Role of sclerostin in the occurrence and development of uveal melanoma and its biological mechanism / 中华实验眼科杂志

Objective:To investigate the effects of sclerostin (SOST) and WNT/CTNNB1 signaling pathway on the cell cycle, migration and invasion of human uveal melanoma (UM) cells and its related mechanism.Methods:UM tissues from 20 cases of epithelioid UM and 16 cases of spindle cell type UM were collected.The contents of SOST, Wnt-1 and Catenin beta-1 proteins in the collected tissues were detected by immunohistochemical staining.Three human UM tissue derived cell lines OCM-1 (primary spindle cell type), Mum-2B (metastatic epithelioid) and Mum-2C (metastatic spindle cell type) were selected and divided into three groups, blank control group not transfected, empty vector group transfected with SOST negative control vector and SOST siRNA group transfected with SOST siRNA.After 24-hour transfection, the mRNA and protein expression levels of SOST, CTNNB1, WNT protein family 1 (WNT1), CCND1, matrix metalloproteinase (MMP)2 and MMP9 were detected by real-time fluorescence quantitative PCR and Western blot, respectively.The invasion and migration ability of the transfected cells were measured by transwell method, and the cell cycle distribution was detected by flow cytometry.Another 9 female BALB/c nude mice were selected and randomized into OCM-1 group, OCM-1 empty vector group and SOST shRNA group, inoculated with OCM-1 without lentivirus infection, OCM-1 with blank lentivirus infection and OCM-1 with SOST shRNA lentivirus infection, respectively.Six weeks after inoculation, the in situ formation of tumor was observed.The interaction between SOST and low density lipoprotein receptor related protein(LRP)-5/6 in OCM-1 cells was explored by co-immunoprecipitation assay.The study protocol was approved by the Ethics Committee of Tianjin Medical University Eye Hospital (2018KY[L]-20).Results:Immunohistochemical staining results showed that the SOST expression level was higher and the expression levels of Wnt-1 and Catenin beta-1 were lower in spindle cell type UM tissues than in epithelioid UM tissues, and the differences were all statistically significant (all at P<0.01). The real-time fluorescence quantitative PCR results showed that the relative expression of SOST mRNA was significantly lower and the relative expressions of CCND1, WNT1 and MMP9 mRNA were significantly higher in SOST siRNA groups than in corresponding empty vector groups in the three cell lines (all at P<0.05). In OCM-1 and Mum-2C cell lines, the relative expressions of CTNNB1 mRNA were significantly higher in SOST siRNA groups than in empty vector groups (all at P<0.01). Western blot results showed that the relative expression of SOST protein was significantly lower and the relative expressions of Wnt-1, Catenin beta-1, cyclin-D1, MMP2 and MMP9 proteins were significantly higher in SOST siRNA groups than in empty vector groups (all at P<0.01). Transwell assay showed that the cell invasion and migration ability of SOST siRNA group was significantly higher than that of blank control group and empty vector group in the three cell lines (all at P<0.01). Flow cytometry showed that the proportion of G1-phase cells and the G1/S-phase ratio were significantly lower in SOST siRNA group than in blank control groups and empty vector groups (all at P<0.01). The eyeball volume of OCM-1 group, OCM-1 empty vector group and SOST shRNA group was (42.7±4.6), (49.0±22.9) and (135.2±32.7)mm 3, respectively, showing a significant overall difference ( F=19.963, P<0.01). The eyeball volume of SOST shRNA group was larger than that of OCM-1 group and OCM-1 empty vector group, and the differences were statistically significant (both at P<0.05). Co-immunoprecipitation results showed that SOST could interact with LRP-5 and LRP-6 by binding to them, respectively. Conclusions:Silencing SOST can promote the invasion and migration of UM cells, and increase the proportion of UM cells in the division phase.Silencing SOST can promote tumor growth in eyes of nude mice.SOST may play this function by interacting with the membrane receptor LRP-5/LRP-6 and then regulating the WNT/CTNNB1 signal pathway.

