Objective With eukaryote expression vector pEGFP-C2, to establish the HepG2 cell line which may stably express the P22e of hepatitis B virus (HBV), and may be used to study the existed relationship between HBVP22e and hepatitis B. Methods HBVP22e cDNA obtained by PCR from HBV adr subtype 1.2 copies genome plasmid p3.8II was inserted into universal vector pMD18-T for identification. The plasmids were transfected into HepG2 cells via liposome, while EGFP-HBVP22e was analyzed by Western blot, and observed with fluorescence microscope in HepG2 cells. Results Expression vectors of recombinant pEGFP-C2HBV P22e were constructed and expressed steadily in HepG2 cells. Conclusion The results can be used to explore biological activity of HBVP22e in hepatitis B.

Objective To evaluate the impact of hepatitis B e antigen (HBeAg) states and hepatitis B virus DNA loads on the prognosis of chronic severe hepatitis B.Methods A total of 406 hospitalized patients with chronic severe hepatitis B in Nanfang Hospital of Southern Medical University from January 2002 to December 2007 were retrospectively analyzed.The impact of HBeAg states and HBV DNA loads on the prognosis was evaluated.The measurement data were compared by t test and rates were compared by chi square test.Results Of all the 406 patients with chronic severe hepatitis B,208 (51.2%) patients were HBeAg-positive and the remaining 198 (48.8%) were HBeAgnegative.There was no significant difference of constituent ratio of male and female,average peak value of total bilirubin and average valley value of prothrombin activity between HBeAg-positive group and HBeAgnegative group.However,the average age of HBeAg-negative patients was (46.7±12.8) years old,which was significantly higher than that (38.3±13.5) years old in HBeAg-positive group (t = 6.43,P＜0.01 )the proportion of patients with liver cirrhosis in HBeAg-negative group (67.7%) was much higher than that in HBeAg-positive group (45.7%) (X2=19.97,P＜0.01);the improved rate in HBeAg-negative group (32.3%) was significant lower than that in HBcAg-positive group (44.7%) (X2=6.56,P＜0.05).Increasing HBV DNA levels was associated with lower improved rate in both 208 HBeAg-positive and 198 HBeAg-negative patients(X2=22.98,26.04,respectively,both P＜0.01 ).Conclusions HBeAg-negative patients with chronic severe hepatitis B has worse prognosis than HBeAg-positive patients;and the prognosis is getting worse with the increasing HBV DNA level regardless of the HBeAg status.

Objective: To investigate the characteristics of 25(OH)D level in children with ASD and its correlation with clinical features. Methods: A total of 196 children with ASD who received outpatient and inpatient rehabilitation training from January 2017 to January 2019 were included in ASD group, and 178 healthy children who visited the hospital during the same period were included in healthy control group. Differences in 25(OH)D levels and general data between study group and healthy control group were compared. In addition, ASD group was divided into 25(OH)D normal group and abnormal group in accordance with 25(OH)D level (≥30 ng/mL). Differences in general data, total score of CARS scale and factor scores were compared between two groups. Finally, the correlation between serum 25(OH)D level and CARS total score and factor scores of children with ASD was evaluated. Results: 25(OH)D level in ASD group was significantly lower than that in healthy control group (P<0.01). The incidence of sleep disorder, dietary bias, vomiting, constipation and diarrhea in children with ASD was statistically significant compared with that of healthy children (P<0.01); there were statistically significant differences in breastfeeding, sleep disorder, dietary bias and diarrhea between 25(OH)D normal group and abnormal group (χ2=4.97，8.69,6.67,3.98,P<0.05); there were statistically significant differences in 10 aspects including CARS total score, interpersonal relationship, imitation, emotional response, physical use ability, relationship with inanimate objects, adaptation to environmental changes, visual response, auditory response and general impression (P<0.05); there was a significant negative correlation between serum 25(OH)D level and CARS total score in children with ASD (r=-0.32, P<0.01). Conclusion Breastfeeding could reduce the risk of 25(OH)D abnormalities in children with ASD. 25(OH)D reduction would cause sleep disorder, dietary bias and gastrointestinal problems, while dietary bias and gastrointestinal problems would affect 25(OH)D uptake and absorption. 25(OH)D might be related to the occurrence of ASD in children. Serum 25(OH)D level could be used as a reference index for the severity of ASD in children.

