Objective To investigate the effects of inhibiting KDM2A gene on the proliferation,invasion and migration of liver cancer cells and its possible regulatory mechanism.Methods Forty pairs of HCC tissues and their adjacent normal counterparts were collected from 2014 to 2017 in Tongji Hospital,Tongji Medical College Affiliated to Huazhong University of Science and Technology.Human liver cancer cell lines HepG2,Huh7,HCCLM3,MHCC-97H and normal liver cells LO2 were cultured in vitro.The mRNA and protein expression levels of KDM2A in HCC tissues and cells were detected by qRT-PCR and Western blotting.Huh7 cells were taken and set up as follows:(1)si-NC group(transfected with si-NC)and si-KDM2A group(transfected with si-KDM2A);(2)mimic-NC group(transfected with mimic-NC),miRNA-29a-3p mimic group(transfected with miRNA-29a-3p mimic),inhibitor-NC group(transfected with inhibitor-NC)and miRNA-29a-3p inhibitor group(transfected with miRNA-29a-3p inhibitor).The mRNA and protein expression levels of KDM2A were detected by qRT-PCR and Western blotting.The invasion and migration of cells were detected by Transwell,the proliferation of cells was detected by CCK-8 methods.The online databases TargetScan,miRDIP,miRWalk,Starbase and miRDB were used to predict the binding sites of KDM2A and miR-29a-3p.The KDM2A 3'-UTR(WT)or KDM2A 3'-UTR(MUT)report plasmid was co-transfected with NC-miRNA or miR-29a-3p mimics respectively for 48 h in 293T cells,and the luciferase activity was detected by the luciferase reporter gene detection system.Results Compared with adjacent normal counterparts,the relative mRNA and protein expression levels of KDM2A in HCC tissues increased significantly(P＜0.05).Compared with LO2,the relative mRNA and protein expression levels of KDM2A in HepG2,Huh7,HCCLM3 and MHCC-97H increased significantly(P＜0.05).Compared with si-NC group,the proliferation,invasion and migration of Huh7 cells in si-KDM2A group decreased significantly(P＜0.05 or P＜0.01).The analysis results of TargetScan,miRDIP,miRWalk,Starbase and miRDB showed that there were binding sites between KDM2A and miR-29a-3p.The results of the dual luciferase reporter assay showed that miR-29a-3p mimic significantly reduced KDM2A-MUT luciferase activity(P＜0.01).After overexpression of miRNA-29a-3p,the relative mRNA and protein expression levels of KDM2A were decreased(P＜0.01),the proliferation,invasion and migration abilities were decreased(P＜0.05)in Huh7 cells.After inhibiting the expression of miRNA-29a-3p,the relative mRNA and protein expression levels of KDM2A were increased(P＜0.05),the proliferation,invasion and migration abilities were enhanced(P＜0.05)in Huh7 cells.Conclusion Inhibiting the expression of KDM2A can reduce the proliferation and migration ability of Huh7 cells.miR-29a-3p may be the upstream regulator of KDM2A and participate in the occurrence and development of hepatocellular carcinoma.

Objective To observe the cage subsidence after oblique lateral interbody fusion(OLIF)for lumbar spondylo-sis,summarize the characteristics of the cage subsidence,analyze causes,and propose preventive measures.Methods The data of 144 patients of lumbar spine lesions admitted to our hospital from October 2015 to December 2018 were retrospectively ana-lyzed.There were 43 males and 101 females,and the age ranged from 20 to 81 years old,with an average of(60.90±10.06)years old.Disease types:17 patients of lumbar intervertebral disc degenerative disease,12 patients of giant lumbar disc hernia-tion,5 patients of discogenic low back pain,33 patients of lumbar spinal stenosis,26 patients of lumbar degenerative spondy-lolisthesis,28 patients of lumbar spondylolisthesis with spondylolisthesis,11 patients of adjacent vertebral disease after lumbar internal fixation,7 patients of primary spondylitis in the inflammatory outcome stage,and 5 patients of lumbar degenerative scoliosis.Preoperative dual-energy X-ray bone mineral density examination showed 57 patients of osteopenia or osteoporosis,and 87 patients of normal bone density.The number of fusion segments:124 patients of single-segment,11 patients of two-seg-ment,8 patients of three-segment,four-segment 1 patient.There were 40 patients treated by stand-alone OLIF,and 104 patients by OLIF combined with posterior pedicle screw.Observed the occurrence of fusion cage settlement after operation,conducted monofactor analysis on possible risk factors,and observed the influence of fusion cage settlement on clinical results.Results All operations were successfully completed,the median operation time was 99 min,and the median intraoperative blood loss was 106 ml.Intraoperative endplate injury occurred in 30 patients and vertebral fracture occurred in 5 patients.The mean follow-up