Objective To investigate the neuroprotective effects of recombinant adeno-associated virus (rAAV) expressing vascular endothel ial growth factor (VEGF) on traumatic spinal cord injury (SCI) of rat and its mechanisms. Methods The 144 male Sprague Dawley rats were randomly divided into 4 groups, and each group contained 36 rats. The rats in sham group (group A) received dorsal laminectomy without SCI and microinjection, the rats in model control group (group B), rAAV-green fluorescent protein (GFP) group (group C), and rAAV-hVEGF165-GFP group (group D) received dorsallaminectomy with SCI and injection of 20 μL sal ine, rAAV-GFP viruses, or rAAV-hVEGF165-GFP viruses, respectively. At 3 and 7 days after operation, Basso-Beattie-Bresnahan (BBB) score was used to evaluate the neurologic function. At 7 days after operation, Nissl’s body staining was used to evaluate the histopathological changes; apoptosis was confirmed by transmission electron microscope examination and TUNEL staining; the expression of aquaporin 4 (AQP-4) was detected by Western blot assay. At 1, 3, 5, and 7 days, ELISA assay was used to detect the VEGF165 protein expression. Results According to BBB scores, the neurologic function in group D was significantly better than those in groups B and C at 3 and 7 days after operation (P lt; 0.05). Nissl’s body staining showed that tissue damage in group D was significantly milder than those in groups B and C at 7 days after operation (P lt; 0.05). ELISA results showed that VEGF165 protein expression was slowly-released in low dose in group D, and the expression in group D was significantly higher than that in groups A, B, and C at 3, 5, and 7 days after operation (P lt; 0.05). The results of transmission electron microscope and TUNEL staining showed that apoptosis rate of spinal cord neurons in group D was significantly lower than that in groups B and C at 7 days after operation (P lt; 0.05). The results of Western blot showed that AQP-4 expression in group D was significantly decreased when compared with that in groups B and C at 7 days after operation (P lt; 0.05). Conclusion TherAAV expressing VEGF has neuroprotective effects by inhibiting apoptosis of spinal cord neurons and relieving spinal cord edema.
Objective To investigate the expression levels of osteoprotegerin (OPG) and receptor activator of NF-κB l igand (RANKL) mRNAs in bone tissues of the femoral head of the patients suffering glucocorticoid-induced osteonecrosisof the femoral head (ONFH), and to discuss the relationship between OPG/RANKL and ONFH. Methods Between March2007 and March 2008, bone tissues of the femoral head were collected as the experimental material from 35 patients suffering ONFH (experimental group) and from 21 patients suffering fracture of femoral neck (control group). The ratio of men to women in both groups was 4 ∶ 3, whose age was 41-70 years old (55.34 on average in the experimental group and 55.33 on average in the control group). The experimental group received over 3 weeks’ glucocorticoid treatment or more than 1 week’ s high-dose glucocorticoid treatment in recent 2 years, while the control group never received more than 1 week’s hormone treatment. In the two groups, the microstructure of bone tissues of the femoral head was detected by HE staining and the bone tissue total RNA was extracted, and then the expression levels of OPG mRNA and RANKL mRNA were examined by realtime quantitative PCR (RTQ-PCR) for each sample. Results HE staining: bone trabeculae and bone units were replaced by interrupted bone fragments, which were surrounded by many inflammatory granulation tissues and few osteocytes were seen in bone lacunae in the experimental group. In the control group, bone trabeculae and bone units were made by complete lamellar bones which surrounded blood vessels and osteocytes were seen in lacunae. RTQ-PCR testing: in the experimental group, OPG mRNA and RANKL mRNA were 1.35 ± 0.42 and 4.36 ± 1.35, respectively, while in the control group they were 1.78 ± 0.63 and 3.49 ± 1.02, respectively. The expression level of OPG mRNA in the experimental group was significantly lower than that in the control group, and the expression level of RANKL mRNA of the former was significantly higher than the latter. The OPG mRNA/ RANKL mRNA ratio in the xperiment group (0.34 ± 0.16) was significantly lower than that in the control group (0.54 ± 0.20), and there was significant difference (P lt; 0.05). Conclusion The glucocorticoid-induced ONFH may be related to the expression levels of OPG mRNA/RANKL mRNA in bone tissues.
