ObjectiveTo investigate the effect and mechanism of transplantation of human umbilical cord mesenchymal stem cell （hUC-MSC） with overexpression of the Numb gene in the treatment of cholestatic liver fibrosis （CLF）. MethodsThe technique of lentiviral transfection was used to induce the overexpression of the Numb gene in hUC-MSC （hUC-MSC^Numb-OE）， and hUC-MSC transfected with empty vector （hUC-MSC^OE-EV） was used as negative control. Bile duct ligation （BDL） was performed to establish a rat model of CLF， and then the rats were randomly divided into BDL group， hUC-MSC group， hUC-MSC^OE-EV group， and hUC-MSC^Numb-OE group， while a sham-operation group was also established. The rats in the intervention groups were given a single splenic injection of the corresponding cells after BDL， and samples were collected at the end of week 4. Related indicators were measured， including serum biochemistry， liver histopathology， the content of hydroxyproline （Hyp） in the liver， hepatic stellate cell activation， ductular reaction， liver regeneration， and the expression levels of key molecules in the Numb-p53 signaling axis. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the BDL group， the hUC-MSC group and the hUC-MSC^OE-EV group had significant reductions in the levels of serum biochemical parameters （aspartate aminotransferase， gamma-glutamyl transpeptidase， total bile acid， total bilirubin， and direct bilirubin）， liver fibrosis markers （the content of Hyp and the expression levels of alpha-smooth muscle actin， tumor necrosis factor-α， and transforming growth factor-beta 1）， and ductular reaction markers （the expression levels of CK7 and CK19） （all P <0.05）， and compared with the hUC-MSC^OE-EV group， the hUC-MSC^Numb-OE group had significantly greater improvements in the above indicators （all P <0.05）. In addition， compared with the hUC-MSC^OE-EV group， the hUC-MSC^Numb-OE group had significant improvements in the expression levels of liver regeneration-related markers （albumin and hepatocyte nuclear factor 4α） and the molecules associated with the Numb-p53 signaling axis （Numb， pNumb， Mdm2， and p53） （all P <0.05）. ConclusionOverexpression of the Numb gene can enhance the therapeutic effect of hUC-MSC on CLF， possibly by activating the Numb-PTB^L-p53-HNF4α axis， promoting the hepatic differentiation of hUC-MSCs and subsequently enhancing liver regeneration.

Objective:To use metagenomic sequencing to compare the differences in intestinal microbiota species and metabolic pathways in patients with hepatitis B cirrhosis with or without ascites and further explore the correlation between the differential microbiota and clinical indicators and metabolic pathways.Methods:20 hepatitis B cirrhosis cases [10 without ascites (HBLC-WOA), 10 with ascites (HBLC-WA), and 5 healthy controls (HC)] were selected from the previously studied 16S rRNA samples. Metagenome sequencing was performed on the intestinal microbiota samples. The Kruskal-Wallis rank sum test and Spearman test were used to identify and analyse differential intestinal microbiota populations, metabolic pathways, and their correlations.Results:(1) The overall structure of the intestinal microbiota differed significantly among the three groups ( R = 0.19, P = 0.018). The HC group had the largest abundance of Firmicutes and the lowest abundance of Proteobacteria at the genus level. Firmicutes abundance was significantly decreased ( Pfdr < 0.01), while Proteobacteria abundance was significantly increased ( Pfdr < 0.01) in patients with cirrhosis accompanied by ascites; (2) LEfSe analysis revealed that 29 intestinal microbiota (18 in the HBLC-WA group and 11 in the HBLC-WOA group) played a significant role in the disease group. The unclassified Enterobacteriaceae and Klebsiella species in the HBLC-WA group and Enterobacteriaceae in the HBLC-WOA group were positively correlated with the Child-Turcotte-Pugh (CTP) score, prothrombin time, and international normalized ratio score and negatively correlated with albumin and hemoglobin levels ( P < 0.05). Escherichia and Shigella in the HBLC-WA group were positively correlated with CTP scores ( P < 0.05); (3) The correlation analysis results between the KEGG pathway and 29 specific intestinal microbiota revealed that Enterobacteriaceae and arachidonic acid, α-linolenic acid, glycerolipid metabolism, and fatty acid degradation were positively correlated in the lipid metabolism pathway, while most Enterobacteriaceae were positively correlated with branched-chain amino acid degradation and negatively correlated with aromatic amino acid biosynthesis in the amino acid metabolic pathway. Conclusion:A significant increment of Enterobacteriaceae in the intestines of HBLC-WA patients influenced hepatic reserve function and was associated with amino acid and lipid metabolic pathways. Therefore, attention should be paid to controlling the intestinal microbiota to prevent complications and improve the prognosis in patients with hepatitis B cirrhosis, especially in those with ascites.