Comparison of the effects of balanced orbital decompression and endoscopic transnasal inferomedial wall decompression with the high orbital pressure / 中华实验眼科杂志

Objective:To explore the surgical outcomes and surgery-related complications of balanced orbital decompression and endoscopic transnasal inferomedial wall decompression for Grave's ophthalmopathy (GO).Methods:A cohort study was performed.The 56 GO patients who underwent balanced orbital decompression or endoscopic transnasal inferomedial wall decompression in the Tianjin Medical University Eye Hospital from December 2016 to December 2019 were enrolled.The follow-up time was 6 months.Patients were divided into two groups according to the operation modes.Thirty-three eyes of 24 subjects were given deep lateral wall rim-sparing orbital decompression and transcaruncular medial wall decompression, and 51 eyes of 36 cases were given endoscopic transnasal inferomedial wall decompression.The demographics, surgical details, imaging data, postoperative changes of exophthalmos, best corrected visual acuity (BCVA), orbital pressure and diplopia, surgery-related complications and further treatment were analyzed and compared.This study followed the Declaration of Helsinki and was approved by the Ethics Committee of Tianjin Medical University Eye Hospital [No.2020KY(L)-39]. All subjects signed informed consent.Results:The exophthalmos was (21.03±3.11)mm before operation, which was significantly higher than (17.06±2.55)mm after operation in the balanced orbital decompression group ( P<0.05). The exophthalmos was (20.51±3.53)mm before operation, which was significantly higher than (16.28±2.96)mm after operation in the endoscopic transnasal inferomedial wall decompression group ( P<0.05). No significant difference in the mean reduction of proptosis was found between the two groups ( P>0.05). All the subjects were accompanied with increase of intraorbital pressure before operation.The intraorbital pressure returned to normal at 6 months after operation.The postoperative BCVA of subjects with dysthyroid optic neuropathy (DON) were significantly higher than preoperative values ( Z=-3.524, -4.376; both at P<0.01). The postoperative improvement values of BCVA were 0.48 (0.25, 0.67) and 0.72 (0.40, 0.80) in the balanced orbital decompression group and the endoscopic transnasal inferomedial wall decompression group, respectively, with a significant difference between the two groups ( Z=-2.481, P=0.016). The incidence of complications in the balanced orbital decompression group was 21.2% (7/33), which was significantly lower than 47.0% (24/51) in the endoscopic transnasal inferomedial wall decompression group ( χ2=5.748, P=0.017). Conclusion:The two kinds surgical methods can effectively reduce the degree of exophthalmos and orbital pressure.Endoscopic transnasal inferomedial wall decompression can provide better improvement of visual function in patients with DON, but has a higher risk of surgery-related complications in comparison with the balanced orbital decompression.

Biocompatibility research of a novel pH sensitive ion exchange resin microsphere

The main objective of this study was to investigate biocompatibility and provide in vivo pharmacological and toxicological evidence for further investigation of the possibility of pH sensitive ion exchange resin microsphere for clinical utilizations. Acute toxicity study and general pharmacological studies were conducted on the pH sensitive ion exchange resin microsphere we prepared. The general pharmacological studies consist of the effects of the pH sensitive ion exchange resin microsphere on the nervous system of mice, the functional coordination of mice, the hypnosis of mice treated with nembutal at subliminal dose, the autonomic activities of tested mice, and the heart rate, blood pressure, ECG and breathing of the anesthetic cats. The LD50 of pH sensitive ion exchange resin microsphere after oral administration was more than 18.84 g kg[-1]. Mice were orally administered with 16 mg kg[-1], 32 mg kg[-1] and 64 mg kg[-1] of pH sensitive ion exchange resin microsphere and there was no significant influence on mice nervous system, general behavior, function coordination, hypnotic effect treated with nembutal at subliminal dose and frequency of autonomic activities. Within the 90 min after 5 mg kg[-1], 10 mg kg[-1], 20 mg kg[-1] pH sensitive ion exchange resin microsphere was injected to cat duodenum, the heart rate, blood pressure, breathing and ECG of the cats didn't make significant changes in each experimental group compared with the control group. The desirable pharmacological and toxicological behaviors of the pH sensitive ion exchange resin microsphere exhibited that it has safe biocompatibility and is possible for clinical use

