Background Allograft rejection is a main cause of failure of penetrating keratoplasty,especially in the patient with high risk of rejection condition.Previous study on allograft rejection mechanism focused on limbal and corneal neovascularization,but these factors did not explain all the phenomena of allograft rejection.Research found that immune cells appeared in iris and ciliary body when rejection occurred,but the relationship between these immune cells and allograft rejection is unclear Objective This study was to evaluate the relationship between diversity of vascular permeability in the iris and ciliary body and allograft rejection after penetrating keratoplasty.Methods Seventy clean eight-week-old BALB/c mice were divided into allogeneic corneal transplantation group (60 mice) and blank control group (10 mice).Allogeneic corneal transplantation was performed with the same age of C57BL/6 mice as donor and BALB/c mice as the recipients.The grafts were examined under the slit lamp microscope and scored based on the criteria of Hegde.The mice were sacrificed and iris and ciliary tissue were obtained 5,10 days and rejection after surgery.Immunohistochemistry and reverse transcription PCR (RT-PCR) was used respectively to detect the expression diversities of occludin,zonula occludens protein-1 (ZO-1),matrix metalloproteinase-9(MMP-9),major histocompatibility complex-Ⅱ (MHC-Ⅱ),and CCR5,CCR7 and their mRNA in iris and ciliary body.Image-J image analysis software was used to calculate the quantity of positive cells on iris wholemount,and absorbance of target genes (A values).The use and care of the experimental animals complied the ARVO Resolution on the Use of Animals in Research.Results The mean survival time of corneal gratts was (17±3) days after operation.The mean score was 0.6 in 5 days and 0.5 in 10 days,and 3.3 in 18 days after operation.Expression of ZO-1 reduced significantly,and that of MMP-9 increased obviously at the time of rejection.MHC Ⅱ + cells were scattered in iris and ciliary body in normal mice,and the number of the positive cells (cells/field) was increased after operation with a peak value when rejection occurred.A significant difference was seen between normal mice and rejection mice (1559.67±350.29 vs.4021.83±495.18) (P=0.000).The expressions of occludin mRNA and ZO-1 mRNA in the iris and ciliary body decreased obviously in the rejection mice.Compared with normal mice,theA value of ZO-1 and occluding were 36.74±3.13 vs.110.11±11.88 and 57.54±3.41 vs.59.90±3.50respectively,with significant differences between them (all P＜0.05).The expressions of MMP-9 mRNA,CCR5 mRNA and CCR7 mRNA in the iris and ciliary body increased gradually with the time lapse after operation and peaked when the rejection appeared.The A value of MMP-9 mRNA,CCR5 mRNA and CCR7 mRNA were significantly higher than those of normal mice (20.29±1.19 vs.2.77±0.85 for MMP-9 mRNA; 35.43±2.56 vs.9.11±0.29 for CCR5 mRNA,and 60.83±0.87 vs.0.89 ±0.95 for CCR7 mRNA) respectively (all P＜0.05).Conclusions The permeability of vascules in the iris and ciliary body increase during the allograft rejection after penetrating keratoplasty.Increased antigen presenting cells were also detected.

Objective To explore the value of color doppler ultrasonography by bed side in the early diagnosis of HIE in full term neonates.Methods The changes of cerebral parenchymal and cerebral arterial blood stream parameter on 35 cases of neonates clinically diagnosed HIE of mild and moderate degree and 40 cases of normal newborns on the 24,48 and 72 hours after birth were observed by color doppler ultrasonography by bed side.Results 1.The cerebral parenchyma was even echo in normal newborns,but it was maldistributed and reinforced in mild asphyxia neonates and it was more serious in moderate degree.The echo of cerebral parenchyma in mild degree was near normal in 48 hours after birth,while the echo of cerebral parenchyma in moderate degree was still maldistributed and reinforced in 48 and 72 hours after birth.2.There was obvious changes in the cerebral arterial blood stream parameter and hemodynamics of the asphyxia newborns compared with normals.The systolic peak velocity(Vs)and end diastolic velocity(Vd)of the cerebral arteries in mild and moderate degree were obviously lower than that of control group in 24,48 hours after birth(Pa0.05).3.Resistance index(RI)of the cerebral arteries in mild and moderate degree were higher than that of control group in 24,48 hours after birth(Pa0.05).Conclusion Color doppler ultrasonography by bed side is a convenient,noninvasive method for diagnosing HIE.

