 groups: the experimental group (group A) and the control group(group B). MCL of all the rats were ruptured to establish the wound models. In group A, 5 μg of PDGF-BB was locally injected in the wound of each rat and then the wound was sutured; but in group B, the wound was only sutured. After 2 weeks, histological evaluations were performed to determine whether PDGF-BB could promote the healing of MCL. Results There were significantly more fibroblasts formed during the ligament healing process in group A than in group B (213.44±15.32 vs. 180.42±12.78, Plt;0.01). The fibroblasts were more mature andmore regularlyarranged in group A than in group B. The type, content, and crosslink of the collagen were improved to a greater extent in group A than in group B (Plt;0.01). Conclusion PDGF can promote the healing of the injured ligament.
Objective To investigate the effect of combined therapy of granulocyte colony stimulating factor (G-CSF) and bone marrow mesenchymal stem cells (BMSCs) carrying hepatocyte growth factor (HGF) gene on the angiogenesis of myocardial infarction (MI) in rats and the mechanisms of the synergistic effect. Methods BMSCs were aspirated from the femur and tibia of 3-week-old Sprague Dawley (SD) male rats. The third generation of BMSCs were harvested and transfectedwith Ad-HGF. The MI models were establ ished in 44 SD male rats (weighing 200-250 g) by l igating the left coronary artery. At 4 weeks after l igation, the shorting fraction (FS) of the left ventricle being below 30% was used as a criteria of model success. The BMSCs (5 × 107/ mL) transfected with Ad-HGF were transplanted into the infarct zone of 12 SD rats, and the expression of HGF protein was detected by Western blot method at 2, 7, and 14 days after transplantation. At 4 weeks, the other 32 SD rats were randomly divided into 4 groups (n=8). The 0.1 mL normal sal ine was injected into the infarct zone in control group; 0.1 mL normal sal ine was injected combined with intraperitoneal injection G-CSF [100 μg/ (kg•d)] for 5 days in G-CSF group; 0.1 mL BMSCs (5 × 107/ mL) transfected with Ad-HGF was injected into the infarct zone in HGF group; 0.1 mL BMSCs (5 × 107/ mL) transfected with Ad-HGF was injected combined with intraperitoneal injection G-CSF [100 μg/ (kg•d)] for 5 days in combined therapy group. At 2 weeks after transplantation, heart function was detected by cardiac ultrasound and hemodynamic analysis, and then myocardial tissue was harvested to analyse the angiogenesis of the infarct zone, and the expression of VEGF protein by immunofluorescence staining. Results The expression of HGF protein in vivo was detected at 2 days and 7 days of BMSCs transfected with Ad-HGF transplantation. There was no significant difference in left ventricular systol ic pressure (LVSP), left ventricular end-diastol ic pressure (LVEDP), dP/dtmax, and FS between G-CSF group and control group (P gt; 0.05). When compared with the control group, LVEDP decreased significantly; LVSP, FS, and dP/dtmax increased significantly (P lt; 0.05) in HGF group and combined therapy group. When compared with HGF group, FS and dP/dtmax increased significantly in combined therapy group (P lt; 0.05). Immunofluorescence staining showed that the vascular endothel ial cells were observed in myocardial infarction border zone. The vascular density and the expression of VEGF protein were significantly higher in combined therapygroup than in other 3 groups (P lt; 0.05). Conclusion The combined therapy of G-CSF and BMSCs carrying HGF gene has a synergistic effect and can enhance infarct zone angiogenesis through inducing the expression of VEGF protein.
【Abstract】Objective This study was conducted to build experimental model of orthotopic liver transplantation in rat (ROLT) with the character of acute rejection; and to study the effect of cytotoxic T lymphocyte antigen 4 immunoglobulin G (CTLA4Ig) on prevention of rejection and the induction of immune tolerance of ROLT. Methods Build model of Wistar→SD ROLT(performed by the two cuff method) with character of acute rejection.Recipients were injected with CTLA4Ig 75 μg per ROLT into abdominal cavity after 2 days of operation. Contrast was made with no treatment group, the clinical characters, the liver function, the transplantated liver pathologic character and the concentrations of TNFα in serum were observed and measured on postoperative day 7. In treatment group, all above observation were done on postoperative month 4. Above all, determination of the effect of CTLA4Ig on preventing acute rejection and inducing tolerance in ROLT was observed.Results ①Recipients (no treatment group) died one by one within 6th～14th days; pathologic character of rejection in transplantation liver could be found; ② In treatment group, on postoperative day 7 and month 4, no clinical rejection character and no pathologic character of rejection in transplantation liver were found and serum concentration of cytokins related to TNFα found lower than that of contrast group(P＜0.05), and serum concentration of ALT、AST、TBIL、DBIL found lower too(P＜0.05); But serum concentration of TP and Alb was found higher than that of contrast group(P＜0.05). Conclusion ① CTLA4Ig treatment alone inhibits the rejection responce in ROLT. ② CTLA4Ig treatment can Prevent rejection and induce immune tolerance in ROLT model with characters of acute rejection; the serum concentration of cytokins related to TNFα can probably be used as evaluation standard of rejection in ROLT rejection.