Objective To study the effect of olfactory ensheathingcells(OECs) transplantation on protecting spinal cord and neurons after peripheral nerve injury. Methods Fifty-five SD rats were randomly divided into blank group (n=5), experimental group (n=25) and control group (n=25). The right sciatic nerves of all the rats were transected. The proximal end was embedded in muscle and treated with OECs (experimental group) and DMEM (control group). No treatment was given to the blank group. The rats were sacrificed 1, 2, 3, 7, and 14 days after the transplantation, the related neurons were observed with histological and TUNEL methods. Results After sciatic nerves were transected, death of neurons occurred in spinal cord and ganglion. One, 2, 3 days after treatment, the neuron survival rate in experimental group was 98.4％±6.5%，97.6％±6.5%，95.2％±6.7% respectively. The neuron survival rate in control group was 97.8％±6.7%，97.4％±6.4%，94.3％±6.8% 1, 2, and 3 days after treatment respectively. There was no significant difference between experimental group and control group. Seven and 14 days after treatment, the neuron survival rate in experimental group was 92.4％±8.9%，87.7％±9.4% respectively. The neuron survival rate in control group was 87.4％±8.6%，83.4％±8.5% 7 and 14 days after treatment respectively. There was significant difference between experimental group and control group. On 1st and 2nd day, no apoptosis was seen in spinal cord anterior horn of the rats in both experimental group and control group. On 3rd, 7th, and 14th day, the apoptosis index of spinal cord anterior horn motoneuron in experimental rats were lower（1.2±0.8，1.4±0.6，4.1±1.3） than that in the control group（2.1±1.1，3.1±1.1，6.1±1.8）（Plt;0.05）. One, 2, and 3 days after the operation, no ganglion neurons apoptosis was observed in all rats. On 7th day the apoptosis index of ganglion neurons in experimental group（2.10±0.32）were lower than thatin control group （4.40±0.56）（Plt;0.05）. On 14th day there was no significant difference in the apoptosis index of ganglion neurons between experimental group （4.30±1.80）and control group（6.70±2.50）(P＜0.05). Conclusion Apoptosis of neurons occur after peripheral nerve injury in spinal cord and ganglion. OECs transplantation is effective in preventing apoptosis.
目的 观察评价盐酸氨基葡萄糖结合塞来昔布治疗膝骨关节炎（KOA）的疗效及安全性。 方法 2001年3月－2012年3月采用随机对照方法，将184例KOA患者随机分为对照组与试验组，各92例。对照组单独给予塞来昔布，试验组给予塞来昔布和盐酸氨基葡萄糖，共治疗8周，停药后继续观察4周。采用Lequesne指数作为疗效评分标准，观察服药前后的膝关节症状变化，包括休息痛、运动痛、压痛、肿胀、晨僵和行走能力的改善程度，纪录不良反应及实验室生化指标等。 结果 两组Lequesne指数在治疗前相比均明显下降，两组治疗8周后Lequesne总指数比较差异均有统计学意义（P＜0.05）。治疗8周后试验组总有效率明显优于对照组。安全性方面两组比较无差异。 结论 盐酸氨基葡萄糖结合塞来昔布治疗KOA，能明显改善患者的临床症状，疗效优于单纯的塞来昔布治疗，且不会增加药物不良反应，具有较好的临床价值。
Objective To study the biological activity of recombinant adeno-associated virus vector (rAAV) coexpressing human vascular endothel ial growth factor165 (hVEGF165) and human bone morphogenetic protein 7 (hBMP-7) genes in vitro so as to provide a new method for the therapeutics of osteonecrosis. Methods The 3rd passage rabbit bone marrow mesenchymal stem cells (BMSCs) were transfected with rAAV-hVEGF165-internal ribosome entry site (IRES)-hBMP-7(experimental group) and green fluorescent protein (GFP) labeled rAAV-IRES-GFP (control group). The expressions ofhVEGF165 and hBMP-7 were detected by ELISA assay at the 1st, 2nd, 3rd, 7th, 14th days and Western blot assay at the14th day after transfection. The expression consistencies of hVEGF165 and hBMP-7 were observed by immunofluorescence assay at the 14th day after transfection. The biological activity of hVEGF165 was assessed by angiopoiesis experiment of the 3rd passage human umbil ical vein endothel ial cells (HUVEC). The biological activity of hBMP-7 was assessed by mineral ization of BMSCs detected by ALP staining and al izarin red staining. Results With infecting time, the hVEGF165 and hBMP-7 expressions increased gradually in two groups, showing significant difference between two groups (P lt; 0.05). The expressions of hVEGF165 and hBMP-7 were positive in experimental group and negative in control group, respectively. Immunofluorescence assay showed positive expressions of hVEGF165 and hBMP-7 in the exprimental group and negative expression in the control group, the expression of hVEGF165 and hBMP-7 had good consistencies. hVEGF165 secreted from BMSCs enhanced HUVEC migration, prol iferation and tube formation in experimental group. There was significant difference in the number of blood vessel between two groups (P lt; 0.05). The ALP staining showed more bly stained granules in experimental group than in control group. There was significant difference in the number of the mineral ized nodules between two groups (P lt; 0.05). Conclusion The rAAV-hVEGF165-IRES-hBMP-7 has good biological activity in vitro.
