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Objective To investigate diagnostic value of miRNAs for esophageal cancer .Methods Blood samples were obtained from patients with esophageal cancer .MiRNAs including miR-30a ,miR-126a ,let-7b were detected and compared with healthy con-trols .After adjusting smoking and insobriety ,Logistic regression was used to judging which was best for clinical guidance .ROC curve was used to analyzed the clinic value of these miRNAs .Results Age of the two groups had no significant difference(P>0 .05) .The levels of smoking ,alcoholism and family history of cancer in the two groups had significant difference (P<0 .05) .Two groups′miR-30a levels in plasma had no significant differences (P>0 .05) .Let-7b and miR-126a levels in plasma in patients with e-sophageal were significantly higher in healthy volunteers (P<0 .05) .After adjustment the factors of smoking and alcohol ,let-7b lev-els were associated with the risk of esophageal cancer (P<0 .05) .Area under the ROC curve was 0 .712 ,which had a certain esoph-ageal cancer diagnostic performance .Conclusion Plasma let-7b could be treated as a biomarker for the diagnosis of esophageal canc-er.
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Objective To investigate diagnostic value of miRNAs for esophageal cancer .Methods Blood samples were obtained from patients with esophageal cancer .MiRNAs including miR-30a ,miR-126a ,let-7b were detected and compared with healthy con-trols .After adjusting smoking and insobriety ,Logistic regression was used to judging which was best for clinical guidance .ROC curve was used to analyzed the clinic value of these miRNAs .Results Age of the two groups had no significant difference(P>0 .05) .The levels of smoking ,alcoholism and family history of cancer in the two groups had significant difference (P<0 .05) .Two groups′miR-30a levels in plasma had no significant differences (P>0 .05) .Let-7b and miR-126a levels in plasma in patients with e-sophageal were significantly higher in healthy volunteers (P<0 .05) .After adjustment the factors of smoking and alcohol ,let-7b lev-els were associated with the risk of esophageal cancer (P<0 .05) .Area under the ROC curve was 0 .712 ,which had a certain esoph-ageal cancer diagnostic performance .Conclusion Plasma let-7b could be treated as a biomarker for the diagnosis of esophageal canc-er.
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Objective:To predict the basic physicochemical properties ,structure and function ,and linear B-cell epitopes of the capsid protein VP1 of coxsackievirus A6(CVA6).Methods: The amino acid sequence of the CVA6 VP1 was analyzed using Bioedit software and various online tools including SubLoc ,TargetP and the others from ExPASy Bioinformatics Resource Portal.Results: The CVA6 VP1 protein was a hydrophilic protein with a relative molecular weight of 33.6 kD and an isoelectric point of 7.92.This protein containsed 24 phosphorylation sites , but no signal peptide , transmembrane domains and possible fatty acylation sites.Its secondary structure was characterized by the richest random coils , and 48.52 percent of its amino acid residues exposed at the solution inter-face.Epitope prediction by Bepipred showed a number of potential B cell epitopes in the protein ,the highest antigenicity index among them located in the region of amino acids residue 155-165.Conclusion:The basic physicochemical properties ,structure and function characteristics ,and potential linear B-cell epitopes of CVA 6 VP1 were successfully predicted , which laid foundations for the further study on the protein and the preparation of vaccines and immunological diagnostic reagents for CVA 6 infection.