Wnt3a is Important in The Differentiation From Neural Stem Cell Into Dopaminergic Neuron In vitro / 生物化学与生物物理进展

Wnt signaling is implicated in the control of cell growth and differentiation during neural stem cell(CNS) development.Wnt3a, one of wnt gene family members, has effect on regeneration neurospheres and differentiation into neurons.Wnt3a inhibits regeneration of neurospheres, and promotes its differentiation. In vitro neurosphere was cultured in a serum-free defined medium DMEM/F12 supplemented with bFGF and EGF. Dissociated cells were plated onto poly-d-lysine-coated coverslips and propagated in medium containing recombined Wnt3a-adenovirus. Plenty of Nurr1 were detected by RT-PCR after 3 days. Wnt3a combined AA would improve NSC differentiation into dopaminergic (DA) neuron. The quantity of DA neuron is obviously more than the AA alone group's. Moreover, the expression of TH mRNA is 1.86 fold in Wnt3a combined AA group. Induced cells were immunostained for TH and DAT. The proportion of TH-positive was (37.42 ? 2.54) % (P

Preparation of human single chain Fv antibody specifically against hepatitis C virus E2 antigen / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To identify human single chain Fv antibody (ScFv) against hepatitis C viral E2 antigen and its value clinically.</p><p><b>METHODS</b>The recombinant phages were panned by E2 antigen which was coated in a microtiter plate. After five rounds of biopanning, 56 phage clones were identified specific to E2 antigen. The affinity and specificity of ScFv were evaluated by ELISA and immunohistochemistry, respectively.</p><p><b>RESULTS</b>The data of E2-ScFv DNA digestion and DNA sequencing showed that the ScFv gene was composed of 750bp. ELISA and immunohistochemistry demonstrated that the human single chain Fv antibody against HCV E2 antigen had a specific combination character with hepatitis C virus E2 antigen.</p><p><b>CONCLUSIONS</b>ScFv, having a sutestantial affinity and specificity and being easy to prepare, is valuable in the detection of HCV E2 antigen.</p>

Screening and application of human-derived HBsAg bacteriophage single chain antibody in clinical diagnosis / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To identify human single chain Fv antibody (ScFv) against hepatitis B viral surface antigen.</p><p><b>METHODS</b>The recombinant phages were panned by HBsAg which was coated in a microtiter plate, after five rounds of biopanning, 56 phage clones were identified specific to HBsAg. The specificity of ScFv was evaluated by ELISA and immunohistochemistry, respectively.</p><p><b>RESULTS</b>The data of HB sAg-ScFv DNA digestion and DNA sequencing showed that the ScFv gene is composed of 750 bp. ELISA and immunohistochemistry demonstrated that the human single chain Fv antibody against hepatitis B surface antigen has a specific combination character with hepatitis B surface antigen of different sources and paraffin-embedded patients tissue specimens, it did not react with normal liver tissue and HCV.</p><p><b>CONCLUSIONS</b>The application of HBsAg specific ScFv in immunohistochemistry was successfully achieved.</p>

Quasispecies and variations of hepatitis B virus: core promotor region as an example / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the HBV quasispecies groups in the patients with chronic HBV infection.</p><p><b>METHODS</b>A set of specific primers was synthesized according to DNA sequence of HBV strain found in China. The whole core promoter (CP) region was amplified by PCR method from the sera of 3 patients with chronic HBV infection, and the PCR products were subcloned into pGEM Teasy vectors. The clones were randomly selected to be sequenced. Sequence comparison of the selected clones was made to find the difference.</p><p><b>RESULTS</b>By comparison, it was found that each sequence of selected clones was different. The point mutation always occurred in TATA-like boxes, especially from T to C replacement on 184 site. There is a hot region (33.3% 5/15) in basic core promoter where deletion mutation frequently happened.</p><p><b>CONCLUSIONS</b>There is a hot deletion region near DR I in CP. The replacement at 184 nt (T to C) in the third TATA-like box may influence the expression of pre-C/C protein. The sequencing results suggest that there are HBV quasispecies groups in chronically infected patients.</p>