Objective To investigate the expression levels of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B l igand (RANKL) mRNAs in BMSCs in patients suffering glucocorticoid-induced necrosis of the femoral head (GNFH), and to discuss the relationshi p between OPG/RANKL system and GNFH. Methods The bone tissue and BMSCs of femoral head were collected from 35 patients suffering GNFH (experimental group) and from 21 patients suffering fracture of femoral neck (control group). The ratio of men to women was 4 ∶ 3 in two groups, aged 41 to 70 years (mean 55.34years in the experimental group and mean 55.33 years in the control group). The patients of experimental group received over 3 weeks’ glucocorticoid treatment or more than 1 week’s high-dose glucocorticoid therapy in recent 2 years, but patients of the control group did not receive more than 1 week’s hormone therapy. In 2 groups, the microstructure of bone tissue of femoral head was detected by HE staining. The BMSCs were isolated and cultured by adherent-wall method; the expression levels of OPG and RANKL mRNAs were examined by real-time quantitative polymerase chain reaction and the ratio of OPG mRNA to RANKL mRNA was caculated. Results Bone trabeculae and bone units were replaced by interrupted bone fragments, which were surrounded by inflammation and granulation tissue and few osteocytes were seen in bone lacunae in the experimental group. In control group, bone trabeculae and bone units were made by complete lamellar bone which surrounded blood vessels and osteocytes were seen in lacunae. The expression levels of OPG mRNA in the experimental group (0.37 ± 0.12) was significantly lower than that in the control group (0.47 ± 0.13), and the levels of RANKL mRNA in the experimental group (1.12 ± 0.39) was significantly higher than that in the control group (0.84 ± 0.24), showing statistically significant difference (P lt; 0.05). The ratio of OPG mRNA to RANKL mRNA in the experimental group (0.37 ± 0.17) was significantly lower than that in the control group (0.61 ± 0.26, P lt; 0.05). Conclusion The GNFH may be related to the expression levels of OPG mRNA and RANKL mRNA in BMSCs.
To construct the recombinant adeno-associated virus (rAAV) vector co-expressinghVEGF165 and hBMP-7 depending on internal ribosome entry site (IRES) sequence, to measure the virus titer and to ver ify the correct recombination. Methods The AAV helper-free system was used to generate the rAAV co-expressing hVEGF165 and hBMP-7 genes. The IRES sequence from the bicistronic eukaryotic expression plasmid pIRES was cut down and subcloned into the ITR/MCS containing vector pAAV-MCS to get pAAV-MCS A-IRES-MCS B, in which upstream MCS A and downstream MCS B was constructed. The hVEGF165 and hBMP-7 genes were ampl ified by PCR and inserted into MCS A and MCS B respectively. The recombinant expression plasmid pAAV-hVEGF165-IRES-hBMP-7 was co-transfected into AAV-293 cells with pHelper and pAAV-RC for packaging of recombinant AAV. The green fluorescent protein (GFP) labeled rAAVIRES GFP was simultaneously packaged by using the parallel plasmid pAAV-IRES-GFP. The efficiency of rAAV packagingwas monitored under fluorescent microscope and recombinant viral particles were harvested from infected AAV-293 cells.The virus titer was measured through infecting AAV-HT1080 cells, and the recombinant rAAV-hVEGF165-IRES-hBMP-7was verified by PCR of the exogenous interest genes of hVEGF165 and hBMP-7. Results The recombinant plasmid pAAVhVEGF165- IRES-hBMP-7 was verified by double digestion. Using the AAV helper-free system, GFP expression could be observed under fluorescent microscope 72 hours after triple plasmid co-transfection and the system provided a high packing ratio of 95%-100%. The rAAV has a high purity and high titer of 5.5 × 1011vp/mL, and AAV-HT1080 cell could be infected at a ratio of 90%. The recombinant virus was confirmed by PCR of exogenous hBMP-7 and hVEGF165 genes. Conclusion Re combinant rAAV-hVEGF165-IRES-hBMP-7 was successfully constructed with a high virus titer, which may offer the basement