Objective To investigate the therapeutic effect of traditional Chinese medicine (TCM) on portal vein thrombosis (PVT) in patients with liver cirrhosis and its medication characteristics. Methods A retrospective analysis was performed for 89 patients with liver cirrhosis and PVT who were hospitalized and treated in Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, and according to whether TCM treatment was applied in combination, they were divided into TCM group with 59 patients and control group with 30 patients. Related data were collected for the two groups, including demographic data, laboratory examination, radiological examination, gastroscopy, history of surgery, portal hypertension-related complications, medication, and follow-up data. The independent samples t -test was used for comparison of normally distributed continuous data between two groups, and the Mann-Whitney U rank sum test was used for comparison of non-normally distributed continuous data between two groups; the chi-square test or the Fisher's exact test was used for comparison of categorical data between two groups. An ordinal polytomous Logistic regression analysis was used for multivariate analysis. TCM Inheritance Computing Platform (V3.0) was used to perform a drug effect cluster analysis of TCM prescriptions. Results The multivariate logistic regression analysis showed that esophageal and gastric varices (odds ratio [ OR ]=3.144, 95% confidence interval [ CI ]: 1.221-8.094), PVT involving the portal vein (PV) and the superior mesenteric vein (SMV) ( OR =51.667, 95% CI : 3.536-754.859), PVT involving PV+spleen vein (SV)+SMV ( OR =13.271, 95% CI : 2.290-76.928), cavernous transformation of the portal vein ( OR =11.896, 95% CI : 1.172-120.696), and TCM intervention ( OR =0.348, 95% CI : 0.129-0.938) were influencing factors for the outcome of PVT in liver cirrhosis. Follow-up results showed that compared with the control group, the TCM group had a significantly lower progression rate (16.95% vs 56.67%, P < 0.001) and a significantly lower incidence rate of variceal rupture and bleeding (8.47% vs 33.33%, P < 0.001). Effective TCM drugs with a relatively high frequency of use included deficiency-tonifying drugs (359 times, 34.6%), blood-activating and stasis-resolving drugs (202 times, 19.5%), and diuresis-inducing and dampness-draining drugs (180 times, 17.3%); the TCM drugs with a relatively high frequency of use included Astragalus membranaceus (57 times, 8.7%), Angelica sinensis (50 times, 7.6%), and leech (48 times, 7.3%); TCM drug combinations with a relatively high frequency of use included Astragalus membranaceus+Angelica sinensis, Astragalus membranaceus+leech, Angelica sinensis+leech, and Astragalus membranaceus+Angelica sinensis+leech. Conclusion Qi-tonifying, blood-activating, and stasis-breaking drugs, such as Astragalus membranaceus, Angelica sinensis, and leech, can promote the stabilization or recanalization of PVT in liver cirrhosis and reduce the incidence rate of bleeding events due to portal hypertension.