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Objective To investigate the expressions of B7-H1 and PD-1 on peripheral leukocytes from patients with Henoch-Schtmlein purpura(HSP)and their significance.Methods Peripheral leukocytes were obtained from 36 patients with HSP(HSP group)and 24 healthy human controls(control group).Immunofluorescent staining and flow cytometry were used to measure the expressions of B7-H1 and PD-1 on peripheral leukocytes.The expression of both two molecules was compared between the HSP group and control group as well as between patients with Henoch-Sch(o)nlein purpura nephritis(HSPN)(n=9)and those without(n=27).SPSS 12.0 for Windows was used for data analysis.Results The expression rate of B7-H1 on monocytes significantly increased(24.43%±25.79%vs 7.69%±8.31%,t=3.62,P＜0.011),while that of PD-1 decreased(0.84%±1.96%vs 2.28%±1.95%,t=2.78,P＜0.01)in HSP group compared with those in the control group.No significant difference was revealed in the expression of B7-H1 or PD-1 on lymphocytes between HSP group and control group(both P＞0.05).There was a significant increase in the expression of B7-H1 on monocytes(44.81%±12.36%vs 17.63%±25.63%,t=3.05,P＜0.01)and lymphocytes(8.78%±2.10%vs 5.65%±3.96%,t=2.25,P＜0.05)in patients with HSPN compared with those without.Conclusion There is a high expression of B7-H1.but low expression of PD-1 on peripheral blood monocytes from patients with HSP.suggesting that B7-H1 and PD-1 may play a certain role in the Dathogenesis of HSP.
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To obtain single chain variable fragment (scFv) and bivalent single chain variable fragment (bsFv) against transferrin receptor, up-stream and down-stream primers were designed according to the complementary sequences of FR1 region of variable heavy (VH) and FR4 of variable light (VL), respectively, which contained inter-linker G4S and the restriction endonuclease SfiI, AscI and NotI. Two pieces of scFv fragments were first amplified through PCR and then inserted into plasmid pAB1, which could express scFv protein once induced by IPTG in the host bacteria. To express scFv and bsFv, E. coli TG1 was cultured in LB broth and was induced by IPTG. The restriction enzyme digestion map and DNA sequencing demonstrated that scFv and bsFv genes were successfully inserted into the expression plasmid. SDS-PAGE and Western blotting revealed the protein band at 35kD and 60kD, which were consistent with the molecular weight of scFv and bsFv respectively. Flow cytometry showed that scFv and bsFv harbored the specific binding activity with TfR expressed in various tumor cells, and the avidity of bsFv was higher than that of the parent scFv.
Subject(s)
Base Sequence , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Genetic Vectors/genetics , Hep G2 Cells , K562 Cells , Molecular Sequence Data , Receptors, Transferrin/immunology , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Single-Chain Antibodies/biosynthesis , Single-Chain Antibodies/geneticsABSTRACT
The purpose of this study was to construct expression vectors of idiotype (Id) SmIg in patients with B-chronic lymphocytic leukemia and to express them in E.coli to obtain recombinant Id, and to investigate the effect of the protein on the proliferation and secretion of IL-2 and IFN-gamma of stimulated peripheral blood mononuclear cells (PBMC) in vitro. Light chain gene and Fd fragment of heavy chain gene were inserted into fd-tet-DOG2 vector to construct fd-tet-DOG2-Fab. Fab gene was further cloned into expression vector pHEN2 to construct the soluble expression vector pHEN2-Fab. After induction by IPTG, Fab protein was purified by Ni-NTA-chromatography. MTT was used to determine the effects of purified protein on the proliferation of stimulated PBMC in vitro and the concentrations of IL-2 and IFN-gamma in the culture supernatants were detected by ELISA. The results showed that recombinant pHEN2-Fab expression vector was constructed successfully. Fab protein was expressed in positive clone after induced by IPTG and two specific bands at 24-25 kD position were observed by SDS-PAGE electrophoresis. Proliferation of PBMC could be induced by purified Fab and the concentrations of IL-2 and IFN-gamma in culture supernatants were increased significantly after induction. It was suggested that the expression vector of SmIg Fab fragment was constructed successfully, and expressed and secreted from E. coli. The Fab protein could induce proliferation of PBMC and promote secretion of IL-2 and IFN-gamma.
