OBJECTIVE: To prepare decellularized nerve grafts from alpha-1, 3-galactosyltransferase (GGTA1) gene-edited pigs and explore their biocompatibility for xenotransplantation. METHODS: The sciatic nerves from wild-type pigs and GGTA1 gene-edited pigs were obtained and underwent decellularization. The alpha-galactosidase (α-gal) content in the sciatic nerves of GGTA1 gene-edited pigs was detected by using IB4 fluorescence staining and ELISA method to verify the knockout status of the GGTA1 gene, and using human sciatic nerve as a control. HE staining and scanning electron microscopy observation were used to observe the structure of the nerve samples. Immunofluorescence staining and DNA content determination were used to evaluate the degree of decellularization of the nerve samples. Fourteen nude mice were taken, and subcutaneous capsules were prepared on both sides of the spine. Decellularized nerve samples of wild-type pigs ( n=7) and GGTA1 gene-edited pigs ( n=7) were randomly implanted in the subcutaneous capsules. Blood was drawn at 1, 3, 5, and 7 days after implantation to detect neutrophil counting. RESULTS: IB4 fluorescence staining and ELISA detection showed that GGTA1 gene was successfully knocked out in the nerves of GGTA1 gene-edited pigs. HE staining showed that the structure of the decellularized nerve from GGTA1 gene-edited pigs was well preserved; the nerve basement membrane tube structure was visible under scanning electron microscopy; no cell nuclei was observed, and the extracellular matrix components was retained in the nerve grafts by immunofluorescence staining; and the DNA content was significantly reduced when compared with the normal nerves ( P<0.05). In vivo experiments showed that the number of neutrophils in the two groups were similar at 1, 3, and 7 days after implantation, with no significant difference ( P>0.05); only at 5 days, the number of neutrophils was significantly lower in the GGTA1 gene-edited pigs than in the wild-type pigs ( P<0.05). CONCLUSION The decellularized nerve grafts from GGTA1 gene-edited pigs have well-preserved nerve structure, complete decellularization, retain the natural nerve basement membrane tube structure and components, and low immune response after xenotransplantation through in vitro experiments.
Animals ; Transplantation, Heterologous ; Galactosyltransferases/genetics* ; Sciatic Nerve/immunology* ; Swine ; Tissue Engineering/methods* ; Humans ; Graft Rejection/prevention & control* ; Gene Editing ; Mice ; Mice, Nude ; Heterografts/immunology* ; Animals, Genetically Modified ; Tissue Scaffolds ; Decellularized Extracellular Matrix

Objective To study the development of conus medullaris by quantitative three-dimensional ultrasound in the second and third trimesters of pregnancy,and to analyze the value of terminal caudal vertebral ossification center count in diagnosis of closed spina bifida.Methods A total of 108 normal fetuses in the second and third trimesters of pregnancy who were admitted to Baoding No.1 Central Hospital from January 2019 to March 2024 were selected and assigned to the normal group.The distance between the fetal conus medullaris and the first sacral vertebra,the distance between the end of the conus medullaris and the end of sacrum,and the number of corresponding vertebrae were measured by three-dimensional ultrasound at different gestational weeks.Meanwhile,54 fetuses who were confirmed to have closed spina bifida after delivery were enrolled into the abnormal group.The number of ossification centers on the median sagittal section of the caudal spine was counted and compared between the two groups.The receiver operating characteristic(ROC)curve was drawn to analyze the value of ossification center count of distal caudal vertebrae in diagnosis of closed spina bifida.Results With the increase of gestational weeks,the distance between the fetal conus medullaris and the first sacral vertebra and the distance between the end of the conus medullaris and the end of the sacral bone were gradually increased.The conus medullaris of normal fetuses was mainly located in L1.5,L2 and L2.5,accounting for 85.19%(92/108).The ossification center count of distal caudal vertebra was slightly increased with the increase of gestational weeks in the normal group.At 17-20 gestational weeks,85.71%(12/14)of the ossification center count of distal caudal vertebra was 5 to 7.At 21-28 gestational weeks,100.00%(45/45)of the ossification center count was 6 and more.At week 29 and above,100.00%(45/45)of the ossification center count was more than 7.In the abnormal group,96.30%(52/54)of the fetuses had the ossification center less than 6,and 85.71%(42/49)of the ossification center count was≤5 at 21-32 gestational weeks.There were significant differences in the ossification center count between the two groups at 17-20,21-24,25-28,29-32,and 33-36 gestational weeks(P<0.05).ROC curve showed that,at 17-20,21-24,25-28,29-32,and 33-36 gestational weeks,the area under the curve(AUC)in prediction of closed spina bifida was 0.804,0.744,0.776,0.819,and 0.722 when the ossification center count of distal caudal vertebra was 5.31,4.59,4.81,4.67,and 5.49 as the cut-off value,respectively.Conclusion The conus medullaris moves upward with the increase of gestational weeks in the second and third trimesters of pregnancy.The ossification center count of the distal caudal vertebra is less than 6 in most fetuses with closed spina bifida,which is conducive to screening for closed spina bifida.