Objective To investigate the effect of olfactory ensheathing cell culture medium (OECCM) on the growth of spinal cord neurons and its protective effect on the injured neurons by H2O2, and to disscuss the probable protective mechanisms of olfactory ensheathing cells (OECs). Methods The primary olfactory ensheathing cells (OECs) were isolated from olfactory bulb of adult SD rat, and OECCM were prepared. The morphology of OECs was observed by inverted phase contrast microscope, identified by rabbit-antiratlow-affinity nerve growth factor p75 (NGFRp75), and its purity were calculated.Primary spinal cord neurons were cultured from 15 to 17 days pregnant SD rats, and injury model of neurons were prepared by H2O2. OECCM and control culture medium were added into the normal spinal neurons (groups A, B). OECCM and control culture medium were added into the injured spinal neurons by H2O2 (groups C, D). In groups A and C, 200 μL of control culture medium was used; in groups B and D, 100 μL of control culture medium and 100 μL of OECCM were used. Then the growth index such as average diameter of neuron body, the number and length of neuron axons were measured. The viabil ities of normal and injured neurons were assessed by MTT. Results OECs showed bipolar or tripolar after 6-9 days of culture. Primary spinal cord neurons were round and bigger, and neuron axons grew significantly and showed bipolar after 5-7 days of culture. The immunocytochemisty of OECs by NGFRp75 showed that membrane were stained. The degree of purity was more than 90%. Primary spinal cord neurons grew well after 6-9 days of culture, and compared with group A, neurons of group B grew b, whose cell density and diameter were bigger. The average diameter of neuron body, the number and length of neuron axons were (33.38 ± 6.80) D/μm, (1.67 ± 0.80), and (91.19 ± 62.64) L/μm in group A, and (37.39 ± 7.28) D/μm, (1.76 ± 0.82), and (121.33 ± 81.13) L/μm in group B; showing statistically significant differences (P lt; 0.05). The absorbency (A) value of neurons was 0.402 0 ± 0.586 9 in group A and 0.466 0 ± 0.479 0 in group B; showing statistically significant difference (P lt; 0.01). After 2 hours of injury by H2O2, the cell density of spinal cord neurons decreased, and neuron axons shortened. The A value of injured neurons was 0.149 0 ± 0.030 0 in group C and 0.184 0 ± 0.052 0 in group D, showing statistically significant difference (P lt; 0.01). Conclusion The results above suggest that OECCM could improve the growth of spinal cord neurons and protectthe injured neurons. The neurotrophic factors that OECs secrete play an important role in the treatment of spinal cord injury.
Objective Neuron purification is essential to procedure of various nerve cell experimental research, however, at present there is few reports on the effect of various factors on neural axons during purification. To find out a simple method of neuron purification, and to investigate the influence factors of corresponding purification culture in dorsal root gangl ion (DRG) tissue culture on β3-tubul in positive axon. Methods The DRGs were obtained from the 3 days neonatal SD rat microscopically and were made into cell suspension. Then, the amount of attached DRG neurons and non neuronal cells in poly-D-lysine (PDL) group, PDL/Laminin (PDL/LN) group and collagen-I (Col I) group was observed from 10 to 100 minutes. Then, the extension and arborization of β3-tubul in positive axons were observed after 72 hours completely randomised DRG tissue culture for the research of the influences among culture substrates (PDL, PDL/LN, and Col I), FBS (0, 5%, and 10%), 5 fluorouracil (5-Fu, 0, 20, and 40 μmol/L), and cytrarabine (Ara-C, 0, 10, and 20 μmol/L). Results Adherent cells were observed instantly after inoculation by inverted phase contrast microscope and inverted fluoresence microscope; after cell suspension was removed, adherent growth of DRGn cells and non-DRGn cells were still seen. In PDL group, the amount of NSE negative cells was significantly higher than that of NSE positive cells at 10 and 30 minutes (P lt; 0.05); the amount of NSE positive cells was significantly higher than that of NSE negative cells at 80, 90 and 100 minutes (P lt; 0.05). In PDL/LN gruop, there was no significant difference (P gt; 0.05) in the amount of NSE negative cells and NSE positive cells at 10, 20, 30, 40, and 50 minutes; the amount of NSE positive cells was significantly higher (P lt; 0.05) than that of NSE negative cells at 60, 70, 80, 90, 100 minutes. In Col I group, the amount of NSE negative cells was higher than that of NSE positive cells at 10-40 minutes, but showing no significant difference (P gt; 0.05); the amount of NSE positive cells was significantly higher (P lt; 0.05) than that of NSE negative cells at 70-100 minutes. At 72 hours after DRG tissue culture, the best result of β3-tubul in positive axon extension and arborization was obtained when the substrate level was PDL/LN, and the average length of PDL/LN level was significantly larger than that of other two substrates (P lt; 0.05). The highest number of β3-tubul in positive axon distal end was obtained at 5% concentration level of FBS (P lt; 0.05), but showing no significant differences in β3-tubul in positive axon length among three levels (P gt; 0.05). Both the most of β3-tubul in positive axon distal ends and the longest β3-tubul in positive axon average length were obtained at 0 μmol/L concentration level of 5-Fu, showing significant differences between 0 μmol/L level and 20, 40 μmol/L levels (P lt; 0.05). A similar result of β3-tubul in positive axon distal end was got at the 0 μmol/L level and 10 μmol/L level of Ara-C, which was significantly higher than that of 20 μmol/L level (Plt; 0.05). Conclusion? A purified DRG neuron suspension for neuron culture could be obtained via PDL differential attachment for 30 minutes. When DRG neuron culture, neuron special medium, PDL/LN substrate and 10 μmol/L Ara-C are recommended in β3-tubul in positive axon research.