OBJECTIVETo study the effect and mechanism of Danhong injection on isolated mesenteric arterial rings in rats.

METHODAn isolated vascular ring experiment was conducted to determine the changes in tension of vascular rings with a biological signal collection and analytical system.

RESULTDanhong injection had no impact on the tension of vascular rings. Danhong injection showed a significant vasodilatation effect on treated arteria rings of norepinephrine, and no remarkable impact was made on the effect without endothium. It showed notable effect on blood vessels treated with Ca(2+) and no significant impact on those treated with caffeine. It could inhibit NE-induced intracellular calcium from releasing and external calcium from inflowing. No effects of potassium channel blockers on aorta ring tensile force were found.

CONCLUSIONDanhong injection shows significant vasodilation effect, which mainly works through vascular smooth muscle. Its vasodilation effect may be related to inhibitory receptor, voltage-dependent Ca(2+)-release and IP3 receptor-mediated Ca(2 +)-influx.

Animals ; Calcium ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; In Vitro Techniques ; Injections ; Mesenteric Arteries ; drug effects ; physiology ; Rats ; Rats, Sprague-Dawley ; Vasodilation ; drug effects

OBJECTIVETo study the antiviral constituents in the stems and leaves of Pithecellibium clypearia.

METHODThe constituents of P. clypearia were systematically separated with various chromatographic techniques in combination with antiviral activity monitoring. Their structures were elucidated by physical and chemical properties and spectral data.

RESULTSix compounds were isolated from P. clypearia and were identified as: tricetiflavan (5, 7, 3', 4', 5'-pentahydroxylflavan) (1), myricitrin (myricetin-3-O-alpha-L-rhamnopyranoside) (2), quercitrin (quercetin-3-O-alpha-L-rhamnopyranoside) (3), quereetin (4), methyl gallate (5) and gallic acid (6).

CONCLUSIONCompound 1 approximately 5 were obtained from this plant for the first time. Compound 4 was found to show an obvious anti-respiratory syncytial virus (RSV) activity.

Antiviral Agents ; chemistry ; isolation & purification ; pharmacology ; Fabaceae ; chemistry ; Flavonoids ; chemistry ; isolation & purification ; pharmacology ; Gallic Acid ; analogs & derivatives ; chemistry ; isolation & purification ; pharmacology ; Inhibitory Concentration 50 ; Plant Leaves ; chemistry ; Plant Stems ; chemistry ; Plants, Medicinal ; chemistry ; Quercetin ; chemistry ; isolation & purification ; pharmacology ; Respiratory Syncytial Viruses ; drug effects

Objective: To prepare monoclonal antibodies (McAb) with cardiac troponin I (cTnI) which was purified from fresh human cardiac muscle within 6 h. Methods: (1) Extraction and purification of human cTnI: cTnI was purified by high salt extraction, saltless precipitation, 65℃ treatment, ammonium sulfate fractionation and DEAE-cellulose chromatography, etc. (2) Preparation of anti human cTnI McAb: The purified cTnI was injected into the spleen of BALB/c mice. The cTnI-primed spleen cells were fused with Sp2/0 myoloma cell. The McAbs anti human cTnI were obtained by screening with indirect ELISA and 3 times clone. (3)The identification of anti cTnI McAb. Results: Five hybridoma cell lines, named 3A7，3A11，3D2，3F10 and 1H9 were developed, which could secret McAb stably. The 5 McAbs all were demonstrated to be IgG2a by double gel diffusion test. The number of hybridoma chromosomes was between 92 to 110 and the chromosomes were mainly telocentric. Five kinds of ascites had no cross-reaction to LDH，CK，CK-MB ，AST and cardiac troponin T(cTnT), and their titers were between 3.2×10-6 to 1.6×10-7. Conclusion: 3D2，3F10 and 3A7,3A11,1H9 react to different epitopes of cTnI.