Cloning and analysis of the genomic DNA sequence of augmenter of liver regeneration from rat / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To search for genomic DNA sequence of the augmenter of liver regeneration (ALR) of rat.</p><p><b>METHODS</b>Polymerase chain reaction (PCR) with specific primers was used to amplify the sequence from the rat genome.</p><p><b>RESULTS</b>A piece of genomic DNA sequence and a piece of pseudogene of rat ALR were identified. The lengths of the gene and pseudogene are 1508 bp and 442 bp, respectively. The ALR gene of rat includes 3 exons and 2 introns. The 442 bp DNA sequence may represent a pseudogene or a ALR-related peptide. Predicted amino acid sequence analysis showed that there were 14 different amino acid residues between the gene and pseudogene. ALR-related peptide is 84 amino acid residues in length and relates closely to ALR protein.</p><p><b>CONCLUSION</b>There might be a multigene family of ALR in rat.</p>

THE STUDY ON QUASISPECIES OF HEPATITIS B VIRUS : PRE-CORE/CORE GENE AS AN EXAMPLE / 解放军医学杂志

To investigate the HBV quasispecies groups in the patients with chronic HBV infection. A set of specific primers was synthesized according to HBV DNA sequence of the Chinese strain, and the whole pre core/core region was amplified by PCR method from the serum of 4 patients with chronic HBV infection.Then the PCR products were subcloned into pGEM Teasy vectors. Clones were selected to be sequenced,and then sequence comparison was made to find the difference. Each sequence of selected clones was found to be different. The homology of the nucleic acid sequence from the same patient was above 98 0%. There were many mutation types in this region, including point substitution, core region internal deletion, as well as frame shift reading. The results indicated that there were HBV quasispecies groups in chronically infected patients. The mutation in this region may influence the prognosis of HBV infection.

CLONING AND ANALYSIS OF THE CODING SEQUENCE FOR HEPATITIS B VIRUS PRES1 BINDING PROTEIN BY T7 cDNA PHAGE DISPLAY SYSTEM / 解放军医学杂志

T7 cDNA phage display method was employed to find the binding protein of PreSl protein. PreSl protein was coated in a 96-well ELISA plate, and then T7 cDNA library phages were bound to the target protein. Phages which did not bind to garget protein were washed away and the binding phages were eluted. Insertions from different clones were sequenced, and the deduced amino acid sequences were analyzed by Vector 6.0 software. Using BLAST software in GenBank, whole length of amino acid sequence of binding protein was obtained. After 4 rounds of biopanning, recombinant T7 phages with binding ablity were amplifed by infection to E. coli. One piece of amino acid sequence was found to be amino terminal of product of glioma tumor suppressor candidate region gene 2 (GLTSCR2). There was a binding domain KxPxKSGxxxL in these clones. T7 cDNA phage display technique can be used bo find the ligand. GLTSCR2 coding protein may be the binding protein to preSl protein of HBV.\;

EXPRESSION OF SOLUBLE HUMAN ScFv AGAINST CORE PROTEIN OF HEPATITIS C VIRUS IN E COLI / 解放军医学杂志

To construct expressive vector for human ScFv against core protein of hepatitis C virus (HCV core ScFv),and to express soluble HCV core ScFv in E.coli JM109. Using phage display technique, the recombinant phages were panned by recombinant core antigen which was coated in a microtiter plate, and after five rounds of biopanning, 86 clones were identified specific to core antigen. 750 bp fragment could be released from the plasmid of positive phage clones, and the sequence analysis indicated that we have obrained the ScFv DNA fragment. Then DNA fragment was inserted into the expressive vector pCANTAB5E, and E. coli host JM109 was transformed and induced by IPTG. The specificity of ScFv in the culture medium was evaluated by enzyme linked immunosorbent assay (ELISA).The molecular weight of expressed HCV core ScFv protein is 28 000 dalton as determined with the aid of SDS polyacrymide gel electrophoresis (PAGE). The expressed HCV core ScFv protein will be useful in the immunohistochemical study of liver tissue from patients with hepatitis C and gene therapy against HCV infection.