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 study the time effect of the gene expression of recombinant adeno-associated virus (rAAV) vector co-expressing human vascular endothel ial growth factor 165 (hVEGF165) and human bone morphogenetic protein 7 (hBMP-7) genes so as to lay a theoretical foundation for gene therapy of osteonecrosis. Methods The best multipl icity of infection (MOI) of BMSCs transfected with rAAV was detected by fluorescent cell counting. The 3rd generation rabbit bone 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), respectively. The expression of GFP was observed by inverted fluorescent microscope. The expressions of hVEGF165 and hBMP-7 were assessed by RT-PCR assay and Western blot assay in vitro. The transfected cells in 2 groups were prepared into suspension with 5 × 106 cells/mL, and injected into the rabbit thigh muscles of experimental group 1 (n=9) and control group 1 (n=9), respectively. The muscle injected with rAAV-IRES-GFP was sl iced by frozen section method and the expression of GFP protein was observed by inverted fluorescent microscope. The expressions of hVEGF165 and hBMP-7 were assessed by Western blot assay and ELISA assay in vivo. Results The best MOI of BMSCs transfected with rAAV was 5 × 104 v.g/cell. In vitro, the expressions of GFP, hVEGF165, and hBMP-7 genes started at 1 day after transfection, the expressions obviously increased at 14 days after transfection, and the expression maintained the b level at 28 days after transfection. In vivo, the expressions of GFP, hVEGF165, and hBMP-7 genes could be detected at 2 weeks after injection, and b expressions were shown at 6 to 8 weeks after injection. The values of hVEGF165 and hBMP-7 were (248.67 ± 75.58) pg/mL and (4.80 ± 0.61) ng/mL respectively in experimental group 1, and were (32.28 ± 8.42) pg/mL and (0.64 ± 0.42) ng/mL respectively in control group 1; showing significant differences between 2 groups (P lt; 0.05). Conclusion The rAAV-hVEGF165-IRES-hBMP-7 has efficient gene expression ability.
OBJECTIVE: To study the allograft antigenicity of human ear cartilage and the effect of the cell extraction on antigenicity. METHODS: The human ear cartilage was acellular by cell extraction with Triton X-100. Then the cartilage and the acellular cartilage were analyzed by anti-MHC-I immunohistochemical staining, the reaction of the peripheral blood mononuclear(PBM) cells to the cartilage and the acellular cartilage and the migration of the PBM cells toward the cartilage and the acellular cartilage. RESULTS: The result of human ear cartilage was positive for the anti-MHC-I immunohistochemical staining, whereas that of the acellular cartilage was negative for the staining. The reactive proliferation of the PBM cells was more when they were co-cultured with human ear cartilage than that when they were cultured alone in vitro(P lt; 0.05), but the acellular cartilage did not show the same phenomena (P gt; 0.05); when the cartilage and the acellular cartilage were co-cultured with the PBM cells, the PBM cells migrated to the cartilage much more than that to acellular cartilage(P lt; 0.01). CONCLUSION: Human ear cartilage has allograft antigenicity and its antigenicity can be removed by cell extraction with Triton X-100.
Objective To investigate the effectiveness of facial nerve-sublingual nerve parallel bridge anastomosis for facial nerve injury resulting from closed temporal bone fractures. Methods Between January 2017 and December 2019, 9 patients with facial nerve injury resulting from closed temporal bone fracture caused by head and face trauma were treated. Among them, 5 patients were treated with facial nerve-sublingual nerve parallel bridge anastomosis (operation group), and 4 patients were treated with neurotrophic drugs combined with rehabilitation exercise (conservative group). There was no significant difference in gender, age, side, cause of injury, duration of facial nerve injury before surgery, House-brackmann grading (hereinafter referred to as HB grading) of facial nerve injury, and other general information between 2 groups (P>0.05). HB grading was used to evaluate the improvement of facial nerve function before and after treatment. At the same time, facial nerve neuroelectrophysiological test was performed to evaluate the electrical activity of facial muscles before and after treatment. Tongue function, atrophy, and tongue deviation were evaluated after nerve anastomosis according to the tongue function scale proposed by Martins et al. Results Patients in both groups were followed up 12-30 months, with an average of 25 months. None of the 5 patients in the operation group showed symptoms such as tongue muscle atrophy, tongue extension deviation, hypoglossal nerve dysfunction (mainly including slurred speech, choking with water), postoperative infection, bleeding, lower limb muscle atrophy or lower limb motor dysfunction after sural nerve injury. Postoperative skin sensory disturbance in lateral malleolus area was found, but gradually recovered to normal. During the follow-up, facial nerve and sublingual motor neurons were innervated to paralyzed facial muscle in the operation group. At last follow-up, the HB grading of 5 patients in the operation group improved from preoperative grade Ⅴ in 2 cases, grade Ⅵ in 3 cases to grade Ⅱ in 3 cases, grade Ⅲ in 1 case, and grade Ⅳ in 1 case. And in the conservative group, there were 1 patient with grade Ⅴ and 3 patients with grade Ⅵ before operation, facial asymmetry continued during follow-up, and only 2 patients improved from grade Ⅵ to grade Ⅴ at last follow-up. There was significant difference in prognosis HB grading between the two groups (t=5.693, P=0.001). In the operation group, the amplitude and frequency of F wave were gradually improved, and obvious action potential could be collected when the facial muscle was vigorously contracted. On the contrary, there was no significant difference in neuroelectrophysiological results before and after treatment in the conservative group. ConclusionFacial nerve-sublingual nerve parallel bridge anastomosis can effectively retain the integrity of the facial nerve, while introducing the double innervation of the sublingual nerve opposite nerve, which is suitable for the treatment of severe incomplete facial nerve injury caused by closed fracture.