Objective: To investigate the relationship between P27，P53 and PCNA expression in human gastric carcinoma tissues and clinicopathological parameters. Methods: The expression of P27，P53 and PCNA in 62 human gastric carcinoma tissues was examined with immunohistochemistry SP method. Results: Positive rates of P27，P53 and PCNA expression were 37.1％, 40.4%，83.9％. P27 expression was related with Bormann type, infiltrative depth, lymph node and distant metastasis and clinical stage. P53 expression was related with sex of patients, distant metastasis and clinical stage. PCNA expression was related with age of patients and infiltrative depth of tumor. P27 positive expression group was higher than negative group as to 5-year survival. P27 expression was in reverse relation with PCNA expression. Conclusion: The expression of P27, P53 and PCNA may be regarded as an important marker in judging malignant degree of gastric carcinoma,distant metastasis and prognosis.

OBJECTIVETo investigate the clinical and histological characteristics of fibrosing cholestatic hepatitis (FCH) and the therapeutic effect of lamivudine.

METHODSBy retrospective analysis, 17 cases developed severe jaundice in 794 renal-transplanted recipients, and of them, FCH was clinically suggested in 11 and confirmed by liver biopsy in 6 cases.

RESULTSThe prevalence of chronic HBV infection in renal transplantation patients was 9.3%, of whom the FCH occurred in 22.9%. In 6 liver-biopsied cases, the onset was within 1.5-22 months. Two cases remitted who had early received lamivudine and 4 cases who were treated with the drug before transplantation did not develop the disease. All patients received large amounts of multiple immuno-suppressors after transplantation. About one fifth of HBV-infected cases gradually developed cholestatic hepatitis and some of them rapidly proceeded to hepatic failure. All had very high serum level of HBV DNA. The histology revealed unique lesion combination. The hepatocytes had widespread ballooning change and some ground-glass appearance. There were liver cytolysis and focal cell loss, bile stasis, periportal fibrosis, while only mild lymphocytic infiltration.

CONCLUSIONSFibrosing cholestatic hepatitis may happen following renal transplantation. Lamivudine has marked therapeutic effect for FCH.

Adult ; Anti-HIV Agents ; therapeutic use ; Cholestasis, Intrahepatic ; drug therapy ; etiology ; pathology ; DNA, Viral ; blood ; genetics ; Female ; Fibrosis ; Hepatitis B ; blood ; complications ; Hepatitis B virus ; genetics ; Hepatocytes ; drug effects ; pathology ; Humans ; Kidney Transplantation ; adverse effects ; Lamivudine ; therapeutic use ; Male ; Middle Aged ; Retrospective Studies

Objective To investigate the efficacy of individualized interferon (IFN)-alpha therapy in HBeAg-negative chronic hepatitis B patients. Methods Seventy- six Chinese HBeAg-negative chronic hepatitis B patients proven by liver biopsy were treated with 5 MU recombinant IFN-alpha 1b subcutaneously thrice every week. All the patients were followed up for at least 24 months the combined responses were defined as normalization of serum alanine transaminase (ALT) and HBV DNA＜3 log10 copy/mL. An intention-to-treat (ITT) analysis was used in this paper in which all 76 patients were included. Results Six patients were lost. Treatment duration was in the range 2-24 months with a median of 8.5 months, and combined responses were achieved at a median of 6.0 months (range 2-19 months) of treatment duration.Seventy-five-percentile of treatment duration to endpoints was 10.0 months. The combined response rate was 46.1% (35/76) at the end of treatment, 43.3% (33/76) at 12-month follow-up and 40.8% (31/76) at 24-month follow-up. The relapse rate was 20. 0% (7/35) and 25. 7% (9/35) at 12-month and 24-month follow-up, respectively. Higher necroinflammatory activity in liver biopsy predicted a good response, while gender, age, liver fibrosis, baseline ALT, aspartate aminotransferase levels and baseline HBV DNA levels were not impact factors of therapeutic effects by binary Logistic regression analysis.Conclusion Individualized prolonged IFN-alpha regimen lead to considerable sustained disease suppression in patients with HBeAg-negative chronic hepatitis B.

