OBJECTIVE: To investigate the bionomics of the olfactory ensheathing cells (OECs) of human olfactory mucosa. METHOD: To separate and cultivate the OECs of human and rat olfactory mucosa. To observe the cell growth, cell grouping and cell migration in vitro of the two types of OECs. RESULT: Successfully separated and cultivated the OECs of human and rat olfactory mucosa. OECs of the human and rat olfactory mucosa had the similar cell growth, cell grouping and cell migration ability in vitro. CONCLUSION OECs of the human and rat olfactory mucosa have the similar bionomics in vitro, as a result, OECs of the human olfactory mucosa could be a reliable source of cell transplant for nerve injury.
Animals ; Cell Culture Techniques ; Cell Movement ; Cells, Cultured ; Humans ; Olfactory Mucosa ; cytology ; Rats

Objective To establish a doxycycline-controlled immortalized pre-cartilaginons stem cells (IPCSCs) strains, clone parathroid hormone-related peptide[PTHrP(1-36)] gene and construct re- sponsive plasmid, pTRE-PTHrP (1-36). Methods Plasmid pTet-on was transfected into IPCSCs by using LipoinfectaminTM 2000 and then the stable clones were obtained by G418 screening. The doxycyc- line was added into the medium of monoclonal cells that were transiently transfected with plasmid pTRE- 2Hyg-Lue. The total RNA was extracted from PCSCs and the PTHrP(1-36) gene obtained by RT-PCR method. Then, the PTHrP (1-36) gene was subcloned to plasmids of Tet-responsive element with the se- lection marker of hygromycin pTRE-2Hyg to construct recombinant eukaryotic expression plasmid pTRE- PTHrP(1-36). After transferred into E. coli-DH5α, the clone was amplified, the recombinant plasm0ids were purified and identified by double-enzyme digestion. Results The doxycycline induced IPCSCs line was obtained, with 50 times higher than the non-induced cell line. Double enzyme digestion analysis and sequencing showed that the target gene was cloned into recombinant plasmid. Conclusions The induced IPCSCs line can be used to highly express alien genes. The responsive plasmid containing PTHrP (1-36) gene may be premising for rigorous control of PTHrp (1-36) gene expression.

Objective To explore the clinical effect of training assisted by a lower limb rehabilitation robot on the bladder and intestinal function of paraplegic spinal cord injury survivors. Methods Thirty-eight paraplegic patients with spinal cord injury were divided according to their admission order into an experimental group ( n=19) and a control group (n=19). Both groups were given conventional rehabilitation training, while the experimental group was additionally provided with robot-assisted lower limb training in three stages:adaptation, training and con-solidation. It lasted 30 minutes daily, 5 days per week for 12 weeks. Before and after the training, an urodynamics examination system was used to evaluate the maximum urine flow, bladder capacity, residual urine volume, bladder pressure and detrusor pressure. Colon transit time, mean rectal pressure and intestinal function were measured using the colon transit test, a mean rectal pressure test, and the Functional Independence Measure ( FIM) scale respective-ly. Results The average bladder volume, maximum urine flow rate, average urine flow rate, detrusor pressure, bladder compliance, average rectal pressure and intestinal FIM score of the robot training group after training were all significantly better than before the training, as were the average residual urine volume and colon transit time. After the training, the average bladder volume, maximum urine flow rate, average urine flow rate, detrusor pressure, bladder compliance and average rectal pressure of the robot training group were all significantly higher than those of the control group, while the average residual urine volume and colon transit time were significantly smaller. Then, 32％ of the patients in the experimental group achieved no less than 6 points for their average FIM score, significantly higher than in the control group. Conclusion Robot-assisted lower limb training combined with comprehensive rehabilitation training can effectively improve the bladder and intestinal function of paraplegic patients after a spinal cord injury.