Objective To investigate the effects of 3 methods (suture removal, suture removal with epineurium neurolysis, and l igated femoral nerve resection with end-end suture) in repairing femoral nerve injury after l igation in different periods so as to provide a reference for cl inical use of repairing iatrogenic l igation injury of the peri pheral nerve. Methods A total of 120 adult female Sprague Dawley rats, weighing (200 ± 20) g, were used to prepare the animal models of left femoralnerve l igation, and were divided into groups A (n=40), B (n=40), and C (n=40) according different repairing methods. Atimmediate, 1, 3, and 5 months (10 rats each time point) after l igation, suture removal was performed in group A, suture removal with epineurium neurolysis in group B, and l igated femoral nerve resection with end-end suture in group C. At 3 months after operation, the foot-base angle (FBA) and the heels-tail angle (HTA), action potential and conduction velocity of femoral nerve, and wet weight of quadriceps femoris muscle (QFM) were measured; the samples of quadriceps femoris and femoral nerve were harvested for histological observation, muscle fiber count, and nerve fiber passing rate measuring. Results The FBA in group A was significant smaller than that in group C at immediate, 1, 3, and 5 months (P lt; 0.05), but there was no significant difference between groups A and B (P gt; 0.05). The HTA in group A was significantly smaller than that in group C at immediate, 1, 3, and 5 months (P lt; 0.05), and the THA in group B was significantly smaller than that in group C at 1, 3, and 5 months (P lt; 0.05). The wet weight of QFM in group B was significantly higher than that in group C at immediate, 3, and 5 months (P lt; 0.05), and the wet weight of QFM in group A was significantly higher than that in group C at immediate and 3 months (P lt; 0.05), but no significant difference was found between groups A and B at immediate, 1, and 3 months (P gt; 0.05). There was significant difference in the action potential of femoral nerve between group A and groups B and C at immediate and 1 month (P lt; 0.05), but there was no significant difference between other groups at 3 and 5 months (P gt; 0.05) except between groups A and C at 5 months (P lt; 0.05). The conduction velocity of femoral nerve in group A was significantly faster than that in group C at immediate, 1, and 5 months (P lt; 0.05), and it was significantly faster in group A than in group B at immediate and 1 month (P lt; 0.05), but no significant difference was found between groups A and B at 3 and 5 months (P gt; 0.05), between groups B and C at other time points (P gt; 0.05) except at immediate (P lt; 0.05). The count of muscle fibre of the quadriceps femoris was significantly more in groups A and B than in group C at immediate (P lt; 0.05); it was significantly more in group A than in group B at 5 months (P lt; 0.05). The passing rate of the femoral nerve fiber was significantly higher in group A than in groups B and C at 3 months (P lt; 0.05), but no significant difference was found between the other groups (P gt; 0.05). Conclusion After femoral nerve l igation, suture removal method has the best effect at early term, the next is epineurium neurolysis method, and the worst is the l igation femoral nerve resection with end-end suture repair.
Objective Dexamethasone is one of the basic agents which could induce osteogenic differentiation of mesenchymal stem cells. To investigate the optimal concentration of dexamethasone in osteogenic differentiation of adiposederivedstem cells (ADSCs) so as to provide the theoretical basis for further bone tissue engineering researches. Methods FiveNew Zealand rabbits (2-3 kg) of clean grade, aged 3 months and male or female, were obtained. ADSCs were isolated from the subcutaneous adipose tissue of inguinal region, and cultured with collagenase digestion, then were detected and identified by CD44, CD106 immunofluorescence staining and adi pogenic differentiation. ADSCs at passage 3 were used and the cell density was adjusted to 1 × 105 cells/mL, then the cells were treated with common cultural medium (group A) and osteogenic induced medium containing 0 (group B), 1 × 10-9 (group C), 1 × 10-8 (group D), 1 × 10-7 (group E), 1 × 10-6 (group F), and 1 × 10-5 mol/ L (group G) dexamethasone, respectively. The cell prol iferation and the mRNA expressions of osteocalcin (OC) and core binding factor α1 (Cbfα1) were detected by MTT and RT-PCR, respectively. The activity of alkal ine phosphatase (ALP) was measured, and the percentage of mineral area was calculated. The mineral nodules were also detected by al izarin red staining. Results ADSCs mostly presented fusiform and polygon shape with positive expression of CD44 and negative expression of CD106. The result of oil red O staining was positive after ADSCs treated with adipogenic induced medium. The result of MTT revealed that the absorbance (A) value decl ined with the ascending of the concentration of dexamethasone, and there was significant difference in A value between groups D and E at 5 and 7 days after osteogenic induction (P lt; 0.05). The mRNA expressions of OC and Cbfα1 reached the peak in groups E and D at 7 days after osteogenic induction, respectively. The activity of ALP and the percentage of mineral area had the maximum value in group D at 14 days, then decl ined gradually. There was no significant difference in the mRNA expressions of OC and Cbfα1, the activity of ALP, and the percentage ofmineral area between groups D and E (P gt; 0.05), but significant differences were found between groups D and E and other groups (P lt; 0.05). After 14 days, the cells of group G died, and the result of al izarin red staining was positive in groups B, C, D, E, and F. Conclusion When the concentration of dexamethasone in osteogenic medium is 1 × 10-8 mol/L, it could not only reduce the inhibitive effect on cells prol iferation, but also induce osteogenic differentiation of ADSCs more efficiently.