Objective To investigate the disease assessment and prognosis value of serum copeptin level in patients with acute cerebral infarction (ACI). Methods One hundred first diagnosed ACI patients were selected as ACI group. According to the National Institutes of Health stroke score (NIHSS), the ACI patients were divided into mild (NIHSS<7 scores), moderate (NIHSS 7-15 scores) and severe (NIHSS>15 scores). Sixty cases of healthy subjects were selected as control group. The serum copeptin level was measured by double antibody sandwich enzyme linked immunosorbent assay method in control group and ACI group (onset within 24 h). The NIHSS, Alberta stroke program early CT score (ASPECTS) and modified Rankin score (mRS) onset within 24 h and 14 d were evaluated in patients with ACI, and the mRS 90 d and 180 d after ACI were evaluated. The neurological impairment was assessed by mRS 180 d after ACI, mRS ≤ 2 scores was good prognosis, ≥ 3 scores was poor prognosis. The correlation was analyzed. Results Among the 100 patients with ACI, mild was in 52 cases, moderate in 34 cases, and severe in 14 cases; good prognosis was in 79 cases and poor prognosis in 21 cases. The serum copeptin levels within 24 h of ACI in mild, moderate and severe patients of ACI group were significantly higher than that in control group:(4.82 ± 1.25), (6.39 ± 2.21) and (9.28 ± 3.82) pmol/L vs. (1.95 ± 0.28) pmol/L. The serum copeptin level within 24 h of ACI in moderate patients was significantly higher than that in mild patients, in severe patients was significantly higher than that in moderate patients, and there were statistical differences (P<0.05). Within 24 h of ACI , the ASPECTS in moderate and severe patients were significantly lower than that in mild patients:(10.02 ± 2.10) and (6.24 ± 3.05) scores vs. (12.16 ± 0.84) scores, in severe patients was significantly lower than that in moderate patients, and there were statistical differences (P<0.05). The NIHSS in moderate and severe patients were significantly higher than that in mild patients:(10.68 ± 3.14) and (16.20 ± 4.26) scores vs. (4.35 ± 1.52) scores, in severe patients was significantly higher than that in moderate patients, and there were statistical differences (P<0.05). The serum copeptin levels within 24 h of ACI and NIHSS in each time point in good prognosis patients were significantly lower than those in poor prognosis patients:(3.52 ± 1.26) pmol/L vs. (8.68 ± 3.06) pmol/L and (5.68 ± 2.11) scores vs. (15.36 ± 3.25) scores, (4.85 ± 1.86) scores vs. (12.60 ± 3.89) scores, (3.68 ± 1.21) scores vs. (6.35 ± 2.96) scores, (2.16 ± 0.75) scores vs. (5.21 ±1.96) scores, and the ASPECTS within 24 h of ACI was significantly higher than that in poor prognosis patients:(11.38 ± 2.21) scores vs. (7.86 ± 2.49) scores, and there were statistical differences (P<0.05). The single factor Logistic regression analysis results showed that the age, ASPECTS, NIHSS and serum copeptin level were the influencing factors of severity of illness in patients with ACI (OR = 1.21, 5.36, 5.61 and 6.62;95%CI 0.99-1.39, 3.34-9.21, 2.86-7.52 and 1.38-12.64;P=0.04, 0.01, 0.01 and 0.00), and the influencing factors of poor prognosis (OR=1.32, 5.21, 4.86 and 6.82;95%CI 0.84-1.43, 3.52-8.39, 2.62-5.35 and 2.67-11.85;P=0.04, 0.01, 0.01 and 0.00). ROC analysis results showed that the area under curve of NIHSS, serum copeptin level and ASPECTS in predicting poor prognosis in patients with ACI were 0.926, 0.863 and 0.624. In the mild, moderate and severe patients, the serum copeptin level was negative correlated with ASPECTS ( r=-0.682,-0.594 and-0.572;P<0.01), and the serum copeptin level was positively correlated with NIHSS ( r = 0.652, 0.614 and 0.586; P<0.01). Conclusions The serum copeptin level in patients with ACI is significantly elevated. The serum copeptin level is positively correlated with neurologic impairment severity and prognosis in patients with ACI, and it has important significance in evaluating pathogenetic condition and prognosis.

Objective To investigate the dynamic changes of serum interleukin(IL)-18,IL-1β and copeptin in acute cerebral infarction(ACI),and analyze the relationship with the serious degree of ACI.Methods The levels of serum IL-18,IL-1β and copeptin were measured by Double-antibody sanduicb enzyme-linked immunosorbent assay(ELISA)in 83 patients with ACI who were admitted to the hospital within 24 h,and neurological impairment were evaluated by European stroke scale(ESS)at the 1st,3rd and 7th day respectively after hospitalization.At the same time,the patients with ACI were compared with 32 normal adults.Results The levels of serum IL-18,IL-1β and copeptin at the 1st,3rd and 7th day after hospitalization were(131.30±31.62),(168.30±28.12),(141.26±24.23)ng/L,(0.35±0.04),(0.82±0.10),(0.52±0.21)μ g/L,and(3.64±0.26),(4.18±0.53),(3.26±2.41)μ g/L respectively.There were significant differences among different times respectively(P＜0.05),furthermore they were higher than those in normal adults[(119.12±27.42)ng/L,(0.21±0.08)μ g/L,(2.63±0.23)μ g/L](P＜0.05).Univariate Logistic regression analysis showed that age,copeptin,IL-18,IL-1β and ESS score was the influencing factor in the serious degree of ACI.There was no significant difference in assessing the serious degree of ACI by receiver operating characteristic(ROC)curve between IL-18 and age,copeptin,IL-1 β,ESS score(P＞0.05).Conclusions The serum levels of IL-18,IL-1β and copeptin are increasing after ACI,indicating that the inflammatory and immune factors may be involved in the development process of ACI.The serum levels of IL-18,IL-1β and copeptin can reflect the serious degree of ACI.

The extract content and in vitro releasing content from different processed products of Squama Manitis were determined using protein as index.The result indicates that the releasing rate of both pro- eessed products are obviously higher than that of the crude.The in vitro study on releasing rate of the same processed product shows that the dissolving rate in vitro has tight relation to its particle size.