Objective:To evaluate clinical and pathological factors related to the actual 5-year survival of patients with hilar cholangiocarcinoma (HCC).Methods:A total of 94 HCC patients who underwent radical surgery at the Department of Hepatobiliary and Pancreatic Surgery, Li Huili Hospital of Ningbo Medical Center from Jan 2000 to Jun 2015 were enrolled in this study.Patients were divided into two groups: postoperative survival group beyond 5 years and death group within 5 years. The clinical and pathological features of the two groups were analyzed.Results:Of the 94 patients, 19 (20.2%) had a postoperative survival time of more than 5 years. The actual 5-year overall survival rate of HCC patients (20.2%) was lower than that estimated by Kaplan-Meier survival analysis (22.2%). Gender, age, CEA value, CA199 value, total bilirubin, Child-Pugh classification, Bismuth classification and preoperative jaundice reduction were not significantly different between the two groups nor there were significant difference between two groups in operation time, blood loss, surgical procedure, combined caudate lobectomy, combined pancreaticoduodenectomy, combined resection of surrounding organs, vascular reconstruction and number of bile duct orifices in remnant liver surface. There were significant differences between two groups in the variables of pathological phenotype ( P=0.012), lymph node metastasis ( P=0.001) and resection level ( P=0.048). Conclusion:Non-papillary type, lymph node metastasis and R 1 resection are the independent risk factors of the actual 5-year survival.

Objective:To investigate the changes of non-specific and HBV core antigen (HBcAg)-specific Th9 cells, and intereleukin-9 (IL-9) in HBV-infected patients, and to assess the influence of Th9 cells on CD8 + T cell function. Methods:Twelve patients with acute hepatitis B (AHB) and 58 with chronic hepatitis B (CHB), who were hospitalized in the First Affiliated Hospital of Xinxiang Medical University between January 2018 and January 2019, were enrolled in this study. Twenty healthy subjects negative for HBsAg were selected as controls. Peripheral blood mononuclear cells (PBMCs) and plasma samples were isolated. Non-specific Th9 cells (CD3 + CD4 + IL-9 + ) and HBcAg-specific Th9 cells were analyzed by flow cytometry. Plasma IL-9 level was measured by enzyme linked immunosorbent assay. CHB patients received tenofovir disoproxil fumarate (TDF) antiviral therapy. The changes of non-specific Th9 cells, HBcAg-specific Th9 cells and plasma IL-9 level were assessed 48 weeks after TDF therapy. CD4 + CCR4 -CCR6 -CXCR3 -(Th9) cells and CD8 + T cells were isolated from 12 HLA-A2 restricted CHB patients and co-cultured with HepG2.2.15 cells with the presence of anti-IL-9 neutralizing antibody. The percentage of dead HepG2.2.15 cells and the levels of IFN-γ and TNF-α were detected. Student′s t test, one-way analysis of variance or SNK- q test was used for statistical comparison between groups. Results:There were no significant differences in non-specific Th9 cells or plasma IL-9 level among AHB patients, CHB patients and healthy controls ( P>0.05). HBcAg-specific Th9 cells was down-regulated in CHB patients when compared with AHB patients [(2.49±0.61)% vs (3.19±0.62)%, P<0.001]. The percentage of HBcAg-specific Th9 cells was negatively correlated with HBV DNA ( r=-0.385, P=0.003), but not correlated with ALT ( P>0.05) in CHB patients. TDF therapy for 48 weeks remarkably elevated the HBcAg-specific Th9 cells [(2.94±0.48)%, P<0.001], however, did not affect non-specific Th9 cells or plasma IL-9 level ( P>0.05) in CHB patients. The cytotoxicity of HBcAg-specific Th9 cells was low in CHB patients. However, HBcAg-specific Th9 cells could induce enhanced cytotoxicity of CD8 + T cells to HepG2.2.15 cells, which manifested as increased percentage of dead HepG2.2.15 cells and higher levels of IFN-γ and TNF-α. Anti-IL-9 neutralizing antibody reduced the enhancement of CD8 + T cell cytotoxicity by HBcAg-specific Th9 cells ( P<0.001). Conclusions:Chronic HBV infection might suppress the level and function of HBcAg-specific Th9 cells, resulting in persistent infection.