Objective To evaluate the effectiveness of open wedge high tibial osteotomy (OWHTO) in treatment of medial unicompartmental knee osteoarthritis (MUKOA). Methods A clinical data of 61 cases with MUKOA who were treated with OWHTO between January 2015 and January 2017 were retrospectively analyzed. There are 14 males and 47 females with an average age of 52.8 years (mean, 44-60 years). The body mass index ranged from 19.1 to 34.7 kg/m2 (mean, 25.3 kg/m2). Twenty-seven cases were left side and 34 cases were right side. The disease duration was 1-9 years (mean, 5.3 years). The MUKOA was rated as stage Ⅱ in 33 cases and stage Ⅲ in 28 cases. Preoperative Hospital for Special Surgery (HSS) score was 56.0±3.7. Walking visual analogue scale (VAS) score was 4.6±1.0. Results The operation time was 49-85 minutes (mean, 66.5 minutes). The length of incision was 10-13 cm (mean, 11.0 cm). The total overt blood loss was 80-210 mL (mean, 139.1 mL). The postoperative bed-rest time was 1-10 days (mean, 4.7 days). All patients were followed up 12-24 months (mean, 17.3 months). The bearing area of tibial platform at 3 months after operation was 60.3%-66.8%, with an average of 63.4%. At 3 and 6 months after operation, the HSS score was 79.1±4.2 and 85.3±3.1 respectively, and the VAS score was 1.7±0.7 and 0.6±0.5 respectively, all showing significant differences (P<0.05). Conclusion OWHTO is an ideal choice for treating MUKOA with less postoperative complications. The force line could be corrected by OWHTO. However, the preoperative preparations are very important, especially that the open angle should be measured accurately.
ObjectiveTo compare the short-term effectiveness between arthroscopic cystectomy and internal drainage combined with cystectomy in popliteal cyst.MethodsBetween March 2014 and March 2017, 56 patients with symptomatic popliteal cyst were enrolled in the study, randomized block design was used to divided the patients into trial group (arthroscopic cystectomy combined with internal drainage group, n=28) and control group (arthroscopic internal drainage group, n=28). Excluding those who had incomplete follow-up and received surgery for other diseases postoperatively, 26 patients in the experimental group and 27 patients in the control group were finally enrolled in the study. There was no significant difference in gender, age, side, course of disease, maximum diameter and grade of popliteal cyst, and associated diseases between two groups (P>0.05). The operation time, duration of popliteal ecchymosis and the middle back of calf tenderness were observed postoperatively. The circumference of calf at 1 day, 1 week, and 2 weeks after operation were measured and the differences were calculated with the measurement before operation. Lower extremity venous thrombosis was observed by color doppler ultrasonography at 1 week after operation. The effectiveness was evaluated by Rauschning and Lindgren grading criteria. And MRI was used to observe whether the popliteal cyst disappeared or decreased and measured its maximum diameter at 1 year after operation.ResultsPatients in both groups were followed up 12-14 months, with an average of 12.5 months. The operation time, duration of popliteal ecchymosis, and the middle back of calf tenderness of the trial group were all longer than those in the control group (P<0.05), the differences of circumference of calf at 1 day, 1 week, and 2 weeks after operation of the trial group were greater than those in the control group (P<0.05). Color doppler ultrasonography of the lower extremity at 1 week after operation found that the intermuscular venous thrombosis occurred in 2 cases of the trial group, while no lower extremity thrombosis was found in the control group; and the difference between two groups was not significant (P=0.236). According to the Rauschning and Lindgren grading criteria, there were 16 cases of grade 0, 6 cases of grade 1, and 4 cases of grade 2 in the trial group, and 17 cases of grade 0, 4 cases of grade 1, and 6 cases of grade 2 in the control group at 1 year after operation. There was no significant difference between 2 groups (Z=–1.872, P=0.078). Nine cases (34.62%) of the trial group and 13 cases (48.15%) of the control group still have residual cysts by MRI, the maximum diameter of which was less than 2 cm. The cysts disappeared in the remaining patients in both groups, and there was no recurrence during the follow-up. There was no significant difference in cyst residual rate between 2 groups (χ2=2.293, P=0.852).ConclusionCompared with arthroscopic internal drainage, the short-term effectiveness of the arthroscopic internal drainage combined with cystectomy had no significant improvement, and the operation time was prolonged, the postoperative complications were obviously increased.