Objective: To study the incidence and clinicopathological features of intermediate fibroblastic/myofibroblastic tumors(IF/MFT) in infants and the young children. Methods: All available cases with soft tissue tumors in infants and children were retrieved from the files of Women and Children′s Hospital Affiliated to Qingdao University, from January 2012 to December 2017.The incidence rate of IF/MFT was observed.Cases of IF/MFT were identified and investigated by light microscopy and immunohistochemistry by reviewing the related literature. Results: Among 290 soft tissue tumors, 15 cases(5.2%) were IF/MFT, accounted for 88.2%(15/17 cases) of borderline soft tissue tumors.Twelve cases were male, 3 cases were female, the median age was 8 months, and 4 cases were congenital.Clinically, 11 cases were presented with slow-growing painless masses located in the trunk or extremities.According to histopathology, 9 cases(60.0%) were categorized as infantile fibromatosis(IFM), including 5 cases(33.3%) desmoid-type and 4 cases(26.7%) diffuse-type; 3 cases(20.0%) as lipofibromatosis(LFM); 2 cases(13.3%) as infantile fibrosarcoma(IFS) and 1 case(6.7%) as giant cell fibroblastoma(GCF). All 15 tumors were characterized by the presence of spindle fibroblasts and myofibroblasts with infiltration of the surrounding structures.Immunohistochemically, all the 15 cases were diffusely positive for Vimentin(Vim), but negative for Myogenin, MyoD1, Desmin and S-100.Smooth muscle actin(SMA), β-catenin and Bcl-2 were positive in some cases to a certain degree.The Ki-67 proliferation index was higher in diffuse-type IFM and IFS, the former was 5.0%-20.0%, and the latter was about 20.0%, however, the other cases showed Ki-67 <5.0%.The main clinical treatment was complete or extensive excision. Conclusions IF/MFT accounts for a high proportion of intermediate soft tissue tumors in infants and young children, mostly seen in male children, and IFM and LFM are the main types.The clinical signs and symptoms associated with these tumors are often nonspecific, and their histopathologic manifestations may overlap.The final diagnosis of IF/MFT must depend on the characteristics of age, location, histopathologic changes and immunohistoche-mical findings.

Objective To investigate clinical effect and safety of eccentric reamer technique foraminoplasty during percutaneous endoscopic lumbar discectomy for the treatment of lumbar lateral recess stenosis.Methods All of 45 cases diagnosed of lumbar lateral recess stenosis between Apr 2010 to Jun 2012 were analyzed in our study.Among these cases,28 cases were male and 17 were female.The age of enrolled cases was 39-62 years old and the average age was 49.23±23.96 years old.All the cases were one-level,25 cases in L4,5 and 20 cases in L5S1.All the patients underwent percutaneous endoscopic lumbar discectomy.During surgery,eccentric reamer technique was applied for foraminoplasty.Perioperative data included surgical segment,surgical duration and blood loss during surgery.Oswestry Disability Index (ODI) and Visual analogue scale (VAS) for lower back and leg pain were recorded for the evaluation of surgical effect.All the data were recorded before surgery,1 day,1 month after surgery and final follow-up.Recurrent lumbar disc herniation,spinal canal hematoma,intervertebral disc infection,nerve root injury and post-operative dysthesia (POD) were recorded for complications.Results All the cases finished surgery and none of the cases was changed to other surgery method and no other electronic drill were used in these patients.Surgical duration was 45-76 min and average time was 61.23± 13.89 min.Blood loss during surgery was 10-25 ml and average blood loss was 15.02±4.33 ml.Post-operative hospitalization days were 1-3 days and 2.80±1.57 days.The follow-up time was 60-84 months and the average time was 73.23±8.47 months.ODI in one-day after surgery was 9.34±6.54％,significantly lower than 68.06±24.14％ before surgery and no statistically change had been observed in one-month after surgery and final follow-up.The VAS for lower back was 1.54±0.97 in one-day after surgery,significantly lower than 3.57±3.12 before surgery.The VAS for leg pain was 1.14±0.79 in one-day after surgery,significantly lower than 6.24±2.35 before surgery.During follow-up,1 case suffered from recurrent lumbar disc herniation (2.22％) and no intervertebral disc infection,spinal canal hematoma and POD was observed.Conclusion Eccentric reamer technique foraminoplasty in percutaneous endoscopic lumbar discectomy is an effective and safe surgical method for the treatment of lumbar lateral recess stenosis.