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Objective To construct vector expressing soluble mPDL1-hIgGFc and study its effect on the proliferation and apeptosis of cells in vitro. Methods The extrncellular domain of mPDL1 gene was amplified from pmPDL1 vector by PCR and inserted into phIgGFc vector. The recombinant pmPDL1-hIgGFc was transfected into CHO cells by LipofectAMINETM2000, and the transfected cells were named as CHOp. The expression of mPDL1-hIgGFc in the culture supernatants of CHOp was assayed by ELISA and Western blot. The effects of CHOp culture supernatants on mixed lymphocyte culture(MLC) was analysed by Flowm-etry. Results The extracellular domain of mPDL1 gene were obtained from PCR. DNA sequencing and the identification of digestion by HindⅢ and KpnⅠ indicated the recombinant plasmid pmPDL1-hIgGFc was suc-cessfully constructed. ELISA and Western blot analysis proved that the CHOp could express mPDL1-hIgG-Fc. CHOp culture supernatants could inhibit lymphocyte proliferation and induce the apoptosis of the activa-ted T cells in MLC in vitro in a dose-dependent manner. Conclusion The mPDL1-hIgGFc protein could in-hibit lymphocyte proliferation and induce the apoptosis of the activated T cells.
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In order to investigate the expression of endothelin receptor B (ETR-B) in human malignant melanoma (MM) cells A375 and SK-mel-1 and the proliferative effects of endothelin 3 (ET3) on A375 cells, RT-PCR was applied to detect the expression of ETR-B gene in human MM cells A375 and SK-mel-1. MTT method was used to evaluate the growth enhancing effects of ET3 on A375 cell line in vitro. The results showed that ETR-B gene was expressed in both MM A375 and SK-mel-1 cells. ET3 had stronger ability to enhance the proliferation of A375 cells in vitro in a concentration-dependent manner. It was suggested that ET3/ETR-B might play an important proliferative role in MM.
Subject(s)
Cell Line, Tumor , Cell Proliferation/drug effects , Endothelin-3/pharmacology , Melanoma/metabolism , Melanoma/pathology , Receptor, Endothelin B/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
In order to investigate the expression of endothelin receptor B (ETR-B) in human ma-lignant melanoma (MM) cells A375 and SK-mel-1 and the proliferative effects of endothelin 3 (ET3) on A375 cells, RT-PCR was applied to detect the expression of ETR-B gene in human MM cells A375 and SK-mel-1. MTT method was used to evaluate the growth enhancing effects of ET3 on A375 cell line in vitro. The results showed that ETR-B gene was expressed in both MM A375 and SK-mel-1 cells. ET3 had stronger ability to enhance the proliferation of A375 cells in vitro in a con- centration-dependent manner. It was suggested that ET3/ETR-B might play an important proliferative role in MM.
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Objective To explore the effects of SLC concentration gradient on suppression of tumor immune escape. Methods According to different SLC concentration, there were six groups. The HLA-Ⅰexpression and apoptosis of MCF-7 were detected with FCM,and intracellular BCL-2 expression was analyzed by western blot. The production of TGF-? was detected with ELISA. Results In a certain range of concentration gradient, following SLC increase, HLA-Ⅰexpression level on MCF-7 was improved, and apoptosis was induced but BCL-2 expression was enhanced. Moreover, the secretion of TGF-? was suppressed. Conclusion SLC inhibites tumor immune escape.