was(14.57±7.14)months from 6 to 30 months.During the follow-up,except for the patients of primary lumbar interstitial in-flammation and some patients of lumbar spondylolisthesis with spondylolisthesis,the others all had different degrees of cage subsidence.Cage subsidence classification:119 patients were normal subsidence,and 25 patients were abnormal subsidence(23 patients were grade Ⅰ,and 2 patients were grade Ⅱ).There was no loosening or rupture of the pedicle screw system.The height of the intervertebral space recovered from the preoperative average(9.48±1.84)mm to the postoperative average(12.65±2.03)mm,and the average(10.51±1.81)mm at the last follow-up.There were statistical differences between postop-erative and preoperative,and between the last follow-up and postoperative.The interbody fusion rate was 94.4％.The low back pain VAS decreased from the preoperative average(6.55±2.2 9)to the last follow-up(1.40±0.82),and there was statistically significant different.The leg pain VAS decreased from the preoperative average(4.72±1.49)to the final follow-up(0.60± 0.03),and the difference was statistically significant(t=9.13,P＜0.000 1).The ODI index recovered from the preoperative av-erage(38.50±6.98)％to the latest follow-up(11.30±3.27)％,and there was statistically significant different.The complication rate was 31.3％(45/144),and the reoperation rate was 9.72％(14/144).Among them,8 patients were reoperated due to fusion cage subsidence or displacement,accounting for 57.14％(8/14)of reoperation.The fusion cage subsidence in this group had obvious characteristics.The monofactor analysis showed that the number of abnormal subsidence patients in the osteopenia or osteoporosis group,Stand-alone OLIF group,2 or more segments fusion group,and endplate injury group was higher than that in the normal bone mass group,OLIF combined with pedicle screw fixation group,single segment fusion group,and no endplate injury group,and the comparison had statistical differences.Conclusion Cage subsidence is a common phenomenon after 0-LIF surgery.Preoperative osteopenia or osteoporosis,Stand-alone OLIF,2 or more segments of fusion and intraoperative end-plate injury may be important factors for postoperative fusion cage subsidence.Although there is no significant correlation be-tween the degree of cage subsidence and clinical symptoms,there is a risk of cage migration,and prevention needs to be strengthened to reduce serious complications caused by fusion of cage subsidence,including reoperation.

Objective:Human leukocyte antigen(HLA)B27 is a susceptibility allele of ankylosing spondylitis(AS),and HLA-B27 antigen typing is an important indicator for clinical diagnosis of AS,but current typing methods such as sequence specific primer polymerase chain reaction(PCR-SSP)still possess limitation.Therefore,this study aims to analyze the correlation between B27 subtypes and susceptibility to AS in Hunan Province by applying high-resolution polymerase chain reaction-sequence-based typing(PCR-SBT). Methods:Peripheral blood of 116 patients with suspected AS(suspected AS group)and 121 healthy volunteers(control group)admitted to the Second Xiangya Hospital from January 2020 to December 2020 were collected for HLA-B genotyping by PCR-SBT.Among the patients in the suspected AS group,23 patients were finally diagnosed with AS(confirmed AS group),and the remaining 93 undiagnosed patients served as the non-confirmed AS group.PCR-SBT and PCR-SSP were used to detect HLA-B27 typing in 116 patients with suspected AS,and the results of the 2 methods were compared. Results:The HLA-B27 allele frequency in the suspected AS group was significantly higher than that in the control group[11.63%vs 2.48%;P<0.001,odds ratio(OR)=5.18,95%confidence interval(CI)2.097 to 12.795].B*27:04,B*27:05,B*27:06,and B*27:07 were detected in the suspected AS group and the control group.The frequency of the B*27:04 allele in the suspected AS group was significantly higher than that in the control group(9.48%vs 1.24%;P<0.001,OR=8.346,95%CI 2.463 to 28.282).The positive rate of B27 in the suspected AS group and the confirmed AS group(B27+/+ and B27+/-)was significantly higher than that in the control group(χ2=16.579,P<0.001;χ2=94.582,P<0.001,respectively).Among the confirmed AS group,21 were HLA-B27 carriers,and the B27 positive rate in the confirmed AS group was 91.3%.PCR-SBT could achieve high resolution typing of the HLA-B gene locus,with higher sensitivity,specificity,positive predictive value,negative predictive value,and accuracy than PCR-SSP. Conclusion:PCR-SBT typing analysis shows a strong correlation between HLA-B * 27:04 and AS in Hunan province.The PCR-SBT method can be used as the preferred option for the auxiliary diagnosis of clinical AS.