Objective: To prepare monoclonal antibodies (McAb) with cardiac troponin I (cTnI) which was purified from fresh human cardiac muscle within 6 h. Methods: (1) Extraction and purification of human cTnI: cTnI was purified by high salt extraction, saltless precipitation, 65℃ treatment, ammonium sulfate fractionation and DEAE-cellulose chromatography, etc. (2) Preparation of anti human cTnI McAb: The purified cTnI was injected into the spleen of BALB/c mice. The cTnI-primed spleen cells were fused with Sp2/0 myoloma cell. The McAbs anti human cTnI were obtained by screening with indirect ELISA and 3 times clone. (3)The identification of anti cTnI McAb. Results: Five hybridoma cell lines, named 3A7，3A11，3D2，3F10 and 1H9 were developed, which could secret McAb stably. The 5 McAbs all were demonstrated to be IgG2a by double gel diffusion test. The number of hybridoma chromosomes was between 92 to 110 and the chromosomes were mainly telocentric. Five kinds of ascites had no cross-reaction to LDH，CK，CK-MB ，AST and cardiac troponin T(cTnT), and their titers were between 3.2×10-6 to 1.6×10-7. Conclusion: 3D2，3F10 and 3A7,3A11,1H9 react to different epitopes of cTnI.

OBJECTIVE: To study the expression and diagnostic value of plasma miR-145 and miR-183 in children with lupus nephritis (LN). METHODS: A total of 92 children with LN who were admitted from January 2016 to May 2019 were enrolled as the LN group, among whom 17 had type II LN, 15 had type III LN, 36 had type IV LN, 18 had type V LN, and 6 had type VI LN. Forty healthy children who underwent physical examination were enrolled as the healthy control group. According to Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), the 92 children with LN were further divided into a stable LN group with 34 children (SLEDAI score <10) and an active LN group with 58 children (SLEDAI score ≥10). RT-PCR was used to measure the expression of miR-145 and miR-183 in plasma. The receiver operating characteristic (ROC) curve was used to analyze the value of plasma miR-145, miR-183, and anti-dsDNA antibody in the diagnosis of LN. Pearson correlation analysis was used to investigate the correlation of the expression levels of miR-145 and miR-183 in plasma with laboratory markers. RESULTS: The LN, active LN, and stable LN groups had significantly higher levels of anti-dsDNA antibody, C-reactive protein, serum creatinine (Scr), and blood urea nitrogen (BUN) than the control group (P<0.05). The active LN group had significantly higher SLEDAI score, anti-dsDNA antibody, Scr, and BUN than the stable LN group (P<0.05). The LN, active LN, and stable LN groups had significantly lower levels of complement C3, complement C4, and serum albumin (Alb) than the control group (P<0.05). The active LN group had a significantly lower level of Alb than the stable LN group (P<0.05). The LN, active LN, and stable LN groups had significantly lower plasma levels of miR-145 and miR-183 than the control group (P<0.01). The active LN group had significantly lower plasma levels of miR-145 and miR-183 than the stable LN group (P<0.01). The children with difference types of LN had significantly lower plasma levels of miR-145 and miR-183 than the control group (P<0.01), and the type V-VI group and the type IV group had significantly lower plasma levels of miR-145 and miR-183 than the type II-III group (P<0.01). The ROC curve analysis showed that the optimal cut-off values of plasma miR-145, miR-183, and anti-dsDNA antibody were 1.05, 0.62, and 186.30 IU/mL respectively, in the diagnosis of LN, and the combination of these three indices had the largest area under the ROC curve of 0.896 (95%CI: 0.835-0.955), with a sensitivity of 90.5% and a specificity of 84.2%. In the children with LN, the plasma levels of miR-145 and miR-183 were negatively correlated with SLEDAI score, anti-dsDNA antibody, Scr, and BUN (P<0.05) and were positively correlated with complement C3, complement C4, and Alb (P<0.05). CONCLUSIONS There are significant reductions in the expression levels of miR-145 and miR-183 in plasma in children with LN, which are correlated with the activity level and pathological typing of LN. Combined measurement of miR-145, miR-183, and anti-dsDNA antibody has a high value in the diagnosis of LN.
Biomarkers ; Child ; Complement C4 ; Humans ; Lupus Nephritis ; genetics ; MicroRNAs ; genetics ; ROC Curve

OBJECTIVETo investigate the effect of the COX-2 gene on the oncogenesis and development of the hepatocellular carcinoma and the influence of COX-2 gene on the expression of P-gp protein.

METHODFifty-two pieces of the hepatocellular carcinoma samples and 20 cases of normal liver samples were collected from the patients operated from October 2003 to June 2005. RT-PCR and immunohistochemistry staining were employed to detect the COX-2 mRNA as well as P-gp protein in the normal liver tissues and the carcinoma tissues. Meanwhile, the expression of the mdr 1 mRNA in the carcinoma tissues was also determined and the correlation between the expressions of the COX-2 and P-gp was investigated.