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The expression vector of SmIg scFv fragment was constructed in patient with B cell chronic lymphocyte leukemia (B-CLL) and expressed in E. coli to obtain scFv fragment, and the effect of the protein on the proliferation of stimulated peripheral blood mononuclear cells (PBMC) was investigated in vitro. Two pairs of primers were designed, and variable region genes of light chain and heavy chain were amplified by PCR respectively from the pGEM-T vectors previously constructed in our laboratory which containing light chain gene or Fd fragment of heavy chain gene. The PCR product was digested, purified and inserted into pHEN2 vector to construct the soluble expression vector pHEN2-scFv. After the induction by IPTG, the scFv protein was identified by SDS-PAGE electrophoresis and purified by Ni-NTA-Chromatography. MTT was used to determine the effect of purified protein on the proliferation of stimulated PBMC in vitro. Plasmid PCR and restriction enzyme digestion of pHEN2-scFv revealed the pHEN2-scFv vector was constructed successfully. Id-scFv protein was expressed in positive clone after induced by IPTG. SDS-PAGE analysis showed that the relative molecular weight of fusion protein was about 30 kD (1 kD= 0.9921 ku), which was consistent with the theoretically predicted value. Proliferation of PBMC could be induced by purified Id-scFv. It was suggested that the expression vector of SmIg scFv fragment was constructed successfully, and scFv protein was expressed and secreted from E. coli, which could induce proliferation of PBMC. This may lay an experimental foundation for further research of Id-HSP complex vaccine for B-CLL.
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AIM: To analyze curative effect of four regulating-intestines prescriptions (Wumei Wan, Baitouweng Tang, Shenling Baizhu San and Tongxie Yao-fang) on the treatment of ulcerative colitis (UC) through summing up the previous animal experimental results.METHODS: We collected the conclusions in the papers related to four regulating-intestines prescriptions for the treatment of UC which were published recently, made comparisons from the aspectsof symptoms, physical sign, pathological change, cytokine and its protein expression, blood adhesion molecule, cell apoptosis and controlling gene and analyzed the pathogenesis of UC and action mechanism of four regulating-intestines prescriptions. Four regulating-intestines prescriptions included Wumei Wan,Baitouweng Tang, Shenling Baizhu San and Tongxie Yaofang. Wumei Wan is from Treatise on Exogenous Febrile Diseases and Synopsis of the Golden Chamber, which consists of dark plum, asarum herb, dried ginger, Chinese goldthread, Chinese angelica root, aconite root, pricklyash peel, cassia twig,ginseng (sun-dried ginseng) and bark of cork tree and has marked effect in treating colic caused by ascariasis and persistent dysentery. Baitouweng Tang, from Treatise on Exogenous Febrile Diseases, consists of medicinal herbs such as pulsatilla root, Chinese goldthread, bark of cork tree and ash bark, which has functions of clearing away the heat-evil, expelling superficial evils and relieving dysentery. In addition, it has marked effect in treating heat-type dysentery. Shenling Baizhu San, from Prescriptions of Peaceful benevolent Dispensary, consists of medicinal herbs such as pulp of lotus seed, coix seed, amomum fruit, balloon flower root, white hyacinth bean, poria, ginseng (sun-dried ginseng), glycyrrhiza, bighead atractylodes and rhizoma dioscoreae, which has the nature of replenishing qi to invigorate the spleen and eliminate wetness to arrest diarrhea and has marked effect on treating diarrhea due to the hypofunction of spleen. Tongxie Yao-fang which is from The Complete Works of Zhang Jingyue consists of four herbals of agehead atractylodes, root of herbaceous peony, dried tangerine peel and ledebouriella root and has the functions of soothing liver and invigorating spleen and stopping diarrhea, and has marked effect on treating liver sthenia and deficient spleen, borborygmus and abdominal pain and diarrhea.RFSULTS: ① Because UC is a chronic protracted dysentery with deficiency of vital energy and existing of evil energy, the vital energy will be harmed if its treatment is specialized in removing and dissolving the stagnation, evil energy will continue to exist and stagnation will continue to accumulate if its treatment is specialized in strengthening vital energy and inducing astringency. Only supporting healthy energy and expelling evil energy is the correct therapy method. This is in accordance with the main treatment of Wumei Wan ②Eliminating dampness and pathogenic heat from the blood to treat diarrhea is the main treatment method of Baitouweng Tang, and this is incompletely suitable for the treatment of UC.