OBJECTIVE: To explore the feasibility and clinical effect of Stand-alone oblique lateral interbody fusion (OLIF) in the treatment of lumbar intervertebral disc degeneration with Modic changes and endplate sclerosis. METHODS: A retrospective analysis was performed on 16 cases with lumbar intervertebral disc degeneration with Modic changes and endplate sclerosis admitted to three medical centers from January 2015 to December 2018. There were 6 males and 10 females, the age ranged from 45 to 67 years old with an average of (55.48±8.07) years old, the medical history ranged from 36 to 240 months with an average of (82.40±47.68) months. The lesion sites included L_2,3 in 2 cases, L_3,4 in 5 cases, and L_4,5 in 9 cases. All patients presented with chronic low back pain with lower limb neurological symptoms in 3 cases. All patients were treated by Stand-alone oblique lateral lumbar interbody fusion. Clinical and radiological findings and complications were observed. RESULTS: There was no vascular injury, endplate injury and vertebral fracture during the operation. The mean incision length, operation time, and intraoperative blood loss were(4.06±0.42) cm, (45.12±5.43) min, (33.40±7.29) ml, respectively. The mean visual analogue scale (VAS) of the incision pain was (1.14±0.47) at 72 hours after operation. There was no incision skin necrosis, poor incision healing or infection in patients. Sympathetic chain injury occurred in 1 case, anterolateral pain and numbness of the left thigh in 2 cases, and weakness of the left iliopsoas muscle in 1 case, all of which were transient injuries with a complication rate of 25%(4/16). All 16 patients were followed up from 12 to 36 months with an average of (20.80±5.46) months. The intervertebral space height was significantly recovered after operation, with slight lost during the follow-up. Coronal and sagittal balance of the lumbar spine showed good improvement at the final follow-up. There was no obvious subsidence or displacement of the cage, and the interbody fusion was obtained. At the final follow-up, Japanese Orthopaedic Association(JOA) score and Oswestry disability index(ODI) were significantly improved. CONCLUSION As long as the selection of case is strict enough and the preoperative examination is sufficients, the use of Stand-alone OLIF in the treatment of lumbar intervertebral disc degeneration with Modic changes and endplate sclerosis has a good results, with obvious clinical advantages and is a better surgical choice.
Male ; Female ; Humans ; Child, Preschool ; Intervertebral Disc Degeneration/surgery* ; Retrospective Studies ; Sclerosis ; Treatment Outcome ; Lumbar Vertebrae/surgery* ; Spinal Fusion/methods*

Objective:To investigate the diagnostic accuracy of serological indicators and evaluate the diagnostic value of a new established combined serological model on identifying the minimal hepatic encephalopathy (MHE) in patients with compensated cirrhosis.Methods:This prospective multicenter study enrolled 263 compensated cirrhotic patients from 23 hospitals in 15 provinces, autonomous regions and municipalities of China between October 2021 and August 2022. Clinical data and laboratory test results were collected, and the model for end-stage liver disease (MELD) score was calculated. Ammonia level was corrected to the upper limit of normal (AMM-ULN) by the baseline blood ammonia measurements/upper limit of the normal reference value. MHE was diagnosed by combined abnormal number connection test-A and abnormal digit symbol test as suggested by Guidelines on the management of hepatic encephalopathy in cirrhosis. The patients were randomly divided (7∶3) into training set ( n=185) and validation set ( n=78) based on caret package of R language. Logistic regression was used to establish a combined model of MHE diagnosis. The diagnostic performance was evaluated by the area under the curve (AUC) of receiver operating characteristic curve, Hosmer-Lemeshow test and calibration curve. The internal verification was carried out by the Bootstrap method ( n=200). AUC comparisons were achieved using the Delong test. Results:In the training set, prevalence of MHE was 37.8% (70/185). There were statistically significant differences in AMM-ULN, albumin, platelet, alkaline phosphatase, international normalized ratio, MELD score and education between non-MHE group and MHE group (all P<0.05). Multivariate Logistic regression analysis showed that AMM-ULN [odds ratio ( OR)=1.78, 95% confidence interval ( CI) 1.05-3.14, P=0.038] and MELD score ( OR=1.11, 95% CI 1.04-1.20, P=0.002) were independent risk factors for MHE, and the AUC for predicting MHE were 0.663, 0.625, respectively. Compared with the use of blood AMM-ULN and MELD score alone, the AUC of the combined model of AMM-ULN, MELD score and education exhibited better predictive performance in determining the presence of MHE was 0.755, the specificity and sensitivity was 85.2% and 55.7%, respectively. Hosmer-Lemeshow test and calibration curve showed that the model had good calibration ( P=0.733). The AUC for internal validation of the combined model for diagnosing MHE was 0.752. In the validation set, the AUC of the combined model for diagnosing MHE was 0.794, and Hosmer-Lemeshow test showed good calibration ( P=0.841). Conclusion:Use of the combined model including AMM-ULN, MELD score and education could improve the predictive efficiency of MHE among patients with compensated cirrhosis.