T cells engineered with chimeric antigen receptor (CAR) have been successfully applied to treat advanced refractory B cell malignancy.However,many challenges remain in extending its application toward the treatment of solid tumors.The immunosuppressive nature of tumor microenvironment is considered one of the key factors limiting CAR-T efficacy.One negative regulator of T cell activity is lymphocyte activation gene-3 (LAG-3).We successfully generated LAG-3 knockout T and CAR-T cells with high efficiency using CRISPR-Cas9 mediated gene editing and found that the viability and immune phenotype were not dramatically changed during in vitro culture.LAG-3 knockout CAR-T cells displayed robust antigen-specific antitumor activity in cell culture and in murine xenograft model,which is comparable to standard CAR-T cells.Our study demonstrates an efficient approach to silence immune checkpoint in CAR-T cells via gene editing.

OBJECTIVETo compare the expressions of hypoxia-inducible factor 1 alpha(HIF-1α) and Semaphorin 4D(Sema4D) in colorectal carcinoma and normal colorectal tissues, and to investigate their correlation and clinical significance.

METHODSThe expressions of HIF-1α and Sema4D were examined in 86 cases of colorectal carcinoma and 52 normal colorectal tissues by SP immunohistochemical staining. Correlation between these two expressions and association of the expressions with clinicopathological characters and prognosis were analyzed.

RESULTSThe positive rates of HIF-1α and Sema4D protein in colorectal carcinoma tissues were significantly higher than those in normal colorectal tissues(58.1% vs. 7.7%, χ(2)=34.624, P<0.01; 60.5% vs. 11.5%, χ(2)=31.839, P<0.01). HIF-1α and Sema4D protein expressions were closely associated with colorectal carcinoma histological types(P=0.003, P=0.010), TNM staging (P=0.003, P=0.017) and lymphatic metastasis (P=0.003, P=0.020), and a significant correlation was observed between the expressions of HIF-1α and Sema4D protein (r=0.567, P<0.01). The 5-year overall survival rate was 37%. Univariate analysis showed that 5-year survival rates of patients with positive and negative HIF-1α protein expression were 24% and 56%(P=0.003), and those with positive and negative Sema4D protein expression were 23% and 59%(P=0.001). Multivariate Cox analysis showed that expression of Sema4D was an independent prognostic factor of colorectal cancer patients(P=0.026), while expression of HIF-1α was not(P=0.501).

CONCLUSIONCombined detection of HIF-1α and Sema4D has the potential to predict the development trend of colorectal carcinoma and prognosis of patients.

Antigens, CD ; metabolism ; Colorectal Neoplasms ; metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Lymphatic Metastasis ; Neoplasm Staging ; Prognosis ; Semaphorins ; metabolism ; Survival Rate

BACKGROUNDHypoxia promotes tumor angiogenesis and hypoxia-inducible factor-1 alpha (HIF-1α) plays a pivotal role in this process. Recently identified pro-angiogenic factor, semaphorin4D (Sema4D) also promotes angiogenesis and enhances invasive proliferation in some tumors. Furthermore, tumor-associated macrophages (TAMs) can increase the expression of HIF-1α and Sema4D in cancer cells and thus influence tumor growth and progression. The purpose of this study was to evaluate the effect of TAMs on the expression of Sema4D and HIF-1α and the impact of biologic behavior in colon cancer cells.

METHODSImmunohistochemistry was used to analyze HIF-1α and Sema4D expression in 86 curatively resected colon cancer samples and 52 normal colon tissues samples. The relationship between their expression and clinicopathological factors was analyzed. Furthermore, macrophage-tumor cell interactions, such as metastasis, angiogenesis, were also studied using in vitro co-culture systems. Statistical analysis was performed using SPSS 17.0 software (SPSS Inc., USA). Differences between two groups were analyzed with Student's t test.

RESULTSHIF-1α (58%) and Sema4D (60%) were expressed at a significantly higher level in tumors than in normal tissues (P < 0.01, for both). Furthermore, HIF-1α and Sema4D expression was significantly correlated with lymphatic metastasis, specific histological types and TNM stages (P < 0.05), but not with age and tumor size (P > 0.05). Sema4D expression was correlated with that of HIF-1α (r = 0.567, P < 0.01). TAMs markedly induced HIF-1α and Sema4D expression in colon cancer cells and subsequently increased their migration and invasion.