③Shenling Baizhu San had the effect on soothing liver and invigorating spleen and stopping diarrhea, and this is also incompletely suitable for the treatment of UC. ④ The prescription of Tongxie Yaofang is used for treating diarrhea caused by deficient spleen and liver sthenia, and spleen controlled by liver, and abnormal ascent and descent. It accords with main pathogenesis of UC, deficient spleen, excessive dampness of deficient spleen and it is weaker in invigorating the spleen in catabasis of UC than Shenling Baizhu San. Therefore, Wumei Wan has the best curative effect,Baitouweng Tang the second, Shengling Baizhu San the third and Tongxie Yaofang a little poor.CONCLUSION: UC belongs to recurrent dysentery, and its pathogenesis is in accordance with the main treatment of Wumei Wan, but not the other three prescriptions, so Wumei Wan is the most efficient prescription in treating ulcerative colitis. Diagnosis and treatment based on an overall analysis of signs and symptoms of TCM is the premise of obtaining the best curative effect. Modeling of animal tests must be consistent with the type of syndrome of the traditional Chinese medicine. Ating ulcerative colitis (UC) from aspects such as symptom, physical sign, pathological changes, adherence factor, cytokine and its protein expression, apoptosis and its controlling gene by means of modeling, which proves their functions and effects are different and their curative effect are also different due to their different ingredient though they all have the functions of treating. Results of this test show Wumei Wan has the best curative effect, Baitouweng Tang the second, Shenling Baizhu San the third and Tongxie Yaofang a little poor[1-11] Mechanism of four regulating-intestines prescription in the treatment of ulcerative colitis is discussed from the viewpoints of traditional Chinese medicine as follows.
ABSTRACT
The expression vector of SmIg scFv fragment was constructed in patient with B cell chronic lymphocyte leukemia (B-CLL) and expressed in E. coli to obtain scFv fragment, and the effect of the protein on the proliferation of stimulated peripheral blood mononuclear cells (PBMC) was investigated in vitro. Two pairs of primers were designed, and variable region genes of light chain and heavy chain were amplified by PCR respectively from the pGEM-T vectors previously constructed in our laboratory which containing light chain gene or Fd fragment of heavy chain gene. The PCR product was digested, purified and inserted into pHEN2 vector to construct the soluble expression vector pHEN2-scFv. After the induction by IPTG, the scFv protein was identified by SDSPAGE electrophoresis and purified by Ni-NTA-Chromatography. MTT was used to determine the effect of purified protein on the proliferation of stimulated PBMC in vitro. Plasmid PCR and restriction enzyme digestion of pHEN2-scFv revealed the pHEN2-scFv vector was constructed successfully. Id-scFv protein was expressed in positive clone after induced by IPTG. SDS-PAGE analysis showed that the relative molecular weight of fusion protein was about 30 kD (1 kD=0.9921 ku),which was consistent with the theoretically predicted value. Proliferation of PBMC could be induced by purified Id-scFv. It was suggested that the expression vector of SmIg scFv fragment was constructed successfully, and scFv protein was expressed and secreted from E. coli, which could induce proliferation of PBMC. This may lay an experimental foundation for further research of IdHSP complex vaccine for B-CLL.
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The relationship between bone mineral density (BMD) and Zn, Cu, Ca levels in the meal and hair of urban and rural elderly people were studied. 470 subjects above 60 years old (urban 205 and rural 265), 178 males with an average age of 65.70 +/- 3.48 and 292 females with an average age of 65.90 +/- 4.02, were inquired. The BMD and Zn, Cu, Ca levels in the meal and hair were measured. The detected BMD in urban and rural female old people was significantly lower than that of the males; The contents of Ca and Zn in the meal of the urban females were significantly lower than those of the urban males; The Ca, Zn in the meal and Zn in the hair of the rural females were significantly lower than those of rural males (P < 0.05 or 0.01). The BMD, Ca intakes, Ca and Zn in the hair of the rural old people were significantly lower than those of the urban old people (P < 0.05 or 0.01). There was a correlation between BMD with the Ca, Zn of the hair and dietary Ca, Zn, Cu or between dietary Zn with Ca, Zn in the hair and Ca, Cu intakes. The Zn, Cu and Ca levels in the meal nutrients were correlated with BMD to some degrees. Lack of Ca and Zn in the meal can cause the reduction of BMD.