Objective To explore the effects of miR-221 on tumor cell proliferation, migration and invasion in nonsmall cell lung cancer (NSCLC) xenograft model mice, and to preliminarily analyze its possible mechanism of regulating Akt/ mammalian target of rapamycin(mTOR) signaling pathway by targeting tissue inhibitor of metalloproteinase-2 (TIMP-2) on tumor cells in non-small cell lung cancer (NSCLC) through tumor-bearing nude mice. Methods The A549 cells were divided into control group, mimic group, TIMP-2 group and mimic+TIMP-2 group. The mimic group and TIMP-2 group were transfected with miR-221 mimic and TIMP-2 overexpression plasmids, respectively. The mimic + TIMP-2 group was simultaneously transfected with miR-221 mimic and TIMP-2 overexpression plasmids. The control group was transfected with the same amount of negative control plasmid. After transfection, the cells of each group were injected subcutaneously into the left forelimb to construct the corresponding 4 groups of NSCLC mouse models. The proliferation-related protein (Ki67) was detected by immunohistochemical staining to detected the effect of cell proliferation ability. Matrix metalloproteinase-2 (MMP-2) and N-cadherin proteins in each group were tested by Western blotting to assess and compare the abilities of migration and invasion. The levels of miR-221, TIMP-2 and Akt/ mTOR pathways in bone marrow and tumor tissues were detected by Real-time PCR and Western blotting. Results When co-transfected with wild type(WT)-TIMP-2 and miR-221 mimic, the relative luciferase activity in the cells reduced significantly (P<0. 05). Compared with the control group, the tumor mass, volume, Ki67, MMP-2 and N-cadherin protein expression levels, miR-221 and Akt/ mTOR pathway levels were increased significantly, while the levels of TIMP-2 mRNA and protein were significantly reduced in the mimic group (P<0. 05). Compared with the control group, the levels of TIMP-2 mRNA and protein in the TIMP-2 group increased significantly, while the other indicators decreased significantly (P<0. 05). Tumor tissue mass, volume, Ki67, MMP-2, Ncadherin, miR-221 and Akt/ mTOR pathway levels in mimic+TIMP-2 group were significantly lower than those in the mimic group and significantly higher than those in the TIMP-2 group, while TIMP-2 mRNA and protein levels were significantly higher than those in the mimic group and significantly lower than those in the TIMP-2 group (P<0. 05). Conclusion In the NSCLC transplanted tumor mouse model, miR-221 may mediate the Akt/ mTOR pathway by targeting the expression of TIMP-2 protein to promote cell proliferation, migration and invasion.

Since the implementation of drug registration in China, the classification of Chinese medicine has greatly met the needs of public health and effectively guided the transformation, inheritance, and innovation of research achievements on traditional Chinese medicine(TCM). In the past 30 years, the development of new Chinese medicine has followed the registration transformation model of " one prescription for single drug". This model refers to the R&D and registration system of modern drugs, and approximates to the " law-abiding" medication method in TCM clinic, while it rarely reflects the sequential therapy of syndrome differentiation and comprehensive treatment with multiple measures. In 2017, Opinions on Deepening the Reform of Review and Approval System and Encouraging the Innovation of Drugs and Medical Devices released by the General Office of the CPC Central Committee and the General Office of the State Council pointed out that it is necessary to " establish and improve the registration and technical evaluation system in line with the characteristics of Chinese medicine, and handle the relationship between the traditional advantages of Chinese medicine and the requirements of modern drug research". Therefore, based on the development law and characteristics of TCM, clinical thinking should be highlighted in the current technical requirements and registration system of research and development of Chinese medicine. Based on the current situation of registration supervision of Chinese medicine and the modern drug research in China, the present study analyzed limitations and deficiency of " one prescription for single drug" in the research and development of Chinese medicine. Additionally, a new type of " series prescriptions" was proposed, which was consistent with clinical thinking and clinical reality. This study is expected to contribute to the independent innovation and high-quality development of the TCM industry.