CONCLUSIONSHIF-1α and Sema4D expression are closely related to lymphatic metastasis, specific histological types and TNM stages in colon cancer. Furthermore, TAMs promote migration and invasion of colon cancer cells and endothelial tube formation, possibly through up-regulation of HIF-1α and Sema4D.

Adult ; Aged ; Antigens, CD ; genetics ; metabolism ; Cell Line, Tumor ; Colonic Neoplasms ; genetics ; metabolism ; Female ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Immunohistochemistry ; Macrophages ; immunology ; metabolism ; Male ; Middle Aged ; Neoplasm Metastasis ; genetics ; pathology ; Semaphorins ; genetics ; metabolism

Objective To compare the expressions of hypoxia-inducible factor 1 alpha (HIF-1α) and Semaphorin 4D (Sema4D) in colorectal carcinoma and normal colorectal tissues, and to investigate their correlation and clinical significance. Methods The expressions of HIF-1α and Sema4D were examined in 86 cases of colorectal carcinoma and 52 normal colorectal tissues by SP immunohistochemical staining. Correlation between these two expressions and association of the expressions with clinicopathological characters and prognosis were analyzed. Results The positive rates of HIF-1α and Sema4D protein in colorectal carcinoma tissues were significantly higher than those in normal colorectal tissues (58.1% vs. 7.7%, χ2=34.624,P<0.01; 60.5% vs. 11.5%, χ2=31.839, P<0.01). HIF-1α and Sema4D protein expressions were closely associated with colorectal carcinoma histological types (P=0.003,P=0.010), TNM staging (P=0.003,P=0.017) and lymphatic metastasis (P=0.003,P=0.020), and a significant correlation was observed between the expressions of HIF-1α and Sema4D protein (r=0.567, P<0.01). The 5-year overall survival rate was 37%. Univariate analysis showed that 5-year survival rates of patients with positive and negative HIF-1α protein expression were 24% and 56%(P=0.003), and those with positive and negative Sema4D protein expression were 23% and 59%(P=0.001). Multivariate Cox analysis showed that expression of Sema4D was an independent prognostic factor of colorectal cancer patients(P=0.026), while expression of HIF-1α was not(P=0.501). Conclusion Combined detection of HIF-1α and Sema4D has the potential to predict the development trend of colorectal carcinoma and prognosis of patients.

Objective To compare the expressions of hypoxia-inducible factor 1 alpha (HIF-1α) and Semaphorin 4D (Sema4D) in colorectal carcinoma and normal colorectal tissues, and to investigate their correlation and clinical significance. Methods The expressions of HIF-1α and Sema4D were examined in 86 cases of colorectal carcinoma and 52 normal colorectal tissues by SP immunohistochemical staining. Correlation between these two expressions and association of the expressions with clinicopathological characters and prognosis were analyzed. Results The positive rates of HIF-1α and Sema4D protein in colorectal carcinoma tissues were significantly higher than those in normal colorectal tissues (58.1% vs. 7.7%, χ2=34.624,P<0.01; 60.5% vs. 11.5%, χ2=31.839, P<0.01). HIF-1α and Sema4D protein expressions were closely associated with colorectal carcinoma histological types (P=0.003,P=0.010), TNM staging (P=0.003,P=0.017) and lymphatic metastasis (P=0.003,P=0.020), and a significant correlation was observed between the expressions of HIF-1α and Sema4D protein (r=0.567, P<0.01). The 5-year overall survival rate was 37%. Univariate analysis showed that 5-year survival rates of patients with positive and negative HIF-1α protein expression were 24% and 56%(P=0.003), and those with positive and negative Sema4D protein expression were 23% and 59%(P=0.001). Multivariate Cox analysis showed that expression of Sema4D was an independent prognostic factor of colorectal cancer patients(P=0.026), while expression of HIF-1α was not(P=0.501). Conclusion Combined detection of HIF-1α and Sema4D has the potential to predict the development trend of colorectal carcinoma and prognosis of patients.