Subject(s)
Bone Density , Cadmium/analysis , Copper/analysis , Diet Surveys , Hair/chemistry , Nutritional Status , Osteoporosis/prevention & control , Rural Health , Zinc/analysisABSTRACT
The relationship between bone mineral density (BMD) and Zn, Cu, Ca levels in the meal and hair of urban and rural elderly people were studied. 470 subjects above 60 years old (urban 205 and rural 265), 178 males with an average age of 65.70±3.48 and 292 females with an average age of 65.90±4.02, were inquired. The BMD and Zn, Cu, Ca levels in the meal and hair were measured. The detected BMD in urban and rural female old people was significantly lower than that of the males; The contents of Ca and Zn in the meal of the urban females were significantly lower than those of the urban males; The Ca, Zn in the meal and Zn in the hair of the rural females were significantly lower than those of rural males (P＜ 0.05 or 0.01). The BMD, Ca intakes, Ca and Zn in the hair of the rural old people were significantly lower than those of the urban old people (P＜0.05 or 0.01). There was a correlation between BMD with the Ca, Zn of the hair and dietary Ca,Zn, Cu or between dietary Zn with Ca, Zn in the hair and Ca, Cu intakes. The Zn, Cu and Ca levels in the meal nutrients were correlated with BMD to some degrees. Lack of Ca and Zn in the meal can cause the reduction of BMD.
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The variable heavy chain region (VH) genes of 3 untreated patients with B-cell chronic lymphocytic leukemia (B-CLL) were cloned and analyzed. The VH family used was VH3-11, VH3-72 and VH3-33. More than 2% difference from the corresponding germline gene was detected in all the 3 obtained potential functional genes (average 16.7). Mutation pattern analysis indicated evidence of antigen selective pressure observed in 1 of 3 cases. Our findings suggested that the tumor cells originate from post-GC cells.
Subject(s)
Amino Acid Sequence , Gene Rearrangement , Immunoglobulin Variable Region/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Molecular Sequence Data , MutationABSTRACT
In order to further investigate the effect of annexin II (Ann-II) on tissue plasminogen activator (t-PA)-dependent plasminogen (PLG) activation and its interactive mechanism, recombinant native Ann-II bound t-PA, PLG and plasmin with high affinity was examined. The flow cytometric assay showed that the ann-II expression rate was higher in the human umbilical vein endothelial cell (HUVEC) (87.65%) than in the HL-60 cells as controls (35.79%). Two irrelevant proteins, bovine serum albumin (BSA) and equine IgG (EIG) had no effect on the production of plasmin. Ann-II-mediated enhancement of t-PA-dependent PLG activation was inhibited by epsilon-aminocaproic acid or by pretreatment of Ann-II with carboxypeptidase B with the inhibitive rate being 77.8% and 77.0%, respectively. It was revealed that the effect of Ann-II on PLG activation was specific for t-PA. Urokinase didn't bind to Ann-II, demonstrating the role of receptor-related lysine residues on activation of PLG, showing that the Ann-II-PLG interaction was dependent upon carboxyl-terminal lysine residues. These findings suggest that annexin II-mediated co-assembly of t-PA and PLG may promote plasmin generation and play a key role in modulating fibrinolysis on the endothelial surface.