China ; Drugs, Chinese Herbal/therapeutic use* ; Medicine, Chinese Traditional ; Prescriptions ; Public Health

Objective: To examine the significance and prognostic value of the classification of hilar cholangiocarcinoma based on actual anatomical location. Methods: A retrospective study was conducted including 120 patients of hilar cholangiocarcinoma treated at the Second Affiliated Hospital,Zhejiang University School of Medicine and Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine from January 2019 to December 2021. Patients with hilar cholangiocarcinoma were classified for seven types according to the site of tumor location. The clinicopathological and prognostic data of 120 patients were retrospectively analyzed(There were 57 males and 63 females,the age (M(IQR)) was 61(22)years(range:42 to 85 years)). All patients received radical resection without visible intraoperative tumor residue and negative bile duct resection margin according to intraoperative pathological biopsy. The classification variables were analyzed by Pearson χ² test or Fisher's exact probability test,one-way ANOVA or Kruskal-Wallis rank sum test.Kaplan-Meier method was used for survival analysis. Cox proportional risk model was used for prognostic factors. Results: The coincidence rate of preoperative surgical planning and actual operational styles was verified in 33 cases. Twenty-six cases were consistent,and 7 cases were inconsistent,with a coincidence rate of 78.8%. According to the actual anatomical location,patients in type of secondary branch experienced a significantly longer operation duration,a higher portal vein resection rate,margin positive rate and more advanced T stage(all P<0.05). The median overall survival time of the unilateral main trunck group was 27.0 months,and the bilateral group was 17.0 months. Survival analysis based on the tumor classification of the actual anatomical location showed that the unilateral or main trunck group predicted less aggressive clinical features and favorable outcomes(HR=1.931,95%CI:1.066 to 3.499,P<0.05). Multivariate analysis demonstrated that the actual anatomical location of the tumor type(HR=2.269,95%CI:1.333 to 3.861,P=0.003),combined liver resection(HR=0.464,95%CI:0.253 to 0.848,P=0.013) and N stage(HR=6.317,95%CI:3.083 to 12.944,P<0.01) were independent factors affecting the prognosis of patients. Conclusion: The classification based on the actual anatomy can be used as a promising scheme in refining patient stratification and predicting survival in hilar cholangiocarcinoma,and it can guide the selection of surgical methods,and predict operative safety and radical resection rate.
Bile Duct Neoplasms/surgery* ; Bile Ducts, Intrahepatic/pathology* ; China ; Cholangiocarcinoma/surgery* ; Female ; Humans ; Klatskin Tumor/surgery* ; Male ; Retrospective Studies

Aim To investigate the protective effect of 1, 8-Cineole on the injury of human aortic endothelial cells (HAECs) induced by high glucose (HG) via regulating autophagy. Methods Cells were incubated with different doses of 1, 8-Cineole followed by exposing to HG for 60 h, and MTT assay was used to analyse cell viability, lactate dehydrogenase (LDH) was used to detect cytotoxicity, and Western blot was used to detect Beclin1, LC3-II/I, p62, caspase-3 and caspase-9 expressions. Autophagy inhibitor (chloroquine, CQ) was treated on HAECs, and the expressions of Beclinl, LC3-II/I, p62, caspase-3 and caspase-9 were measured by Western blot. Results 1, 8-Cineole increased cell viability, reduced the content of LDH, activated autophagy and inhibited apoptosis. Compared with control group, the expression of Beclinl, LC3-II/I, p62, caspase-3 and caspase-9 in CQ group increased. Simultaneously, the expression of above-mentioned between CQ + HG group and CQ + HG + CH group. Conclusions 1, 8-Cineole has protective effect on the injury of HAECs induced by high glucose, and its effect is related to improving autophagy flux.