OBJECTIVETo study the clinical and histological features of chronic hepatitis B (CHB) with negative hepatitis B e-antigen (HBeAg).

METHODSA total of 743 in-patients with chronic hepatitis B were recruited into the study and divided into two groups according to the HBeAg status. The correlation among alanine transaminase (ALT) levels, hepatitis B virus (HBV) DNA semiquantification, and the liver histopathological data were detected.

RESULTSOf the 743 successive in-patients, 267 (35.9%) were HBeAg-negative. The HBDAG-negative group had significantly lower serologic HBV DNA levels (63.0% of < 100 pg/ml) vs HBeAg-positive (42.6%, P < 0.001), while more sever inflammation (58.1% of inflammatory scores of histological activity index (HAIinf > or = 9) vs HBeAg-positive group (46.0%, P < 0.001) and severe fibrosis (45.3% of fibrosis scores of histological activity index (HAIfib > or = 3) vs HBeAg-positive group (27.9%, P < 0.001) of liver histology. In HBeAg-positive patients, increasing ALI levels were significantly associated with high inflammation and fibrosis scores and low HBV DNA levels. However, it was not the case in the HBeAg-negative cases. In HBeAg-positive patients, 91.3% of them had HAIinf > or = 9 and 65.7% had HAIfib > or = 3 with HBV DNA > 100 pg/ml, while 8.2% of them had HAIinf > or = 9 and 12.3% had HAIfib > or = 3 with HBV DNA < 20 pg/ml, indicating an obverse correlation between HBV DNA levels and histology scores.

CONCLUSIONSAs regards clinical and histological background, the chronic HBeAg-negative hepatitis B is a different subpopulation from the HBeAg-positive counterpart.

DNA, Viral ; Fibrosis ; pathology ; Hepatitis B ; immunology ; pathology ; Hepatitis B e Antigens ; analysis ; Hepatitis B virus ; genetics ; Hepatitis, Chronic ; Humans ; Liver ; pathology