Subject(s)
Annexin A2/pharmacology , Cells, Cultured , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Fibrinolysis , Plasminogen/metabolism , Recombinant Proteins/pharmacology , Tissue Plasminogen Activator/metabolism , Umbilical Veins/cytologyABSTRACT
In order to further investigate the effect of annexin II (Ann-II) on tissue plasminogen activator (t-PA)-dependent plasminogen (PLG) activation and its interactive mechanism, recombinant native Ann-II bound t-PA, PLG and plasmin with high affinity was examined. The flow cytometric assay showed that the ann-II expression rate was higher in the human umbilical vein endothelial cell (HUVEC) (87.65%) than in the HL-60 cells as controls (35.79%). Two irrelevant proteins, bovine serum albumin (BSA) and equine IgG (EIG) had no effect on the production of plasmin. Ann-II-mediated enhancement of t-PA-dependent PLG activation was inhibited by epsilon-aminocaproic acid or by pretreatment of Ann-II with carboxypeptidase B with the inhibitive rate being 77.8% and 77.0%, respectively. It was revealed that the effect of Ann-II on PLG activation was specific for t-PA. Urokinase didn't bind to Ann-II, demonstrating the role of receptor-related lysine residues on activation of PLG, showing that the Ann-II-PLG interaction was dependent upon carboxyl-terminal lysine residues. These findings suggest that annexin II-mediated co-assembly of t-PA and PLG may promote plasmin generation and play a key role in modulating fibrinolysis on the endothelial surface.
Subject(s)
Humans , Annexin A2 , Pharmacology , Cells, Cultured , Endothelium, Vascular , Cell Biology , Metabolism , Fibrinolysis , Plasminogen , Metabolism , Recombinant Proteins , Pharmacology , Tissue Plasminogen Activator , Metabolism , Umbilical Veins , Cell BiologyABSTRACT
The variable heavy chain region (VH) genes of 3 untreated patients with B-cell chronic lymphocytic leukemia (B-CLL) were cloned and analyzed. The VH family used was VH3-11, VH3-72 and VH3-33. More than 2% difference from the corresponding germline gene was detected in all the 3 obtained potential functional genes (average 16.7). Mutation pattern analysis indicated evidence of antigen selective pressure observed in 1 of 3 cases. Our findings suggested that the tumor cells originate from post-GC cells.
Subject(s)
Humans , Amino Acid Sequence , Gene Rearrangement , Immunoglobulin Variable Region , Genetics , Leukemia, Lymphocytic, Chronic, B-Cell , Genetics , Molecular Sequence Data , MutationABSTRACT
In order to study the role of annexin II, a recombinant expression vector, pZeoSV2(+) ANN II, containing the annexin II cDNA, was developed. The 1.1-kb-length annexin II cDNA was inserted into a expression vector, PZeoSV(+) and transfected into HL-60 cells which had low baseline expression of Ann- II. pZeoSV(+) ANN II was analyzed by restriction mapping and the Ann- II sequence identified. The ability of the transfected cells, non-transfected and mock-transfected cells to stimulate t-PA-depend plasminogen activation was compared. The results showed that HL-60 with pZeoSV(+) ANN II transfection could significantly increase the plasminogen activation (8.9 +/- 1.2 U) in vitro with the difference being significant as compared with non-transfected (1.5 +/- 0.4 U) and mock-transfected cells (4.2 +/- 0.9 U), respectively. Antiannexin II oligonucleotides significantly inhibited the binding ability of t-PA and plasminogen to annexin II, and obviously reduced the plasminogen activation in vitro. The above findings showed human umbilical vein endothelial cells (HUVECs) treated with sense or missense oligonucleotides indicated no significant change in binding of t-PA and PLG. Treatment of HUVECs with antiannexin II oligonucleotides could significantly reduce the plasminogen activation by 2.4 +/- 0.3 U as compared with sense oligonucleotide group in binding of t-PA and PLG. These results, therefore, suggest that Ann- II can bind plasminogen and participate in the stimulation of t-PA-dependent activation of plasminogen, and that interference with Ann-II mRNA by antisense oligonucleotide may be a new strategy for the therapy of bleeding in patients with hyperfibrinolysis.