RESULTSNo expression of the COX-2 in the normal liver tissue was detected. The positive expression of COX-2 in the low and middle differentiated carcinoma was elevated significantly as compared with that in the high differentiated carcinoma tissue (x2 = 6.80, P less than 0.01). The positive expression of the COX-2 in the HBSAg (+) carcinoma tissue was significantly higher as compared with that in the HBSAg (-) carcinoma (x2 = 4.70, P less than 0.05), and the carcinoma in combination with cirrhosis also showed significantly higher in expression of COX-2 than the carcinoma without cirrhosis (x2 = 7.51, P less than 0.01). The mdr1 mRNA was found both expressed in the normal and carcinoma tissues. The expression of COX-2 mRNA was found in the carcinoma, but not found in the normal tissues. The COX-2 mRNA and mdr1 mRNA was found both expressed in the normal and carcinoma tissues. The correlation coefficient between COX-2 and mdr1 mRNA was 0.563 ( P less than 0.01).

CONCLUSIONThe results indicated that Cox-2 gene might involved in the multidrug resistance of the hepatocellular carcinoma mediated by P-gp.

ATP Binding Cassette Transporter, Sub-Family B ; ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Adult ; Aged ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Cyclooxygenase 2 ; metabolism ; Drug Resistance, Multiple ; genetics ; Drug Resistance, Neoplasm ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Male ; Middle Aged

OBJECTIVETo analyze the molecular genetic basis for a new B112 allele of ABO blood group and the pedigree of the proband.

METHODSThe ABO group antigens on red cells of the proband were identified by monoclonal antibodies. The ABO antibody in serum was detected by using standard A, B and O cells. The exons 5-7 of ABO gene for the proband was amplified by polymerase chain reaction and the amplified products were digested with double enzymes and sequenced for exons 6 and 7. A magnetic bead-based, haplotype specific extraction was used to separate the diploid sample of the proband into its haploid components. The exons 6 and 7 of the two single ABO haplotypes were then amplified and sequenced separately. The samples of the parents of the proband were collected, and the blood group serological test and sequence analysis for exons 6 and 7 of ABO gene were performed.

RESULTSThe serum characteristic of the proband was consisted with the normal B phenotype. The DNA sequencing of exons 6 and 7 showed 261G/del, 297A/G, 526C/G, 559C/T, 657C/T, 703G/A, 796C/A, 803G/C and 930G/A heterozygotes and was assigned for B/O genotype. After separation of the two single strands of the proband with haplotype specific extraction, a B112 and an O01 allele were identified after sequencing. The B112 allele had one nucleotide change (C to T) at position 559 compared with B101, which resulted in an amino acid change at position 187 (Arg to Cys). The B112 in the proband was identified to inherit from his mother after pedigree analysis, the ABO blood group serological characteristics and sequences of exons 6 and 7 of the mother were identical to that of the proband.

CONCLUSIONA novel B112 allele of ABO group system with 559C>T was identified. It had normal B antigen expression, suggesting that Arg118Cys of alpha-1, 3 galactosyltransferase did not affect its enzyme activity.

ABO Blood-Group System ; genetics ; Alleles ; Asian Continental Ancestry Group ; genetics ; Exons ; genetics ; immunology ; Female ; Genotype ; Humans ; Male ; Mutation, Missense ; genetics ; Pedigree ; Phenotype

OBJECTIVETo observe the injury on micro-skin induced by a self designed micro-skin machine.

METHODSMicro-skin was produced either with the machine or by hand. Cells at the edge of micro-skin were observed by transmission electron microscope. succinic dehydrogenase activity in supernatant of cultivated cells was analyzed, and the cell proliferation of micro-skin was assessed by (3)H-TdR. Twenty patients were enrolled in the study for the observation of the wound healing time between the two groups of micro-skin after being grafted.

RESULTSTransmission electron microscope examination revealed that the cellular injury at the edge of the micro-skin in machine-made group was mild compared with that in man-made group. (3)H-TdR rate was elevated but the activity of succinic dehydrogenase in the supernatant of cultured cells decreased in supernatant of cultured cells of machine produced micro-skin. Wound healing time was shortened in machine made group. (P < 0.05).

CONCLUSIONThe cellular injury at the edge of micro-skin in the machine made group was mild when compared with that in the man-made group with cell proliferation accelerated and wound healing time shortened.

Burns ; surgery ; Cell Division ; Epithelium ; pathology ; Humans ; Microscopy, Electron ; Skin ; ultrastructure ; Skin Transplantation ; methods ; Wound Healing