Objective:To analyse the risk factors of biliary leakage after surgical resection in patients with perihilar cholangiocarcinoma (PHCC).Methods:The medical data on 179 patients who underwent surgical resection for PHCC at the Department of Hepatopancreatobiliary Surgery, Ningbo Medical Centre Lihuili Hospital, Ningbo University from April 2000 to April 2020 were collected, and 160 patients were finally enrolled into this study. There were 86 males and 74 females, aged (63.4±10.8) years. The 44 patients with class B biliary leakage and the 5 patients with class C biliary leakage were classified into the biliary leakage group, while the remaining 111 patients were classified into the control group. Risk factors of biliary leakage were analysed by univariate and multivariate logistic regression analyses.Results:Operation time ≥360 min, resection and reconstruction of hepatic hilar vessels on the preserved side of liver and number of bile duct openings of >3 in remnant liver were significantly higher in the biliary leakage than the control group (all P<0.05). Multivariate analysis showed that resection and reconstruction of hepatic hilar vessels on the preserved side ( OR=2.322, 95% CI: 1.078-5.002, P=0.028) and 3 or more bile duct openings in the remnant liver ( OR=2.656, 95% CI: 1.198-5.892, P=0.016) were significantly associated with biliary leakage. Conclusion:Resection and reconstruction of hepatic hilar vessels on the preserved side of liver and 3 or more bile duct openings in remnant liver were independent risk factors for biliary leakage after PHCC resection.

Objective To assess the association of single nucleotide polymorphisms (SNPs)of biliverdin reductase A (BLVRA) with neonatal hyperbilirubinemia from Fujian area.Methods A total of 286 patients with neonatal hyperbilirubinemia and 250 healthy controls were enrolled.Genotypes of 5 SNPs within BLVRA gene including rs699512,rs1802846,rs7738,rs1637530 and rs2302032 were determined with matrix-assisted laser desorption ionization/time of flight mass spectrometer.The frequencies of genotype,allele,haplotype and their differentiations were analyzed.Results All 5 SNPs had conformed to Hardy-Weinberg equilibrium (all P ＞ 0.05).rs699512 and rs1637530 showed a significant difference between the 2 groups in both allelic and genotypic frequencies (all P ＜ 0.05),but no significant differences were found in the other SNPs(all P ＞ O.05).In recessive model,the frequency of rs699512 GG genotype of patients was significantly lower than that of the healthy control group(OR =0.494,95％ CI:0.276-0.886,P =0.018),while in dominant model,the frequencies of rs699512 GG + AG and rs1637530 TT + CT genotype of patients were significantly lower than that of the healthy control group(OR =0.678,0.627;95％ CI:0.482-0.954,0.444-0.885;P =0.026,0.008).Based on linkage disequilibrium analysis and haplotype construction,rs1637530,rs2302032,rs699512 and rs1802846 locus in the same area.Based on haplotype CGAT,TGGT,CTAT and CGGT had significant differences between the 2 groups (all P ＜ 0.05),and could reduce the risk of high blood bilirubin (OR =0.588,0.687,0.501;95％ CI:0.434-0.797,0.496-0.952,0.250-1.004).Conclusions rs699512 and rs1637530 may be associated with neonatal hyperbilirubinemia,A allele in rs699512 and C allele in rs1637530 may be associated with significantly increased risk of neonatal hyperbilirubinemia.

ObjectiveTo observe the effect of enriched environment (EE) combined with acupuncture at head point (HA) on behavior in rats with autism spectrum disorder. MethodsHealthy female Wistar rats were given peritoneal injection of sodium valproate at 12.5 days of gestation. Twenty-four male offspring rats were randomly selected and then randomly divided into model group (n = 6), EE group (n = 6), HA group (n = 6) and EE combined with HA group (the combined group, n = 6). Six male offspring rats born from female mice injected with the same amount of saline intraperitoneally were as control group. After four weeks of treatment, all the five groups were tested with three-chamber test and marble burying test, and the sociability index, the social novelty index and the number of buried marbles were recorded. The levels of interleukin (IL)-1β and IL-6 in peripheral blood were determined by enzyme-linked immunosorbent assay (ELISA). ResultsAfter treatment, compared with the model group, the sociability index and the social novelty index improved (P < 0.05), the number of buried marbles reduced (P < 0.05), and the levels of IL-6 and IL-1β in peripheral blood decreased in EE group, HA group and the combined group (P < 0.05); while the combined group was the best (P < 0.01). ConclusionBoth EE or acupuncture at HA could improve behavioral symptoms, and reduce the expression of inflammatory factors in rats with autism spectrum disorder. The combination of the two methods showed the best result.