Objective:To evaluate the volume changes of cervical longus and cervical extensor after anterior cervical discectomy and fusion (ACDF), and the correlation with the clinical efficacy of patients.Methods:All of 57 patients with cervical spondylotic myelopathy who underwent single-segment ACDF surgery from January 2013 to December 2018 were analyzed. The follow-up time was 23.0±4.8 months (range 16-34 months). All included subjects underwent MR examination within 1 week before operation and 3rd, 12th months after operation and at the last follow-up. The axial section cross section area (AxCSA) of the cervical longus and the ratio of length to short diameter line (RLS) at the level of each disc of C 2-C 7 were measured on the axial T2WI. Calculate the volume of the cervical longus based on the layer thickness. At the same time, measure the cervical extensor cross-sectional area (CESA) of the same level including the multifidus, cervical semispinous muscle, semispinous head, splinter head, and cervical splinter muscles, and compare CESA with the corresponding vertebral cross-sectional area (VBA). The ratio is analyzed as the volume of the neck extensor muscle, namely CESA/VBA. At the 3rd and 12th months after operation and at the last follow-up, the axial pain was assessed by visual analogue scale (VAS) for assessing pain, and the modified Japanese Orthopedic Association score (mJOA) and the neck dysfunction index (NDI) were used to assess the functional status of the cervical spine. Analyze the morphological changes of thecervical longus and extensor cervical muscles before and after the operation and during the follow-up period, and analyze the correlation with VAS, mJOA, and NDI. Results:Compared with the preoperative period, the average AxCSA of the surgical segment decreased at the 3rd and 12th months after the operation and at the last follow-up. The difference was statistically significant ( F=24.113, P<0.05), which was changed from 140.84±19.51 mm 2 respectively reduce to 117.74±17.15 mm 2 ( t=6.714, P<0.05), 116.37±18.67 mm 2 ( t=6.841, P<0.05) and 116.27±18.65 mm 2 ( t=6.873, P<0.05). Compared with preoperatively, they were reduced by 16.40%, 17.37% and 17.45%, respectively, while the average RLS of surgical segments increased slightly, and the difference was statistically significant ( F=22.612, P<0.05), which increased from preoperative 1.97±0.67 to 2.73±0.60 (38.58% increased, t=6.380, P<0.05), 2.82±0.64 (43.15% increased, t=6.926, P<0.05) and 2.74±0.62 (39.09% increased, t=6.368, P<0.05). The volume of thecervical longus of the patients decreased after the operation, and the difference was statistically significant ( F=64.511, P<0.05), which decreased from 8853.48±458.65 mm 3 before the operation to 7834.53±461.59 mm 3 (11.51% decreased, t=11.822, P<0.05), 7926.42±456.24 mm 3 (10.47% decreased, t=10.819, P<0.05), 7892.38±450.78 mm 3 (10.86% decreased, t=11.283, P<0.05). There were no statistically significant differences in the non-surgical segment AxCSA, RLS and the volume of thecervical longus at the 3rd and 12th months after surgery and the last follow-up ( P>0.05). There was no statistically significant difference of CESA and CESA/VBA compared to preoperative in the surgical segment and non-surgical segment ( P>0.05). Pearson correlation analysis showed that the volume of cervical longus and VAS at the 3rd month ( r=-0.308, P<0.05), the 12th month ( r=-0.210, P<0.05) and the last follow-up ( r=-0.404, P<0.05) were negatively correlated; Among the volume of cervical longus and NDI in the 3rd month ( r=-0.511, P<0.05), 12th month ( r=-0.518, P<0.05) and the last follow-up ( r=-0.352, P<0.05), there was a negative correlation; However, there was no statistically significant correlation between the cervical longus muscle volume and mJOA at each follow-up time point ( P>0.05); There was no significant correlation between CESA/VBA and VAS, NDI, and mJOA at the 3rd, 12th and last follow-up ( P>0.05). Conclusion:The volume and morphology of cervical longus after ACDF was significantly reduced compared with that before the operation, but the volume and morphology of the cervical extensor muscle did not change significantly. ACDF surgery mainly affects the cervical longus corresponding to the surgical segment, and the volume is negatively correlated with the VAS and NDI during follow-up.