Objective To establish a reliable rats model of orthotopic liver transplantation with non-heart beating donors. Methods The model was established with modified double-cuff method. According to obtain pre-liver warm ischemia time experiencing non-heart-beat the rats were divided into 3 groups: 10 min (R10 group), 20 min (R20 group) and 30 min (R30 group), then one week survival after operation was compared in rats. Results The operative time of donor was 30 min approximately except warm ischemia time and the cold preservation time of donor liver was 1 h. The anastomotic time for suprahepatic vena cava was 12-22 min (mean 15 min). The anastomotic time for portal vein and infrahepatic vena cava was about 2 min and 1 min, respectively. The anhepatic phase sustained 14-24 min (mean 19 min). The operative time of receptor was 50-65 min (mean 60 min). Twelve rats died at 24 h after operation, which was considered as operative failure. The success rates of operation in R10 group, R20 group, and R30 group were 95% (19/20), 80% (16/20), and 65% (13/20), respectively. After one week the survival rate was 95% (18/19), 81% (13/16), and 54% (7/13), respectively. Conclusions Improved non-heart donor liver transplantation model of rat on the basis of Kamada’s “twocuff technique” acts as a good simulation in clinical non-heart-donor liver transplantation. This study showes that rat liver can tolerate warm ischemia time less than 30 min, the short-term survival after transplantation can reach satisfactory results. However, long-term survival requires further study.
Objective Bone marrow mesenchymal stem cells (BMSCs), as replacement cells of Schwann cells, can increase the effect of peripheral nerve repair. However, it has not yet reached any agreement to add the appropriate number of seeded cells in nerve scaffold. To investigate the effect of different number of BMSCs on the growth of rat dorsal root gangl ia(DRG). Methods Three 4-week-old Sprague Dawley (SD) rats (weighing 80-100 g) were selected to isolate BMSCs, whichwere cultured in vitro. Three 1- to 2-day-old SD rats (weighing 4-6 g) were selected to prepare DRG. BMSCs at passage 3 were used to prepare BMSCs-fibrin glue complex. According to different number of BMSCs at passage 3 in fibrin glue, experiment was divided into group A (1 × 103), group B (1 × 104), group C (1 × 105), and group D (0, blank control), and BMSCs were cocultured with rat DRG. The axon length of DRG, Schwann cell migration distance, and axon area index were quantitatively evaluated by morphology, neurofilament 200, and Schwann cells S-100 immunofluorescence staining after cultured for 48 hours. Results Some long cell processes formed in BMSCs at 48 hours; migration of Schwann cells and axons growth from the DRG were observed, growing in every direction. BMSCs in fibrin glue had the biological activity and could effect DRG growth. The axon length of DRG and Schwann cell migration distance in groups A, B, and C were significantly greater than those in group D (P lt; 0.05). The axon length of DRG and Schwann cell migration distance in group C were significantly less than those in group B (P lt; 0.05), but there was no significant difference between group A and group C, and between group A and group B (P gt; 0.05). The axon area index in groups A and B was significantly greater than that in group D (P lt; 0.05), but there was no significant difference between group C and group D (P gt; 0.05); there was no significant difference in groups A, B, and C (P gt; 0.05). Conclusion In vitro study on DRG culture experiments is an ideal objective neural model of nerve regeneration. The effect of different number of BMSCs in fibrin glue on the growth of DRG has dose-effect relationship. It can provide a theoretical basis for the appropriate choice of the BMSCs number for tissue engineered nerve.