Objective:To investigate the effects of exosomes of human nucleus pulposus cells (NPCs) on the differentiation of urine derived stem cells (USCs) into nucleus pulposus-like cells.Methods:USCs and NPCs were isolated and cultured in vitro. The exosomes of NPCs were extracted and detected by Western-blot. USCs cytoplasm was transfected with GFP lentivirus, while nucleus was transfected with DAPI dye. The NPCs exosomes were transfected with PKH26 dye. After co-incubation for 12 h, USCs and NPCs exosomes were observed by macroscopy. USCs differentiation was induced by NPCs exosomes and non-contact co-culture methods. The relative expression of marker gene mRNA of nucleus pulposus cells in each group and the absorbance at 450 nm wavelength were detected.Results:The isolated USCs had the ability to differentiate into osteocytes, adipocytes and chondrocytes with high expression of marker CD29 (99.57%), CD44 (97.46%) and CD73 (97.71%) and with low expression of negative proteins CD31 (0.59%) and CD45 (0.19%). The isolated NPCs highly expressed nuclear pulposus cell marker COL2A1, ACAN and SOX-9. The exosomes extracted from NPCs showed high expression of exosome marker CD63, CD81 and Tsg101. After 12 h co-incubation, NPCs exosomes fused with USCs membrane and appeared in the cytoplasm of USCs. At 3, 5 and 7 days of co-culture, the absorbance value of USCs cells in exosome group (0.44±0.004, 0.76±0.004, 0.82±0.006) was higher than that in co-culture group (0.39±0.022, 0.63±0.035, 0.69±0.012) ( P<0.05). The mRNA relative expression of USCs nucleus pulposus marker genes ACAN (1.80±0.31, 3.50±0.21, 5.35±0.31, 7.46±0.12), COL2A1 (1.43±0.15, 4.33±0.23, 6.89±0.22, 8.11±0.31), SOX-9 (2.21±0.13, 3.13±0.11, 3.96± 0.14, 4.52±0.26) and HIF-1α (1.45±0.16, 2.14±0.21, 4.31±0.41, 4.01±0.25) in exosomes group were significantly higher than those in the control group ( P<0.05) at the 3rd, 7th, 14th and 21st days. The mRNA relative expression of USCs nucleus pulposus marker genes ACAN (5.69±0.21, 6.69±0.13), COL2A1 (6.33±0.17, 7.89±0.15), SOX-9 (4.19±0.29, 4.38±0.12), HIF-1α (4.49±0.32, 4.96±0.26) in exosomes group were significantly higher than those ACAN (3.69±0.35, 5.13±0.23), COL2A1 (3.40±0.16, 6.79±0.19), SOX-9 (2.26±0.32, 3.69±0.26), HIF-1α (2.39±0.11, 3.96±0.13) in non-contact co-culture group ( P<0.05) at the 14th and 21st days. Conclusion:Human nucleus pulposus exosomes could induce differentiation of human USCs into nucleus pulposus-like cells in vitro. Compared with non-contact co-culture, exosomes have higher induction efficiency and can better maintain the proliferation activity of nucleus pulposus-like cells

Our previous report has demonstrated that 5-formylhonokiol (FH), a derivative of honokiol (HK), exerts more potent anti-proliferative activities than honokiol in several tumor cell lines. In present study, we first explored the antiangiogenic activities of 5-formylhonokiol on proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) for the first time in vitro. Then we investigated the in vivo antiangiogenic effect of 5-formylhonokiol on zebrafish angiogenesis model. In order to clarify the underlying molecular mechanism of 5-formylhonokiol, we investigated the signaling pathway involved in controlling the angiogenesis process by western blotting assay. Wound-healing results showed that 5-formylhonokiol significantly and dose-dependently inhibited migration of cultured human umbilical vein enthothelial cells. The invasiveness of HUVEC cells was also effectively suppressed at a low concentration of 5-formylhonokiol in the transwell assay. Further F-actin imaging revealed that inhibitory effect of 5-formylhonokiol on invasion may partly contribute to the disruption of assembling stress fiber. Tube formation assay, which is associated with endothelial cells migration, further confirmed the anti-angiogenesis effect of 5-formylhonokiol. In in vivo zebrafish angiogenesis model, we found that 5-formylhonokiol dose-dependently inhibited angiogenesis. Furthermore, western blotting showed that 5-formylhonokiol significantly down-regulated extracellular signal-regulated kinase (ERK) expression and inhibited the phosphorylation of ERK but not affecting the total protein kinase B (Akt) expression and related phosphorylation, suggesting that 5-formylhonokiol might exert anti-angiogenesis capacity via down-regulation of the ERK signal pathway. Taken together, these data suggested that 5-formylhonokiol might be a viable drug candidate in antiangiogenesis and anticancer therapies.
Actins/metabolism ; Angiogenesis Inhibitors/*pharmacology ; Animals ; Antineoplastic Agents, Phytogenic/pharmacology ; Biphenyl Compounds/*pharmacology ; Blotting, Western ; Cell Line, Tumor ; Cell Movement/drug effects ; Cell Proliferation/drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; Embryo, Nonmammalian/drug effects/metabolism ; Endothelium, Vascular/*drug effects/metabolism ; Extracellular Signal-Regulated MAP Kinases/*antagonists & inhibitors/metabolism ; Humans ; Lignans/*pharmacology ; Neovascularization, Physiologic/*drug effects ; Signal Transduction/*drug effects ; Umbilical Veins/cytology ; Wound Healing ; Zebrafish/embryology/metabolism